| Title;Studies of diagnosis and therapy of intractable disease state in systemic autoimmune disease. Study of clinical and pathogenic significance of anti-GAPDH antibody. |
| Author; TAKASAKI YOSHINARI (Juntendo Univ., School of Medicine, JPN) FURUHATA NAOKO (Juntendo Univ., School of Medicine, JPN)YAMADA HIROSHI (Juntendo Univ., School of Medicine, JPN) NAWATA MASUYUKI (Juntendo Univ., School of Medicine, JPN) IKEDA KEIGO (Juntendo Univ., School of Medicine, JPN) MATSUSHITA MASAKAZU (Juntendo Univ., School of Medicine, JPN) MATSUDAIRA RAN(Juntendo Univ., School of Medicine, JPN) KANEDA KAZUHIKO (Juntendo Univ., School of Medicine, JPN) ASANO MASANAO (Juntendo Univ., School of Medicine, JPN) |
| Journal Title;Zenshinsei Jiko Men'eki Shikkan ni okeru Nanchisei Byotai no Shindan to Chiryoho ni kansuru Kenkyu Heisei 16 Nendo Sokatsu, Buntan Kenkyu Hokokusho |
| Journal Code:N20051360 |
| ISSN: |
| VOL.;NO.;PAGE.42-45(2005) |
| Figure&Table&Reference;FIG.4, REF.4 |
| Pub. Country;Japan |
| Language;Japanese |
| Abstract;The involvement of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in autoantibody production for the proliferating cell nuclear antigen (PCNA) protein complex and the clinical and pathogenic significance of the anti-GAPDH antibody were examined. The subjects were systemic lupus erythematosus of 76 cases, scleroderma of 21 cases, articular rheumatism of 24 cases, multiple myositis/dermatomyositis of 18 cases, mixed connective tissue disease of 20 cases and Sjoegren syndrome of 18 cases. A possibility that GAPDH is one of the pivots inducing immune response for PCNA protein complex and a possibility that GAPDH is involved in disease state formation of renal lesion were indicated. |
Immunobiology. 2011 Jun 12.
Source
Zentrum fuer Zahn-, Mund- und Kieferheilkunde, Justus-Liebig-Universitaet Giessen, Department of Periodontology, Schlangenzahl 14, 35392 Giessen, Germany.
Abstract
The up-regulation of the B7-H1 receptors in host cells might influence the chronicity of inflammatory disorders that frequently precede the development of human cancers. B7-H1 expression has been detected in the majority of human cancers, leading to anergy and apoptosis of activated T cells, and enabling tumor cells to overcome host response. Porphyromonas gingivalis (P. gingivalis), a putative periodontal pathogen, is an etiologic agent of periodontitis and expresses a variety of virulence factors. In this study, the expression of B7-H1 and B7-DC receptors on squamous cell carcinoma cells SCC-25 and BHY and primary human gingival keratinocytes (PHGK) was analyzed after infection with two virulent P. gingivalis strains in vitro. After 48h, the cells were stained with antibodies for human B7-H1 and B7-DC and further analyzed by flow cytometry. RNA was extracted and gene expression of B7-H1 or B7-DC was quantified by real time PCR. After infection with P. gingivalis, both B7-H1 and B7-DC receptors were up-regulated. The mean fluorescence intensity (MFI) increased from 4.5 to 9.9 (B7-H1) and from 6.9 to 15.0 (B7-DC) (p<0.05, respectively) in SCC-25 cells. PHGK showed an increase from 4.8 to 12.4 (B7-H1) and from 5.5 to 15.6 (B7-DC) (p<0.05, respectively). Streptococcus salivarius K12, a commensal bacterium, caused no up-regulation. After 24h, the expression of B7H1 and B7-DC mRNA in infected cells, normalized to GAPDH and in relation to non-infected cells, was 6.4 fold (B7-H1) and 8.6 fold (B7-DC) higher. In PHGK B7-H1/DC mRNA expression increased 8.2 fold (B7-H1) and 5.9 fold (B7DC) (p<0.05) respectively. The results of the study demonstrate that in contrast to S. salivarius K12 virulent P. gingivalis strains are able to induce the expression of the B7-H1 and B7-DC receptors in squamous carcinoma cells and human gingival keratinocytes, which might facilitate immune evasion by oral cancers.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2011 Apr;29(2):199-202.
[Article in Chinese]
Source
Research Center for Stomatology, Stomatological Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an 710004, China.
Abstract
OBJECTIVE:
To clone the glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli.
METHODS:
GAPDH was obtained by PCR and was inserted into cloning vector pMD-18-T to construct clone recon. Double enzymes digest the recon pMD18-T-GAPDH and the prokaryotic expression vector pET-32a and then connect to get the expressing recon pET-32a-GAPDH. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 and induced the expression of GAPDH with isopropyl beta-D-1-thiogalactopyranoside (IPTG) of different density.
RESULTS:
DNA sequencing showed that the fragment was 99.802% the same to the sequence published in NCBI. Under the best density, IPTG could be highly expressed.
CONCLUSION:
The GAPDH had been successfully cloned and expressed in E. coli which gets ready for the following experiment to study the immunity of GAPDH and the homologues antibody preparation.
Microbiology. 2011 Aug;157(Pt 8):2328-38. Epub 2011 May 5.
Source
Dresden University of Technology, Medical Faculty Carl Gustav Carus, Institute of Medical Microbiology and Hygiene, Fetscherstrasse 74, D-01307 Dresden, Germany.
Abstract
In different, phylogenetically unrelated micro-organisms, glycolytic enzymes play a dual role. In the cytosol they are involved in metabolic reactions whereas the surface-localized fraction of the enzymes contributes to adhesion and virulence. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a typical member of this group of multifunctional proteins. In this study, we characterized the GAPDH of Mycoplasma pneumoniae, a common pathogen of the human respiratory mucosa. Full-length GAPDHof M. pneumoniae was successfully expressed and used to produce a polyclonal antiserum. By immunofluorescence, colony blot and ELISA experiments with different fractions of the M. pneumoniae proteins, GAPDH was demonstrated to be present in the cytosol and at even higher concentrations at the surface of mycoplasmas. Nevertheless, antibodies against recombinant GAPDH were not detected in sera of immunized animals or of patients with confirmed M. pneumoniae infection. RecombinantGAPDH bound to different human cell lines in a concentration-dependent manner, and binding was inhibited by specific anti-GAPDH serum. In contrast, this antiserum did not significantly influence the adherence of M. pneumoniae to HeLa cells. When different human extracellular matrix proteins were tested in Western blot assays, GAPDH bound to fibrinogen. The results showed that the GAPDH of M. pneumoniae is a member of the family of cytosol-localized glycolytic enzymes, which also occur at the surface of the bacterium, and mediates interactions with the extracellular matrix proteins of the human host. Thus, the surface-exposed fraction of GAPDH may be a factor that contributes to the successful colonization of the human respiratory tract by M. pneumoniae.
J Bacteriol. 2011 Jul;193(13):3356-66. Epub 2011 Apr 29.
Source
College of Veterinary Medicine, Gyeongsang National University, Gyeongnam 660-701, South Korea.
Abstract
Although Streptococcus parauberis is known as a bacterial pathogen associated with bovine udder mastitis, it has recently become one of the major causative agents of olive flounder (Paralichthys olivaceus) streptococcosis in northeast Asia, causing massive mortality resulting in severe economic losses. S. parauberis contains two serotypes, and it is likely that capsular polysaccharide antigens serve to differentiate the serotypes. In the present study, the complete genome sequence of S. parauberis (serotype I) was determined using the GS-FLX system to investigate its phylogeny, virulence factors, and antigenic proteins. S. parauberis possesses a single chromosome of 2,143,887 bp containing 1,868 predicted coding sequences (CDSs), with an average GC content of 35.6%. Whole-genome dot plot analysis and phylogenetic analysis of a 60-kDa chaperonin-encoding gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding gene showed that the strain was evolutionarily closely related to Streptococcus uberis. S. parauberis antigenic proteins were analyzed using an immunoproteomic technique. Twenty-one antigenic protein spots were identified in S. parauberis, by reaction with an antiserum obtained from S. parauberis-challenged olive flounder. This work provides the foundation needed to understand more clearly the relationship between pathogen and host and develops new approaches toward prophylactic and therapeutic strategies to deal with streptococcosis in fish. The work also provides a better understanding of the physiology and evolution of a significant representative of the Streptococcaceae.
J Orthop Res. 2011 Aug;29(8):1284-90. doi: 10.1002/jor.21406. Epub 2011 Mar 8.
Yurube T, Takada T, Hirata H, Kakutani K, Maeno K, Zhang Z, Yamamoto J, Doita M, Kurosaka M,Nishida K.
Source
Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
Abstract
House-keeping genes (HKGs) are generally used as endogenous controls for molecular normalization in quantitative PCR analysis. However, whether all the so-called HKGs are useful for intervertebral disc research is controversial. Our objective was, using a prevalidated rat tail static compression loading-induced disc degeneration model, to clarify the feasibility of common HKGs for gene-quantification in the nucleus pulposus cells. In real-time RT-PCR for five HKGs [β-actin, β-glucuronidase, β-2 microglobulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and lactate dehydrogenase A (LDHA)], static compression at 1.3 MPa for up to 56 days demonstrated messenger RNA (mRNA) expression levels of consistent β-2 microglobulin and GAPDH, slightly up-regulated β-glucuronidase, and fairly down-regulated β-actin and LDHA. Especially, β-actin had a drastic suppression to 0.15-fold in the loaded relative to unloaded discs at 7 days. In immunofluorescence, β-actin showed a significant down-regulation to almost undetectable levels from 28 days, while GAPDH was constantly detected throughout. β-Actin mRNA and protein-distribution are thought to be affected by the loading treatment, however, GAPDH mRNA and protein-distribution can retain relatively stable expressions. Under prolonged static compression, β-actin and probably LDHA are inappropriate, and GAPDH is a feasible HKG as internal references in the disc cells.
Copyright © 2011 Orthopaedic Research Society.
Anticancer Res. 2011 Feb;31(2):731-7.
Source
Department of Tumor Biology, Kagawa Prefectural University of Health Sciences, Takamastu 761-0123, Japan. hata@chs.pref.kagawa.jp
Abstract
AIM:
A monoclonal antibody that targeted vascular endothelial growth factor (VEGF) resulted in a dramatic suppression of tumor growth in vivo, which led to the development of bevacizumab, a humanized variant of anti-VEGF antibody, as an anticancer agent. The aims of this study were to clarify the significance of VEGF gene expression in relation to clinicopathological parameters and to identify potential candidates for anti-VEGF therapy with bevacizumab.
PATIENTS AND METHODS:
VEGF gene expression was analyzed by real-time quantitative reverse transcription-polymerase chain reaction in 178 surgical epithelial ovarian cancer specimens. This gene expression was correlated with clinicopathological parameters and patient survival.
RESULTS:
The median VEGF gene expression level and range relative to GAPDH were 0.147 and 0.016-2.44, respectively. Patients were dichotomized into two groups with low and high expression levels by using the median value as the cutoff. VEGF gene expression did not affect prognosis of patients overall (p = 0.541). Although statistical significance was not noted, we found the prognosis of patients with high VEGF gene expression tended to be worse than that of those with low VEGF gene expression by univariate Cox regression analysis (p = 0.085) in patients with stage III-IV cancer. Macroscopic residual disease (positive; p = 0.012) was significantly associated with poor prognosis in univariate Cox regression analysis in patients with stage III-IV cancer. Moreover, presence of macroscopic residual disease was positively associated with VEGF gene expression (p = 0.030) in patients with stage III-IV cancer.
CONCLUSION:
Patients with epithelial ovarian cancer with tumors with positive macroscopic residual disease and high VEGF gene expression could be potential candidates for anti-VEGF therapy with bevacizumab.
Oral Dis. 2011 May;17(4):433-42. doi: 10.1111/j.1601-0825.2010.01778.x. Epub 2011 Mar 2.
Source
Department of Oral and Maxillofacial Surgery, University of Erlangen-Nuremberg, Erlangen, Germany. falk.wehrhan@uk-erlangen.de
Abstract
OBJECTIVES:
Bone-destructive disease treatments include bisphosphonates and antibodies against receptor activator for nuclear factor κB ligand (aRANKL). Osteonecrosis of the jaw (ONJ) is a side-effect. Aetiopathology models failed to explain their restriction to the jaw. The osteoproliferative transcription factor Msx-1 is expressed constitutively only in mature jaw bone. Msx-1 expression might be impaired in bisphosphonate-related ONJ. This study compared the expression of Msx-1, Bone Morphogenetic Protein (BMP)-2 and RANKL, in ONJ-affected and healthy jaw bone.
MATERIAL AND METHODS:
An automated immunohistochemistry-based alkaline phosphatase-anti-alkaline phosphatase method was used on ONJ-affected and healthy jaw bone samples (n = 20 each): cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed to quantitatively compare Msx-1, BMP-2, RANKL and GAPDH mRNA levels.
RESULTS:
Labelling indices were significantly lower for Msx-1 (P < 0.03) and RANKL (P < 0.003) and significantly higher (P < 0.02) for BMP-2 in ONJ compared with healthy bone. Expression was sevenfold lower (P < 0.03) for Msx-1, 22-fold lower (P < 0.001) for RANKL and eightfold higher (P < 0.02) for BMP-2 in ONJ bone.
CONCLUSIONS:
Msx-1, RANKL suppression and BMP-2 induction were consistent with the bisphosphonate-associated osteopetrosis and impaired bone remodelling in BP- and aRANKL-induced ONJ. Msx-1 suppression suggested a possible explanation of the exclusivity of ONJ in jaw bone. Functional analyses of Msx-1- RANKL interaction during bone remodelling should be performed in the future.
© 2011 John Wiley & Sons A/S.
J BUON. 2010 Oct-Dec;15(4):758-62.
Tomuleasa C, Soritau O, Kacso G, Fischer-Fodor E, Cocis A, Ioani H, Timis T, Petrescu M, Cernea D,Virag P, Irimie A, Florian IS.
Source
Department of Cancer Immunology - Ion Chiricuta Oncology Institute, Cluj Napoca, Romania. ciprian.tomuleasa@gmail.com
Abstract
PURPOSE:
glioblastoma multiforme (GBM) still bears a very dismal prognosis even with complete resection followed by adjuvant chemoradiation. The aim of the current study was to evaluate in vitro the antitumor efficacy of arsenic trioxide (ATO) in combination with ionizing radiation plus temozolomide and bevacizumab against cultured glioblastoma stem-like cells, as possible way to increase the therapeutic index in patients diagnosed with recurrent, therapy-refractory GBM.
METHODS:
stem-like tumor cells isolated from a GBM biopsy were established by cell proliferation assays and upregulation of stem cell markers, as proven by reverse transcription - polymerase chain reaction (RT-PCR). Low concentrations of ATO were added prior to temozolomide, bevacizumab and ionizing irradiation.
RESULTS:
molecular analysis showed that cells expressed CXCR4, Oct-3/4 and GAPDH when compared to placental mesenchymal stem cells, as well as nestin, GFAP and neurofilament protein. Low concentrations of ATO led to morphologic differentiation, with fewer stem cells in Go state and differentiation-associated cytochemical features, like increased sensitivity to cytostatic drugs and radiotherapy.
CONCLUSION:
ATO exposure before conventional postoperative chemoradiotherapy for GBM might increase treatment efficacy. Further in vivo experiments on laboratory animals and analysis of absorption rate and side effects are required.
Mol Immunol. 2011 Feb;48(5):733-45. Epub 2010 Dec 30.
Source
Dept. of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, 91120 Jerusalem, Israel. laskov@md.huji.ac.il
Abstract
Epstein-Barr virus transforms human peripheral B cells into lymphoblastoid cell lines (LCL) that secrete specific antibodies. Our previous studies showed that a monoclonal LCL that secretes a rheumatoid factor expressed activation-induced cytidine deaminase (AID) and displayed an ongoing process of somatic hypermutation (SHM) at a frequency of 1.7×10⁻³ mut/bp in its productively rearranged IgVH gene. The present work shows that SHM similarly affects the nonproductive IgVH allele of the same culture. Sequencing of multiple cDNA clones derived from cellular subclones of the parental culture, showed that both alleles exhibited an ongoing mutational process with mutation rates of 2-3×10⁻⁵ mut/bp×generation with a high preference for C/G transition mutations and lack of a significant strand bias. About 50% of the mutations were targeted to the underlined C/G bases in the WRCH/DGYW and RCY/RGY hotspot motifs, indicating that they were due to the initial phase of AID activity. Mutations were targeted to the VH alleles and not to the Cμ or to the GAPDH genes. Genealogical trees showed a stepwise accumulation of only 1-3 mutations per branch of the tree. Unexpectedly, 27% of all the mutations in the two alleles occurred repeatedly and independently within certain sites (not necessarily the canonical hotspot motifs) in cellular clones belonging to different branches of the lineage tree. Furthermore, some of the mutations seem to arise as recurrent mutational clusters, independently generated in different cellular clones. Statistical analysis showed that it is very unlikely that these clusters were due to random targeting of equally accessible hotspots, indicating the presence of 'hypermutable sites' that generate recurring mutational clusters in the IgVH alleles. Intrinsic hypermutable sites may enhance affinity maturation and generation of effective mutated antibody repertoires against invading pathogens.
Copyright © 2010 Elsevier Ltd. All rights reserved.
Clin Exp Rheumatol. 2010 Jul-Aug;28(4 Suppl 60):S31-8. Epub 2010 Sep 23.
Source
Department of Dermatology, Yonsei University College of Medicine, Seoul, Korea. sbcho@yuhs.ac
Abstract
OBJECTIVES:
We evaluated the reactivity of sera from Behçet's disease (BD), systemic lupus erythematosus (SLE), dermatomyositis (DM), rheumatoid arthritis (RA), and Takayasu's arteritis (TA) patients against human α-enolase and streptococcal α-enolase, and identified additional streptococcal antigens.
METHODS:
Enzyme-linked immunosorbent assay (ELISA) and immunoblotting were performed using sera from patients with BD, SLE, DM, RA, and TA and healthy volunteers (control) against human α-enolase and streptococcal α-enolase. Immunoblot analysis and matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry were used to identify and recombine other streptococcal antigens.
RESULTS:
Specific positive signals against recombinant human α-enolase were detected by IgM ELISA of serum samples from 50% of BD, 14.3% of SLE, 57.1% of DM, 42.9% of RA, and 57.1% of TA patients. Specific positive signals against streptococcal α-enolase were detected from 42.9% of BD, 14.3% of DM, and 14.3% of TA patients. No SLE and RA sera reacted against streptococcal α-enolase antigen. Streptococcal proteins reacting with sera were identified as hypothetical protein (HP) for SLE and DM patients, acid phosphatase (AP) for RA patients, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for TA patients.
CONCLUSIONS:
We observed that RA patients did not present serum reactivity against either HP orGAPDH though BD, SLE, DM, and TA patients did. Also, AP reacted with sera from BD, SLE, DM, RA, and TA patients.
Gen Physiol Biophys. 2010 Dec;29(4):396-401.
Source
Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava, Slovakia.
Abstract
Expression of drug-transporting P-glycoprotein (P-gp, an integral protein of the plasma membrane) in neoplastic cells confers multidrug resistance and also involves alteration of cell sensitivity to inhibitors of the sarco/endoplasmic reticulum calcium pump thapsigargin (Th). Mouse leukaemia L1210 cell sublines that overexpress P-gp due to selection with vincristine (R) or stable transfection with a gene encoding human P-gp (T) were less sensitive to Th than the parental cell line (S). Th at a concentration of 0.1 μmol/l did not induce alterations in the amount of P-gp mRNA in R or T cells (S cells did not contain any measurable amount of this transcript as assessed by RT-PCR) or in the amount of calnexin (CNX) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in all three cell sublines. However, when using a concentration of 10 μmol/l, Th decreases the amounts of CNX, GAPDH (in S, R and T cells) and P-gp (in R and T cells) mRNAs. In contrast to R and T cells (which contain abundant P-gp), S cells did not contain any P-gp detectable by the c219 antibody on a Western blot. Th at a concentration of 0.1 μmol/l induced a reduction in the amount of P-gp present in R and T cells, particularly in isoforms with higher molecular weights (i.e., mature fully glycosylated isoforms). Similar results were observed when Th was used at a concentration of 10 μmol/l. R and T cells contained lower levels of CNX than S cells. While Th at a lower concentration did not alter the levels of CNX in S, R or T cells, a higher concentration of this substance induced a measurable decrease in the amount of CNX. S, R and T cells did not differ with respect to GAPDH content, but Th induced a reduction in the amount of this protein in all cell sublines. More pronounced results were observed when Th was applied at a concentration of 10 μmol/l comparing with a concentration of 0.1 mmol/l. These changes may be involved together with the Th efflux activity of P-gp in Th-resistance associated with the P-gp-mediated multidrug resistance of R and T cells.
J Biomed Sci. 2010 Nov 13;17:86.
Source
Department of Pharmacy, Faculty of Medicine and Health Sciences, International Medical University, No 126 Jalan 19/155B Bukit Jalil, Kuala Lumpur, 57000 Malaysia.
Abstract
BACKGROUND:
Bacillus thuringiensis (Bt), an ubiquitous gram-positive spore-forming bacterium forms parasporal proteins during the stationary phase of its growth. Recent findings of selective human cancer cell-killing activity in non-insecticidal Bt isolates resulted in a new category of Bt parasporal protein called parasporin. However, little is known about the receptor molecules that bind parasporins and the mechanism of anti-cancer activity. A Malaysian Bt isolate, designated Bt18 produces parasporal protein that exhibit preferential cytotoxic activity for human leukaemic T cells (CEM-SS) but is non-cytotoxic to normal T cells or other cancer cell lines such as human cervical cancer (HeLa), human breast cancer (MCF-7) and colon cancer (HT-29) suggesting properties similar to parasporin. In this study we aim to identify the binding protein for Bt18 in human leukaemic T cells.
METHODS:
Bt18 parasporal protein was separated using Mono Q anion exchange column attached to a HPLC system and antibody was raised against the purified 68-kDa parasporal protein. Receptor binding assay was used to detect the binding protein for Bt18 parasporal protein in CEM-SS cells and the identified protein was sent for N-terminal sequencing. NCBI protein BLAST was used to analyse the protein sequence. Double immunofluorescence staining techniques was applied to localise Bt18 and binding protein on CEM-SS cell.
RESULTS:
Anion exchange separation of Bt18 parasporal protein yielded a 68-kDa parasporal protein with specific cytotoxic activity. Polyclonal IgG (anti-Bt18) for the 68-kDa parasporal protein was successfully raised and purified. Receptor binding assay showed that Bt18 parasporal protein bound to a 36-kDa protein from the CEM-SS cells lysate. N-terminal amino acid sequence of the 36-kDa protein was GKVKVGVNGFGRIGG. NCBI protein BLAST revealed that the binding protein was Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Double immunofluorescence staining showed co-localisation of Bt18 and GAPDH on the plasma membrane of the CEM-SS cells.
CONCLUSIONS:
GAPDH has been well known as a glycolytic enzyme, but recently GAPDH was discovered to have roles in apoptosis and carcinogenesis. Pre-incubation of anti-GAPDH antibody with CEM-SS cells decreases binding of Bt18 to the susceptible cells. Based on a qualitative analysis of the immunoblot and immunofluorescence results, GAPDH was identified as a binding protein on the plasma membrane of CEM-SS cells for Bt18 parasporal protein.
BMC Microbiol. 2010 Nov 9;10:280.
Source
Molecular Bacteriology and Immunology Group, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, UK.
Abstract
BACKGROUND:
Glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDHenzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility toantibodies was dependent on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion.
RESULTS:
In this study, expression of GapA-1 was shown to be well conserved across diverse isolates of Neisseria species. Flow cytometry confirmed that GapA-1 could be detected on the cell surface, but only in a siaD-knockout (capsule-deficient) background, suggesting that GapA-1 is inaccessible toantibody in in vitro-grown encapsulated meningococci. The role of GapA-1 in meningococcal pathogenesis was addressed by mutational analysis and functional complementation. Loss of GapA-1 did not affect the growth of the bacterium in vitro. However, a GapA-1 deficient mutant showed a significant reduction in adhesion to human epithelial and endothelial cells compared to the wild-type and complemented mutant. A similar reduction in adhesion levels was also apparent between a siaD-deficient meningococcal strain and an isogenic siaD gapA-1 double mutant.
CONCLUSIONS:
Our data demonstrates that meningococcal GapA-1 is a constitutively-expressed, highly-conserved surface-exposed protein which is antibody-accessible only in the absence of capsule. Mutation of GapA-1 does not affect the in vitro growth rate of N. meningitidis, but significantly affects the ability of the organism to adhere to human epithelial and endothelial cells in a capsule-independent process suggesting a role in the pathogenesis of meningococcal infection.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2010 Nov;26(11):1108-10.
[Article in Chinese]
Source
Department of Pathology, Kunming General Hospital of Chengdu Command, Kunming 650032, China. wenxing-zhao@sohu.com
Abstract
AIM:
To develop a SYBR Green I real-time fluorescence quantitative PCR for the detection of lymphocyte immune function.
METHODS:
The primers of NKG2D, perforin, granzyme B and keeping-home gene GAPDH were designed and synthesized according to NCBI gene sequences, and the real-time fluorescence quantitative PCR was established. The gene expressions of NKG2D, perforin and granzyme B in lymphocytes from cancer patients and CIK induced from the cancer patients lymphocytes in vitro were quantified by real-time fluorescence quantitative PCR.
RESULTS:
NKG2D, perforin and granzyme B mRNA could be specifically amplified and quantitatively detected by the SYBR Green I real-time fluorescence quantitative PCR according to agarose gel electrophoresis and melt curve analysis. The mRNA expression of granzyme B was reduced in lymphocytes from cancer patients, however the mRNA expressions of perforin and granzyme B were increased in CIK induced by cytokines and monoclone antibody compared with their lymphocytes(P<0.01).
CONCLUSION:
The SYBR Green I real-time fluorescence quantitative PCR is a useful method for the quantitative detection of NKG2D, perforin and granzyme B mRNA to investigate cellular immune function.
J Transl Med. 2010 Oct 13;8:96.
Source
Department of Oral and Maxillofacial Surgery University of Erlangen-Nuremberg, Germany. Falk.Wehrhan@uk-erlangen.de
Abstract
BACKGROUND:
Bone-destructive disease treatments include bisphosphonates and antibodies against the osteoclast differentiator, RANKL (aRANKL); however, osteonecrosis of the jaw (ONJ) is a frequent side-effect. Current models fail to explain the restriction of bisphosphonate (BP)-related and denosumab (anti-RANKL antibody)-related ONJ to jaws. Msx-1 is exclusively expressed in craniofacial structures and pivotal to cranial neural crest (CNC)-derived periodontal tissue remodeling. We hypothesised that Msx-1 expression might be impaired in bisphosphonate-related ONJ. The study aim was to elucidate Msx-1 and RANKL-associated signal transduction (BMP-2/4, RANKL) in ONJ-altered and healthy periodontal tissue.
METHODS:
Twenty ONJ and twenty non-BP exposed periodontal samples were processed for RT-PCR and immunohistochemistry. An automated staining-based alkaline phosphatase-anti-alkaline phosphatase method was used to measure the stained cells:total cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed on ONJ-affected and healthy jaw periodontal samples (n = 20 each) to quantitatively compare Msx-1, BMP-2, RANKL, and GAPDH mRNA levels.
RESULTS:
Semi-quantitative assessment of the ratio of stained cells showed decreased Msx-1 and RANKL and increased BMP-2/4 (all p < 0.05) expression in ONJ-adjacent periodontal tissue. ONJ tissue also exhibited decreased relative gene expression for Msx-1 (p < 0.03) and RANKL (p < 0.03) and increased BMP-2/4 expression (p < 0.02) compared to control.
CONCLUSIONS:
These results explain the sclerotic and osteopetrotic changes of periodontal tissue following BP application and substantiate clinical findings of BP-related impaired remodeling specific to periodontal tissue. RANKL suppression substantiated the clinical finding of impaired bone remodelling in BP- and aRANKL-induced ONJ-affected bone structures. Msx-1 suppression in ONJ-adjacent periodontal tissue suggested a bisphosphonate-related impairment in cellular differentiation that occurred exclusively jaw remodelling. Further research on developmental biology-related unique features of jaw bone structures will help to elucidate pathologies restricted to maxillofacial tissue.
Biochim Biophys Acta. 2010 Sep;1799(9):642-52. Epub 2010 Aug 26.
Source
Institute of Biomedical and Biomolecular Science, University of Portsmouth, Portsmouth, UK.
Abstract
Antibodies to the six chicken histone H1 subtypes and the variant histone H5 have been used in immunoprecipitations of crosslinked chromatin fragments (xChIPs) to map linker histones across the β-globin locus and the widely expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and carbonic anhydrase (CA) genes in three cell types: 15-day embryo chicken erythrocytes, 15-day embryo chicken brain and the early erythroid cell line HD24. In erythrocytes, where the β-adult and β-hatching genes are active, the H1.01, H1.11L and H1.11R subtypes are substantially depleted throughout the β-globin locus and the neighboring heterochromatin, in contrast to the other four subtypes, in particular the more abundant H5. Active genes therefore carry high levels of some but not all linker histone subtypes. The situation is similar in HD24 cells, except that substantial depletions are found at the promoters of the adult β(A) and embryonic β(ρ) and β(ε) genes, despite these genes not yet being active in HD24 cells. The distributions in the brain tissue are characterised by the absence of H1.02, H1.03 and H5 from the hypersensitive site HS3 and from the β-adult 3' enhancer for the H1.11L and H1.11R subtypes. The data show that although linker histone subtypes play distinct cell-type specific roles in gene regulation, their widespread distribution indicates they are not intrinsically inhibitory to basic chromatin transactions.
Copyright © 2010 Elsevier B.V. All rights reserved.
Oncol Rep. 2010 Sep;24(3):677-85.
Source
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, CEP 31.270-901 Belo Horizonte, MG, Brazil.
Abstract
Breast cancer is a major health burden, responsible for >10% of all cases of cancer worldwide. Advances in breast cancer diagnosis and treatment have contributed to an improved rate of survival, although mortality rates remains significantly high. The establishment of breast cancer cell lines is an important model for understanding biological processes involved in this disease and for identifying potential therapeutic targets. The novel human breast cancer cell lines, MACL-1 and MGSO-3, were used in this study to identify possible surface antigens by antibodies directed against two commercial breast cancer cell lines MCF-7 and MDA-MB-231. We purified a 37 kDa antigen by affinity chromatography from MDA-MB-231, and its N-terminal amino acid sequence was homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Therefore, immunohistochemical experiments, using specific monoclonal antibodies, evidenced a co-localization of GAPDH and Na+/K+-ATPase on the surface of commercially available and recently established breast cancer cell lines. It is of note that Na+/K+-ATPase was used as a plasma membrane marker. This finding opens new perspectives for breast cancer diagnosis and treatment since GAPDH could be used as a biomarker or as a potential therapeutic target in breast cancer.
Bioorg Khim. 2010 May-Jun;36(3):312-8.
[Article in Russian]
Abstract
Haponin (HLDF-alike protein) was previously identified from the human promyelocytic leukemia HL-60 cell line. For the functional study of this protein, we obtained recombinant haponin with an N-terminal hexahistidine tag using a baculovirus expression system. Antibodies against 6xHis-haponin were produced, and the expression of endogenous haponin was demonstrated in mammalian cell lines of different origin. Using affinity chromatography and immunoprecipitation methods, we have shown that in CHO-K1 cells haponin interacts with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is one of the vital glycolytic enzymes with a diverse set of noncanonical functions.
J Immunol. 2010 Aug 1;185(3):1968-75. Epub 2010 Jul 7.
Source
Department of Neurology, University of California at Irvine, Irvine, CA 92697, USA.
Abstract
We have previously shown that B cells and Abs reactive with GAPDH and antitriosephosphate isomerase (TPI) are present in lesions and cerebrospinal fluid (CSF) in multiple sclerosis (MS). In the current study, we studied the effect of anti-GAPDH and anti-TPI CSF IgG on the glycolytic enzyme activity of GAPDH and TPI after exposure to intrathecal IgG from 10 patients with MS and 34 patients with other neurologic diseases. The degree of inhibition of GAPDH activity by CSF anti-GAPDH IgG in the seven MS samples tested varied from 13 to 98%, which seemed to correlate with the percentage of anti-GAPDH IgG in the CSF IgG (1-45%). Inhibition of GAPDH activity (18 and 23%) by CSF IgG was seen in two of the 34 patients with other neurologic diseases, corresponding to the low percentage of CSF anti-GAPDH IgG (1 and 8%). In addition, depletion of anti-GAPDH IgG from CSF IgG, using immobilized GAPDH, removed the inhibitory effect of the IgG on GAPDH. No inhibition of GAPDH activity was seen with CSF samples not containing anti-GAPDH IgG. No inhibition of TPI activity was seen with any purified CSF IgG sample. These findings demonstrate an increased percentage of anti-GAPDHAbs in the CSF of patients with MS that can inhibit GAPDH glycolytic enzyme activity and may contribute to neuroaxonal degeneration.
Int J Legal Med. 2010 Sep;124(5):397-404. Epub 2010 Jun 11.
Source
Department of Forensic Pathology, China Medical University School of Forensic Medicine, No.92, Beier Road, Heping District, Shengyang, Liaoning Province, 110001, People's Republic of China.
Abstract
The expression of the cannabinoid receptor type 2 (CB2R) was investigated by immunohistochemistry, Western blotting, and RT-PCR during wound healing of contused skeletal muscle in rats with attempt of its applicability to skeletal muscle wound age estimation. Furthermore, Macrophage Marker (MAC387) was utilized to identify macrophages recruited into injured skeletal muscle tissue. Co-localization of CB2R with Macrophage Marker was detected by confocal laser scanning microscopy. A total of 50 Sprague-Dawley male rats were divided into control and contusion groups (3 h, 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury). In the uninjured controls, immunoreactivity of CB2R was detected in the sarcolemma and sarcoplasm of normal myofibers. In the contusion groups, a few polymorphonulcear cells, a large number of macrophages, and spindle-shaped fibroblastic cells showed a positive staining for CB2R in wounded zones. By Western blotting analysis, the average of CB2R to GAPDH ratios in 5-7 days post-injury groups was highest, and all the samples had ratios of >2.60. In the other groups, no samples showed ratios of >2.60 and the CB2R toGAPDH ratios ranged from 1.19 to 2.59. The expression tendency was also confirmed by RT-PCR. From the viewpoint of forensic pathology, these observations suggested that the ratio markedly exceeding 2.60 strongly indicated a wound age of 5-7 days. In conclusion, dynamic distribution and expression of CB2R suggest that CB2R be involved in modulating macrophages in response to inflammatory event in rat skeletal muscle wound healing and CB2R be available as a marker for wound age determination.
J Cell Biochem. 2011 Jan;112(1):59-70.
Source
Center for Nanomedicine Research, National Health Research Institutes, Zhunan, Miaoli County, Taiwan.
Abstract
Astrocytes, the major glial population in the central nervous system (CNS), can secrete thrombospondin (TSP)-1 that plays the role in synaptogenesis and axonal sprouting during CNS development and tissue repair. However, little is known about the regulation of TSP-1 expression in astrocytes under oxidative stress condition. Here, a hypoxic mimetic reagent, cobalt chloride (CoCl(2)), was used to initiate hypoxia-induced oxidative stress in primary rat astrocytes. CoCl(2) at the concentration range of 0.1-0.5 mM was found to cause no significant cell death in primary rat astrocytes. However, CoCl(2) at 0.2-0.5 mM increased intracellular reactive oxygen species (ROS) levels and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression that is known as a hallmark for oxidative damage. We further found that TSP-1 mRNA expression in astrocytes was inhibited dose- and time-dependently by CoCl(2). TSP-1 mRNA levels were increased in CoCl(2)-exposed astrocytes in the presence of the inhibitors (U0126 and PD98059) of mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK), when compared to that detected in the culture only exposed to CoCl(2). Moreover, the inhibition in TSP-1 mRNA expression by CoCl(2) was blocked by the addition of the potent antioxidant, N-acetylcysteine (NAC). Thus, we conclude that CoCl(2) inhibits TSP-1 mRNA expression in astrocytes via a ROS mechanism possibly involving MAPK/ERK. This inhibition may occur after CNS injury and impair the supportive function of astrocytes on neurite growth in the injured CNS tissues.
Antioxid Redox Signal. 2011 Jan 1;14(1):49-60. Epub 2010 Aug 30.
Source
Cardiovascular Division, King's College London, The Rayne Institute, St. Thomas' Hospital, London, United Kingdom.
Abstract
Protein sulfenic acids (SOHs) are the principal oxidation products formed when redox active proteins interact with peroxide molecules. We have developed a new antibody reagent that detects protein SOHs derivatized with dimedone. Using this new antibody, we found that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is the predominant protein sulfenate present in isolated rat ventricular myocytes under basal conditions. During oxidative stress with hydrogen peroxide (H(2)O(2)), GAPDHSOH labeling is lost, but a number of secondary dimedone-reactive protein sulfenates then appear. As the sulfenate labeling is lost, the Cys-149 sulfinic/sulfonic acid oxidation states of GAPDH appear. This hyperoxidized GAPDH is associated with both the inhibition of glycolysis and its ability to reduce H(2)O(2). We examined whether inactivation of GAPDH was causative in the generation of secondary protein sulfenates that coincide with its hyperoxidation. The selective GAPDH inhibitor koningic acid (which functions by forming a covalent adduct at Cys-149) fully prevented basal SOH labeling, as well as subsequent peroxide-induced hyperoxidation. However, koningic acid-mediated inhibition ofGAPDH alone did not induce the formation of intracellular H(2)O(2) or secondary protein sulfenates and also failed to potentiate their peroxide-induced formation. Overall, GAPDH appears to have peroxidase-like properties, but its inhibition failed to impact on downstream oxidant signaling involving secondary protein sulfenation.
J Neurotrauma. 2010 Aug;27(8):1477-87.
Source
Department of Pharmacology, Chang Gung University, Tao-Yuan, Taiwan.
Abstract
Heme oxygenase-1 (HO-1), a kind of stress protein, is critical for the protection against ischemic stroke and cerebrovascular endothelium damage. However, the effects of HO-1 on trauma-induced brain injury are still unknown. Hence, we attempted to use a cold injury-induced brain trauma (CIBT) model in mice, which provides for a well-established approach for assessing brain edema and blood-brain barrier breakdown. Additionally, we explored cultured mouse brain endothelial cells (bEnd.3) to investigate the protective effects of HO-1. HO-1 was induced by infection with a recombinant adenovirus carrying the human HO-1 gene or an inducer of HO-1 activity, cobalt protoporphyrin IX (CoPP). The recombinant adenovirus (3.5 x 10(7) PFU/mouse, i.v.) or CoPP (10 mg/kg, i.v.) significantly increased HO-1 protein expression and HO-1 enzyme activity in the cerebral cortex of the mice. We found that overexpression of HO-1 protected against cold injury-induced secondary damage and behavioral impairment. Up-regulation of HO-1 decreased brain edema and neutrophil infiltration induced by cold injury. These HO-1-dependent protecting effects were abrogated by pretreatment with the HO-1 inhibitor, zinc protoporphyrin IX (ZnPP; 3 mg/kg, i.v.). HO-1 expression in the cerebral endothelium was observed by immunofluorescent staining. CoPP-induced (1 muM, 24 h) HO-1 protein expression was determined by western blotting in bEnd.3 cells. Enhanced HO-1 also protected against cold injury-induced cell loss and damage, which were respectively determined by GAPDH leakage into the cell medium and XTT assay in bEnd.3 cells. In summary, HO-1 overexpression appears to offer an effective neuroprotection against cold-induced secondary brain injury.
BMC Res Notes. 2010 Apr 14;3:101.
Source
Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Av, Brasil 2950, Valparaíso, Chile. andrea.pena@ucv.cl.
Abstract
BACKGROUND:
Due to the limited number of species specific antibodies against fish proteins, differential gene expression analyses are vital for the study of host immune responses. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most powerful tools for this purpose. Nevertheless, the accuracy of the method will depend on the careful selection of genes whose expression are stable and can be used as internal controls for a particular experimental setting.
FINDINGS:
The expression stability of five commonly used housekeeping genes [beta-actin (ACTB), elongation factor 1-alpha (EF1A), ubiquitin (UBQ), glyceraldehyd-3-phosphate dehydrogenase (GAPDH) and tubulin alpha (TUBA)] were monitored in salmonid cell lines CHSE-214 and RTS11 after infection with two of the most fastidious fish pathogens, the facultative bacterium Piscirickettsia salmonis and the aquabirnavirus IPNV (Infectious Pancreatic Necrosis Virus). After geNorm analysis, UBQ and EF1A appeared as the most stable, although EF1A was slightly upregulated at late stages of P. salmonis infection in RTS11. ACTB instead, showed a good performance in each case, being always considered within the three most stable genes of the panel. In contrast, infection-dependent differential regulation of GAPDH and TUBA was also demonstrated.
CONCLUSION:
Based on the data presented here with the cell culture models CHSE-214 and RTS11, we suggest the initial choice of UBQ, ACTB and EF1A as reference genes in qRT-PCR assays for studying the effect of P. salmonis and IPNV on the host immune response.
Source
Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Japan.
Abstract
Loose interaction between the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) was visualized in living CHO-K1 cells by fluorescence resonance energy transfer (FRET), using time-domain fluorescence lifetime imaging microscopy. FRET between active tetrameric subunits of GAPDH linked to cerulean or citrine was observed, and this FRET signal was significantly attenuated by coexpression of PGK. Also, direct interaction betweenGAPDH-citrine and PGK-cerulean was observed by FRET. The strength of FRET signals between them was dependent on linkers that connect GAPDH to citrine and PGK to cerulean. A coimmunoprecipitation assay using hemagglutinin-tagged GAPDH and FLAG-tagged PGK coexpressed in CHO-K1 cells supported the FRET observation. Taken together, these results demonstrate that a complex of GAPDH and PGK is formed in the cytoplasm of living cells.
Cancer Genomics Proteomics. 2010 Jan-Feb;7(1):17-23.
Suzuki A, Iizuka A, Komiyama M, Takikawa M, Kume A, Tai S, Ohshita C, Kurusu A, Nakamura Y,Yamamoto A, Yamazaki N, Yoshikawa S, Kiyohara Y, Akiyama Y.
Source
Shizuoka Cancer Center Research Institute, Nagaizumi-cho, Sunto-gun, Japan.
Abstract
BACKGROUND:
Melanoma is an intractable cancer with a poor prognosis and increasing prevalence worldwide. Specific biomarkers for early diagnosis have yet to be found.
MATERIALS AND METHODS:
Serum samples from melanoma patients and healthy volunteers were utilized for identifying melanoma marker proteins using a serological proteome approach. Specifically, G361 cell protein spots separated by 2-dimensional gel electrophoresis and transferred to a membrane were incubated with patient sera, and positive spots that reacted with more than 5 serum samples were identified using time of flight mass spectrometry.
RESULTS:
Only patient sera showed many spots reacted in G361 gels. A total of 13 positive spots were detected and 5 proteins were identified: eukaryotic elongation factor2 (EEF2), enolase1 (ENO1), aldolase A (ALDOA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heterogeneous nuclear ribonucleoproteins (HNRNP) A2B1. The mRNAs of four proteins (EEF2, ENO1, ALDOA and HNRNPA2B1) were highly expressed in G361 cells compared with melanocytes. EEF2, ENO1 and ALDOA mRNAs were also frequently expressed in other melanoma cell lines.
CONCLUSION:
The autoantibody-based proteomic approach was effective for investigating melanoma biomarkers. This study might contribute to the development of a diagnostic device for the early detection of cancer.
Mol Cancer. 2010 Jan 26;9:14.
Source
Centre for Translational and Applied Genomics, British Columbia Cancer Agency, Provincial Health Services Authority Laboratories, 600 West 10th Avenue, Vancouver, British Columbia, V5Z 4E6, Canada.
Abstract
BACKGROUND:
We have previously reported a novel constitutively overexpressed 21 kDa protein in Hodgkin Lymphoma (HL) and aggressive Non-Hodgkin Lymphomas (NHL). The objective of the current study was to 1) identify this protein using two independent methods, 2) study the expression of the protein and its encoding mRNA in reactive lymph nodes, normal lymphocytes and CD34+ bone marrow precursor cells, 3) analyse patterns of expression of the protein in tissue microarrays assembled from a large number of diagnostic clinical biopsies from patients with HL, and 4) determine the copy number variation and mutation status of the encoding gene in HL cell lines.
RESULTS:
Peptide sequencing by LC-MS/MS and protein identification by protein array screening identified a single protein, CYB5B. No mutations were detected in the CYB5B gene in HL cell lines. Quantitative PCR showed CYB5B gene expression was increased in HL and NHL cell lines. Array CGH using a submegabase resolution tiling array revealed gains in the CYB5B locus in HL cell lines KMH2 and L428. Membrane expression was seen in Reed-Sternberg cells in clinical biopsies from patients with HL but not in reactive lymph nodes. Bone marrow CD34+ precursor cells were CYB5B negative on the cell surface. RT-PCR assays of RNA extracted from T and B cell enriched fractions obtained from normal peripheral blood mononuclear cells, reactive lymph nodes, tonsils and normal bone marrow samples showed no evidence of increased mRNA levels of CYB5B in comparison to housekeeping gene GAPDH.
CONCLUSIONS:
The 21 kDa protein overexpressed in HL and aggressive NHL is identical to CYB5B. CYB5B gene expression is increased in a subset of HL and NHL cell lines tested. This is associated with CYB5B gene amplification in HL cell lines KMH2 and L428. CYB5B may be a potential target forantibody-based therapy of HL and aggressive NHL as although cytoplasmic expression is present in reactive lymphocytes, it is not expressed on the cell surface of non-neoplastic lymphocytes or bone marrow precursor cells.
J Appl Physiol. 2010 Apr;108(4):923-32. Epub 2010 Jan 21.
Exercise training normalizes ACE and ACE2 in the brain of rabbits with pacing-induced heart failure.
Source
Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5850, USA.
Abstract
Exercise training (EX) normalizes sympathetic outflow and plasma ANG II in chronic heart failure (CHF). The central mechanisms by which EX reduces this sympathoexcitatory state are unclear, but EX may alter components of the brain renin-angiotensin system (RAS). Angiotensin-converting enzyme (ACE) may mediate an increase in sympathetic nerve activity (SNA). ACE2 metabolizes ANG II to ANG-(1-7), which may have antagonistic effects to ANG II. Little is known concerning the regulation of ACE and ACE2 in the brain and the effect of EX on these enzymes, especially in the CHF state. This study aimed to investigate the effects of EX on the regulation of ACE and ACE2 in the brain in an animal model of CHF. We hypothesized that the ratio of ACE to ACE2 would increase in CHF and would be reduced by EX. Experiments were performed on New Zealand White rabbits divided into the following groups: sham, sham + EX, CHF, and CHF + EX (n = 5 rabbits/group). The cortex, cerebellum, medulla, hypothalamus, paraventricular nucleus (PVN), nucleus tractus solitarii (NTS), and rostral ventrolateral medulla (RVLM) were analyzed. ACE protein and mRNA expression in the cerebellum, medulla, hypothalamus, PVN, NTS, and RVLM were significantly upregulated in CHF rabbits (ratio of ACE toGAPDH: 0.3 +/- 0.03 to 0.8 +/- 0.10 in the RVLM, P < 0.05). EX normalized this upregulation compared with CHF (0.8 +/- 0.1 to 0.4 +/- 0.1 in the RVLM). ACE2 protein and mRNA expression decreased in CHF (ratio of ACE2 to GAPDH: 0.3 +/- 0.02 to 0.1 +/- 0.01 in the RVLM). EX increased ACE2 expression compared with CHF (0.1 +/- 0.01 to 0.8 +/- 0.1 in the RVLM). ACE2 was present in the cytoplasm of neurons and ACE in endothelial cells. These data suggest that the activation of the central RAS in animals with CHF involves an imbalance of ACE and ACE2 in regions of the brain that regulate autonomic function and that EX can reverse this imbalance.
Transfusion. 2010 May;50(5):1050-6. Epub 2010 Jan 15.
Source
Institute for Transfusion Medicine, Pittsburgh, Pennsylvania 15213, USA. lqu@itxm.org
Abstract
BACKGROUND:
Human herpesvirus 8 (HHV-8) is a gamma-herpesvirus that causes Kaposi's sarcoma. The prevalence of HHV-8 genomes in US blood donors has not been systemically studied.
STUDY DESIGN AND METHODS:
A sensitive and quantitative real-time polymerase chain reaction (PCR) assay was used to detect HHV-8 DNA from purified CD19+ B lymphocytes from randomly selected US whole blood donors. Cellular target for the GAPDH gene was used to quantify cell-equivalent DNA. HHV-8 PCR was run in duplicate for each donor specimen along with an HHV-8 genomic copy standard. HHV-8 antibodies were detected by an indirect immunofluorescence assay.
RESULTS:
Specimens were obtained from 962 blood donors. DNA from more than 3 x 10(5) B-cell equivalents were obtained from 684 donors. No HHV-8 DNA was detected from any of the blood donor specimens. For the 684 donors, HHV-8 genomes were not detected in the DNA equivalent of 3 x 10(5) to 6 x 10(5) CD19+ B cells with real-time PCR, which has a detection limit of eight copies (95% confidence interval, 0-3/684). Negative results from the remaining 220 donors were potentially confounded by insufficient input DNA into the PCR procedures. Antibodies to HHV-8 were detected in 7.3% (70/962) of the donors. HHV-8 genomes were not detected from 40 of 70 HHV-8-seropositive donor B-cell DNA samples.
CONCLUSION:
The results indicate that the prevalence of detectable HHV-8 genomes in healthy blood donors is very low.
J Microbiol Biotechnol. 2009 Dec;19(12):1635-43.
Source
Laboratoire de Chimie des Substances Naturelles, EA 1069, Antenne IUT, Département Génie Biologique, U. Limoges, France.
Abstract
The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDHactivity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.
Vet Parasitol. 2010 Apr 19;169(1-2):82-92. Epub 2009 Dec 22.
Source
Laboratory of Immunology and Molecular Parasitology, Department of Microbiology and Medical Zoology, School of Medicine, University of Puerto Rico, San Juan, P.R., Puerto Rico. ana.espino1@upr.edu
Abstract
Use of the rabbit as disease model has long been hampered by a lack of immunological assays specific to this species. In the present study we developed a SYBR Green-based, real-time RT-PCR protocol to quantitate cytokine mRNA in freshly harvested rabbit peripheral mononuclear cells. The method was validated in the course of a vaccination trial in which animals vaccinated with the recombinant antigen FhSAP2 were challenged with Fasciola hepatica metacercariae. Changes in the levels of rabbit interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha (TNFalpha), and interferon-gamma (IFNgamma) mRNA were determined. Messenger RNA from the universally expressed housekeeping gene GAPDH was used as an amplification control and allowed for correction of variations in the efficiencies of RNA extraction and reverse transcription. Rabbits vaccinated with FhSAP2 showed an 83.3% reduction in liver fluke burden after challenge infection when compared to non-vaccinated controls. All cytokine mRNAs were found at detectable levels; however, the levels of IFNgamma, TNFalpha, IL-2 and IL-10 were significantly higher in the vaccinated group compared to the non-vaccinated group. These results suggest that protection conferred by FhSAP2 protein could be associated with a mixed Th1/Th2 immune response in which Th1 cytokines are dominant. The real-time RT-PCR method described herein can be a useful tool for monitoring changes in basic immune functions in the rabbit model of fascioliasis and may also aid in studies of human diseases for which the rabbit is an important experimental model.
Clin Endocrinol (Oxf). 2010 Oct;73(4):522-8. doi: 10.1111/j.1365-2265.2009.03753.x.
Source
Department of Medicine, Nepean Clinical School, The University of Sydney, Penrith, NSW, Australia.
Abstract
BACKGROUND:
Graves' Ophthalmopathy (GO) is a complex eye and orbital disorder that is uniquely linked to Graves' Hyperthyroidism (GH) and has traditionally been considered a cross-reactive immune response against the thyroid stimulating hormone receptor (TSHR) in orbital tissue. However, because there is no direct evidence, such as specific TSHR antibodies or T lymphocytes targeting the orbital tissues in patients with GO compared to those without eye disease, it is important to consider alternative hypotheses for the pathogenesis of GO. The aim of this study was to identify differentially expressed genes within the thyroid of patients with GO and GH as a possible explanation for a thyroid initiated orbital autoimmunity.
METHODS:
RNA was extracted from thyroid glands of patients with GO (n = 10) and GH (n = 8) post-total thyroidectomy. RNA samples were arrayed on Illumina® Human Ref-8 Expression BeadChips™ representing 20,589 genes. Microarray results of selected genes were validated by quantitative PCR (qPCR) and levels of protein translation measured by Western blot analysis.
FINDINGS:
Two hundred and ninety-five genes were differentially expressed between patients with GO and GH. Of these, the cardiac calsequestrin gene (CASQ2) was the most highly expressed gene in GO (2.2-fold increase, P < 0.05). The succinate dehydrogenase flavoprotein subunit gene (SDHA) was also significantly up-regulated in GO (P < 0.05), 1.4-fold, while genes encoding the thyroid antigens thyroglobulin, thyroid peroxidase and TSHR were not differentially expressed. qPCR verified up-regulation of CASQ2 and down-regulation BMP7, CD80, IGFBP5, and MYD88 genes in GO. Western blot analysis showed that the average CASQ/GAPDH protein expression ratios for GH and GO were 1.04 and 1.03, respectively. t-Test analysis of data generated a P-value of 0.26, therefore no significant difference was found for CASQ protein expression in thyroid tissue between GH and GO.
INTERPRETATION:
The skeletal and cardiac calsequestrin proteins share 68.4% amino acid homology. Previous work has shown that RNA levels of skeletal muscle calsequestrin are 4.7 times higher in extraocular muscle (EOM) than in masticatory skeletal muscle (jaw), and cardiac calsequestrin is expressed 2.7 times more in EOM. We postulate that up-regulation of casq2 gene in the thyroid of patients with GH may lead to the production of autoantibodies and sensitized T-lymphocytes, which cross-react with calsequestrin in the EOM of patients who develop ophthalmopathy.
© 2010 Blackwell Publishing Ltd.
Vet Microbiol. 2010 May 19;142(3-4):346-51. Epub 2009 Oct 20.
Guimaraes AM, Brandão PE, Moraes W, Kiihl S, Santos LC, Filoni C, Cubas ZS, Robes RR, Marques LM, Neto RL, Yamaguti M, Oliveira RC, Catão-Dias JL, Richtzenhain LJ, Messick JB, Biondo AW,Timenetsky J.
Source
Departmento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo (USP), São Paulo, SP, Brazil.
Abstract
Although antibodies to Bartonella henselae have been described in all neotropical felid species, DNA has been detected in only one species, Leopardus wiedii. The aim of this study was to determine whether DNA of Bartonella spp. could be detected in blood of other captive neotropical felids and evaluate risk factors and hematological findings associated with infection. Blood samples were collected from 57 small felids, including 1 Leopardus geoffroyi, 17 L. wiedii, 22 Leopardus tigrinus, 14 Leopardus pardalis, and 3 Puma yagouaroundi; 10 blood samples from Panthera onca were retrieved from blood banks. Complete blood counts were performed on blood samples from small felids, while all samples were evaluated by PCR. DNA extraction was confirmed by amplification of the cat GAPDHgene. Bartonella spp. were assessed by amplifying a fragment of their 16S-23S rRNA intergenic spacer region; PCR products were purified and sequenced. For the small neotropical felids, risk factors [origin (wild-caught or zoo-born), gender, felid species, and flea exposure] were evaluated using exact multiple logistic regression. Hematological findings (anemia, polycythemia/hyperproteinemia, leukocytosis and leukopenia) were tested for association with infection using Fisher's exact test. The 635bp product amplified from 10 samples (10/67=14.92%) was identified as B. henselae by sequencing. Small neotropical felid males were more likely to be positive than females (95% CI=0.00-0.451, p=0.0028), however other analyzed variables were not considered risk factors (p>0.05). Hematological abnormalities were not associated with infection (p>0.05). This is the first report documenting B. henselae detection by PCR in several species of neotropical felids.
Copyright 2009 Elsevier B.V. All rights reserved.
Clin Chim Acta. 2010 Jan;411(1-2):67-71. Epub 2009 Oct 12.
Source
Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong.
Abstract
BACKGROUND:
Early growth response-1 (Egr-1) is expressed in human airways and its polymorphisms have been associated with total IgE and atopy in asthmatic patients. We investigated the effects of Chinese-tagging single nucleotide polymorphism (SNP) of Egr-1 and its mRNA expression on allergic rhinitis (AR) traits.
METHODS:
Among 214 Chinese AR adults and 259 controls, tag SNP -4071 A-->G was genotyped and mRNA expression in peripheral blood was quantified by real-time PCR.
RESULTS:
Egr-1 mRNA expression was significantly higher in patients than controls (median of 0.23 vs 0.15 fold GAPDH expression; p<0.001). Its expression was not associated with -4071 polymorphism. However, significant correlations were found between -4071 A-->G with increased plasma total IgE (p=0.028) and atopy (p=0.030) in patients. Logistic regression confirmed the association (p=0.034) with age and gender adjusted. Patients homozygous for the A allele had a 2.3-fold and 1.9-fold risks, respectively of having increased plasma total IgE and atopy than those G allele carriers.
CONCLUSIONS:
We showed high levels of Egr-1 mRNA expression and demonstrated a significant association of polymorphism with increased plasma total IgE and atopy in AR patients. It may be useful to explore the pharmacogenetics of Egr-1 inhibitors.
J Microbiol Biotechnol. 2009 Sep;19(9):982-6.
Source
Department of Biotechnology, Pukyong National University, Busan 608-737, Korea.
Abstract
For the production of ghost bacteria vaccine to prevent the streptococcal disease in aquaculture fish species, a double cassettes vector was constructed and cloned in Escherichia coli DH5alpha. Ghost bacteria vaccine production from Escherichia coli DH5alpha/pHCE-InaN-GAPDH-Ghost 37 SDM (SIG) was maximized at a glucose concentration of 1 g/l, agitation of 300 rpm, and aeration of 1 vvm. The maximal efficiency of ghost bacteria formation was obtained at the mid-exponential phase (OD600=2.0) with the concentration of 0.77 g/l for SIG. The molecular mass of GAPDH was detected at 67 kDa with the insoluble fraction, by SDS-PAGE and Western blot. The protective efficacy of ghost bacteria vaccine was evaluated by challenge test using olive flounder. The cumulative mortalities of the positive control, formalin-killed cell (FKC) vaccine, and SIG vaccine immunized groups were 91% , 74% , and 57% , respectively. These results suggest that SIG vaccine showed efficacy as a vaccine and had a higher potential to induce protective antibodies than did FKC vaccine.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2009 Jul;25(7):581-3.
[Article in Chinese]
Source
Overseas Creative Team, Kunming Institute of Zoology, Kunming 650223, China. zhangping5021@163.com
Abstract
AIM:
To amplify the CD1d and analyze its tissue distribution in Rhesus macaque.
METHODS:
Extracting total RNA from different tissues of Rhesus macaque. CD1d amplification was carried using specific primers and tissue distribution was analyzed by semi-quantification RT-PCR, using the house-keeping gene GAPDH as an internal control.
RESULTS:
The CD1d coding region was obtained in this experiment and tissue distribution was analyzed: a highest level of CD1d expression was detected in heart, liver and spleen, lower level in blood, lung, small intestine and stomach, and least in brain.
CONCLUSION:
The successful amplification of CD1d is useful to produce anti-CD1d antibody and CD1d-tetramer to study NKT cells in Rhesus macaque. The expression profile of CD1d mRNA in Rhesus macaque is in accordance with the tissue distribution and the functions of NKT cells in other species. These results will provide important insights for future research on the relationship of CD1d/NKT cells and their roles in different tissues.
Mol Biol Rep. 2011 Jan;38(1):675-81. Epub 2009 Aug 21.
Source
Molecular and Cell Biology Laboratory, Department of Physiology and Cell Biology, University of Health Sciences (UHS), Khayaban-e-Jamia Punjab, Lahore, 54600, Pakistan. ranacemb@yahoo.com
Erratum in
· Mol Biol Rep. 2011 Jan;38(1):683. Imran, Muhammad [removed].
Abstract
IgE high affinity receptor (FcεRI) plays an important role in triggering type I allergic reactions. In this study, we have investigated the ability of four synthetic and sequence-specific RNA interfering antisense oligodeoxynucleotides (AS-ODNs) to reduce the expression of FcεRIα gene in granulocytes of allergy sufferers in vitro. Only AS1 out of four AS-ODNs specifically inhibited the FcεRIα gene expression and the dose response assay revealed that AS1 was capable of specific inhibition of target mRNA expression over a linear concentration range without affecting the expression of house keeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Together, these results indicate that sequence-specific RNA interfering ODNs can be effectively used to silence the expression of key genes like IgE high affinity receptor that are involved in chronic inflammatory diseases.
J Vet Diagn Invest. 2009 Jul;21(4):504-9.
Source
National Research Institute of Aquaculture, Fisheries Research Agency, Minami-Ise, Mie 516-0193, Japan. tasakai@affrc.go.jp
Abstract
Edwardsiella tarda is a fish pathogen that causes systemic infections in fresh water and marine fish. Determining the antigenic proteins is important for the development of an immunodiagnostic tests and a vaccine for effective infection control in fish. In the current study, antigens were detected by immunoblotting and affinity column chromatography using a Japanese flounder (Paralichthys olivaceus) antibody produced by experimental infection with E. tarda. GroEL, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), outer membrane protein A, filament protein, 30S ribosomal protein S6, 50S ribosomal protein L9, cold shock protein, and carbon storage protein were identified as antigens of E. tarda through biochemical analyses of the molecular weights, isoelectric points, and N-terminal amino-acid sequences. These proteins can be easily detected in flounder infected with E. tarda and are potential diagnostic markers.
Vet Microbiol. 2009 Oct 20;139(1-2):113-20. Epub 2009 Jun 6.
Source
Laboratory of Aquatic Animal Diseases, Research Institute of Life Science, College of Veterinary Medicine, Gyeongsang National University, Jinju, Gyeongnam 660-701, Republic of Korea.
Abstract
Lactococcus garvieae is an important etiological agent of lactococcosis in various fish species including olive flounder (Paralichthys olivaceus). In this study, proteomic and immunoproteomic analyses were employed to compare the antigenic profiles of strains KG9408, MS93003, and NSS9310 strains of L. garvieae. Proteomic analysis using two-dimensional gel electrophoresis (2-DE) revealed differences in five protein spots among the different L. garvieae strains. In immunoproteomic analysis, there was a significant difference in the 2-DE immunoblot profiles of the L. garvieae strains using sera collected from fish surviving infection with either L. garvieae strains KG9408 or NSS9310. These sera reacted with 8 and 7 unique antigenic protein spots, respectively. Heat shock protein (HSP) 70 and DNA-directed RNA polymerase were among the specific antigens recognized by the anti-NSS9310 serum. In addition, the anti-NSS9310 and anti-KG9408 olive flounder sera reacted with 25 common antigenic protein spots of all the L. garvieae strains, which included elongation factor (EF)-Tu, arginine deiminase (AD), inosine-5'-monophosphate dehydrogenase (IMPD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphomannomutase (PMM), L-lactate dehydrogenase (L-LDH), 6-phosphofructokinase and UDP-galactose 4-epimerase (UDP-galactose). Based on the present results, the 8 antigens recognized by the anti-KG9408 serum and the 25 common antigens recognized by both sera may serve as potential markers for developing an effective vaccine against this bacterium.
J Virol Methods. 2009 Jul;159(1):40-6. Epub 2009 Mar 4.
Source
Robert Koch-Institute, Nordufer 20, D-13353 Berlin, Germany.
Abstract
Specific, effective and rapid neutralization assays are crucial for the development of an HIV vaccine based on the stimulation of neutralizing antibodies and the development of such an assay for the human immunodeficiency virus-2 (HIV-2) is described. Virus neutralization was measured as the reduction of provirus integration using a duplex real-time PCR with high efficiency (99.4%). This PCR uses primers and a probe specific for the proviral LTR. Amplification and quantitative analysis of the cellular GAPDH gene was carried out in parallel to control for toxic or growth-inhibitory components in the sera. The neutralization assay was used to screen sera from 23 HIV-2 infected patients. 21 sera were able to neutralize HIV-2(60415K), 20 sera neutralized HIV-2(7312A) and 7 sera cross-neutralized HIV-1 IIIB. In contrast, when 14 of these sera were tested in parallel with a conventional neutralization assay based on a p27Gag capture ELISA, only one was found to neutralize HIV-2(60415K) and 11 to neutralize HIV-2(7312A) compared with 12 and 13 sera respectively using the PCR-based assay.
Acta Biochim Biophys Sin (Shanghai). 2009 May;41(5):399-406.
Source
Laboratoire de Biochimie et Biologie Moleculaire, Universite Hassan II-Ain Chock, Faculte des Sciences Ain Chock, km 8 route d'El Jadida BP. 5366, Maarif, Casablanca, Morocco. drissmountassif@yahoo.fr
Abstract
A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from human erythrocytes. The comparison between this rapid method (one step) and the traditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time. The characterization of the purifiedGAPDH reveals that the native enzyme is a homotetramer of ~150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide (NAD(+)). Western blot analysis using purified monospecific polyclonal antibodies raised against the purified GAPDH showed a single 36 kDa band corresponding to the enzyme subunit. Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 43 degrees C and 8.5, respectively. The kinetic parameters were also calculated: the Vmax was 4.3 U/mg and the Km values against G3P and NAD(+) were 20.7 and 17.8 muM, respectively. The new protocol described represents a simple, economic, and reproducible tool for the purification of GAPDH and can be used for other proteins.
J Neurosci Res. 2009 Oct;87(13):2881-9.
Identification of major S-nitrosylated proteins in murine experimental autoimmune encephalomyelitis.
Source
Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico. obizzozero@salud.unm.edu
Abstract
Nitrosative stress has been implicated in the pathophysiology of several CNS disorders, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). We have recently shown that protein nitrosothiols (PrSNOs) accumulate in the brain of MS patients, and there is indirect evidence that PrSNO levels are also increased in EAE. In this study we sought to identify the major PrSNOs in the spinal cord of EAE animals prepared by active immunization of C57/BL6 mice with MOG(35-55) peptide. For this purpose, PrSNOs from control and EAE mice at various disease stages were derivatized with HPDP-biotin, and the biotinylated proteins were isolated with streptavidin-agarose. Proteins from total and streptavidin-bound fractions were then analyzed by Western blotting using antibodies against the major S-nitrosylated substrates of CNS tissue. With this approach we found that the proportion of S-nitrosylated neurofilament proteins, NMDA receptors, alpha/beta-tubulin, beta-actin, and GAPDH is increased in EAE. Other potential substrates either were not S-nitrosylated in vivo (HCN3, HSP-72, CRMP-2, gamma-actin, calbindin) or their S-nitrosylation levels were unaltered in EAE (Na/K ATPase, hexokinase, glycogen phosphorylase). We also discovered that neuronal specific enolase is the major S-nitrosylated protein in acute EAE. Given that S-nitrosylation affects protein function, it is likely that the observed changes are significant to the pathophysiology of inflammatory demyelination.
2009 Wiley-Liss, Inc.
Infect Immun. 2009 Jul;77(7):2703-11. Epub 2009 Apr 20.
Source
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164, USA. alderete@wsu.edu
Abstract
Trichomonas vaginalis colonizes the urogenital tract of humans and causes trichomonosis, the most prevalent nonviral sexually transmitted disease. We have shown an association of T. vaginalis with basement membrane extracellular matrix components, a property which we hypothesize is important for colonization and persistence. In this study, we identify a fibronectin (FN)-binding protein of T. vaginalis. A monoclonal antibody (MAb) from a library of hybridomas that inhibited the binding of T. vaginalis organisms to immobilized FN was identified. The MAb (called ws1) recognized a 39-kDa protein and was used to screen a cDNA expression library of T. vaginalis. A 1,086-bp reactive cDNA clone that encoded a protein of 362 amino acids with identity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained. The gapdh gene was cloned, and recombinant GAPDH(rGAPDH) was expressed in Escherichia coli cells. Natural GAPDH and rGAPDH bound to immobilized FN and to plasminogen and collagen but not to laminin. MAb ws1 inhibited binding to FN. GAPDH was detected on the surface of trichomonads and was upregulated in synthesis and surface expression by iron. Higher levels of binding to FN were seen for organisms grown in iron-replete medium than for organisms grown in iron-depleted medium. In addition, decreased synthesis of GAPDH by antisense transfection of T. vaginalis gave lower levels of organisms bound to FN and had no adverse effect on growth kinetics. Finally, GAPDH did not associate with immortalized vaginal epithelial cells (VECs), and neither GAPDH nor MAb ws1 inhibited the adherence of trichomonads to VECs. These results indicate that GAPDH is a surface-associated protein of T. vaginalis with alternative functions.
Mol Cancer Ther. 2009 Apr;8(4):864-72.
Source
Temple University School of Pharmacy, 3307 North Broad Street, Philadelphia, PA 19140, USA.
Abstract
The identification of new molecular components of the DNA damage signaling cascade opens novel avenues to enhance the efficacy of chemotherapeutic drugs. High-mobility group protein 1 (HMGB1) is a DNA damage sensor responsive to the incorporation of nonnatural nucleosides into DNA; several nuclear and cytosolic proteins are functionally integrated with HMGB1 in the context of DNA damage response. The functional role of HMGB1 and HMGB1-associated proteins (high-mobility group protein B2, HMGB2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; protein disulfide isomerase family A member 3, PDIA3; and heat shock 70 kDa protein 8, HSPA8) in DNA damage response was assessed in human carcinoma cells A549 and UO31 by transient knockdown with short interfering RNAs. Using the cell proliferation assay, we found that knockdown of HMGB1-associated proteins resulted in 8-fold to 50-fold decreased chemosensitivity of A549 cells to cytarabine. Western blot analysis and immunofluorescent microscopy were used to evaluate genotoxic stress markers in knocked-down cancer cells after 24 to 72 hours of incubation with 1 micromol/L of cytarabine. Our results dissect the roles of HMGB1-associated proteins in DNA damage response: HMGB1 and HMGB2 facilitate p53 phosphorylation after exposure to genotoxic stress, and PDIA3 has been found essential for H2AX phosphorylation (no gamma-H2AX accumulated after 24-72 hours of incubation with 1 micromol/L of cytarabine in PDIA3 knockdown cells). We conclude that phosphorylation of p53 and phosphorylation of H2AX occur in two distinct branches of the DNA damage response. These findings identify new molecular components of the DNA damage signaling cascade and provide novel promising targets for chemotherapeutic intervention.
J Biomed Sci. 2009 Apr 15;16:40.
Source
Institute of Biotechnology and Department of Life Science, National Dong Hwa University, Taiwan, ROC. hsb8971111@msn.com
Abstract
Replication of the Japanese encephalitis virus (JEV) genome depends on host factors for successfully completing their life cycles; to do this, host factors have been recruited and/or relocated to the site of viral replication. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cellular metabolic protein, was found to colocalize with viral RNA-dependent RNA polymerase (NS5) in JEV-infected cells. Subcellular fractionation further indicated that GAPDH remained relatively constant in the cytosol, while increasing at 12 to 24 hours postinfection (hpi) and decreasing at 36 hpi in the nuclear fraction of infected cells. In contrast, the redistribution patterns of GAPDH were not observed in the uninfected cells. Co-immunoprecipitation of GAPDH and JEV NS5 protein revealed no direct protein-protein interaction; instead, GAPDH binds to the 3' termini of plus- and minus-strand RNAs of JEV by electrophoretic mobility shift assays. Accordingly, GAPDH binds to the minus strand more efficiently than to the plus strand of JEV RNAs. This study highlights the findings that infection of JEV changes subcellular localization of GAPDH suggesting that this metabolic enzyme may play a role in JEV replication.
Invest Ophthalmol Vis Sci. 2009 Sep;50(9):4237-43. Epub 2009 Mar 25.
Source
Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, USA.
Abstract
PURPOSE:
To ascertain the expression pattern of alpha(2)-adrenergic receptors in the ciliary body (CB) and determine the effect of brimonidine on matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in ciliary body smooth muscle (CBSM) cells.
METHODS:
Qualitative RT-PCR was performed to detect the mRNA of the alpha(2)-adrenergic receptor subtypes alpha(2)A, alpha(2)B, and alpha(2)C in CB and CBSM cultures. Immunohistochemistry and immunoblot analysis were performed to further investigate alpha(2)A receptor expression in CB tissue and CBSM cells. CBSM cells from 15 different human donors received control or brimonidine tartrate (45 nM) for 1, 3, or 7 days. Changes in pro-MMP-1, -2, -3, -9, and -24 and TIMP-1, -2, -3, and -4 levels were evaluated by Western blot, with GAPDH as the endogenous control. Zymography was used to assess the activity of MMP-1, -2, -3, and -9.
RESULTS:
The mRNA of alpha(2)A, alpha(2)B, and alpha(2)C were detected in CB tissue and CBSM cells. Immunohistochemistry localized alpha(2)A receptors within the CB stroma. Immunoblot analysis demonstrated production by CBSM cells. Brimonidine increased pro-MMP-9 an average of 116% +/- 34% (P = 0.0360); enzymatic activity of MMP-9 was unchanged. TIMP-4 decreased an average of 25% +/- 8% (P = 0.0329) in conditioned medium, but increased 70% +/- 13% (P = 0.0057) in cell lysates.
CONCLUSIONS:
The presence of alpha(2)A, alpha(2)B, and alpha(2)C in CB tissue and CBSM cells indicates the possibility that brimonidine affects uveoscleral outflow. However, the changes in MMP-9 and TIMP-4 without significant changes in MMP-9 activity suggest that a role of the MMP/TIMP system in outflow is unlikely.
Anal Chem. 2009 Apr 15;81(8):2832-9.
Source
Seoulin Bioscience Institute, 452-2 Seongnae-dong, Gangdong-gu, Seoul 134-030, Republic of Korea.
Abstract
Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting association of individual proteins with specific genomic regions; the technique requires several complex steps and is tedious. In this paper, we develop a microbead-packed microfluidic chip which eliminates most of the laborious, time-consuming, and skill-dependent processes of the ChIP procedure. A computational fluid dynamics model was established to analyze fluidic behavior in a microbead-packed microchannel. With the use of the new chip, a ChIP procedure was performed to purify the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene from human embryonic kidney cells (cell line 293). The ChIP capability of the microfluidic chip was evaluated and compared with that of a commercial assay kit; the precipitation performance of both methods was almost identical as shown by quantitative measurement of DNA. However, our chip offers the advantage of low resin volume, and the experimental time is greatly reduced. In addition, our method is less dependent on individual technical skill. Therefore, we expect that our microfluidic chip-based ChIP method will be widely used in DNA-, gene-, and protein-related research and will improve experimental efficiency.
Mol Cell Neurosci. 2009 Jun;41(2):206-18. Epub 2009 Mar 11.
Source
Zentrum für Molekulare Neurobiologie, Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany.
Abstract
We have identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a binding partner for the cell adhesion molecule L1. GAPDH binds to sites within the extracellular domain of L1, namely the immunoglobulin-like domains I-VI and the fibronectin type III homologous repeats 4-5. ExtracellularGAPDH was detected at the cell surface of neuronal cells by surface biotinylation and immunocytochemistry. Addition of GAPDH antibodies to cultured cerebellar neurons inhibited L1-dependent neurite outgrowth in the presence of ATP, while the application of exogenous GAPDHpromoted L1-dependent neurite outgrowth. Pre-treatment of substrate-coated L1-Fc with ATP andGAPDH, which phosphorylates L1, subsequently led to an enhanced neurite outgrowth. Furthermore, aggregation of L1-Fc carrying beads was enhanced in the presence of both GAPDH and ATP. L1-dependent neurite outgrowth and aggregation of L1 were diminished in the presence of alkaline phosphatase or a protein kinase inhibitor. Our results show that GAPDH-dependent phosphorylation of L1 is a novel mechanism in regulating L1-mediated neurite outgrowth.
J Neurol. 2009 May;256(5):774-82. Epub 2009 Feb 25.
Source
Multiple Sclerosis Research Center of New York, 521 West 57th Street, 4th Fl., New York, NY 10019, USA.
Abstract
In an amyotrophic lateral sclerosis (ALS) patient who also had an IgA gammopathy, autopsy studies identified the IgA in the surviving motor neurons. Further, the IgA bound the surface of isolated bovine motor neurons and inhibited neuronal proliferation in culture. To determine the pathologic basis of this IgA interaction with motor neurons, a neuroblastoma cDNA library was generated and screened with the IgA monoclonal antibody. Reactive clones were identified as flavin adenine dinucleotide (FAD) synthetase. To extend this finding to ALS in general, quantitative RT-PCRs were performed on blood samples from 26 ALS and 30 control blood samples to determine mRNA expression levels of FAD synthetase and other electron transport chain proteins, specifically riboflavin kinase (RFK), cytochrome C1 (CYC1), and succinate dehydrogenase complex subunit B (SDHB). All expression levels were measured against a control enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression levels for a non-respiratory chain protein (beta-actin) were also measured. We found that FAD synthetase expression levels were decreased in ALS samples compared to expression levels in controls (P = 0.0151). Expression levels for RFK, CYC1, and SDHB were also significantly decreased in the ALS group (P = 0.0025, P = 0.0002, and P < 0.0001, respectively). As control, expression levels for beta-actin did not show a significant difference between ALS and control groups (P = 0.2118). Our data show that a reduction in electron transport proteins, namely FAD synthetase, RFK, CYC1, and SDHB, is seen in patients with ALS. It is possible that this may have an effect on oxygen-dependent metabolic pathways. Human motor neurons may be particularly susceptible to injury if there is sub-optimal oxidative metabolism.
Exp Cell Res. 2009 Mar 10;315(5):760-8. Epub 2008 Dec 30.
Source
Medical Research Service, 151G Durham VAMC, 508 Fulton Street, Durham, NC 27705, USA.
Abstract
Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for beta-actin andGAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.
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