RSSMass spectrometric characteristics and kinetics of iron release in visceral mass of Saccostrea cucullata.
Source
State Key Laboratory of Stress Cell Biology (Xiamen University), School of Life Sciences, Xiamen University, Xiamen, China.
Abstract
Ferritins with electrophoretic homogeneity were prepared from the visceral mass of Saccostrea cucullata in batch. The native PAGE approach showed similar electrophoretic mobility among pig pancreatic ferritin, liver ferritin of Dasyatis akajei, and visceral mass ferritin of Saccostrea cucullata. SDS-PAGE indicated that the Saccostrea cucullata visceral ferritin (SCVF) consisted of a single subunit type and had a molecular weight (MW) of approximately 20 kDa, suggesting that the protein shell in SCVF was composed of a single subunit. In addition, peptide mass fingerprinting and transmission electron microscopy were used to identify SCVF further, and to observe its molecular structure. We found that the molecular structure in SCVF was similar to that of most mammalian ferritins, which are composed of a protein shell and an iron core. The results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry under the assistance of an acidic matrix, sinapic acid, also showed that SCVF was composed of a single subunit type and its subunit MW was calculated to be 19871.042 Da in the absence of heme. Kinetics analysis revealed that the complete process of iron release fitted the law of a first-order reaction, which is similar to that of most ferritins in mammals. Similar to bacterial ferritin, studies indicated that the shell consisted of a single subunit type and showed similar kinetics of iron release, suggesting that this subunit plays two important roles in iron release and storage, and that it shows different stability and intensity of interaction in carrying out its physiological functions in SCVF.
Copyright © 2011 John Wiley & Sons, Ltd.
Two types of calmodulin play different roles in Pacific white shrimp (Litopenaeus vannamei) defenses against Vibrio parahaemolyticus and WSSV infection.
Source
Key Laboratory of Science and Technology for Aquaculture and Food Safety of Fujian Province University, Jimei University, Xiamen 361021, China.
Abstract
Calmodulin (CaM) plays an important role in calcium-dependent signal transduction pathways. In the present study, two alternative splicing isoforms of CaM (named LvCaM-A and LvCaM-B) cDNA were cloned from the Pacific white shrimp, Litopenaeus vannamei. LvCaM-A was of 1101 bp, including a 5'-terminal untranslated region (UTR) of 70 bp, a 3'-terminal UTR of 581 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with a calculatedmolecular weight (Mw) of 17 kDa and pI of 4.41. LvCaM-B was 689 bp, including a same 5'-UTR of 70 bp, a 3'-terminal UTR of 109 bp and an ORF of 510 bp encoding a polypeptide of 169 amino acids with a calculated Mw of 19 kDa and pI of 4.36. Both LvCaM-A and LvCaM-B contained 4 conservative EF-hand motifs. Quantitative real-time reverse transcription PCR analysis revealed LvCaM-A to be expressed in most shrimp tissues, with the predominant expression in nerve and the weakest expression in heart. However, LvCaM-B expression level was much weaker than those of LvCaM-A in all the tested tissues with main expression in hepatopancreas. The expression of LvCaM-A and LvCaM-B after challenge with Vibrio parahaemolyticus and WSSV were tested in hemocytes, hepatopancreas and nerve. The results indicated that LvCaM-A and LvCaM-B transcripts could be significantly induced in hemocytes and hepatopancreas respectively by injection with V. parahaemolyticus. The highest expression of LvCaM-A was in the hemocytes with 2.3 times (at 48 h) greater expression than in the control (p < 0.05). However, sharp down-regulation of both LvCaM-A and LvCaM-B were detected in nerve after Vibrio- and WSSV injection (p < 0.05). These results suggested that CaM might play an important role in shrimp's defense against pathogenic infection.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Cloning and sequence analysis of a novel xylanase gene, Auxyn10A, from Aspergillus usamii.
Source
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, People's Republic of China.
Abstract
A full-length cDNA sequence, encoding a novel endo-1,4-β-D: -xylanase (AuXyn10A) of Aspergillus usamii, was obtained by using rapid amplification of cDNA ends (RACE) methods and cloned into the pUCm-T vector, followed by DNA sequencing. The cDNA gene, designated as Auxyn10A, is 1,235 bp in length harboring 5'- and 3'-non-encoding regions, as well as an ORF of 984 bp that encodes a 19-aa signal peptide, a 6-aa propeptide and a 302-aa mature peptide with acalculated MW of 32,756 Da. The AuXyn10A displays high similarity to the xylanases of Aspergillus niger, Aspergillus kawachii and Aspergillus niger, members of the glycoside hydrolase family 10. Its three-dimensional structure was predicted using http://swiss-model.expasy.org/on-line programs based on the crystal structure of Penicillium simplicissimum xylanase (1B30_A) from the family 10. The complete DNA gene was cloned from the genomic DNA of A. usamii using conventional PCR and hairpin structure-mediated PCR techniques. The DNA gene is 2,255 bp in length, containing a 510 bp of 5'-flanking promoter region and a 1,745 bp of downstream fragment that consists of ten exons and nine short introns ranging from 52 to 62 bp.
© Springer Science+Business Media B.V. 2011
A novel approach to oral delivery of insulin by conjugating with low molecular weight chitosan.
Source
School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea.
Abstract
A new oral delivery system for insulin was developed aiming to improve bioavailability based on a conjugate between insulin and low molecular weight chitosan (LMWC) of narrow molecular weight distribution. The conjugate was synthesized from the reaction between site-specifically modified insulin at the lysine residue of the B-chain and sulfhydryl-modified LMWC. To investigate the effect of MWs of LMWC on oral bioavailability of insulin, various LMWCs (3, 6, 9, and 13k average MW) with narrow MW distribution were used to synthesize LMWC-insulin conjugates. The content of insulin in the LMWC-insulin conjugates was calculated by UV spectrophotometer: 62%, 44%, 38%, and 29% for 3, 6, 9, and 13 kDa LMWC, respectively. The biological activity of insulin in LMWC(6k)-insulin conjugate in vivo was 43 ± 0.7%. LMWC-insulin conjugates after oral administration to diabetic rat models could control blood glucose levels effectively for several hours. Of those conjugates, LMWC(9k)-insulin exhibited the highest pharmacodynamic bioavailability of 3.7 ± 0.3% relative to that of subcutaneously (s.c.) injected insulin (100%).
Dopamine and cAMP regulated phosphoprotein MW 32 kDa is overexpressed in early stages of gastric tumorigenesis.
Source
Department of Surgery, Section of Surgical Sciences, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Abstract
BACKGROUND:
Gastric adenocarcinoma is a leading cause of cancer mortality. The role of dopamine and cAMP regulated phosphoprotein MW 32 kDa (DARPP-32) overexpression in the gastric tumorigenesis cascade remains unclear.
METHODS:
The expression of DARPP-32 in the multistep carcinogenesis cascade was examined using immunohistochemistry analysis on 533 samples. The contribution of DARPP-32 in cellular transformation and molecular signaling was investigated using NIH3T3, AGS, and SNU16 cells.
RESULTS:
The composite expression score (CES), calculated from immunostaining patterns, increased significantly from normal or gastritis to metaplasia, dysplasia, and adenocarcinoma (P < .001). In patients with normal stomach or gastritis and tumor samples, a 76% and 77% chance, respectively, was found (P < .001) that CES was higher in the tumor. High median CES correlated with well- or moderately differentiated (P = .03) gastric adenocarcinomas. NIH3T3 cells transfected with DARPP-32 demonstrated increased levels of phospho-AKT and a 5-fold increase in the number of foci as compared with the control (P = .02). DARPP-32 expression in AGS cells led to increased protein levels of phospho-AKT and BCL-2. For validation, the knockdown of endogenous DARPP-32 expression in SNU16 cells using shRNA resulted in decreased levels of phospho-AKT phosphorylation and BCL-2.
CONCLUSION:
Our results suggest that DARPP-32 overexpression may participate in the transition to intestinal metaplasia and in the progression to neoplasia. The ability of DARPP-32 to transform NIH3T3 cells and to regulate AKT and BCL-2 underscores its possible oncogenic potential.
Copyright 2010 Mosby, Inc. All rights reserved.
A long form of shrimp astakine transcript: molecular cloning, characterization and functional elucidation in promoting hematopoiesis.
Source
Institute of Zoology, National Taiwan University, Taipei 10617, Taiwan, ROC.
Abstract
Hemocytes play important roles in crustacean immune responses. Generation of new hemocytes (hematopoiesis) is thus necessary to maintain homeostasis which is vital to crustaceans. In vertebrates, certain cytokines have been demonstrated to regulate hematopoiesis and immune responses. In invertebrates, however, little is known about cytokines related to hematopoiesis. In the present study, we cloned an astakine molecule from hemocytic cDNA of tiger shrimp (Penaeus monodon) which was 1509 bp in length with a 5'-UTR of 143 bp, a coding region of 375 bp and a 3'-UTR of 991 bp. The present clone (GenBank accession no. EU980444) showed to be a longer form of astakine transcript with an extra insert of 671 bp in the 3'-UTR than the NCBI-recorded shrimp astakine cDNA sequence (GenBank accession no. AY787657). The deduced protein had 124 amino acid residues, including a signal peptide and one prokineticin domain. The calculated molecular weight (MW) of the mature peptide was 11,295 Da and pI was 5.2. Phylogenetically, this molecule is most similar to astakine-related molecules of arthropod including tiger shrimp astakine, crayfish astakines 1, 2a and 2b, aphid astakine-like molecule and parasitic wasp astakine-like molecule. Nested RT-PCR showed that astakine mRNA is expressed in many tissues and organs of the shrimp such as eyestalk, subcuticular epithelium, gills, heart, hepatopancreas, lymphoid organ, intestine, muscle, nerve and hemocytes. Real-time PCR further revealed that astakine mRNA is expressed mainly in the hemocytes. The astakine transcript is not inducible in the hemocytes until 24 h post LPS injection of shrimp. The recombinant protein of shrimp astakine (rPmAst) was synthesized using insect cell-baculovirus expression system. The authenticity of rPmAst protein was examined by MALDI-MS/MS spectrometry. Using ESI-MS it was determined that the MW of C-terminally histidine-tagged recombinant protein is 12,107 Da. It is 10 Da less than the computer-predicted MW (12,117 Da), allowing the formation of five pairs of disulfide bonds. Using BrdU incorporation assay it was demonstrated that the injection of rPmAst to the shrimp promoted cell proliferation in hematopoietic tissues. Therefore, we conclude that shrimp astakine functions as a cytokine that influences cell proliferation in the hematopoietic tissues.
Copyright 2009 Elsevier Ltd. All rights reserved.
Peak compression factor of proteins.
Source
Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600, USA.
Abstract
An experimental protocol is proposed in order to measure with accuracy and precision the band compression factor G(12)(2) of a protein in gradient RPLC. Extra-column contributions to bandwidth and the dependency of both the retention factor and the reduced height equivalent to a theoretical plate (HETP) on the mobile phase composition were taken into account. The band compression factor of a small protein (insulin, MW kDa) was measured on a 2.1mm x 50mm column packed with 1.7 microm C(4)-bonded bridged ethylsiloxane BEH-silica particles, for 1 microL samples of dilute insulin solution (<0.05g/L). A linear gradient profile of acetonitrile (25-28% acetonitrile in water containing 0.1% trifluoroacetic acid) was applied during three different gradient times (5, 12.5, and 20 min). The mobile phase flow rate was set at 0.20 mL/min in order to avoid heat friction effects (maximum column inlet pressure 180 bar). The band compression factor of insulin is defined as the ratio of the experimental space band variance measured under gradient conditions to the reference space band variance, which would be observed if no thermodynamic compression would take place during gradient elution. It was 0.56, 0.71, and 0.76 with gradient times of 5, 12.5, and 20 min, respectively. These factors are 20-30% smaller than the theoretical band compression factors (0.79, 0.89, and 0.93) calculated from an equation derived from the well-known Poppe equation, later extended to any retention models and columns whose HETP depends on the mobile phase composition. This difference is explained in part by the omission in the model of the effect of the pressure gradient on the local retention factor of insulin during gradient elution. A much better agreement is obtained for insulin when this effect is taken into account. For lower molecular weight compounds, the pressure gradient has little effect but the finite retention of acetonitrile causes a distortion of the gradient shape during the migration of its breakthrough front along the column. This phenomenon should be taken into account in the theoretical models.
[Influence of amino acid and dipeptide composition on protein stability of piezophilic microbes].
Source
Department of Bioengineering and Biotechnology, Huaqiao University, Xiamen 361021, China. zhgyghh@hqu.edu.cn
Abstract
OBJECTIVE:
To compare the amino acid and dipeptide composition of proteins from piezophilic and non-piezophilic microbes is of great significance for understanding the stability of piezophilic protein and directed mutation of them.
METHODS:
The amino acids of different secondary structure and the dipeptides of 639 orthologs proteins were counted and the deviation of them were calculated.
RESULTS:
The amino acid composition based on secondary structure and the dipeptide composition reveals some common trends. The amino acids vary widely in beta sheet and coil. In beta sheet, piezophilic proteins contain more amino acids such as Val, Ile and Leu, whereas less Arg, Lys and Asp; in coil, piezophilic proteins contained more amino acids such as Val and Ile, whereas less Gly and Pro. On the other hand, piezophilic proteins contain more dipeptides such as YM, MN, KD, QC, CI, MW, MM, CY, WQ, HC, RC and QH, whereas less dipeptides such as TW, MS, VD, DH, YE, CT, MW, CF, CK, CM, MY, QI, TH, MQ, QQ and MC.
CONCLUSION:
Dipeptide contains more structure and sequence information than amino acid, and it will be more helpful for understanding the mechanism of piezophilic adaptation and guiding the engineering of proteins.
- PMID:
- 19445175
- [PubMed - indexed for MEDLINE]
Estimation of uncertainty in size-exclusion chromatography with a double detection system (light-scattering and refractive index).
Source
Dpto. Ingeniería Química y Tecnología Farmacéutica, Facultad de Farmacia, Universidad de La Laguna, Avda. Fco. Sanchez, s/n, La Laguna, 38200, La Laguna, Tenerife, Spain. amoliva@ull.es
Abstract
Size-exclusion chromatography (SEC) coupled with online laser light-scattering (LS) and refractive index (RI) detection provides an excellent approach to determine the molecular weights (Mw) of proteins by the "two-detector" approach. Mwis determined only at the maximum of a peak, using either peak heights or area ratio from the two detectors. However, proper calibration of the SEC/LS/RI system is critical to obtain high precision. Today, an essential part of any analysis is to evaluate the uncertainty associated with the method. Basically, it is possible to distinguish between factors related to signal nature, precision and those due to signal processing. Given the signal of interest is the peak height or area ratio from two detectors, the signal ratio uncertainty was calculated using the random propagation of error formula. In this case, the effect of signal correlation was evaluated to avoid the uncertainty overestimation. In the second case, the sources of uncertainty affecting analytical measurement were estimated with the information from the precision assessment. For this, two designs with two-factor fully nested were followed for each method. Finally, the contributions from various uncertainty sources related with calibration are also analysed in detail. There are in fact only three main sources of measurement uncertainty: intermediate precision, calibration and repeatability. Of these, method precision is always the greatest, regardless of approach. For all proteins and peptides studied, the Mw calculated using both methods are close to the theoretical results, independently of the design, but the contributions of individual terms to combined uncertainty depend on both the design and method used. For example, the combined uncertainty varied between 223 and 813.2 Da for carbonic anhydrase, although higher values were found for human insulin and ovalbumin dimer. Other considerations that can have a significant impact on the results are discussed. The reproducibility of the two methods versus that based on ASTRA software used as reference method was performed using the concordance correlation coefficient. The methods' reproducibility depends on the permitted losses in precision and accuracy.
Light-dependent magnetoreception: quantum catches and opponency mechanisms of possible photosensitive molecules.
Source
Biology Department, Duke University, Durham, NC 27708, USA. sjohnsen@duke.edu
Abstract
Dozens of experiments on magnetosensitive, migratory birds have shown that their magnetic orientation behavior depends on the spectrum of light under which they are tested. However, it is not certain whether this is due to a direct effect on the magnetoreceptive system and which photosensitive molecules may be involved. We examined 62 experiments of light-dependent magnetoreception in three crepuscular and nocturnal migrants (48 for the European robin Erithacus rubecula, ten for the silvereye Zosterops lateralis, and four on the garden warbler Sylvia borin). For each experiment, we calculated the relative quantum catches of seven of the eight known photosensitive molecules found in the eyes of passerine birds: a short- (SW), medium- (MW) and long-wavelength (LW) cone pigment, rhodopsin, melanopsin, and cryptochrome in its fully-oxidized and semiquinone state. The following five opponency processes were also calculated: LW-SW, LW-MW, MW-SW, LW-(MW+SW), and cryptochrome-semiquinone. While the results do not clearly show which receptor system may be responsible for magnetoreception, it suggests several candidates that may inhibit the process. The two significant inhibitors of magnetoreceptive behavior were overall irradiances (from 400 to 700 nm) higher than those found at sunset and high quantum catch by the LW receptor. The results were also consistent with the hypothesis that high quantum catch by the semiquinone form of cryptochrome inhibits magnetoreception. The opponency mechanism that best separated oriented from non-oriented behavior was LW-MW, where a difference above a certain level inhibited orientation. Certain regions of experimental spectral space have been over-sampled, while large regions have not been sampled at all, including: (1) from 440 to 500 nm at all irradiance levels, (2) for wavelengths longer than 570 nm from 10(12) to 3x10(12) photons s(-1) cm(-2) and (3) for wavelengths less than 560 nm from 10(12) to 3x10(12) photons s(-1) cm(-2) and below 5x10(11) photons s(-1) cm(-2). Experiments under these conditions are needed to draw further conclusions.
[Relationship between cytokine concentration and activities of daily living in rehabilitation patients with stroke].
Source
Fujita Memorial Nanakuri Institute, Fujita Health University, Tsu.
Abstract
Stroke rehabilitation is effective in some patients, however not so effective in others. Our ultimate aim is to use the clinical laboratory assessment as a tool for effectiveness discrimination in rehabilitation. Subjects were 15 stroke patients (68.1 +/- 12.7 years old) who were admitted to our convalescent rehabilitation wards. Fasting blood samples were analyzed for serum concentrations of hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and insulin-like growth factor-I (IGF-I) which are considered to be involved in hypermyotrophy using ELISA methods on admission and at discharge. Sixteen healthy control subjects (63.0 +/- 7.6 years old) were also employed. As accuracy control of these analyses, decrease of serum HGF after keeping at -20 degrees C for 499 days were measured. The concentration was 0.66ng/mL from 0.71 ng/mL and residual ratio was 94.0%. Reaction specificity to MW 60 kDa HGF antibody using the Western blot method was confirmed. Average HGF and VEGF were higher in stroke patients than those in control subjects. Average IGF-I was lower in stroke patients. The correlations between HGF, VEGF, and IGF-I and the score of activities of daily living expressed by the Functional Independence Measure (FIM) were calculated. Highest correlation coefficient of 0.67 (p < 0.01) was obtained between HGF at discharge and the FIM efficiency (the gain of the FIM during hospitalization divided by length of stay). The correlation coefficients related to VEGF or IGF showed lower value. High FIM efficiency denotes rapid recovery with vigorous exercise. HGF at discharge would reflect the result of high activity.
- PMID:
- 17657984
- [PubMed - indexed for MEDLINE]
Intercellular imaging by a polyarginine derived cell penetrating peptide labeled magnetic resonance contrast agent, diethylenetriamine pentaacetic acid gadolinium.
Source
Imaging Center, Second Affiliated Hospital of Medical School, Xi'an Jiao Tong University, Xi'an 710004, China. cjr.guoyoumin@vip.163.com
Abstract
BACKGROUND:
The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance (MR) contrast agent. Cell penetrating peptide (CPP) is used to internalize proteins, peptides, and radionuclide. This study was undertaken to assess the value of a new intracellular MR contrast medium, CPP labeled diethylenetriamine pentaacetic acid gadolinium (Gd-DTPA) in molecular imaging in vitro.
METHODS:
Fluorescein-5-isothiocyanate (FITC) and Gd-DTPA respectively labeled with CPP (FITC-CPP, Gd-DTPA-CPP) were synthesized by the solid-phase method. Human hepatic cancer cell line-HepG2 was respectively stained by FITC-CPP and FITC to observe the uptake and intracellular distribution. HepG2 was respectively incubated with 100 nmol/ml Gd-DTPA-CPP for 0, 10, 30, 60 minutes, and imaged by MR for studying the relationship between the incubation time and T(1)WI signal. The cytotoxicity to NIH3T3 fibroblasts cells was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide reduction assay (MTT).
RESULTS:
The molecular weights of CPP labeled imaging agents, which were determined by MALDI mass spectrometry (FITC-CPP MW = 2163.34, Gd-DTPA-CPP MW = 2285.99), were similar to the calculated molecular weights. Confocal microscopy suggested HepG2 translocated FITC-CPP in cytoplasm and nucleus independent with the incubation temperature. MR images showed HepG2 uptaken Gd-DTPA-CPP had a higher T(1) weighted imaging (T(1)WI) signal, and that the T(1)WI signal intensity was increasing in a time-dependent manner (r = 0.972, P = 0.001), while the signal intensity between the cells incubated by Gd-DTPA for 60 minutes and the controlled cells was not significantly different (P = 0.225). By MTT, all concentrations from 50 nmol/ml to 200 nmol/ml had no significant (F = 0.006, P = 1.000) effect on cell viability of mouse NIH3T3 fibroblasts, compared with the control group.
CONCLUSIONS:
The newly constructed CPP based on polyarginine can translocate cells by carrying FITC and MR contrast agent Gd-DTPA, and the intracellular concentrations are readily detectable by MR imaging, suggesting a new way for MR molecular imaging.
Cell uptake, cytoplasmic diffusion and nuclear access of a 6.5 nm diameter dendrimer.
Source
Centre for Drug Delivery Research, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1 AX, UK. pakatip.ruenraroengsak@pharmacy.ac.uk
Abstract
Macromolecular crowding and the presence of organelles in the cytosol present barriers to particle mobility, such that it is unclear how nano-carriers can deliver their active agents to the nucleus. In this work a sixth generation amino terminated polyamide polylysine dendrimer (Gly-Lys(63) (NH(2))(64)) (MW 8149, diameter 6.5 nm) which is fluorescent allowed the study of nuclear uptake and mobility in living lung carcinoma (SK/MES-1) and colon adenocarcinoma (Caco-2) cells. The dendrimer is found within 25-45 min of incubation inside the cell nuclei. Living cells were then used to develop a method for the dynamic nuclear uptake study using confocal microscopy. The dynamic uptake of the dendrimer demonstrated here allowed the apparent cytoplasmic diffusion coefficient (D) of the dendrimer to becalculated. Values were found in the range 5.99 x 10(-11)cm(2)s(-1) (SK/MES-1 cells) to 9.82 x 10(-11)cm(2)s(-1) (Caco-2 cells). The difference must reflect variation in the intracellular architecture of the cell types.
Pharmacokinetics of intravenous rifampicin (rifampin) in neonates.
Source
Department of Clinical Pharmacy, University Hospital of Maastricht, The Netherlands. jpul@kfls.azm.nl
Abstract
Few reports have addressed neonatal rifampicin plasma concentrations and data on neonatal rifampicin pharmacokinetics are completely lacking. Therefore, plasma concentrations of rifampicin and its main metabolite 25-O-desacetylrifampicin (DES) were measured in 123 surplus plasma samples from routine vancomycin monitoring in 21 neonates using reversed-phase HPLC. Rifampicin peak and trough plasma concentrations were 4.66 +/- 1.47 mg/L and 0.21 +/- 0.20 mg/L, respectively, after a dose of 8.5 +/- 2.1 (mean +/- SD) mg/kg per day. A significant linear relationship between rifampicin dose and peak plasma concentrations was found, but inter-patient variability was high. Pharmacokinetic parameters of rifampicin were calculated according to a one-compartment open model with iterative two-stage Bayesian fitting (MW\PHARM 3.60, Mediware, The Netherlands). First-order elimination constant, volume of distribution corrected for weight, total body clearance corrected for weight (CL/W), and elimination half-life were 0.16 +/- 0.06 h(-1), 1.84 +/- 0.59 L/kg, 0.28 +/- 0.11 Lkg(-1) h(-1), and 4.9 +/- 1.7 h, respectively. A high Pearson correlation was found between CL/W rifampicin and the covariates plasma creatinine and CL/W gentamicin of a preceding gentamicin treatment course, r = 0.728 (n = 17) and r = 0.837 (n = 12), respectively. DES was detected in each plasma sample. Therefore, rifampicin seems to be eliminated by both renal and metabolic pathways in neonates. In 8 study patients, plasma concentrations of rifampicin and DES were measured again after two weeks of therapy. CL/W rifampicin was significantly higher (67 +/- 50%). The authors suggest maintaining the current dose regimen of 10 mg/kg once a day. Because of the large inter-patient variability in rifampicin plasma concentrations and CL/W increase during therapy, the authors suggest monitoring rifampicin peak and trough plasma concentrations to avoid low plasma concentrations. More research is needed to determine well-founded dosing guidelines.
Estimation of the identity of proteolytic aggrecan fragments using PAGE migration and Western immunoblot.
Source
Department of Clinical Sciences, Orthopaedics, Lund University, Lund, Sweden. andre.struglics@med.lu.se
Abstract
OBJECTIVE:
To develop calculation models, using Western immunoblot, as a tool for the estimation of proteolytic human aggrecan fragment identity.
METHOD:
Seven human aggrecan fragments (calibrators), purified by CsCl gradient centrifugation and identified by Western immunoblot of N- and C-terminals, were used to develop calculation models. The models were used for identification of unknown aggrecan fragments each having one of their N- or C-terminals identified.
RESULTS:
The calibrator molecular weights (Mw) from sodium dodecyl sulfate (SDS)-gels (m), the Mw of amino acids (a) and the Mw of their carbohydrate substitution (g) were expressed as K = m/(a+g), or as K = 1.085m/(a+g) when compensation for the G1 domain was required. Using these models together with average K-values, 12 out of the 17 immuno-detected aggrecan fragments were calculated to a known protease cleavage site, while five were identified to domain levels.
CONCLUSIONS:
With six neoepitope antibodies together with antibodies against the G1- and G3-domain it was possible to predict the identity of several proteolytic fragments from different regions within the aggrecan monomer.
Recombinant generation of two fragments of the rat complement inhibitory factor H [FH(SCR1-7) and FH(SCR1-4)] and their structural and functional characterization in comparison to FH isolated from rat serum.
Source
Department of Immunology, Georg-August University of Göttingen, Germany.
Abstract
Factor H (FH) is the predominant soluble inhibitor of the complement system. With a concentration of 200-800 microg/ml in human and rat plasma it acts as a cofactor for the soluble factor I (FI)-mediated cleavage of the component C3b to iC3b. Furthermore it competes with factor B for binding to C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. FH is a monomer of about 155 kDa which comprises 20 short consensus repeats (SCR), each of which is composed of approximately 60 amino acid (aa) residues. Two functional fragments of FH comprising the SCR1-4 or SCR1-7 were generated using either the Baculovirus system or stably transfected human embryonal kidney cells, respectively. These fragments, as well as FH purified from rat serum, were first analyzed for their relative molecular weights (Mr) using non-reducing or reducing SDS-PAGE. The Mr of the FH variants differed by about 20% depending on the experimental conditions employed. Only the Mr of proteins separated under reducing conditions were in accordance with the MW calculated from the aa sequence. Analyses of the glycosylation patterns using PAS-staining showed a lack of staining of the recombinant variants (SCR1-4 and SCR1-7) in contrast to FH(SCR1-20) from serum. Using a complement hemolysis assay (CH50-assay) all three variants exhibited a molar complement inhibitory activity of FH(1-20)/FH(1-7)/FH(1-4) of about 3/1/1. These data support the postulated model of FH bearing three binding sites for its ligand C3b, from which one is located in the SCR1-4, whereas the other two are located in the SCR8-20.
Determination of potato carboxypeptidase inhibitor in African Green Monkey plasma using 96-well SPE and LC-MS/MS.
Source
Merck Research Laboratories, West Point, PA 19486, USA. marose@amgen.com
Abstract
Potato carboxypeptidase inhibitor (CPI), a peptide with multiple isoforms (MW>4000 Da) was determined from African Green Monkey plasma using a PE Sciex API-3000 LC-MS/MS in the positive ionization mode with the turbo ionspray interface (450 degrees C). Samples were prepared using an Oasis MCX 96-well solid phase extraction plate and chromatographed on an Allure C18 HPLC Column (50 mm x 1.0 mm, 5 microm) using gradient elution. Upon analysis of the extracts using LC-MS/MS, the concentration of CPI was calculated using a single MS/MS transition (m/z 830.5-->221.0) that was reflective of the mass concentration (microg/mL) of main the CPI isoforms present in plasma from monkeys after they were given an intravenous dose of CPI. The assay was linear for CPI over concentrations of 0.05-10 microg/mL when extracting 200-microL aliquots of African Green Monkey plasma. The assay was applied to the determination of CPI in African Green Monkey plasma samples in two separate analytical runs (correlation of standard curves, r1=0.9991 and r2=0.9953). Quality control (QC) samples were run at 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 microg/mL for each assay. Average ranges (n=12) for accuracy and precision for all concentrations of QCs during the two runs were 92.0-102.0% of expected potency and 10.4-21.8% (coefficient of variations), respectively.
Synthesis, characterization and cytotoxicity of poly(ethylene glycol)-graft-trimethyl chitosan block copolymers.
Source
Department of Pharmaceutics and Biopharmacy, Philipps-University of Marburg, Ketzerbach 63, D-35032 Marburg, Germany.
Abstract
PEGylated trimethyl chitosan (TMC) copolymers were synthesized in an attempt to both increase the solubility of chitosan in water, and improve the biocompatibility of TMC. A series of copolymers with different degrees of substitution were obtained by grafting activated poly(ethylene glycol)s (PEG) of different MW onto TMC via primary amino groups. Structure of the copolymers was characterized using 1H, 13C NMR spectroscopy and GPC. Solubility experiments demonstrated that PEG-g-TMC copolymers were completely water-soluble over the entire pH range of 1-14 regardless of the PEG MW, even when the graft density was as low as 10%. Using the methyl tetrazolium (MTT) assay, the effect of TMC molecular weight, PEGylation ratio, PEG and TMC molecular weight in the copolymers, and complexation with insulin on the cytotoxicity of TMC was examined, and IC50 values were calculated with L929 cell line. All polymers exhibited a time- and dose-dependent cytotoxic response that increased with molecular weight. PEGylation can decrease the cytotoxicity of TMC to a great extent in the case of low molecular weight TMCs. According to the cytotoxicity results, PEG 5 kDa is superior for PEGylation when compared to PEG 550 Da at similar graft ratios. Complexation with insulin further increased cell viability. In addition, Lactate dehydrogenase (LDH) assays were performed to quantify the membrane-damaging effects of the copolymers, which is in line with the conclusion drawn from MTT assay. Moreover, the safety of the copolymers was corroborated by observing the morphological change of the cells with inverted phase contrast microscopy. Based upon these results PEG-g-TMC merits further investigations as a drug delivery vehicle.
Dynamic heterodimer-functionalized surfaces for endothelial cell adhesion.
Source
Department of Bioengineering, University of Pennsylvania, 3320 Smith Walk, 120 Hayden Hall, Philadelphia, PA 19104, USA.
Abstract
The functionalization of hydrogels for receptor-mediated cell adhesion is one approach for targeted cell and tissue engineering applications. In this study, polyacrylamide gel surfaces were functionalized with specific cell adhesion ligands via the self-assembly of a peptide-based heterodimer. The system was comprised of a cysteine-terminated monomer, A (MW approximately 5400), grafted to the polyacrylamide gels and a complementary ligand presenting monomer, B(X) (MW approximately 5800) that was designed to heterodimerize with A. Two ligand presenting monomers were synthesized: one presenting the RGDS ligand, B(D), for receptor-mediated cell adhesion, and the other, a control monomer presenting the nonadhesive RGES ligand, B(E). Assembly of the peptide pair A-B(X) by association of the monomers into a coiled coil was verified by circular dichroism in solution. Binding studies were conducted to determine the dissociation constant of the pair A-B(X), which was found to be K(D) approximately 10(-8) m. Polyacrylamide gels functionalized with A-B(X) heterodimers were evaluated for cell adhesion using bovine aortic endothelial cells (BAECs). Endothelial cells cultured on the A-B(D) functionalized surfaces demonstrated typical cell morphologies and expected spreading behavior as a function of the density of RGDS ligand, calculated as the amount of B(D) associated with grafted A on the surface of the gels. In contrast, A-B(E) linked surfaces supported no cell adhesion. The adhesion of the substrate was dynamically altered through the reassembly of A-B(X) dimers as B(D) molecules in the solution replaced B(E) molecules at the substrate. The molecular constructs described here demonstrate the potential to design a broad family of switchable peptides that impart the dynamic control of biofunctionality at an interface, which would be useful for precise manipulation of cell physiology.
Isolation and characterization of a heparin with high anticoagulant activity from the clam Tapes phylippinarum: evidence for the presence of a high content of antithrombin III binding site.
Source
Department of Biologia Animale, University of Modena and Reggio Emilia, Via Campi 213/D, 41100 Modena, Italy.
Abstract
Heparin with high anticoagulant activity (activated partial thromboplastin time of 347 +/- 56.4 and anti-Xa activity of 317 +/- 48.3) was isolated from the marine clam species Tapes phylippinarum in an amount of approximately 2.1 mg/g dry animals. Agarose-gel electrophoresis showed a high content of the slow-moving heparin component (22 +/- 6.8%) and 78 +/- 5.4% of the fast-moving species. An average molecular mass of 13,600 was calculated by PAGE analysis, whereas a number average molecular weight Mn value of 10,700, a weight average molecular weight Mw of 14,900, and a dispersity index Mn/Mw of 1.386 were obtained by high-performance size-exclusion chromatography. Structural analysis of clam heparin, performed by depolymerizing heparin samples with heparinase (EC 4.2.2.7) and then separating the resulting unsaturated oligosaccharides by strong anion exchange-HPLC revealed the presence of large amounts (more than 130% than standard pharmaceutical heparin obtained from bovine intestine) of the oligosaccharide sequence bearing part of the ATIII-binding region, DeltaUA2S (1-->4)-alpha-D-GlcN2S6S (1-->4)-alpha-L-IdoA (1-->4)-alpha-D-GlcNAc6S (1-->4)-beta-D-GlcA (1-->4)-alpha-D-GlcN2S3S6S in the T. phylippinarum heparin, in comparison with bovine mucosal heparin and a sample of porcine mucosal heparin previously published. Furthermore, as expected from the oligosaccharide compositional analysis, due to the presence of a great mol % (80.6%) of the trisulfated disaccharide DeltaUA2S(1-->4)-alpha-D-GlcN2S6S, mollusc heparin is a more sulfated polysaccharide than bovine mucosal heparin (73.5%) and a sample of porcine mucosal (72.8%) heparin previously reported. To our knowledge, this is the first article describing a clam heparin having the ATIII binding site mainly identical to that of human and porcine intestinal mucosal heparins and bovine intestinal mucosal heparin but different from that found in beef lung heparin.
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