Characterization of the secreted proteome of rat hepatocytes cultured in collagen sandwiches

Analysis of proteins in biological samples opens up the possibility of discovering new markers of toxicity. The liver is one of the primary targets of drug-induced toxicity, and it also secretes many plasma proteins, which can be measured clinically. Most of the plasma proteins produced by the liver are secreted by hepatocytes, but there is little information regarding the protein profile secreted by these cells. The purpose of this study was to analyze the secreted proteome of primary rat hepatocytes in a collagen gel sandwich configuration by a gel-LC-MS/MS procedure. We identified over 600 peptides corresponding to more than 200 proteins. The protein profile included over 50 plasma proteins, suggesting that the cultured hepatocytes secrete many of the proteins that they produce in vivo. Our data also suggests that the hepatocytes are actively remodeling their environment, since we identified several structural extracellular matrix proteins as well as some proteins known to be secreted specifically during liver regeneration. We also identified two proteins, alpha1-antitrypsin and alpha2-macroglobulin, whose secretions appear to be down-regulated in cells exposed to aflatoxin B1. It was noted that a 15 nM dose of aflatoxin B1 led to substantially diminished levels of these proteins and that day 6 of incubation was the ideal time point for medium collection. These data suggest that proteins in the conditioned medium of hepatocyte sandwich culture might lead to the discovery of biomarkers for drug-induced chemical toxicity.     

Partial purification and characterisation of a synovial fluid inhibitor of osteoblasts.

A polypeptide inhibitor of osteoblast proliferation is described which occurs in synovial effusions of patients with rheumatoid arthritis. Partial purification of the inhibitor showed a molecular weight of approximately 81,000 by gel electrophoresis. This polypeptide seems to be unique as no inhibitor of osteoblasts of similar molecular weight has been previously described in rheumatoid synovial effusions.     

Vibrionaceae from cases of acute diarrhoea and their antimicrobial sensitivity pattern - a five year prospective study

Over a five year period, stool samples were screened for Vibrionaceae from cases of acute diarrhoea, to study their isolation rate and their antimicrobial sensitivity pattern. All the isolates were identified by standard laboratory techniques. A total of 323 species belonging to Vibrionaceae were isolated from 4492 stool samples tested over five year period (1996-2000), giving a positivity rate of 7.2%. Maximum isolation was during the months of May to August (62.5%). Out of 323 isolates, Vibrio spp. comprised 252 and 93.3% of them were Vibrio cholerae O1 biotype El Tor. Aeromonas spp. were isolated from 71 samples and 64.8% of them were A. hydrophila. V. cholerae showed 86.8% sensitivity to amikacin followed by 73.8% to cefotaxime. Tetracycline sensitivity was only 39.6%. Aeromonas spp. also showed maximum sensitivity to amikacin (70.4%). Isolation of Vibrio spp. have increased over the years, whereas Aeromonas spp. have decreased. Amikacin sensitivity has remained within 70-80% over the years, cefotaxime sensitivity has increased and tetracycline sensitivity has decreased.     

Formulation and evaluation of acyclovir microcapsules using bakers yeast

ObjectiveTo formulate and evaluate acyclovir microcapsules using bakers yeast.MethodsAcyclovir, pretreated yeast and deionised water were taken at a volumetric ratio of 1:2:4 respectively. This suspension was agitated in a magnetic stirrer at 25°C, 30°C, 35°C, and 40°C for 4 hours. The suspension was then centrifuged for 10 minutes at 2 000 rpm. The supernatant solution was decanted and the cells were washed 5 times with deionised water. Then the suspended drug entrapped yeast cells were dried in a lyophillizer for 48 hours. The yield was noted.ResultsThe first four formulations were done with 200 mg of the drug, followed by 400 mg for the next four formulations and 800 mg the last four formulations. SEM showed that the surface of the microcapsules was intact, with no burst characteristics. FTIR showed no interaction between acyclovir and the cell wall. DSC showed that the peak was within the standard values. The mean particle size for all the samples was 8 μm in diameter. The dissolution studies were done for all the twelve samples and showed a Fickian model of diffusion.ConclusionsFrom the results it is inferred that the samples prepared at 40°C (FY-4, FY-8, FY-12) show better entrapment and release. So these samples are formulated in the form of a suspension and compared with marketed acyclovir suspension using HPLC technique. The formulated suspensions with FY-4, FY-8 and FY-12 shows drug content in accordance with the standards of the pharmacopoeial limits.     

Truncating mutation in the NHS gene: phenotypic heterogeneity of Nance-Horan syndrome in an asian Indian family

PURPOSE: A four-generation family containing eight affected males who inherited X-linked developmental lens opacity and microcornea was studied. Some members in the family had mild to moderate nonocular clinical features suggestive of Nance-Horan syndrome. The purpose of the study was to map genetically the gene in the large 57-live-member Asian-Indian pedigree. METHODS: PCR-based genotyping was performed on the X-chromosome, by using fluorescent microsatellite markers (10-cM intervals). Parametric linkage analysis was performed by using two disease models, assuming either recessive or dominant X-linked transmission by the MLINK/ILINK and FASTLINK (version 4.1P) programs (http:www.hgmp.mrc.ac.uk/; provided in the public domain by the Human Genome Mapping Project Resources Centre, Cambridge, UK). The NHS gene at the linked region was screened for mutation. RESULTS: By fine mapping, the disease gene was localized to Xp22.13. Multipoint analysis placed the peak LOD of 4.46 at DSX987. The NHS gene mapped to this region. Mutational screening in all the affected males and carrier females (heterozygous form) revealed a truncating mutation 115C-->T in exon 1, resulting in conversion of glutamine to stop codon (Q39X), but was not observed in unaffected individuals and control subjects. conclusions. A family with X-linked Nance-Horan syndrome had severe ocular, but mild to moderate nonocular, features. The clinical phenotype of the truncating mutation (Q39X) in the NHS gene suggests allelic heterogeneity at the NHS locus or the presence of modifier genes. X-linked families with cataract should be carefully examined for both ocular and nonocular features, to exclude Nance-Horan syndrome. RT-PCR analysis did not suggest nonsense-mediated mRNA decay as the possible mechanism for clinical heterogeneity.     

Potential radiotherapy improvements with respiratory gating

Gating is a relatively new and potentially useful therapeutic addition to external beam radiotherapy applied to regions affected by intra-fraction motion. The impact was of gating on treatment margins, image artifacts, and volume and positional accuracy was investigated by CT imaging of sinusoidally moving spheres. The motion of the spheres simulates target motion. During the CT imaging of dynamically moving spheres, gating reproduced the static volume to within 1%, whereas errors of over 20% were observed where gating was not used. Using a theoretical analysis of margins, gating alone or in combination with an electronic portal imaging device may allow a 2-11 mm reduction in the CTV to PTV margin, and thus less healthy tissue need be irradiated. Gating may allow a reduction of treatment margins, an improvement in image quality, and an improvement in positional and volumetric accuracy of the gross tumor volume.     

A pilot assessment of oxidative stress byproducts and antioxidant activities among Indian patients with various stages of hypertension

In humans, hypertension is considered a state of oxidative stress that can contribute to the development of atherosclerosis and other hypertension-induced organ damages. The objective of this study was to evaluate oxidative status, antioxidant activities, and oxidative stress by-products among Indian patients with various stages of hypertension. Lipid profile, enzymatic and non-enzymatic antioxidants, lipid peroxidation as thiobarbituric acid reactive substances (TBARS), C-reactive protein, electrolytes, and minerals were analyzed in the blood of newly diagnosed prehypertensives, stage I and II hypertensives (n = 20 in each group) and were compared to their age-matched normotensives. Elevated levels of lipid profile (except high density lipoprotein cholesterol [HDL-C]) were observed in stage I and II hypertensive patients. Enzymatic and non-enzymatic antioxidants were significantly (P < 0.05) lower, while TBARS and C-reactive protein were higher in prehypertensives, and stage I and II hypertensives. Significant (P <0.05) changes were also observed in the plasma Na(+) and K(+) concentrations among the hypertensive groups. Serum levels of zinc, copper, and magnesium were significantly (P < 0.05) lower in prehypertensives, and stage I and II hypertensives as compared to normotensives. The study indicated a strong association between blood pressure (BP) and oxidative stress-related parameters and suggests a possible role of oxidative stress in the development of elevated BP.