Characterization of Lung Fibroblasts More than Two Decades after Mustard Gas Exposure

Purpose

In patients with short-term exposure to the sulfur mustard gas, the delayed cellular effects on lungs have not been well understood yet. The lung pathology shows a dominant feature consistent with obliterative bronchiolitis, in which fibroblasts play a central role. This study aims to characterize alterations to lung fibroblasts, at the cellular level, in patients with delayed respiratory complications after short-term exposure to the sulfur mustard gas.

Methods

Fibroblasts were isolated from the transbronchial biopsies of patients with documented history of exposure to single high-dose sulfur mustard during 1985–7 and compared with the fibroblasts of control subjects.

Results

Compared with controls, patients’ fibroblasts were thinner and shorter, and showed a higher population doubling level, migration capacity and number of filopodia. Sulfur mustard decreased the in vitro viability of fibroblasts and increased their sensitivity to induction of apoptosis, but did not change the rate of spontaneous apoptosis. In addition, higher expression of alpha smooth muscle actin showed that the lung''s microenvironment in these patients is permissive for myofibroblastic differentiation.

Conclusions

These findings suggest that in patients under the study, the delayed pulmonary complications of sulfur mustard should be considered as a unique pathology, which might need a specific management by manipulation of cellular components.     

Molecular charcterization of tatD DNAse gene from Ralstonia paucula RA4T soil bacterium

Ralstonia paucula strain RA4^T, a gram negative, non-spore forming, motile bacterium having positive catalase and oxidase test, was isolated from surface soil. Twin arginine translocation protein type D (TatD) is shown to be located in cytoplasm and exhibits magnesium-dependent DNase. A tatD DNase gene was isolated and cloned from Ralstonia paucula RA4^T genome. Nucleotide sequence analysis of the gene revealed 813 nucleotides encoding a protein of 270 amino acid residues. The tatD gene showed a high similarity to homolog gene from Ralstonia pickettii strain 12D. The deduced polypeptide sequence of TatD DNase from R. paucula RA4^T had a typical catalytic site, HHPLDEHRHDP, and its calculated molecular mass and predicted isoelectric point were 29616 Da and 5.33, respectively. The deduced amino acid sequence showed a high degree of similarity to TatD DNase isoforms from Ralstonia genus and other sources. Predicted three-dimensional structure of TatD confirmed the presence of active site and theoretical function as DNase.     

Efficient isolation of high quality nucleic acids from different tissues of <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.     

Association and impact of <Emphasis Type="Italic">XPG Asp</Emphasis> 1104 <Emphasis Type="Italic">His</Emphasis> gene polymorphism in HIV 1 disease progression to AIDS among north Indian HIV seropositive individuals

Various efforts made to stop the deadly epidemic of HIV since its discovery in 1983 remain unsuccessful and this virus still continues to claim the lives of millions of individuals every year. The viral effect in the cell is complicated and the overall disease outcome is the result of interaction between a few viral proteins and complex host immune response. Because it has been reported that XPG (Xeroderma pigementesum group G) gene does play a role in reducing UV induced apoptosis and participate in Nucleotide Excision Repair (NER) process of DNA damage, it was hypothesized that polymorphism in this gene may have a role in HIV 1 disease progression to AIDS. The aim of the present study, therefore, was to find out the association between XPG gene polymorphism and its effect on the rate of HIV 1 disease progression to AIDS. 300 HIV seropositive cases and an equal number of age and sex matched controls were recruited for the study from north Indian population. The PCR-RFLP method was utilized to genotype 600 study subject for the XPG Asp ¹¹⁰⁴ His gene polymorphism. There was significant difference in the frequency of the His/His variant genotype (OR 1.95, 95% CI = 1.93–3.63, P = 0.04) between cases and controls indicating a probable role of this gene in host viral interactions.     

Whole-body to tissue concentration ratios for use in biota dose assessments for animals

Environmental monitoring programs often measure contaminant concentrations in animal tissues consumed by humans (e.g., muscle). By comparison, demonstration of the protection of biota from the potential effects of radionuclides involves a comparison of whole-body doses to radiological dose benchmarks. Consequently, methods for deriving whole-body concentration ratios based on tissue-specific data are required to make best use of the available information. This paper provides a series of look-up tables with whole-body:tissue-specific concentration ratios for non-human biota. Focus was placed on relatively broad animal categories (including molluscs, crustaceans, freshwater fishes, marine fishes, amphibians, reptiles, birds and mammals) and commonly measured tissues (specifically, bone, muscle, liver and kidney). Depending upon organism, whole-body to tissue concentration ratios were derived for between 12 and 47 elements. The whole-body to tissue concentration ratios can be used to estimate whole-body concentrations from tissue-specific measurements. However, we recommend that any given whole-body to tissue concentration ratio should not be used if the value falls between 0.75 and 1.5. Instead, a value of one should be assumed.     

Sperm status and DNA dose play key roles in sperm/ICSI‐mediated gene transfer in caprine

In relation to the growing recent interest in the establishment of sperm‐mediated gene transfer (SMGT) technology as a convenient and effective method for the simple production of transgenic animals, in this study the possibility of using SMGT to produce transgenic caprine embryos was investigated for the first time. Buck sperm were directly incubated with different concentrations (0–500 ng) of pcDNA/his/Lac‐Z plasmid and used for IVF or ICSI. Sperm used for ICSI were categorized into motile or live‐immotile group before being injected into oocytes. In a separate experiment, dead sperm prepared by repeated freezing/thawing were used for DNA‐incubation before ICSI. Sham injection was carried out by intracytoplasmic injection of approximately the same volume of media containing different doses of DNA using an ICSI needle. Transgene expression and transmission were detected by X‐Gal staining and PCR analysis of developed embryos, respectively. A reasonable blastocyst rate was observed in all the groups. Only embryos in the sham group were negative for transgene transmission. Transgene expression was completely dependent on the delivery technique and status of sperm, and was only observed in the live‐immotile and dead ICSI groups. The results of this study showed that the technique (IVF vs. ICSI vs. sham injection), sperm status (motile vs. live‐immotile vs. dead) and to some extent DNA concentration affect embryo development, transgene transmission and expression. Mol. Reprod. Dev. 77:868–875, 2010. © 2010 Wiley‐Liss, Inc.     

Shotgun strategy-based proteome profiling analysis on the head of silkworm Bombyx mori

Insect head is comprised of important sensory systems to communicate with internal and external environment and endocrine organs such as brain and corpus allatum to regulate insect growth and development. To comprehensively understand how all these components act and interact within the head, it is necessary to investigate their molecular basis at protein level. Here, the spectra of peptides digested from silkworm larval heads were obtained from liquid chromatography tandem mass spectrometry (LC–MS/MS) and were analyzed by bioinformatics methods. Totally, 539 proteins with a low false discovery rate (FDR) were identified by searching against an in-house database with SEQUEST and X!Tandem algorithms followed by trans-proteomic pipeline (TPP) validation. Forty-three proteins had the theoretical isoelectric point (pI) greater than 10 which were too difficult to separate by two-dimensional gel electrophoresis (2-DE). Four chemosensory proteins, one odorant-binding protein, two diapause-related proteins, and a lot of cuticle proteins, interestingly including pupal cuticle proteins were identified. The proteins involved in nervous system development, stress response, apoptosis and so forth were related to the physiological status of head. Pathway analysis revealed that many proteins were highly homologous with the human proteins which involved in human neurodegenerative disease pathways, probably implying a symptom of the forthcoming metamorphosis of silkworm. These data and the analysis methods were expected to be of benefit to the proteomics research of silkworm and other insects.     

Application of one-step liquid chromatography–electrospray tandem MS/MS and collision-induced dissociation to quantification of ezetimibe and identification of its glucuronated metabolite in human serum: A pharmacokinetic study

A new one-step liquid chromatography–electrospray tandem MS/MS method is described to quantify ezetimibe (EZM) a novel lipid lowering drug in human serum. Also using collision-induced dissociation (CID) of the analyte, identification and chromatographic separation of its major metabolite, ezetimibe glucuronide (EZM-G) is achieved in this study. A thawed serum aliquot of 100 μL was deproteinated by addition of 500 μL methanol containing omeprazole as internal standard (I.S.). Separation of the drug, its metabolite and the I.S. were achieved using acetonitrile–water (70:30, v/v) as mobile phase at flow rate of 0.5 mL/min on a MZ PerfectSil target C18 column. Multiple reaction monitoring (MRM) mode of precursor–product ion transition (408.7 → 272.0 for EZM and 345 → 194.5 for the I.S.) was applied for detection and quantification of the drug while, EZM-G was chromatographically separated and identified using CID. The analytical method was linear over the concentration range of 1–32 ng/mL of EZM in human serum with a limit of quantification of 1 ng/mL. The coefficient variation values of both inter- and intra-day analysis were less than 8% whereas the percentage error was less than 3.7. The validated method was applied in a randomized cross-over bioequivalence study of two different EZM preparations in 24 healthy volunteers.     

Localization of synaptic proteins involved in neurosecretion in different membrane microdomains

A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Ca(v)2.1 (P/Q-type) channels and soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Ca(v)2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-beta-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery.     

Simultaneous purification and polymerization method for bovine serum albumin preparation

Bovine serum albumin (BSA) has various applications in blood group serology and different research purposes. In this study purification of BSA has been compared with human serum albumin (HSA) using modified ethanol precipitation method based on the method of Cohn. The purification process was carried out under controlled conditions, particularly of ethanol concentration, pH, ionic strength and temperature. It was revealed that the produced BSA and HSA have purity more than 95%. It is obvious that HSA can be used, as a drug when the amount of its polymers is less than 5% whereas polymer generation is required in order to enhance the potentiating properties of BSA in agglutination of red cells. We propose here a simple and rapid two-step method for simultaneously purification and polymerization of BSA. By this method simply BSA with desired amount of polymers was obtained by 40% ethanol concentration.