Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp. from the chemostat.

Pseudomonas sp. strain IST 103 (PCP103) capable of utilizing pentachlorophenol (PCP) was determined by utilization of a carbon source and release of the hydroxylating enzyme PCP-4 monooxygenase. The metabolites were extracted from the culture medium and analyzed by high-performance liquid chromatography. The enzyme purified to apparent homogeneity from an extract of PCP-grown cells indicated that a fraction of DEAE-cellulose ion exchange chromatography of molecular size of 30,000 kDa determined by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis was responsible for dechlorination of PCP. The plasmid isolated from the bacterium was subjected to Shotgun cloning by restriction digestion by BamHI, HindIII, and SalI, ligated to pUC19 vector, and transformed into Escherichia coli XLBlue1alpha. The recombinant clones having higher potentiality to degrade PCP were selected by utilization of a carbon source and release of intermediary metabolites during degradation of PCP as the sole source of carbon and energy. The recombinant clones, which contained an insert of 3.0 kb of SalI and HindIII sites, were sequenced and compared with gene sequences deposited in GenBank by BLAST search; this indicated homology with the thdf gene of monooxygenase of thiophene and furan. Southern blot analysis performed by developing gene probes indicated the presence of the PCP monooxygenase gene in plasmids of the bacterium.     

Physiological correlates of calcium-accumulating properties of mitochondria: Fish-muscle mitochondria

The mitochondria isolated from the muscles of fish acclimated to grow in different salinities have been studied with reference to their Ca²⁺ uptake capacity and compared to those isolated from fresh-water fish muscle. The results show a drastic response by the mitochondria with reference to their Ca²⁺ uptake function soon after exposure to the stress. Evidence is also presented to suggest an alteration in conformation. This perturbation appears to be the initial response to the stress since the normal state (as that of the fresh-water fish) is restored in course of time. Further, so far there is no indication that the electron transport function and ATP production are affected by the ionic stress conditions. This would support the physiological relevance of the mitochondrial capacity for Ca²⁺ uptake.     

Protective role of ascorbic acid against lipid peroxidation and myocardial injury

Ascorbic acid (AH₂) is a potential scavenger of superoxide radical and singlet oxygen. In the guinea pig, marginal AH₂ deficiency results in intracellular oxidative damage in the cardiac tissue as evidenced by lipid peroxidation, formation of fluorescent pigment and loss of structural integrity of the microsomal membranes. The oxidative damage does not occur due to lack of enzymatic scavengers of reactive oxygen species such as superoxide dismutase, catalase and glutathione peroxidase. Also, glutathione transferase activity is not decreased in AH₂ deficiency. Lipid peroxidation, fluorescent pigment formation and protein modification disappear after AH₂ therapy. These results, if extra-polated to human beings, would indicate that chronic subclinical AH₂ deficiency may result in progressive oxidative damage which in the long run may lead to permanent degenerative diseases in the heart.     

Intrapatient diversity and its correlation with viral setpoint in human immunodeficiency virus type 1 CRF02_A/G-IbNG infection

The human immunodeficiency virus type 1 (HIV-1) viral setpoint during the disease-free interval has been strongly associated with future risk of disease progression. An awareness of the correlation between viral setpoint and HIV-1 genetic evolution over time is important in the understanding of viral dynamics and infection. We examined genetic diversity in HIV-1 CRF02_A/G-IbNG-infected seroincident women in Dakar, Senegal; determined whether a viral setpoint kinetic pattern existed for CRF02_A/G-IbNG during the disease-free interval; and correlated viral load level and diversity. Samples were drawn during the disease-free interval from consenting CRF02_A/G-IbNG-infected, antiretroviral therapy-na?ve female commercial sex workers in Dakar, Senegal. Based on sequential plasma RNA values, low and high viral setpoint groups were established. Intrapatient diversity and divergence over time was determined from earlier and later time point DNA samples from each person. Most individuals followed the viral setpoint paradigm. For each 1/-/log(10) copy/ml of plasma increase in viral load, intrapatient diversity increased by 1.4% (P = 0.028). A greater diversification rate was observed in the high viral setpoint group than in the low viral setpoint group (P = 0.01). Greater nucleotide (P = 0.015) and amino acid (P = 0.048) divergences and a greater nucleotide divergence rate (P = 0.03) were found in the high viral setpoint group. There was no difference between the groups in the ratio of the number of nonsynonymous substitutions per nonsynonymous site to the number of synonymous substitutions per synonymous site. The greater intrapatient diversity, divergence, and diversification rates observed in the high viral setpoint group supports the notion that diversity is driven by cycles of viral replication resulting in accumulated mutations. Recognizing diversity potential based on viral load levels in individuals may inform the design of vaccines and therapies.     

Calmodulin-Mediated Signal Transduction Pathways in Arabidopsis Are Fine-Tuned by Methylation

Calmodulin N-methyltransferase (CaM KMT) is an evolutionarily conserved enzyme in eukaryotes that transfers three methyl groups to a highly conserved lysyl residue at position 115 in calmodulin (CaM). We sought to elucidate whether the methylation status of CaM plays a role in CaM-mediated signaling pathways by gene expression analyses of CaM KMT and phenotypic characterization of Arabidopsis thaliana lines wherein CaM KMT was overexpressed (OX), partially silenced, or knocked out. CaM KMT was expressed in discreet spatial and tissue-specific patterns, most notably in root tips, floral buds, stamens, apical meristems, and germinating seeds. Analysis of transgenic plants with genetic dysfunction in CaM KMT revealed a link between the methylation status of CaM and root length. Plants with suppressed CaM methylation had longer roots and CaM KMT OX lines had shorter roots than wild type (Columbia-0). CaM KMT was also found to influence the root radial developmental program. Protein microarray analyses revealed a number of proteins with specificity for methylated forms of CaM, providing candidate functional intermediates between the observed phenotypes and the target pathways. This work demonstrates that the functionality of the large CaM family in plants is fine-tuned by an overarching methylation mechanism.     

ASSESSMENT OF Cryptococcus albidus FOR BIOPULPING OF EUCALYPTUS

Cryptococcus albidus shows delignification activity in nature. It was used for the biopulping of eucalyptus wood (Eucalyptus grandis) to access its potential for industrial application in the pulp and paper industry. Enzyme analysis on days 15, 30, and 60 showed the presence of laccase and xylanase as key enzymes. The production of endo-glucanase (CMCase) and exo-glucanase (FPase) was very low. Scanning electron microscopy (SEM) showed the surface colonization of wood and loosening of wood fibers in C. albidus-treated samples. Fourier-transformation infrared spectroscopy (FT-IR) indicated the chemical modification of eucalyptus wood. Denaturing gradient gel electrophoresis (DGGE) analysis on days 15, 30, and 60 confirmed the presence of C. albidus throughout the experiments. Cryptococcu albidus was able to suppress the growth of a native population. Further, after 60 days both the control and treated eucalyptus wood chips were given kraft pulping treatment. The kappa number of pulp of control wood was 21 and for treated wood was 17. Kappa number is considered a measure of lignin content in wood; hence the treatment of eucalyptus by C. albidus (biopulping) was effective in reducing its lignin content and can be used for biopulping in the pulp and paper industry.     

Analysis of Aβ (1-40) and Aβ (1-42) monomer and fibrils by capillary electrophoresis

A method based on capillary electrophoresis (CE) with UV absorbance detection is presented to characterize synthetic amyloid beta (Aβ) peptide preparations at different aggregation states. Aggregation of Aβ (1-40) and Aβ (1-42) is closely linked to Alzheimer's disease (AD), and studying how Aβ peptides self-assemble to form aggregates is the focus of intense research. Developing methods capable of identifying, characterizing and quantifying a wide range of Aβ species from monomers to fully formed fibrils is critical for AD research and is a major analytical challenge. Monomer and fibril samples of Aβ (1-40) and Aβ (1-42) were prepared and characterized for this study. The monomer-equivalent concentration for each sample was determined by HPLC-UV, and aggregate formation was confirmed and characterized by transmission electron microscopy. The same samples were studied using CE with UV absorbance detection. Analysis by mass spectrometry of collected CE fractions was used to confirm the presence of Aβ for some CE-UV peaks. The CE-UV method reported here clearly indicates that monomeric and aggregated Aβ were electrophoretically separated, and substantial differences in the electrophoretic profiles between samples of Aβ (1-40) and Aβ (1-42) were observed. This CE-UV method can differentiate between Aβ monomer, oligomeric intermediates, and mature fibrils.     

Development and validation of highly sensitive method for determination of misoprostol free acid in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry: application to a clinical pharmacokinetic study

A highly sensitive, selective and evaporation free SPE extraction, ESI-LC-MS/MS method has been developed for estimation of misoprostol free acid in human plasma using misoprostol acid-d(5) as an internal standard (IS). The analyte was separated using isocratic mobile phase on reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M-H] anions, m/z 367-249 for misoprostol acid and m/z 372-249 for the IS. The total run time was 5.0 min and the elution of misoprostol acid and misoprostol acid-d(5) (IS) occurred at 3.6 min. The developed method was validated in human plasma with a lower limit of quantification of 2.5 pg/mL. A linear response function was established for the range of concentrations 2.5-1200 pg/mL (r>0.998) for misoprostol acid in human plasma. The intra and inter-day precision values for misoprostol acid met the acceptance as per FDA guidelines. Misoprostol acid was stable in the battery of stability studies viz., bench-top, auto-sampler and freeze/thaw cycles. The developed assay method was applied to an oral pharmacokinetic study in humans.     

Impact of low oxygen on the secretome of human adipose-derived stromal/stem cell primary cultures

Tissue fibrosis can lead to organ dysfunction, patient morbidity, and mortality. Adipose-derived Stromal/stem Cells (ASCs) represent a potential therapeutic. Immediately following grafting, ASCs would reside in a lower O₂ environment. ASC secretome was examined under 5% O₂ (“low O₂”) and 21% O₂ (“ambient O₂”) culture conditions. ASCs from five female donors were cultured in low or ambient O₂ conditions for 3 days and pooled conditioned medium was compared by two-dimensional liquid chromatography and tandem mass spectrometry (2D-LC–MS/MS). Of 71 proteins identified, five proteins involved in extracellular matrix (ECM) remodeling exhibited ≥2-fold decrease under low O₂ culture and were confirmed by Western immunoblot and qRT-PCR: fibronectin 1, TGF-β1-induced protein (βig-h3), osteonectin, and collagens type 1α₁ and α₂. ELISAs performed using 10 donors also confirmed significant decreases during low O₂ culture in 4–6 ASC donors. For low abundant proteins, a 36 cytokine/chemokine array was performed. Fifteen cytokines/chemokines including Type 2 cytokines IL-13, MCP-1, and CD40 ligand were detected in ambient O₂ ASC medium. IL-6 was detected in low O₂ but not ambient O₂ ASC medium. These findings demonstrate that low O₂ ASC exposure resulted in reduced ECM protein and Type 2 cytokine secretions that are significant with regard to inflammation in fibrosis.