Structure and Energetics of Encapsidated DNA in Bacteriophage HK97 Studied by Scanning Calorimetry and Cryo-electron Microscopy

Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When DNA-filled capsids (heads) are scanned in buffer containing 1 mM Mg²⁺, DNA melting and capsid denaturation both contribute to the complex thermal profile between 82 °C and 96 °C. In other conditions (absence of Mg²⁺ and lower ionic strength), DNA melting shifts to lower temperatures and the two events are resolved. Heads release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. We suggest that, on heating, the internal pressure increases, causing the DNA to exit—probably via the portal vertex-while the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Heads differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal that is embedded in the capsid wall, whereupon the structure of the portal and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion.     

Antibody expression in protease-deficient strains of the methylotrophic yeast Ogataea minuta

When human antibody genes were expressed in the methylotrophic yeast Ogataea minuta, the secreted antibody became partially degraded. To suppress the degradation, a vacuolar protease-deficient strain was constructed and its antibody production was evaluated. Although antibody productivity was improved in the vacuolar protease-deficient strain, the secreted antibody still became partially degraded. Peptide sequencing revealed that the cleavage occurred in the CH1 region of the heavy chain, implying that the cleavage was caused by an aspartic protease, Yps1p. To inhibit this cleavage, Yps1p-deficient strains were constructed and their antibody production was evaluated. As a result, the partial degradation of the antibody was suppressed in the O. minuta multiple-protease-deficient strains.     

Overexpression and characterization of two human salivary proline rich proteins

Proline rich proteins (PRP) are among major human saliva constituents and are known to interact with wine tannins that are involved in astringency. To characterize these interactions, a human salivary proline rich pro-protein, PRB4S, was overexpressed in Pichia pastoris. Six recombinant proteins resulting from maturation in bioreactor were detected by SDS-PAGE analysis between 15 and 45 kDa (apparent molecular weight). Two of them, the 45 and the 15 kDa ones, were isolated from culture supernatant by adsorption and permeation chromatography. They were characterized by N-terminal sequencing and MALDI-TOF analysis after trypsic digestion. The 45 kDa protein is glycosylated while the 15 kDa one was obtained after a furin-like proteolysis. Both of them are similar to human whole saliva PRP resulting from proteolysis of PRB4S pro-protein in Golgi network and known as II-1 and IB-5. Because of their sensitivity to proteolysis or their unusual mobility on SDS-PAGE gel, these recombinant proteins seem to be intrinsically unstructured proteins.     

Progesterone receptors in the human uterus and their possible role in parturition

An overview is given on the role of progesterone in parturition in the human. Progesterone withdrawal is considered to be a major event for the beginning of parturition. However, in the human, no evidence exists in favour of a decline in placental progesterone production prior to labour. Progesterone actions are mediated by two functionally different but structurally highly related intranuclear proteins, progesterone receptor (PR) A and PRB. In the human, functional progesterone withdrawal is thought to play a role. This may be mediated by a change in the expression of the two isoforms of the PR, with an increase in the PRA:PRB ratio, and this is accompanied by an increase in the expression of the estrogen receptor. These mechanisms are considered to be critical for the endocrine control of parturition.     

The effect of multiple primer–template mismatches on quantitative PCR accuracy and development of a multi-primer set assay for accurate quantification of pcrA gene sequence variants

Quantitative PCR (qPCR) is a critical tool for quantifying the abundance of specific organisms and the level or expression of target genes in medically and environmentally relevant systems. However, often the power of this tool has been limited because primer–template mismatches, due to sequence variations of targeted genes, can lead to inaccuracies in measured gene quantities, detection failures, and spurious conclusions. Currently available primer design guidelines for qPCR were developed for pure culture applications, and available primer design strategies for mixed cultures were developed for detection rather than accurate quantification. Furthermore, past studies examining the impact of mismatches have focused only on single mismatches while instances of multiple mismatches are common. There are currently no appropriate solutions to overcome the challenges posed by sequence variations. Here, we report results that provide a comprehensive, quantitative understanding of the impact of multiple primer–template mismatches on qPCR accuracy and demonstrate a multi-primer set approach to accurately quantify a model gene pcrA (encoding perchlorate reductase) that has substantial sequence variation. Results showed that for multiple mismatches (up to 3 mismatches) in primer regions where mismatches were previously considered tolerable (middle and 5′ end), quantification accuracies could be as low as ~ 0.1%. Furthermore, tests were run using a published pcrA primer set with mixtures of genomic DNA from strains known to harbor the target gene, and for some mixtures quantification accuracy was as low as ~ 0.8% or was non-detect. To overcome these limitations, a multiple primer set assay including minimal degeneracies was developed for pcrA genes. This assay resulted in nearly 100% accurate detection for all mixed microbial communities tested. The multi-primer set approach demonstrated herein can be broadly applied to other genes with known sequences.     

Dexamethasone down-regulates ABCG2 expression levels in breast cancer cells

The breast cancer resistance protein ABCG2 effluxes a variety of drugs and is believed to play an important role in multidrug resistance to chemotherapy. We show here for the first time that dexamethasone (DEX) and progesterone (PROG) are able to strongly inhibit ABCG2 expression in progesterone receptor (PR)-positive MCF7 and PR-negative MDA-MB-231 breast cells. In contrast, in the latter cells stably-transfected with progesterone receptor isoforms A and B, ABCG2 expression was strongly up-regulated by DEX and PROG. In addition, two other ligands of Pregnane X Receptor (PXR) and/or Glucocorticoid Receptor (GR) were also able to down-regulate ABCG2 expression in PXR- and GR-positive MCF7 cells. ABCG2 expression regulation by DEX likely resulted from the activation of PR-, PXR-, and/or GR-signaling pathways. ABCG2 expression inhibition by DEX was associated with increased sensitivity to mitoxantrone, a known ABCG2 substrate. The findings suggest that DEX may be useful in improving drug efficacy under certain conditions.     

Isolation,Characterization and Kinetics of Perchlorate Reducing Magnetospirillum Species

Perchlorate reducing bacteria reduce perchlorate to chlorate (ClO₃?), which, in turn, is reduced to chlorite (ClO₂?) and ultimately to chloride (Cl^?). Magnetospirillum strains are reported to use chlorate/perchlorate as electron acceptors. This study describes the perchlorate reducing property of strain VITRJS5, a Magnetopsirillum isolated from freshwater sediment collected from Chelur freshwater lake, Kerala, India. The strain was microaerophile and was phylogenetically related to a Magnetospirillum sp., a member of the α-subclass of the class Proteobacteria. The placement of the isolate in the genus Magnetospirillum has further confirmed the presence of four key magnetosome membrane genes. PCR amplification and phylogenetic analysis of central metabolic genes such as nifH (nitrogenase) and cbbM (type II RubisCo) displayed the highest similarity (97% and 81%, respectively) with Magnetospirillum sp. BB-1 The growth kinetic parameters of the isolate were studied with acetate as the electron donor in batch experiments. Monod's substrate utilization model has been established with oxygen, nitrate and perchlorate as electron acceptors separately. The maximum specific growth rate (µ_max) and half-saturation constant (k_s^conc) for the bacterium varied while utilizing different electron acceptors. The maximum specific growth rate was 0.226, 0.190 and 0.096 per hour and half-velocity constant K_s was 25.09, 33.36 and 65.37 mg acetate/l for oxygen, nitrate and perchlorate, respectively. The reduction of perchlorate has been analyzed using kinetic studies of the substrate uptake by the bacteria and the half-velocity constant K_s was found to be 52.8 mg/l. The results indicate that the strain VITRJS5 effectively reduces perchlorate by using it as an electron acceptor.     

Evaluation of Three Electron-Donor Permeable Reactive Barrier Materials for Enhanced Reductive Dechlorination of Trichloroethene

Understanding the fate of complex electron-donor materials is important for developing efficient biostimulation strategies to treat ground water contamination by chlorinated ethenes (CEs). The fermentation product distributions and H₂ production of common permeable reactive barrier (PRB) carbon substrates (dairy whey, sodium lactate syrup, and Hydrogen Release Compound [HRC]) were monitored as measures of substrate efficiency in aquifer microcosms spiked with trichloroethene (TCE). In long-term experiments, the fermentation of PRB substrates to slow-degrading organic acids maintained low H₂ partial pressures (≤ 10^?3.5) that, as previous studies suggest, may give competitive advantage to dechlorinators over hydrogenotrophic methanogens. Whey-amended and lactate-amended microcosms exhibited faster complete dechlorination and, according to organic acid carbon flow, higher rates of fermentation to acetate. In HRC-amended microcosms, propionate appeared to serve as a carbon sink that prolonged dechlorination. Upon complete dechlorination, whey microcosms contained the highest percentage of organic acid carbon. Native Dehalococcoides populations increased by 3 orders of magnitude (per g sediment) in whey-amended microcosms. Whey's efficiency improved in microcosms prepared with aquifer sediment and water from within a downgradient whey PRB. Results suggested whey loading values of 0.2 kg/m³ may be appropriate under sufficiently reducing conditions to efficiently stimulate hydrogenotrophic and potentially actetotrophic dechlorinating populations. Renewal of whey PRBs may, however, be required. Implications for further long-term study of cost-efficiencies are discussed.