Ion transit pathways and gating in ClC chloride channels

ClC chloride channels possess a homodimeric structure in which each monomer contains an independent chloride ion pathway. ClC channel gating is regulated by chloride ion concentration, pH and voltage. Based on structural and physiological evidence, it has been proposed that a glutamate residue on the extracellular end of the selectivity filter acts as a fast gate. We utilized a new search algorithm that incorporates electrostatic information to explore the ion transit pathways through wild-type and mutant bacterial ClC channels. Examination of the chloride ion permeation pathways supports the importance of the glutamate residue in gating. An external chloride binding site previously postulated in physiological experiments is located near a conserved basic residue adjacent to the gate. In addition, access pathways are found for proton migration to the gate, enabling pH control at hyperpolarized membrane potentials. A chloride ion in the selectivity filter is required for the pH-dependent gating mechanism.     

Specific and unspecific responses of plants to cold and drought stress

Different environmental stresses to a plant may result in similar responses at the cellular and molecular level. This is due to the fact that the impacts of the stressors trigger similar strains and downstream signal transduction chains. A good example for an unspecific response is the reaction to stressors which induce water deficiency e.g. drought, salinity and cold, especially frost. The stabilizing effect of liquid water on the membrane bilayer can be supported by compatible solutes and special proteins. At the metabolic level, osmotic adjustment by synthesis of low-molecular osmolytes (carbohydrates, betains, proline) can counteract cellular dehydration and turgor loss. Taking the example of Pinus sylvestris, changes at the level of membrane composition, and concomitantly of photosynthetic capacity during frost hardening is shown. Additionally the effect of photoperiod as measured via the phytochrome system and the effect of subfreezing temperatures on the incidence of frost hardening is discussed. Extremely hydrophilic proteins such as dehydrins are common products protecting not only the biomembranes in ripening seeds (late embryogenesis abundant proteins) but accumulate also in the shoots and roots during cold adaptation, especially in drought tolerant plants. Dehydrins are characterized by conserved amino acid motifs, called the K-, Y-or S-segments. Accumulation of dehydrins can be induced not only by drought, but also by cold, salinity, treatment with abscisic acid and methyl jasmonate. Positive effects of the overexpression of a wild chickpea (Cicer pinnatifidum) dehydrin in tobacco plants on the dehydration tolerance is shown. The presentation discusses the perception of cold and drought, the subsequent signal transduction and expression of genes and their products. Differences and similarities between the plant responses to both stressors are also discussed.     

Staphylococcus aureus ClpC ATPase is a late growth phase effector of metabolism and persistence

Staphylococcus aureus Clp ATPases (molecular chaperones) alter normal physiological functions including an aconitase‐mediated effect on post‐stationary growth, acetate catabolism, and entry into death phase (Chatterjee et al., J. Bacteriol. 2005, 187, 4488–4496). In the present study, the global function of ClpC in physiology, metabolism, and late‐stationary phase survival was examined using DNA microarrays and 2‐D PAGE followed by MALDI‐TOF MS. The results suggest that ClpC is involved in regulating the expression of genes and/or proteins of gluconeogenesis, the pentose‐phosphate pathway, pyruvate metabolism, the electron transport chain, nucleotide metabolism, oxidative stress, metal ion homeostasis, stringent response, and programmed cell death. Thus, one major function of ClpC is balancing late growth phase carbon metabolism. Furthermore, these changes in carbon metabolism result in alterations of the intracellular concentration of free NADH, the amount of cell‐associated iron, and fatty acid metabolism. This study provides strong evidence for ClpC as a critical factor in staphylococcal energy metabolism, stress regulation, and late‐stationary phase survival; therefore, these data provide important insight into the adaptation of S. aureus toward a persister state in chronic infections.     

Combined exposure to cigarette smoke and nontypeable Haemophilus influenzae drives development of a COPD phenotype in mice

Background

Cigarette smoke (CS) is the major etiologic factor of chronic obstructive pulmonary disease (COPD). CS-exposed mice develop emphysema and mild pulmonary inflammation but no airway obstruction, which is also a prominent feature of COPD. Therefore, CS may interact with other factors, particularly respiratory infections, in the pathogenesis of airway remodeling in COPD.

Methods

C57BL/6 mice were exposed to CS for 2 h a day, 5 days a week for 8 weeks. Mice were also exposed to heat-killed non-typeable H. influenzae (HK-NTHi) on days 7 and 21. One day after the last exposure to CS, mice were sacrificed and lung inflammation and mechanics, emphysematous changes, and goblet cell metaplasia were assessed. Mice exposed to CS or HK-NTHi alone or room air served as controls. To determine the susceptibility to viral infections, we also challenged these mice with rhinovirus (RV).

Results

Unlike mice exposed to CS or HK-NTHi alone, animals exposed to CS/HK-NTHi developed emphysema, lung inflammation and goblet cell metaplasia in both large and small airways. CS/HK-NTHi-exposed mice also expressed increased levels of mucin genes and cytokines compared to mice in other groups. CS/HK-NTHi-exposed mice infected with RV demonstrated increased viral persistence, sustained neutrophilia, and further increments in mucin gene and chemokine expression compared to other groups.

Conclusions

These findings indicate that in addition to CS, bacteria may also contribute to development of COPD, particularly changes in airways. Mice exposed to CS/HK-NTHi are also more susceptible to subsequent viral infection than mice exposed to either CS or HK-NTHi alone.     

Genes expressed during sexual differentiation of Chlamydomonas reinhardtii

Four genes specifically expressed during gametogenesis of Chlamydomonas reinhardtii have been cloned and their expression patterns analyzed. mRNAs encoded by these gamete-specific genes (gas) were absent or present only at very low levels in vegetative cells and mature zygotes. In young zygotes 2 h after gamete fusion, the mRNAs of three gas genes still persisted. The gas mRNAs accumulated during gametic differentiation. The temporal patterns of accumulation of individual mRNAs differed; some started to increase early during gametogenesis, others accumulated in the late phase. The accumulation of one of the late mRNAs (gas28) was stricly light-dependent. To illustrate the utility of the genes cloned in the analysis of sexual differentiation in Chlamydomonas reinhardtii we show that in a gametogenesis-defective mutant, the expression of late genes is prevented while that of early genes is normal.     

Glc1.8 from Microplitis demolitor bracovirus induces a loss of adhesion and phagocytosis in insect high five and S2 cells

Polydnaviridae is a unique family of DNA viruses that are symbiotically associated with parasitoid wasps. Upon oviposition, wasps inject these viruses into their hosts, where they cause several physiological alterations, including suppression of the cellular immune response. Here we report that expression of the glc1.8 gene from Microplitis demolitor bracovirus (MdBV) causes a loss of adhesion by two hemocyte-like cell lines, namely, High Five cells from the lepidopteran Trichoplusia ni and S2 cells from the dipteran Drosophila melanogaster. The expression of recombinant Glc1.8 also greatly reduced the ability of these cells to phagocytize foreign targets. Glc1.8 is characterized by a signal peptide at its N terminus, an extracellular domain comprised of five nearly perfect tandem repeats of 78 amino acids, and a C-terminal hydrophobic domain that encodes a putative membrane anchor sequence. The expression of a Glc1.8 mutant lacking the anchor sequence resulted in a secreted protein that had no effect on adhesion or phagocytosis. In contrast, sequential deletion of the repeats in the extracellular domain resulted in a progressive reduction in immunosuppressive activity. Since each repeat and its associated glycosylation sites are nearly identical, these results suggested that adhesion-blocking activity depends more on the overall number of repeats in the extracellular domain than on the specific determinants within each repeat. While it severely compromised adhesion and phagocytic functions, Glc1.8 did not cause cell death. Collectively, these results indicate that Glc1.8 is a major pathogenic determinant of MdBV that is involved in suppression of the insect cellular immune response.     

Ghrelin, macronutrient intake and dietary preferences in long-evans rats

Ghrelin is a recently discovered peptide that is primarily produced by the stomach. As a ligand of the growth hormone (GH) secretagogue (GHS) receptor, it stimulates GH secretion but it also stimulates feeding and has adipogenic effects in rodents. Although its circulating levels are modulated by fasting and refeeding, its relationship with diet composition is not known. In the present paper, we measured plasma ghrelin as well as two important hormones (leptin and insulin) in Long-Evans rats placed in two different feeding situations, e.g., either with imposed diets or with food choice. In the first case, the rats were fed unbalanced diets (either high-carbohydrate (HC) or high-fat (HF) diets) for 14 weeks, whereas in the second situation, they had the choice between these 2 diets for 2 weeks and were selected for their fat or carbohydrate preference. The intake of the HF diet for 14 weeks was associated with lower levels of ghrelin (-30% vs control diet; P < 0.01). These levels increased when the percentage of carbohydrate in the diet increased (+26 to +42% vs control diet; P < 0.01 or less). Ghrelin was inversely correlated with plasma leptin (r = -0.55; P < 0.003) and blood glucose (r = -0.58; P < 0.001) as well as with body weight (r = -0.63; P < 0.0001) and body fat content estimated by the sampling of specific fat pads (r = -0.62; P < 0.0001). In the food choice experiment, fat-preferring rats had plasma ghrelin levels lower than the carbohydrate-preferring rats (-33%; P < 0.0002). Ghrelin secretion was therefore very sensitive to the diet composition. Its down-regulation by fat ingestion might serve as a counterregulatory mechanism to limit the development of dietary-induced adiposity. Ghrelin may signal when a high calorie diet is ingested.     

Assessment of RainDrop BS-seq as a method for large-scale,targeted bisulfite sequencing

We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics.