Calcium-regulating peptide hormones and blood electrolytic balance in chronic heart failure

Calcium-regulating system is important for the functional activity of myocardium. However, little is known about the role of this system in the pathogenesis of cardiovascular diseases. Blood samples from the patients with chronic heart failure (CHF) caused by ischaemic disease (coronary artery disease) (NYHA class I-IV) were used to analyze the levels of calcium, inorganic phosphate, sodium, potassium, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP). The heart beat rate and arterial blood pressure were chosen as additional tests for the functional status of cardiovascular system. The alteration of electrolytes homeostasis was found dependent on the severity of the pathology being maximally expressed in the NYHA class IV patients. Similar tendency was demonstrated for circulating PTH and PTHrP with the highest blood concentrations observed in patients of the NYHA class III and IV. The extent of these changes was found more pronounced in the female patients. It is suggested that the calcium-regulating hormonal system is involved in the pathogenesis of the ischaemic heart disease; however the sharp increase of PTH and PTHrP at the severe stages of pathology may play a compensatory role in maintaining the heart function.     

The blockade of endothelin A receptor protects astrocytes against hypoxic injury: common effects of BQ-123 anderythropoietin on the rejuvenation of the astrocyte population

In the present study the role of endothelin (ET) and its receptors (ETA-R and ETB-R) in cellular mechanisms underlying the resistance of astroglial cells to low oxygen level and development of hypoxia has been investigated. To define the influences of ET and its receptors on survival and on antigenic as well as morphologic differentiation of rat astroglial cells in normoxic (NC) and hypoxic culture (HC) the selective antagonists of ETA-R (BQ-123) and ETB-R (BQ-788) were used. Treatment of HC with BQ-123 caused an increase in cell number and inhibited the hypoxia-induced apoptosis by 37%. BQ-123 decreased the hypoxia-induced cytotoxicity in HC. These effects of BQ-123 were abolished in cultures simultaneously treated with BQ-123 and BQ-788. Administration of BQ-788 alone decreased the number of living cells in NC, but not in HC. The activity of caspase-3/-7 was not changed by exposure of NC and HC to BQ-788. The protection provided by BQ-123 to astroglial cells against cytotoxicity in NC and HC was similar to that of erythropoietin (EPO), a cytokine with established neuroprotective effects. The functional improvement of astroglial cells and slowing down of their differentiation under exposure to BQ-123, or EPO, or BQ-123 + EPO has been evidenced by an increased number of nestin+/glial fibrillary acidic protein-positive (GFAP+) astrocytes accompanied by decrease of nestin-/GFAP+ cells. The simultaneous treatment with BQ-123 and EPO additionally decreased the activities of caspase-3/-7 (64%) and release of LDH into the medium (94%). The benefits in the functional states of astrocytes obtained by combined treatment of HC with BQ-123 and EPO suggest a new therapeutic strategy in treatment of hypoxic brain injury.     

Determining the quality and complexity of next-generation sequencing data without a reference genome

We describe an open-source kPAL package that facilitates an alignment-free assessment of the quality and comparability of sequencing datasets by analyzing k-mer frequencies. We show that kPAL can detect technical artefacts such as high duplication rates, library chimeras, contamination and differences in library preparation protocols. kPAL also successfully captures the complexity and diversity of microbiomes and provides a powerful means to study changes in microbial communities. Together, these features make kPAL an attractive and broadly applicable tool to determine the quality and comparability of sequence libraries even in the absence of a reference sequence. kPAL is freely available at https://github.com/LUMC/kPAL.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0555-3) contains supplementary material, which is available to authorized users.     

Epistatic Effects of the Protease/Chaperone HflB on Some Damaged Forms of the Escherichia coli Ammonium Channel AmtB

The Escherichia coli ammonium channel AmtB is a trimer in which each monomer carries a pore for substrate conduction and a cytoplasmic C-terminal extension of ∼25 residues. Deletion of the entire extension leaves the protein with intermediate activity, but some smaller lesions in this region completely inactivate AmtB, as do some lesions in its cytoplasmic loops. We here provide genetic evidence that inactivation depends on the essential protease HflB, which appears to cause inactivation not as a protease but as a chaperone. Selection for restored function of AmtB is a positive selection for loss of the ATPase/chaperone activity of HflB and reveals that the conditional lethal phenotype for hflB is cold sensitivity. Deletion of only a few residues from the C terminus of damaged AmtB proteins seems to prevent HflB from acting on them. Either yields the intermediate activity of a complete C-terminal deletion. HflB apparently “tacks” damaged AmtB tails to the adjacent monomers. Knowing that HflB has intervened is prerequisite to determining the functional basis for AmtB inactivation.Amt proteins concentrate the hydrated gas NH₄⁺ against a gradient and appear to be “active” channels (Andrade and Einsle 2007; Fong et al. 2007; Ludewig et al. 2007). Each monomer of the trimer carries a pore for substrate conduction and a C-terminal extension of variable length. The ordered C terminus of Escherichia coli AmtB is a long α-helix interrupted in the middle by a sharp kink, a fold very similar to that of the C-terminal region of Amt-1 from Archaeoglobus fulgidis (Andrade et al. 2005). The C terminus binds precisely to short cytoplasmic loop regions within the monomer to which it is covalently attached and also the adjacent monomer. It completes the cytoplasmic vestibule of the adjacent monomer to link the two (Conroy et al. 2007; Gruswitz et al. 2007). Deletion of the entire C terminus of E. coli AmtB yields a trimeric form of the protein with partial activity (Coutts et al. 2002; Severi et al. 2007): Uptake of [¹⁴C]methylammonium by a strain carrying AmtB^ΔC-term is between that of wild type and an amtB null strain.In organisms that are sensitive to the ammonium analog methylammonium, selection for resistance yields lesions in Amt proteins (Monahan et al. 2002; J. Hsu, W. B. Inwood and S. Kustu, unpublished results). These include some lesions that change residues in the C-terminal kink. Hence we introduced changes into the kink of the E. coli AmtB protein. All three that we tried—G393A, L394A, and the combination of the two—completely inactivated the protein, indicating that something had occurred beyond loss of function of the C terminus (Coutts et al. 2002; Severi et al. 2007). Likewise, we changed charged residues in the cytoplasmic loops of AmtB to alanine and this, too, inactivated the protein in several cases. By selecting for growth at NH₃ concentrations <50 nm, where unmediated diffusion is limiting, we isolated strains carrying a large number of mutations that suppressed the growth defect caused by these C-terminal and loop lesions. This work characterizes the intragenic suppressor mutations that affected the C terminus of AmtB, which were about a quarter of the total, and all of the extragenic suppressor mutations, which were ∼40%. It provides genetic evidence that the protease/chaperone HflB attempts to fold damaged C termini unsuccessfully and that this results in loss of AmtB activity. In the absence of intervention by HflB, the mutant C termini mimic a C-terminal delete.HflB (also called FtsH, which is unfortunately a misnomer because the hflB lesion was not responsible for the filamentation that was observed) is a membrane-bound protease that is the only essential ATP-dependent protease in E. coli (Ito and Akiyama 2005). It acts as a processive endopeptidase to release peptides of ∼20 residues. To digest inner-membrane proteins, it requires an N- or C-terminal cytoplasmic extension of about this length. HflB is divided into three regions: an N-terminal membrane-bound region containing two transmembrane segments separated by a large periplasmic loop (residues 1–143), an ATPase segment (AAA⁺ class; residues 144–398), and an unusual metalloprotease segment (residues 399–649) (Krzywda et al. 2002; Ito and Akiyama 2005; Bieniossek et al. 2006). Although the conditional lethal phenotype for hflB was long thought to be heat sensitivity, this has been questioned (Ogura et al. 1999). A deletion of hflB is tolerated in the presence of a suppressor mutation in fabZ that increases FabZ activity and restores the balance between phospholipid and lipopolysaccharide synthesis. The deletion is likewise tolerated in lpxA or lpxD backgrounds that decrease lipopolysacharide synthesis. HflB has been considered by some a charonin (Schumann 1999; Ito and Akiyama 2005), but there are few specific reports of its chaperone activity, and it is known to have difficulty unfolding proteins that are thermodynamically stable (Herman et al. 2003).     

Similar protective effects of BQ-123 and erythropoietin on survival of neural cells and generation of neurons upon hypoxic injury

Our recent study [Danielyan et al., 2005. Eur. J. Cell Biol. 84, 567-579] showed an additive protective effect of endothelin (ET) receptor A (ETA-R) blockade and erythropoietin (EPO) on the survival and rejuvenation of rat astroglial cells exposed to hypoxia. Whether the effects observed with rodent astroglial cells can be reproduced in human astrocytes and whether these effects of ETA-R blockade and EPO on astrocytes are associated with neuronal survival remained open. Therefore, in the present study, the effects of the ETA-R antagonist BQ-123 and EPO on the maintenance of the neuronal population and survival of the human fetal astroglial cell line (SV-FHAS) under normoxic and hypoxic conditions (NC and HC, respectively) were investigated. Rat brain primary cultures exposed to BQ-123 and/or EPO revealed an increase in the number of beta-III tubulin-positive neurons under NC. The hypoxia-caused loss of neurons was abolished by administration of BQ-123 or EPO. Simultaneous application of EPO and BQ-123 led to an additive protective effect on the generation of neurons under NC only. By contrast, BQ-788, the selective ETB-R antagonist, diminished the neuronal population both in NC and HC. Both under NC and HC the number of non-differentiated nestin+/GFAP- neural cells increased upon application of EPO or BQ-123. SV-FHAS responded to BQ-123 or EPO by a decrease in LDH activity in the culture medium under NC (35%) and HC (26% LDH decrease). Concomitant effects of EPO and BQ-123 were illustrated in an additional increase in the survival of human astrocytes (33% under NC and 17% under HC). These data hint at a neuroprotective therapeutic potency of ETA-R blockade, which either alone or in combination with EPO may improve the survival of astroglial and neuronal cells upon hypoxic injury.     

A Novel Approach to Evaluating the Iron and Folate Status of Women of Reproductive Age in Uzbekistan after 3 Years of Flour Fortification with Micronutrients

Background

The Uzbekistan 1996 Demographic Health Survey reported 60.4% of women of reproductive age (WRA) had low hemoglobin concentrations (<120 g/L), and anemia was an important public health problem. Fortification of wheat flour was identified as an appropriate intervention to address anemia due to the ubiquitous consumption of wheat flour. A National Flour Fortification Program (NFFP) was implemented in 2005.

Methodology/Principal Findings

After 3-years of the NFFP, a national survey using large country-lot quality assurance sampling was carried out to assess iron, folate, hemoglobin and inflammation status of WRA; the coverage and knowledge of the fortified first grade UzDonMakhsulot (UDM) flour/grey loaf program; and consumption habits of women to investigate the dietary factors associated with anemia. Estimated anemia prevalence was 34.4% (95% CI: 32.0, 36.7), iron depletion 47.5% (95% CI: 45.1, 49.9) and folate deficiency 28.8% (95% CI: 26.8, 30.8); the effect of inflammation was minimal (4% with CRP >5 mg/L). Severe anemia was more prevalent among folate deficient than iron depleted WRA. Presence of UDM first grade flour or the grey loaf was reported in 71.3% of households. Among WRA, 32.1% were aware of UDM fortification; only 3.7% mentioned the benefits of fortification and 12.5% understood causes of anemia. Consumption of heme iron-containing food (91%) and iron absorption enhancers (97%) was high, as was the consumption of iron absorption inhibitors (95%).

Conclusions/Significance

The NFFP coincided with a substantial decline in the prevalence of anemia. Folate deficiency was a stronger predictor of severe anemia than iron depletion. However, the prevalence of iron depletion was high, suggesting that women are not eating enough iron or iron absorption is inhibited. Fortified products were prevalent throughout Uzbekistan, though UDM flour must be adequately fortified and monitored in the future. Knowledge of fortification and anemia was low, suggesting consumer education should be prioritized.     

The TRPM8 Protein Is a Testosterone Receptor: II. FUNCTIONAL EVIDENCE FOR AN IONOTROPIC EFFECT OF TESTOSTERONE ON TRPM8*

Testosterone is a key steroid hormone in the development of male reproductive tissues and the regulation of the central nervous system. The rapid signaling mechanism induced by testosterone affects numerous behavioral traits, including sexual drive, aggressiveness, and fear conditioning. However, the currently identified testosterone receptor(s) is not believed to underlie the fast signaling, suggesting an orphan pathway. Here we report that an ion channel from the transient receptor potential family, TRPM8, commonly known as the cold and menthol receptor is the major component of testosterone-induced rapid actions. Using cultured and primary cell lines along with the purified TRPM8 protein, we demonstrate that testosterone directly activates TRPM8 channel at low picomolar range. Specifically, testosterone induced TRPM8 responses in primary human prostate cells, PC3 prostate cancer cells, dorsal root ganglion neurons, and hippocampal neurons. Picomolar concentrations of testosterone resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Furthermore, acute applications of testosterone on human skin elicited a cooling sensation. Our data conclusively demonstrate that testosterone is an endogenous and highly potent agonist of TRPM8, suggesting a role of TRPM8 channels well beyond their well established function in somatosensory neurons. This discovery may further imply TRPM8 channel function in testosterone-dependent behavioral traits.     

Auto-regulation of DNA degrading bacteriocins: molecular and ecological aspects

Colicins, proteinaceous antibiotics produced by Escherichia coli, specifically target competing strains killing them through one of a variety of mechanisms, including pore formation and nucleic acid degradation. The genes encoding colicins display a unique form of expression, which is tightly regulated, involving the DNA damage response regulatory system (the SOS response system), confined to stressful conditions and released by degradation of the producing cell. Given their lethal nature, colicin production has evolved a sophisticated system for repression and expression. While exploring the expression of 13 colicins we identified a novel means of induction unique to strains that kill by DNA degradation: these colicinogenic strains mildly poison themselves inflicting DNA damage that induces their DNA repair system (the SOS system), and their own expression. We established that among the four known DNase colicins (E2, E7, E8 and E9), three act to induce their own production. Using different stresses we show that this form of self-regulation entails high cost when growth conditions are not optimal, and is not carried out by individual cells but is a population-mediated trait. We discuss this novel form of colicins’ regulation and expression, and its possible molecular mechanism and evolutionary implications.     

Electromechanical stimulation ameliorates inactivity-induced adaptations in the medial gastrocnemius of adult rats.

The efficacy of high-load, short-duration isometric contractions, delivered as one vs. two sessions per day, on blunting inactivity-induced adaptations in the medial gastrocnemius (MG) were compared. Adult rats were assigned to a control (Con) or spinal cord-isolated (SI) group where one limb was stimulated (SI-Stim) while the other served as a SI control (SI-C). One bout of stimulation (BION microstimulator) consisted of a 100-Hz, 1-s stimulus, delivered every 30 s for 5 min with a 5-min rest period. This bout was repeated six times consecutively (SI-Stim1) or with a 9-h rest interval after the third bout (SI-Stim2) for 30 consecutive days. MG weights (relative to body weight) were 63, 72, and 79% of Con in SI-C, SI-Stim1, and SI-Stim2, respectively. Mean fiber size was 56% smaller in SI-C than in Con, and it was 19 and 31% larger in SI-Stim1 and SI-Stim2, respectively, compared with SI-C. Maximum tetanic tension was 42, 60, and 73% of Con in SI-C, SI-Stim1, and SI-Stim2, respectively. Specific tension was 77% of Con in SI-C, and at Con levels in both SI-Stim groups. SI increased the percent IIb myosin heavy chain composition (from 49 to 77%) and IIb+ fibers (from 63 to 79%): these adaptations were prevented by both Stim paradigms. These results demonstrate that 1) brief periods of high-load isometric contractions are effective in reducing inactivity-induced atrophy, functional deficits, and phenotypic adaptations in a fast hindlimb extensor, and 2) the same amount of stimulation distributed in two compared with one session per day is more effective in ameliorating inactivity-related adaptations.     

Dynamic Adaptation of Liver Mitochondria to Chronic Alcohol Feeding in Mice: BIOGENESIS,REMODELING, AND FUNCTIONAL ALTERATIONS*

Liver mitochondria undergo dynamic alterations following chronic alcohol feeding to mice. Intragastric alcohol feeding to mice resulted in 1) increased state III respiration (109% compared with control) in isolated liver mitochondria, probably due to increased levels of complexes I, IV, and V being incorporated into the respiratory chain; 2) increased mitochondrial NAD⁺ and NADH levels (∼2-fold), with no change in the redox status; 3) alteration in mitochondrial morphology, with increased numbers of elongated mitochondria; and 4) enhanced mitochondrial biogenesis in the liver, which corresponded with an up-regulation of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α). Oral alcohol feeding to mice, which is associated with less liver injury and steatosis, slightly enhanced respiration in isolated liver mitochondria (30.8% compared with control), lower than the striking increase caused by intragastric alcohol feeding. Mitochondrial respiration increased with both oral and intragastric alcohol feeding despite extensive N-acetylation of mitochondrial proteins. The alcohol-induced mitochondrial alterations are probably an adaptive response to enhance alcohol metabolism in the liver. Isolated liver mitochondria from alcohol-treated mice had a greater rate of acetaldehyde metabolism and respiration when treated with acetaldehyde than control. Aldehyde dehydrogenase-2 levels were unaltered in response to alcohol, suggesting that the greater acetaldehyde metabolism by isolated mitochondria from alcohol-treated mice was due to increased mitochondrial respiration that regenerated NAD⁺, the rate-limiting substrate in alcohol/acetaldehyde metabolism. Overall, our work suggests that mitochondrial plasticity in the liver may be an important adaptive response to the metabolic stress caused by alcohol intake and could potentially play a role in many other vital functions performed by the liver.