Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis

The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu515) to the beginning of S6 (Ala560). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro527 and Ile541 was compatible with the presence of a alpha-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions -1, 0, +1, +2 around Asp542 (high Ca2+ affinity site) were non-functional. Whole cell currents of cysteine mutants at +4 and +5 positions were however covalently inhibited by external MTSET and MTSES. Altogether, the pattern of covalent modification by MTS reagents globally supports a KcsA homology-based three-dimensional model whereby the external vestibule in ECaC-TRPV5 encompasses three structural domains consisting of a coiled structure (Glu515 to Tyr526) connected to a small helical segment of 15 amino acids (527PTALFSTFELFLT539) followed by two distinct coiled structures Ile540-Pro544 (selectivity filter) and Ala545-Ile557 before the beginning of S6.     

新疆马鹿塔里木亚种的现状与保护

新疆马鹿塔里木亚种高度适应荒漠生境, 自20 世纪50 年代以来, 由于自然和人为因素的影响, 种群数量急剧下降。栖息地退缩和片段化不仅导致种群遗传多样性的丧失, 而且也因遗传漂变和近亲繁殖, 不断地加剧了该亚种的濒危程度。为有效的保护该物种, 特提出可行的保护措施建议。     

Antidepressant-like effect of a Ginkgo biloba extract (EGb761) in the mouse forced swimming test: role of oxidative stress

EGb761 is a well-defined mixture of active compounds extracted from Ginkgo biloba leaves. This extract is used clinically due to its neuroprotective effects, exerted probably via its potent antioxidant or free radical scavenger action. Previous studies suggest that oxidative stress, via free radical production, may play an important role in depression and animal models for depression-like behavior. Preclinical studies have suggested that antioxidants may have antidepressants properties. The aim of this study was to investigate the antidepressant-like of EGb761 due to its antioxidant role against oxidative stress induced in the forced swimming test, the most widely used preclinical model for assessing antidepressant-like behavior. Male BALB/c mice were pretreated with EGb761 (10 mg/kg, ip) daily for 17 days followed by the forced swimming test and spontaneous locomotor activity. Animals were sacrificed to evaluate lipid peroxidation, different antioxidant enzyme activities, serotonin and dopamine content in midbrain, hippocampus and prefrontal cortex. EGb761 significantly decreased the immobility time (39%) in the forced swimming test. This antidepressant-like effect of EGb761 was associated with a reduction in lipid peroxidation and superoxide radical production (indicated by a downregulation of Mn-superoxide dismutase activity), both of which are indicators of oxidative stress. The protective effect of EGb761 is not related to excitatory or inhibitory effects in locomotor activity, and was also associated with the modulation of serotonergic and dopaminergic neurotransmission. It is suggested that EGb761 produces an antidepressant-like effect, and that an antioxidant effect against oxidative stress may be partly responsible for its observed neuroprotective effects.     

NO-cGMP Signaling and Regenerative Medicine Involving Stem Cells

Nitric oxide (NO) is a short lived diatomic free radical species synthesized by nitric oxide synthases (NOS). The physiological roles of NO depend on its local concentrations as well as availability and the nature of downstream target molecules. At low nanomolar concentrations, activation of soluble guanylyl cyclase (sGC) is the major event initiated by NO. The resulting elevation in the intracellular cyclic GMP (cGMP) levels serves as signals for regulating diverse cellular and physiological processes. The participation of NO and cGMP in diverse physiological processes is made possible through cell type specific spatio-temporal regulation of NO and cGMP synthesis and signal diversity downstream of cGMP achieved through specific target selection. Thus cyclic GMP directly regulates the activities of its downstream effectors such as Protein Kinase G (PKG), Cyclic Nucleotide Gated channels (CNG) and Cyclic nucleotide phosphodiesterases, which in turn regulate the activities of a number of proteins that are involved in regulating diverse cellular and physiological processes. Localization and activity of the NO-cGMP signaling pathway components are regulated by G-protein coupled receptors, receptor and non receptor tyrosine kinases, phosphatases and other signaling molecules. NO also serves as a powerful paracrine factor. At micromolar concentrations, NO reacts with superoxide anion to form reactive peroxinitrite, thereby leading to the oxidation of important cellular proteins. Extensive research efforts over the past two decades have shown that NO is an important modulator of axon outgrowth and guidance, synaptic plasticity, neural precursor proliferation as well as neuronal survival. Excessive NO production as that evoked by inflammatory signals has been identified as one of the major causative reasons for the pathogenesis of a number of neurodegenerative diseases such as ALS, Alzheimers and Parkinson diseases. Regenerative therapies involving transplantation of embryonic stem cells (ES cells) and ES cell derived lineage committed neural precursor cells have recently shown promising results in animal models of Parkinson disease (PD). Recent studies from our laboratory have shown that a functional NO-cGMP signaling system is operative early during the differentiation of embryonic stem cells. The cell type specific, spatio-temporally regulated NO-cGMP signaling pathways are well suited for inductive signals to use them for important cell fate decision making and lineage commitment processes. We believe that manipulating the NO-cGMP signaling system will be an important tool for large scale generation of lineage committed precursor cells to be used for regenerative therapies. Special issue dedicated to John P. Blass.     

Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members

Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/betaKlotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between beta strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the beta1-beta2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/betaKlotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors.     

Physiologically based cord clamping for infants ≥32+0 weeks gestation: A randomised clinical trial and reference percentiles for heart rate and oxygen saturation for infants ≥35+0 weeks gestation

BackgroundGlobally, the majority of newborns requiring resuscitation at birth are full term or late-preterm infants. These infants typically have their umbilical cord clamped early (ECC) before moving to a resuscitation platform, losing the potential support of the placental circulation. Physiologically based cord clamping (PBCC) is clamping the umbilical cord after establishing lung aeration and holds promise as a readily available means of improving early newborn outcomes. In mechanically ventilated lambs, PBCC improved cardiovascular stability and reduced hypoxia. We hypothesised that PBCC compared to ECC would result in higher heart rate (HR) in infants needing resuscitation, without compromising safety.Methods and findingsBetween 4 July 2018 and 18 May 2021, infants born at ≥32⁺⁰ weeks’ gestation with a paediatrician called to attend were enrolled in a parallel-arm randomised trial at 2 Australian perinatal centres. Following initial stimulation, infants requiring further resuscitation were randomised within 60 seconds of birth using a smartphone-accessible web link. The intervention (PBCC) was to establish lung aeration, either via positive pressure ventilation (PPV) or effective spontaneous breathing, prior to cord clamping. The comparator was early cord clamping (ECC) prior to resuscitation. The primary outcome was mean HR between 60 to 120 seconds after birth, measured using 3-lead electrocardiogram, extracted from video recordings blinded to group allocation. Nonrandomised infants had deferred cord clamping (DCC) ≥120 seconds in the observational study arm.Among 508 at-risk infants enrolled, 123 were randomised (n = 63 to PBCC, n = 60 to ECC). Median (interquartile range, IQR) for gestational age was 39.9 (38.3 to 40.7) weeks in PBCC infants and 39.6 (38.4 to 40.4) weeks in ECC infants. Approximately 49% and 50% of the PBCC and ECC infants were female, respectively. Five infants (PBCC = 2, ECC = 3, 4% total) had missing primary outcome data. Cord clamping occurred at a median (IQR) of 136 (126 to 150) seconds in the PBCC arm and 37 (27 to 51) seconds in the ECC arm. Mean HR between 60 to 120 seconds after birth was 154 bpm (beats per minute) for PBCC versus 158 bpm for ECC (adjusted mean difference −6 bpm, 95% confidence interval (CI) −17 to 5 bpm, P = 0.39). Among 31 secondary outcomes, postpartum haemorrhage ≥500 ml occurred in 34% and 32% of mothers in the PBCC and ECC arms, respectively. Two hundred ninety-five nonrandomised infants (55% female) with median (IQR) gestational age of 39.6 (38.6 to 40.6) weeks received DCC. Data from these infants was used to create percentile charts of expected HR and oxygen saturation in vigorous infants receiving DCC. The trial was limited by the small number of infants requiring prolonged or advanced resuscitation. PBCC may provide other important benefits we did not measure, including improved maternal–infant bonding and higher iron stores.ConclusionsIn this study, we observed that PBCC resulted in similar mean HR compared to infants receiving ECC. The findings suggest that for infants ≥32⁺⁰ weeks’ gestation who receive brief, effective resuscitation at closely monitored births, PBCC does not provide additional benefit over ECC (performed after initial drying and stimulation) in terms of key physiological markers of transition. PBCC was feasible using a simple, low-cost strategy at both cesarean and vaginal births. The percentile charts of HR and oxygen saturation may guide clinicians monitoring the transition of at-risk infants who receive DCC.Trial registrationAustralian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12618000621213.

Shiraz Badurdeen and colleagues evaluate whether physiologically-based cord clamping provides physiological benefits over early cord clamping for infants requiring resuscitation at birth.     

Follistatin Protein Enhances Satellite Cell Counts in Reinnervated Muscle

Background Muscle recovery following peripheral nerve repair is sup-optimal. Follistatin (FST), a potent muscle stimulant, enhances muscle size and satellite cell counts following reinnervation when administered as recombinant FST DNA via viral vectors. Local administration of recombinant FST protein, if effective, would be more clinically translatable but has yet to be investigated following muscle reinnervation. Objective  The aim of this study is to assess the effect of direct delivery of recombinant FST protein on muscle recovery following muscle reinnervation. Materials and Methods  In total, 72 Sprague-Dawley rats underwent temporary (3 or 6 months) denervation or sham denervation. After reinnervation, rats received FST protein (isoform FS-288) or sham treatment via a subcutaneous osmotic pump delivery system. Outcome measures included muscle force, muscle histomorphology, and FST protein quantification. Results  Follistatin treatment resulted in smaller muscles after 3 months denervation ( p  = 0.019) and reduced force after 3 months sham denervation ( p  < 0.001). Conversely, after 6 months of denervation, FST treatment trended toward increased force output ( p  = 0.066). Follistatin increased satellite cell counts after denervation ( p  < 0.001) but reduced satellite cell counts after sham denervation ( p  = 0.037). Conclusion  Follistatin had mixed effects on muscle weight and force. Direct FST protein delivery enhanced satellite cell counts following reinnervation. The positive effect on the satellite cell population is intriguing and warrants further investigation.     

Chronology and pattern of replication in the bone marrow chromosomes of Gallus domesticus

The duration of the cell generation, the chronology, and the pattern of chromosome duplication was studied in the bone marrow of Gallus domesticus. The duration of the phases of the cell cycle is: cell generation 17.5 hours, S period 9 hours. G₂ period plus prophase stage 2.5 hours, G₁ period 6 hours. Chromosome replication begins at many sites. During middle S it extends to the whole complement and finally finishes in small, late replicating regions of the macrochromosomes. Interchromosomal asynchrony of duplication at the initiation or at the end of the S period was not observed. Z-chromosomes begin and finish DNA synthesis synchronously with the other macrochromosomes. The W-chromosome in females is the last microchromosome to finish replication. However it ends DNA synthesis at about the same time as the macrochromosomes. Similarities and differences between chromosome replication in Aves and Mammalia are considered.     

Separation and Purification of Antioxidant Peptides from Enzymatically Prepared Scorpion (Buthus martensii Karsch) Protein Hydrolysates

International Journal of Peptide Research and Therapeutics - The purpose of this study was to separate and purify antioxidant peptides from the scorpion (Buthus martensii Karsch) protein...