Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

South African founder mutations in the low-density lipoprotein receptor gene causing familial hypercholesterolemia in the Dutch population

In South African Afrikaners, three point mutations in the gene coding for the low-density lipoprotein (LDL)-receptor are responsible for more than 95% of the cases of familial hypercholesterolemia (FH). To investigate whether one or more of these mutations originated in The Netherlands, a large group of Dutch heterozygous FH patients was screened for the presence of these three mutations. Of these, a missense mutation in exon 9 of the LDL-receptor gene, resulting in a substitution of Met for Val⁴⁰⁸, responsible for 15% of FH in Afrikaners, was found in 19 (1.5%) of 1268 FH patients of Dutch descent. Nine of the patients carrying the exon 9 mutation on one allele shared the LDL-receptor DNA haplotype with an FH patient from South Africa, who was homozygous for the same mutation. This would suggest that the mutation in these patients and in the South African patient have a common ancestral background. The remaining ten FH patients all shared a common haplotype, partly identical to the Afrikaner haplotype, which chould have arisen from a single recombinational event. This mutation has not been described in persons other than of Dutch ancestry and supports the hypothesis that this mutation in exon 9 originated in The Netherlands and, in all likelihood, was introduced into South Africa by early Dutch settlers in the seventeenth century.     

Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability

We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Na+ and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Shaw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.      

Anabolic steroid effects on immune function: differences between analogues

As an untoward effect of chronic anabolic steroid use, immunologic alterations may be induced. To evaluate this possibility five commercially available steroids with various types of structural differences were studied in male Sprague-Dawley rats. Animals were divided into five groups and treated with testosterone (Group 1), testosterone propionate (Group 2), testolactone (Group 3), oxandrolone (Group 4), and stanozolol (Group 5). Androgenic anabolic steroids were administered daily, subcutaneously dissolved in oil, at a dose of 1.1 mg/kg. Immune alterations were assessed by skin-test responses to phytohemagglutinin. After five days of treatment (1.1 mg/kg/day) a significant immuno-suppression was observed with all groups. However, by day 10, groups 3, 4, and 5 showed an immuno-stimulation. Using oxandrolone as the model stimulant, serum testosterone levels were significantly suppressed, while castration abolished the stimulatory effect. These observations indicate that immune alterations do occur with anabolic steroids which are immuno-suppressive when the steroid nucleus is intact and immuno-stimulatory with nuclear alterations. It appears that these changes are associated with altered gonadal testosterone release.     

Intensified Tuberculosis Case Finding among Malnourished Children in Nutritional Rehabilitation Centres of Karnataka,India: Missed Opportunities

Background

Severe acute malnutrition (SAM) is the most serious form of malnutrition affecting children under-five and is associated with many infectious diseases including Tuberculosis (TB). In India, nutritional rehabilitation centres (NRCs) have been recently established for the management of SAM including TB. The National TB Programme (NTP) in India has introduced a revised algorithm for diagnosing paediatric TB. We aimed to examine whether NRCs adhered to these guidelines in diagnosing TB among SAM children.

Methods

A cross-sectional study involving review of records of all SAM children identified by health workers during 2012 in six tehsils (sub-districts) with NRCs (population: 1.8 million) of Karnataka, India.

Results

Of 1927 identified SAM children, 1632 (85%) reached NRCs. Of them, 1173 (72%) were evaluated for TB and 19(2%) were diagnosed as TB. Of 1173, diagnostic algorithm was followed in 460 (37%). Among remaining 763 not evaluated as per algorithm, tuberculin skin test alone was conducted in 307 (41%), chest radiography alone in 99 (13%) and no investigations in 337 (45%). The yield of TB was higher among children evaluated as per algorithm (4%) as compared to those who were not (0.3%) (OR: 15.3 [95%CI: 3.5-66.3]). Several operational challenges including non-availability of a full-time paediatrician, non-functioning X-ray machine due to frequent power cuts, use of tuberculin with suboptimal strength and difficulties in adhering to a complex diagnostic algorithm were observed.

Conclusion

This study showed that TB screening in NRCs was sub-optimal in Karnataka. Some children did not reach the NRC, while many of those who did were either not or sub-optimally evaluated for TB. This study pointed to a number of operational issues that need to be addressed if this collaborative strategy is to identify more TB cases amongst malnourished children in India.     

Similar Organization of the Lipopolysaccharide-Binding Protein (LBP) and Phospholipid Transfer Protein (PLTP) Genes Suggests a Common Gene Family of Lipid-Binding Proteins

The transfer of lipids in aqueous environments such as serum has been attributed to a recently characterized class of proteins. Abnormal regulation of serum lipids by these proteins is thought to be a key event in the pathophysiology of cardiovascular diseases. Lipopolysaccharide (endotoxin) binding protein (LBP) was identified by virtue of its ability to bind bacterial lipid A. We have analyzed the exon–intron organization of the LBP gene and the nucleotide sequence of its approximately 20 kb spanning 5′- and 3′-untranslated regions. When comparing the genomic organization of LBP with that of two other genes coding for lipid transfer proteins, significant homologies were found. The LBP gene includes 15 exons, and the 2-kb promoter contains recognition elements of acute phase-typical reactants and a repetitive 12-mer motif with an as yet unknown protein-binding property. Detailed sequence comparison revealed a closer relatedness of LBP with PLTP than with CETP as demonstrated by an almost identical intron positioning. This high degree of similarity supports functional studies by others suggesting that like LBP, PLTP may also be able to bind and transport bacterial lipopolysaccharide.     

Synthesis and conformational analysis of an expanded cyclic ketoxime‐hexapeptide

In this work the synthesis of a linear hexapeptide with a hydroxylamine functionality at the N‐terminus and a ketone instead of the carboxylic acid at the C‐terminus is described. Cyclization by ketoxime formation yields the 19‐membered ring‐expanded cyclic hexapeptide cyclo[Goly‐Val‐Ala‐Pro‐Leu‐Kly] which adopts a main conformer with two intramolecular hydrogen bonds. The hydrolytic stability of a ketoxime lies between the inert amide and the labile imine. The substitution of an amide bond for an iminium bond transforms the irreversible macrocyclization into a reversible process, but macrocyclic imines are difficult to isolate because they are prone to hydrolysis. The enhanced chemical stability of the ketoxime justifies its application in ligation protocols. The detailed NMR analysis of a ketoxime linkage presented here identifies its local conformational preferences in a constrained peptide environment. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibition of fungal ABC transporters by unnarmicin A and unnarmicin C, novel cyclic peptides from marine bacterium

Novel inhibitors of fungal ATP-binding cassette transporters were obtained by screening compounds and crude extracts from marine-derived fungi and bacteria using disk diffusion assays of Saccharomyces cerevisiae strains overexpressing a variety of fungal multi-drug efflux pumps. The cyclodepsipeptides unnarmicin A and unnarmicin C were able to sensitize cells overexpressing azole drug pumps ScPdr5p, CaCdr1p, CgCdr1p, and CgPdh1p to sub-MIC concentrations of fluconazole without affecting the growth of CaCdr2p and CaMdr1p overexpressing cells. Unnarmicin A and unnarmicin C were potent inhibitors of rhodamine 6G efflux of CaCdr1p expressing cells with IC₅₀ values of 3.61 and 5.65 μM, respectively. They inhibited the in vitro CaCdr1p ATPase activity at IC₅₀ values of 0.495 and 0.688 μM, respectively. And most importantly, they were able to sensitize azole-resistant Candida albicans clinical isolates to fluconazole. Unnarmicin A and unnarmicin C are candidate efflux pump inhibitors with the potential to be used as adjuvants for antifungal chemotherapy.     

Altered contribution of RhoA/Rho kinase signaling in contractile activity of myometrium in leptin receptor-deficient mice

In late gestation, enhanced myometrial contractility is mediated in part through increased Rho/Rho kinase. Since leptin, which is elevated in pregnancy and obesity, can directly depress myometrial function, we hypothesized that in leptin receptor-deficient mice, myometrial contractility would be greater in late pregnancy due to increased Rho/Rho kinase activity. To test this, we correlated RhoA and Rho kinase expression to contractility in myometrium from nonpregnant (NP) and late-pregnant (P18) heterozygous leptin receptor-deficient mice (db/+) vs. wild-type (WT) mice. In NP mice, KCl-induced contractions were similar between WT and db/+ myometrium. However, the Rho kinase-dependent component of the contractions was greater in db/+ mice, along with an increased expression of Rho kinase. KCl-induced contractions increased in strength in myometrium from P18 WT and db/+ compared with NP. Although the contribution of Rho kinase to contractions was unchanged in P18 WT mice, it was decreased in P18 db/+ mice. The decrease in Rho kinase-dependent contractions in P18 db/+ mice coincided with reduced RhoA and Rho kinase expression relative to NP db/+. Addition of high-fat-induced abnormal glucose utilization prevented changes in Rho kinase function. We conclude that abnormal leptin signaling increases expression and function of Rho kinase to maintain contractile function in NP myometrium and that during pregnancy the contribution of RhoA and Rho kinase expression to myometrial function is reduced despite an increase in myometrial contractility. Thus, other signaling mechanisms appear to compensate when leptin signaling is reduced to maintain contractile function during pregnancy.