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.     

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.      

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.     

Ontogenetic shifts in functional morphology of dragonfly legs (Odonata: Anisoptera)

Anisopteran leg functions change dramatically from the final larval stadium to the adult. Larvae use legs mainly for locomotion, walking, climbing, clinging, or burrowing. Adults use them for foraging and grasping mates, for perching, clinging to the vegetation, and for repelling rivals. In order to estimate the ontogenetic shift in the leg construction from the larva to the adult, this study quantitatively compared lengths of fore, mid, and hind legs and the relationships between three leg segments, femur, tibia, and tarsus, in larval and adult Anisoptera of the families Gomphidae, Aeshnidae, Cordulegastridae, Corduliidae, and Libellulidae, represented by two species each. We found that leg segment length ratio as well as ontogenetic shift in length ratios was different between families, but rather similar within the families. While little ontogenetic shift occurred in Aeshnidae, there were some modifications in Corduliidae and Libellulidae. The severest shift occurred in Gomphidae and Cordulegastridae, both having burrowing larvae. These two families form a cluster, which is in contrast to their taxonomic relationship within the Anisoptera. Cluster analysis implies that the function of larval legs is primarily responsible for grouping, whereas adult behavior or the taxonomic relationships do not explain the grouping. This result supports the previous hypothesis about the convergent functional shift of leg characters in the dragonfly ontogenesis.     

Processive lipid galactosyl/glucosyltransferases from Agrobacterium tumefaciens and Mesorhizobium loti display multiple specificities

The glycosyltransferase family 21 (GT21) includes both enzymes of eukaryotic and prokaryotic organisms. Many of the eukaryotic enzymes from animal, plant, and fungal origin have been characterized as uridine diphosphoglucose (UDP-Glc):ceramide glucosyltransferases (glucosylceramide synthases [Gcs], EC 2.4.1.80). As the acceptor molecule ceramide is not present in most bacteria, the enzymatic specificities and functions of the corresponding bacterial glycosyltransferases remain elusive. In this study, we investigated the homologous and heterologous expression of GT21 enzymes from Agrobacterium tumefaciens and Mesorhizobium loti in A. tumefaciens, Escherichia coli, and the yeast Pichia pastoris. Glycolipid analyses of the transgenic organisms revealed that the bacterial glycosyltransferases are involved in the synthesis of mono-, di- and even tri-glycosylated glycolipids. As products resulting from their activity, we identified 1,2-diacyl-3-(O-beta-D-galacto-pyranosyl)-sn-glycerol, 1,2-diacyl-3-(O-beta-D-gluco-pyranosyl)-sn-glycerol as well as higher glycosylated lipids such as 1,2-diacyl-3-[O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol, 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol, 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->6)-O-beta-D-gluco-pyranosyl]-sn-glycerol, and the deviatingly linked diglycosyldiacylglycerol 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->3)-O-beta-D-galacto-pyranosyl]-sn-glycerol. From a mixture of triglycosyldiacylglycerols, 1,2-diacyl-3-[O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol could be separated in a pure form. In vitro enzyme assays showed that the glycosyltransferase from A. tumefaciens favours uridine diphosphogalactose (UDP-Gal) over UDP-Glc. In conclusion, the bacterial GT21 enzymes differ from the eukaryotic ceramide glucosyltransferases by the successive transfer of up to three galactosyl and glucosyl moieties to diacylglycerol.     

Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi.

Glucosylceramides are membrane lipids in most eukaryotic organisms and in a few bacteria. The physiological functions of these glycolipids have only been documented in mammalian cells, whereas very little information is available of their roles in plants, fungi, and bacteria. In an attempt to establish appropriate experimental systems to study glucosylceramide functions in these organisms, we performed a systematic functional analysis of a glycosyltransferase gene family with members of animal, plant, fungal, and bacterial origin. Deletion of such putative glycosyltransferase genes in Candida albicans and Pichia pastoris resulted in the complete loss of glucosylceramides. When the corresponding knock-out strains were used as host cells for homologous or heterologous expression of candidate glycosyltransferase genes, five novel glucosylceramide synthase (UDP-glucose:ceramide glucosyltransferase) genes were identified from the plant Gossypium arboreum (cotton), the nematode Caenorhabditis elegans, and the fungi Magnaporthe grisea, Candida albicans, and P. pastoris. The glycosyltransferase gene expressions led to the biosynthesis of different molecular species of glucosylceramides that contained either C18 or very long chain fatty acids. The latter are usually channeled exclusively into inositol-containing sphingolipids known from Saccharomyces cerevisiae and other yeasts. Implications for the biosynthesis, transport, and function of sphingolipids will be discussed.     

Identification of a haem domain in human soluble adenylate cyclase

The second messengers cAMP and cGMP mediate a multitude of physiological processes. In mammals, these cyclic nucleotides are formed by related Class III nucleotidyl cyclases, and both ACs (adenylate cyclases) and GCs (guanylate cyclases) comprise transmembrane receptors as well as soluble isoforms. Whereas sGC (soluble GC) has a well-characterized regulatory HD (haem domain) that acts as a receptor for the activator NO (nitric oxide), very little is known about the regulatory domains of the ubiquitous signalling enzyme sAC (soluble AC). In the present study, we identify a unique type?of HD as a regulatory domain in sAC. The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer. Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide). Splice variants of sAC comprising this domain are expressed in testis and skeletal muscle, and the HD displays an activating effect on the sAC catalytic core. Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.