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.     

Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78

White rot fungi efficiently degrade lignin, a complex aromatic polymer in wood that is among the most abundant natural materials on earth. These fungi use extracellular oxidative enzymes that are also able to transform related aromatic compounds found in explosive contaminants, pesticides and toxic waste. We have sequenced the 30-million base-pair genome of Phanerochaete chrysosporium strain RP78 using a whole genome shotgun approach. The P. chrysosporium genome reveals an impressive array of genes encoding secreted oxidases, peroxidases and hydrolytic enzymes that cooperate in wood decay. Analysis of the genome data will enhance our understanding of lignocellulose degradation, a pivotal process in the global carbon cycle, and provide a framework for further development of bioprocesses for biomass utilization, organopollutant degradation and fiber bleaching. This genome provides a high quality draft sequence of a basidiomycete, a major fungal phylum that includes important plant and animal pathogens.     

Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET

LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3beta-p-tolyl-8-aza-bicyclo[3.2.1]octane-2beta-carboxylic acid methyl ester), a cocaine derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic acid derivative, the latter used as precursor for labelling both with tritium and the positron-emitter carbon-11 (half-life: 20.38 min), were synthesized from (R)-cocaine. [(3)H]LBT-999 (>99% radiochemically pure, specific radioactivity of 3.1 TBq/mmol) was prepared from [(3)H]methyl iodide, allowing its in vitro pharmacological evaluation (K(D): 9 nM for DAT and IC(50) > 1000 nM for SERT and NET). Routine production batches of 4.5-9.0 GBq of iv injectable solutions of [(11)C]LBT-999 (with specific radioactivities ranging from 30 to 45 GBq/mumol) were prepared in 25-30 min (HPLC purification and formulation included) using the efficient methylation reagent [(11)C]methyl triflate. The preliminary in vivo pharmacological evaluation of [(11)C]LBT-999, using both biodistributions in rats and brain imaging in monkeys with positron emission tomography (PET), clearly illustrates that this ligand is an excellent candidate for quantification with PET of DAT in humans.     

A selective radiobrominated cocaine analogue for imaging of dopamine uptake sites: pharmacological evaluation and PET experiments

(E)-N-(3-bromoprop-2-enyl)-2beta-carbomethoxy-3beta-4'-tolyl -nortropane or PE2Br, an analogue of cocaine was labelled with the positron emitter 76Br (T1/2=16 h) for pharmacological evaluation in the rat and PET investigation in the monkey. [76Br]PE2Br was obtained by electrophilic substitution from the tributylstannyl precursor with radiochemical yield of 80%. In vivo biodistribution studies of [76Br]PE2Br (20 MBq/nmol) in rats showed a high uptake in the striatum (2.2% ID/g tissue at 15 min p.i.). The striatum to cerebellum radioactivity ratio was 6 at 1 hour p.i. Striatal uptake of [76Br]PE2Br was almost completely prevented by pretreatment with GBR 12909, but citalopram and maprotiline had no effect, confirming the selectivity of the radioligand for the dopamine transporter. PET imaging of the biodistribution of [76Br]PE2Br in the baboon demonstrated rapid and high uptake in the brain (5% ID at 3 min p.i.). The striatal radioactivity concentration reached a plateau at 20 min p.i. (7% ID/100 mL). The uptake in the cortex and cerebellum was very low. A significantly higher uptake in the thalamus was observed. At 1h p.i., the striatum to cerebellum ratio and thalamus to cerebellum ratio were 8 and 1.9 respectively. In competition experiments the radioactivity in the striatum and the thalamus was displaced by 5 mg/kgof cocaine and 5 mg/kg of GBR 12909, but citalopram and maprotiline had no effect. These results showed that [76Br]PE2Br is in vivo a potent and selective radioligand suitable for PET imagingof the dopamine transporter.     

Single-nucleotide polymorphism,linkage disequilibrium and geographic structure in the malaria parasite <Emphasis Type="Italic">Plasmodium vivax</Emphasis>: prospects for genome-wide association studies

Background

The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite P. vivax remain little characterized.

Results

We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of P. vivax in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for ~40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of P. vivax. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the pvmdr-1 locus, putatively associated with drug resistance.

Conclusion

These findings support the feasibility of genome-wide association studies in carefully selected populations of P. vivax, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.See commentary: http://www.biomedcentral.com/1741-7007/8/90

     

Synthesis of tropane and nortropane analogues with phenyl substitutions as serotonin transporter ligands.

The effects of structural modifications of 2 beta-carbomethoxy-3 beta-phenyl tropane analogues were evaluated on in vitro affinity to the dopamine (DAT) and serotonin (5-HTT) transporters in rat brain tissue. The introduction of a large alkyl group at the 4'-position of the phenyl ring, affording 2 beta-carbomethoxy-3 beta-(4'-alkylphenyl) tropane, diminished the affinity for the DAT whereas moderate 5-HTT affinity was obtained. The introduction of an iodine at the 3'-position of the 4'-alkylphenyl, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) tropane, and N-demethylation, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) nortropane, improved affinity and specificity for the 5-HTT. It could be assumed from these results that the combination of these three modifications of tropane structure yielded highly selective compounds for the 5-HTT. Of the new compounds synthesized, the most selective cocaine derivative, 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-isopropylphenyl) nortropane (8d) labeled with iodine-123 or carbon-11, could be a potential ligand for exploration of the 5-HT transporter by SPET or PET.