Species‐specific detection of Candida tropicalis using evolutionary conserved intein DNA sequences

Inteins (internal proteins) are self‐splicing transportable genetic elements present in conserved regions of housekeeping genes. The study highlights the importance of intein as a potential diagnostic marker for species‐specific identification of Candida tropicalis, a rapidly emerging opportunistic human pathogen. Initial steps of primer validation, sequence alignment, phylogenetic tree analysis, gel electrophoresis and real‐time polymerase chain reaction (PCR) assays were performed to confirm the specificity of the designed primers. The primers were selective for C. tropicalis with 100% inclusivity and showed no cross‐species or cross‐genera matches. The established technique is a prototype for developing multifaceted PCR assays and for point‐of‐care testing in near future.

Significance and Impact of the Study

Development of molecular markers for specific detection of microbial pathogens using real‐time polymerase chain reaction (PCR) is an appealing and challenging technique. A real‐time PCR is an emerging technology frequently used to detect the aetiologic agents. In recent times, designing species‐specific primers for pathogen detection is gaining momentum. The method offers rapid, accurate and cost‐effective strategy to identify the target, thus providing sufficient time to instigate appropriate chemotherapy. The study highlights the use of intein DNA sequence as molecular markers for species‐specific identification of Candida tropicalis. The study also offers a prototype model for developing multifaceted PCR assays using intein DNA sequences, and provides a developmental starting point for point‐of‐care testing in near future.     

Movement response of Orange-Vaal largemouth yellowfish (<Emphasis Type="Italic">Labeobarbus kimberleyensis</Emphasis>) to water quality and habitat features in the Vaal River,South Africa

Many threatened fish species that utilize riverine habitats are faced with habitat degradation and subsequent deterioration in their ecological surroundings. Habitat degradation is a consequence of water quality parameters associated with anthropogenic activities including mining, industrial, agricultural and urban activities. We examined how the movement behaviour of radio-tracked Orange-Vaal largemouth yellowfish (Labeobarbus kimberleyensis) responded to a suite of water quality chemical parameters and habitat features in the Vaal River, South Africa. We found that the probability of their movement increased with a decrease in water clarity, presence of emergent and overhanging marginal vegetation and fast flowing rapids. High mobility in conditions of low water clarity was probably related to low prey capture success of this piscivorous fish. High movement of largemouth yellowfish in emergent and overhanging marginal vegetation areas and rapid habitat biotopes were attributed to accessibility of prey within these important cover features. When water quality parameters were considered, the probability of largemouth yellowfish movement increased with increasing levels of dissolved chloride (Cl) and silicon (Si), whereas movement decreased with high phosphate (PO₄) concentrations and increased with alkalinity levels in the river. High nutrient levels associated with eutrophication caused reductions in the movement of largemouth yellowfish. The association of increased movement of largemouth yellowfish with increasing Cl and Si is indicative of degraded habitat condition in the Vaal River system. Our study showed the importance of monitoring fish behavioural movement patterns to multiple environmental parameters, as these fish are important ecological indicators when appropriate conservation and management plans of freshwater ecosystems are required.     

On the genetics of prelingual deafness.

In view of the many discordant findings in previous studies regarding the genetics of prelingual deafness, family data (133 nuclear families and 25 pedigrees) were gathered from India. Analysis of these data has revealed that the defect is primarily genetic, which is in agreement with earlier findings. Segregation analysis was performed to compare various autosomal diallelic one-locus and multilocus models. Our analysis revealed that the most parsimonious model for prelingual deafness is that it is controlled by recessive genes at a pair of unlinked diallelic autosomal loci. Individuals are affected if and only if they are recessive homozygous at both loci. The likelihood of the present data under this two-locus multiple recessive homozygosis model is at least 10(8) times higher than that of the one-locus models that were examined in previous studies. This model is also the best-fitting model among other plausible two-locus models.     

Physico-chemical studies of micelle formation on sepia cartilage collagen solutions in acetate buffer and its interaction with ionic and nonionic micelles. Hydrodynamic and thermodynamic studies

Sepia cartilage collagen (pepsin-extracted) in acetate buffer (pH = 2.98) forms micelles at a particular concentration below which they do not normally form. The critical micelle concentration (cmc) of the collagen was determined in buffer as well as in SDS, cetyltrimethylammonium bromide (CTAB) and Tween-80 micellar environments at different temperatures. Mutual interaction of collagen micelles with the ionic and nonionic micelles through the formation of the mixed micelle concept has also been found. The cmc of collagen decreased in the presence of SDS and Tween-80 micelles whereas it increased in the presence of CTAB micelles. This clearly suggests that the micelle formation of collagen is facilitated by the presence of SDS and Tween-80 and hindered by CTAB micelles. The various thermodynamic parameters were estimated from viscosity measurements and the transfer of collagen into the micelles of various surfactants and the reverse phenomenon was analyzed. This analysis has also been modelled conceptually as a different phase and the results have supported the above phenomenon. Our thermodynamic results are also able to predict the exact denaturation temperature as well as the structural order of water in the collagen in various environments. The hydrated volumes, Vh, of collagen in the above environments and intrinsic viscosity were also calculated. The low intrinsic viscosity, [eta], of collagen in an SDS environment compared to buffer and other surfactant environments suggested more workable systems in cosmetic and dermatological skin care preparations. The one and two-hydrogen-bonded models of this collagen in various environments have been analyzed. The calculated thermodynamic parameters varied with the concentration of collagen. The change of thermodynamic parameters from coil-coil to random-coil conformation upon denaturation of collagen were calculated from the amount of proline and hydroxyproline residues and compared with viscometric results. Thermodynamic results suggest that the stability of the collagen in the additive environments is in the following order: SDS greater than Tween-80 greater than buffer greater than CTAB.     

Heritability estimates of citral content in East Indian lemongrass

Summary Citral content in oil obtained from 80 representative clones, chosen randomly from a population raised from open-pollinated seeds of clonally propagated East Indian lemongrass variety SD-68 and their half-sib progenies was estimated by gas chromatography. Heritability estimates were determined by variance component and parent-offspring regression analyses. Broad-sense heritability was 0.49 while narrow sense heritabilities by variance and regression analyses were 0.37 and 0.24, respectively. Phenotypic and genotypic correlations between citral content in oil and oil content were very low both among the parental clones (-0.01 and -0.01, respectively) and among their progenies (0.13 and 0.08, respectively) indicating that selection for either of these traits would not have much effect on the other.     

Purification and characterization of two trypsin inhibitors from the hemolymph of Manduca sexta larvae

Trypsin inhibitory activity from the hemolymph of the tobacco hornworm (Manduca sexta) was purified by affinity chromatography on immobilized trypsin and resolved into two fractions with molecular weights of 14,000 (M. sexta hemolymph trypsin inhibitor (HLTI) A) and 8,000 (HLTI B) by molecular sieve chromatography on Sephadex G-75. Electrophoresis of these inhibitors under reducing conditions on polyacrylamide gels gave molecular weight estimates of 8,300 for HLTI A and 9,100 for HLTI B, suggesting that HLTI A is a dimer and HLTI B is a monomer. Isoelectrofocusing on polyacrylamide gels focused HLTI A as a single band with pI 5.7, whereas HLTI B was resolved into two components with pI values of 5.3 and 7.1. Both inhibitors were stable at 100 degrees C and pH 1.0 for at least 30 min. HLTIs A and B inhibited serine proteases such as trypsin, chymotrypsin, and plasmin, but did not inhibit elastase, papain, pepsin, subtilisin BPN', and thermolysin. In fact, subtilisin BPN' completely inactivated both inhibitors. Both inhibitors formed low-dissociation complexes with trypsin in a 1:1 molar ratio. The inhibition constant for trypsin inhibition by HLTI A was estimated to be 1.45 x 10(-8) M. The HLTI A-chymotrypsin complex did not inhibit trypsin; similarly, the HLTI A-trypsin complex did not inhibit chymotrypsin, indicating that HLTI A has a common binding site for both trypsin and chymotrypsin. The amino-terminal amino acid sequences of HLTIs A and B revealed that both these inhibitors are homologous to bovine pancreatic trypsin inhibitor (Kunitz).     

Detection of point mutations associated with genetic diseases by an exon scanning technique

A major challenge in genetics is identifying the basis of human heritable disease. We describe an "exon scanning" technique which surveys exons in genomic DNA for sequence alterations. By hybridizing genomic DNA to RNA probes derived from cDNAs, we can use RNase A to survey entire coding regions, comprising exons spread across extensive regions of genomic DNA, for mutations associated with genetic disease. Exon scanning of the beta-globin locus in the DNA of patients with 12 different hemoglobinopathies detected all of the culpable single base substitutions and deletions, but not single base insertions. Our analysis also revealed unsuspected polymorphisms and corrected a diagnosis originally based on hemoglobin electrophoresis. Exon scanning of the ornithine aminotransferase gene in a gyrate atrophy patient detected and localized a mutation in the sixth exon. Subsequent PCR amplification and sequencing characterized this as a missense mutation (proline----glutamine). Exon scanning of genomic DNA for sequence alterations, in combination with PCR amplification and sequencing, should be a generally useful strategy for evaluating suspect genes in disorders of unknown etiology, as well as for clinical diagnosis.