Enzymatic synthesis and isolation of thymidine diphosphate-6-deoxy-D-xylo-4-hexulose and thymidine diphosphate-L-rhamnose. Production using cloned gene products and separation by HPLC.

A two-step enzymatic synthesis of dTDP-L-rhamnose is developed using enzymes from sonicated extracts of cultures of Escherichia coli K12 strains harboring plasmids containing different parts of the rfb gene cluster of Salmonella enterica LT2. The intermediate dTDP-6-deoxy-D-xylo-4-hexulose was isolated after a 1-h reaction, using only dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 89%). In a two-step reaction using dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase in the first step, and with NADPH, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase in the second hour of incubation, the dTDP-D-glucose was fully converted to dTDP-L-rhamnose. The hexoses of both products were identified by mass spectroscopy. The molar yield of dTDP-L-rhamnose, after protein precipitation, anion-exchange chromatography and desalting by gel chromatography, was 62%, corresponding to more than 150 mg, starting from 250 mg of dTDP-D-glucose. When stored lyophilysed under nitrogen, these products were found to be stable for several months. Both dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose have light absorption maxima at 267 nm, with molar absorption coefficients close to that of dTMP. However, the absorption coefficient of dTDP-6-deoxy-D-xylo-4-hexulose at the absorption maximum of 320 nm (specific for sugars containing keto groups) was found to be approximately 20% higher than values presented earlier. Furthermore, an HPLC technique is presented for determining the net activity of dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase, based on separation of dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose. The HPLC technique is also suitable for determination of all the nucleotide components involved in the synthesis.     

Anomalous side chain amidation in plasma membrane proteins of simian virus 40-transformed lymphocytes indicated by isoelectric focussing and laser Raman spectroscopy.

Plasma membranes isolated from normal hamster lymphocytes and lymphoid cells transformed by SV40¹ have been compared. Isoelectric focussing in 1% Triton $\text{X-100}\text{8M}$ urea reveals higher isoelectric points than normal for the non-glycosylated proteins in the membranes of transformed cells. This suggests greater amidation of membrane aspartates and/or glutamates. The focussing patterns also reveal a shift to lower pH for the isoelectric points of most glycosylated proteins suggesting increased silalylation. The Amide I – Amide II laser Raman spectra of the two membrane categories are consistent with greater side chain amidation in the membranes of neoplastic lymphocytes.     

Withania somnifera (L.) Dunal whole-plant extracts exhibited anti-sporotrichotic effects by destabilizing peripheral integrity of Sporothrix globosa yeast cells

Chronic topical cases of Sporotrichosis, a chronic fungal infection caused by the ubiquitously present cryptic members of the Sporothrix species complex, are treated with oral administrations of itraconazole. However, severe pulmonary or disseminated cases require repeated intra-venous doses of amphotericin B or even surgical debridement of the infected tissue. The unavoidable adverse side-effects of the current treatments, besides the growing drug resistance among Sporothrix genus, demands exploration of alternative therapeutic options. Medicinal herbs, due to their multi-targeting capacity, are gaining popularity amidst the rising antimicrobial recalcitrance. Withania somnifera is a well-known medicinal herb with reported antifungal activities against several pathogenic fungal genera. In this study, the antifungal effect of the whole plant extract of W. somnifera (WSWE) has been explored for the first time, against an itraconazole resistant strain of S. globosa. WSWE treatment inhibited S. globosa yeast form growth in a dose-dependent manner, with IC₅₀ of 1.40 mg/ml. Minimum fungicidal concentration (MFC) was found to be 50 mg/ml. Sorbitol protection and ergosterol binding assays, revealed that anti-sporotrichotic effects of WSWE correlated well with the destabilization of the fungal cell wall and cell membrane. This observation was validated through dose-dependent decrease in overall ergosterol contents in WSWE-treated S. globosa cells. Compositional analysis of WSWE through high performance liquid chromatography (HPLC) exhibited the presence of several anti-microbial phytochemicals like withanone, withaferin A, withanolides A and B, and withanoside IV and V. Withanone and withaferin A, purified from WSWE, were 10–20 folds more potent against S. globosa than WSWE, thus, suggesting to be the major phytocompounds responsible for the observed anti-sporotrichotic activity. In conclusion, this study has demonstrated the anti-sporotrichotic property of the whole plant extract of W. somnifera against S. globosa that could be further explored for the development of a natural antifungal agent against chronic Sporotrichosis.     

Bacteriophage MB78 DNA synthesis is specifically inhibited by the chelating agent ethylene diamine tetraacetic acid

The growth of bacteriophage MB78, a virulent phage of Salmonella typhimurium is extremely sensitive to the chelating agent EDTA. Other chelating agents like EGTA, a specific chelator for Ca²⁺ and orthophenanthroline which chelates Zn²⁺ and Fe²⁺ have no effect. EDTA stops phage MB78 DNA synthesis while synthesis of host DNA and other Salmonella phage DNA are not affected in presence of such low concentrations of EDTA. The present report indicates that some early phage function(s) and most probably the phage DNA synthesis are sensitive to EDTA which is probably due to chelation of Mg²⁺.     

Construction of a Genetic Linkage Map and Identification of QTLs for Seed Weight and Seed Size Traits in Lentil (Lens culinaris Medik.)

Seed weight and seed size both are quantitative traits and have been considered as important components of grain yield, thus identification of quantitative trait loci (QTL) for seed traits in lentil (Lens culinaris) would be beneficial for the improvement of grain yield. Hence the main objective of this study was to identify QTLs for seed traits using an intraspecific mapping population derived from a cross between L. culinaris cv. Precoz (seed weight-5.1g, seed size-5.7mm) and L. culinaris cv. L830 (seed weight-2.2g, seed size-4mm) comprising 126 F₈-RILs. For this, two microsatellite genomic libraries enriched for (GA/CT) and (GAA/CTT) motif were constructed which resulted in the development of 501 new genomic SSR markers. Six hundred forty seven SSR markers (including 146 previously published) were screened for parental polymorphism and 219 (33.8%) were found to be polymorphic among the parents. Of these 216 were mapped on seven linkage groups at LOD4.0 spanning 1183.7cM with an average marker density of 5.48cM. Phenotypic data from the RILs was used to identify QTLs for the seed weight and seed size traits by single marker analysis (SMA) followed by composite interval mapping (CIM) which resulted in one QTL each for the 2 traits (qSW and qSS) that were co-localized on LG4 and explained 48.4% and 27.5% of phenotypic variance respectively. The current study would serve as a strong foundation for further validation and fine mapping for utilization in lentil breeding programs.