Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi

The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity hydrolase involved in the catabolism of glycoconjugates, presumably in the digestive vacuole. We have cloned the alpha-mannosidase gene from a T.cruzi epimastigote genomic library. The alpha-mannosidase gene was determined to be single copy by Southern analysis, and similar sequences were not detected in genomic digests of either Trypanosoma brucei or Leishmania donovani. The coding region was subcloned into the Pichia pastoris expression vector pPICZ, and alpha-mannosidase activity was detected in the medium of induced cultures. The recombinant alpha- mannosidase demonstrated a pH optimum, inhibition by swainsonine, Km, and substrate specificity consistent with the characteristics of the alpha-mannosidase previously purified from T.cruzi epimastigotes. The recombinant enzyme was purified 103-fold from the culture medium of Pichia pastoris and had a native molecular mass of 359 kDa by gel filtration. A combination of SDS-PAGE, deglycosylation with endo H, and NH2-terminal sequencing indicates that the enzyme is originally synthesized as a homodimeric polypeptide that is subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa subunits. A polyclonal antibody raised to the recombinant enzyme was shown to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and will be used in future immunolocalization studies.      

Protein targets in red complex pathogens for catechin

The development of antimicrobial drug resistance has encouraged scientists to develop alternate methods to combat infectious pathogens associated with dental diseases. Therefore, it is of interest to predict interactions for catechin (a plant derived compound) with protein targets in the red complex pathogens using computer aided network tools. However, in vitro and in vivo studies are warranted to confirm the antimicrobial effect of catechin (gallocatechin, epicatechin, epigallactocatechin (EGC) and gallolyl catechins) on the dental pathogens.     

Identification of potentially safe promising fungal cell factories for the production of polyketide natural food colorants using chemotaxonomic rationale

Background  

Colorants derived from natural sources look set to overtake synthetic colorants in market value as manufacturers continue to meet the rising demand for clean label ingredients – particularly in food applications. Many ascomycetous fungi naturally synthesize and secrete pigments and thus provide readily available additional and/or alternative sources of natural colorants that are independent of agro-climatic conditions. With an appropriately selected fungus; using in particular chemotaxonomy as a guide, the fungal natural colorants could be produced in high yields by using the optimized cultivation technology. This approach could secure efficient production of pigments avoiding use of genetic manipulation.     

SIMPROT: Using an empirically determined indel distribution in simulations of protein evolution

Background  

General protein evolution models help determine the baseline expectations for the evolution of sequences, and they have been extensively useful in sequence analysis and for the computer simulation of artificial sequence data sets.     

A method for the enrichment of auxotrophic mutants of Serratia marcescens with the antibiotic azthreonam

A new method for the enrichment of cultures of Serratia marcescens for auxotrophic mutants has been developed. The method is based on the formation of filaments by growing cells in minimal medium M70 containing azthreonam. Auxotrophic mutants unable to grow in M70 do not form filaments. Mutants are collected from the culture by filtration.     

Water Balance in Cucumis Plants, Measured by Nuclear Magnetic Resonance, II

Nuclear magnetic resonance (NMR) was used to investigate theeffects of changes in root temperature, of changes in the areaof root in contact with culture solution and of day/night rhythmon the water balance of a cucumber and a gherkin plant. Resultsare discussed in terms of water potential, flow rate and resistanceusing a previously presented model of water balance. As longas water uptake alone is varied, flow rate and water content(or potential) will change in the same direction. In contrast,from that model it is predicted that changes in transpirationwill affect flow rate and water content in opposite ways. Anexperimental verification of this prediction was given in theprevious paper. Results obtained by the NMR method are comparedto those determined using a dendrometer. The results demonstratethat the NMR method is a valuable tool to study plant waterbalance and that it can serve as a technique for discriminatingbetween changes in plant water balance that are due to changesin water uptake by roots and those due to changes in transpiration. Key words: Water balance model, Cucumis satious L., flow, water content, NMR, water balance measurement     

Mutation screening by a combination of biotin-SSCP and direct sequencing

We have developed a mutation detection strategy that combines single strand conformational polymorphism (SSCP) analysis of one strand of a double-stranded amplification product with direct sequencing of the other. Using this strategy, which we find economical of both time and resources, we have identified a G to A transition, which substitutes a serine for glycine residue at position 862 in the major helix of the 1 chain of Type I collagen. We use this mutation, which causes a lethal form of osteogenesis imperfecta, to illustrate the technique.