Production of Aspergillus terreus alpha-L-rhamnosidase by solid state fermentation

AIMS: The study of production of Aspergillus terreus CECT 2663 alpha-L-rhamnosidase in solid state fermentation using wheat bran, washed sugar cane bagasse and polyurethane foam as substrates or supports for the enzyme production. METHODS AND RESULTS: Cultures were carried out in Petri dishes under controlled temperature and humidity. Naringin or rhamnose were the enzyme inducers and carbon sources. The enzyme activity to inducer ratio was appreciably greater when using sugar cane bagasse or polyurethane foam than wheat bran. The influence of inoculum size, inducer, airflow, humidity and temperature were determined. Under optimum conditions, about four units of enzyme per ml nutrient solution were obtained after 4-6 d. CONCLUSIONS: The activity to inducer ratio was higher, and the cultivation time was shorter in solid state fermentation than those observed in submerged cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Solid cultures, using naringin as inducer, can be appropriate alpha-L-rhamnosidase production.     

Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC

von Hippel-Lindau (VHL) disease is caused by loss of function of the VHL tumor suppressor protein. Here, we demonstrate that the folding and assembly of VHL into a complex with its partner proteins, elongin B and elongin C (herein, elongin BC), is directly mediated by the chaperonin TRiC/CCT. Association of VHL with TRiC is required for formation of the VHL-elongin BC complex. A 55-amino acid domain of VHL is both necessary and sufficient for binding to TRiC. Importantly, mutation or deletion of this domain is associated with VHL disease. We identified two mutations that disrupt the normal interaction with TRiC and impair VHL folding. Our results define a novel role for TRiC in mediating oligomerization and suggest that inactivating mutations can impair polypeptide function by interfering with chaperone-mediated folding.     

Cloning, characterization, and functional expression in Escherichia coli of chaperonin (groESL) genes from the phototrophic sulfur bacterium Chromatium vinosum.

A recombinant lambda phage which was able to propagate in groE mutants of Escherichia coli was isolated from a Chromatium vinosum genomic DNA library. A 4-kbp SalI DNA fragment, isolated from this phage and subcloned in plasmid vectors, carried the C. vinosum genes that allowed lambda growth in these mutants. Sequencing of this fragment indicated the presence of two open reading frames encoding polypeptides of 97 and 544 amino acids, respectively, which showed high similarity to the molecular chaperones GroES and GroEL, respectively, from several eubacteria and eukaryotic organelles. Expression of the cloned C. vinosum groESL genes in E. coli was greatly enhanced when the cells were transferred to growth temperatures that induce the heat shock response in this host. Coexpression in E. coli of C. vinosum groESL genes and the cloned ribulose bisphosphate carboxylase/oxygenase genes from different phototrophic bacteria resulted in an enhanced assembly of the latter enzymes. These results indicate that the cloned DNA fragment encodes C. vinosum chaperonins, which serve in the assembly process of oligomeric proteins. Phylogenic analysis indicates a close relationship between C. vinosum chaperonins and their homologs present in pathogenic species of the gamma subdivision of the eubacterial division Proteobacteria.     

Carbon tetrachloride activation in liver microsomes from rats induced with 3-methylcholantrene

The irreversible binding of¹⁴C from¹⁴CCl₄ to microsomal lipids is decreased in animals treated with 3-methylcholantrene (3-MC), while it is increased in animals induced with phenobarbital (PB). CCl₄-induced lipid peroxidation in 3-MC treated rats is as intense as in controls. Destruction of glucose 6-phosphatase (G6P-ase) by CCl₄ is smaller in 3-MC treated rats than in controls. Destruction of total cytochrome P-450 (P-450 + P₁-450) by CCl₄ is smaller in 3-MC treated than in PB treated rats but similar to that obtained in controls. Results would indicate that P-450 would participate in CCl₄ activation much more effectively than P₁-450.     

Distribution,growth and reproduction of the limpet Nacella (Patinigera) concinna (Strebel 1908) in relation to potential food availability,in Esperanza Bay (Antarctic Peninsula)

The present work addresses the effect of environmental factors (icing, water temperature, food availability) on the ecology of the patellid limpet Nacella (Patinigera) concinna, in a bay on the Antarctic Peninsula. Sampling was conducted at three depths (intertidal, 5m, 10m) from February 1987 to January 1988. Temperature was recorded and concentrations of Chlorophyll a were measured on the bottom, in the water and in the ice-water layer. The limpets were measured, weighed and a condition coefficient for somatic and gonadal mass was calculated. Their ages were estimated through size frequency distribution analysis and a seasonalized von Bertalanffy growth model was applied. The intertidal subpopulation migrated to deeper levels at the beginning of the icing season and recolonized the intertidal zone after ice retreat. The growth rate was very low (von Bertalanffy K 0.08). Growth rates showed important seasonal variations, with maxima during December and January. Nacella (P.) concinna spawns once a year and spawning coincided with raising water temperature (from -1.33°C to-0.84°C), and probably was also related to increasing spring food availability. Body mass increased during periods of high standing stock of microphytobenthos, revealing that ice-algae and phytoplankton were of minor importance as food sources for limpets in Esperanza Bay.     

Consistency of population genetics parameters estimated from isozyme and RAPDs dataset in species of genus <Emphasis Type="Italic">Prosopis</Emphasis> (Leguminosae,Mimosoideae)

Genetic variability, population structure and differentiation among 17 populations of 5 species and 2 natural interspecific hybrids of section Algarobia of genus Prosopis were analyzed from data of 23 isozyme and 28 RAPD loci. Both markers indicated that the studied populations are highly variable. P. alba populations in average showed lower values of genetic variability estimates from isozyme data, but this trend was not observed for RAPD markers. The hierarchical analyses of the distribution of genetic variability showed that the highest proportion of variation occurred within populations, the differentiation among species was intermediate and the lowest component was observed among populations within species. The consistency between results from both dataset implies that they are not biased and reflect the actual genetic structure of the populations analyzed. The matrices of Euclidean distances obtained from the two sets of markers were highly correlated according to Mantel test. In both cases the corresponding phenogram and MDS plot tended to cluster conspecific populations while hybrid populations were not intermediate between putative parents. Some disagreements between isozyme and RAPD phenograms were observed mainly in the affinities of hybrid populations. Such inconsistencies might result from reticular rather than dichotomic evolutionary relationships. The phenetic associations retrieved gave no support to the division of the section Algarobia into series.     

Hemolytic activity of venoms from snakes of the genera Bothrop, Lachesis, Crotalus, and Micrurus (Serpentes: Viperidae and Elapidae]

Hemolytic activity of eight Peruvian snake venoms from the families Viperidae and Elapidae (Bothrops atrox, B. pictus, B. hyoprorus, B. bilineatus, B. neuwedii, Lachesis m. muta, Crotalus d. terrificus, Micrurus tschudi), and three Brazilian viperids (B. jararacussu, B. alternatus and C. d. collilineatus) is described. None of the venoms caused direct lysis on washed human erythrocytes. However, all of them caused indirect hemolysis provided that the incubation medium contains an exogenous source of lecithin. Venom of Micrurus tschudi was the most hemolytic (HD50 2.8 ug/ml) while that of B. bilineatus was the least (HD50 681.3 ug/ml). Only six of eleven venoms showed parallel curves of hemolytic activity, and the HD50 varied from 198 to 681 ug/ml and the following decreasing order of hemolytic activity was obtained: L. muta, C. d. terrificus, C. d. collilineatus, B. hyoprorus, B. bilineatus, B. alternatus.     

Monitoring HIV Viral Load in Resource Limited Settings: Still a Matter of Debate?

Introduction

Consequences of lack of viral monitoring in predicting the effects of development of HIV drug resistance mutations during HAART in resource-limited settings (RLS) is still a matter of debate.

Design

To assess, among HIV+ patients receiving their first-line HAART, prevalence of virological failure and genotypic resistance mutations pattern in a Médécins Sans Frontières/Ministry of Health programme in Busia District (Kenya).

Methods

Patients with HAART treatment for ≥12 months were eligible for the study and those with HIV-RNA ≥5000 copies/ml underwent genotypic study. Total HIV-1 RNA from Dried Blood Spots was extracted using Nuclisens method.

Results

926 patients were included. Among 274 (29.6%) patients with detectable viral load, 55 (5.9%) experienced treatment failure (viral load >5.000 copies/ml); 61.8% were female and 10 (18.2%) had clinical failure. Median CD4 cell count was 116 cell/mm3 (IQR: 54–189). Median HIV-RNA was 32,000 copies/ml (IQR: 11000–68000). Eighteen out of 55 (33%) samples could be sequenced on PR and RT genes, with resistance associated mutations (RAMs) in 15 out of 18 samples (83%). Among patients carrying RAMs, 12/15 (81%) harboured RAMs associated to thymidine analogues (TAMs). All of them (100%) showed M184V resistance associated mutation to lamivudine as well as NNRTI''s RAMS.

Conclusions

Virological failure rate in resource-limited settings are similar to those observed in developed countries. Resistance mutation patterns were concordant with HAART received by failing patients. Long term detectable viral load confers greater probability of developing resistance and as a consequence, making difficult to find out a cost-effective subsequent treatment regimen.     

Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

In the human fungal pathogen Cryptococcus neoformans, Ras signaling mediates sexual differentiation, morphogenesis, and pathogenesis. By studying Ras prenylation and palmitoylation in this organism, we have found that the subcellular localization of this protein dictates its downstream signaling specificity. Inhibiting C. neoformans Ras1 prenylation results in the defective general membrane targeting of this protein and the loss of all Ras function. In contrast, palmitoylation mediates localization of Ras1 to the plasma membrane and is required for normal morphogenesis and survival at high temperatures. However, palmitoylation and plasma membrane localization are not required for Ras-dependent sexual differentiation. Likely as a result of its effect on thermotolerance, Ras1 palmitoylation is also required for the pathogenesis of C. neoformans. These data support an emerging paradigm of compartmentalized Ras signaling. However, our studies also demonstrate fundamental differences between the Ras pathways in different organisms that emphasize the functional flexibility of conserved signaling cascades.