Calliandra selloi Macbride trypsin inhibitor: isolation, characterization, stability, spectroscopic analyses

A trypsin inhibitor was purified from Calliandra selloi Macbride seeds (CSTI). SDS-PAGE under non-reducing conditions showed a single band of approximately 20,000 Da, while under reducing conditions two bands of 16,000 and 6000 Da were observed, indicating that CSTI consists of two polypeptide chains. Molecular masses of 20,078 and 20,279 were obtained by mass spectrometry, although only one pI of 4.0 was observed and one peak was obtained by reversed phase chromatography. Amino-terminal sequence analysis showed homology to Kunitz-type inhibitors. CSTI was able to inhibit trypsin (Ki 2.21 x 10(-7)M), alpha-chymotrypsin (Ki 4.95 x 10(-7)M) and kallikrein (Ki 4.20 x 10(-7)M) but had no effect on elastase. Trypsin inhibitory activity was stable over a wide range of pH and temperature. CSTI was particularly susceptible to DTT treatment, followed by addition of iodoacetamide. Far-UV circular dichroism measurements revealed that CSTI is a beta-II protein. Thermal unfolding showed a two-state transition with a midpoint at 68 degrees C. Far-UV CD spectra of CSTI at pH extremes showed little changes, while more pronounced differences in near-UV CD spectra were detected. Remarkably, treatment with 1mM DTT caused very slight changes in the far-UV CD spectrum, and only after carbamidomethylation was there was a marked loss observed in secondary structure.     

Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway

Dengue virus (DENV) relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of Aedes aegypti and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP) plays an important role in regulating infectious DENV production. Using RNA interference studies, we show in vivo that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector.     

Genetic differentiation in Chilean hake Merluccius gayi gayi (Pisces: Merlucciidae)

The genetic structure of Chilean hake Merluccius gayi gayi, was analyzed using starch gel protein electrophoresis. Samples were collected from four localities along the coast off Chile. A total of 1500 specimens sampled from Coquimbo, San Antonio, Talcahuano and Puerto Montt were used in the study. Genetic information was obtained for six allozyme loci Pgi-1, Pgi-2, Pgm, Idh-1, Idh-2 and Aat. The results of the analysis showed no significant allelic differences among samples from various localities, with an average F_ST value of 0.007 for all loci and samples. A heterogeneity test for all loci and specimens from the four localities showed only two significant values. Thus, Chilean hake populations along the coast of Chile appear to be genetically homogeneous.     

Apseudopsis adami, a new species of tanaidacean (Crustacea: Peracarida) from the NW Iberian Peninsula: postmarsupial development and remarks on morphological characters

A new species of apseudomorph tanaidacean, Apseudopsis adami sp. nov., is described, including intraspecific variation, from specimens collected in the seagrass meadows of O Grove inlet (NW Iberian Peninsula, NE Atlantic). The new species is characterized by the unique combination of the following characters: pointed rostrum with rounded shoulders, four ventral propodal spines on pereopod 1, and posterolateral pointed apophyses on pereonites 2–5, regardless of the developmental stage of the individual. Two juvenile stages were identified before reaching maturity; early juvenile limbs are scarcely ornamented in comparison to the adults. Advanced juveniles represent the vast majority of the studied population, and the limb ornamentation is similar to that of the adults. Males show two different cheliped morphologies. After a preparatory stage, females pass through a sequence of copulatory instars followed by intermediate stages in which the female loses the ovisac after manca release. The number of segments of the antennular and antennal flagella and uropod endopods, as well as the development and ornamentation of the mandibular palp, are meristic changes that occur during development and allow rapid identification of the developmental instar.     

Mitochondrial Respiration Chain Enzymatic Activities in the Human Brain: Methodological Implications for Tissue Sampling and Storage

Neurochemical Research - Mitochondrial respiratory chain complexes enzymatic (MRCCE) activities were successfully evaluated in frozen brain samples. Epilepsy surgery offers an ethical opportunity...     

Fate of Mycobacterium avium subsp. paratuberculosis after application of contaminated dairy cattle manure to agricultural soils

Details regarding the fate of Mycobacterium avium subsp. paratuberculosis (basonym, Mycobacterium paratuberculosis) after manure application on grassland are unknown. To evaluate this, intact soil columns were collected in plastic pipes (lysimeters) and placed under controlled conditions to test the effect of a loamy or sandy soil composition and the amount of rainfall on the fate of M. paratuberculosis applied to the soil surface with manure slurry. The experiment was organized as a randomized design with two factors and three replicates. M. paratuberculosis-contaminated manure was spread on the top of the 90-cm soil columns. After weekly simulated rainfall applications, water drainage samples (leachates) were collected from the base of each lysimeter and cultured for M. paratuberculosis using Bactec MGIT ParaTB medium and supplements. Grass was harvested, quantified, and tested from each lysimeter soil surface. The identity of all probable M. paratuberculosis isolates was confirmed by PCR for IS900 and F57 genetic elements. There was a lag time of 2 months after each treatment before M. paratuberculosis was found in leachates. The greatest proportions of M. paratuberculosis-positive leachates were from sandy-soil lysimeters in the manure-treated group receiving the equivalent of 1,000 mm annual rainfall. Under the higher rainfall regimen (2,000 mm/year), M. paratuberculosis was detected more often from lysimeters with loamy soil than sandy soil. Among all lysimeters, M. paratuberculosis was detected more often in grass clippings than in lysimeter leachates. At the end of the trial, lysimeters were disassembled and soil cultured at different depths, and we found that M. paratuberculosis was recovered only from the uppermost levels of the soil columns in the treated group. Factors associated with M. paratuberculosis presence in leachates were soil type and soil pH (P < 0.05). For M. paratuberculosis presence in grass clippings, only manure application showed a significant association (P < 0.05). From these findings we conclude that this pathogen tends to move slowly through soils (faster through sandy soil) and tends to remain on grass and in the upper layers of pasture soil, representing a clear infection hazard for grazing livestock and a potential for the contamination of runoff after heavy rains.