The Activities of Six Exo- and Endopeptidases in the Substantia Nigra,Neostriatum, and Cortex of the Rat Brain

We determined the enzymatic activity and crude subcellular distribution of four exopeptidases: Dipeptidylaminopeptidase IV (DAP-IV), Alanyl aminopeptidase (AAP), Prolyl aminopeptidase (PAP) and -Glutamyl transpeptidase (GTP), and two endopeptidases: Postproline endopeptidase (PEP) and Trypsin-like peptidase (T-L P) in pars compacta (SNPC) and pars reticulata (SNPR) of substantia nigra, caudate-putamen (CAU) and cerebral cortex (CC) of the rat brain. We found: 1) DAP-IV activity is comparatively higher in SNPC and it is equally distributed in the postmitochondrial precipitate (PR) and supernatant (SN) fractions of SNPC, CAU and CC but higher in the SN from SNPR. 2) CC shows the highest activity of AAP and its activity is mainly located in the SN from all areas. 3) The activity of PAP is comparatively higher in SNPC and it is exclusively located in the SN from all areas. 4) GTP activity is similar in all areas but its predominance is in the SN for SNPC and SNPR, and in the PR for CAU and CC. 5) CAU has higher PEP activity (higher in the PR) than CC (higher in the SN); no activity is detected in the substantia nigra. 6) The activity of a Trypsin-like peptidase is the highest in SNPC and SNPR; this activity have some predominance in the SN and higher predominance in the same fraction from CAU and CC.     

Diversity of Bacillus thuringiensis strains from Latin America with insecticidal activity against different mosquito species

The characterization of selected Bacillus thuringiensis strains isolated from different Latin America countries is presented. Characterization was based on their insecticidal activity against Aedes aegypti, Culex quinquefasciatus, and Anopheles albimanus larvae, scanning electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and plasmid profiles as well as PCR analysis using novel general and specific primers for cry and cyt genes encoding proteins active against mosquitoes (cyt1, cyt2, cry2, cry4A, cry4B, cry10, cry11, cry17, cry19, cry24, cry25, cry27, cry29, cry30, cry32, cry39, and cry40). Strains LBIT315, LBIT348, and IB604 showed threefold higher mosquitocidal activity against A. aegypti and C. quinquefasciatus larvae than B. thuringiensis subsp. israelensis and displayed high similarities with the B. thuringiensis subsp. israelensis used in this study with regard to protein and plasmid profiles and the presence of cry genes. Strain 147-8906 has activity against A. aegypti similar to that of B. thuringiensis subsp. israelensis but has different protein and plasmid profiles. This strain, harboring cry11, cry30, cyt1, and cyt2 genes, could be relevant for future resistance management interventions. Finally, the PCR screening strategy presented here led us to identify a putative novel cry11B gene.     

Expanded bed adsorption processing of mammalian cell culture fluid: comparison with packed bed affinity chromatography

A comparison between expanded bed adsorption and conventional packed bed Protein A Fast Flow to purify the anti-rHBsAg mAbs from feedstock is presented in this work. Direct capture by STREAMLINE expanded bed adsorption chromatography resulted in 92% product recovery and sevenfold more concentrated product with similar purity levels compared to that obtained by the standard packed method. The process time and buffer consumption were reduced in the expanded bed adsorption method not only with the binding-elution conditions but also with the use of NaOH during the cleaning-in-place step. The latter is the most widely accepted agent in downstream processing, being a cost effective technique that provides not only efficient cleaning but also sanitizes complete column systems and destroys pirogens.     

Characterization of urea transport in Bufo arenarum oocytes

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water.     

Effects of an optical brightener and an abrasive on iridescent virus infection and development of Aedes aegypti

The insecticidal properties of certain entomopathogenic viruses can be greatly improved in mixtures with substances that affect the integrity of the insect peritrophic membrane, particularly optical brighteners. We aimed to determine the effect of an optical brightener, Blankophor BBH, and an abrasive compound, silicon carbide, alone and in mixtures, on the prevalence of patent and covert infection of Aedes aegypti (L.) (Diptera: Culicidae) by Invertebrate iridescent virus 6 (IIV‐6) (Iridoviridae). The prevalence of patent infection by IIV‐6 was < 1.5% in all treatments involving virus. Contrary to predictions, there were significantly fewer patent infections in virus treatments involving Blankophor with or without silicon carbide compared with controls. Covert infection of adults detected by insect bioassay was between 6.7 and 12.2%, although no significant differences were observed between treatments. Exposure to IIV‐6 alone or silicon carbide alone did not significantly increase larval mortality compared to the controls, whereas exposure to Blankophor alone, or in any combination with IIV‐6 or silicon carbide, clearly increased larval mortality. These effects did not carry‐over to the pupal stage. Adult females emerged ～1.5 days later than males. Compared to control insects, female development rate was extended by 11.4 and 12.6% in the treatments involving IIV‐6 alone and silicon carbide alone, respectively. The sex ratio at adult emergence did not differ significantly between control insects and those of other treatments. These results support the hypothesis that the gut is unlikely to represent the principal point of infection of mosquito larvae by iridescent viruses.     

Infection of Anastrepha ludens following soil applications of Heterorhabditis bacteriophora in a mango orchard

To determine the efficacy of Heterorhabditis bacteriophora Poinar (Nematoda: Heterorhabditidae) for control of Anastrepha ludens (Loew) (Diptera: Tephritidae), field experiments were performed in a mango orchard with soil temperatures of 24–29 °C. The density of third‐instar A. ludens (50–500 larvae per plot) released into 0.25 m² wood‐framed experimental plots containing soil (16% wt/wt moisture) previously treated with 125 infective juveniles per square centimetre soil surface did not significantly influence the prevalence of infection by H. bacteriophora. In subsequent experiments, the percentages of infection of fly pupae were positively correlated with the concentration of infective stages applied to soil plots. The highest average percentage of infection (74% at 250 infective juveniles per square centimetre) was observed when fly larvae were released simultaneously onto soil, compared to larvae that emerged from laboratory‐infested mangoes over a period of 8 days (52% infection at 500 infective juveniles per square centimetre). Double applications of infective juveniles at an interval of 4 days did not greatly improve the prevalence of infection (～10% higher) compared to single applications. Between 9 and 15% of larvae that remained within infested mangoes became infected by nematodes, irrespective of the concentration of nematodes applied to each experimental plot. We conclude that effective control of A. ludens requires very high densities of H. bacteriophora. The successful use of this nematode for biocontrol of A. ludens will depend on identifying ways of overcoming the fly's ability to avoid infection.     

Evaluation of the SCP from Opuntia juice

Summary In deseeded opuntia juice medium batch and continuous cultivation ofCandida utilis produced 12 and 19.9gl^–1 yield of the SCP respectively. Its biological value(72), digestibility coefficient(70) and protein score(58) indicated it to be a good protein.     

Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus thuringiensis HD-73 was transformed with the endochitinase gene chiA74 under the control of a strong promoter (pcytA) and a 5′ mRNA stabilizing (STAB-SD) sequence (HD-73-pEBchiA74). Expression levels were compared with those observed from the wild type strain (HD-73) and the recombinant HD-73 strain expressing chiA74 under the control of its native promoter (HD-73-pEHchiA74). The chitinolytic activity of HD-73-pEBchiA74 was markedly elevated, being ~58- and 362-fold higher than, respectively, HD-73-pEHchiA74 and parental HD-73, representing the highest levels of chitinase expression in recombinant B. thuringiensis reported to date. Parasporal crystals measured under transmission electron microscopy showed that HD-73 produced crystals of 1.235 (±0.214) and 1.356 (±0.247) μm in length when the bacterium was grown in respectively, NBS and NBS with glucose. Otherwise, HD-73-pEBchiA74 synthesized crystals of 1.250 (±0.222) and 1.139 (±0.202) μm in length when cultivated in NBS and NBS with glucose, respectively, values that showed a diminution of ~10 and 20% compared with crystals produced by HD-73-pEHchiA74 grown under the same conditions. Comparison of viable spore counts per ml showed that HD-73-pEBchiA74 produced fewest viable spores (1.5 × 10⁹, 1.3 × 10⁹), compared to HD-73-pEHchiA74 (4.9 × 10⁹, 5.3 × 10⁹) and HD-73 (6.8 × 10⁹, 8.8 × 10⁹) when grown in NBS and NBS supplemented with glucose, respectively. No change in cellular protease activity was observed despite the overproduction of the chitinase.     

Metalloproteases Secreted by <Emphasis Type="Italic">Actinobacillus suis</Emphasis>

Actinobacillus suis secretes metalloproteases into its medium. These secreted proteins, when concentrated by precipitation with 70% (NH₄)₂SO₄ or methanol, displayed proteolytic activity at >200 kDa molecular mass bands in 10% polyacrylamide gels copolymerized with bovine casein (1%). They showed activity in a broad pH range (from pH 5 to pH 10) and were inhibited by 20 mM EDTA or EGTA, but could be reactivated by calcium. They were found heat stable at 40°C, 50°C, 60°C, and 70°C, but their activity diminished at 80°C or higher. They degraded pig and bovine IgG and cross-reacted with a polyclonal serum against a high molecular mass secreted protease from A. pleuropneumoniae. Extracellular proteases could play a role in diseases caused by A. suis.