Induction and characterization of streptomycin-resistant mutants in Capsicum praetermissum

Streptomycin-resistant mutants were isolated from mutagenised cotyledon explants of Capsicum praetermissum Heiser & Smith. The explants were mutagenised with N-ethyl-N-nitrosourea, which resulted in a high frequency of streptomycin-resistant mutants (18.0%) and a low frequency of chlorophyll-deficient (albino) mutants (8.0%). Complete streptomycin-resistant plantlets were obtained after rooting of the regenerated green shoots on rooting medium containing 1.0 mg L-1 IAA and 500 mg L-1 streptomycin sulphate. Leaf-segment assay of these plantlets revealed that they were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, and spectinomycin. Reciprocal crosses between streptomycin-resistant and -sensitive plants showed a non-Mendelian transmission of resistance by female parents.     

Synthesis and evaluation of photolabile insulin prodrugs

We have developed two photolabile insulin prodrugs, insulin-2P and insulin-3P. These prodrugs were synthesized by protecting GlyA1 (N(alphaA1)), and one or both of the PheB1 (N(alphaB1)) and LysB29 (N(epsilonB29)) amino groups in insulin using 5'-(alpha-methyl-nitro-piperonyl)oxy-carbonyl as the protecting group. These insulin prodrugs were efficiently activated by exposure to longwave UV light to produce insulin quantitatively. Using 2-deoxyglucose uptake assays, both di- and tri-protected compounds were less active than native insulin in the protected state, and showed comparable activity to native insulin upon photoactivation.     

Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries

Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.     

On invariants of a 2-D proteome map derived from neighborhood graphs

We consider the problem of the construction of invariants for characterization of 2-D maps, such as 2-D proteome maps, 2-D NMR spectral maps, etc., that in addition to facilitating cataloguing such maps, can be used for comparison of maps and numerical evaluation of their degree of similarity. A novel approach, based on the concept that the nearest neighborhood of points (spots) on a map are sufficiently flexible to allow one not only to vary the number of points used for characterization of the map but also the density of information on their relative positions, is put forward. The method is illustrated with the Coomassie brilliant blue stained 2-D gel electrophoresis patterns of the proteomes from liver cells of healthy male Fisher F344 rats and the rats treated with four peroxisome proliferators.     

Differential protection and recovery of 5-HT1A receptors from N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) inactivation in regions of rat brain

The effect of N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ) on 5-HT_1A receptors was studied in Sprague Dawley rats. A single dose of EEDQ (4 mg/kg body wt., i.p.) significantly inactivated 5-HT_1A receptors, as measured by [³H]8-hydroxy-2-[di-n-propylamino]-tetralin ([³H]8-OH-DPAT), in cortex (64%, p < 0.0001) and hippocampus (48%, p < 0.0001). A significant (p < 0.01) increase in the affinity of 5-HT_1A receptors for radioligand was observed in both regions. A dose dependent protection of cortical 5-HT_1A receptors from EEDQ inactivation with pre-treatment of different doses of 8-OH-DPAT (4–20 mg/kg) was observed, along with recovery of affinity of [³H]8-OH-DPAT for 5-HT_1A receptors in both regions. Although, a dose of 4 mg/kg of 8-OH-DPAT failed to attenuate the effect of EEDQ on hippocampal 5-HT_1A receptors, a significant protection of these receptors was observed with 10 and 20 mg/kg of 8-OH-DPAT. Displacement studies revealed that EEDQ has more affinity for cortical (Ki = 101.3 ± 11.8 nM) than hippocampal (Ki = 133.5 ± 25.8 nM) 5-HT_1A receptors. A time dependent natural recovery of 5-HT_1A receptors from inactivation by a single dose of EEDQ (4 mg/kg) was observed more in cortex compared to hippocampus over a period from 1 day to 14 days. The results of this study suggest that 8-OH-DPAT inhibited EEDQ inactivation of cortical and hippocampal 5-HT_1A receptors in a concentration dependent manner. The synthesis and turnover of 5-HT_1A receptors differ in cortex and hippocampus, as evident by earlier recovery in the cortex.     

Development of Veterinary Laboratory Networks for Avian Influenza and Other Emerging Infectious Disease Control: The Southeast Asian Experience

The outbreak of highly pathogenic H5N1 avian influenza, with its international spread, confirmed that emerging infectious disease control must be underpinned by effective laboratory services. Laboratory results are the essential data underpinning effective surveillance, case diagnosis, or monitoring of responses. Importantly, laboratories are best managed within national and international networks of technological support rather than in isolation. A well planned laboratory network can deliver both a geographical spread of testing capacity and also a cost effective hierarchy of capability. Hence in the international context regional networks can be particularly effective. Laboratories are an integral part of a country’s veterinary services and their role and function should be clearly defined in the national animal health strategy and supporting government policies. Not every laboratory should be expected to deliver every possible service, and integration into regional and broader international networks should be a part of the overall strategy. The outputs required of each laboratory should be defined and then ensured through accredited quality assurance. The political and scientific environment in which laboratories operate changes continuously, not only through evolving national and regional animal health priorities but also through new test technologies and enhancements to existing technologies. Active networks help individual laboratories to monitor, evaluate, and respond to such challenges and opportunities. The end result is enhanced emerging infectious disease preparedness across the region.