pHG165: A pBR322 copy number derivative of pUC8 for cloning and expression

During the construction of the Messing pUC plasmid series, the rop(rom) gene of pBR322 which mediates the activity of RNAI was deleted. This has resulted in an elevated copy number for the pUC plasmids which makes the expression of beta-galactosidase activity constitutive in a host containing the Iqtss lac repressor. We describe the construction of a new series of vectors which retain the pUC multiple cloning site (MCS) but in which copy number control has been recovered. In addition, the lac alpha/lac promoter expression region has been inserted into a HpaI cassette. This facilitates the movement of recombinant DNA clones within the MCS. It also increases the complementation activity of the lac alpha peptide by an order of magnitude, allowing selection of recombinants by their Lac- phenotype on MacConkey agar.     

Eicosanoids in insect immunity: bacterial infection stimulates hemocytic phospholipase A2 activity in tobacco hornworms

Intracellular phospholipase A(2) (PLA(2)) is responsible for releasing arachidonic acid from cellular phospholipids, and is thought to be the first step in eicosanoid biosynthesis. Intracellular PLA(2)s have been characterized in fat body and hemocytes from tobacco hornworms, Manduca sexta. Here we show that bacterial challenge stimulated increased PLA(2) activity in isolated hemocyte preparations, relative to control hemocyte preparations that were challenged with water. The increased activity was detected as early as 15 s post-challenge and lasted for at least 1 h. The increased activity depended on a minimum bacterial challenge dose, and was inhibited in reactions conducted in the presence of oleyoxyethylphosphorylcholine, a site-specific PLA(2) inhibitor. In independent experiments with serum prepared from whole hemolymph, we found no PLA(2) activity was secreted into serum during the first 24 h following bacterial infection. We infer that a hemocytic intracellular PLA(2) activity is increased immediately an infection is detected. The significance of this enzyme lies in its role in launching the biosynthesis of eicosanoids, which mediate cellular immune reactions to bacterial infection.     

Adolescent Sexual and Reproductive Health Services and Implications for the Provision of Voluntary Medical Male Circumcision: Results of a Systematic Literature Review

Background

Voluntary medical male circumcision (VMMC) is a critical HIV prevention tool. Since 2007, sub-Saharan African countries with the highest prevalence of HIV have been mobilizing resources to make VMMC available. While implementers initially targeted adult men, demand has been highest for boys under age 18. It is important to understand how male adolescents can best be served by quality VMMC services.

Methods and Findings

A systematic literature review was performed to synthesize the evidence on best practices in adolescent health service delivery specific to males in sub-Saharan Africa. PubMed, Scopus, and JSTOR databases were searched for literature published between January 1990 and March 2014. The review revealed a general absence of health services addressing the specific needs of male adolescents, resulting in knowledge gaps that could diminish the benefits of VMMC programming for this population. Articles focused specifically on VMMC contained little information on the adolescent subgroup. The review revealed barriers to and gaps in sexual and reproductive health and VMMC service provision to adolescents, including structural factors, imposed feelings of shame, endorsement of traditional gender roles, negative interactions with providers, violations of privacy, fear of pain associated with the VMMC procedure, and a desire for elements of traditional non-medical circumcision methods to be integrated into medical procedures. Factors linked to effective adolescent-focused services included the engagement of parents and the community, an adolescent-friendly service environment, and VMMC counseling messages sufficiently understood by young males.

Conclusions

VMMC presents an opportune time for early involvement of male adolescents in HIV prevention and sexual and reproductive health programming. However, more research is needed to determine how to align VMMC services with the unique needs of this population.     

Inhibition of hepatitis C virus NS2/3 processing by NS4A peptides. Implications for control of viral processing

The NS2/3 protease of hepatitis C virus is responsible for a single cleavage in the viral polyprotein between the nonstructural proteins NS2 and NS3. The minimal protein region necessary to catalyze this cleavage includes most of NS2 and the N-terminal one-third of NS3. Autocleavage reactions using NS2/3 protein translated in vitro are used here to investigate the inhibitory potential of peptides likely to affect the reaction. Peptides representing the cleaved sequence have no effect upon reaction rates, and the reaction rate is insensitive to dilution. Both results are consistent with prior suggestions that the NS2/3 cleavage is an intramolecular reaction. Surprisingly, peptides containing the 12-amino acid region of NS4A responsible for binding to NS3 inhibit the NS2/3 reaction with K(i) values as low as 3 microM. Unrelated peptide sequences of similar composition are not inhibitory, and neither are peptides containing incomplete segments of the NS4A region that binds to NS3. Inhibition of NS2/3 by NS4A peptides can be rationalized from the organizing effect of NS4A on the N terminus of NS3 (the NS2/3 cleavage point) as suggested by the known three-dimensional structure of the NS3 protease domain (Yan, Y., Li, Y., Munshi, S., Sardana, V., Cole, J. L., Sardana, M., Steinkuhler, C., Tomei, L., De Francesco, R., Kuo, L. C., and Chen, Z. (1998) Protein Sci. 7, 837-847). These findings may imply a sequential order to proteolytic maturation events in hepatitis C virus.     

Rigid structural requirement of the 5' terminus of mRNA for translational activity.

Oxidation by sodium periodate of the ribose cis-diol of the 5′ terminal of liver mRNA to the corresponding dialdehyde virtually destroyed its template activity in the wheat germ translation system. The rigid structural requirement for the ribose cis-diol is indicated by the failure of reduction of the dialdehyde to the corresponding primary alcohols to restore the template activity of the mRNA. Sodium periodate alone inhibited the translational system at concentrations above 0.25 mM. Purification of the periodate oxidized mRNA by sucrose density gradient centrifugation or exclusion on Sephadex G-100 did not increase its template activity. Periodate oxidized mRNA was not inhibitory to translation of untreated mRNA.