A unique mRNA species for a regulatory subunit of cAMP-dependent protein kinase is specifically induced in haploid germ cells

AbstractCyclic AMP (cAMP) and its action by way of cAMP-dependent protein kinase is important for sperm motility. Previous studies on germ cells have demonstrated a selective decrease in the amount of type I cAMP-dependent protein kinase during spermatid development, and that type II was the major form present in elongating spermatids and in mature sperm. This would indicate activation of a gene in haploid germ cells, encoding a regulatory subunit of type II protein kinase. However, haploid expression of such a gene has so far not been shown. In the present study we demonstrate high-levelled expression of a unique mRNA species for a specific regulatory subunit of type II cAMP-dependent protein kinase at late stages of spermatogenesis, i.e. during spermatid elongation

Activation mechanism of rabbit skeletal muscle myosin light chain kinase 5′-p-Fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme

Abstract5′-p-Fluorosulfonylbenzoyl adenosine (FSBA), an ATP-like affinity labelling reagent, reacted with rabbit skeletal muscle myosin light chain kinase (skMLCK) and its calmodulin complex in a site-specific manner. Reaction was dependent upon the presence of the adenosine moiety of FSBA, saturated with increasing FSBA, was inhibited by MgATP, and was accompanied by stoichiometric incorporation of [14C]FSBA. The kinetic constants describing the reaction were similar for skMLCK and its calmodulin complex: k3= −0.040 min−1 and −0.038 mint-1, and Ki=0.18 mM and 0.40 mM, respectively. It is concluded that the MgATP-binding site on skMLCK remains accessible at all times and maintains a near constant conformation

Substrate recognition by casein kinase-II: The role of histidine-160

AbstractCasein kinase-II (CK-II) belongs to the protein kinases recognizing serine/threonine in proximity to acidic residues in protein substrates. Crystallography and mutagenesis studies on the cAMP-dependent protein kinase (PKA) disclosed that glutamic acid-170 (E170), is important for interaction of substrates with the enzyme. At a position corresponding to E170 in PKA most Ser/Thr kinases have an aspartic or glutamic acid, while CK-II has a histidine residue (H160). In order to examine the relevance of this substitution for CK-II substrate specificity, a mutant of the catalytic α subunit (H160D), in which H160 was changed to aspartic acid, was made. Our results show that H160 is not primarily involved in canonical substrate recognition, but does interact with an acidic residue located at position −2 with respect to the target Ser/Thr

Dynamic MAIT cell response with progressively enhanced innateness during acute HIV-1 infection.

Mucosa-associated invariant T (MAIT) cell loss in chronic HIV-1 infection is a significant insult to antimicrobial immune defenses. Here we investigate the response of MAIT cells during acute HIV-1 infection utilizing the RV217 cohort with paired longitudinal pre- and post-infection samples. MAIT cells are activated and expand in blood and mucosa coincident with peak HIV-1 viremia, in a manner associated with emerging microbial translocation. This is followed by a phase with elevated function as viral replication is controlled to a set-point level, and later by their functional decline at the onset of chronic infection. Interestingly, enhanced innate-like pathways and characteristics develop progressively in MAIT cells during infection, in parallel with TCR repertoire alterations. These findings delineate the dynamic MAIT cell response to acute HIV-1 infection, and show how the MAIT compartment initially responds and expands with enhanced function, followed by progressive reprogramming away from TCR-dependent antibacterial responses towards innate-like functionality

An Observational Cohort Comparison of Facilitators of Retention in Care and Adherence to Anti-Eetroviral Therapy at an HIV Treatment Center in Kenya

BACKGROUND: Most HIV treatment programs in resource-limited settings utilize multiple facilitators of adherence and retention in care but there is little data on the efficacy of these methods. We performed an observational cohort analysis of a treatment program in Kenya to assess which program components promote adherence and retention in HIV care in East Africa. METHODS: Patients initiating ART at A.I.C. Kijabe Hospital were prospectively enrolled in an observational study. Kijabe has an intensive program to promote adherence and retention in care during the first 6 months of ART that incorporates the following facilitators: home visits by community health workers, community based support groups, pharmacy counseling, and unannounced pill counts by clinicians. The primary endpoint was time to treatment failure, defined as a detectable HIV-1 viral load; discontinuation of ART; death; or loss to follow-up. Time to treatment failure for each facilitator was calculated using Kaplan-Meier analysis. The relative effects of the facilitators were determined by the Cox Proportional Hazards Model. RESULTS: 301 patients were enrolled. Time to treatment failure was longer in patients participating in support groups (448 days vs. 337 days, P<0.001), pharmacy counseling (480 days vs. 386 days, P = 0.002), pill counts (482 days vs. 189 days, P<0.001) and home visits (485 days vs. 426 days, P = 0.024). Better adherence was seen with support groups (89% vs. 82%, P = 0.05) and pill counts (89% vs. 75%, P = 0.02). Multivariate analysis using the Cox Model found significant reductions in risk of treatment failure associated with pill counts (HR = 0.19, P<0.001) and support groups (HR = 0.43, P = 0.003). CONCLUSION: Unannounced pill counts by the clinician and community based support groups were associated with better long term treatment success and with better adherence