Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice

Phospholemman (FXYD1), a 72-amino acid transmembrane protein abundantly expressed in the heart and skeletal muscle, is a major substrate for phosphorylation in the cardiomyocyte sarcolemma. Biochemical, cellular, and electrophysiological studies have suggested a number of possible roles for this protein, including ion channel modulator, taurine-release channel, Na(+)/Ca(2+) exchanger modulator, and Na-K-ATPase-associated subunit. We have generated a phospholemman-deficient mouse. The adult null mice exhibited increased cardiac mass, larger cardiomyocytes, and ejection fractions that were 9% higher by magnetic resonance imaging compared with wild-type animals. Notably, this occurred in the absence of hypertension. Total Na-K-ATPase activity was 50% lower in the phospholemman-deficient hearts. Expression (per unit of membrane protein) of total Na-K-ATPase was only slightly diminished, but expression of the minor alpha(2)-isoform, which has been specifically implicated in the control of contractility, was reduced by 60%. The absence of phospholemman thus results in a complex response, including a surprisingly large reduction in intrinsic Na-K-ATPase activity, changes in Na-K-ATPase isoform expression, increase in ejection fraction, and increase in cardiac mass. We hypothesize that a primary effect of phospholemman is to modulate the Na-K-ATPase and that its reduced activity initiates compensatory responses.     

Discovery of GlyT1 inhibitors with improved pharmacokinetic properties

Glycine transporter 1 (GlyT1) represents a novel target for the treatment of schizophrenia via the potentiation of glutamatergic NMDA receptors. The discovery of 4,4-disubstituted piperidine inhibitors of GlyT1 which exhibit improved pharmacokinetic properties, including oral bioavailability, is discussed.     

Metabolism of inositol 1,4,5-trisphosphate in guinea-pig hepatocytes.

Metabolism of inositol 1,4,5-trisphosphate was investigated in permeabilized guinea-pig hepatocytes. The conversion of [3H]inositol 1,4,5-trisphosphate to a more polar 3H-labelled compound occurred rapidly and was detected as early as 5 s. This material co-eluted from h.p.l.c. with inositol 1,3,4,5 tetrakis[32P]phosphate and is presumably an inositol tetrakisphosphate. A significant increase in the 3H-labelled material co-eluting from h.p.l.c. with inositol 1,3,4-trisphosphate occurred only after a definite lag period. Incubation of permeabilized hepatocytes with inositol 1,3,4,5-tetrakis[32P]phosphate resulted in the formation of 32P-labelled material that co-eluted with inositol 1,3,4-trisphosphate; no inositol 1,4,5-tris[32P]phosphate was produced, suggesting the action of a 5-phosphomonoesterase. The half-time of hydrolysis of inositol 1,3,4,5-tetrakis[32P]phosphate of approx. 1 min was increased to 3 min by 2,3-bisphosphoglyceric acid. Similarly, the rate of production of material tentatively designed as inositol 1,3,4-tris[32P]phosphate from the tetrakisphosphate was reduced by 10 mM-2,3-bisphosphoglyceric acid. In the absence of ATP there was no conversion of [3H]inositol 1,4,5-trisphosphate to [3H]inositol tetrakisphosphate or to [3H]inositol 1,3,4-trisphosphate, which suggests that the 1,3,4 isomer does not result from isomerization of inositol 1,4,5-trisphosphate. The results of this study suggest that the origin of the 1,3,4 isomer of inositol trisphosphate in isolated hepatocytes is inositol 1,3,4,5-tetrakisphosphate and that inositol 1,4,5-trisphosphate is rapidly converted to this tetrakisphosphate. The ability of 2,3-bisphosphoglyceric acid, an inhibitor of 5-phosphomonoesterase of red blood cell membrane, to inhibit the breakdown of the tetrakisphosphate suggests that the enzyme which removes the 5-phosphate from inositol 1,4,5-trisphosphate may also act to convert the tetrakisphosphate to inositol 1,3,4-trisphosphate. It is not known if the role of inositol 1,4,5-trisphosphate kinase is to inactivate inositol 1,4,5-trisphosphate or whether the tetrakisphosphate product may have a messenger function in the cell.     

Polymorphism and evolution in the constant region of the T-cell receptor beta chain in an advanced teleost fish

Sequence, PCR and Southern data are presented as evidence that, as in mammals, two gene loci encode C regions of the TCR beta chain in the bicolor damselfish, Stegastes partitus. The loci are distinguished by an insertion of ten amino acids in the c-d loop at one locus and by a high interlocus divergence of the third intron and fourth exon sequences. Unlike their mammalian counterparts, the damselfish TCRBC genes encode highly polymorphic regions. None of the eight complete cDNA or four partial genomic DNA sequences presented from a single animal are identical; and three of the four animals examined are heterozygous at both loci, suggesting high heterozygosity in the damselfish population. Coding regions of the eight cDNA clones differ by up to 12% at the DNA level and 23% at the amino acid level. Polymorphism is concentrated primarily in the less evolutionarily conserved regions, suggesting that this variation may be selectively neutral. However, a comparison of the variation between synonymous and non-synonymous sites suggests that at least a portion of the observed variation results from selection. As in mammals, a gradient of sequence homogenization between the two loci is observed. Data presented here suggest that both interlocus homogenization and the sharing of polymorphic segments are likely achieved by partial gene conversion.     

Manipulation of heterogeneous hybridoma cultures for overproduction of monoclonal antibodies.

In searching for ways to manipulate heterogeneous hybridoma cell cultures (ATCC HB124) to obtain increased production of monoclonal antibodies (IgG2a), we have selected for a higher secreting but slower growing subpopulation using the level of fluorescent surface-associated antibodies and a fluorescence-activated cell sorter. Cell surface fluorescence was found to be correlated with specific antibody secretion rate over the short term but not with intracellular antibody content. Also, the specific secretion rate of a heterogeneous population of hybridoma cells grown in batch culture has been shown to be inversely correlated with an increase in either the initial cell concentration or the medium antibody concentration. Several experiments suggest that an upper limit exists for medium antibody concentration, above which antibody is degraded at the same rate at which it is produced. Should other cell lines behave similarly, strategies for overproduction of monoclonal antibodies suggested herein could be profitably used in industry.     

Pseudomonas syringae effector HopZ3 suppresses the bacterial AvrPto1–tomato PTO immune complex via acetylation

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1_Psy-triggered immunity. HopZ3 acetylates AvrPto1_Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1_Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1_Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3’s targets such as AvrPto1_Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3’s unusual capacity to modify histidine in addition to serine, threonine and lysine residues.     

Gender inequity norms are associated with increased male-perpetrated rape and sexual risks for HIV infection in Botswana and Swaziland

Background

There is limited empirical research on the underlying gender inequity norms shaping gender-based violence, power, and HIV risks in sub-Saharan Africa, or how risk pathways may differ for men and women. This study is among the first to directly evaluate the adherence to gender inequity norms and epidemiological relationships with violence and sexual risks for HIV infection.

Methods

Data were derived from population-based cross-sectional samples recruited through two-stage probability sampling from the 5 highest HIV prevalence districts in Botswana and all districts in Swaziland (2004–5). Based on evidence of established risk factors for HIV infection, we aimed 1) to estimate the mean adherence to gender inequity norms for both men and women; and 2) to model the independent effects of higher adherence to gender inequity norms on a) male sexual dominance (male-controlled sexual decision making and rape (forced sex)); b) sexual risk practices (multiple/concurrent sex partners, transactional sex, unprotected sex with non-primary partner, intergenerational sex).

Findings

A total of 2049 individuals were included, n = 1255 from Botswana and n = 796 from Swaziland. In separate multivariate logistic regression analyses, higher gender inequity norms scores remained independently associated with increased male-controlled sexual decision making power (AORmen = 1.90, 95%CI:1.09–2.35; AORwomen = 2.05, 95%CI:1.32–2.49), perpetration of rape (AORmen = 2.19 95%CI:1.22–3.51), unprotected sex with a non-primary partner (AORmen = 1.90, 95%CI:1.14–2.31), intergenerational sex (AORwomen = 1.36, 95%CI:1.08–1.79), and multiple/concurrent sex partners (AORmen = 1.42, 95%CI:1.10–1.93).

Interpretation

These findings support the critical evidence-based need for gender-transformative HIV prevention efforts including legislation of women''s rights in two of the most HIV affected countries in the world.     

Informing Comprehensive HIV Prevention: A Situational Analysis of the HIV Prevention and Care Context,North West Province South Africa

Objective

Building a successful combination prevention program requires understanding the community’s local epidemiological profile, the social community norms that shape vulnerability to HIV and access to care, and the available community resources. We carried out a situational analysis in order to shape a comprehensive HIV prevention program that address local barriers to care at multiple contextual levels in the North West Province of South Africa.

Method

The situational analysis was conducted in two sub-districts in 2012 and guided by an adaptation of WHO’s Strategic Approach, a predominantly qualitative method, including observation of service delivery points and in-depth interviews and focus groups with local leaders, providers, and community members, in order to recommend context-specific HIV prevention strategies. Analysis began during fieldwork with nightly discussions of findings and continued with coding original textual data from the fieldwork notebooks and a select number of recorded interviews.

Results

We conducted over 200 individual and group interviews and gleaned four principal social barriers to HIV prevention and care, including: HIV fatalism, traditional gender norms, HIV-related stigma, and challenges with communication around HIV, all of which fuel the HIV epidemic. At the different levels of response needed to stem the epidemic, we found evidence of national policies and programs that are mitigating the social risk factors but little community-based responses that address social risk factors to HIV.

Conclusions

Understanding social and structural barriers to care helped shape our comprehensive HIV prevention program, which address the four ‘themes’ identified into each component of the program. Activities are underway to engage communities, offer community-based testing in high transmission areas, community stigma reduction, and a positive health, dignity and prevention program for stigma reduction and improve communication skills. The situational analysis process successfully shaped key programmatic decisions and cultivated a deeper collaboration with local stakeholders to support program implementation.     

Global macroecology of bird assemblages in urbanized and semi‐natural ecosystems

Aim Despite the increasing pace of urbanization, little is known about how this process affects biodiversity globally. We investigate macroecological patterns of bird assemblages in urbanized areas relative to semi‐natural ecosystems. Location World‐wide. Methods We use a database of quantitative bird surveys to compare key assemblage structure parameters for plots in urbanized and semi‐natural ecosystems controlling for spatial autocorrelation and survey methodology. We use the term ‘urbanized’ instead of ‘urban’ ecosystems as many of the plots were not located in the centre of towns but in remnant habitat patches within conurbations. Results Some macroecological relationships were conserved in urbanized landscapes. Species–area, species–abundance and species–biomass relationships did not differ significantly between urbanized and non‐urbanized environments. However, there were differences in the relationships between productivity and assemblage structure. In forests, species richness increased with productivity; in both forests and open habitats, the evenness of species abundances declined as productivity increased. Among urbanized plots, instead, both species richness and the evenness of species abundances were independent of variation in productivity. Main conclusions Remnant habitats within urbanized areas are subject to many ecological alterations, yet key macroecological patterns differ remarkably little in urbanized versus non‐urbanized plots. Our results support the need for increased conservation activities in urbanized landscapes, particularly given the additional benefits of local experiences of biodiversity for the human population. With increasing urbanization world‐wide, broad‐scale efforts are needed to understand and manage the effects of this driver of change on biodiversity.