Structural and Biochemical Basis for the Inhibitory Effect of Liprin-α3 on Mouse Diaphanous 1 (mDia1) Function

Diaphanous-related formins are eukaryotic actin nucleation factors regulated by an autoinhibitory interaction between the N-terminal RhoGTPase-binding domain (mDia_N) and the C-terminal Diaphanous-autoregulatory domain (DAD). Although the activation of formins by Rho proteins is well characterized, its inactivation is only marginally understood. Recently, liprin-α3 was shown to interact with mDia1. Overexpression of liprin-α3 resulted in a reduction of the cellular actin filament content. The molecular mechanisms of how liprin-α3 exerts this effect and counteracts mDia1 activation by RhoA are unknown. Here, we functionally and structurally define a minimal liprin-α3 core region, sufficient to recapitulate the liprin-α3 determined mDia1-respective cellular functions. We show that liprin-α3 alters the interaction kinetics and thermodynamics of mDia_N with RhoA·GTP and DAD. RhoA displaces liprin-α3 allosterically, whereas DAD competes with liprin-α3 for a highly overlapping binding site on mDia_N. Liprin-α3 regulates actin polymerization by lowering the regulatory potency of RhoA and DAD on mDia_N. We present a model of a mechanistically unexplored and new aspect of mDia_N regulation by liprin-α3.     

Kinesin-5 is not essential for mitotic spindle elongation in Dictyostelium

The proper assembly and operation of the mitotic spindle is essential to ensure the accurate segregation of chromosomes and to position the cytokinetic furrow during cell division in eukaryotes. Not only are dynamic microtubules required but also the concerted actions of multiple motor proteins are necessary to effect spindle pole separation, chromosome alignment, chromatid segregation, and spindle elongation. Although a number of motor proteins are known to play a role in mitosis, there remains a limited understanding of their full range of functions and the details by which they interact with other spindle components. The kinesin-5 (BimC/Eg5) family of motors is largely considered essential to drive spindle pole separation during the initial and latter stages of mitosis. We have deleted the gene encoding the kinesin-5 member in Dictyostelium, (kif13), and find that, in sharp contrast with results found in vertebrate, fly, and yeast organisms, kif13(-) cells continue to grow at rates indistinguishable from wild type. Phenotype analysis reveals a slight increase in spindle elongation rates in the absence of Kif13. More importantly, there is a dramatic, premature separation of spindle halves in kif13(-) cells, suggesting a novel role of this motor in maintaining spindle integrity at the terminal stages of division.     

Exploring the Prevalence of Disrespect and Abuse during Childbirth in Kenya

Background

Poor quality of care including fear of disrespect and abuse (D&A) perpetuated by health workers influences women’s decisions to seek maternity care. Key manifestations of D&A include: physical abuse, non-consented care, non-confidential care, non-dignified care, discrimination, abandonment, and detention in facilities. This paper describes manifestations of D&A experienced in Kenya and measures their prevalence.

Methods

This paper is based on baseline data collected during a before-and-after study designed to measure the effect of a package of interventions to reduce the prevalence of D&A experienced by women during labor and delivery in thirteen Kenyan health facilities. Data were collected through an exit survey of 641 women discharged from postnatal wards. We present percentages of D&A manifestations and odds ratios of its relationship with demographic characteristics using a multivariate fixed effects logistic regression model.

Results

Twenty percent of women reported any form of D&A. Manifestations of D&A includes: non-confidential care (8.5%), non-dignified care (18%), neglect or abandonment (14.3%), Non-consensual care (4.3%) physical abuse (4.2%) and, detainment for non-payment of fees (8.1). Women aged 20-29 years were less likely to experience non-confidential care compared to those under 19; OR: [0.6 95% CI (0.36, 0.90); p=0.017]. Clients with no companion during delivery were less likely to experience inappropriate demands for payment; OR: [0.49 (0.26, 0.95); p=0.037]; while women with higher parities were three times more likely to be detained for lack of payment and five times more likely to be bribed compared to those experiencing there first birth.

Conclusion

One out of five women experienced feeling humiliated during labor and delivery. Six categories of D&A during childbirth in Kenya were reported. Understanding the prevalence of D&A is critical in developing interventions at national, health facility and community levels to address the factors and drivers that influence D&A in facilities and to encourage clients’ future facility utilization.     

Separating Instability from Aggregation Propensity in γS-Crystallin Variants

Molecular dynamics (MD) simulations, circular dichroism (CD), and dynamic light scattering (DLS) measurements were used to investigate the aggregation propensity of the eye-lens protein γS-crystallin. The wild-type protein was investigated along with the cataract-related G18V variant and the symmetry-related G106V variant. The MD simulations suggest that local sequence differences result in dramatic differences in dynamics and hydration between these two apparently similar point mutations. This finding is supported by the experimental measurements, which show that although both variants appear to be mostly folded at room temperature, both display increased aggregation propensity. Although the disease-related G18V variant is not the most strongly destabilized, it aggregates more readily than either the wild-type or the G106V variant. These results indicate that γS-crystallin provides an excellent model system for investigating the role of dynamics and hydration in aggregation by locally unfolded proteins.     

MRE11/RAD50/NBS1: complex activities

The MRE11/RAD50/NBS1 complex (Mre11 complex) is a central player in most aspects of the cellular response to DNA double-strand breaks, including homologous recombination, non-homologous end joining, telomere maintenance and DNA damage checkpoint activation. Several of these findings are explained by the unusual enzymatic activities and macromolecular structure of the Mre11 complex. The Mre11 complex possesses an ATP-stimulated nuclease to process heterogeneous DNA ends and long coiled-coil tails to link DNA ends and/or sister chromatids. However, the mechanistic role of the Mre11 complex in checkpoint activation has been unclear until recently. New data suggest that the Mre11 complex can directly activate the ATM checkpoint kinase at DNA breaks. These findings, together with newly determined functional interactions, identify the Mre11 complex as an architectural and mechanistic keystone of cellular response events emerging from DNA breaks.     

Terminal deoxynucleotidyltransferase deficiency decreases autoimmune disease in diabetes-prone nonobese diabetic mice and lupus-prone MRL-Fas(lpr) mice

The wide diversity of the T and B Ag receptor repertoires becomes even more extensive postneonatally due to the activity of TdT, which adds nontemplated N nucleotides to Ig and TCR coding ends during V(D)J recombination. In addition, complementarity-determining region 3 sequences formed in the absence of TdT are more uniform due to the use of short sequence homologies between the V, D, and J genes. Thus, the action of TdT produces an adult repertoire that is both different from, and much larger than, the repertoire of the neonate. We have generated TdT-deficient nonobese diabetic (NOD) and MRL-Fas(lpr) mice, and observed a decrease in the incidence of autoimmune disease, including absence of diabetes and decreased pancreatic infiltration in NOD TdT(-/-) mice, and reduced glomerulonephritis and increased life span in MRL-Fas(lpr) TdT(-/-) mice. Using tetramer staining, TdT(-/-) and TdT(+/+) NOD mice showed similar frequencies of the diabetogenic BDC 2.5 CD4(+) T cells. We found no increase in CD4(+)CD25(+) regulatory T cells in NOD TdT(-/-) mice. Thus, TdT deficiency ameliorates the severity of disease in both lupus and diabetes, two very disparate autoimmune diseases that affect different organs, with damage conducted by different effector cell types. The neonatal repertoire appears to be deficient in autoreactive T and/or B cells with high enough affinities to induce end-stage disease. We suggest that the paucity of autoreactive specificities created in the N region-lacking repertoire, and the resultant protection afforded to the newborn, may be the reason that TdT expression is delayed in ontogeny.     

Peroxisomal localization of sulfite oxidase separates it from chloroplast-based sulfur assimilation

Recently, we isolated the sulfite oxidase (SO) gene from Arabidopsis thaliana and characterized the purified SO protein. The purpose of the present study was to determine the subcellular localization of this novel plant enzyme. Immunogold electron-microscopic analysis showed the gold labels nearly exclusively in the peroxisomes. To verify this finding, green fluorescent protein was fused to full-length plant SO including the putative peroxisomal targeting signal 1 (PTS1) 'SNL' and expressed in tobacco leaves. Our results showed a punctate fluorescence pattern resembling that of peroxisomes. Co-labelling with MitoTracker-Red excluded that the observed fluorescence was due to mitochondrial sorting. By investigation of deleted or mutated PTS1, no functional peroxisomal targeting signal 2 (PTS2) could be detected in plant SO. This conclusion is supported by expression studies in Pichia pastoris mutants with defined defects either in PTS1- or PTS2-mediated peroxisomal import.     

Amaranth Globulin Polypeptide Heterogeneity

The polypeptides integrating amaranth globulin-p and 11S-globulin were characterized by two-dimensional electrophoresis, ion-exchange chromatography and RP-HPLC. All polypeptides exhibited charge and hydrophobic heterogeneity. Almost all acid (A, pI 5–7) and basic (B, pI 9–10) polypeptides were present in both globulins, and the same happened with the unprocessed M polypeptides with pI in the range of 7–7.5 which fits well with a sequence containing both the A and B polypeptides. There were other polypeptides only present in 11S-globulin, like some of 41 and 16 kDa, which might come from another precursor or be the products of a different processing of the propolypeptide. These results suggested that, although amaranth subunits from different subfamilies are interchangeable in different oligomers, some structural differences between them might affect the assembly of globulin molecules. Structural differences arising from this behavior could account for the different physicochemical properties of globulin molecules.     

UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 as a new immunohistochemical breast cancer marker.

Mucin O-glycosylation is characterized in cancer by aberrant expression of immature carbohydrate structures (Tn, T, and sialyl-Tn antigens). The UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-T) family enzymes regulate the initial steps of mucin O-glycosylation and could be responsible for the altered glycosylation observed in cancer. Considering that we recently found the ppGalNAc-T6 mRNA expressed in breast carcinomas, we produced a highly specific monoclonal antibody (MAb T6.3) to assess the expression profile of ppGalNAc-T6 protein product in breast tissues. The expression of ppGalNAc-T6 by breast carcinoma cells was confirmed on MCF-7 and T47D cell lines. In formalin-fixed tissues, ppGalNAc-T6 expression was observed in 60/74 (81%) breast cancers, 21/23 (91.3%) adjacent ductal carcinoma in situ (DCIS), 4/20 benign breast lesions (2/2 sclerosing adenosis and 2/13 fibroadenoma), and in 0/5 normal breast samples. We observed a statistically significant association of ppGalNAc-T6 expression with T1 tumor stage. This fact, as well as the observation that ppGalNAc-T6 was strongly expressed in sclerosing adenosis and in most DCIS, suggests that ppGalNAc-T6 expression could be an early event during human breast carcinogenesis. Considering that an abnormal O-glycosylation greatly contributes to the phenotype and biology of breast cancer cells, ppGalNAc-T6 expression could provide new insights about breast cancer glycobiology.