Submaximal inhibition of protein kinase C restores ADP-induced dense granule secretion in platelets in the presence of Ca2+

Protein kinase C (PKC) is a family of serine/threonine kinases that play isoform-specific inhibitory and stimulatory roles in platelet activation. We show here that the pan-PKC inhibitor Ro31-8220 can be used to dissect these events following platelet activation by ADP. Submaximal concentrations of Ro31-8220 potentiated aggregation and dense granule secretion to ADP in plasma anticoagulated with citrate, in D-Phe-Pro-Arg-chloromethyl ketone-anticoagulated plasma, which has physiological levels of Ca(2+), and in washed platelets. Potentiation was retained on inhibition of cyclooxygenase and was associated with an increase in intracellular Ca(2+). Potentiation of aggregation and secretion was abolished by a maximally effective concentration of Ro31-8220, consistent with a critical role of PKC in secretion. ADP-induced secretion was potentiated in the presence of an inhibitor of PKCβ but not in the presence of available inhibitors of other PKC isoforms in human and mouse platelets. ADP-induced secretion was also potentiated in mouse platelets deficient in PKCε but not PKC. These results demonstrate that partial blockade of PKC potentiates aggregation and dense granule secretion by ADP in association with increased Ca(2+). This provides a molecular explanation for the inability of ADP to induce secretion in plasma in the presence of physiological Ca(2+) concentrations, and it reveals a novel role for PKC in inhibiting platelet activation by ADP in vivo. These results also demonstrate isoform-specific inhibitory effects of PKC in platelets.     

Poly(carboxybetaine methacrylamide)-modified nanoparticles: a model system for studying the effect of chain chemistry on film properties, adsorbed protein conformation, and clot formation kinetics

Nonfouling polymer architectures are considered important to the successful implementation of many biomaterials. It is thought that how these polymers induce conformational changes in proteins upon adsorption may dictate the fate of the device being utilized. Herein, oxidized silicon nanoparticles (SiNP) were modified with various forms of poly(carboxybetaine methacrylamide) (PCBMA) for the express purpose of understanding how polymer chemistry affects film hydration, adsorbed protein conformation, and clot formation kinetics. To this end, carboxybetaine monomers differing in intercharge separating spacer groups were synthesized, and nitroxide-mediated free radical polymerization (NMP) was conducted using alkoxyamine initiators with hydrophobic (TEMPO) and hydrophilic (β-phosphonate) terminal groups. The physical properties (surface composition, thickness, grafting density, etc.) of the resulting polymer-SiNP conjugates were quantified using several techniques, including Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and thermogravimetric analysis (TGA). The effect of spacer group on the surface charge density was determined using zeta potential measurements. Three proteins, viz., lysozyme, bovine α-lactalbumin, and human serum albumin, were used to evaluate the effect film properties (charge, hydration, end-group) have on adsorbed protein conformation, as determined by circular dichroism (CD), fluorescence spectroscopy, and fluorescence quenching techniques. Hemocompatibility of these surfaces was observed by measuring clot formation kinetics using the plasma recalcification time assay. It was found that chain chemistry, as opposed to end-group chemistry, was a major determiner for water structure, adsorbed protein conformation, and clotting kinetics. It is thought that the systematic evaluation of how both chain (internal) and end-group (external) polymer properties affect film hydration, protein conformation, and clot formation will provide valuable insight that can be applied to all engineered surfaces for biomedical applications.     

Localization of the Fanconi anemia complementation group D gene to a 200-kb region on chromosome 3p25.3

Fanconi anemia (FA) is a rare autosomal recessive disease manifested by bone-marrow failure and an elevated incidence of cancer. Cells taken from patients exhibit spontaneous chromosomal breaks and rearrangements. These breaks and rearrangements are greatly elevated by treatment of FA cells with the use of DNA cross-linking agents. The FA complementation group D gene (FANCD) has previously been localized to chromosome 3p22-26, by use of microcell-mediated chromosome transfer. Here we describe the use of noncomplemented microcell hybrids to identify small overlapping deletions that narrow the FANCD critical region. A 1.2-Mb bacterial-artificial-chromosome (BAC)/P1 contig was constructed, bounded by the marker D3S3691 distally and by the gene ATP2B2 proximally. The contig contains at least 36 genes, including the oxytocin receptor (OXTR), hOGG1, the von Hippel-Lindau tumor-suppressor gene (VHL), and IRAK-2. Both hOGG1 and IRAK-2 were excluded as candidates for FANCD. BACs were then used as probes for FISH analyses, to map the extent of the deletions in four of the noncomplemented microcell hybrid cell lines. A narrow region of common overlapping deletions limits the FANCD critical region to approximately 200 kb. The three candidate genes in this region are TIGR-A004X28, SGC34603, and AA609512.     

Salmonella maintains the integrity of its intracellular vacuole through the action of SifA

A method based on the Competitive Index was used to identify Salmonella typhimurium virulence gene interactions during systemic infections of mice. Analysis of mixed infections involving single and double mutant strains showed that OmpR, the type III secretion system of Salmonella pathogenicity island 2 (SPI-2) and SifA [required for the formation in epithelial cells of lysosomal glycoprotein (lgp)-containing structures, termed Sifs] are all involved in the same virulence function. sifA gene expression was induced after Salmonella entry into host cells and was dependent on the SPI-2 regulator ssrA. A sifA(-) mutant strain had a replication defect in macrophages, similar to that of SPI-2 and ompR(-) mutant strains. Whereas wild-type and SPI-2 mutant strains reside in vacuoles that progressively acquire lgps and the vacuolar ATPase, the majority of sifA(-) bacteria lost their vacuolar membrane and were released into the host cell cytosol. We propose that the wild-type strain, through the action of SPI-2 effectors (including SpiC), diverts the Salmonella-containing vacuole from the endocytic pathway, and subsequent recruitment and maintenance of vacuolar ATPase/lgp-containing membranes that enclose replicating bacteria is mediated by translocation of SifA.     

Does Pet Ownership in Infancy Lead to Asthma or Allergy at School Age? Pooled Analysis of Individual Participant Data from 11 European Birth Cohorts

Objective

To examine the associations between pet keeping in early childhood and asthma and allergies in children aged 6–10 years.

Design

Pooled analysis of individual participant data of 11 prospective European birth cohorts that recruited a total of over 22,000 children in the 1990s.

Exposure definition

Ownership of only cats, dogs, birds, rodents, or cats/dogs combined during the first 2 years of life.

Outcome definition

Current asthma (primary outcome), allergic asthma, allergic rhinitis and allergic sensitization during 6–10 years of age.

Data synthesis

Three-step approach: (i) Common definition of outcome and exposure variables across cohorts; (ii) calculation of adjusted effect estimates for each cohort; (iii) pooling of effect estimates by using random effects meta-analysis models.

Results

We found no association between furry and feathered pet keeping early in life and asthma in school age. For example, the odds ratio for asthma comparing cat ownership with “no pets” (10 studies, 11489 participants) was 1.00 (95% confidence interval 0.78 to 1.28) (I² = 9%; p = 0.36). The odds ratio for asthma comparing dog ownership with “no pets” (9 studies, 11433 participants) was 0.77 (0.58 to 1.03) (I² = 0%, p = 0.89). Owning both cat(s) and dog(s) compared to “no pets” resulted in an odds ratio of 1.04 (0.59 to 1.84) (I² = 33%, p = 0.18). Similarly, for allergic asthma and for allergic rhinitis we did not find associations regarding any type of pet ownership early in life. However, we found some evidence for an association between ownership of furry pets during the first 2 years of life and reduced likelihood of becoming sensitized to aero-allergens.

Conclusions

Pet ownership in early life did not appear to either increase or reduce the risk of asthma or allergic rhinitis symptoms in children aged 6–10. Advice from health care practitioners to avoid or to specifically acquire pets for primary prevention of asthma or allergic rhinitis in children should not be given.     

Teratogenic evaluation of terpenoid derivatives

Eight terpenoid derivatives with juvenile hormone activity were tested for teratogenicity. Three of the derivatives were teratogenic when administered to mice as a single intraperitoneal injection of 1 mg/gm on either day 9 or day 10 of pregnancy. However, intubation at 5 mg/gm of the two most teratogenic derivatives resulted in one of the derivatives marginally increasing the incidence of abnormal embryos above peanut oil-intubated controls; the other intubated derivative was without adverse affect. Of the remaining five compounds, two with high juvenile hormone activity were without teratogenic effect when injected at 1 mg/gm. Since teratogenic effects were observed only at a dose above likely exposure levels and by a route of administration not possible in the environment, support is provided for the potential use of terpenoid derivatives in the control of insect pests.     

Effects of N-cadherin overexpression on the adhesion properties of embryonic stem cells

Constitutive overexpression of N-cadherin in mouse embryonic stem cells led to marked changes in the phenotype and adhesion properties of these cells. The changes included the formation of smaller embryonic bodies, elevated mRNA and total protein levels of N-cadherin, and increased amounts of p120 catenin and connexin-43. N-cadherin cells exhibited decreased attachment to non-cell surfaces, while their adhesiveness to each other and to rat neonatal cardiomyocytes was significantly elevated. The findings suggest that N-cadherin overexpression can facilitate electromechanical integration of stem cells into excitable tissues with endogenously high levels of N-cadherin, such as the heart and brain.Key words: stem cells, cardiomyocytes, N-cadherin, connexin 43, gap junctions