PKC epsilon is associated with myosin IIA and actin in fibroblasts

Proteins coimmunoprecipitating with protein kinase C (PKC) epsilon in fibroblasts were identified through matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF m/s). This method identified myosin IIA in PKC epsilon immunoprecipitates, as well as known PKC epsilon binding proteins, actin, beta'Cop and cytokeratin. Myosin is not a substrate for PKC epsilon. Immunofluorescence analysis showed that PKC epsilon is colocalised with actin and myosin in actomyosin stress fibers in fibroblasts. Inhibitors of PKC and myosin ATPase activity, as well as microfilament-disrupting drugs, all inhibited spreading of fibroblasts after passage, suggesting a role for a PKC epsilon-actin-myosin complex in cell spreading.     

Furin cleavage is not a requirement for Drosophila Notch function

Notch (N) is a large transmembrane protein that acts as a receptor in an evolutionarily conserved intercellular signalling pathway. Because of this conservation, it has been assumed that biochemical events mediating N function are identical in all species. For instance, intracellular maturation by furin protease and subunit assembly leading to the formation of a heterodimeric cell surface N receptor are thought to be central to its function in both mammals and flies. However, in Drosophila the majority of N appears to be full-length. It has not been determined whether this full-length N protein is on the cell surface. We describe experiments which indicate that unlike mammalian N, the majority of Drosophila N on the cell surface is full-length and that in Drosophila, in vivo, furin cleavage is not required for biological activity. We further show that the behaviour of fly and mouse N can be interchanged simply by swapping the regions in which the mammalian furin-like cleavage site is located.     

Effect of the environment on genotypic diversity of Actinomyces naeslundii and Streptococcus oralis in the oral biofilm

The genotypic diversity of Actinomyces naeslundii genospecies 2 (424 isolates) and Streptococcus oralis (446 isolates) strains isolated from two sound approximal sites in all subjects who were either caries active (seven subjects) or caries free (seven subjects) was investigated by using the repetitive extragenic palindromic PCR. The plaque from the caries-active subjects harbored significantly greater proportions of mutans streptococci and lactobacilli and a smaller proportion of A. naeslundii organisms than the plaque sampled from the caries-free subjects. These data confirmed that the sites of the two groups of subjects were subjected to different environmental stresses, probably determined by the prevailing or fluctuating acidic pH values. We tested the hypothesis that the microfloras of the sites subjected to greater stresses (the plaque samples from the caries-active subjects) would exhibit reduced genotypic diversity since the sites would be less favorable. We found that the diversity of A. naeslundii strains did not change (chi2 = 0.68; P = 0.41) although the proportional representation of A. naeslundii was significantly reduced (P < 0.05). Conversely, the diversity of the S. oralis strains increased (chi2 = 11.71; P = 0.0006) and the proportional representation of S. oralis did not change. We propose that under these environmental conditions the diversity and number of niches within the oral biofilm that could be exploited by S. oralis increased, resulting in the increased genotypic diversity of this species. Apparently, A. naeslundii was not able to exploit the new niches since the prevailing conditions within the niches may have been deleterious and not supportive of its proliferation. These results suggest that environmental stress may modify a biofilm such that the diversity of the niches is increased and that these niches may be successfully exploited by some, but not necessarily all, members of the microbial community.     

CFTR directly mediates nucleotide-regulated glutathione flux

Studies have shown that expression of cystic fibrosis transmembrane conductance regulator (CFTR) is associated with enhanced glutathione (GSH) efflux from airway epithelial cells, implicating a role for CFTR in the control of oxidative stress in the airways. To define the mechanism underlying CFTR-associated GSH flux, we studied wild-type and mutant CFTR proteins expressed in Sf9 membranes, as well as purified and reconstituted CFTR. We show that CFTR-expressing membrane vesicles mediate nucleotide-activated GSH flux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Further, we reveal that purified CFTR protein alone directly mediates nucleotide-dependent GSH flux. Interestingly, although ATP supports GSH flux through CFTR, this activity is enhanced in the presence of the non-hydrolyzable ATP analog AMP-PNP. These findings corroborate previous suggestions that CFTR pore properties can vary with the nature of the nucleotide interaction. In conclusion, our data demonstrate that GSH flux is an intrinsic function of CFTR and prompt future examination of the role of this function in airway biology in health and disease.     

ALFRED: an allele frequency database for diverse populations and DNA polymorphisms--an update

ALFRED (the ALelle FREquency Database) is designed to store and disseminate frequencies of alleles at human polymorphic sites for multiple populations, primarily for the population genetics and molecular anthropology communities. Currently ALFRED has information on over 180 polymorphic sites for more than 70 populations. Since our initial release of the database we have focussed on increasing the quantity and quality of data, making reciprocal links between ALFRED and other related databases, and providing useful tools to make the data more comprehensible to the end user. ALFRED is accessible from the Kidd Lab home page (http://info.med.yale. edu/genetics/kkidd/) or from ALFRED directly (http://alfred.med.yale. edu/alfred/index.asp).     

Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the roundabout receptor

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Little is known about the signaling mechanisms which function downstream of Robo to mediate repulsion. Here, we present genetic and biochemical evidence that the Abelson (Abl) tyrosine kinase and its substrate Enabled (Ena) play direct and opposing roles in Robo signal transduction. Genetic interactions support a model in which Abl functions to antagonize Robo signaling, while Ena is required in part for Robo's repulsive output. Both Abl and Ena can directly bind to Robo's cytoplasmic domain. A mutant form of Robo that interferes with Ena binding is partially impaired in Robo function, while a mutation in a conserved cytoplasmic tyrosine that can be phosphorylated by Abl generates a hyperactive Robo receptor.     

Schwann cell myelination requires timely and precise targeting of P(0) protein

This report investigated mechanisms responsible for failed Schwann cell myelination in mice that overexpress P(0) (P(0)(tg)), the major structural protein of PNS myelin. Quantitative ultrastructural immunocytochemistry established that P(0) protein was mistargeted to abaxonal, periaxonal, and mesaxon membranes in P(0)(tg) Schwann cells with arrested myelination. The extracellular leaflets of P(0)-containing mesaxon membranes were closely apposed with periodicities of compact myelin. The myelin-associated glycoprotein was appropriately sorted in the Golgi apparatus and targeted to periaxonal membranes. In adult mice, occasional Schwann cells myelinated axons possibly with the aid of endocytic removal of mistargeted P(0). These results indicate that P(0) gene multiplication causes P(0) mistargeting to mesaxon membranes, and through obligate P(0) homophilic adhesion, renders these dynamic membranes inert and halts myelination.     

Spectrometric studies on stability of tenuazonic acid (TeA) solution in organic solvents

The stability of tenuazonic acid solution at different temperatures and storage times was studied using methanol, methanol-water (8:2 v/v), benzene and benzene-acetonitrile (98:2 v/v) as solvents. Solutions were analysed by a spectrometric method TeA U.V.-spectrum was recorded. Results indicated that the optimum temperature for long-time storage period of tenuazonic acid solution in any solvent assayed is -20°C. Benzene and benzene-acetonitrile (98:2 v/v) could be advised to make tenuazonic acid solution which will be stored less than 2 months at 4°C. Methanol and methanolwater (8:2 v/v) are not recommended because a low stability of TeA solution in this solvents.     

Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

Background  

The involvement of placental lactogen (PL) in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL) concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG) via a foetal catheter (in vivo study). The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions.     

lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

Background

The spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually override mitotic arrest by a process known as mitotic slippage or adaptation. This is a problem for cancer chemotherapy using microtubule poisons.

Results

Here we describe mitotic slippage in yeast bub2?? mutant cells that are defective in the repression of precocious telophase onset (mitotic exit). Precocious activation of anaphase promoting complex/cyclosome (APC/C)-Cdh1 caused mitotic slippage in the presence of nocodazole, while the SAC was still active. APC/C-Cdh1, but not APC/C-Cdc20, triggered anaphase progression (securin degradation, separase-mediated cohesin cleavage, sister-chromatid separation and chromosome missegregation), in addition to telophase onset (mitotic exit), during mitotic slippage. This demonstrates that an inhibitory system not only of APC/C-Cdc20 but also of APC/C-Cdh1 is critical for accurate chromosome segregation in the presence of insufficient kinetochore-microtubule attachments.

Conclusions

The sequential activation of APC/C-Cdc20 to APC/C-Cdh1 during mitosis is central to accurate mitosis. Precocious activation of APC/C-Cdh1 in metaphase (pre-anaphase) causes mitotic slippage in SAC-activated cells. For the prevention of mitotic slippage, concomitant inhibition of APC/C-Cdh1 may be effective for tumor therapy with mitotic spindle poisons in humans.