Phosphorylation of the MAPKKK regulator Ste50p in Saccharomyces cerevisiae: a casein kinase I phosphorylation site is required for proper mating function

The Ste50 protein of Saccharomyces cerevisiae is a regulator of the Ste11p protein kinase. Ste11p is a member of the MAP3K (or MEKK) family, which is conserved from yeast to mammals. Ste50p is involved in all the signaling pathways that require Ste11p function, yet little is known about the regulation of Ste50p itself. Here, we show that Ste50p is phosphorylated on multiple serine/threonine residues in vivo. Threonine 42 (T42) is phosphorylated both in vivo and in vitro, and the protein kinase responsible has been identified as casein kinase I. Replacement of T42 with alanine (T42A) compromises Ste50p function. This mutation abolishes the ability of overexpressed Ste50p to suppress either the mating defect of a ste20 ste50 deletion mutant or the mating defect of a strain with a Ste11p deleted from its sterile-alpha motif domain. Replacement of T42 with a phosphorylation-mimetic aspartic acid residue (T42D) permits wild-type function in all assays of Ste50p function. These results suggest that phosphorylation of T42 of Ste50p is required for proper signaling in the mating response. However, this phosphorylation does not seem to have a detectable role in modulating the high-osmolarity glycerol synthesis pathway.     

Glucose Decouples Intracellular Ca2+ Activity from Glucagon Secretion in Mouse Pancreatic Islet Alpha-Cells

The mechanisms of glucagon secretion and its suppression by glucose are presently unknown. This study investigates the relationship between intracellular calcium levels ([Ca²⁺]_i) and hormone secretion under low and high glucose conditions. We examined the effects of modulating ion channel activities on [Ca²⁺]_i and hormone secretion from ex vivo mouse pancreatic islets. Glucagon-secreting α-cells were unambiguously identified by cell specific expression of fluorescent proteins. We found that activation of L-type voltage-gated calcium channels is critical for α-cell calcium oscillations and glucagon secretion at low glucose levels. Calcium channel activation depends on K_ATP channel activity but not on tetrodotoxin-sensitive Na⁺ channels. The use of glucagon secretagogues reveals a positive correlation between α-cell [Ca²⁺]_i and secretion at low glucose levels. Glucose elevation suppresses glucagon secretion even after treatment with secretagogues. Importantly, this inhibition is not mediated by K_ATP channel activity or reduction in α-cell [Ca²⁺]_i. Our results demonstrate that glucose uncouples the positive relationship between [Ca²⁺]_i and secretory activity. We conclude that glucose suppression of glucagon secretion is not mediated by inactivation of calcium channels, but instead, it requires a calcium-independent inhibitory pathway.     

Suppression of arbuscular mycorrhizal colonization and nodulation in split-root systems of alfalfa after pre-inoculation and treatment with Nod factors

Roots of legumes establish symbiosis with arbuscular mycorrhizal fungi (AMF) and nodule-inducing rhizobia. The existing nodules systemically suppress subsequent nodule formation in other parts of the root, a phenomenon termed autoregulation. Similarly, mycorrhizal roots reduce further AMF colonization on other parts of the root system. In this work, split- root systems of alfalfa (Medicago sativa) were used to study the autoregulation of symbiosis with Sinorhizobium meliloti and the mycorrhizal fungus Glomus mosseae. It is shown that nodulation systemically influences AMF root colonization and vice versa. Nodules on one half of the split-root system suppressed subsequent AMF colonization on the other half. Conversely, root systems pre-colonized on one side by AMF exhibited reduced nodule formation on the other side. An inhibition effect was also observed with Nod factors (lipo-chito-oligosaccharides). NodSm-IV(C16:2, S) purified from S. meliloti systemically suppressed both nodule formation and AMF colonization. The application of Nod factors, however, did not influence the allocation of (14)C within the split-root system, excluding competition for carbohydrates as the regulatory mechanism. These results indicate a systemic regulatory mechanism in the rhizobial and the arbuscular mycorrhizal association, which is similar in both symbioses.     

SIMS microscopy in the biomedical field.

We attempted to indicate the requirements for biomedical applications of SIMS microscopy. Sample preparation methodology should preserve both the structural and the chemical integrity of the tissue. Furthermore, it is often necessary to correlate ionic and light microscope images. This implies a common methodological approach to sample preparation for both microscopes. The use of low or high mass resolution depends on the elements studied and their concentrations. To improve the acquisition and processing of images, digital imaging systems have to be designed and require both ionic and optical image superimposition. However, the images do not accurately reflect element concentration; a relative quantitative approach is possible by measuring secondary ion beam intensity. Using an internal reference element (carbon) and standard curves the results are expressed in micrograms/mg of tissue. Despite their limited lateral resolution (0.5 microns) the actual SIMS microscopes are very suitable for the resolution of biomedical problems posed by action modes and drug localization in human pathology. SIMS microscopy should provide a new tool for metabolic radiotherapy by facilitating dose evaluation. The advent of high lateral resolution SIMS imaging (less than 0.1 microns) should open up new fields in biomedical investigation.     

Sulfur dioxide inhibits the sucrose carrier of the plant plasma membrane.

Plasma membrane vesicles were prepared by phase partition from a microsomal fraction of broad bean (Vicia faba L.) leaf. In order to study the effects of sodium sulfite on active uptake of sucrose, the vesicles were artificially energized by a transmembrane pH gradient (delta pH) and/or a transmembrane electrical gradient (delta psi). At 1 mM, sulfite strongly inhibited sucrose uptake but did not affect the two components of the proton motive force, delta pH (measured by dimethyloxazolidine dione) and delta psi (measured by tetraphenylphosphonium). Moreover, sulfite did not inhibit the proton-pumping ATPase of the plasma membrane vesicles. These data demonstrate that sulfite may inhibit transport of photoassimilates in plant by a direct inhibition of the sucrose carrier of the plasma membrane.     

Compartment-Specific Biosensors Reveal a Complementary Subcellular Distribution of Bioactive Furin and PC7

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Evolution of putative barrier loci at an intermediate stage of speciation with gene flow in campions (Silene)

Understanding the origin of new species is a central goal in evolutionary biology. Diverging lineages often evolve highly heterogeneous patterns of genetic differentiation; however, the underlying mechanisms are not well understood. We investigated evolutionary processes governing genetic differentiation between the hybridizing campions Silene dioica (L.) Clairv. and S. latifolia Poiret. Demographic modelling indicated that the two species diverged with gene flow. The best‐supported scenario with heterogeneity in both migration rate and effective population size suggested that a small proportion of the loci evolved without gene flow. Differentiation (F _ST) and sequence divergence (d _XY) were correlated and both tended to peak in the middle of most linkage groups, consistent with reduced gene flow at highly differentiated loci. Highly differentiated loci further exhibited signatures of selection. In between‐species population pairs, isolation by distance was stronger for genomic regions with low between‐species differentiation than for highly differentiated regions that may contain barrier loci. Moreover, differentiation landscapes within and between species were only weakly correlated, suggesting that linked selection due to shared recombination and gene density landscapes is not the dominant determinant of genetic differentiation in these lineages. Instead, our results suggest that divergent selection shaped the genomic landscape of differentiation between the two Silene species, consistent with predictions for speciation in the face of gene flow.     

Congenital Dislocation of the Hip: Prognostic Implications of Early Diagnosis

A retrospective analysis of 62 patients with 78 congenital dislocations of the hip (CDH) referred to Children''s Hospital at Stanford was done to ascertain whether CDH was being diagnosed as early as had been thought possible and whether various treatments were effective. In this series there were a substantial number of cases of CDH that were not diagnosed within the first 48 hours of life. At Children''s Hospital the Pavlik harness was the most effective form of treatment of congenital dislocations of the hip in the perinatal period.     

Study protocol of the YOU CALL - WE CALL TRIAL: impact of a multimodal support intervention after a "mild" stroke

Background

More than 60% of new strokes each year are "mild" in severity and this proportion is expected to rise in the years to come. Within our current health care system those with "mild" stroke are typically discharged home within days, without further referral to health or rehabilitation services other than advice to see their family physician. Those with mild stroke often have limited access to support from health professionals with stroke-specific knowledge who would typically provide critical information on topics such as secondary stroke prevention, community reintegration, medication counselling and problem solving with regard to specific concerns that arise. Isolation and lack of knowledge may lead to a worsening of health problems including stroke recurrence and unnecessary and costly health care utilization. The purpose of this study is to assess the effectiveness, for individuals who experience a first "mild" stroke, of a sustainable, low cost, multimodal support intervention (comprising information, education and telephone support) - "WE CALL" compared to a passive intervention (providing the name and phone number of a resource person available if they feel the need to) - "YOU CALL", on two primary outcomes: unplanned-use of health services for negative events and quality of life.

Method/Design

We will recruit 384 adults who meet inclusion criteria for a first mild stroke across six Canadian sites. Baseline measures will be taken within the first month after stroke onset. Participants will be stratified according to comorbidity level and randomised to one of two groups: YOU CALL or WE CALL. Both interventions will be offered over a six months period. Primary outcomes include unplanned use of heath services for negative event (frequency calendar) and quality of life (EQ-5D and Quality of Life Index). Secondary outcomes include participation level (LIFE-H), depression (Beck Depression Inventory II) and use of health services for health promotion or prevention (frequency calendar). Blind assessors will gather data at mid-intervention, end of intervention and one year follow up.

Discussion

If effective, this multimodal intervention could be delivered in both urban and rural environments. For example, existing infrastructure such as regional stroke centers and existing secondary stroke prevention clinics, make this intervention, if effective, deliverable and sustainable.

Trial Registration

ISRCTN95662526