1alpha,25(OH)2D3 regulates chondrocyte matrix vesicle protein kinase C (PKC) directly via G-protein-dependent mechanisms and indirectly via incorporation of PKC during matrix vesicle biogenesis.

Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1alpha,25(OH)(2)D(3). Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1alpha,25(OH)(2)D(3) in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1alpha,25(OH)(2)D(3) during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1alpha,25(OH)(2)D(3), PKCzeta in matrix vesicles was inhibited, while PKCalpha in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCzeta in matrix vesicles was increased, while PKCalpha in plasma membranes was unchanged. The effect of 1alpha,25(OH)(2)D(3) was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1alpha,25(OH)(2)D(3) membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1alpha,25(OH)(2)D(3)-dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1alpha,25(OH)(2)D(3) in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or G(i)/G(s) had no effect. However, inhibition of G(q) blocked the effect of 1alpha,25(OH)(2)D(3). The rapid effect of 1alpha,25(OH)(2)D(3) also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1alpha,25(OH)(2)D(3) at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid.     

Association of Bcr-Abl with the proto-oncogene Vav is implicated in activation of the Rac-1 pathway.

Vav is a guanine nucleotide exchange factor for the Rho/Rac family predominantly expressed in hematopoietic cells and implicated in cell proliferation and cytoskeletal organization. The oncogenic tyrosine kinase Bcr-Abl has been shown to activate Rac-1, which is important for Bcr-Abl induced leukemogenesis. Previous studies by Matsuguchi et al. (Matsuguchi, T., Inhorn, R. C., Carlesso, N., Xu, G., Druker, B., and Griffin, J. D. (1995) EMBO J. 14, 257-265) describe enhanced phosphorylation of Vav in Bcr-Abl-expressing Mo7e cells yet fail to demonstrate association of the two proteins. Here, we report the identification of a direct complex between Vav and Bcr-Abl in yeast, in vitro and in vivo. Furthermore, we show tyrosine phosphorylation of Vav by Bcr-Abl. Mutational analysis revealed that the SH2 domain and the C-terminal SH3 domain as well as a tetraproline motif directly adjacent to the N-terminal SH3 domain of Vav are important for establishing this phosphotyrosine dependent interaction. Activation of Rac-1 by Bcr-Abl was abrogated by co-expression of the Vav C terminus encoding the SH3-SH2-SH3 domains as a dominant negative construct. Bcr-Abl transduced primary bone marrow from Vav knock-out mice showed reduced proliferation in a culture cell transformation assay compared with wild-type bone marrow. These results suggest, that Bcr-Abl utilizes Vav as a guanine nucleotide exchange factor to activate Rac-1 in a process that involves a folding mechanism of the Vav C terminus. Given the importance of Rac-1 activation for Bcr-Abl-mediated leukemogenesis, this mechanism may be crucial for the molecular pathogenesis of chronic myeloid leukemia and of importance for other signal transduction pathways leading to the activation of Rac-1.     

suREJ3, a polycystin-1 protein, is cleaved at the GPS domain and localizes to the acrosomal region of sea urchin sperm.

The sea urchin sperm acrosome reaction (AR) is a prerequisite for sperm-egg fusion. This report identifies sea urchin sperm receptor for egg jelly-3 (suREJ3) as a new member of the polycystin-1 family (the protein mutated in autosomal dominant polycystic kidney disease). suREJ3 is a multidomain, 2,681-amino acid, heavily glycosylated orphan receptor with 11 putative transmembrane segments (TMS) that localize to the plasma membrane covering the sperm acrosomal vesicle. Like the latrophilins and other members of the secretin family of G-protein-coupled receptors, suREJ3 is cleaved at the consensus GPS (G-protein-coupled receptor proteolytic site) domain. Antibodies to the extracellular 1,455-residue NH(2)-terminal portion identify a band at 250 kDa that shifts in electrophoretic mobility to 180 kDa upon glycosidase digestion. Antibodies to the 1,226-residue COOH-terminal portion identify a band at 150 kDa that shifts to 140 kDa after glycosidase treatment. Antibodies to both portions of suREJ3 localize exclusively to the plasma membrane over the acrosomal vesicle. Immunoprecipitation shows that both portions of suREJ3 are associated in detergent extracts. This is the first report showing that a polycystin family member is cleaved at the GPS domain. Localization of suREJ3 to the acrosomal region provides the first suggestion for the role of a polycystin-1 protein (components of nonselective cation channels) in a specific cellular process.     

The influence of coastal upwelling on the distribution of Calanus chilensis in the Mejillones Peninsula (northern Chile): implications for its population dynamics

A field experiment was carried out in October 1998 during active upwelling in a coastal area off the Mejillones Peninsula (23° S). Zooplankton was sampled at day and night, during two subsequent days at 4 stations inside and outside of the upwelling plume. Three depth strata were sampled: 0–20 m, 20–80 m and 80–200 m. Oceanographic data were obtained in a grid of 23 stations using a CTDO, a fluorometer and a Doppler current meter. Calanus chilensis was mostly represented by late stages, i.e. copepodid C5 and adult males and females. There were no day/night effects on vertical distribution, and abundance was significantly higher inside the upwelling plume in the upper 20-m layer at nearly 14 ind. m^–3, compared to ca. 5 ind. m^–3 outside the upwelling plume. Temperature at 10 m depth and biomass, estimated from stage numbers and their mean dry weights, were used to estimate growth and daily production of Calanus at temperature-dependent rates. The potential loss of biomass from the upwelling center because of advection in the upwelling plume was estimated from current data in the Ekman layer and biomass density. The mean cross-shelf component of the current was estimated at 10.4 km d^–1 within the upwelling plume. This yielded a loss of biomass of 9.7 mg dry weight m^–2 . Production, estimated by a temperature-dependent approach, ranged between 44 and 35 mg dry weight m^–2 d^–1, at mean temperatures of 14.6 °C and 15.8 °C inside and outside of the upwelling plume respectively. Within the plume, as much as 22% of daily production may be advected offshore. However, a higher concentration of biomass in the upwelling plume allowed a greater production compared to surrounding areas. A mass balance approach suggests that advective losses may not have a major impact on the C. chilensis population, because of very high daily production at temperature-dependent rates.     

Differential effects of stretch and compression on membrane currents and [Na]c in ventricular myocytes

Mechano-electrical feedback was studied in the single ventricular myocytes. A small fraction (approximately 10%) of the cell surface could be stretched or compressed by a glass stylus. Stretch depolarised, shortened the action potential and induced extra systoles. Stretch activated non-selective cation currents (I_ns) showed a linear voltage dependence, a reversal potential of 0 mV, a pure cation selectivity, and were blocked by 8 μM Gd³⁺ or 30 μM streptomycin. Stretch reduced Ca²⁺ and K⁺ (I_K) currents. Local compression of broadwise attached cells activated I_K but not I_ns. Cytochalasin D or colchicin, thought to disrupt the cytoskeleton, suppressed the mechanosensitivity of I_ns and I_K. During stretch, the cytosolic sodium concentration increased with spatial heterogeneities, local hotspots with [Na⁺]_c>24 mM appeared close to surface membrane and t-tubules (pseudoratiometric imaging using Sodium Green fluorescence). Electronprobe microanalysis confirmed this result and indicated that stretch increased total sodium [Na] in cell compartments such as mitochondria, nuclear envelope and nucleus. Our results obtained by local stretch differ from those obtained by end-to-end stretch (literature). We speculate that channels may be activated not only by axial but also by shear stress, and, that stretch can activate channels outside the deformed sarcomeres via second messenger.     

Cladogenesis and reticulation in the Hawaiian endemic mints (Lamiaceae)

The Hawaiian endemic mints, which comprise 58 species of dry‐fruited Haplostachys and fleshy‐fruited Phyllostegia and Stenogyne, represent a major island radiation that likely originated from polyploid hybrid ancestors in the temperate North American Stachys lineage. In contrast with considerable morphological and ecological diversity among taxa, sequence variation in the nrDNA 5S non‐transcribed spacer was found to be remarkably low, which when analyzed using standard parsimony resulted in a lack of phylogenetic resolution among accessions of insect‐pollinated Phyllostegia and bird‐pollinated Stenogyne. However, many within‐individual nucleotide polymorphisms were observed, and under the assumption that they could contain phylogenetic information, these ambiguities were recoded as new character states. Substantially more phylogenetic structure was obtained with these data, including the resolution of most Stenogyne species into a monophyletic group with an apparent recent origin on O’ahu (3.0 My) or the Maui Nui island complex (2.2 My). Subsequent diversification appears to have involved multiple inter‐island dispersal events. Intergeneric placements for a few morphotypes, seemingly misplaced within either Phyllostegia or Stenogyne, may indicate reticulation as one polymorphism‐generating force. For a finer scale exploration of hybridization, preliminary AFLP fragment data were examined among putative hybrids of Stenogyne microphylla and S. rugosa from Mauna Kea, Hawai’i, that had been identified based on morphology. Cladistic analysis (corroborated by multivariate correspondence analysis) showed the morphologically intermediate individuals to group in a strongly supported monophyletic clade with S. microphylla. Therefore, reticulation could be both historic and active in Stenogyne, and perhaps a force of general importance in the evolution of the Hawaiian mints. The relatively greater extent of lineage‐sorted polymorphisms in Stenogyne may indicate selective differentiation from other fleshy‐fruited taxa, perhaps through the agency of highly specialized bird pollinators that restricted gene flow with other Hawaiian mint morphotypes.     

Dynamic Localization of the Swe1 Regulator Hsl7 During the Saccharomyces cerevisiae Cell Cycle

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Delta cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Delta cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.