New pollen-specific receptor kinases identified in tomato,maize and Arabidopsis: the tomato kinases show overlapping but distinct localization patterns on pollen tubes

We previously characterized LePRK1 and LePRK2, pollen-specific receptor kinases from tomato (Muschietti et al., 1998). Here we identify a similar receptor kinase from maize, ZmPRK1, that is also specifically expressed late in pollen development, and a third pollen receptor kinase from tomato, LePRK3. LePRK3 is less similar to LePRK1 and LePRK2 than either is to each other. We used immunolocalization to show that all three LePRKs localize to the pollen tube wall, in partially overlapping but distinct patterns. We used RT-PCR and degenerate primers to clone homologues of the tomato kinases from other Solanaceae. We deduced features diagnostic of pollen receptor kinases and used these criteria to identify family members in the Arabidopsis database. RT-PCR confirmed pollen expression for five of these Arabidopsis candidates; two of these are clearly homologues of LePRK3. Our results reveal the existence of a distinct pollen-specific receptor kinase gene family whose members are likely to be involved in perceiving extracellular cues during pollen tube growth.     

Low sequence variation among isolates of infectious hypodermal and hematopoietic necrosis virus (IHHNV) originating from Hawaii and the Americas

A 2.9 kb fragment of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) genome, which contains the coding sequence of putative non-structural and capsid proteins, was amplified and sequenced from each of 14 IHHNV isolates collected from cultured penaeid shrimp stocks in Hawaii and various sites in the Americas between 1982 and 1997. The sequence comparison indicates that the IHHNV genome is very stable, with 99.6 to 100% similarity among these 14 isolates. Only nucleotide substitutions were found. The percentage of substitution was higher in the putative capsid proteins region (1.3%) than in the putative non-structural proteins region (0.6%). Out of 25 substitutions found, 14 resulted in amino acid changes. There is no apparent association between clinical outcomes and particular amino acid substitutions. Based on genetic distances, the isolates were clustered into 3 groups that generally correspond with their geographic origins.     

Effects of different peptide fragments derived from proadrenomedullin on gene expression of adrenomedullin gene

Primary culture of vascular smooth muscle cells (VSMC) from rat aorta was used for the study of the effect of different peptides derived from proadrenomedullin on the expression of adrenomedullin (ADM) gene. ADM and preproADM(22-41) (PAMP) secreted by VSMC were measured by radioimmunoassay, and ADM mRNA in VSMC was determined by quantitative RT-PCR. After the incubation of VSMC in 10(-7)M ADM for 24h, PAMP in the medium and ADM mRNA in the VSMC were decreased by 34 and 41.3%, respectively, and cAMP concentration in the VSMC was increased by 385%. After the incubation of VSMC in 10(-7)M PAMP for 24h, ADM in the medium and ADM mRNA in the VSMC were decreased by 12.2 and 39.1%, respectively, and cAMP concentration in the VSMC was increased by 67%. The decreased ADM mRNA in VSMC induced by the ADM and PAMP treatment was completely reversed by the pre-treatment of the cells in 10(-7)M protein kinase inhibitor for 30 min. After the incubation of VSMC in 10(-7)M preproADM(153-185) (ADT) for 24h, however, ADM in the medium and ADM mRNA in the VSMC were increased by 21 and 35.2%. The increased ADM mRNA in VSMC induced by the ADT treatment was partially blocked by the co-incubation in ADM and ADT, and was totally blocked by the co-incubation in PAMP+ADM and ADT, but was not blocked by the co-incubation in PAMP and ADT. Our results suggest that the four peptides derived from proadrenomedullin may have different effects, possibly through a cAMP-dependent pathway, on the expression of ADM gene.     

Differentiation of embryonic stem cell to astrocytes visualized by green fluorescent protein

Green fluorescent protein (GFP) gene was transfected and expressed in murine embryonic stem (ES) cells under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Stably transfected cells were characterized by immunohistochemistry and by fluorescence microscopy. Cells containing GFP were differentiated to Type I and Type II astrocytes after induction by all-trans retinoic acid. Differentiated cells were expressed GFP and visualized by fluorescence microscopy. Differentiated cells expressed GFP were correlated with the expression of GFAP and morphological change. It demonstrates that the cell line expressed GFP can be used to trace the morphological changes of astrocytes during differentiation, and further for the isolation of astrocytes from the mixed cells differentiated from ES cell.     

Nitric oxide decreases the sensitivity of pulmonary endothelial cells to LPS-induced apoptosis in a zinc-dependent fashion

We hypothesized that: (a) S-nitrosylation of metallothionein (MT) is a component of pulmonary endothelial cell nitric oxide (NO) signaling that is associated with an increase in labile zinc; and (b) NO mediated increases in labile zinc in turn reduce the sensitivity of pulmonary endothelium to LPS-induced apoptosis. We used microspectrofluorometric techniques to show that exposing mouse lung endothelial cells (MLEC) to the NO-donor, S-nitrosocysteine, resulted in a 45% increase in fluorescence of the Zn²⁺-specific fluorophore, Zinquin, that was rapidly reversed by exposure to the Zn²⁺ chelator, NNNN-tetrakis-(2-pyridylmethyl)ethylenediamine; TPEN). The absence of a NO-mediated increase in labile Zn²⁺ in MLEC from MT-I and -II knockout mice inferred a critical role for MT in the regulation of Zn²⁺ homeostasis by NO. Furthermore, we found that prior exposure of cultured endothelial cells from sheep pulmonary artery (SPAEC), to the NO-donor, S-nitroso-N-acetylpenicillamine (SNAP) reduced their sensitivity to lipopolysaccharide (LPS) induced apoptosis. The anti-apoptotic effects of NO were significantly inhibited by Zn²⁺ chelation with low doses of TPEN (10 M). Collectively, these data suggest that S-nitrosylation of MT is associated with an increase in labile (TPEN chelatable) zinc and NO-mediated MT dependent zinc release is associated with reduced sensitivity to LPS-induced apoptosis in pulmonary endothelium.     

Nitrate uptake,nitrate reductase distribution and their relation to proton release in five nodulated grain legumes

Nitrate uptake, nitrate reductase activity (NRA) and net proton release were compared in five grain legumes grown at 0.2 and 2 mM nitrate in nutrient solution. Nitrate treatments, imposed on 22-d-old, fully nodulated plants, lasted for 21 d. Increasing nitrate supply did not significantly influence the growth of any of the species during the treatment, but yellow lupin (Lupinus luteus) had a higher growth rate than the other species examined. At 0.2 mM nitrate supply, nitrate uptake rates ranged from 0.6 to 1.5 mg N g(-1) d(-1) in the order: yellow lupin > field pea (Pisum sativum) > chickpea (Cicer arietinum) > narrow-leafed lupin (L angustifolius) > white lupin (L albus). At 2 mM nitrate supply, nitrate uptake ranged from 1.7 to 8.2 mg N g(-1) d(-1) in the order: field pea > chickpea > white lupin > yellow lupin > narrow-leafed lupin. Nitrate reductase activity increased with increased nitrate supply, with the majority of NRA being present in shoots. Field pea and chickpea had much higher shoot NRA than the three lupin species. When 0.2 mM nitrate was supplied, narrow-leafed lupinreleased the most H+ per unit root biomass per day, followed by yellow lupin, white lupin, field pea and chickpea. At 2 mM nitrate, narrow-leafed lupin and yellow lupin showed net proton release, whereas the other species, especially field pea, showed net OH- release. Irrespective of legume species and nitrate supply, proton release was negatively correlated with nitrate uptake and NRA in shoots, but not with NRA in roots.     

Synaptosome-associated protein of 25 kilodaltons modulates Kv2.1 voltage-dependent K(+) channels in neuroendocrine islet beta-cells through an interaction with the channel N terminus

Insulin secretion is initiated by ionic events involving membrane depolarization and Ca(2+) entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the beta-cell voltage-dependent K(+) channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Kv2.1. SNAP-25 reduced currents from recombinant Kv2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K(+) currents from rat beta-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward K(+) currents in beta-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other beta-cell Kv channels. This study demonstrates that SNAP-25 can regulate Kv2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.     

Neuritogenesis: polarization of constitutive exocytosis by effectors of Rho-family GTPases?

The sprouting of neurites from a neuron represents a highly specialized form of cellular morphogenesis that must involve coordinated changes in two major cellular processes at two membrane locations: reorganization of the cytoskeleton and redirection of membrane traffic from the trans-Golgi network to the plasma membrane of the growth tip. How exactly are these two processes linked and how is spatial and temporal coordination achieved at the first instance of neurite sprouting? Recent advances may have already revealed some, if not most of the pieces in the puzzle. We discuss below, with some extrapolations, of what has recently come to light, and what more is needed to construct a coherent picture.