Identification of G-proteins in rat parotid gland plasma membranes and granule membranes: presence of distinct components in granule membranes

We have identified by immunoblotting and ADP-ribosylation by cholera toxin and pertussis toxin the presence of Mr 43 and 46 KDa G_s, and 39 and 41 KDa G_i;.. subunits in rat parotid gland plasma membranes but not in granule membranes. A Mr 28 KDa polypeptide that served as substrate for ADP-ribosylation by both cholera toxin and pertussis toxin was present exclusively in granule membranes. Photoaffinity crosslinking of [-³²P]GTP showed the presence of high molecular weight GTP-binding proteins (Mr 160,100 KDa) in granule membranes. Six low molecular weight GTP-binding proteins (Mr 21–28 KDa) were differentially distributed in both plasma membranes and granule membranes. The present study identifies various GTP-binding proteins in rat parotid gland plasma membranes and granule membranes, and demonstrates the presence of distinct molecular weight GTP-binding proteins in granule membranes. These granule-associated GTP-binding proteins may be involved in secretory processes.     

Variation in the strength of inbreeding depression across environments: Effects of stress and density dependence

In what types of environments should we expect to find strong inbreeding depression? Previous studies indicate that inbreeding depression, δ, is positively correlated with the stressfulness of the environment in which it is measured. However, it remains unclear why stress, per se, should increase δ. To our knowledge, only “competitive stress” has a logical connection to δ. Through competition for resources, better quality (outbred) individuals make the environment worse for lower quality (inbred) individuals, accentuating the differences between them. For this reason, we expect inbreeding depression to be stronger in environments where the fitness of individuals is more sensitive to the presence of conspecifics (i.e., where fitness is more density dependent). Indeed, some studies suggest a role for competition within environments, but this idea has not been tested in the context of understanding variation in δ across environments. Using Drosophila melanogaster, we estimated δ for viability in 22 different environments. These environments were simultaneously characterized for (1) stressfulness and (2) density dependence. Although stress and density dependence are moderately correlated with each other, inbreeding depression is much more strongly correlated with density dependence. These results suggest that mean selection across the genome is stronger in environments where competition is intense, rather than in environments that are stressful for other reasons.     

Minimal Mesoscale Model for Protein-Mediated Vesiculation in Clathrin-Dependent Endocytosis

In eukaryotic cells, the internalization of extracellular cargo via the endocytic machinery is an important regulatory process required for many essential cellular functions. The role of cooperative protein-protein and protein-membrane interactions in the ubiquitous endocytic pathway in mammalian cells, namely the clathrin-dependent endocytosis, remains unresolved. We employ the Helfrich membrane Hamiltonian together with surface evolution methodology to address how the shapes and energetics of vesicular-bud formation in a planar membrane are stabilized by presence of the clathrin-coat assembly. Our results identify a unique dual role for the tubulating protein epsin: multiple epsins localized spatially and orientationally collectively play the role of a curvature inducing capsid; in addition, epsin serves the role of an adapter in binding the clathrin coat to the membrane. Our results also suggest an important role for the clathrin lattice, namely in the spatial- and orientational-templating of epsins. We suggest that there exists a critical size of the coat above which a vesicular bud with a constricted neck resembling a mature vesicle is stabilized. Based on the observed strong dependence of the vesicle diameter on the bending rigidity, we suggest that the variability in bending stiffness due to variations in membrane composition with cell type can explain the experimentally observed variability on the size of clathrin-coated vesicles, which typically range 50–100 nm. Our model also provides estimates for the number of epsins involved in stabilizing a coated vesicle, and without any direct fitting reproduces the experimentally observed shapes of vesicular intermediates as well as their probability distributions quantitatively, in wildtype as well as CLAP IgG injected neuronal cell experiments. We have presented a minimal mesoscale model which quantitatively explains several experimental observations on the process of vesicle nucleation induced by the clathrin-coated assembly prior to vesicle scission in clathrin dependent endocytosis.     

Interplant communication: airborne methyl jasmonate is essentially converted into JA and JA-Ile activating jasmonate signaling pathway and VOCs emission

Methyl jasmonate (MeJA) was identified as an airborne signal involved in mediating interplant defense response communications over a decade ago. However, how MeJA activates plant defense systems and what becomes of the compound after it has done so has, thus far, remained unknown. To investigate this, Achyranthes bidentata plants were exposed to deuterated methyl jasmonate (d₂MeJA) followed by absolute quantification of metabolic products of d₂MeJA, and emissions of volatile organic compound (VOC) as defensive markers. We found that d₂MeJA was metabolized mainly into deuterated jasmonic acid (d₂JA) and jasmonoyl isoleucine (d₂JA-Ile), and to a much lesser extent, deuterated jasmonoyl leucine (d₂JA-Leu). Increases in d₂JA-Ile/Leu and also endogenous JA-Ile/Leu were tightly co-related with, and significantly influenced the pattern and amount of, VOC emissions. The amount of accumulated d₂JA-IIe was 13.1-fold higher than d₂JA-Leu, whereas the amounts of JA-IIe and JA-Leu accumulated were almost identical. This study demonstrates that exogenous MeJA activates defensive systems (such as VOC emissions) in receiver plants by essentially converting itself into JA and JA-IIe and initiating a signal transduction leading to VOC emissions and induction of endogenous JA-IIe and JA-Leu, which in turn cause further amplification of VOC emissions.     

Molecular cloning and mRNA expression analysis of a novel rice (Oryzasativa L.) MAPK kinase kinase,OsEDR1, an ortholog of Arabidopsis AtEDR1, reveal its role in defense/stress signalling pathways and development

Mitogen-activated protein kinase (MAPK) cascade(s) is important for plant defense/stress responses. Though MAPKs have been identified and characterized in rice (Oryza sativa L.), a monocot cereal crop research model, the first upstream component of the kinase cascade, namely MAPK kinase kinase (MAPKKK) has not yet been identified. Here we report the cloning of a novel rice gene encoding a MAPKKK, OsEDR1, designated based on its homology with the Arabidopsis MAPKKK, AtEDR1. OsEDR1, a single copy gene in the genome of rice, encodes a predicted protein with molecular mass of 113046.13 and a pI of 9.03. Using our established two-week-old rice seedling in vitro model system, we show that OsEDR1 has a constitutive expression in seedling leaves and is further up-regulated within 15 min upon wounding by cut, treatment with the global signals jasmonic acid (JA), salicylic acid (SA), ethylene (ethephon, ET), abscisic acid, and hydrogen peroxide. In addition, protein phosphatase inhibitors, fungal elicitor chitosan, drought, high salt and sugar, and heavy metals also dramatically induce its expression. Moreover, OsEDR1 expression was altered by co-application of JA, SA, and ET, and required de novo synthesized protein factor(s) in its transient regulation. Furthermore, using an in vivo system we also show that OsEDR1 responds to changes in temperature and environmental pollutants-ozone and sulfur dioxide. Finally, OsEDR1 expression varied significantly in vegetative and reproductive tissues. These results suggest a role for OsEDR1 in defense/stress signalling pathways and development.     

Evidence that insulin-like growth factor-1 requires protein kinase C-epsilon, PI3-kinase and mitogen-activated protein kinase pathways to protect human vascular smooth muscle cells from apoptosis

Insulin-like growth factor (IGF)-1 has been implicated in the development of occlusive vascular lesions. Although its role in vascular smooth muscle cell (VSMC) growth and migration are fairly well characterized, anti-apoptotic signals of IGF-1 in human VSMC remain largely unknown. In this study, we examined IGF-1 signals that protect human and rat VSMC from staurosporine (STAU)- and c-myc- induced apoptosis, respectively. Treatment with STAU resulted in apoptotic DNA fragmentation, phosphatidylserine externalization and cell shrinkage, but only occasional VSMC 'blebbing'. STAU-induced death and IGF-1-mediated survival were concentration dependent, while time-lapse video microscopy showed that IGF-1 inhibited c-myc-induced apoptosis by 90%. Pretreatment with mitogen-activated protein kinase/extracellular signal regulated kinase kinase (MEK) inhibitors UO126 and PD098059, or with the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, reversed IGF-1-mediated human VSMC survival by 25-27% and 66%, respectively. Translocation studies showed that IGF-1 activated protein kinase C (PKC)-epsilon, but not PKC-alpha or PKC-delta, even in the presence of STAU, while pharmacological PKC inhibition (Ro-318220 or Go6976) implicated PKC-zeta or a novel PKC isozyme in IGF-1-mediated survival. Transient expression of activated PKC-epsilon but not activated PKC-zeta decreased myc-induced apoptosis in rat VSMC. In human VSMC, antisense oligodeoxynucleotides to PKC-epsilon partially reversed IGF-1-induced survival. In addition, IGF-1 elicited a mild but sustained activation of extracellular signal regulated kinase (ERK)1/2 in human VSMC that was abolished after 1 h in the presence of STAU. PKC downregulation reversed both IGF-1- and PMA-induced ERK activity, but platelet-derived growth factor (PDGF)-induced activity was unchanged. These results indicate for the first time that IGF-1 can protect human VSMC via multiple signals, including PKC-epsilon, PI3-K and mitogen-activated protein kinase pathways.