Thinning Jeffrey pine stands to reduce susceptibility to bark beetle infestations in California,U.S.A.

• 1 Bark beetles (Coleoptera: Curculionidae, Scolytinae) are commonly recognized as important tree mortality agents in coniferous forests of the western U.S.A.
• 2 High stand density is consistently associated with bark beetle infestations in western coniferous forests, and therefore thinning has long been advocated as a preventive measure to alleviate or reduce the amount of bark beetle‐caused tree mortality.
• 3 The present study aimed to determine the effectiveness of thinning to reduce stand susceptibility to bark beetle infestations over a 10‐year period in Pinus jeffreyi forests on the Tahoe National Forest, California, U.S.A. Four treatments were replicated three times within 1‐ha square experimental plots. Treatments included thinning from below (i.e. initiating in the smallest diameter classes) to a residual target basal area (cross‐sectional area of trees at 1.37 m in height) of: (i) 18.4 m²/ha (low density thin); (ii) 27.6 m²/ha (medium density thin); (iii) 41.3 m²/ha (high density thin); and (iv) no stand manipulation (untreated control).
• 4 Throughout the present study, 107 trees died as a result of bark beetle attacks. Of these, 71% (75 trees) were Abies concolor killed by Scolytus ventralis; 20.6% (22 trees) were Pinus ponderosa killed by Dendroctonus ponderosae; 4.7% (five trees) were P. jeffreyi killed by Dendroctonus jeffreyi; 1.8% (two trees) were P. jeffreyi killed by Ips pini; 0.9% (one tree) were P. jeffreyi killed by Orthotomicus (= Ips) latidens; 0.9% (one tree) were P. ponderosa killed by both Dendroctonus brevicomis and D. ponderosae; and 0.9% (one tree) were P. jeffreyi killed by unknown causes.
• 5 In the low density thin, no pines were killed by bark beetles during the 10‐year period. Significantly fewer trees (per ha/year) were killed in the low density thin than the high density thin or untreated control. No significant treatment effect was observed for the percentage of trees (per year) killed by bark beetles.

     

Why clinically used tazobactam and sulbactam are poor inhibitors of OXA-10 beta-lactamase: Raman crystallographic evidence

The clinically used inhibitors tazobactam and sulbactam are effective in the inhibition of activity of class A beta-lactamases, but not for class D beta-lactamases. The two inhibitors exhibit a complex multistep profile for their chemistry of inhibition with class A beta-lactamases. To compare the inhibition profiles for class A and D enzymes, the reactions were investigated within OXA-10 beta-lactamase (a class D enzyme) crystals using a Raman microscope. The favored reaction pathway appears to be distinctly different from that for class A beta-lactamases. In contrast to the case of class A enzymes that favor the formation of a key enamine species, the OXA-10 enzyme forms an alpha,beta-unsaturated acrylate (acid or ester). Quantum mechanical calculations support the likely product as the adduct of Ser115 to the acrylate. Few enamine-like species are formed by sulbactam or tazobactam with this enzyme. Taken together, our results show that the facile conversion of the initial imine, formed upon acylation of the active site Ser67, to the cis- and/or trans-enamine is disfavored. Instead, there is a significant population of the imine that could either experience cross-linking to a second nucleophile (e.g., Ser115) or give rise to the alpha,beta-unsaturated product and permanent inhibition. Alternatively, the imine can undergo hydrolysis to regenerate the catalytically active OXA-10 enzyme. This last process is the dominant one for class D beta-lactamases since the enzyme is not effectively inhibited. In contrast to sulbactam and tazobactam, the reactions between oxacillin or 6alpha-hydroxyisopropylpenicillinate (both substrates) and OXA-10 beta-lactamase appear much less complex. These compounds lead to a single acyl-enzyme species, the presence of which was confirmed by Raman and MALDI-TOF experiments.     

Activation of the phosphatidylinositol 3-kinase Vps34 by a G protein alpha subunit at the endosome

In the yeast Saccharomyces cerevisiae, the G protein beta gamma subunits are essential for pheromone signaling. The Galpha subunit Gpa1 can also promote signaling, but the effectors in this pathway are not well characterized. To identify candidate Gpa1 effectors, we expressed the constitutively active Gpa1(Q323L) mutant in each of nearly 5000 gene-deletion strains and measured mating-specific responses. Our analysis reveals a requirement for both the catalytic (Vps34) and regulatory (Vps15) subunits of the sole phosphatidylinositol 3-kinase in yeast. We demonstrate that Gpa1 is present at endosomes, where it interacts directly with both Vps34 and Vps15 and stimulates increased production of phosphatidylinositol 3-phosphate. Notably, Vps15 binds to GDP-bound Gpa1 and is predicted to have a seven-WD repeat structure similar to that of known G protein beta subunits. These findings reveal two new components of the pheromone signaling pathway. More remarkably, these proteins appear to comprise a preformed effector-G beta subunit assembly and function at the endosome rather than at the plasma membrane.     

Temperature dependence of fluid phase endocytosis coincides with membrane properties of pig platelets

In previous studies we have shown that platelets take up low molecular weight molecules from the medium by fluid phase endocytosis, a phenomenon that we previously have used to load trehalose into human platelets, after which we have successfully freeze-dried them. We now extend those findings to a species to be used in animal trials of freeze-dried platelets:pigs. Further, we report results of studies aimed at elucidating the mechanism of the uptake. Temperature dependence of fluid-phase endocytosis was determined in pig platelets, using lucifer yellow carbohydrazide (LY) as a marker. A biphasic curve of marker uptake versus temperature was obtained. The activation energy was significantly higher above 22 degrees C (18.7+/-1.8 kcal/mol) than below that critical temperature (7.5+/-1.5 kcal/mol). The activation energy of fluid phase endocytosis in human platelets was 24.1+/-1.6 kcal/mol above 15 degrees C. In order to establish a correlation between the effect of temperature on fluid phase endocytosis and the membrane physical state, Fourier transform infrared spectroscopy (FTIR) and fluorescence anisotropy experiments were conducted. FTIR studies showed that pig platelets exhibit a main membrane phase transition at approximately 12 degrees C, and two smaller transitions at 26 and 37 degrees C. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed a major transition at 8 degrees C and smaller transitions at 26 and 35 degrees C. In order to investigate the relative roles of known participants in fluid phase endocytosis, the effects of several chemical inhibitors were investigated. LY uptake was unaffected by colchicine, methylamine, and amiloride. However, disruption of specific microdomains in the membrane (rafts) by methyl-beta-cyclodextrin reduced uptake of LY by 35%. Treatment with cytochalasin B, which inhibits actin polymerization, reduced the uptake by 25%. We conclude that the inflection point in the LY uptake versus temperature plot at around 22 degrees C is correlated with changes in membrane physical state, and that optimal LY internalization requires an intact cytoskeleton and intact membrane rafts.     

Diminished growth and enhanced glucose metabolism in triple knockout mice containing mutations of insulin-like growth factor binding protein-3, -4, and -5

IGF-I and IGF-II are essential regulators of mammalian growth, development and metabolism, whose actions are modified by six high-affinity IGF binding proteins (IGFBPs). New lines of knockout (KO) mice lacking either IGFBP-3, -4, or -5 had no apparent deficiencies in growth or metabolism beyond a modest growth impairment (approximately 85-90% of wild type) when IGFBP-4 was eliminated. To continue to address the roles of these proteins in whole animal physiology, we generated combinational IGFBP KO mice. Mice homozygous for targeted defects in IGFBP-3, -4, and -5 remain viable and at birth were the same size as IGFBP-4 KO mice. Unlike IGFBP-4 KO mice, however, the triple KO mice became significantly smaller by adulthood (78% wild type) and had significant reductions in fat pad accumulation (P < 0.05), circulating levels of total IGF-I (45% of wild type; P < 0.05) and IGF-I bioactivity (37% of wild type; P < 0.05). Metabolically, triple KO mice showed normal insulin tolerance, but a 37% expansion (P < 0.05) of beta-cell number and significantly increased insulin secretion after glucose challenge, which leads to enhanced glucose disposal. Finally, triple KO mice demonstrated a tissue-specific decline in activation of the Erk signaling pathway as well as weight of the quadriceps muscle. Taken together, these data provide direct evidence for combinatorial effects of IGFBP-3, -4, and -5 in both metabolism and at least some soft tissues and strongly suggest overlapping roles for IGFBP-3 and -5 in maintaining IGF-I-mediated postnatal growth in mice.     

An active form of Vav1 induces migration of mammary epithelial cells by stimulating secretion of an epidermal growth factor receptor ligand

Background

Vav proteins are guanine nucleotide exchange factors (GEF) for Rho family GTPases and are activated following engagement of membrane receptors. Overexpression of Vav proteins enhances lamellipodium and ruffle formation, migration, and cell spreading, and augments activation of many downstream signaling proteins like Rac, ERK and Akt. Vav proteins are composed of multiple structural domains that mediate their GEF function and binding interactions with many cellular proteins. In this report we examine the mechanisms responsible for stimulation of cell migration by an activated variant of Vav1 and identify the domains of Vav1 required for this activity.

Results

We found that expression of an active form of Vav1, Vav1Y3F, in MCF-10A mammary epithelial cells increases cell migration in the absence or presence of EGF. Vav1Y3F was also able to drive Rac1 activation and PAK and ERK phosphorylation in MCF-10A cells in the absence of EGF stimulation. Mutations in the Dbl homology, pleckstrin homology, or cysteine-rich domains of Vav1Y3F abolished Rac1 or ERK activation in the absence of EGF and blocked the migration-promoting activity of Vav1Y3F. In contrast, mutations in the SH2 and C-SH3 domains did not affect Rac activation by Vav1Y3F, but reduced the ability of Vav1Y3F to induce EGF-independent migration and constitutive ERK phosphorylation. EGF-independent migration of MCF-10A cells expressing Vav1Y3F was abolished by treatment of cells with an antibody that prevents ligand binding to the EGF receptor. In addition, conditioned media collected from Vav1Y3F expressing cells stimulated migration of parental MCF-10A cells. Lastly, treatment of cells with the EGF receptor inhibitory antibody blocked the Vav1Y3F-induced, EGF-independent stimulation of ERK phosphorylation, but had no effect on Rac1 activation or PAK phosphorylation.

Conclusion

Our results indicate that increased migration of active Vav1 expressing cells is dependent on Vav1 GEF activity and secretion of an EGF receptor ligand. In addition, activation of ERK downstream of Vav1 is dependent on autocrine EGF receptor stimulation while active Vav1 can stimulate Rac1 and PAK activation independent of ligand binding to the EGF receptor. Thus, stimulation of migration by activated Vav1 involves both EGF receptor-dependent and independent activities induced through the Rho GEF domain of Vav1.