PCR-RFLP is used to investigate relations among species in the entomopathogenic genera Eryniopsis and Entomophaga

The shape and nucleation of primary conidia are important characters in the classification of the Entomophthoraceae (Zygomycetes). The five species in the genus Eryniopsis vary in the shapes of primary conidia, although within most genera in the order Entomophthorales species have the same shapes of primary conidia. Using PCR-RFLP, we investigated two species in Eryniopsis, Ery. caroliniana with oblong-ovoid primary conidia and Ery. ptychopterae with pear-shaped primary conidia, with five species of Entomophaga, all having pear-shaped conidia. Molecular results merged with morphological data indicate that Ery. ptychopterae belongs in the genus Entomophaga while Ery. caroliniana clearly differs from Entomophaga. Ery. ptychopterae and Ery. transitans are transferred to the genus Entomophaga. Our results support the idea that morphology of primary conidia is of major importance in defining entomophthoralean genera. These results also show that such studies can be conducted with species that have not been isolated, if fungal-filled cadavers can be obtained.     

Ala31-Aib32: identification of the key motif for high affinity and selectivity of neuropeptide Y at the Y5-receptor

The turn-inducing sequence Ala-Aib introduced into positions 31 and 32 of neuropeptide Y (NPY) and its analogues has been identified as the key structure for Y(5)-receptor selectivity. Analogues of NPY and PP/NPY chimera containing the motif Ala-Aib were prepared; these peptides turned out to be selective for the Y(5)-receptor. The affinity of the NPY-based peptides was in the range of 6-150 nM, while the affinity of three (Ala-Aib)-containing PP/NPY chimera was in the range of 0.2-0.9 nM. The circular dichroism spectra of the Aib analogues in aqueous solution were all characteristic of an alpha helix; however, they had different intensities of the two negative bands at 220 and 208 nm. Affinity and selectivity for the Y(5)-receptor were correlated with the ratio of the ellipticity at 220 nm versus the one at 208 nm (R), which indicates the presence of a pronounced helix (R > 1) versus a less stabile one (R < 1). When R was in the range 0.74-0.96, the affinity at the Y(5)-receptor was in the range >5 nM, while there was complete loss of affinity at the Y(4)-receptor. R > 1.15 was associated with very high affinity at the Y(5)-receptor and weak affinity at the Y(4)-receptor. These results suggest that the selectivity of the Ala(31)-Aib(32) motif for the Y(5)-receptor derives from a specific conformation that must be correlated with the bioactive conformation of NPY at this subtype.     

Microsatellite diversity and genetic structure of fragmented populations of the rare,fire-dependent shrub Grevillea macleayana

Recent habitat loss and fragmentation superimposed upon ancient patterns of population subdivision are likely to have produced low levels of neutral genetic diversity and marked genetic structure in many plant species. The genetic effects of habitat fragmentation may be most pronounced in species that form small populations, are fully self-compatible and have limited seed dispersal. However, long-lived seed banks, mobile pollinators and long adult lifespans may prevent or delay the accumulation of genetic effects. We studied a rare Australian shrub species, Grevillea macleayana (Proteaceae), that occurs in many small populations, is self-compatible and has restricted seed dispersal. However, it has a relatively long adult lifespan (c. 30 years), a long-lived seed bank that germinates after fire and is pollinated by birds that are numerous and highly mobile. These latter characteristics raise the possibility that populations in the past may have been effectively large and genetically homogeneous. Using six microsatellites, we found that G. macleayana may have relatively low within-population diversity (3.2-4.2 alleles/locus; Hexp = 0.420-0.530), significant population differentiation and moderate genetic structure (FST = 0.218) showing isolation by distance, consistent with historically low gene flow. The frequency distribution of allele sizes suggest that this geographical differentiation is being driven by mutation. We found a lack mutation-drift equilibrium in some populations that is indicative of population bottlenecks. Combined with evidence for large spatiotemporal variation of selfing rates, this suggests that fluctuating population sizes characterize the demography in this species, promoting genetic drift. We argue that natural patterns of pollen and seed dispersal, coupled with the patchy, fire-shaped distribution, may have restricted long-distance gene flow in the past.     

Hypercholesterolemia promotes a CD36-dependent and endothelial nitric-oxide synthase-mediated vascular dysfunction

Numerous studies have implicated either the presence or absence of CD36 in the development of hypertension. In addition, hypercholesterolemia is associated with the loss of nitric oxide-induced vasodilation and the subsequent increase in blood pressure. In the current study, we tested the hypothesis that diet-induced hypercholesterolemia promotes the disruption of agonist-stimulated nitric oxide generation and vasodilation in a CD36-dependent manner. To test this, C57BL/6, apoE null, CD36 null, and apoE/CD36 null mice were maintained on chow or high fat diets. In contrast to apoE null mice fed a chow diet, apoE null mice fed a high fat diet did not respond to acetylcholine with a decrease in blood pressure. Caveolae isolated from in vivo vessels did not contain endothelial nitric-oxide synthase and were depleted of cholesterol. Age-matched apoE/CD36 null mice fed a chow or high fat diet responded to acetylcholine with a decrease in blood pressure. The mechanism underlying the vascular dysfunction was reversible because vessels isolated from apoE null high fat-fed mice regained responsiveness to acetylcholine when incubated with plasma obtained from chow-fed mice. Further analysis demonstrated that the plasma low density lipoprotein fraction was responsible for depleting caveolae of cholesterol, removing endothelial nitric-oxide synthase from caveolae, and preventing nitric oxide production. In addition, the pharmacological removal of caveola cholesterol with cyclodextrin mimicked the effects caused by the low density lipoprotein fraction. We conclude that the ablation of CD36 prevented the negative impact of hypercholesterolemia on agonist-stimulated nitric oxide-mediated vasodilation in apoE null mice. These studies provide a direct link between CD36 and the early events that underlie hypercholesterolemia-mediated hypertension and mechanistic linkages between CD36 function, nitric-oxide synthase activation, caveolae integrity, and blood pressure regulation.     

Leucine-derived cyano glucosides in barley

Barley (Hordeum vulgare) seedlings contain five cyano glucosides derived from the amino acid L-leucine (Leu). The chemical structure and the relative abundance of the cyano glucosides were investigated by liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses using spring barley cultivars with high, medium, and low cyanide potential. The barley cultivars showed a 10-fold difference in their cyano glucoside content, but the relative content of the individual cyano glucosides remained constant. Epiheterodendrin, the only cyanogenic glucoside present, comprised 12% to 18% of the total content of cyano glucosides. It is proposed that the aglycones of all five cyano glucosides are formed by the initial action of a cytochrome P450 enzyme of the CYP79 family converting L-Leu into Z-3-methylbutanal oxime and subsequent action of a less specific CYP71E enzyme converting the oxime into 3-methylbutyro nitrile and mediating subsequent hydroxylations at the alpha-, as well as beta- and gamma-, carbon atoms. Presence of cyano glucosides in the barley seedlings was restricted to leaf tissue, with 99% confined to the epidermis cell layers of the leaf blade. Microsomal preparations from epidermal cells were not able to convert L-[(14)C]Leu into the biosynthetic intermediate, Z-3-methylbutanal-oxime. This was only achieved using microsomal preparations from other cell types in the basal leaf segment, demonstrating translocation of the cyano glucosides to the epidermal cell layers after biosynthesis. A beta-glucosidase able to degrade epiheterodendrin was detected exclusively in yet a third compartment, the endosperm of the germinating seed. Therefore, in barley, a putative function of cyano glucosides in plant defense is not linked to cyanide release.     

A zebrafish orthologue (whnb) of the mouse nude gene is expressed in the epithelial compartment of the embryonic thymic rudiment

The cloning and characterization of the zebrafish orthologue of the mouse nude (Whn/Foxn1) gene, whnb are reported. A previously described Whn-like gene from zebrafish, now designated as whna, is shown to be the orthologue of the mouse Foxn4 gene. The whnb gene is specifically expressed in the thymic rudiment of zebrafish embryos at day 3 after fertilization, whereas the whna gene is expressed in eye and brain structures. Whnb expression is maintained in cloche mutants, where endothelial and haematopoietic cell differentiation is defective, but absent in casanova mutants where endoderm formation is impaired. In adult thymi, whnb is expressed throughout cortical and medullary areas, whereas whna expression is observed in rare cell clusters only. Our results provide the first specific marker for the epithelial compartment of the zebrafish thymus.     

Regulation of cell separation in the dimorphic fungus Ustilago maydis

During its haploid phase the dimorphic fungus Ustilago maydis grows vegetatively by budding. We have identified two genes, don1 and don3, which control the separation of mother and daughter cells. Mutant cells form tree-like clusters in liquid culture and grow as ring-like (donut-shaped) colonies on solid medium. In wild-type U. maydis cells, two distinct septa are formed during cytokinesis and delimit a fragmentation zone. Cells defective for either don1 or don3 display only a single septum and fail to complete cell separation. don1 encodes a guanine nucleotide exchange factor (GEF) of the Dbl family specific for Rho/Rac GTPases. Don3 belongs to the germinal-centre-kinase (GC) subfamily of Ste20-like protein kinases. We have isolated the U. maydis homologues of the small GTP binding proteins Rho2, Rho3, Rac1 and Cdc42. Out of these, only Cdc42 interacts specifically with Don1 and Don3 in the yeast two-hybrid system. We propose that Don1 and Don3 regulate the initiation of the secondary septum, which is required for proper cell separation.     

Wound closure in sheared endothelial cells is enhanced by modulation of vascular endothelial-cadherin expression and localization

We previously demonstrated that laminar shear stress enhances human coronary artery endothelial cell (HCAEC) wound closure via the mechanisms of cell spreading and migration. Because cell-cell junctional proteins such as vascular endothelial cell cadherin (VE-cadherin) are critical to cell-cell adhesion and motility, we tested the hypothesis that modulation of VE-cadherin expression under shear stress may be linked to this enhancement in wound closure. HCAEC monolayers were preconditioned to attain cellular alignment by shearing at 12 dynes/cm(2) for 18 hr in a parallel-plate flow chamber. Subsequently, they were divided into the following three groups: (i) control; (ii) treated with anti-cadherin-5 antibody; or (iii) treated with the calcium chelating agent EGTA. Next, the monolayers were wounded with a metal spatula and resheared at 20 dynes/cm(2) or left static. Time-lapse imaging was performed during the first 3 hr after imposition of these conditions. Immunocytochemistry or Western blot analyses for VE-cadherin expression were performed on all wounded monolayers. Deconvolution microscopy, three-dimensional cell-cell junctional reconstruction images, and histogram analyses of interendothelial junction signal intensities were performed on cells at the wound edge of a monolayer. Under shear, HCAEC demonstrated increased VE-cadherin immunofluorescence and protein expression despite an enhancement in wound closure compared with static conditions. In separate experiments, application with anti-cadherin-5 antibody or treatment with EGTA attenuated VE-cadherin expression and further enhanced wound closure compared with control shear and all static conditions. In addition, the pattern of VE-cadherin localization with these treatments became more intracellular and nuclear in appearance. These findings of changes in this junctional adhesion protein expression and localization may further our understanding of laminar shear stress-induced endothelial repair in the coronary circulation.     

Chemoimmunotherapy for melanoma with dacarbazine and 2,4-dinitrochlorobenzene elicits a specific T cell-dependent immune response

An empirically established chemoimmunotherapy for metastatic melanoma combines the systemic administration of the chemotherapeutic agent dacarbazine (DTIC) with the epifocal application of the contact sensitizer 2,4-dinitrochlorobenzene (DNCB) on cutaneous metastases. Although this therapy yields high response rates resulting in prolonged survival, the mechanisms involved remain unknown. Here, we investigated whether treatment of tumor-bearing mice with DTIC and DNCB resulted in a specific immune response against the tumor. Subcutaneous (s.c.) tumors and lung metastases were induced in C57BL/6 mice by injecting syngeneic B16-melanoma cells s.c. or into the lateral tail vein, respectively. Mice were treated with intraperitoneal injections of DTIC followed by epifocal application of DNCB. This therapeutic approach significantly reduced the growth of s.c. tumors as well as lung metastases. Our data showed that the effector mechanisms involved are dependent on T cells. No therapeutic effect was observed in immunodeficient RAG-1(-/-) mice, or when the contact sensitizer DNCB was replaced by skin irritants (croton oil or tributyltin). Splenic lymphocytes obtained from treated mice displayed a three-fold higher specific cytolytic activity against B16 cells than in tumor-bearing controls. Both CD8(+) and CD4(+) T cells were able to lyse B16 cells. No changes were observed in natural killer (NK) cell activity. Likewise, tumor-infiltrating lymphocytes (TIL) of treated mice showed higher cytolytic activity than that of controls. Analysis of cytokine expression in s.c. tumors revealed increased mRNA levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) in treated tumors. Together, these findings demonstrate the ability of DTIC/DNCB treatment to induce an effective T cell-dependent host immune response against a syngeneic tumor.     

Structural and functional criteria reveal a new nuclear import sequence on the 5-lipoxygenase protein

Leukotrienes are lipid mediators with important roles in immunity. The enzyme 5-lipoxygenase initiates leukotriene synthesis; nuclear import of 5-lipoxygenase modulates leukotriene synthetic capacity. In this study, we used structural and functional criteria to identify potential nuclear import sequences. Specifically, we sought basic residues that 1) were common to different 5-lipoxygenases but not shared with other lipoxygenases, 2) were found on random coil/loop structures, and 3) could be replaced without eliminating catalytic activity. Application of these criteria to the putative bipartite nuclear import sequence of 5-lipoxygenase revealed that this region formed an alpha-helix rather than a random coil, that the critical residue arginine 651 serves a structural role, and that mutation of this residue eliminated catalytic activity. A previously unrecognized region corresponding to residues 518-530 on human 5-lipoxygenase was found to be unique to 5-lipoxygenase and on a random coil. This region alone was sufficient to drive import of green fluorescent protein to the same degree as complete 5-lipoxygenase. Replacement of basic residues in this region of the complete protein was capable of eliminating nuclear import without abolishing catalytic activity. Surprisingly, two subpopulations of cells expressing 5-lipoxygenase with this mutated region could be discerned: those with strongly impaired import and those with normal import. Taken together, these results show that the previously identified region with a bipartite motif is not a functional import sequence, whereas the newly identified basic region constitutes a true nuclear import sequence. Moreover, we suggest that another sequence that can mediate nuclear import of 5-lipoxygenase remains to be identified.