Neuropeptides in perisympathetic organs of Manduca sexta: specific composition and changes during the development

We used a combination of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and immunocytochemistry to investigate the peptides from abdominal perisympathetic organs of Manduca sexta. Altogether three mass peaks, detected in mass spectra from single abdominal perisympathetic organs were identical with already known neuropeptides, namely CAP(2b), CCAP, and Manduca-allatotropin. Only CAP(2b) was found throughout the postembryonic development. In larvae, perisympathetic organs of the abdominal ganglia 1 and 7 do not accumulate neuropeptides. During the metamorphosis, the number of putative hormones stored in the abdominal perisympathetic organs, increases dramatically. Not a single substance, however, obtained in mass spectra of larval perisympathetic organs disappeared in the respective adult neurohemal organs. Peptides from abdominal perisympathetic organs are different from those of thoracic perisympathetic organs and the retrocerebral complex. Manduca-FLRFa-2 and -3 are enriched in thoracic perisympathetic organs; FLRFa-1, corazonin and adipokinetic hormone are abundant peptides of the retrocerebral complex. The majority of ion signals, however, represent unknown substances. An antiserum which recognized CAP(2b) allowed the morphological characterization of a median neurosecretory system in the abdominal ventral nerve cord of M. sexta, which resembles that of cockroach embryos. Double stainings confirmed that crustacean cardioactive peptide (CCAP) becomes colocalized with CAP(2b) in median neurosecretory cells during the last larval instar. This colocalization continues in adult insects.     

Medaka dmY/dmrt1Y is not the universal primary sex-determining gene in fish

An outstanding candidate for a primary male-determining gene equivalent to Sry of mammals has been recently described from a non-mammalian vertebrate, the medaka fish (Oryzias latipes). However, the universality of dmY/dmrt1Y as the master sex-determining gene in fish is questionable. Phylogenetic analysis shows that dmY/dmrt1Y is an evolutionarily young Y chromosome-specific duplicate of a gene involved in testis development in vertebrates, and that this duplicate cannot be the primary sex-determining gene in most other fish species. Study of alternative fish models will probably uncover new genetic strategies controlling sexual dimorphism in vertebrates.     

Ecdysteroids and other constituents from Sida spinosa L

Two compounds (3 and 10) were isolated from the aerial parts of Sida spinosa L. Their structures have been established as glyceryl-1-eicosanoate and 20-hydroxy, 24-hydroxymethylecdysone by 1D and 2D-NMR techniques. In addition 12 known compounds (1, 2, 4-9 and 11-14) have been isolated and identified.     

A conserved tyrosine in the neck of a fungal kinesin regulates the catalytic motor core

The neck domain of fungal conventional kinesins displays characteristic properties which are reflected in a specific sequence pattern. The exchange of the strictly conserved Tyr 362, not present in animals, into Lys, Cys or Phe leads to a failure to dimerize. The destabilizing effect is confirmed by a lower coiled-coil propensity of mutant peptides. Whereas the Phe substitution has only a structural effect, the Lys and Cys replacements lead to dramatic kinetic changes. The steady state ATPase is 4- to 7-fold accelerated, which may be due to a faster microtubule-stimulated ADP release rate. These data suggest that an inhibitory effect of the fungal neck domain on the motor core is mediated by direct interaction of the aromatic ring of Tyr 362 with the head, whereas the OH group is essential for dimerization. This is the first demonstration of a direct influence of the kinesin neck region in regulation of the catalytic activity.     

Proteasome inhibition results in TRAIL sensitization of primary keratinocytes by removing the resistance-mediating block of effector caspase maturation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhibition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-kappaB activation. Here we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is removed by proteasome inhibitors independent of NF-kappaB in primary human keratinocytes. This sensitization was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 maturation. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.     

Single eubacterial origin of eukaryotic sulfide:quinone oxidoreductase,a mitochondrial enzyme conserved from the early evolution of eukaryotes during anoxic and sulfidic times

Mitochondria occur as aerobic, facultatively anaerobic, and, in the case of hydrogenosomes, strictly anaerobic forms. This physiological diversity of mitochondrial oxygen requirement is paralleled by that of free-living alpha-proteobacteria, the group of eubacteria from which mitochondria arose, many of which are facultative anaerobes. Although ATP synthesis in mitochondria usually involves the oxidation of reduced carbon compounds, many alpha-proteobacteria and some mitochondria are known to use sulfide (H2S) as an electron donor for the respiratory chain and its associated ATP synthesis. In many eubacteria, the oxidation of sulfide involves the enzyme sulfide:quinone oxidoreductase (SQR). Nuclear-encoded homologs of SQR are found in several eukaryotic genomes. Here we show that eukaryotic SQR genes characterized to date can be traced to a single acquisition from a eubacterial donor in the common ancestor of animals and fungi. Yet, SQR is not a well-conserved protein, and our analyses suggest that the SQR gene has furthermore undergone some lateral transfer among prokaryotes during evolution, leaving the precise eubacterial lineage from which eukaryotes obtained their SQR difficult to discern with phylogenetic methods. Newer geochemical data and microfossil evidence indicate that major phases of early eukaryotic diversification occurred during a period of the Earth's history from 1 to 2 billion years before present in which the subsurface ocean waters contained almost no oxygen but contained high concentrations of sulfide, suggesting that the ability to deal with sulfide was essential for prokaryotes and eukaryotes during that time. Notwithstanding poor resolution in deep SQR phylogeny and lack of a specifically alpha-protebacterial branch for the eukaryotic enzyme on the basis of current lineage sampling, a single eubacterial origin of eukaryotic SQR and the evident need of ancient eukaryotes to deal with sulfide, a process today germane to mitochondrial quinone reduction, are compatible with the view that eukaryotic SQR was an acquisition from the mitochondrial endosymbiont.     

Differences in the methyl ester distribution of homogalacturonans from near-isogenic wheat lines resistant and susceptible to the wheat stem rust fungus

Plants possess an efficient nonself surveillance system triggering induced disease resistance mechanisms upon molecular recognition of microbial invaders. Successful pathogens have evolved strategies to evade or counteract these mechanisms, e.g., by the generation of suppressors. Pectic fragments produced during host cell wall degradation can act as endogenous suppressors of the hypersensitive response in wheat leaves. We have isolated and characterized homogalacturonans from cell walls of two wheat cultivars susceptible to the stem rust fungus, Puccinia graminis f. sp. tritici, namely cvs. Prelude and Marquis, and from near-isogenic lines of both cultivars containing the Sr5-gene for hypersensitive rust resistance. Two independent approaches were used to compare their methyl esterification: i) immunochemistry using the monoclonal antibodies JIM5, JIM7, PAM1, and LM7 and ii) chromatography of oligogalacturonides representing stretches of contiguous nonmethyl-esterified GalA residues. The results clearly indicate a significant difference in the homogalacturonans from susceptible and resistant wheat lines. The difference can best be explained by assuming a nonrandom and more blockwise distribution of the methyl esters in the homogalacturonans of susceptible wheat cultivars as compared with a presumably more random distribution in the near-isogenic resistant lines. Possible consequences of this difference for the enzymatic generation of endogenous suppressors are discussed.     

HIV-1 and its transmembrane protein gp41 bind to different Candida species modulating adhesion

Oral candidiasis in HIV-1-infected individuals is widely believed to be triggered by the acquired T-lymphocyte immunodeficiency. Recently, binding of the HIV-1 envelope protein gp160 and its subunit gp41, and also of the whole virus itself, to Candida albicans has been shown. The present study shows that, in addition to C. albicans, HIV-1 gp41 also binds to yeast and hyphal forms of Candida dubliniensis, a species which is closely related to C. albicans, and to Candida tropicalis but not to Candida krusei, Candida glabrata or Saccharomyces cerevisiae. The previous finding that gp41 binding to C. albicans augments fungal virulence in vitro is supported by the observation that the yeast showed an enhanced adhesion to HIV-infected H9 cells in comparison to uninfected cells. In line with these results soluble gp41 itself reduced binding of C. albicans to both endothelial and epithelial cell lines, confirming a dominant role of the gp41 binding moiety on the surface of Candida for adhesion. Surface-associated secreted aspartic proteinases (Saps) play an important role in candidial adhesion, but are not likely to be involved in the interaction as gp41 binding to the C. albicans parental wild-type strain was comparable to that of three different isogenic Sap deletion mutants. Furthermore, gp41 binding to the yeast killer toxin-susceptible C. albicans strain 10S was not inhibitable by an anti-YKT receptor antibody. In conclusion, HIV-1 interacts with different clinically important Candida spp., and may thereby affect the outcome of the respective fungal infection.     

Interaction of sertraline with Candida species selectively attenuates fungal virulence in vitro

This study investigated whether the interaction between isolates of Candida albicans (n=7), Candida parapsilosis (n=3), Candida krusei (n=2), Candida dubliniensis (n=1) and sertraline, a typical selective serotonin reuptake inhibitor, alters candidal virulence. Sertraline treatment of Candida spp. significantly (P<0.05) affected hyphal elongation, phospholipase activity, production of secreted aspartyl proteinases and fungal viability. In addition, monocyte-derived macrophages (MDMs) treated with sertraline reduced inhibition of blastoconidia germination in comparison to MDMs alone. In conclusion, our findings suggest that the interaction between sertraline and Candida spp. may also diminish the virulence properties of this fungal pathogen in vivo.