Prostaglandin E(1) inhibits abnormal follicle rupture and restores ovulation in indomethacin-treated rats

In the presence of indomethacin, an inhibitor of prostaglandin (PG) synthesis, the gonadotropin surge induces abnormal follicle rupture at the basolateral follicle sides, thus preventing effective ovulation in rats. This study was undertaken to analyze whether exogenous prostaglandin administration can overcome the antiovulatory action of indomethacin. Cycling rats were treated with vehicle (olive oil) or indomethacin (1 mg/rat) on the morning of proestrus. Rats treated with indomethacin were injected with different doses (50, 250, or 500 micro g/rat) of PGE(1), PGE(2), PGF(2alpha), or vehicle (saline) at 1900 h in proestrus. The ovulatory response was analyzed on the morning of estrus by evaluating follicle rupture and the location of the oocytes in serially sectioned ovaries. The number of oocytes in the oviducts was also counted in rats treated with the highest prostaglandin doses. In indomethacin-treated rats, most newly formed corpora lutea showed abnormal follicle rupture at the basolateral sides. In addition, invasion of the ovarian stroma and blood and lymphatic vessels by granulosa cells and follicular fluid was observed. Prostaglandins of the E series, and especially PGE(1), inhibited abnormal follicle rupture and restored ovulation, although the number of oocytes in the oviducts were significantly decreased. PGF(2alpha) was only partially effective in inhibiting abnormal follicle rupture and restoring ovulation. These data suggest that prostaglandins of the E series, and particularly PGE(1), play a crucial role in ovulation by determining the targeting of follicle rupture at the apex, thus allowing release of oocytes to the periovarian space.     

Mammalian Pex14p: membrane topology and characterisation of the Pex14p-Pex14p interaction

Peroxisomal biogenesis is a complex process requiring the action of numerous peroxins. One central component of this machinery is Pex14p, an intrinsic peroxisomal membrane protein probably involved in the docking of Pex5p, the receptor for PTS1-containing proteins (peroxisomal targeting signal 1-containing proteins). In this work the membrane topology of mammalian Pex14p was studied. Using a combination of protease protection assays and CNBr cleavage, we show that the first 130 amino acid residues of Pex14p are highly protected from exogenously added proteases by the peroxisomal membrane itself. Data indicating that this domain is responsible for the strong interaction of Pex14p with the organelle membrane are presented. All the other Pex14p amino acid residues are exposed to the cytosol. The properties of recombinant human Pex14p were also characterised. Heterologous expressed Pex14p was found to be a homopolymer of variable stoichiometry. Finally, in vitro binding assays indicate that homopolymerisation of Pex14p involves a domain comprising amino acid residues 147-278 of this peroxin.     

Reversible unfolding of FtsZ cell division proteins from archaea and bacteria. Comparison with eukaryotic tubulin folding and assembly

The stability, refolding, and assembly properties of FtsZ cell division proteins from Methanococcus jannaschii and Escherichia coli have been investigated. Their guanidinium chloride unfolding has been studied by circular dichroism spectroscopy. FtsZ from E. coli and tubulin released the bound guanine nucleotide, coinciding with an initial unfolding stage at low denaturant concentrations, followed by unfolding of the apoprotein. FtsZ from M. jannaschii released its nucleotide without any detectable secondary structural change. It unfolded in an apparently two-state transition at larger denaturant concentrations. Isolated FtsZ polypeptide chains were capable of spontaneous refolding and GTP-dependent assembly. The homologous eukaryotic tubulin monomers misfold in solution, but fold within the cytosolic chaperonin CCT. Analysis of the extensive tubulin loop insertions in the FtsZ/tubulin common core and of the intermolecular contacts in model microtubules and tubulin-CCT complexes shows a loop insertion present at every element of lateral protofilament contact and at every contact of tubulin with CCT (except at loop T7). The polymers formed by purified FtsZ have a distinct limited protofilament association in comparison with microtubules. We propose that the loop insertions of tubulin and its CCT-assisted folding coevolved with the lateral association interfaces responsible for extended two-dimensional polymerization into microtubule polymers.     

Type II phosphatidylinositol 4-kinase beta is a cytosolic and peripheral membrane protein that is recruited to the plasma membrane and activated by Rac-GTP

Phosphoinositides have a pivotal role as precursors to important second messengers and as bona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIalpha and PI4KIIbeta. PI4KIIalpha was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIbeta and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIbeta is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIalpha is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIbeta recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIbeta translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIalpha further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIbeta activation, because microsome-bound PI4KIIbeta is 16 times more active than cytosolic PI4KIIbeta. Membrane-associated PI4KIIbeta is as active as membrane-associated PI4KIIalpha and has essentially identical kinetic properties. We conclude that PI4KIIalpha and PI4KIIbeta may have partially overlapping, but not identical, functions. PI4KIIbeta is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells.     

Unusual acetylenic sex pheromone of grape leaffolder (Lepidoptera: Pyralidae)

The female-produced sex pheromone of grape leaffolder, Desmia_funeralis (Hübner), has been identified as a blend of (Z,Z)-11,13-hexadecadienal, 11-hexadecynal, and (Z)-11-hexadecenal. The first two components were essential for attraction of male moths, whereas the third compound was not essential, but increased trap catches approximately twofold when added in appropriate doses to the optimum blend of the other two components. In field tests, male moths were trapped equally well in traps baited with pheromone doses of 0.2 to >6 mg, and lures remained attractive for at least 5 wk.     

Scedosporium apiospermum keratitis

A case of Scedosporium apiospermum keratitis is reported in a 65-year-old farmer referred for treatment of an extensive corneal ulcer in the left eye. Direct examination of scrapes revealed abundant filamentous septate hyphae; all cultures were consistently positive for the same fungus, identified later as Scedosporium apiospermum. The patient successfully responded to treatment with amphotericin B.     

<Emphasis Type="Italic">Rhizopus microsporus</Emphasis> var.<Emphasis Type="Italic"> rhizopodiformis</Emphasis>: a thermotolerant fungus with potential for production of thermostable amylases

The effect of several nutritional and environmental parameters on growth and amylase production from Rhizopus microsporus var. rhizopodiformis was analysed. This fungus was isolated from soil of the Brazilian "cerrado" and produced high levels of amylolytic activity at 45°C in liquid medium supplemented with starch, sugar cane bagasse, oat meal or cassava flour. Glucose in the culture medium drastically repressed the amylolytic activity. The products of hydrolysis were analysed by thin layer chromatography, and glucose was detected as the main component. The amylolytic activity hydrolysed several substrates, such as amylopectin, amylase, glycogen, pullulan, starch, and maltose. Glucose was always the main end product detected by high-pressure liquid chromatography analysis. These results indicated that the amylolytic activity studied is a glucoamylase, but there were also low levels of -amylase. As compared to other fungi, R. microsporus var. rhizopodiformis can be considered an efficient producer of thermostable amylases, using raw residues of low cost as substrates. This information is of technological value, considering the importance of amylases for industrial hydrolysis.