EndoPDI,a novel protein-disulfide isomerase-like protein that is preferentially expressed in endothelial cells acts as a stress survival factor

We have identified a novel protein-disulfide isomerase and named it endothelial protein-disulfide isomerase (EndoPDI) because of its high expression in endothelial cells. Isolation of the full-length cDNA showed EndoPDI to be a 48 kDa protein that has three APWCGHC thioredoxin motifs in contrast to the two present in archetypal PDI. Ribonuclease protection and Western analysis has shown that hypoxia induces EndoPDI mRNA and protein expression. In situ hybridization analysis showed that EndoPDI expression is rare in normal tissues, except for keratinocytes of the hair bulb and syncytiotrophoblasts of the placenta, but was present in the endothelium of tumors and in other hypoxic lesions such as atherosclerotic plaques. We have compared the function of EndoPDI to that of PDI in endothelial cells using specific siRNA. PDI was shown to have a protective effect on endothelial cells under both normoxia and hypoxia. In contrast, EndoPDI has a protective effect only in endothelial cells exposed to hypoxia. The loss of EndoPDI expression under hypoxia caused a significant decrease in the secretion of adrenomedullin, endothelin-1, and CD105; molecules that protect endothelial cells from hypoxia-initiated apoptosis. The identification of an endothelial PDI further extends this increasing multigene family and EndoPDI, unlike archetypal PDI, may be a molecule with which to target tumor endothelium.     

Heparan sulfate proteoglycans as regulators of fibroblast growth factor-2 signaling in brain endothelial cells. Specific role for glypican-1 in glioma angiogenesis

Fibroblast growth factor-2 (FGF2) is a potent angiogenic factor in gliomas. Heparan sulfate promotes ligand binding to receptor tyrosine kinase and regulates signaling. The goal of this study was to examine the contribution of heparan sulfate proteoglycans (HSPGs) to glioma angiogenesis. Here we show that all brain endothelial cell HSPGs carry heparan sulfate chains similarly capable of forming a ternary complex with FGF2 and fibroblast growth factor receptor-1c and of promoting a mitogenic signal. Immunohistochemical analysis revealed that glypican-1 was overexpressed in glioma vessel endothelial cells, whereas this cell-surface HSPG was consistently undetectable in normal brain vessels. To determine the effect of increased glypican-1 expression on FGF2 signaling, we transfected normal brain endothelial cells, which express low base-line levels of glypican-1, with this proteoglycan. Glypican-1 expression enhanced growth of brain endothelial cells and sensitized them to FGF2-induced mitogenesis despite the fact that glypican-1 remained a minor proteoglycan. In contrast, overexpression of syndecan-1 had no effect on growth or FGF2 sensitivity. We conclude that the glypican-1 core protein has a specific role in FGF2 signaling. Glypican-1 overexpression may contribute to angiogenesis and the radiation resistance characteristic of this malignancy.     

Assignment of paternity groups without access to parental genotypes: multiple mating and developmental plasticity in squid

We present a novel approach to investigating sibling relationships and reconstructing parental genotypes from a progeny array. The Bayesian method we have employed is flexible and may be applicable to a variety of situations in addition to the one presented here. While mutation rates and breeding population allele frequencies can be taken into account, the model requires relatively few loci and makes few assumptions. Paternity of 270 veined squid (Loligo forbesi) hatchlings from three egg strings collected from one location was assigned using five microsatellite loci. Paternal and maternal genotypes reconstructed for each of the three strings were identical, strongly indicating that a single female produced the strings that were fertilized by the same four males. The proportion of eggs fertilized was not equal between males in all three strings, with male 1 siring most offspring (up to 68% in string 1), through to male 4 siring the least (as low as 2.4% in string 1). Although temperature had a profound effect on incubation time, paternity did not affect this trait at 12 degrees C or 8 degrees C.     

Metabolites of dietary 1,8-cineole in the male koala (Phascolarctos cinereus)

The in vivo metabolic fate of 1,8-cineole was investigated in six male koalas. Koalas were fed ad lib a diet of Eucalyptus cephalocarpa leaf with a 1,8-cineole concentration of 2.53+/-0.70% dry mass of leaf, corresponding to a 1,8-cineole intake of 2.4+/-1.1 mmol/kg (3.1+/-1.3 g). Urine and faeces were collected for 24 h and metabolites identified by GC-MS and LC-MS. Metabolites were quantified before and after hydrolysis with beta-glucuronidase to give free and total levels, respectively. Fractional recovery of ingested 1,8-cineole was 1.3+/-0.4 and 1.4+/-0.4 (mean+/-S.D.) for free and total measurements, respectively. Seven metabolites were identified and quantified: 9- and 7-hydroxycineole, 9- and 7-cineolic acid, 7-hydroxy-9-cineolic acid, 9-hydroxy-7-cineolic acid and 7,9-dicineolic acid. The hydroxycineolic acids dominated the metabolite profile (85%). 7,9-Dicineolic acid, a novel metabolite of 1,8-cineole, accounted for almost 10% of the recovered dose making it the second most abundant metabolite after 7-hydroxy-9-cineolic acid (77%). Together, the less oxidised metabolites, the hydroxycineoles and cineolic acid, accounted for only 5% of the cineole consumed. Significant conjugation only occurred with four minor, less oxidised, alcohol and carboxylic acid metabolites. We have shown that the koala detoxifies and eliminates 1,8-cineole primarily by extensive oxidation without utilising conjugation pathways.     

Septum volume and food-storing behavior are related in parids

The hippocampal formation (HF) of food-storing birds is larger than non-storing species, and the size of the HF in food-storing Black-Capped Chickadees (Poecile atricapillus) varies seasonally. We examined whether the volume of the septum, a medial forebrain structure that shares reciprocal connections with the HF, demonstrates the same species and seasonal variation as has been shown in the HF. We compared septum volume in three parid species; non-storing Blue Tits (Parus caeruleus) and Great Tits (Parus major), and food-storing Black-Capped Chickadees. We found the relative septum volume to be larger in chickadees than in the non-storing species. We also compared septum and nucleus of the diagonal band (NDB) volume of Black-Capped Chickadees at different times of the year. We found that the relative septum volume varies seasonally in food-storing birds. The volume of the NDB does not vary seasonally. Due to the observed species and seasonal variation, the septum, like the hippocampal formation of food-storing birds, may be specialized for some aspects of food-storing and spatial memory.     

Monoamines in the albumen gland,plasma, and central nervous system of the snail Biomphalaria glabrata during egg-laying

The potential role of selected biogenic monoamines and related compounds in the reproductive physiology of the freshwater snail Biomphalaria glabrata was investigated. Extracts of the albumen gland (AG), plasma, and central nervous system (CNS) were subjected to high pressure liquid chromatography with electrochemical detection (HPLC-ED), and under the extraction and separation conditions employed the following amines were detected: tyrosine, dihydroxyphenylalanine (DOPA), dopamine, and tryptophan in the AG; DOPA, tyrosine, and tryptophan in the plasma; DOPA, tyrosine, dopamine and 5-hydroxytryptamine in the CNS. These compounds were then quantified in individual samples taken from snails known to be in a particular stage of the egg-laying process. AG dopamine levels were highest in snails in the first stage of the reproductive process, when the AG is secreting perivitelline fluid around each fertilized ovum. Significant decreases in AG protein content during the later stages of the egg-laying process were also evident. Plasma tyrosine and DOPA levels were lowest in snails that contained a fully packaged egg mass, while no changes in monoamine content were observed in the CNS. These data provide insights into the role(s) that monoamines, especially catecholamine-related compounds, may play in B. glabrata reproductive physiology.     

Preliminary investigation: the impact of the NCAA Wrestling Weight Certification Program on weight cutting

Historically, wrestling is a sport dependent on weight. Three tragic deaths in late 1997 prompted the National Collegiate Athletic Association (NCAA) to make a Wrestling Weight Certification Program (WWCP) mandatory to foster a safe competitive environment. One institution examined the impact of this program on weight cutting. Thirty-two NCAA Division I wrestlers completed the WWCP in the 1998-1999 season and 29 in 1999-2000. Eighteen (56%) of 32 wrestlers in 1998-1999 weighed in 10 or more pounds above the previous year's competition weight. Whereas, 28% weighed in 20 or more pounds above the previous year's competition weight. Weekly weight loss for the wrestlers in 1998-1999 revealed a substantial loss during the first week, possibly demonstrating the use of time-tested techniques for weight loss. However, in 1999-2000, the first week weight loss was less pronounced, with 65.8% of the weight being lost during the second half of the WWCP. Therefore, these wrestlers may be breaking the sport historic cycle of weight fluctuations through the WWCP.     

Structure-based design of a potent purine-based cyclin-dependent kinase inhibitor

Aberrant control of cyclin-dependent kinases (CDKs) is a central feature of the molecular pathology of cancer. Iterative structure-based design was used to optimize the ATP- competitive inhibition of CDK1 and CDK2 by O(6)-cyclohexylmethylguanines, resulting in O(6)-cyclohexylmethyl-2-(4'- sulfamoylanilino)purine. The new inhibitor is 1,000-fold more potent than the parent compound (K(i) values for CDK1 = 9 nM and CDK2 = 6 nM versus 5,000 nM and 12,000 nM, respectively, for O(6)-cyclohexylmethylguanine). The increased potency arises primarily from the formation of two additional hydrogen bonds between the inhibitor and Asp 86 of CDK2, which facilitate optimum hydrophobic packing of the anilino group with the specificity surface of CDK2. Cellular studies with O(6)-cyclohexylmethyl-2-(4'- sulfamoylanilino) purine demonstrated inhibition of MCF-7 cell growth and target protein phosphorylation, consistent with CDK1 and CDK2 inhibition. The work represents the first successful iterative synthesis of a potent CDK inhibitor based on the structure of fully activated CDK2-cyclin A. Furthermore, the potency of O(6)-cyclohexylmethyl-2-(4'- sulfamoylanilino)purine was both predicted and fully rationalized on the basis of protein-ligand interactions.     

In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: Review of the literature

Voriconazole (Vfend™) is a new triazole that currently is undergoing phase III clinical trials. This review summarizes the published data obtained by NCCLS methods on the in vitro antifungal activity of voriconazole in comparison to itraconazole, amphotericin B, fluconazole, ketoconazole and flucytosine. Voriconazole had fungistatic activity against most yeasts and yeastlike species (minimum inhibitory concentrations [MICs] <2 μg/ml) that was similar or superior to those of fluconazole, amphotericin B, and itraconazole. Against Candida glabrata and C. krusei, voriconazole MIC ranges were 0.03 to 8 and 0.01 to >4 μg/ml, respectively. For four of the six Aspergillus spp. evaluated, voriconazole MICs (< 0.03 to 2 μg/ml) were lower than amphotericin B (0.25 to 4 μg/ml) and similar to itraconazole MICs. Voriconazole fungistatic activity against Fusarium spp. has been variable. Against F. oxysporum and solani, most studies showed MICs ranging from 0.25 to 8 μg/ml. Voriconazole had excellent fungistatic activity against five of the six species of dimorphic fungi evaluated (MIC₉₀s < 1.0 μg/ml). The exception was Sporothrix schenckii (MIC₉₀s and geometric mean MICs ≥ 8 μg/ml). Only amphotericin B had good fungistatic activity against the Zygomycetes species (voriconazole MICs ranged from 2 to >32 μg/ml). Voriconazole showed excellent in vitro activity (MICs < 0.03 to 1.0 μg/ml) against most of the 50 species of dematiaceous fungi tested, but the activity of all the agents was poor against most isolates of Scedosporium prolificans and Phaeoacremonium parasiticum (Phialophora parasitica). Voriconazole had fungicidal activity against most Aspergillus spp., B. dermatitidis, and some dematiaceous fungi. In vitro/in vivo correlations should aid in the interpretation of these results. This revised version was published online in June 2006 with corrections to the Cover Date.     

Increased levels of cytosolic phospholipase A2 in dyslexics

Research findings are increasingly reporting evidence of physiological abnormalities in dyslexia and sites for dyslexia have been identified on three chromosomes. It has been suggested that genetic inheritance may cause phospholipid abnormalities in dyslexia somewhat similar to those found in schizophrenia. A key enzyme in phospholipid metabolism, Type IV, or cytosolic, phospholipase A2 (cPLA2), releases arachidonic acid (AA), a 20-carbon fatty acid, which is the major source of production of prostaglandins and leukotrienes. An entirely new assay, which for the first time has enabled determination of the amount of the enzyme rather than its activity, was used to measure cPLA2 in dyslexic-type adults and controls and the two groups were found to differ significantly, the dyslexic-types having more of the enzyme. A report elsewhere of schizophrenics having even greater amounts of the enzyme suggests that dyslexia may be on a continuum with schizophrenia, as may be other neurodevelopmental disorders - which have also been described as phospholipid spectrum disorders.