Hemoglobin-degrading, aspartic proteases of blood-feeding parasites - Substrate specificity revealed by homology models

Blood-feeding parasites, including schistosomes, hookworms, and malaria parasites, employ aspartic proteases to make initial or early cleavages in ingested host hemoglobin. To better understand the substrate affinity of these aspartic proteases, sequences were aligned with and/or three-dimensional, molecular models were constructed of the cathepsin D-like aspartic proteases of schistosomes and hookworms and of plasmepsins of Plasmodium falciparum and Plasmodium vivax, using the structure of human cathepsin D bound to the inhibitor pepstatin as the template. The catalytic subsites S5 through S4' were determined for the modeled parasite proteases. Subsequently, the crystal structure of mouse renin complexed with the nonapeptidyl inhibitor t-butyl-CO-His-Pro-Phe-His-Leu [CHOHCH2]Leu-Tyr-Tyr-Ser-NH2 (CH-66) was used to build homology models of the hemoglobin-degrading peptidases docked with a series of octapeptide substrates. The modeled octapeptides included representative sites in hemoglobin known to be cleaved by both Schistosoma japonicum cathepsin D and human cathepsin D, as well as sites cleaved by one but not the other of these enzymes. The peptidase-octapeptide substrate models revealed that differences in cleavage sites were generally attributable to the influence of a single amino acid change among the P5 to P4' residues that would either enhance or diminish the enzymatic affinity. The difference in cleavage sites appeared to be more profound than might be expected from sequence differences in the enzymes and hemoglobins. The findings support the notion that selective inhibitors of the hemoglobin-degrading peptidases of blood-feeding parasites at large could be developed as novel anti-parasitic agents.

Gulliver, a long terminal repeat retrotransposon from the genome of the oriental blood fluke Schistosoma japonicum

We characterized the consensus sequence and structure of a long terminal repeat (LTR) retrotransposon from the genome of the human blood fluke, Schistosoma japonicum, and have earned this element, Gulliver. The full length, consensus Gulliver LTR retrotransposon was 4788 bp, and it was flanked at its 5'- and 3'-ends by LTRs of 259 bp. Each LTR included RNA polymerase II promoter sequences, a CAAT signal and a TATA box, Gulliver exhibited features characteristic of a functional LTR retrotransposon including two read through (termination) ORFs encoding retroviral gag and pol proteins of 312 and 1071 amino acid residues, respectively. The gag ORF encoded motifs conserved in nucleic acid binding proteins, while the pol ORF encoded conserved domains of aspartic protease, reverse transcriptase (RT), RNaseH and integrase, in that order, a pol pattern conserved in the gypsy lineage of LTR retrotransposons. Whereas the sequence and structure of Gulliver was similar to that of gypsy, phylogenetic analysis revealed that Gulliver did not group particularly closely with the gypsy family. Rather, its closest relatives were a LTR retrotransposon from Caenorhabditis elegans, mag from Bombyx mori and, to a lesser extent, easel from the salmon Oncorhynchus keta. Dot blot hybridizations indicated that Gulliver was present at between 100 and several thousand copies in the S. japonicum genome, and Southern hybridization analysis suggested its probable presence in the genome of Schistosoma mansoni. Transcripts encoding the RT domain of Gulliver were detected by RT-PCR in larval and adult stages of S. japonicum, indicating that (at least) the RT domain of Gulliver is transcribed. This is the first report of the sequence and structure of an LTR retrotransposon from any schistosome or indeed from any species belonging to the phylum Platyhelminthes. (C) 2001 Elsevier Science B.V. All rights reserved.

Postoperative vaginal vault brachytherapy for node-negative Stage II (occult) endometrial carcinoma

Objective. The aim of this study is to look at the efficacy of extended surgical staging and postoperative vaginal vault brachytherapy in patients with Stage II (occult) endometrial carcinoma. Methods. Between January 1989 and December 1997, there were 30 patients with Stage II (occult) endometrial carcinoma who received postoperative vaginal vault brachytherapy as the only adjuvant treatment. The study group consisted of 15 of these patients who had extended surgical staging (including lymphadenectomy). Results. At a median follow-up of 36 months (range 17 to 113 months), there has been no recurrence. There were no major complications from surgery. Only 1 patient had mild rectal bleeding following vaginal vault brachytherapy and there were no grade 3 or 4 bowel toxicities. Conclusions. Extended surgical staging and postoperative vaginal vault brachytherapy for Stage II (occult) endometrial carcinoma is associated with minimal morbidity and excellent survival. (C) 2001 Academic Press.

Peritoneal cytology: impact on disease-free survival in clinical stage I endometrioid adenocarcinoma of the uterus

The prognostic significance of positive peritoneal cytology in endometrial carcinoma has led to the incorporation of peritoneal cytology into the current FIGO staging system, While cytology was shown to be prognostically relevant in patients with stage II and III disease, conflicting data exists about its significance in patients who would have been stage I but were classified as stage III solely and exclusively on the basis of positive peritoneal cytology (clinical stage I). Analysis was based on the data of 369 consecutive patients with clinical stage I endometrioid adenocarcinoma of the endometrium. Standard treatment consisted of an abdominal total hysterectomy, bilateral salpingo-oophorectomy with or without pelvic lymph node dissection. Peritoneal cytology was obtained at laparotomy by peritoneal washing of the pouch of Douglas and was considered positive if malignant cells could be detected regardless of the number of malignant cells present. Disease-free survival (DFS) was considered the primary statistical endpoint. In 13/369 (3.5%) patients, positive peritoneal cytology was found. The median follow-up was 29 months and 15 recurrences occurred. Peritoneal cytology was independent of the depth of myometrial invasion and the grade of tumour differentiation, Patients with negative washings had a DFS of 96'7e at 36 months compared with 67% for patients with positive washings (log-rank P < 0.001). The presence of positive peritoneal cytology in patients with clinically stage I endometrioid adenocarcinoma of the endometrium is considered an adverse prognostic factor. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Immunoelectron microscopy provides evidence for the presence of mitochondrial heat shock 10-kDa protein (chaperonin 10) in red blood cells and a variety of secretory granules

Hsp10 (10-kDa heat shock protein, also known as chaperonin 10 or Cpn10) is a co-chaperone for Hsp60 in the protein folding process. This protein has also been shown to be identical to the early pregnancy factor, which is an immunosuppressive growth factor found in maternal serum. In this study we have used immunogold electron microscopy to study the subcellular localization of Hsp10 in rat tissues sections embedded in LR Gold resin employing polyclonal antibodies raised against different regions of human Hsp10. In all rat tissues examined including liver, heart, pancreas, kidney, anterior pituitary, salivary gland, thyroid, and adrenal gland, antibodies to Hsp10 showed strong labeling of mitochondria. However, in a number of tissues, in addition to the mitochondrial labeling, strong and highly specific labeling with the Hsp10 antibodies was also observed in several extramitochondrial compartments. These sites included zymogen granules in pancreatic acinar cells, growth hormone granules in anterior pituitary, and secretory granules in PP pancreatic islet cells. Additionally, the mature red blood cells which lack mitochondria, also showed strong reactivity with the Hsp10 antibodies. The observed labeling with the Hsp10 antibodies, both within mitochondria as well as in other compartments/cells, was abolished upon omission of the primary antibodies or upon preadsorption of the primary antibodies with the purified recombinant human Hsp10. These results provide evidence that similar to a number of other recently described mitochondrial proteins (viz., Hsp60, tumor necrosis factor receptor-associated protein- 1, P32 (gC1q-R) protein, and cytochrome c), Hsp10 is also found at a variety of specific extramitochondrial sites in normal rat tissue. These results raise important questions as to how these mitochondrial proteins are translocated to other compartments and their possible function(s) at these sites. The presence of these proteins at extramitochondrial sites in normal tissues has important implications concerning the role of mitochondria in apoptosis and genetic diseases.

SOX18 and the transcriptional regulation of blood vessel development

SOX18 is a transcription factor that is transiently expressed in nascent endothelial cells during embryonic development and adult neovascularization. This protein belongs to the SOX family of transcription factors, ih,which are proving to be some of the key regulators of cell-type specification in the vertebrate embryo. Natural mutations in the Sox18 gene have been shown to result to cardiovascular dysfunction, in some cases leading to death. Available evidence thus implicates Sox18 as an important regulator of vascular development, most likely playing a key role in endothelial cell specification. However; the genetic knockout of Sox18 in mice has produced a confounding result that complicates our understanding of the molecular mode of action of the SOX18 protein. We speculate that Sox18 inky act in a redundant fashion with closely related genes such as Sox7 and/or Sox17. (C) 2001, Elsevier Science Inc.

Sox18 expression in blood vessels and feather buds during chicken embryogenesis

Sox18 encodes a transcription factor known to be important for the development of blood vessels and hair follicles in mice. In order to study the functional conservation of this gene through evolution, we have isolated and characterized Sox18 in chickens. cSox18 shows a high degree of sequence homology to both the mouse and human orthologues, particularly in the high mobility group DNA-binding domain and to a lesser extent in the transcriptional activation domain. A region of unusually high sequence conservation at the C-terminus may represent a further, previously unrecognized functional domain. Both the chicken and human proteins appear to be truncated at the N-terminus relative to mouse SOX18. In situ hybridization analyses showed expression in the developing vasculature and feather follicles, consistent with reported expression in the mouse embryo. In addition, cSox18 mRNA was observed in the retina and claw beds. (C) 2001 Elsevier Science B.V. All rights reserved.

Repeated blood pressure measurements in a sample of Swedish twins: heritabilities and associations with polymorphisms in the renin-angiotensin-aldosterone system Iliadou, Anastasia, Lichtenstein, Paul, Morgenstern, Ralf, Forsberg, Lena, Svensson, Richard, de Faire, Ulf, Martin, Nicholas, Pedersen, Nancy

Background Twin and family studies have shown that genetic effects explain a relatively high amount of the phenotypic variation in blood pressure. However, many studies have not been able to replicate findings of association between specific polymorphisms and diastolic and systolic blood pressure. Methods In a structural equation-modelling framework the authors investigated longitudinal changes in repeated measures of blood pressures in a sample of 298 like-sexed twin pairs from the population-based Swedish Twin Registry. Also examined was the association between blood pressure and polymorphisms in the angiotensin-I converting enzyme and the angiotensin 11 receptor type 1 with the 'Fulker' test Both linkage and association were tested simultaneously revealing whether the polymorphism is a Quantitative Trait Locus (QTL) or in linkage disequilibrium with the QTL. Results Genetic influences explained up to 46% of the phenotypic variance in diastolic and 63% of the phenotypic variance in systolic blood pressure. Genetic influences were stable over time and contributed up to 78% of the phenotypic correlation in both diastolic and systolic blood pressure. Non-shared environmental effects were characterised by time specific influences and little transmission from one time point to the next. There was no significant linkage and association between the polymorphisms and blood pressure. Conclusions There is a considerable genetic stability in both diastolic and systolic blood pressure for a 6-year period of time in adult life. Non-shared environmental influences have a small long-term effect Although associations with the polymorphisms could not be replicated, results should be interpreted with caution due to power considerations. (C) 2002 Lippincott Williams Wilkins.

Estimating Two-Stage Models for Genetic Influences on Alcohol, Tobacco or Drug Use Initiation and Dependence Vulnerability in Twin and Family Data

Genetic research on risk of alcohol, tobacco or drug dependence must make allowance for the partial overlap of risk-factors for initiation of use, and risk-factors for dependence or other outcomes in users. Except in the extreme cases where genetic and environmental risk-factors for initiation and dependence overlap completely or are uncorrelated, there is no consensus about how best to estimate the magnitude of genetic or environmental correlations between Initiation and Dependence in twin and family data. We explore by computer simulation the biases to estimates of genetic and environmental parameters caused by model misspecification when Initiation can only be defined as a binary variable. For plausible simulated parameter values, the two-stage genetic models that we consider yield estimates of genetic and environmental variances for Dependence that, although biased, are not very discrepant from the true values. However, estimates of genetic (or environmental) correlations between Initiation and Dependence may be seriously biased, and may differ markedly under different two-stage models. Such estimates may have little credibility unless external data favor selection of one particular model. These problems can be avoided if Initiation can be assessed as a multiple-category variable (e.g. never versus early-onset versus later onset user), with at least two categories measurable in users at risk for dependence. Under these conditions, under certain distributional assumptions., recovery of simulated genetic and environmental correlations becomes possible, Illustrative application of the model to Australian twin data on smoking confirmed substantial heritability of smoking persistence (42%) with minimal overlap with genetic influences on initiation.

Update on intrapartum fetal pulse oximetry

This article examines the current status of fetal pulse oximetry (FPO) as a means of intrapartum assessment of fetal wellbeing. FPO has been developed to a stage where it is a safe and accurate indicator of intrapartum fetal oxygenation. In general, sliding the FPO sensor along the examiner's fingers and through the cervix, to lie alongside the fetal cheek or temple is easy The recent publication of a randomised controlled trial (RCT) of FPO versus conventional intrapartum monitoring has validated its use to reduce caesarean section rates for nonreassuring fetal status. An Australian multicentre RCT is currently underway. Maternal satisfaction rates with FPO are high. FPO may be used during labour when the electronic fetal heart rate trace is nonreassuring or when conventional monitoring is unreliable, such as with fetal arrhythmias. If the fetal oxygen saturation (FSpO(2)) values are < 30%, prompt obstetric intervention is indicated, such as fetal scalp blood sampling or delivery FSpO(2) monitoring should not form the sole basis of intrapartum fetal welfare assessment. Rather, the whole clinical picture should be considered.

Vascular dysfunction and heart failure: Epiphenomenon or etiologic agent?

Endothelial function plays a key role in the local regulation of vascular tone. Alterations in endothelial function may result in impaired release of endothelium-derived relaxing factors or increased release of endothelium-derived contracting factors. Heart failure may impair endothelial function by means of reduced synthesis and release of nitric oxide (NO) or by increased degradation of NO and increased production of endothelin-1. Endothelial dysfunction may worsen heart function by means of peripheral effects, causing increased afterload and central effects such as myocardial ischemia and inducible nitric oxide synthase (iNOS)-induced detrimental effects. Evidence from clinical studies has suggested that there is a correlation between decreased endothelial function and increasing severity of congestive heart failure (CHF). Treatments that improve heart function may also improve endothelial dysfunction. The relationship between endothelial dysfunction and heart failure may be masked by the stage of endothelial dysfunction, the location of vessels being tested, and the state of endothelial-dependent vasodilatation response.

Quantification and stability of everolimus (SDZ RAD) in human blood by high-performance liquid chromatography-electrospray tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug, everolimus (SDZ RAD), using HPLC-tandem mass spectrometry. Whole blood samples (500 mul) were prepared by protein precipitation, followed by C-18 solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface operating in positive ionization mode. The assay was linear from 0.5 to 100 mug/l (r(2) > 0.996, n = 9). The analytical recovery and inter-day imprecision, determined using whole blood quality control samples (n = 5) at 0.5, 1.2, 20.0, and 75.0 mug/l, was 100.3-105.4% and less than or equal to7.6%, respectively. The assay had a mean relative recovery of 94.8 +/- 3.8%. Extracted samples were stable for up to 24 h. Fortified everolimus blood samples were stable at -80 degreesC for at least 8 months and everolimus was found to be stable in blood when taken through at least three freeze-thaw cycles. The reported method provides accurate, precise and specific measurement of everolimus in blood over a wide analytical range and is currently supporting phase 11 and III clinical trials. (C) 2002 Elsevier Science B.V. All rights reserved.

Application of spatial analysis techniques to adjust for fertility trends and identify interplot competition in early stage sugarcane selection trials

Most sugarcane breeding programs in Australia use large unreplicated trials to evaluate clones in the early stages of selection. Commercial varieties that are replicated provide a method of local control of soil fertility. Although such methods may be useful in detecting broad trends in the field, variation often occurs on a much smaller scale. Methods such as spatial analysis adjust a plot for variability by using information from immediate neighbours. These techniques are routinely used to analyse cereal data in Australia and have resulted in increased accuracy and precision in the estimates of variety effects. In this paper, spatial analyses in which the variability is decomposed into local, natural, and extraneous components are applied to early selection trials in sugarcane. Interplot competition in cane yield and trend in sugar content were substantial in many of the trials and there were often large differences in the selections between the spatial and current method used by the Bureau of Sugar Experiment Stations. A joint modelling approach for tonnes sugar per hectare in response to fertility trends and interplot competition is recommended.