Treatment of Refractory Gastrointestinal Bleeding in Patients With Portal Hypertension: A Case Series and Treatment Algorithm.

In patients with portal hypertension, ectopic varices can develop at any site along the gastrointestinal tract outside the classically described gastroesophageal location. Like esophageal variceal hemorrhage, bleeding from ectopic varices can be life-threatening. Diagnosis and treatment of ectopic varices can be challenging; to date, no effective treatment algorithm has been described. A systematic teamwork approach to diagnosing and treatment of ectopic varices is required to successfully manage hemorrhage from ectopic varices.

Competition for space among sessile marine invertebrates: changes in HSP70 expression in two Pacific cnidarians.

The role of stress proteins-either constitutive (HSC) or inducible (HSP)-of the HSP70 family in intra- and interspecific competition for space was examined in two sessile Pacific cnidarians. Anthopleura elegantissima, an intertidal anemone, and Corynactis californica, a subtidal corallimorpharian, express HSP70 in the absence of apparent physical stress. HSP70 protein expression is concentrated in the tentacles of A. elegantissima when the animal is exposed to contact with other benthic organisms. Under the same conditions, however, HSP concentrations are similar in the body and tentacles of C. californica. When two different clones of A. elegantissima interact in the field, the outside polyps (warriors) express more HSP70 than the inside ones (2.4 versus 0.6 ng HSP70/microg Protein). When different C. californica clones interact, HSP70 expression in the outside and inside polyps is similar (1.5 versus 1.8 ng HSP70/microg P) and is fairly constant in the corallimorpharian in the different interspecific encounters. HSP70 expression is related to the different kinds of aggression encountered by both cnidarians. HSP70 expression may be involved in the recovery of tissues damaged by the allelochemical, cytotoxical, or corrosive substances produced by different enemies. C. californica clones appear prepared for war, as evidenced by the high constant expression of HSP70 in the polyps. A. elegantissima exhibits differential HSP70 expression depending on the identity of each neighboring intra- or interspecific sessile competitor. We propose that stress proteins can be used to quantify space competition or aggression among sessile marine invertebrates.

Environmental endocrine disruption in decapod crustacean larvae: hormone titers, cytochrome P450, and stress protein responses to heptachlor exposure.

A variety of enzymes and other proteins are produced by organisms in response to xenobiotic exposures. Cytochrome P450s (CYP) are one of the major phase I-type classes of detoxification enzymes found in terrestrial and aquatic organisms ranging from bacteria to vertebrates. One of the primary functions of stress proteins (HSPs) is to aid in the recovery of damaged proteins by chaperoning their refolding. These and other biomarkers of xenobiotic exposure and resulting effects have not been studied in crustacean larvae. This information is of potential importance for environmental management and risk assessment. In this work, we have given Homarus americanus larvae single 24 h exposures to the cyclodiene pesticide heptachlor, a known environmental endocrine disruptor (EDC) on different days of the 1st larval instar. We followed these larvae during the first larval stage for effects on timing of ecdysis to 2nd stage, ecdysteroid molting hormone titers, and alterations in the levels of cytochrome P450 CYP45 and HSP70 proteins. Delays in ecdysis were correlated with alterations in ecdysteroid levels. This result provides clues that this pesticide may function as an environmental endocrine disruptor in crustaceans. CYP45 and HSP70 levels were significantly elevated for several days following heptachlor exposure. The elevation in HSP70 was prolonged depending on the day of pesticide exposure and this was directly related to the increase in mortality. These results demonstrate the utility of these measurements as potential biomarkers in crustacean larval developmental toxicology and EDC effects research.

Neuroactive steroids modulate crustacean locomotor activity.

We examined the effects of neuroactive steroids known to modulate gamma-aminobutyric acid(A) (GABA)(A) receptor activity, on locomotor activity in a submerged circular open-field apparatus. Juvenile male lobsters, Homarus americanus, were treated with a single administration of an agonist, 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone, 3alpha,5alpha-TH PROG), an antagonist, pregnenolone sulfate (PREGS), or vehicle alone. 3alpha,5alpha-TH PROG treatment (125 and 250 microg) significantly reduced while PREGS significantly elevated locomotor activity in a dose-dependent manner similar to diazepam. PREGS increased locomotor activity at 30 and 60 microg, while diminishing such activity and altering locomotor patterns at 120 microg. These results suggest that neuroactive steroids may affect crustacean GABA receptors in a fashion similar to the GABA(A) type found in the vertebrates, and that they may be involved in the regulation of locomotor behavior.

Hypoxia/ischemia depletes the rat perinatal subventricular zone of oligodendrocyte progenitors and neural stem cells.

Cerebral hypoxia/ischemia of the newborn has a frequency of 4/1,000 births and remains a major cause of cerebral palsy, epilepsy, and mental retardation. Despite progress in understanding the pathogenesis of hypoxic-ischemic injury, the data are incomplete regarding the mechanisms leading to permanent brain injury. Here we tested the hypothesis that cerebral hypoxia/ischemia damages stem/progenitor cells in the subventricular zone (SVZ), resulting in a permanent depletion of oligodendrocytes. We used a widely accepted rat model and examined animals at recovery intervals ranging from 4 h to 3 weeks. Within hours after the hypoxic-ischemic insult 20% of the total cells were deleted from the SVZ. The residual damaged cells appeared necrotic. During 48 h of recovery deaths accumulated; however, these later deaths were predominantly apoptotic. Many apoptotic SVZ cells stained with a marker for immature oligodendrocytes. At 3 weeks survival, the SVZ was smaller and markedly less cellular, and it contained less than 1/4 the normal complement of neural stem cells. The corresponding subcortical white matter was dysmyelinated, relatively devoid of oligodendrocytes and enriched in astrocytes. We conclude that neural stem cells and oligodendrocyte progenitors in the SVZ are vulnerable to hypoxia/ischemia. Consequently, the developmental production of oligodendrocytes is compromised and regeneration of damaged white matter oligodendrocytes does not occur resulting in failed regeneration of CNS myelin in periventricular loci. The resulting dysgenesis of the brain that occurs subsequent to perinatal hypoxic/ischemic injury may contribute to the cognitive and motor dysfunction that results from asphyxia of the newborn.

Induction of marine mollusc stress proteins by chemical or physical stress.

The cellular stress responses of most organisms in part involve the induction of a class of proteins called heat shock or stress proteins (HSPs) as a result of damage to existing proteins. Cellular proteins can be damaged by chemical exposures known to induce various HSPs. In these experiments, we examine the HSP responses of mussel (Mytilus galloprovincialis) and abalone (Haliotis rufescens) tissues to both thermal and chemical exposures. HSP70 isoforms, HSP60, and HSP90 all show varying induction capabilities. The results demonstrate that the extent of stress exposure as both a time- and dose-dependent phenomena can be ascertained by examining changes in mollusc HSP protein levels. We also examined the relationship between HSP induction and levels of a mussel cytochrome P450 (CYP4Y1) mRNA in dose-response experiments with the products of biologically degraded weathered crude oil. The increases in HSP70 isoforms and HSP90 were correlated with decreases in CYP4Y1 expression levels in a dose-dependent manner. HSP responses may therefore be a valuable part of a suite of biomarkers in biomonitoring for hydrocarbon exposures in nearshore environments.

Malignancy in endometriosis: frequency and comparison of ovarian and extraovarian types.

One thousand consecutive cases of surgically proven endometriosis were reviewed to evaluate the frequency and types of pelvic cancers that were associated with ovarian and extraovarian endometriosis. The frequency and types of histologic abnormalities present in the eutopic endometrium when cancers were noted in endometriosis were also evaluated. In the large subset of cases for which the authors were the primary pathologists and all foci of endometriosis were recorded, the frequency of malignancy was 10.8%. In contrast, the frequency was only 3.2% in cases diagnosed by others previously in our institution. Cancers were more commonly found in ovaries when endometriosis was present in that ovary (5%) compared to when endometriosis was present at other sites (1%). Clear cell and endometrioid carcinomas were the malignancies most commonly seen in ovaries containing endometriosis, while clear cell adenocarcinoma and adenosarcoma were most commonly seen in conjunction with extraovarian endometriosis. The association of endometriosis with endometrioid and clear cell carcinoma was much stronger than that of serous and mucinous tumors (p < .01). Concurrent endometrial pathology was commonly seen in cases of malignant transformation of endometriosis (32% of cases).

Inducible P450s of the CYP9 family from larval Manduca sexta midgut.

Several related cytochrome P450 cDNAs belonging to the CYP9 family have been cloned from the midgut of larval tobacco hornworms, Manduca sexta. The first P450, CYP9A2, was obtained by RT-PCR using degenerate primers. Northern blot analysis of expression in the midgut using the CYP9A2 probe revealed a significant induction by a variety of chemicals. Diets supplemented with the wild tomato compound 2-undecanone caused a dose-dependent induction which peaked after 48 h. Induction was also observed after addition to the diet of indole-3-carbinol, phenobarbital, 2-tridecanone and xanthotoxin. Neither alpha-pinene, clofibrate nor nicotine were effective inducers. The CYP9A2 probe hybridized to two mRNA species, one of 2. 0 kb and another of 4.2 kb, suggesting cross-hybridization to other P450 mRNAs. Additional P450 clones of the CYP9 family were then obtained and sequenced. Northern hybridization revealed that the 4.2 kb band also hybridized to CYP9A4 whereas the 2.0 kb hybridized to CYP9A5. Despite being 91% identical, CYP9A4 and CYP9A5 were induced differentially by clofibrate and xanthotoxin. Multiple P450 genes from various families are therefore induced in Lepidoptera in response to plant allelochemicals or xenobiotics.

The Worldwide Web: a panacea or a nuisance for nurses?

This report describes the use of the Web in many arenas as new venture e-health companies are being created. There is much controversy over whether these companies provide appropriate access to health care information. There also are many debates on how these e-health companies alter the patient-provider relationship. Nurses are frontline caregivers who must understand this new market and the shift in how information is increasingly available to the patient. This information transference change is more apparent daily. How nurses handle this paradigm shift and their attitude toward these changes will result in them viewing the web as a panacea or a nuisance in their practice.

Identification of a new cytochrome P450 family, CYP45, from the lobster, Homarus americanus, and expression following hormone and xenobiotic exposures.

A new cytochrome P450, the first member of the CYP45 family, was identified from the hepatopancreas of the American lobster, Homarus americanus. The lobster CYP45 shares significant sequence homologies to the vertebrate CYP3 and the invertebrate CYP6, CYP9, CYP28, and CYP30 families, perhaps indicating a common ancestor of these P450s. Of seven tissues examined, CYP45 was expressed only in the hepatopancreas, the crustacean equivalent of the vertebrate liver, pancreas, and intestine. Over the course of the lobster molt cycle, CYP45 expression mirrored the hemolymph titer of ecdysteroids, suggesting its potential involvement in molting hormone dynamics. This idea was strengthened further by ecdysteroid treatment of intermolt-stage lobsters during the lowest hemolymph titers and CYP45 expression levels. Significant elevations in hepatopancreas CYP45 mRNA levels were elicited by such injections over a 2- to 4-day interval. Similar experiments were performed by intubating juvenile lobsters with various xenobiotics. Induction of CYP45 expression occurred following phenobarbital and heptachlor administration, but not by beta-naphthoflavone. Hormonal and xenobiotic modulation of lobster CYP45 expression provides a potential pathway for endocrine disruption in lobsters.

Evidence for a diazepam-binding inhibitor (DBI) benzodiazepine receptor-like mechanism in ecdysteroidogenesis by the insect prothoracic gland.

The diazepam-binding inhibitor (DBI) is a 10-kDa highly evolutionarily conserved multifunctional protein. In mammals, one of DBI's functions is in the activation of steroid hormone biosynthesis via binding to a specific outer mitochondrial membrane receptor (benzodiazepine receptor, BZD) and promoting cholesterol transport to the inner membrane. In this work, a multitiered approach was utilized to study the role of this receptor-like activity in ecdysteroidogenesis by larval insect prothoracic glands (PGs). First, both DBI protein and messenger RNA (mRNA) levels were correlated with peak PG ecdysteroid production. In vitro ecdysteroid production was stimulated by the diazepam analogue FGIN 1-27 and inhibited anti-DBI antibodies. The DBI protein was found distributed throughout PG cells, including regions of dense mitochondria, supposed subcellular sites of ecdysteroid synthesis. Finally, a potential mitochondrial BZD receptor in PG cells was demonstrated by photoaffinity labeling. These results suggest an important role for the insect DBI in the stimulation of steroidogenesis by prothoracic glands and indicate that a pathway for cholesterol mobilization leading to the production of steroid hormones appears to be conserved between arthropods and mammals.

Cytochrome P450 enzymes belonging to the CYP4 family from marine invertebrates.

Six new cytochrome P450s assigned to the CYP4 family were identified from marine invertebrates belonging to the arthropod, mollusc, and echinoderm phyla. These are the first reported members of the CYP4 gene family from marine invertebrates, and extends the finding of the CYP4 family of cytochrome P450s to molluscs and echinoderms. Members of each phyla (echinodermata, arthropoda (crustacea), and mollusca) expressed genes belonging to the CYP4C subfamily in their respective digestive tissues. A mollusc, the mussel Mytilus galloprovincialis, expressed a gene belonging to a new CYP4 subfamily, CYP4Y. In Northern blotting experiments with digestive tissues of M. galloprovincialis, the expression of the CYP4Y1 gene was found to be inhibited by increasing concentrations of the hydrocarbon beta-naphthoflavone. Thus, the potential use of marine invertebrate CYP4 genes as biomarkers of xenobiotic exposures may be warranted.

Molecular phylogeny of glutathione-S-transferases.

The glutathione-S-transferase (GST) protein superfamily is currently composed of nearly 100 sequences. This study documents a greater phylogenetic diversity of GSTs than previously realized. Parsimony and distance phylogenetic methods of GST amino acid sequences yielded virtually the same results. There appear to be at least 25 groups (families) of GST-like proteins, as different from one another as are the currently recognized classes. This diversity will require the design of a new nomenclature for this large protein superfamily. There is one well-supported large clade containing the mammalian mu, pi, and alpha classes as well as GSTs from molluscs, helminths, nematodes, and arthropods.

Cellular distribution, levels, and function of the diazepam-binding inhibitor/acyl-CoA-binding protein in last instar Manduca sexta midgut.

The diazepam-binding inhibitor (DBI) was originally isolated as an endogenous competitor of diazepam for mammalian central nervous system binding sites. Later DBI was found to be identical with an intracellular medium-long chain acyl-CoA-ester-binding protein (ACBP). Its phylogenetic distribution was also extended outside vertebrates to insects and fungi. We studied DBI/ACBP biochemistry and ultrastructural distribution to learn more about the potential role(s) of the insect protein in lipid transport in the tobacco hornworm, Manduca sexta. We expressed and purfied the M. sexta DBI/ACBP from E. coli for the production of specific polyclonal antisera. We also showed specific binding of [14C]-oleoyl-CoA to the purified protein, supporting its evolutionarily conserved role as an acyl-CoA-binding protein. With the use of an enzyme-linked immunosorbant assay (ELISA) and Northern analysis, it was determined that the M. sexta ACBP is expressed highest during times of active feeding and lipid transport by the larval midgut. Study of the ultrastructural distribution, by immunocytochemistry and electron microscopy, of ACBP in larval midgut showed that acyl-CoA transport is localized throughout the M. sexta columnar cell.

Causal connection between detoxification enzyme activity and consumption of a toxic plant compound.

Insect herbivores can increase their detoxification activities against a particular plant poison in response to prolonged ingestion of the same compound. For example, larval tobacco hornworms (Manduca sexta) experience a dramatic increase in cytochrome P450 activity against nicotine after ingesting nicotine. While it is generally assumed that this induction process permits increased consumption of toxic plant tissues, we are not aware of any direct experimental support for this assumption. Using a two-tiered approach, we examined the functional significance of P450 induction to M. sexta larvae ingesting a toxic but non-deterrent concentration of nicotine. First, we related the time-course of P450 induction in midgut microsomes to changes in nicotine consumption. When offered a nicotine diet, larvae failed to show a significant increase in consumption before 36 h, which was coincident with the time-course of the induction of midgut P450 activities against aldrin and nicotine. Second, we determined whether inhibiting the induced P450 activities affected nicotine consumption. We found that the increase in nicotine consumption following the induction of nicotine metabolism could be strongly inhibited by treatment with piperonyl butoxide, which by itself did not inhibit consumption. These results provide direct evidence for a causal connection between P450-mediated detoxification activity and consumption of a toxic plant compound.

Expression of cytochrome P450 genes of the CYP4 family in midgut and fat body of the tobacco hornworm, Manduca sexta.

Two conserved regions in the alignment of cytochrome P450 family 4 (CYP4) proteins served as guide to the synthesis of degenerate oligonucleotide primers. The primers were used in PCR from a midgut cDNA library and RT-PCR from fat body mRNA, both from last instar larvae of the tobacco hornworm, Manduca sexta. The PCR products of 443-449 bp were cloned and sequenced. Nine P450 clones representing four new genes were obtained from the midgut. Fifteen P450 clones representing three new genes were obtained from the fat body. Two genes were expressed in both tissues. A number of putative allelic variants were also observed for three of the P450 genes. The resulting sequences of 130-132 amino acids were aligned to generate a parsimony analysis of CYP4 P450 proteins. Two new subfamilies of CYP4 were designated from M. sexta by these procedures, CYP4L and CYP4M. The sequence of a full-length cDNA clone for CYP4M2 (41.2% identity to CYP4C1) confirmed that the PCR products obtained by this method were P450s belonging to the CYP4 family. The developmental expression of the CYP4 genes appeared to be coordinately regulated in both fat body and midgut. In the fat body, CYP4 mRNA levels declined after the first day of the final larval instar, peaked during the wandering stage, and fell again until the prepupal molt. Midgut CYP4 mRNA levels were higher during the active feeding, midwandering, prepupal, and pupal stages. Addition of 2-tridecanone or 2-undecanone to the diet induced several P450s in the midgut and in the fat body. Phenobarbital induced CYP4M1 in the fat body and dietary clofibrate induced the mRNA levels of CYP4M1 and CYP4M3 in the midgut. The results indicate that at least four CYP4 genes are expressed in single tissues of a Lepidopteran insect. Several of these P450 may be involved in tissue responses to xenobiotics.

Glutathione S-transferases from larval Manduca sexta midgut: sequence of two cDNAs and enzyme induction.

Two glutathione S-transferase (GST) clones from a larval midgut cDNA library of the tobacco hornworm, Manduca sexta were sequenced. The nucleotide sequence of the first clone, M. sexta GST1, encoded a protein of 217 amino acids with a predicted molecular weight of 24,644 and isoelectric point of 4.8. The M. sexta GST1 was 45.9-48.6% identical to GSTs from Musca domestica and several Drosophila species. The M. sexta GST2 cDNA encoded a protein of 203 amino acids with a predicted molecular weight of 23,596 and isoelectric point of 5.5. The M. sexta GST2 shared 44.8-50.0% sequence identity to a second cluster of insect GSTs from M. domestica, D. melanogaster and Anopheles gambiae. GST1 and GST2 were only 24.1% identical in amino acid sequence. The divergence of these two classes of insect GSTs occurred before the radiation of Diptera and Lepidoptera. Northern analysis of the expression of these GSTs showed increased GST1 mRNA levels in midguts of larvae fed diets containing 2-undecanone, or phenobarbital. Midgut and fat body cytosolic GST activities were induced when larvae were fed diets containing 2-tridecanone, 2-undecanone, or phenobarbital. Partial purification of midgut GSTs by size-exclusion and glutathione affinity chromatography resulted in a series of isoelectric focusing bands, with the major one corresponding to the predicted isoelectric point of the M. sexta GST1. In summary, two midgut GSTs have been identified on the basis of cDNA sequence and one of these, GST1, was inducible by dietary chemicals.