Search

Pro-Pro-endopeptidases (PPEP, EC 3.4.24.89) are secreted, zinc metalloproteases that have the unusual capacity to cleave a peptide bond between two prolines, a bond that is generally less sensitive to proteolytic cleavage. Two well studied members of the family are PPEP-1 and PPEP-2, produced by Clostridioides difficile, a human pathogen, and Paenibacillus alvei, a bee secondary invader, respectively. Both proteases seem to be involved in mediating bacterial adhesion by cleaving cell surface anchor proteins on the bacterium itself.By using basic alignment and phylogenetic profiling analysis, this work shows that the complete family of proteins that contain a PPEP domain includes proteins from more than 130 species spread over 9 genera. These analyses also suggest that the PPEP domain spread through horizontal gene transfer events between species within the Firmicutes’ classes Bacilli and Clostridia. Bacterial species containing PPEP homologs are found in diverse habitats, varying from human pathogens and gut microbiota to free-living bacteria, which were isolated from various environments, including extreme conditions such as hot springs, desert soil and salt lakes.The phylogenetic tree reveals the relationships between family members and suggests that smaller subgroups could share cleavage specificity, substrates and functional similarity. Except for PPEP-1 and PPEP-2, no cleavage specificity, specific physiological target, or function has been assigned for any of the other PPEP-family members. Some PPEP proteins have acquired additional domains that recognize and bind noncovalently to various elements of the bacterial peptidoglycan cell-wall, anchoring these PPEPs. Secreted or anchored to the cell-wall surface PPEP proteins seem to perform various functions.

Under controlled laboratory conditions, toxicity data tend to be less variable than in more realistic in-field studies and responses may thus differ from those in the natural environment, creating uncertainty. The validation of data under environmental conditions is therefore a major asset in environmental risk assessment of chemicals. The present study aimed to validate the mode of action of a commercial fungicide formulation in the soil invertebrate F. candida, under more realistic exposure scenarios (in-field bioassay), by targeting specific molecular biomarkers retrieved from laboratory experiments. Organisms were exposed in soil cores under minimally controlled field conditions for 4 days to a chlorothalonil fungicide dosage causing 75% reduction of reproduction in a previous laboratory experiment (127 mg a.i. kg−1) and half this concentration (60 mg a.i. kg−1). After exposure, organisms were retrieved and RNA was extracted from each pool of organisms. According to previous laboratorial omics results with the same formulation, ten genes were selected for gene expression analysis by qRT-PCR, corresponding to key genes of affected biological pathways including glutathione metabolism, oxidation-reduction, body morphogenesis, and reproduction. Six of these genes presented a dose-response trend with higher up- or down-regulation with increasing pesticide concentrations. Highly significant correlations between their expression patterns in laboratory and in-field experiments were observed. This work shows that effects of toxicants can be clearly demonstrated in more realistic conditions using validated biomarkers. Our work outlines a set of genes that can be used to assess the early effects of pesticides in a realistic agricultural scenario. Keywords: Biomarkers, Chlorothalonil, Collembola, In situ validation, Fungicides, qPCR

Chemical stressors can induce epigenomic changes, i.e., changes that are transferred to the next generation, even when the stressor is removed. Literature on chemical induced epigenetic effects in environmental species is scarce. We here provide the first results on epigenetic effects caused by nanomaterials with an environmental OECD standard soil model species Enchytraeus crypticus species. We assessed the epigenetic potential in terms of global DNA methylation, gene-specific methylation via bisulfite sequencing and MS-HRM (Methylation Sensitive - High Resolution Melting), and gene expression qPCR for genes involved in DNA methylation, histone modifications, non-coding RNA and stress response mechanisms).We have exposed E. crypticus in a multigenerational (MG) test design to Cu (copper oxide nanomaterials (CuO NMs) and copper salt (CuCl2)). To link possible epigenetic effects to population changes, we used exposure concentrations (ECx) that caused a 10% and 50% reduction in the reproductive output (10% and 50% are the standards for regulatory Risk Assessment), the organisms were exposed for five consecutive generations (F1–F5) plus two generations after transferring to clean media (F5–F7), 7 generations in a total of 224 days. Results showed that MG exposure to Cu increased global DNA methylation and corresponded with phenotypic effects (reproduction). Gene expression analyses showed changes in the epigenetic, stress and detoxification gene targets, depending on the generation and Cu form, also occurring in post-exposure generations, hence indicative of transgenerational effects. There were in general clear differences between organisms exposed to different Cu-forms, hence indicate nanoparticulate-specific effects. Keywords: Methylation, Transgenerational, Methylation sensitive high resolution melting

This paper addresses women's empowerment in agriculture, innovations in its measurement, and emerging evidence. We discuss the evolution of the conceptualization and measurement of women's empowerment and gender equality since 2010. Using a gender and food systems framework and a standardized measure of women's empowerment, the Women's Empowerment in Agriculture Index (WEAI), we review the evidence on "what works" to empower women based on impact evaluations of a portfolio of 11 agricultural development projects with empowerment objectives and a scoping review of livestock interventions. We then review the evidence on associations between empowering women and societal benefits--agricultural productivity, incomes, and food security and nutrition. We conclude with recommendations for measurement and policy., Competing Interests: The authors declare that they have no conflict of interest., (© 2023 The Authors.)

Neuropeptides constitute a large and diverse class of signaling molecules that are produced by many types of neurons, neurosecretory cells, endocrines and other cells. Many neuropeptides display pleiotropic actions either as neuromodulators, co-transmitters or circulating hormones, while some play these roles concurrently. Here, we highlight pleiotropic functions of neuropeptides and different levels of neuropeptide signaling in the brain, from context-dependent orchestrating signaling by higher order neurons, to local executive modulation in specific circuits. Additionally, orchestrating neurons receive peptidergic signals from neurons conveying organismal internal state cues and relay these to executive circuits. We exemplify these levels of signaling with four neuropeptides, SIFamide, short neuropeptide F, allatostatin-A and leucokinin, each with a specific expression pattern and level of complexity in signaling., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We describe here the anti-HBV activity of natural and synthetic retinoids in primary human hepatocytes (PHHs). The most potent compounds inhibited HBsAg, HBeAg, viral RNA and DNA production by HBV infected cells with EC 50 values ranging from 0.4 to 2.6 μM. The activity was independent of PHH donor and HBV genotype used in testing. 13-cis retinoic acid (Accutane) was selected for further evaluation in the PXB chimeric mouse model of HBV infection at doses allowing to achieve Accutane peak serum concentrations near its antiviral EC 90 and exposures ∼5-fold higher than a typical clinical dose. While these supraclinical exposures of 100 mg/kg/day were well-tolerated by regular Balb/c mice, PXB mice were more sensitive and even a lower those of 60 mg/kg/day led to significant weight loss. Despite dosing at this maximal tolerated dose for 28 days, Accutane failed to show any anti-HBV activity. RAR target engagement was verified using transcriptome analysis of liver samples from treated versus vehicle groups. However, gene expression changes in PXB liver samples were vastly muted when compared to the in vitro PHH system. When comparing transcriptional changes associated with the conditioning of fresh hepatocytes toward enabling HBV infection, we also observed a large number of changes. Noticeably, a significant number of genes that were up- or down-regulated by the conditioning process were down- or up-regulated by HBV infected PHH treatment with Accutane, respectively. While the lack of efficacy in the PXB model may have many explanations, the observed, opposing transcriptional changes upon conditioning PHH and treating these cultured, HBV-infected PHH with Accutane allow for the possibility that the PHH system may yield artificial anti-HBV hits., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

It is unclear if endometrial cancer (EC) patients are aware of their modifiable risk factors. We administered a 33-item questionnaire to EC patients at a university-based cancer center to assess their understanding of how comorbidities and lifestyle/sexual behaviors impact their cancer risk. We also inquired about their access to a primary care physician (PCP). Pearson's χ 2 test or Fisher's exact test were used to assess differences in understanding based on a dichotomized Charlson comorbidity score, <7 vs ≥7. Of the 50 surveyed women (81% response rate), 39 reported hypertension (80%) and 36 (72%) diabetes. All had a PCP. Most were aware that obesity contributes to diabetes (43/48, 90%), hypertension (42/48, 88%), and heart attack (42, 88%), but only 19/49 (39%) knew that EC is more common in overweight/obese women. More than half lacked understanding of the following risks including modifiable risk factors-unhealthy diet (31, 62%), hormone replacement therapy (38, 76%), alcohol (30, 60%), and the protective effects of cigarette smoking (38, 76%). Most also incorrectly identified the following sexual health factors as risks for EC: early coitarche (30, 60%), or having an abortion (27, 54%), a sexually transmitted infection (35, 70%) or human immunodeficiency virus (34, 68%). Although EC patients recognize that obesity is linked to comorbidities, less than half are aware that it contributes to their cancer risk. Furthermore, responses to lifestyle/sexual health behaviors suggest women may lack understanding of global differences between endometrial and cervical cancer risk factors., Competing Interests: Dr. McCourt reports UpToDate Ad Hoc section editor for EC. Dr. Thaker reports personal fees from Celsion, personal fees from Stryker, grants and personal fees from Tesaro, grants and personal fees from Merck, personal fees from Abbvie, personal fees from Clovis, personal fees from AstraZeneca outside the submitted work. Dr. Powell reports personal fees from Merck, personal fees from Tesaro, personal fees from Clovis Oncology, personal fees from AstraZeneca, personal fees from Roche/Genentech, personal fees from GOG Foundation, outside the submitted work. Dr. Kuroki reports grants from Washington University Institute of Clinical and Translational Sciences (R25 STRENGTH and KL2) during the conduct of the study. All other authors declare no potential conflict of interest.

The social identity approach has been found very useful for the understanding of a range of phenomena within and across organizations. It has been applied in particular to analyze employees' stress and well-being at work and their reactions to organizational change. In this paper, we argue that there is a mismatch between the theoretical notion of shared identities in teams and organizations and empirical research, which largely focuses on the individual employee's identification with his or her social categories at work. We briefly review the literature in the two areas of stress and change and conclude with an agenda for future research., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Sustainable Development Goal 5 (SDG) on gender equality and women's rights and at least 11 of the 17 SDGs require indicators related to gender dynamics. Despite the need for reliable indicators, stylized facts on women, agriculture, and the environment persist. This paper analyzes four gender myths: 1) 70% of the world's poor are women; 2) Women produce 60 to 80% of the world's food; 3) Women own 1% of the world's land; and 4) Women are better stewards of the environment. After reviewing the conceptual and empirical literature, the paper presents the kernel of truth underlying each myth, questions its underlying assumptions and implications, and examines how it hinders us from developing effective food security policies.

Introduction: Sexual violence (SV) on college campuses is common, especially alcohol-related SV. This is a 2-arm cluster randomized controlled trial to test a brief intervention to reduce risk for alcohol-related sexual violence (SV) among students receiving care from college health centers (CHCs). Intervention CHC staff are trained to deliver universal SV education to all students seeking care, to facilitate patient and provider comfort in discussing SV and related abusive experiences (including the role of alcohol). Control sites provide participants with information about drinking responsibly., Methods: Across 28 participating campuses (12 randomized to intervention and 16 to control), 2292 students seeking care at CHCs complete surveys prior to their appointment (baseline), immediately after (exit), 4months later (T2) and one year later (T3). The primary outcome is change in recognition of SV and sexual risk. Among those reporting SV exposure at baseline, changes in SV victimization, disclosure, and use of SV services are additional outcomes. Intervention effects will be assessed using generalized linear mixed models that account for clustering of repeated observations both within CHCs and within students., Results: Slightly more than half of the participating colleges have undergraduate enrollment of ≥3000 students; two-thirds are public and almost half are urban. Among participants there were relatively more Asian (10 v 1%) and Black/African American (13 v 7%) and fewer White (58 v 74%) participants in the intervention compared to control., Conclusions: This study will offer the first formal assessment for SV prevention in the CHC setting. Clinical Trials #: NCT02355470., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Although Absorb Bioresorbable Vascular Scaffolds (A-BVS) are routinely used in the Asia-Pacific, there is little information on patient selection or deployment technique here. This document investigates the experiences of leading interventional cardiologists from the Asia-Pacific region with a focus on patient characteristics, deployment techniques and management., Methods and Results: A detailed questionnaire was distributed to 28 highly-experienced interventional cardiologists ('Authors') from 13 Asia-Pacific countries. The results were discussed at a meeting on patient selection, technical consideration, deployment practices and patient management. Potential patient benefits of Absorb compared to metallic DES, the learning curve for patient selection and preparation, device deployment, and subsequent patient management approaches are presented., Conclusions: Current practices are derived from guidelines optimized for European patients. Differences in approach exist in the Asia-Pacific context, including limited access to imaging and frequency of occurrence of complex lesions. Nevertheless, the use of the Absorb BVS ('Absorb') in certain Asia-Pacific countries has flourished and practices here are continuing to mature., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Combining various antiretroviral agents into one single dosage form has been a strategy to reduce pill burden and enhance medication adherence among human immunodeficiency virus /AIDS (HIV/AIDS) patients., Objectives: This is a cost-utility study from a health care system's perspective comparing coformulated fixed dose (FXD) strategy versus multiple free dose combination (FRC) in antiretroviral therapy., Method: The Medical Expenditure Panel Survey (MEPS) was used to identify HIV/AIDS patients with ≥2 active antiretroviral medications. Patients on FXD were matched in 1:1 ratio with the FRC group using propensity scores. All medical costs excluding those paid by patients and families were included. Utility was measured using SF-6D scores from the SF-12 questionnaire. Incremental cost-utility ratios (ICURs) were calculated using the mean annual estimates. A cost-effectiveness acceptability curve was determined using a Monte Carlo probabilistic simulation technique., Results: Nine FXD antiretroviral formulations approved by the U.S. Food and Drug Administration by 2005 was included in this study. One hundred seventy HIV/AIDS patients with ≥2 antiretroviral agents were identified from the MEPS database, of which 53% (n=92) were on FXD formulation. On matching, 70 patients from FXD had a match from the FRC group. No differences in sociodemographic and health status variables were observed between the matched groups. The mean annual cost was $15,766.15 for FXD patients and $11,875.21 for FRC patients. The mean utility gained by using FXD over FRC was 0.085; however, this difference was not statistically significant. The ICUR for the FXD treatment over FRC treatment was $45,540.49/quality-adjusted life years (QALYs). Probabilistic sensitivity analysis showed FXD to dominate FRC (>50% probability of being cost-effective) above the $40,000 threshold., Conclusion: Although the cost-effectiveness of a single-pill strategy was within the acceptable willingness-to-pay threshold, the QALY difference were minimal. Further research is recommended to explore the long-term impact of the strategy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Previous research on direct-to-consumer advertising (DTCA) has not focused exclusively on the African American population., Purposes: The purpose of this study was to explore African Americans' attitudes toward proactive health behaviors following exposure to DTCA of atorvastatin calcium (Lipitor, Pfizer Inc)., Materials and Methods: One-hundred fifty African American patients participated in the study. Participants' functional health literacy and health locus of control were assessed. The participants were asked to view a DTCA of Lipitor, followed by face-to-face interviews., Results: After watching the DTCA of Lipitor, 89.4% of participants agreed that they would talk to their physician about their cholesterol, 88.6% agreed that they would ask their physician to test their cholesterol level, and 47.3% agreed that they would ask their physician to write them a prescription for Lipitor. Those who had a history of high cholesterol were more likely to agree to ask their physician to test their cholesterol levels. Low household income, having public health insurance, and prior experience with taking Lipitor were significant positive predictors of patients agreeing to ask their physician to write a prescription of the advertised drug., Conclusions: African American patients showed favorable attitudes toward proactive health behaviors after exposure to DTCA of Lipitor.

Benzoxazinoids are secondary metabolites that are effective in defence and allelopathy. They are synthesised in two subfamilies of the Poaceae and sporadically found in single species of the dicots. The biosynthesis is fully elucidated in maize; here the genes encoding the enzymes of the pathway are in physical proximity. This "biosynthetic cluster" might facilitate coordinated gene regulation. Data from Zea mays, Triticum aestivum and Hordeum lechleri suggest that the pathway is of monophyletic origin in the Poaceae. The branchpoint from the primary metabolism (Bx1 gene) can be traced back to duplication and functionalisation of the alpha-subunit of tryptophan synthase (TSA). Modification of the intermediates by consecutive hydroxylation is catalysed by members of a cytochrome P450 enzyme subfamily (Bx2-Bx5). Glucosylation by an UDP-glucosyltransferase (UGT, Bx8, Bx9) is essential for the reduction of autotoxicity of the benzoxazinoids. In some species 2,4-dihydroxy-1,4-benzoxazin-3-one-glucoside (DIBOA-glc) is further modified by the 2-oxoglutarate-dependent dioxygenase BX6 and the O-methyltransferase BX7. In the dicots Aphelandra squarrosa, Consolida orientalis, and Lamium galeobdolon, benzoxazinoid biosynthesis is analogously organised: The branchpoint is established by a homolog of TSA, P450 enzymes catalyse hydroxylations and at least the first hydroxylation reaction is identical in dicots and Poaceae, the toxic aglucon is glucosylated by an UGT. Functionally, TSA and BX1 are indole-glycerolphosphate lyases (IGLs). Igl genes seem to be generally duplicated in angiosperms. Modelling and biochemical characterisation of IGLs reveal that the catalytic properties of the enzyme can easily be modified by mutation. Independent evolution can be assumed for the BX1 function in dicots and Poaceae.

Tetrathiomolybdate (TM) is a potent anticopper drug developed for Wilson's disease. We have found multiple efficacious results from decreasing copper levels with TM in mouse models of disease, using serum Cp as a surrogate marker of copper status and targeting Cp values of 20% to 50% of baseline. We have found efficacious results of TM therapy in mouse models of fibrosis; inflammation; damage from exogenous agents, such as acetaminophen and doxorubicin; and immune-modulated diseases, such as concanavalin A hepatitis, collagen II-induced arthritis, and the non-obese diabetic (NOD) mouse model of type I diabetes. In the current study, we examine TM efficacy in the EAE mouse model of multiple sclerosis (MS). We find that clinical scores of neurologic damage are significantly inhibited by TM therapy, whether therapy is started before MS-inducing antigen administration or after symptoms from antigen administration develop. Furthermore, we find that experimental autoimmune encephalomyelitis (EAE) treatment produces a marked increase of oxidant damage, as measured by urine isoprostane levels, and TM suppresses these isoprostane increases strongly and significantly. Finally, we find marked increases of inflammatory and immune-related cytokines in this model, and we find that TM strongly and significantly suppresses these increases.

Benzoxazinoids are common defence compounds of the grasses and are sporadically found in single species of two unrelated orders of the dicots. In the three dicotyledonous species Aphelandra squarrosa, Consolida orientalis and Lamium galeobdolon the main benzoxazinoid aglucon is 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA). While benzoxazinoids in Aphelandra squarrosa are restricted to the root, in Consolida orientalis and Lamium galeobdolon DIBOA is found in all above ground organs of the adult plant in concentrations as high as in the seedling of maize. The initial biosynthetic steps in dicots and monocots seem to be identical. Indole is most probably the first specific intermediate that is oxygenated to indolin-2-one by a cytochrome P450 enzyme. C. orientalis has an active indole-3-glycerolphosphate lyase for indole formation that evolved independently from its orthologous function in maize. The properties and evolution of plant indole-3-glycerolphosphate lyases are discussed.

Background: Tetrathiomolybdate (TM) is an oral copper-depleting agent that has been shown to inhibit angiogenesis, and angiogenesis is a predictor of poor prognosis in malignant pleural mesothelioma. We hypothesized that cytoreduction of malignant pleural mesothelioma followed by TM will delay time to progression., Methods: Between November 2000 and August 2003, 30 patients with malignant pleural mesothelioma received postoperative TM beginning 4 to 6 weeks after surgery at a dose adjusted to keep ceruloplasmin between 5 and 15 mg/dL). Time to progression was compared with the 55 stage I and II patients and 109 stage III patients previously treated with cytoreduction by one of us (H.P.)., Results: The 30 patients (25 men, 5 women; 13 stage I and II, 17 stage III), median age 67 years (range, 49-81 years), remained on TM a median of 14.9 months (range, 2 to 57 months). All patients reached target ceruloplasmin levels at a mean of 34 +/- 2 days (95% confidence interval, 30 to 39 days), and vascular endothelial growth factor levels at baseline (ceruloplasmin = 45.2 +/- 2 mg/dL) decreased from 2,086 +/- 390 pg/mL to 1,250 +/- 712 pg/mL (p < 0.002) at target ceruloplasmin (13 +/- 2 mg/dL; p < 0.0001 from baseline). The time to progression for all stage I or II TM patients was 20 months whereas that of 55 stage I or II non-TM-treated patients was 10 months (p = 0.046 versus TM). No differences in time to progression for the stage III TM patents from surgery were seen (7 months)., Conclusions: Tetrathiomolybdate has antiangiogenic effects in malignant pleural mesothelioma patients after resection of gross disease, and exhibits minimal toxicity and comparable efficacy to previous multimodality trials. Tetrathiomolybdate should be evaluated for efficacy in combination with standard malignant pleural mesothelioma regimens, as well as for postsurgical maintenance therapy.

Tetrathiomolybdate (TM) is an anticopper drug under development for treating Wilson's disease. Its mechanism of action involves forming a tight tripartite complex in the blood with serum albumin and available copper. When available copper levels are lowered in animals with TM, strong antiangiogenic and antitumor effects are observed. Similarly, TM has excellent efficacy in animal models of fibrotic, inflammatory, and autoimmune diseases, and it protects against heart damage from doxorubicin (DXR) and liver damage from acetaminophen, carbon tetrachloride, and concanavalin A. Tetrathiotungstate (TT) also forms a similar tripartite complex in the blood and has similar effects to TM on copper. In this article, whether TT had similar antitumor effects, and similar effects in protecting the heart against DXR toxicity, as TM was evaluated. It was found that the 2 drugs were comparable in their effects when doses were used that lowered copper availability to the same extent.

Tetrathiomolybdate (TM), presumably by lowering copper levels and availability, has shown excellent efficacy in animal models of cancer and models of injury that produce fibrotic or inflammatory damage in lung, heart, and liver. Trials in human patients are underway. If the efficacy of TM is indeed through lowering copper levels, other anticopper drugs should be equally efficacious. Zinc is an anticopper drug, with proven efficacy in Wilson's disease, a disease of copper toxicity. In this study, the efficacy of zinc is compared with TM on a mouse tumor model and on the doxorubicin model of heart damage, and it is hypothesized that when copper availability is lowered to an equivalent extent, the 2 drugs would show equivalent efficacy. No effect is found of zinc on inhibiting growth of a tumor that is markedly inhibited by TM, and zinc is found to be less effective than TM in inhibiting cardiac damage from doxorubicin. This study shows that TM's mechanism of action in protecting against doxorubicin toxicity is because of its anticopper effects, as copper supplementation eliminated the protective effect of TM. It is also hypothesized that the differences between TM and zinc may be caused by TM's mechanism of action in which it binds copper already in the body, whereas zinc does not.

Tetrathiomolybdate was originally developed for use in Wilson's disease. However, lowering copper levels to below normal levels with tetrathiomolybdate has been found to have efficacy in cancer, probably by turning down signaling by angiogenic cytokines. More recently, we have shown in animals models that tetrathiomolybdate dramatically inhibits pulmonary and liver fibrosis. In other animal models, we have shown that the drug also inhibits liver damage from concanavalin A and acetaminophen, and heart damage from doxorubicin. These studies are briefly reviewed, and we then present data on tetrathiomolybdate's partially protective effect against diabetes in non-obese diabetic mice, an autoimmune model of type I diabetes. Possible mechanisms of tetrathiomolybdate's protective effect are briefly considered.

Cardiac toxicity is the limiting factor in therapy with doxorubicin, an otherwise useful cancer drug. In this article we detail our study of a mouse model of doxorubicin-induced cardiac toxicity in which, after 4 days' treatment, doxorubicin caused marked increases in plasma concentrations of creatine kinase, lactic dehydrogenase, and troponin I, indicators of cardiac injury; marked increases in the plasma concentrations of tumor necrosis factor-alpha and interleukin-1(beta), both inflammatory cytokines; and a marked increase in the plasma concentration of interleukin-2, an indicator of cytotoxic T-cell activation. Therapy with tetrathiomolybdate, designed to limit copper availability, eliminated almost all of the increases of these six parameters in plasma. The marked protection against cardiac injury by doxorubicin in tetrathiomolybdate-treated animals suggests that tetrathiomolybdate would be of use clinically in helping prevent doxorubicin toxicity in patients. In other preclinical work, it has been shown that tetrathiomolybdate potentiates the chemotherapeutic effect of doxorubicin in cancer, so a double benefit might accrue clinically from the combined use of tetrathiomolybdate and doxorubicin. The mechanism by which tetrathiomolybdate protects against doxorubicin toxicity is of considerable interest. Our working hypothesis, based on the inhibition of interleukin-2 by tetrathiomolybdate as shown here, is that tetrathiomolybdate interrupts the inflammatory cascade at the activated-T-lymphocyte stage.

Case-ready fresh beef is typically packaged in a modified-atmosphere with approximately 80% oxygen and 20% carbon dioxide. Recently, USDA approved distribution of fresh meats in a master bag system using 0.4% carbon monoxide (CO). This study compared effects of packaging system (vacuum, 80% oxygen, 0.4% carbon monoxide), fresh meat storage time (7-21 days) and cooking temperature (49-79 °C) on extent of myoglobin denaturation, color and rancidity in cooked top sirloin steaks. Steaks packaged in 80% oxygen or CO retained desirable red color for 14 and 21 days storage, respectively. Steaks stored in 80% oxygen exhibited the greatest TBA values and myoglobin denaturation at all storage times and cooking temperatures. Steaks stored in high oxygen developed brown interior color at internal temperatures as low as 57 °C, the premature browning effect. Premature browning and rancidity associated with steaks packaged in 80% oxygen was prevented by packaging in 0.4% CO or vacuum.

Tetrathiomolybdate is an anticopper drug with a unique mechanism of action. Tetrathiomolybdate complexes copper to protein and itself, rendering the copper unavailable for cellular uptake. It was originally developed for Wilson's disease, and is now being developed as an antiangiogenic agent for the treatment of cancer. Many angiogenic cytokines require normal levels of copper, and lowered copper levels reduce cytokine signaling while cellular copper requirements are met. Cytokines of fibrosis and inflammation may be similarly copper dependent, since tetrathiomolybdate inhibits bleomycin induced pulmonary inflammation and fibrosis. The basis for this inhibition was evaluated here by examination of tetrathiomolybdate effects on cytokines in lung pathophysiologically important in the bleomycin mouse model of pulmonary damage. Results in mice injected endotracheally with bleomycin confirmed that tetrathiomolybdate therapy was effective in reducing fibrosis. This effect was associated with significant inhibition of bleomycin-induced tumor necrosis factor alpha and transforming growth factor beta expression in lung homogenates. These effects were shown to be independent of one another. This indicates that tetrathiomolybdate therapy can be effective even when fibrosis is at a more chronic stage, wherein inflammatory cytokines are playing a diminishing role. The inhibition of tumor necrosis factor alpha suggests that diseases of tumor necrosis factor alpha overexpression are also potential targets of tetrathiomolybdate therapy.

Tetrathiomolybdate (TM), a drug developed for the treatment of Wilson's disease, produces an antiangiogenic effect by reducing systemic copper levels. Several angiogenic cytokines appear to depend on normal levels of copper for activity. In both animal tumor models and in cancer patients, TM therapy has proved effective in inhibiting the growth of tumors. We have hypothesized that the activities of fibrotic and inflammatory cytokines are also subject to modulation by the availability of copper in a manner similar to angiogenic cytokines. As a first step in evaluating whether TM plays a therapeutic role in diseases of inflammation and fibrosis, we studied the effects of TM on a murine model of bleomycin-induced pulmonary fibrosis. Oral TM therapy resulted in dose-dependent reduction in serum ceruloplasmin, a surrogate marker of systemic copper levels. Significant decreases in systemic copper levels were associated with marked reduction in lung fibrosis as determined on the basis of histopathologic findings and a biochemical measure of fibrosis. The protection afforded by TM was also reflected in significantly reduced bleomycin-induced body-weight loss. In the next phase of this work, we will seek to determine the mechanisms by which TM brings about this therapeutic benefit.

The liver cytosolic enzyme tryptophan 2,3-dioxygenase (TDO) catalyzes the oxidation of L-tryptophan to formylkynurenine and controls the physiological flux of tryptophan into both the serotonergic and kynureninic pathways. This hemoprotein enzyme is composed of four noncovalently bound subunits of equivalent mass and contains two heme moieties per molecule. Electron paramagnetic resonance analyses have indicated that a histidyl nitrogen is involved in heme ligation [Henry et al., (1976) J. Biol. Chem. 251, 1578], but the identity of the His residue(s) is unknown. In an attempt to characterize the active site of the enzyme we have substituted each of the 12 His residues in the rat TDO subunit with Ala, to determine their relative importance in heme binding. Sequence alignment of the rat liver protein with that of known or putative TDO sequences from other organisms reveals that four of the His residues are conserved in eukaryotes, two of which are also conserved in prokaryotes. Our findings indicate that replacement of the evolutionarily conserved His 76 and 328 residues resulted in a dramatic reduction of TDO activity, whereas that of the eukaryotically conserved His70 resulted in a significant reduction relative to that of the wild-type enzyme. On the other hand, replacement of the other eukaryotically conserved His273 residue, while affecting the relative expression of the enzyme, had little effect on its specific activity. Size-exclusion analyses revealed that the His76Ala and His328Ala mutants retained little or no heme, suggesting that these may be key residues in ligating the prosthetic heme moieties. Whether these His residues are both provided by the same TDO subunit or a different TDO subunit remains to be determined., (Copyright 2001 Academic Press.)

The objectives were to evaluate appropriate doses of zinc acetate and its efficacy for the maintenance management of Wilson's disease in pediatric cases. Pediatric patients of 1 to 5 years of age were given 25 mg of zinc twice daily; patients of 6 to 15 years of age, if under 125 pounds body weight, were given 25 mg of zinc three times daily; and patients 16 years of age or older were given 50 mg of zinc three times daily. Patients were followed for efficacy (or over-treatment) until their 19th birthday by measuring levels of urine and plasma copper, urine and plasma zinc and through liver function tests and quantitative speech and neurologic scores. Patients were followed for toxicity by measures of blood counts, blood biochemistries, urinalysis, and clinical follow-up. Thirty-four patients, ranging in ages from 3.2 to 17.7 years of age, were included in the study. All doses met efficacy objectives of copper control, zinc levels, neurologic improvement, and maintenance of liver function except for episodes of poor compliance. No instance of over-treatment was encountered. Four patients exhibited mild and transient gastric disturbance from the zinc. Zinc therapy in pediatric patients appears to have a mildly adverse effect on the high-density lipoprotein/total cholesterol ratio, contrary to results of an earlier large study of primarily adults. In conclusion, zinc is effective and safe for the maintenance management of pediatric cases of Wilson's disease. Our data are strongest in children above 10 years of age. More work needs to be done in very young children, and the cholesterol observations need to be studied further.

Wilson's disease is an inherited disease of copper accumulation caused by a failure of biliary excretion of excess copper. Accumulated copper causes liver disease in these patients, and in perhaps two thirds of patients, it causes brain damage leading to clinical neurologic or psychiatric dysfunction. Maintenance treatment involves reversing the positive copper balance. The earliest approaches have used chelators, such as penicillamine or trientine, which increase the urinary excretion of copper. A more recent approach has used zinc, which blocks the absorption of copper and increases copper excretion in the stool. Because of the high level of endogenously secreted copper in alimentary secretions, the reabsorption of which is partially blocked by zinc therapy, zinc acts to remove accumulated copper from the body as well as prevent its reaccumulation. In the present article we present data on the long-term follow-up (up to 10 years) of maintenance zinc treatment of 141 patients with Wilson's disease. The data presented document that zinc is effective as a sole therapy in the long-term maintenance treatment of Wilson's disease and that it has a low toxicity. The results demonstrate the efficacy of zinc therapy in treating the presymptomatic patient from the beginning of therapy. We also present limited data on the use of zinc in the treatment of pregnant patients and children who have Wilson's disease; these data also indicate efficacy and low toxicity. The median follow-up period for the group as a whole is 4.8 years; for the presymptomatic patients it is 6.5 years; for the children it is 3.6 years.

Background: Uncontrolled variceal haemorrhage is the main indication for transjugular intrahepatic portosystemic shunt. However, mortality is 50% for this high-risk group. We have evaluated clinical and laboratory variables prior to transjugular intrahepatic portosystemic shunt in order to establish predictors of mortality, validated prospectively., Method: Over a 4-year period, 367 patients were admitted with variceal bleeding. In 54 patients endoscopic therapy for acute variceal bleeding failed and they had emergency transjugular intrahepatic portosystemic shunt. Failure of therapy was defined as continued bleeding after 2 endoscopy sessions (n=39) or vasoconstrictor-resistant bleeding from gastric/ectopic varices (n=15). Thirty-three variables were analysed from data available immediately prior to transjugular intrahepatic portosystemic shunt., Results: Twenty-six patients died within 6 weeks. In a multivariate analysis, 6 factors had independent prognostic value: moderate/severe ascites, requirement for ventilation, white cell blood count (WBC), platelet count (PLT), partial thromboplastin time with kaolin (PTTK) and creatinine. A prognostic index (PI) score was derived, in which presence of moderate/severe ascites, or need for ventilation, scored 1: PI=1.54 (Ascites)+1.27 (Ventilation)+1.38 Ln (WBC)+2.48 ln (PTTK)+1.55 Ln (Creat)-1.05 Ln (PLT). Using this equation, 42% (n=10) of deaths occurred in the fifth quintile (PI > or = 18.52), where the mortality was 100%. The score was prospectively validated in a further 31 patients, giving 100% positive predictive value. Eleven further patients died, including all seven with a PI >18.5. No survivors had a PI >18.3., Conclusion: Despite immediate control of bleeding by transjugular intrahepatic portosystemic shunt, patients with uncontrolled variceal haemorrhage have a high mortality, particularly when associated with markers of advanced liver disease, sepsis and multi-organ failure. The use of transjugular intrahepatic portosystemic shunt is probably not justified in this subgroup. Our prognostic index can help identify such patients, and, if validated elsewhere, will help in deciding when to use transjugular intrahepatic portosystemic shunt.

Background: Membranous obstruction of the inferior vena cava is a rare cause of hepatic venous outflow obstruction in Caucasians. There has been much debate in the literature about its aetiology., Methods and Results: We describe a Caucasian with hepatic venous outflow obstruction due to an inferior vena cava web, who was found to have hypercoagulability due to factor V Leiden. Following balloon rupture of the membrane and anticoagulation, his symptoms resolved and he has remained well for a year., Conclusions: The age at presentation in this patient, the presence of hypercoagulability and the excellent response to membrane rupture and anticoagulation suggest that in this case the membrane may have been derived from organised thrombus. Balloon rupture of the membrane and anticoagulation appears to be an effective treatment in such cases.

An improved specific analytical method for ochratoxin A (OA) is presented, combining HPLC separation with enhanced fluorescence detection by post-column addition of ammonia. Commercial immunoaffinity columns (Biocode) were for the first time applied to the cleanup of extracts of body fluids; they could be used up to 20 times for blood serum. The extraction efficiency of OA from human serum and milk as well as its derivatization to esters were studied and improved. The quantitation limit for OA was improved and estimated at 5-10 pg/g for human milk and serum. The mean recovery of OA from serum and milk was estimated at 85%. The overall coefficient of variation for OA determinations in serum, milk and selected foodstuffs was estimated at 10% (concentration range 0.01-5 ng/g). The method was applied to sera of 368 blood donors, 10 pairs of maternal and fetal sera, as well as to 40 human milk samples and selected foodstuffs; the results are discussed.

The sharp demarcation between diseased and normal mucosa often observed in ulcerative colitis remains unexplained by current hypotheses of disease pathogenesis. To investigate whether this demarcation occurs at the watershed of vascular territories, the colonic arterial anatomy of 10 patients with ulcerative colitis was studied by in-vitro angiography and macroscopic and histological examination of total colectomy specimens. Of the 10 perfusion-fixed colectomy specimens studied, 7 had pancolitis associated with a complete marginal artery (Arteria marginalis coli) that spanned the entire length of the large bowel. 3 specimens had sharply demarcated disease in which the marginal artery arose from the inferior mesenteric artery and ended abruptly at the point of mucosal demarcation. The colon proximal to this point was histologically normal. These findings suggest that the proximal extent of colitis is determined by the limit of the marginal artery. We suggest that some characteristic of the mucosal microvasculature in the territory of the inferior mesenteric artery, possibly embryological in origin, predisposes the dependent colon to develop ulcerative colitis.

The siblings of patients with newly diagnosed Wilson's disease are each at 25% risk of also having this autosomal recessive disease. Screening these siblings allows their detection and institution of prophylactic therapy before they become clinically ill. Herein we report the successful treatment of 13 presymptomatic patients with zinc acetate. These patients who received zinc have been followed for 3 to 9 years. In well-complying patients, 24-hour urine copper and non-ceruloplasmin plasma copper levels have decreased over years of follow-up, consistent with the elimination of the excess easily mobilized copper (the potentially toxic copper) of the body. Effect of therapy and compliance are easily monitored by following 24-hour urine zinc and copper levels. The urine copper level is a good reflection of the body's excess load of easily mobilizable copper. It will increase if control is not adequate. A decrease in urine zinc is an early signal that the patient's compliance is not optimal. The levels of hepatic copper in response to several years of zinc therapy may remain the same, go down, or go up temporarily. This is a reflection of zinc induction of hepatic metallothionein, which sequesters copper in a non-toxic pool. Hepatic copper levels should not be used to manage therapy. Liver function is well preserved by zinc therapy, and no zinc toxicity occurred in these 13 patients. No patient developed symptoms related to Wilson's disease. We conclude that zinc acetate is a fully effective and non-toxic therapy for the prophylactic treatment of the presymptomatic Wilson's disease patient.

A considerable body of data is now available indicating the efficacy and lack of toxicity of zinc treatment of Wilson's disease. Dose-response studies have shown that regimens of 50 mg of elemental zinc 3 times a day (50 mg x 3), 25 mg x 3, and 50 mg x 2 are effective, but 25 mg x 2 and 50 mg x 1 are not adequately effective. These studies indicate that 75 mg a day is close to the minimally effective dose, but do not address the question of necessary dose frequency. In the current study, the authors have used the minimally effective daily dose, 75 mg, and studied this daily dose in regimens of 25 mg x 3, 37.5 mg x 2, and 75 mg x 1 in treatment of four patients with Wilson's disease. These data have been supplemented with additional data from 11 patients treated with 25 mg 3 times a day and with data from 2 patients treated with 75 mg once a day. Efficacy was evaluated by 10-day copper balance and absorption of orally administered 64copper. The findings indicate that a daily dose of 75 mg must be divided into at least two doses to be effective, and that the 64copper procedure is more sensitive to zinc dose than copper balance.

cis-1,2,3a,4,5,9b-Hexahydro-3H-benz[e]indoles were synthesized and evaluated for in vitro dopamine D1 and D2 receptor binding affinity. The target compounds 21-25 were readily prepared by reduction of the air-sensitive tricyclic enamines 10-14. Reduction of 10-14 with sodium borohydride, sodium cyanoborohydride, palladium on carbon in ethanol, and platinum oxide in ethanol or acetic acid gave only the cis (3a,9b) 1,2,3a,4,5,9b-hexahydro-3H-benz[e]indoles. The stereochemistry was confirmed by single-crystal X-ray analysis. In the 6-hydroxy series, the binding affinity at D1 and D2 receptors was of the order 22 (N-n-butyl) > 21 (N-n-propyl) > 23 (N-H). The compounds demonstrated greater binding affinity at D2 receptors than at D1 binding sites. In contrast, 8-OH derivatives exhibited affinity only for D2 receptors, with 25 (N-n-butyl) having slightly greater affinity than 24 (N-n-propyl).

Changes in splanchnic blood flow are important in the pathogenesis of portal hypertension, but research in this area is hampered by the difficulty in measuring splanchnic arterial blood flow in man. We therefore investigated the use of intra-arterial Doppler catheters in measuring superior mesenteric artery blood flow in man and assessed the effect of intravenous octreotide on superior mesenteric artery blood flow in a placebo-controlled double-blind study. Nine experiments were performed in a flow model using vessels with internal diameters of 6.5, 4.5 and 3.0 mm, with flow rates ranging from 50 to 700 ml/min. In this model the catheters gave instantaneous, reproducible measurements of blood flow in vessels of 6.5 mm internal diameter with a mean error ranging from +5.3% to +36.4%, compared to electromagnetic flowmetry, but were less accurate in smaller vessels. When used in patients, the catheters provided stable, reproducible measurements of superior mesenteric blood flow, in 16 out of 20 patients studied. In a double-blind placebo-controlled study, including 12 subjects, superior mesenteric artery blood flow was significantly reduced in patients receiving octreotide. We suggest that measurement of splanchnic arterial blood flow using intra-arterial Doppler catheters may be a useful additional investigation in the assessment of splanchnic vascular pathophysiology and pharmacology in man.

Patients with liver disease frequently have impaired blood coagulation. The optimal method for liver biopsy in this situation is not established. To investigate this issue we randomised 117 patients with impaired blood coagulation, in whom liver biopsy was required, to receive either transjugular or plugged-percutaneous biopsy. Seventeen patients were excluded prior to biopsy and a protocol biopsy was performed in 100 patients (44 transjugular, 56 plugged-percutaneous). Liver tissue was obtained in 97 (42 transjugular, 55 plugged-percutaneous). Plugged-percutaneous liver biopsy was quicker and easier than transjugular liver biopsy and the biopsies obtained were significantly larger (12 +/- 5 mm vs. 6 +/- 4 mm; p < 0.001). However, 2 of 56 (3.5%) patients who received plugged-percutaneous biopsy had haemorrhage which required transfusion, while none of the 44 patients who received transjugular biopsy had haemorrhage (not significant). Both methods of liver biopsy were associated with a high success rate and a low incidence of complications. Plugged-percutaneous liver biopsy provides larger biopsies but may be associated with an increased risk of haemorrhage.

There is growing interest in screening to detect symptomless hepatocellular carcinoma (HCC), which should be easier to treat than symptomatic tumours. Combined alpha-fetoprotein and ultrasound monitoring can detect HCCs of 1 cm, and Lipiodol retention can be detected in tumours smaller than 1 cm. A number of treatment options are available. Surgical resection may be curative in selected patients with a single small tumour, but the cirrhotic patient is left with a diseased liver and the risk of tumour recurrence or death from underlying liver dysfunction. Orthotopic liver transplantation is a rational treatment for patients with decompensating cirrhosis and a small HCC, but it is expensive and necessitates immunosuppression. A variety of targeted or local therapies, either individually or in combination, can be used to treat HCC. These include percutaneous alcohol injection into an HCC, which may be an alternative to surgical resection. Tumour necrosis can be seen after targeted Lipiodol chemotherapy or radiotherapy. Transcatheter arterial embolisation selectively embolises the feeding artery, and can be combined with Lipiodol chemotherapy. Small tumours are thus amenable to treatment, even in patients who cannot have surgery. Screening and treatment for symptomless HCC seems justified, unless controlled trials teach us differently.

Ester and amide prodrugs of ibuprofen (1) and naproxen (16) were synthesized and evaluated for anti-inflammatory activity and gastrointestinal toxicity. The chemical structure of the prodrugs was varied in terms of lipophilicity and reactivity toward hydrolysis. Inhibition of acetic acid-induced writhing in mice indicated that prodrugs 7, 15, 19, and 20 exhibited significantly better activity (p less than 0.01) than the parent compounds. The average number of ulcers formed in the gastric mucosa following oral administration of 1 and 16 and prodrugs 5, 18, 21, and 22 was determined in rats. All prodrugs, except the glycine amide 21, were significantly less irritating to the gastric mucosa than either 1 or 16.

Caval compression secondary to hypertrophy of the caudate lobe is a well known complication of Budd-Chiari syndrome. This paper describes the use of an expandable metal stent to overcome external caval compression. Meso-caval shunting was subsequently performed with a good end result.