bluuebunny/crossref_metadata_2025 huggingface dataset:
Congenital nephropathy and ventriculomegaly: a report of four cases
Authors: Jolly M. , Goodburn S. , Cox P. , Loughna P.
Published: February 2003

Congenital nephrotic syndrome with ventriculomegaly and a normal karyotype is a rare association. We report four cases, three of which were conceived consecutively by one couple. All the cases were associated with elevated maternal serum alpha‐fetoprotein. Renal histology in one fetus demonstrated colloid filled cysts distributed in the corticomedullary area. Transmission electron microscopy of the glomeruli showed normally developed foot processes and confirmatory genetic studies excluded Finnish congenital nephrotic syndrome. It is probable that congenital nephropathy in conjunction with ventriculomegaly is the result of an autosomal recessive syndrome. Copyright © 2002 John Wiley & Sons, Ltd.
Current Awareness in Prenatal Diagnosis
Authors:   John Wiley & Sons, Ltd.
Published: February 2003

In order to keep subscribers up‐to‐date with the latest developments in their field, John Wiley & Sons are providing a current awareness service in each issue of the journal. The bibliography contains newly published material in the field of prenatal diagnosis. Each bibliography is divided into 17 sections: 1 Books, Reviews & Symposia; 2 General Interest; 3 Normal Fetal Development; 4 Gametogenesis and Pre‐implantation Diagnosis; 5 First Trimester Diagnosis; 6 Second Trimester Diagnosis; 7 Fetal Diagnosis by Ultrasound and Other Imaging; 8 Maternal Screening; 9 Screening for Carriers of Genetic Abnormality; 10 Technological Developments; 11 Confined Placental Mosaicism and Uniparental Disomy; 12 Molecular Cytogenetics; 13 Fetal Cells in Maternal Circulation; 14 Fetal Therapy; 15 Psychosocial Aspects; 16 Epidemiology and Environmental Factors; 17 Developmental Pathology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted
Secretory Component, J Chain, and Immunoglobulins in Human Embryos and Fetuses of the First Trimester of Pregnancy: Immunohistochemical Study
Authors: Gurevich Pavel , Elhayany Asher , Ben-Hur Herzl , Moldavsky Moisey , Szvalb Sergio , Zandbank Judit , Schneider David F. , Zusman Itshak
Published: February 2003

In our previous studies, we described the development of the secretory (mucosal) immune system (SIS) in human fetuses in the second trimester of pregnancy. In the present study, we examined the presence and distribution of components of this system in human embryos and early fetuses in the first trimester. An immunohistochemical study was performed on 17 embryos and 9 fetuses (4 to 12 wk of development) using antibodies against secretory component (SC), joining (J) chain, immunoglobulins (IgA, IgM, IgG), subsets of T and B lymphocytes, and macrophages. Cells positive for SC, J chain, and IgG were found in epithelial tissues from wk 4 of pregnancy. In the internal organs, such as the myocardium and endocardium, capillary endothelium, epithelium of the kidney tubules and some others, only J chain and immunoglobulins were seen. IgA was weakly reactive in tissues where SC and/or J chain were presented. IgM was very weak or absent. Among the cellular components of the SIS, only macrophages were seen in 4-wk-old embryos. CD3+ and CD20+ lymphocytes were found at wk 7 to 8. IgA- and IgM-positive lymphocytes appeared at the end of wk 9. The SIS is widespread in embryonic and early fetal periods and begins to function before the appearance of the common immune system in the developing organism. The first functional components of the SIS, such as IgG and IgA observed in this study, are most probably of maternal origin.
Cystic Fibrosis and Chiari Type I Malformation: Autopsy Study of Two Infants with a Rare Association
Authors: Rakheja Dinesh , Xu Yin , Burns Dennis K. , Veltkamp Daniel L. , Margraf Linda R.
Published: February 2003

Cystic fibrosis (CF), an epithelial cell transport disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is not generally associated with malformations of the central nervous system (CNS). This report describes two African-American children who died at less than 2 years of age with known Chiari I malformations and were found, unexpectedly at autopsy, to have the classic pancreatic and respiratory changes of CF. Both patients had suffered from failure to thrive that had been attributed to their CNS malformations. One child also had recurrent pneumonia and died with Pseudomonas sepsis. Mutational analysis for > 70 common CFTR mutations identified a single delta F508 mutation in one patient and a single 3120 + 1G to A mutation in the other. Their second CFTR mutations were not identified. The association of CF with Chiari I malformation is not likely to be purely coincidental, as the probability of such an occurrence in African-Americans is greater than one in 7,500,000 patients. It is possible that the CFTR gene may play a previously unrecognized role in CNS development. Alternatively, this CNS abnormality may be acquired due to the metabolic and electrolyte imbalances that characteristically occur in CF.
Conditional performance, portfolio rebalancing, and momentum of small‐cap mutual funds
Authors: Gorman Larry
Published: February 2003

To assess the performance of small‐cap stocks net of transaction costs, we analyze 165 actively managed small‐cap oriented portfolios. Our analysis addresses three areas of interest: (i) performance net of transaction costs, (ii) the magnitude of trading costs incurred when rebalancing an actively managed portfolio, and (iii) the potential for momentum strategy profits when investing in small‐cap stocks.

Using conditional estimation, we find that small‐cap funds have earned a significantly positive abnormal return of about 2% per year in the period January 1986 to December 2000. We also estimate the cost of January rebalancing to be 0.4% of portfolio value, a value that is significant for over 20% of the portfolios under study.

Finally, after trading frictions are taken into account, we find evidence that small‐cap portfolios exhibit significant return patterns, similar in nature to momentum patterns initially documented in a frictionless setting by [J. Finance 48 (1993) 65; J. Finance 56 (2001) 699]. Our findings support recent behavioral models, which attempt to explain these patterns. Consistent with the findings of Jegadeesh and Titman, we find that past “winners” continue to outperform in the next 12 months, followed by a performance reversal.
CONTRACEPTIVE APPLICATIONS OF ESTROGEN
Authors: Likis Frances E.
Published: February 2003

Estrogens are a primary component of several contraceptive methods: combined oral contraceptive pills, a combined injectable contraceptive, the combined contraceptive vaginal ring, the combination transdermal contraceptive patch, and combined emergency contraceptive pills. Contraceptive formulations that contain estrogen are referred to as combined contraceptives because they also contain some form of progestin. This article reviews the contraceptive methods containing estrogen, beginning with a discussion of combined oral contraceptive pills. Formulations and clinical management, mechanisms of action, noncontraceptive benefits of use, therapeutic uses in addition to contraception, side effects, contraindications to use, and drug‐drug interactions are described. Information follows about the newer combined contraceptive products including the injection, vaginal ring, and patch. Finally, combined emergency contraceptive pills are reviewed. Thorough knowledge of the contraceptive methods containing estrogen enables clinicians to provide expert care for women using these products.
ANTIVIRAL AGENTS IN WOMEN'S HEALTH: PHARMACOTHERAPEUTICS OF TREATING INFLUENZA AND HERPES
Authors: Burpo Rebecca H.
Published: February 2003

This article updates the practitioner on the pharmacology of antiviral agents. The mechanism of action of the six different classifications of antiviral agents is described. Because influenza and herpes are the viral infections that are most frequently treated in clinical practice, the pharmacotherapeutics of common antiviral agents for these infections are described. This includes pharmacokinetics, adverse effects, preparations, and dosages. Other recommendations related to prescribing and client education surrounding each of the pharmaceutical agents is reviewed.
CASE REPORT: LABIAL FUSION POSTPARTUM AND CLINICAL MANAGEMENT OF LABIAL LACERATIONS
Authors: Arkin Anne , Hughes Betty Chern
Published: February 2003

Lacerations of the external genitalia of various degrees are a common occurrence in the childbirth process. Evidence‐based management of minor lacerations of the vulva has yet to emerge in the scientific literature. Spontaneous approximation of minor lacerations of the labia may uncommonly result in distorted anatomical healing, with resultant dyspareunia, among other distressing symptoms. Management of this occurrence, including pharmacologic and surgical strategies, is presented in this case study discussion.
FEARS AND COPING STRATEGIES ASSOCIATED WITH PREGNANCY AND CHILDBIRTH IN FINLAND
Authors: Melender Hanna‐Leena
Published: February 2003

The purpose of this study was to describe the causes of fear associated with pregnancy and childbirth and to describe coping strategies of pregnant women who have fears. Data were collected by using semistructured interviews on the maternity units of two university hospitals in Finland. A convenience sample of 20 women served as participants, and data were analyzed by using content analysis techniques. In most cases, the source of fear was either previous experience and knowledge or uncertainty. In some cases, the source of fear was beliefs about childbirth with no clear origin of the belief. Knowledge was described as a means of dealing with fear, as were talking, writing, and reasoning. When seeking help, many participants relied on their social network and on health care professionals, but some sought information independently from books, magazines, etc. Methods for dispelling or alleviating fear included talking about feelings, increasing knowledge, accessing health care services, referring to positive experiences, receiving reassurance from medical screening, and/or feeling the baby move. Knowledge can be a cause of fear, but its ability to decrease fear is of greater consequence. What seemed to matter most was the way in which information is given.
THE MEANING OF SEXUAL SATISFACTION IN PREGNANT TAIWANESE WOMEN
Authors: Lee Jian Tao
Published: February 2003

The aim of this qualitative study was to explore Taiwanese women's experience of sexual satisfaction during pregnancy. Twelve participants were recruited through purposive sampling of outpatients registered in the antepartum clinic of a medical center in northern Taiwan. Data collection consisted of semi‐structured interviews and tape recordings. A modified phenomenologic methodology and narrative analysis of transcripts yielded four essential themes related to the personal meaning of sexually satisfying experiences in pregnancy: 1) improvement of self‐identity, 2) empowerment through sexual relations, 3) strengthening the marital bond, and 4) reinforcement of the value of sexuality. This woman‐centered view of the significance of sexually satisfying experiences in pregnancy can help health care professionals understand and educate their clients about women's sexuality during pregnancy.
CROSSING THE RIVER: KHMER WOMEN'S PERCEPTIONS OF PREGNANCY AND POSTPARTUM
Authors: White Patrice M.
Published: February 2003

In Cambodia, a setting of high maternal mortality, little is known about cultural perceptions of pregnancy. Often, cultural perceptions of what is considered normal or problematic guide a woman's decision to seek care. In some settings, the difference between the emic, or cultural insider's perception, and the biomedical perception of what is a serious obstetrical problem may delay lifesaving care. A qualitative study was undertaken to describe an emic perspective of what Khmer women view as normal and view as complications during pregnancy, birth, and postpartum. Focus group and key informant interviews were held to answer the questions: What do Khmer women and their birth attendants view as complications during pregnancy, birth, and postpartum? How are these complications defined? Eighty‐eight rural and urban women of childbearing age participated in focus groups in three rural provinces and Phnom Penh. In‐depth, semistructured interviews were held with 41 rural and urban women, traditional birth attendants, and trained midwives. Sixty‐six hours of taped interviews were transcribed, translated, and analyzed, and descriptions of emic conditions during pregnancy and postpartum were developed. This report details emic categories of antepartum and postpartum conditions identified by these Khmer women. Specific emic categories of normal pregnancy and postpartum are described in detail as well as abnormal emic conditions occurring during the postpartum period. Recommendations are made for use of traditional emic taxonomies as a foundation for explaining biomedical complications and the need for similar studies to guide the development of safe motherhood programs in areas of high maternal mortality.
IDENTIFYING INTIMATE PARTNER VIOLENCE AT ENTRY TO PRENATAL CARE: CLUSTERING ROUTINE CLINICAL INFORMATION
Authors: Anderson Barbara A. , Marshak Helen Hopp , Hebbeler Donna L.
Published: February 2003

Intimate partner violence (IPV) is the greatest trauma‐related risk to American women. Pregnant women are no exception, and escalation of IPV frequently occurs during pregnancy. Many studies have linked IPV during pregnancy to adverse maternal and fetal outcomes. This study examined IPV at the beginning of prenatal care to identify correlates of routine entry‐to‐care information with responses on a validated IPV screening tool, the Abuse Assessment Screen. The purpose of the study was to identify specific data from routine, standard intake information, which could alert clinicians to the potential of violence even in the presence of a negative IPV score or no formally administered screening tool. The point prevalence of abuse, as measured by the Abuse Assessment Screen at entry to care, was slightly in excess of the national mean, reinforcing the need for continual assessment throughout pregnancy. Abused women in this study were more likely to be young, single, and without family or partner support. These women relied on friends for support, admitted to depression, and desired their pregnancies. The findings are consistent with previous studies. Further research needs to be conducted to determine if this cluster of findings at entry to care, with or without a positive score on an IPV screening tool, are consistent markers for an increased risk of IPV.
A CONCEPTUAL FRAMEWORK FOR RESEARCH ON LIFETIME VIOLENCE, POSTTRAUMATIC STRESS, AND CHILDBEARING
Authors: Seng Julia S.
Published: February 2003

The objective of this theoretical article is to describe a conceptual framework for research on effects of past and current abuse and posttraumatic stress on childbearing women. The proposed framework builds on an earlier framework proposed by the Centers for Disease Control and Prevention (CDC) for research on violence occurring around the time of pregnancy. Two main adaptations are suggested. First, cumulative lifetime history of abuse trauma is added to the framework in addition to violence occurring around the time of pregnancy. Second, posttraumatic stress disorder (PTSD) is given greater emphasis as a potential factor contributing to adverse maternity outcomes based on the theoretical proposition that PTSD could be a plausible mechanism for adverse outcomes via both behavioral and neuroendocrine pathways. More research is needed on the effects of violence and PTSD on childbearing. This framework for research could be used to facilitate design of studies in which investigators want to consider PTSD as a potential mediator between lifetime exposure to violence and negative childbearing processes and outcomes. It is congruent with a CDC framework for research and could be incorporated into studies designed to meet their recommendations.
CLINICAL USES OF MIFEPRISTONE: AN UPDATE FOR WOMEN'S HEALTH PRACTITIONERS
Authors: Beal Margaret W. , Simmonds Kathy
Published: February 2003

In September 2000, the U.S. Food and Drug Administration (FDA) approved the use of mifepristone for the provision of medical abortion. Although mifepristone was developed and marketed because of its potential to effect early first‐trimester medical abortion, it has additional applications to health care, including the treatment of gynecologic conditions, cancer, and Cushing's disease. The controversial nature of abortion has dominated the publicity about mifepristone. The evidence for the safety and efficacy of mifepristone in medical abortion has been overshadowed, and many clinicians are unaware of the other potential uses of the drug. This article provides a discussion of background information on the pharmacology, development of, and research on mifepristone and an update on current and potential uses in health care today. Information on the FDA‐approved regimen and alternative protocols for management of mifepristone in its use in abortion care are presented.
Unequal Public Transfers to the Elderly in Different Countries – Equal Disposable Incomes
Authors: Forssell Åsa , Medelberg Magnus , Ståhlberg Ann-Charlotte
Published: February 2003

This article compares public transfers to the elderly and their disposable income in Denmark, Finland, France, Germany, Norway, the Netherlands, Sweden and the U.K. The public transfer income of retirees depends on the particular administrative arrangements in the different countries. The organisation of public pensions varies widely, and yet there is similarity in the overall income situation of older people. Low compensation from the public system is largely made up for by high payments from other systems of compensation, while high compensation from the public system is complemented by other systems of compensation to a lesser extent.

It is important to note that this does not mean that the different public pension solutions have the same effect on the distribution of income. The effects of pension systems on income distribution cannot be determined without taking into account what individuals have to pay for their pensions in the form of lower earnings during their active working life. Contributions to the pension system generally mean correspondingly lower cash wages and the design of the pension system affects the size of incomes indirectly by means of its impact on e.g. the labour supply and educational choices. In this article we discuss the direct distributive effects by comparing the relation between the contributions made by individuals and the benefits they receive in different systems and countries. It emerges that the public pension systems in Denmark, the Netherlands and the U.K. redistribute income from higher to lower earners, that the public pension system in Germany does not redistribute income, while the direct redistributive effects of the Swedish, Finnish, French and Norwegian public pension systems are ambiguous and can only be determined through empirical studies.
The Welfare State and European Integration
Authors: Schulte Bernd
Published: February 2003

In this article it is argued that there is a case for strengthening European social policy in order to re-establish some sort of equilibrium between economic and monetary integration on the one hand and social integration on the other. In order to retain the ‘social quality’ of Europe, market-correcting social intervention, not only by the Member States but also by the European organs – recognising the principle of subsidiarity – will be necessary. A consolidation of the European Constitution, an empowerment of the European Community in the social field, a written catalogue of civil, political and social rights are, among others, means which are advocated for reaching this aim. At the same time, there is a case for strengthening both social and civil dialogue.
Oxidation-triggered c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways for apoptosis in human leukaemic cells stimulated by epigallocatechin-3-gallate (EGCG): a distinct pathway from those of chemically induced and receptor-mediated apoptosis
Authors: SAEKI Koichi , KOBAYASHI Norihiko , INAZAWA Yuko , ZHANG Hong , NISHITOH Hideki , ICHIJO Hidenori , SAEKI Kumiko , ISEMURA Mamoru , YUO Akira
Published: February 2003

We investigated intracellular signalling pathways for apoptosis induced by epigallocatechin-3-gallate (EGCG) as compared with those induced by a toxic chemical substance (etoposide, VP16) or the death receptor ligand [tumour necrosis factor (TNF)]. EGCG as well as VP16 and TNF induced activation of two apoptosis-regulating mitogen-activated protein (MAP) kinases, namely c-Jun N-terminal kinase (JNK) and p38 MAP kinase, in both human leukaemic U937 and OCI-AML1a cells. In U937 cells, the apoptosis and activation of caspases-3 and −9 induced by EGCG but not VP16 and TNF were inhibited with SB203580, a specific inhibitor of p38, while those induced by EGCG and VP16 but not TNF were inhibited with SB202190, a rather broad inhibitor of JNK and p38. In contrast, the EGCG-induced apoptosis in OCI-AML1a cells was resistant to SB203580 but not to SB202190. Unlike TNF, EGCG did not induce the activation of nuclear factor-κB but rather induced the primary activation of caspase-9. N-Acetyl-l-cysteine (NAC) almost completely abolished apoptosis induced by EGCG under conditions in which the apoptosis induced by VP16 or TNF was not affected. The JNK/p38 activation by EGCG was also potently inhibited by NAC, whereas those by VP16 and TNF were either not or only minimally affected by NAC. In addition, dithiothreitol also suppressed both apoptosis and JNK/p38 activation by EGCG, and EGCG-induced activation of MAP kinase kinase (MKK) 3/6, MKK4 and apoptosis-regulating kinase 1 (ASK1) was suppressed by NAC. Dominant negative ASK1, MKK6, MKK4 and JNK1 potently inhibited EGCG-induced cell death. EGCG induced an intracellular increase in reactive oxygen species and GSSG, both of which were also inhibited by NAC, and the decreased synthesis of glutathione rendered the cell susceptible to EGCG-induced apoptosis. Taken together these results strongly suggest that EGCG executed apoptotic cell death via an ASK1, MKK and JNK/p38 cascade which is triggered by NAC-sensitive intracellular oxidative events in a manner distinct from chemically induced or receptor-mediated apoptosis.
Regulation of nuclear translocation of nuclear factor-kappaB relA: evidence for complex dynamics at the single-cell level
Authors: SCHOOLEY Kenneth , ZHU Ping , DOWER Steven K. , QWARNSTRÖM Eva E.
Published: February 2003

We have analysed activation of nuclear factor-κB (NF-κB) in response to interleukin-1 (IL-1) in human fibroblasts by tracking intracellular distribution and levels of endogenous relA, NF-κB1 and inhibitor of κB (I-κB) α using semi-quantitative confocal microscopy. Nuclear translocation of endogenous relA correlated with I-κBα degradation during stimulation with IL-1, whereas no effects were seen on levels or localization of NF-κB1. During pathway activation, relA was transported up a concentration gradient, resulting in a 3—4-fold increase in nuclear levels, but without any significant decrease in cytoplasmic concentration. IL-1 stimulation caused translocation of only 20% of the relA, but resulted in degradation of up to 70% of the cytoplasmic I-κBα. RelA nuclear translocation in fibroblasts correlated with DNA-binding activity measured by electrophoretic mobility shift assay (EMSA), both with respect to kinetics and IL-1 concentration-dependence. Clonal populations of cells demonstrated a marked degree of heterogeneity in the response to IL-1. The single-cell assay revealed the presence of responder and non-responder subpopulations, with an enhanced proportion of responder cells, and prolonged responses at higher concentrations of IL-1. Comparing different cell types demonstrated that whereas HepG2 cells, as fibroblasts, showed good correlation between nuclear translocation of relA and activation of DNA binding by relA-containing dimers, EL4 thymoma cells showed no effect on relA localization, even during induction of significant levels NF-κB activity, as measured by EMSA. The analysis shows that stimulation by IL-1 results in transient perturbation of the NF-κB system, which cycles between the resting and active states with net redistribution of a minor proportion of its DNA-binding component. In addition, it demonstrates significant cell-to-cell variations, as well as cell-type-specific differences in net relA nuclear transport in response to stimuli. The data are consistent with NF-κB constituting a dynamic and versatile system, regulated to a significant degree by binary events involving bidirectional trafficking between the cytoplasmic and nuclear compartments during pathway activation.
Functional interaction between nuclear inhibitor of protein phosphatase type 1 (NIPP1) and protein phosphatase type 1 (PP1) in Drosophila: consequences of over-expression of NIPP1 in flies and suppression by co-expression of PP1
Authors: PARKER Louise , GROSS Sascha , BEULLENS Monique , BOLLEN Mathieu , BENNETT Daimark , ALPHEY Luke
Published: February 2003

The catalytic subunit of type 1 Ser/Thr protein phosphatases (PP1c) forms complexes with many proteins that target it to particular subcellular locations and regulate its activity towards specific substrates. We report the identification of a Drosophila orthologue of nuclear inhibitor of PP1 (NIPP1Dm) through interaction with PP1c in the yeast two-hybrid system. NIPP1Dm shares many properties with mammalian NIPP1 including inhibition of PP1c in vitro, binding to RNA and PP1c, and localization to nuclear speckles. However, the mechanism controlling interaction of PP1c with NIPP1 is not conserved in Drosophila. NIPP1 can function independently of PP1c as a splicing factor, but the relative importance of this function is unknown. Over-expression of NIPP1Dm in Drosophila is cell-lethal in a range of tissues and developmental stages. The effects of ectopic NIPP1Dm are suppressed by co-expression of PP1c, indicating that the only effect of ectopic NIPP1Dm is to affect PP1c function. Co-expression of NIPP1Dm and PP1c does not have any detectable physiological effect in vivo, suggesting that the NIPP1Dm—PP1c holoenzyme is not normally limiting in Drosophila. These data show that NIPP1Dm and PP1c interact in vivo and suggest that NIPP1's role as a phosphatase regulator is conserved in Drosophila.
Methyl pyruvate stimulates pancreatic β-cells by a direct effect on KATP channels, and not as a mitochondrial substrate
Authors: DÜFER Martina , KRIPPEIT-DREWS Peter , BUNTINAS Linas , SIEMEN Detlef , DREWS Gisela
Published: February 2003

In pancreatic β-cells, methyl pyruvate is a potent secretagogue and is widely used to study stimulus—secretion coupling. In contrast with pyruvate, which barely stimulates insulin secretion, methyl pyruvate was suggested to act as an effective mitochondrial substrate. We show that methyl pyruvate elicited electrical activity in the presence of 0.5mM glucose, in contrast with pyruvate. Accordingly, methyl pyruvate increased the cytosolic free Ca2+ concentration after an initial decrease, similar to glucose. The initial decrease was inhibited by thapsigargin, suggesting that methyl pyruvate stimulates ATP production. This assumption is supported by the observation that methyl pyruvate hyperpolarized the mitochondrial membrane potential, similar to glucose. However, in contrast with glucose, methyl pyruvate even slightly decreased NAD(P)H autofluorescence and did not influence ATP production or the ATP/ADP ratio. This observation questions the suggestion that methyl pyruvate acts as a powerful mitochondrial substrate. The finding that methyl pyruvate directly inhibited a cation current across the inner membrane of Jurkat T-lymphocyte mitochondria suggests that this metabolite may increase ATP production in β-cells by activating the respiratory chains without providing reduction equivalents. We conclude that this mechanism may account for a slight and transient increase in ATP production. We further show that methyl pyruvate inhibited the KATP current measured in the standard whole-cell configuration, an effect that was at least partly antagonized by diazoxide. Accordingly, single-channel currents in inside-out patches were blocked by methyl pyruvate. We conclude that inhibition of KATP channels, and not activation of metabolism, mediates the induction of electrical activity in pancreatic β-cells by methyl pyruvate.
The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein
Authors: BASU Piku , KATTERLE Bettina , ANDERSSON K. Kristoffer , DALTON Howard
Published: February 2003

A protocol has been developed which permits the purification of a membrane-associated methane-oxidizing complex from Methylococcus capsulatus (Bath). This complex has 5 fold higher specific activity than any purified particulate methane mono-oxygenase (pMMO) previously reported from M. capsulatus (Bath). This efficiently functioning methane-oxidizing complex consists of the pMMO hydroxylase (pMMOH) and an unidentified component we have assigned as a potential pMMO reductase (pMMOR). The complex was isolated by solubilizing intracytoplasmic membrane preparations containing the high yields of active membrane-bound pMMO (pMMOm), using the non-ionic detergent dodecyl-β-d-maltoside, to yield solubilized enzyme (pMMOs). Further purification gave rise to an active complex (pMMOc) that could be resolved (at low levels) by ion-exchange chromatography into two components, the pMMOH (47, 27 and 24kDa subunits) and the pMMOR (63 and 8kDa subunits). The purified complex contains two copper atoms and one non-haem iron atom/mol of enzyme. EPR spectra of preparations grown with 63Cu indicated that the copper ion interacted with three or four nitrogenic ligands. These EPR data, in conjunction with other experimental results, including the oxidation by ferricyanide, EDTA treatment to remove copper and re-addition of copper to the depleted protein, verified the essential role of copper in enzyme catalysis and indicated the implausibility of copper existing as a trinuclear cluster. The EPR measurements also demonstrated the presence of a tightly bound mononuclear Fe3+ ion in an octahedral environment that may well be exchange-coupled to another paramagnetic species.
Mutation of the important Tyr-33 residue of chicken avidin: functional and structural consequences
Authors: MARTTILA Ari T. , HYTÖNEN Vesa P. , LAITINEN Olli H. , BAYER Edward A. , WILCHEK Meir , KULOMAA Markku S.
Published: February 2003

The strong interaction between avidin and biotin is so tight (dissociation constant 10-15M) that conditions usually sufficient for protein denaturing fail to dislodge biotin from the avidin—biotin complex. This kind of irreversible binding hinders the use of avidin in applications such as affinity purification or protein immobilization. To address this concern, we have constructed a series of mutants of the strategically positioned Tyr-33 in order to study the role of this residue in biotin binding, and to create avidin variants with more reversible ligand-binding properties. Unexpectedly, an avidin mutant in which Tyr-33 was replaced with phenylalanine (Avm-Y33F) displayed similar biotin-binding characteristics to the native avidin, indicating that the hydrogen bond formed between the hydroxy group of Tyr-33 and the carbonyl oxygen of biotin is not as important for the tight binding of biotin as previously suggested. In terms of the reversibility of biotin binding, Avm-Y33H was the most successful substitution constructed in this study. Interestingly, the binding of this mutant exhibited clear pH-dependence, since at neutral pH it bound to the biotin surface in an irreversible fashion, whereas, at pH9, 50% of the bound protein could be released with free biotin. Furthermore, although Tyr-33 is located relatively distant from the monomer—monomer interfaces, the mutagenesis of this residue also weakened the quaternary structure of avidin, indicating that the high ligand binding and the high stability of avidin have evolved together and it is difficult to modify one without affecting the other.
Copper-mediated dimerization of CopZ, a predicted copper chaperone from Bacillus subtilis
Authors: KIHLKEN Margaret A. , LEECH Andrew P. , BRUN Nick E. LE
Published: February 2003

Understanding the metal-binding properties and solution states of metallo-chaperones is a key step in understanding how they function in metal ion transfer. Using spectroscopic, bioanalytical and biochemical methods, we have investigated the copper-binding properties and association states of the putative copper chaperone of Bacillus subtilis, CopZ, and a variant of the protein lacking the two cysteine residues of the MXCXXC copper-binding motif. We show that copper-free CopZ exists as a monomer, but that addition of copper(I) causes the protein to associate into homodimers. The nature of the copper(I)—CopZ complex is dependent on the level of copper loading, and we report the detection of three distinct forms, containing 0.5, 1.0 and 1.5 copper(I) ions per protein. The presence of excess dithiothreitol has a significant effect on copper(I) binding to CopZ, such that, in its presence, copper(I)—CopZ occurs mainly as a monomer species. Data for copper binding to the double-cysteine variant of CopZ are consistent with an essential role for these residues in tight copper binding in the wild-type protein. We conclude that the complex nature of copper(I) binding to CopZ may underpin mechanisms of protein-to-protein copper(I) transfer.
Comparative characterization of hexose transporters of Plasmodium knowlesi, Plasmodium yoelii and Toxoplasma gondii highlights functional differences within the apicomplexan family
Authors: JOËT Thierry , HOLTERMAN Lennart , STEDMAN Timothy T. , KOCKEN Clemens H.M. , WEL Annemarie van der , THOMAS Alan W. , KRISHNA Sanjeev
Published: February 2003

Chemotherapy of apicomplexan parasites is limited by emerging drug resistance or lack of novel targets. PfHT1, the Plasmodium falciparum hexose transporter 1, is a promising new drug target because asexual-stage malarial parasites depend wholly on glucose for energy. We have performed a comparative functional characterization of PfHT1 and hexose transporters of the simian malarial parasite P. knowlesi (PkHT1), the rodent parasite P. yoelii (PyHT1) and the human apicomplexan parasite Toxoplasma gondii (T. gondii glucose transporter 1, TgGT1). PkHT1 and PyHT1 share >70% amino acid identity with PfHT1, while TgGT1 is more divergent (37.2% identity). All transporters mediate uptake of d-glucose and d-fructose. PyHT1 has an affinity for glucose (Km0.12mM) that is higher than that for PkHT1 (Km0.67mM) or PfHT1 (Km1mM). TgGT1 is highly temperature dependent (the Q10 value, the fold change in activity for a 10°C change in temperature, was >7) compared with Plasmodium transporters (Q10, 1.5—2.5), and overall has the highest affinity for glucose (Km30μM). Using active analogues in competition for glucose uptake, experiments show that hydroxyl groups at the C-3, C-4 and C-6 positions are important in interacting with PkHT1, PyHT1 and TgGT1. This study defines models useful to study the biology of apicomplexan hexose permeation pathways, as well as contributing to drug development.
Endogenous phospholipase D2 localizes to the plasma membrane of RBL-2H3 mast cells and can be distinguished from ADP ribosylation factor-stimulated phospholipase D1 activity by its specific sensitivity to oleic acid
Authors: SARRI Elisabeth , PARDO Raul , FENSOME-GREEN Amanda , COCKCROFT Shamshad
Published: February 2003

We have examined the specificity of oleate as an activator of phospholipase D2 (PLD2) and whether it can be used to study PLD2 localization and its involvement in cell function. Oleate stimulates PLD activity in intact RBL-2H3 mast cells. Comparing PLD1- with PLD2-overexpressing cells, oleate enhanced PLD activity only in PLD2-overexpressing cells. Membranes were also sensitive to oleate and when membranes prepared from PLD1- and PLD2-overexpressing cells were examined, oleate further increased PLD activity only in membranes from PLD2-overexpressing cells. Overexpressed green fluorescent protein (GFP)-PLD2 fusion protein was localized at the plasma membrane and GFP-PLD1 was found in an intracellular vesicular compartment. Oleate was used to examine whether overexpressed PLD2 co-localized with endogenous PLD2. RBL-2H3 mast cell homogenates were fractionated on a linear sucrose gradient and analysed for both oleate-stimulated activity and ADP ribosylation factor 1-stimulated PLD1 activity. The oleate-stimulated activity co-localized with markers of the plasma membrane including the β-subunit of the Fc∊RI and linker for activation of T cells. Fractionation of homogenates from PLD2-overexpressing cells demonstrated that the overexpressed PLD2 fractionated in an identical location to the endogenous oleate-stimulated activity and this activity was greatly enhanced in comparison with control membranes. Examination of membranes prepared from COS-7, Jurkat and HL60 cells indicated a relationship between oleate-stimulated PLD2 activity and PLD2 immunoreactivity. We examined whether oleate could be used to activate secretion and membrane ruffling in adherent RBL-2H3 mast cells. Oleate did not stimulate secretion but did stimulate membrane ruffling, which was short-lived. We conclude that oleic acid is a selective activator of PLD2 and can be used for localization studies, but its use as an activator of PLD2 in intact cells to study function is limited due to toxicity.
Myeloid zinc finger (MZF)-like, Kruppel-like and Ets families of transcription factors determine the cell-specific expression of mouse extracellular superoxide dismutase
Authors: ZELKO Igor N. , FOLZ Rodney J.
Published: February 2003

Extracellular superoxide dismutase (EC-SOD or SOD3) is an important protective enzyme against the toxicity of superoxide radicals that are produced under both physiological and pathophysiological conditions. We have isolated and characterized over 11kb of the mouse EC-SOD gene and its 5′- and 3′-flanking regions. The gene consists of two exons, with the entire coding region located within exon 2. In order to study the mechanism of cell-specific gene regulation for mouse EC-SOD, we characterized 2500bp of its 5′-flanking region using cultured cells derived from mouse lung fibroblasts (MLg), kidney medulla (mIMCD3) and hepatocytes (Hepa 1-6). Real-time PCR showed that basal expression of EC-SOD was considerably higher in MLg cells compared with the other cell types. Reporter-gene assays revealed that the proximal promoter region was sufficient to support this high expression in MLg cells. Although no obvious TATA box was identified, our results show that a highly purine-rich region from −208 to +104 contains active binding sites for both the Kruppel-like and Ets families of transcription factors. Using electrophoretic mobility shift, DNase footprinting and reporter gene assays, we identified myeloid zinc finger 1 and gut-enriched Kruppel-like-factor-like nuclear transcription factors as repressors of EC-SOD expression, whereas nuclear transcription factors from the Ets family, such as Elf-1 and GA-binding protein α and β, were potent activators of EC-SOD transcription. We propose a model that highlights competition between Ets activators and Kruppel-like repressors within the proximal promoter region that determines the level of EC-SOD expression in a particular cell type.
Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis
Authors: Corpechot Christophe , Barbu Veronique , Wendum Dominique , Kinnman Nils , Rey Colette , Poupon Raoul , Housset Chantal , Rosmorduc Olivier
Published: February 2003

Cirrhosis consists of hepatocyte nodules surrounded by a highly vascularized fibrous tissue. We previously showed that the development of biliary cirrhosis in the rat is associated with the occurrence of hepatocellular hypoxia and the induction of hepatic angiogenesis. We herein examined the occurrence of hypoxia in an experimental model of diethylnitrosamine (DEN)-induced cirrhosis. We also determined whether hypoxia directly affects the expression of vascular endothelial growth factor (VEGF), of VEGF receptors (Flt-1, Flk-1), and of type I and type IV collagens in activated hepatic stellate cells (HSCs) and the expression of VEGF in hepatocytes. Our results show that in DEN-treated rats, although the progression of liver fibrosis is associated with hepatocellular hypoxia and angiogenesis, VEGF and Flt-1 expressions in the liver are increased and correlated with the density of microvessels. In vitro, hypoxia induces the expression of VEGF, Flt-1, and type I collagen in activated HSCs and that of VEGF in hepatocytes. In addition, we show that hypoxia-induced type I collagen expression in HSCs may occur independently of transforming growth factor β1 (TGF-β1) overexpression. In conclusion, the present study provides further evidence that hepatocellular hypoxia and angiogenesis progress together with fibrogenesis after liver injury and that hypoxia directly contributes to the progression of liver fibrosis.
M6P/IGF2R tumor suppressor gene mutated in hepatocellular carcinomas in Japan
Authors: Oka Yoshihiko , Waterland Robert A. , Killian Keith J. , Nolan Catherine M. , Jang Hong-Seok , Tohara Keiji , Sakaguchi Seigo , Yao Tsuneyoshi , Iwashita Akinori , Yata Yutaka , Takahara Terumi , Sato Shin-ichiro , Suzuki Kazuyuki , Masuda Tomoyuki , Jirtle Randy L.
Published: February 2003

Mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) tumor suppressor- gene mutation is an early event in human hepatocellular carcinoma (HCC) formation in the United States, but its role in hepatocarcinogenesis in Japan is unclear. We therefore determined M6P/IGF2R mutation frequency in HCCs from patients who resided in the southern, central, and northern regions of Japan. Ten single nucleotide polymorphisms were used to identify HCCs and dysplastic liver nodules with M6P/IGF2R loss of heterozygosity. The retained allele in these tumors was also assessed for point mutations and deletions in the M6P/IGF2R ligand binding domains by direct sequencing of polymerase chain reaction (PCR) amplified DNA products. Fifty-eight percent (54 of 93) of the patients were heterozygous at the M6P/IGF2R locus, and 67% (43 of 64) of the HCCs and 75% (3 of 4) of the dysplastic nodules had loss of heterozygosity. The remaining allele in 21% of the HCCs contained either M6P/IGF2R missense mutations or deletions, whereas such mutations were not found in the dysplastic lesions. In conclusion, M6P/IGF2R is mutated in HCCs from throughout Japan with a frequency similar to that in the United States. Loss of heterozygosity in dysplastic liver nodules provides additional evidence that M6P/IGF2R haploid insufficiency is an early event in human hepatocarcinogenesis.
Dose-dependent inhibition of hepatic fibrosis in mice by a TGF-β soluble receptor: Implications for antifibrotic therapy
Authors: Yata Yutaka , Gotwals Philip , Koteliansky Victor , Rockey Don C.
Published: February 2003

Transforming growth factor (TGF) β isoforms (in particular, TGF-β1) play a central role in the fibrogenic response to injury in many organs, including the liver. Although TGF-β is clearly important in fibrogenesis, a number of issues related to therapeutic antagonism have emerged. For example, the long-term effect of TGF-β antagonism is unknown; furthermore, controversy exists as to appropriate levels of TGF-β inhibition. Therefore, we aimed to examine TGF-β in models of chronic liver injury and to determine whether an in vivo dose-response relationship exists for inhibition of TGF-β. Liver injury was induced in BALB/c mice by administering carbon tetrachloride for 4 or 8 weeks. TGF-β binding was inhibited with a soluble TGF-β type II receptor (STR) construct, administered intraperitoneally over a dose range of 4.0, 1.0, 0.4, or 0.1 mg/kg twice weekly during fibrogenesis. Fibrogenesis was assessed by measurement of type I collagen messenger RNA (mRNA) expression and by quantitative morphometric analysis. In the 4-week study, STR at concentrations of 4.0, 1.0, and 0.1 mg/kg reduced type I collagen mRNA expression by 31%, 49%, and 60% compared with immunoglobulin (Ig) G controls, respectively. In the 8-week study, lower concentrations of STR (0.1 mg/kg) also had the greatest effect on type I collagen mRNA expression. Quantitative morphometrics similarly showed that lower concentrations of STR were the most antifibrogenic. In conclusion, the results confirm the antifibrotic effect of inhibiting TGF-β in chronic hepatic wounding and, moreover, show that its in vivo effect in the mouse is dose dependent. Such findings have major translational implications for therapeutic strategies aimed at TGF-β.
Mallory body—A disease-associated type of sequestosome
Authors: Stumptner Cornelia , Fuchsbichler Andrea , Heid Hans , Zatloukal Kurt , Denk Helmut
Published: February 2003

Mallory bodies (MBs) consist of abnormal keratins, ubiquitin, heat shock proteins, and the protein p62. p62 is encoded by an immediate-early response gene that rapidly responds to a variety of extracellular signals involved in cell proliferation, differentiation, and particularly oxidative stress. It acts as an adapter in signal transduction and binds noncovalently to ubiquitin, possibly being involved in the regulation of the fate of ubiquitinated proteins by segregation (i.e., sequestosome or aggresome formation). The presence of p62 together with ubiquitinated abnormal keratins in the MB characterizes MBs as a disease-associated type of sequestosome. A detailed study on the expression of p62 and its relationship to MB formation in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-treated mouse liver is reported based on immunohistochemical, immunoblot, and Northern blot analyses. The results indicate that p62 is rapidly induced in hepatocytes of intoxicated animals preceding MB formation. As suggested by experiments with short-term DDC-treated naive mice and mice refed DDC after recovery from long-term DDC treatment (primed mice), p62 does not exert an initiating effect on MB formation but the appearance of MBs requires the presence of abnormal keratins, which associate with p62 after ubiquitination. The rapid induction of p62 and its association with MBs further support the role of oxidative stress in MB formation. In conclusion, the constant presence of p62 in MBs suggests that binding of p62 to abnormal keratins may allow hepatocytes to dispose potentially harmful proteins in a biologically inert manner.