bluuebunny/crossref_metadata_2025 huggingface dataset:
Difficulties in establishing a limited liability company in Great Britain during the 1860s and the role of financial information: a case history
Authors: BARNES P. , FIRMAN R. J.
Published: July 2002

As a result of legislation in 1855, 1856 and 1862 that effectively enabled limited liability companies to be formed with minimum difficulty for the first time, there was an explosion of new companies. However, after the collapse of Overend, Gurney Ltd in 1866 they became unpopular. This paper examines the case of a business which failed to raise the necessary funding because of suspicion of exaggerated claims made in public prospectuses and the ways in which it attempted to survive. This gypsum industry case history also illustrates the problems facing the new class of ‘pure’ investors and directors who had little understanding of the industry in which their company was operating and the nature and reliability of the financial information available to them required for their decision-making.
Associations between atmospheric temperature inversions and vertical wind profiles: a preliminary assessment
Authors: Milionis A E , Davies T D
Published: July 2002

Despite the potential importance for air pollution, the association between temperature inversions and vertical wind profiles has been paid scant attention. A preliminary statistical assessment is made, using message data from radiosonde soundings at a station in eastern England for the period 1976–1980, from the surface up to 700 mb. In spite of an expectation among many air pollution meteorologists that inversions and local wind maxima (or vertical wind shears) are ‘typically’ associated, no such typical association between inversions and significant wind levels was identified. This conclusion extended to nocturnal surface inversions and low level jets. This does not mean that there are not clear cases of association, but that either (a) the dataset does not discriminate the associations, and/or (b) the associations are not evident on a climatological, or statistical, basis. Copyright © 2002 Royal Meteorological Society
Environmental controls with corrupt bureaucrats
Authors: Damania Richard
Published: July 2002

Environmental regulations typify a large class of activities in the public sector where government agencies are required to monitor the degree of compliance. These tasks are usually delegated to bureaucrats who, as self-interested agents, may engage in corrupt behavior. Such problems abound, particularly in developing countries, where corruption is regarded as one of the major causes of environmental degradation. This paper investigates the implications of corruption for the optimal design of environmental regulations and analyses the interaction between the prosecution rate, monitoring rate, and fines. It is shown that even if corruption can be deterred the fact that it may occur substantially impedes the ability of a regulator to control environmentally degrading activities.
Measuring the cost of environmentally sustainable industrial development in India: a distance function approach
Authors: Murty M.N. , Kumar Surender
Published: July 2002

This paper attempts to estimate the maintenance cost of water pollution abatement measures to the Indian industry using the methodology of distance function in the theory of production. The distance function is estimated using both programming and stochastic frontier models for a sample of water polluting industries in India. The firm-specific shadow prices for pollutants, measures of efficiency, and scale economies are estimated. Estimates show that on average the cost to the Indian industry for reducing one ton of BOD and COD are respectively, Rs 0.246 and 0.077 million. Large differences in the estimates of firm-specific shadow prices of pollutants reflect the use of inefficient water pollution abatement technologies. The relationships between firm-specific shadow prices or marginal costs of abatement of BOD and COD and the index of compliance (ratio of effluent load to sale value) and the pollution load reductions obtained confirm the earlier empirical results of studies on water pollution abatement in Indian industries. The earlier studies have found increasing marginal costs with respect to reductions in pollution concentrations and decreasing marginal cost with respect to the pollution loads reduced by the firms.
Dynamic have in North American and British Isles English
Authors: Trudgill Peter , Nevalainen Terttu , Wischer Ilse
Published: July 2002

There are two important differences between American English and British English with respect to main verb have. First, American English typically employs do-support in constructions such as Do you have any coffee? while traditional British English does not. Secondly, American English typically does not use have in expressions such as I took a shower whereas British Isles English does: I had a shower. In this article, we discuss the possibility that there is a connection between these two facts. We argue that the connection lies in the failure of have in North American English to acquire the full range of dynamic meanings that it has acquired in other varieties of English, and suggest language contact as one explanation for this phenomenon.
Ablaut reduplication in English: the criss-crossing of prosody
and verbal art
Authors: Minkova Donka
Published: July 2002

The two properties that characterize Ablaut reduplication in English (chit-chat, dilly-dally) are: (1) identical vowel quantity in the stressed syllabic peaks, (2) maximally distinct vowel qualities in the two halves, with [i] appearing most commonly to the left and a low vowel to the right. In addition, Ablaut reduplicatives are described as having a trochaic contour, yet there is a great deal of uncertainty regarding the stress on the second part of the formation. Historically, Ablaut reduplication appeared long after Copy reduplication (boo-boo, yo-yo) and flourished during the Renaissance; its productivity declined sharply in the twentieth century.

This article treats Ablaut reduplicatives as verbal art products, analogs of dipodic poetic meter. The naturalness of the template ensues from the interaction of conflicting segmental and prosodic constraints on identity and markedness. An independently established hierarchy blocks high back vowels from appearing in these forms. The height difference is a response to the principle of INTEREST which favors maximum perceptual differentiation between the stressed vowels. The linear ordering of the vowels correlates with domain-final lengthening. The ambiguity between compound stress and level stress that these words exhibit is related tentatively to the existence of a separate prosodic domain, a dipodic colon. The article provides Optimality-theoretic support for the analytical relevance of gradient phonetic properties and the relevance of the colon as a separate prosodic layer, and potentially enriches the taxonomy of metrical forms in English.
Long‐term effective population size of three endangered Colorado River fishes
Authors: Garrigan Daniel , Marsh Paul C. , Dowling Thomas E.
Published: July 2002

The extant genetic variation of a population is the legacy of both long‐term and recent population dynamics. Most practical methods for estimating effective population size are only able to detect recent effects on genetic variation and do not account for long‐term fluctuations in species abundance. The utility of a maximum likelihood estimator of long‐term effective population size based upon the coalescent theory of gene genealogies is examined for three endangered Colorado River fishes: humpback chub (Gila cypha), bonytail chub (Gila elegans) and razorback sucker (Xyrauchen texanus). Extant mitochondrial DNA (mtDNA) variation in humpback chub suggests this species has retained its historical equilibrium genetic variation despite recent declines in abundance. The mtDNA variation in razorback suckers indicates the population was quite large and expanding prior to recent declines and that rare alleles still survive in the remnant populations. The remaining mtDNA variation in bonytail chub indicates that dramatic, recent declines may have already obliterated a substantial portion of any historical variation. The results from long‐term effective population size analyses are consistent with known natural history and illustrate the utility of the analysis for endangered species management.
Census and monitoring based on individually identifiable vocalizations: the role of neural networks
Authors: Terry Andrew M. R. , McGregor Peter K.
Published: July 2002

Vocal individuality is widely suggested as a method for identifying individuals within a population. But few studies have explored its performance in real or simulated conservation situations. Here we simulated the use of vocal individuality to monitor the calling corncrake (Crex crex), a secretive and endangered land rail. Our data set contained 600 calls from 30 individuals and was used to simulate a population of corncrakes being counted and monitored. We tested three different neural network models for their ability to discriminate between and to identify individuals. Neural networks are non‐linear classification tools widely applied to both biological and non‐biological identification tasks. Backpropagation and probabilistic neural networks were used to simulate the reidentification of members of a known population (monitoring) and a Kohonen network was used to simulate the counting of a population of unknown size (census). We found that both backpropagation and probabilistic networks identified all individuals correctly all the time, irrespective of sample size. Kohonen networks were more variable in performance but estimated population size to within one individual of the actual size. Our results indicate that neural networks can be used effectively together with recordings of vocalizations in census and monitoring tasks.
The use of photographic rates to estimate densities of cryptic mammals: response to Jennelle et al.
Authors: Carbone C. , Christie S. , Conforti K. , Coulson T. , Franklin N. , Ginsberg J. R. , Griffiths M. , Holden J. , Kinnaird M. , Laidlaw R. , Lynam A. , MacDonald D. W. , Martyr D. , McDougal C. , Nath L. , O'Brien T. , Seidensticker J. , Smith J. L. D. , Tilson R. , Wan Shahruddin W.N.
Published: July 2002

Most species‐specific conservation efforts require estimates of population size to establish priorities and to monitor management activities. Yet obtaining reliable estimates of animal populations is often difficult, especially given time and funding limitations experienced by many research programmes. Consequently, there is a great need for practical methods to provide indices of animal density. Ideally, accurate estimates of populations would be obtained through mark‐recapture data collected from recognizable individuals over multiple censuses that cover the entire population range. Such data are rarely available, so conservation biologists have no alternative but to resort to analyses of less perfect data, ranging from permanent‐point censuses from cameras through to transect data on sightings and spoor encounters. The importance of census and monitoring data makes the development, and validation, of new techniques a priority. Because we do not live in a perfect world, there is a need to develop methods that can give an estimate of population sizes. It would be naïve to assume that these will give hugely accurate estimates of population size, but these techniques can prove useful in identifying areas that are likely to benefit from conservation action.
Local Government Development in France: Comparison with Japan
Authors: Grémion Catherine
Published: July 2002

We will not try here to attempt a complete comparison of decentralized local government in France and Japan together with their respective impact on economic development, but rather to focus on some points which appear to be of particular significance for further reflection in order to learn from both cases.
A Politics of Recognition and Respect: Involving People with Experience of Poverty in Decision making that Affects their Lives
Authors: Lister Ruth
Published: July 2002

In 1995, over 100 countries signed up to this statement in the Copenhagen Declaration and Programme of Action at the UN World Summit for Social Development. The second section of this article reflects on an initiative in the UK, which aims to make a reality of the Copenhagen commitment around the demand for the involvement of people in poverty in decision making that affects their lives. The first, longer, section makes a normative theoretical case for such action.
Introduction: Themed Section on Evidence-Based Policy as a concept for Modernising Governance and Social Science Research
Authors: David Miriam E
Published: July 2002

The concept of evidence-based policy and practice has many origins but its relation to the growth of the social sciences is arguably the most important. The uses of the social sciences for both understanding and transforming social policies and political systems has come to be assumed – complex and problematic though these may be. The concept is also closely linked with the concepts of globalisation, technological developments, and the ‘knowledge economy’. Thus the notions of ‘evidence’ and social science research have often been elided with political movements for social and economic change. In other contexts, these notions have been contextualised, so that ‘evidence’ and research are not deemed to be the same. Indeed, it is possible to argue that the notion of legal ‘evidence’ illustrates just how ideological it can be, how it can be used to marshal particular arguments and sustain a specific case rather than present it in a dispassionate manner.
Evidence-based Policy: Rhetoric and Reality
Authors: Packwood Angela
Published: July 2002

The drive in the late 1990s for an evidence-based approach to both policy and practice throughout public services has been encoded in a range of articles, operationalised in funded organisations, and implemented through government policy. This paper intends to review some of those articles, organizations and education policies to give an overview of the way in which the approach has been constructed and to make problematic some of the underlying assumptions.
De novo MECP2 frameshift mutation in a boy with moderate mental retardation, obesity and gynaecomastia
Authors: Kleefstra T , Yntema HG , Oudakker AR , Romein T , Sistermans E , Nillessen W , Van Bokhoven H , De Vries BBA , Hamel BCJ
Published: July 2002

Rett syndrome (RTT) is an X‐linked neurodevelopmental disorder caused by mutations in the MECP2 gene, with apparent lethality in male embryos. However, recent studies indicate that mutations in the MECP2 gene can cause congenital encephalopathy, an Angelman‐like phenotype and even nonspecific mental retardation in males.

We report on a 10‐year‐old boy with moderate mental retardation, hypotonia, obesity and gynaecomastia and a de novo 2‐bp deletion in the MECP2 gene that resulted in a frameshift and premature stop codon. As some of the clinical features were suggestive of the Prader–Willi syndrome, it might be worthwhile screening for MECP2 mutations in patients with an atypical Prader–Willi phenotype but without the characteristic abnormalities on chromosome 15q.

This report contributes to the phenotypic knowledge of male patients with MECP2 mutations. Moreover, this is the first reported male case of a de novo MECP2 mutation.
Angiotensinogen genotype, plasma protein and mRNA concentration in isolated systolic hypertension
Authors: Davis D , Liyou N , Lockwood D , Johnson A
Published: July 2002

Isolated systolic hypertension (ISH) occurs predominantly in the elderly, with a considerable morbidity and mortality. Its etiology is unknown but is likely to involve a significant genetic component. The aim of this study was to examine the angiotensinogen gene in ISH. The M235T and G(− 6)A polymorphisms were genotyped by polymerase chain reaction (PCR) in 86 ISH patients and 120 normotensive controls. Plasma angiotensinogen concentration was determined in 198 subjects by an indirect radioimmunoassay technique. Angiotensinogen mRNA concentration was determined by quantitative competitive reverse transcription (RT)–PCR in subcutaneous adipose tissue from a subset of these patients (n = 8) and controls (n = 6). Both the M235T (p = 0.0015) and G(− 6)A (p = 0.029) polymorphisms were associated with ISH. Plasma angiotensinogen concentration was higher in patients than controls (p < 0.0001), but was not associated with genotype. Angiotensinogen mRNA concentration in adipose tissue from ISH subjects was significantly lower than in adipose tissue from normotensive subjects (p = 0.033). The association of angiotensinogen gene variants with ISH and the elevation of plasma angiotensinogen concentration in these patients suggests a role of the angiotensinogen gene in this form of hypertension. Angiotensinogen gene expression may be altered in ISH, but this requires further examination.
Hepatic Met‐enkephalin immunoreactivity is enhanced in primary biliary cirrhosis
Authors: Bergasa NV , Liau S , Homel P , Ghali V.
Published: July 2002

Abstract:  Background/aims: In contrast to the normal adult liver, the fetal human and rat livers, and the liver of rats with cholestasis secondary to bile duct resection (BDR) express the preproenkephalin (ppENK) mRNA, which codes for the endogenous opioid peptide Met‐enkephalin. In addition, Met‐enkephalin immunoreactivity (MEIR) is detected in hepatocytes and in proliferating bile ductules in the cholestatic rat liver. These data suggest that cholestasis is associated with the resurgence of cells that produce Met‐enkephalin. To explore further the status of opioids in cholestasis, we studied the expression of MEIR in liver tissue.

Methods: The MEIR was sought in paraffin‐preserved liver tissues from patients with primary biliary cirrhosis (PBC) (n = 10).

Results: The MEIR was detected in all the PBC livers. Its intensity varied from weak to strong on hepatocytes and bile ducts and the strongest expression appeared as coarse granules. The MEIR was either absent or only faintly expressed by some hepatocytes from disease and nondisease control biopsies, but absent from bile ducts.

Conclusion: These results suggest that the human liver in cholestasis may be a source of endogenous opioids.
Intrahepatic expression of the hepatic stellate cell marker fibroblast activation protein correlates with the degree of fibrosis in hepatitis C virus infection
Authors: Levy MT , McCaughan GW , Marinos G , Gorrell MD
Published: July 2002

Abstract:  Background: Activated hepatic stellate cells (HSCs), recognised by their α smooth muscle actin immunoreactivity, are primarily responsible for liver fibrosis. However, the presence of α smooth muscle actin positive HSCs is not always associated with the development of liver fibrosis. Recently, other markers of human HSCs including the gelatinase fibroblast activation protein (FAP) and glial fibrillary acidic protein have been identified.

Aims: We examined the relationship between the expression of these HSC markers and the severity of liver injury in patients with chronic hepatitis C virus infection.

Methods: Liver tissue from 27 patients was examined using immunohistochemistry. Linear correlation analysis was used to compare staining scores with the stage and grade of liver injury.

Results–Conclusions: FAP expression, seen at the tissue‐remodelling interface, was strongly and significantly correlated with the severity of liver fibrosis. A weaker correlation was seen between glial fibrillary acidic protein expression and fibrosis stage. This contrasted with the absence of a relationship between α smooth muscle actin and the fibrotic score. A correlation was also observed between FAP expression and necroinflammatory score. In summary, FAP expression identifies a HSC subpopulation at the tissue‐remodelling interface that is related to the severity of liver fibrosis.
Thrombopoietin serum levels are elevated in patients with hepatitis B/C infection compared to other causes of chronic liver disease
Authors: Schöffski Patrick , Tacke Frank , Trautwein Christian , Martin Michael Uwe , Caselitz Martin , Hecker Hartmut , Peter Manns Michael , Ganser Arnold
Published: July 2002

Abstract:  Background/aims:  Thrombocytopenia in patients with advanced liver disease may stem from a deficient hepatic thrombopoietin production.

Methods: We determined the relationship between thrombopoietin, thrombocytopenia, aetiology and extent of liver damage by incorporating serum thrombopoietin measurements in the pretransplant evaluation of 111 patients with liver disease.

Results: The extent of thrombocytopenia was related to the underlying cause of disease. The platelet count directly correlated with factor V, II, fibrinogen, and PTT, and a negative correlation was found for splenic size and Child's stage. The thrombopoietin concentrations were age‐dependent, and no significant difference resulted between the median thrombopoietin level of liver disease patients with age‐matched healthy controls. Thrombopoietin concentrations and platelet counts were not correlated. Although noncirrhotic patients had higher platelet counts than those with Child's A–C cirrhosis (p < 0.001, U‐test), no such difference was found in thrombopoietin levels. Patients with hepatitis B and/or C had lower platelet counts compared to patients with nonviral diseases (p < 0.001), and their median thrombopoietin concentrations were significantly higher (p < 0.001).

Conclusion: We conclude that thrombocytopenia in patients with liver disease is unlikely to be explained only based on a deficient hepatic production of thrombopoietin. Patients with chronic viral hepatitis have significantly elevated thrombopoietin levels; the involved pathomechanisms require further study.
Leptin deficiency reduces but does not eliminate the development of hepatic fibrosis in mice infected with Schistosoma mansoni
Authors: Potter James J. , Mezey Esteban
Published: July 2002

Abstract:  Aims/Background: Leptin, a product of the obese (ob) gene, was demonstrated previously in activated hepatic collagen‐producing stellate cells, but not in quiescent retinol‐storing stellate cells. The role of leptin in fibrogenesis is unknown. This study investigated the possible influence of leptin in the pathogenesis of fibrosis by determination of the amount of fibrosis produced by Schistosoma mansoni infection in leptin deficient male ob/ob mice as compared to control mice.

Methods: The mice were infected percutaneously with cercaria of Schistosoma mansoni and the amount of liver fibrosis determined 12 weeks after infection. The amount of hepatic collagen deposited was quantified by morphometric analysis of liver sections stained with sirius red and by hydroxyproline content.

Results: The amount of histologically detectable fibrosis was greater in the infected controls than in the infected ob/ob mice. In the infected control mice, but not in the ob/ob mice, the fibrosis surrounding the granuloma was broad and extended beyond the portal tracts into the lobule with the formation of fibrous septa.

Conclusions: This study shows that leptin is a potentiating, but not an essential factor, for the development of hepatic fibrosis, because leptin deficiency reduces but does not prevent the development of hepatic fibrosis.
The peroxisomal transporter gene ANT1 is regulated by a deviant oleate response element (ORE): characterization of the signal for fatty acid induction
Authors: ROTTENSTEINER Hanspeter , PALMIERI Luigi , HARTIG Andreas , HAMILTON Barbara , RUIS Helmut , ERDMANN Ralf , GURVITZ Aner
Published: July 2002

Saccharomyces cerevisiae ANT1/YPR128c encodes the peroxisomal adenine nucleotide transporter that provides ATP for intra-peroxisomal activation of medium-chain fatty acids. A lacZ reporter construct comprising the ANT1 promoter was shown to be comparatively more highly expressed in a wild-type strain grown on oleic acid, a long-chain fatty acid, than in pip2Δoaf1Δ mutant cells that are defective in fatty acid induction. The ANT1 promoter was demonstrated to contain a deviant oleate response element (ORE) that could bind the Pip2p-Oaf1p transcription factor and confer activation on a basal CYC1-lacZ reporter gene. Expression of Ant1p as well as other enzymes whose genes are known to be regulated by a canonical ORE was found to be increased in cells grown on lauric acid, a medium-chain fatty acid. We concluded that the signal for induction does not differentiate between long- and medium-chain fatty acids. This signal was independent of β-oxidation or the biogenesis of the peroxisomal compartment where this process occurs, since a pox1Δ strain blocked in the first and rate-limiting step of β-oxidation as well as various pex mutant cells devoid of intact peroxisomes produced sufficient amounts of Pip2p-Oaf1p for binding OREs in vitro and for expressing an ORE-driven reporter gene. The signal's durability was shown to be related to the concentration of fatty acids in the medium, since a pex6Δ strain expressed an ORE-driven reporter gene at high levels for a longer period than did isogenic wild-type cells. Generation of the signal was also independent of protein synthesis, as demonstrated by cycloheximide treatment.
PrpE, a PPP protein phosphatase from Bacillus subtilis with unusual substrate specificity
Authors: IWANICKI Adam , HERMAN-ANTOSIEWICZ Anna , PIERECHOD Marcin , SÉROR Simone J. , OBUCHOWSKI Michał
Published: July 2002

Bacillus subtilis is a Gram-positive bacterium with a relatively large number of protein phosphatases. Previous studies have shown that some Ser/Thr phosphatases play an important role in the life cycle of this bacterium [Losick and Stragier (1992) Nature (London) 355, 601—604; Yang, Kang, Brody and Price (1996) Genes Dev. 10, 2265—2275]. In this paper, we report the biochemical properties of a putative, previously uncharacterized phosphatase, PrpE, belonging to the PPP family. This enzyme shares homology with other PPP phosphatases as well as with symmetrical diadenosine tetraphosphatases related to ApaH (symmetrical Ap4A hydrolase) from Escherichia coli. A His-tagged recombinant PrpE was purified from E. coli and shown to have Ni2+-dependent and okadaic acid-resistant phosphatase activity against a synthetic phosphorylated peptide and hydrolase activity against diadenosine 5′,5′′′-tetraphosphate. Unexpectedly, PrpE was able to remove phosphate from phosphotyrosine, but not from phosphothreonine or phosphoserine.
The human DnaJ homologue (Hdj)-1/heat-shock protein (Hsp) 40 co-chaperone is required for the in vivo stabilization of the cystic fibrosis transmembrane conductance regulator by Hsp70
Authors: FARINHA Carlos M. , NOGUEIRA Paulo , MENDES Filipa , PENQUE Deborah , AMARAL Margarida D.
Published: July 2002

The CFTR (cystic fibrosis transmembrane conductance regulator) gene, defective in cystic fibrosis, codes for a polytopic apical membrane protein functioning as a chloride channel. Wild-type (wt) CFTR matures inefficiently and CFTR with a deletion of Phe-508 (F508del), the most frequent mutation, is substantially retained as a core-glycosylated intermediate in the endoplasmic reticulum (ER), probably due to misfolding that is recognized by the cellular quality control machinery involving molecular chaperones. Here, we overexpressed the heat-shock protein (Hsp) 70 chaperone in vivo and observed no changes in degradation rate of the core-glycosylated form, nor in the efficiency of its conversion into the fully glycosylated form, for either wt- or F508del-CFTR, contrary to previous in vitro studies on the affect of heat-shock cognate (Hsc) 70 on part of the first nucleotide-binding domain of CFTR. Co-transfection of Hsp70 with its co-chaperone human DnaJ homologue (Hdj)-1/Hsp40, however, stabilizes the immature form of wt-CFTR, but not of F508del-CFTR, suggesting that these chaperones act on a wt-specific conformation. As the efficiency of conversion into the fully glycosylated form is not increased under Hsp70/Hdj-1 overexpression, the lack of these two chaperones does not seem to be critical for CFTR maturation and ER retention. The effects of 4-phenylbutyrate and deoxyspergualin, described previously to interfere with Hsp70 binding, were also tested upon CFTR degradation and processing. The sole effect observed was destabilization of F508del-CFTR.
A novel putative insect chitinase with multiple catalytic domains: hormonal regulation during metamorphosis
Authors: ROYER Véronique , FRAICHARD Stéphane , BOUHIN Hervé
Published: July 2002

We have used differential display to identify genes that are regulated by juvenile hormone in the epidermis of the beetle Tenebrio molitor. One of the genes encodes T. molitor chitinase 5 (TmChit5), a chitinase possessing an unusual structure. Sequence analysis of TmChit5 identified five ‘chitinase units’ of approx. 480 amino acids with similarity to chitinase family 18. These units are separated by less conserved regions containing putative PEST (rich in proline, glutamic acid, serine and threonine) sequences, putative chitin-binding domains and mucin domains. Northern-blot analysis identified a single transcript of approx. 9kb, whose abundance correlated with that of 20-hydroxyecdysone during metamorphosis. Injection of pupae with 20-hydroxyecdysone alone, or in combination with cycloheximide, indicated that TmChit5 expression is directly induced by the hormone. Further experiments indicated that methoprene (a juvenile hormone analogue) indirectly induced TmChit5 mRNA expression. On the basis of the present results and previous studies, we propose a molecular mechanism for cuticle digestion during the moulting process.
Biochemical and molecular characterization of two cytidine deaminases in the nematode Caenorhabditis elegans
Authors: THOMPSON Fiona J. , BRITTON Collette , WHEATLEY Isla , MAITLAND Kirsty , WALKER Glenda , ANANT Shrikant , DAVIDSON Nicholas O. , DEVANEY Eileen
Published: July 2002

Two cytidine deaminases (CDDs) from the free-living nematode Caenorhabditis elegans have been cloned and characterized. Both Ce-CDD-1 and Ce-CDD-2 are authentic deaminases and both exhibit RNA-binding activity towards AU-rich templates. In order to study their temporal and spatial expression patterns in the worm, reporter gene constructs were made using approx. 2kb of upstream sequence. Transfection of C. elegans revealed that both genes localized to the cells of the intestine, although their temporal expression patterns were different. Expression of Ce-cdd-1 peaked in the early larval stages, whereas Ce-cdd-2 was expressed in all life cycle stages examined. RNA-interference (RNAi) assays were performed for both genes, either alone or in combination, but only cdd-2 RNAi produced a consistent visible phenotype. A proportion of eggs laid from these worms were swollen and distorted in shape.
Loop variants of the serpin thyroxine-binding globulin: implications for hormone release upon limited proteolysis
Authors: GRASBERGER Helmut , GOLCHER Henriette M.B. , FINGERHUT Anja , JANSSEN Onno E.
Published: July 2002

Thyroxine-binding globulin (TBG) and corticosteroid-binding globulin are unique among non-inhibitory members of the superfamily of serine-proteinase inhibitors (serpins) in undergoing a dramatic increase in stability [stressed-to-relaxed (S→R) transition] after proteolytic cleavage within their exposed reactive-site-loop (RSL) equivalent. This structural rearrangement involves the insertion of the cleaved loop as a new strand into the β-sheet A and is accompanied by a decrease in hormone binding. To define the mechanism that leads to disruption of hormone binding of TBG after proteolytic cleavage, the effect of partial loop deletions and replacements by the α1-proteinase inhibitor homologues of TBG were evaluated. Unexpectedly, deletion of the loop's C-terminus, thought to be important for thyroxine binding, improved the binding affinity over that of normal TBG. Proteolytic cleavage of this variant revealed an intact S→R transition and reduced its binding activity to that of cleaved TBG. In contrast, a chimaera with C-terminal loop extension mimicked the decreased binding affinity of cleaved TBG and had a thermal stability intermediate between that of native and cleaved serpins. This variant was still susceptible to loop cleavage and underwent an S→R transition, yet without changing its binding affinity. Our data exclude a direct involvement of loop residues in thyroxine binding of native TBG. Limited insertion of the RSL into β-sheet A appears to trigger hormone release after proteolytic cleavage. In support of this concept, residues within the hinge region of the TBG loop are phylogenetically highly conserved, suggestive of their physiological role as a functional switch in vivo.
Influence of rapid changes in cytosolic pH on oxidative phosphorylation in skeletal muscle: theoretical studies
Authors: KORZENIEWSKI Bernard , ZOLADZ Jerzy A.
Published: July 2002

Cytosolic pH in skeletal muscle may vary significantly because of proton production/consumption by creatine kinase and/or proton production by anaerobic glycolysis. A computer model of oxidative phosphorylation in intact skeletal muscle developed previously was used to study the kinetic effect of these variations on the oxidative phosphorylation system. Two kinds of influence were analysed: (i) via the change in pH across the inner mitochondrial membrane and (ii) via the shift in the equilibrium of the creatine kinase-catalysed reaction. Our simulations suggest that cytosolic pH has essentially no impact on the steady-state fluxes and most metabolite concentrations. On the other hand, rapid acidification/alkalization of cytosol causes a transient decrease/increase in the respiration rate. Furthermore, changes in pH seem to affect significantly the kinetic properties of transition between resting state and active state. An increase in pH brought about by proton consumption by creatine kinase at the onset of exercise lengthens the transition time. At intensive exercise levels this pH increase could lead to loss of the stability of the system, if not compensated by glycolytic H+ production. Thus our theoretical results stress the importance of processes/mechanisms that buffer/compensate for changes in cytosolic proton concentration. In particular, we suggest that the second main role of anaerobic glycolysis, apart from additional ATP supply, may be maintaining the stability of the system at intensive exercise.
Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2
Authors: CHIOATO Lucimara , OLIVEIRA Arthur H.C. de , RULLER Roberto , SÁ Juliana M. , WARD Richard J.
Published: July 2002

Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca2+-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117—122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I. With the exception of Lys122, these same substitutions did not significantly alter the Ca2+-independent membrane-damaging activity. In contrast, substitution of the positively-charged residues at positions 115, 116 and 122 resulted in reduced Ca2+-independent membrane-damaging activity but, with the exception of Lys122, had no effect on myotoxicity. These results indicate that the two activities are independent and are determined by discrete yet partially overlapping motifs in the C-terminal loop. Results from site-directed mutagenesis of the aromatic residues in the same part of the protein suggest that a region including residues 115—119 interacts superficially with the membrane interface and that the residues around position 125 partially insert into the lipid membrane. These results represent the first detailed mapping of a myotoxic site in a phospholipase A2, and support a model of a Ca2+-independent membrane-damaging mechanism in which the C-terminal region of BthTx-I interacts with and contributes to the perturbation of the phospholipid bilayer.
Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice
Authors: CHANAS Simon A. , JIANG Qing , McMAHON Michael , McWALTER Gail K. , McLELLAN Lesley I. , ELCOMBE Clifford R. , HENDERSON Colin J. , WOLF C. Roland , MOFFAT Graeme J. , ITOH Ken , YAMAMOTO Masayuki , HAYES John D.
Published: July 2002

Mice that lack the Nrf2 basic-region leucine-zipper transcription factor are more sensitive than wild-type (WT) animals to the cytotoxic and genotoxic effects of foreign chemicals and oxidants. To determine the basis for the decrease in tolerance of the Nrf2 homozygous null mice to xenobiotics, enzyme assay, Western blotting and gene-specific real-time PCR (TaqMan®) have been used to examine the extent to which hepatic expression of GSH-dependent enzymes is influenced by the transcription factor. The amounts of protein and mRNA for class Alpha, Mu and Pi glutathione S-transferases were compared between WT and Nrf2 knockout (KO) mice of both sexes under both constitutive and inducible conditions. Among the class Alpha and class Mu transferases, constitutive expression of Gsta1, Gsta2, Gstm1, Gstm2, Gstm3, Gstm4 and Gstm6 subunits was reduced in the livers of Nrf2 mutant mice to between 3% and 60% of that observed in WT mice. Induction of these subunits by butylated hydroxyanisole (BHA) was more marked in WT female mice than in WT male mice. TaqMan® analyses showed the increase in transferase mRNA caused by BHA was attenuated in Nrf2−/− mice, with the effect being most apparent in the case of Gsta1, Gstm1 and Gstm3. Amongst class Pi transferase subunits, the constitutive hepatic level of mRNA for Gstp1 and Gstp2 was not substantially affected in the KO mice, but their induction by BHA was dependent on Nrf2; this was more obvious in female mutant mice than in male mice. Nrf2 KO mice exhibited reduced constitutive expression of the glutamate cysteine ligase catalytic subunit, and, to a lesser extent, the expression of glutamate cysteine ligase modifier subunit. Little variation was observed in the levels of glutathione synthase in the different mouse lines. Thus the increased sensitivity of Nrf2−/− mice to xenobiotics can be partly attributed to a loss in constitutive expression of multiple GSH-dependent enzymes, which causes a reduction in intrinsic detoxification capacity in the KO animal. These data also indicate that attenuated induction of GSH-dependent enzymes in Nrf2−/− mice probably accounts for their failure to adapt to chronic exposure to chemical and oxidative stress.
Expression of the leucocyte common antigen-related (LAR) tyrosine phosphatase is regulated by cell density through functional E-cadherin complexes
Authors: SYMONS Javelle R. , LeVEA Charles M. , MOONEY Robert A.
Published: July 2002

The leucocyte common antigen-related phosphatase (LAR) has been implicated in receptor tyrosine kinase signalling pathways while also displaying cell-density-dependency and localization to adherens junctions. Whereas physiological substrates for LAR have not been identified unequivocally, β-catenin associates with LAR and is a substrate in vitro. With the implication that LAR may play a role in regulating E-cadherin-dependent cell—cell communication and contact inhibition, the relationship of LAR with E-cadherin was investigated. LAR expression increased with cell density in the human breast cancer cell line MCF-7 and in Ln 3 cells derived from the 13762NF rat mammary adenocarcinoma. LAR protein levels decreased rapidly when cells were replated at a low density after attaining high expression of LAR at high cell density. COS-7 cells displayed comparable density-dependent regulation of LAR expression when transiently expressing exogenous LAR under the control of a constitutively active promoter, indicating that the regulation of expression is not at the level of gene regulation. Disrupting homophilic E-cadherin complexes by chelating extracellular calcium caused a marked decrease in LAR protein levels. Similarly, blocking E-cadherin interactions with saturating amounts of E-cadherin antibody (HECD-1) also led to a rapid and pronounced loss of cellular LAR. In contrast, mimicking cell-surface E-cadherin engagement by plating cells at low density on to dishes coated with HECD-1 resulted in a 2-fold increase in LAR expression compared with controls. These results suggest that density-dependent regulation of LAR expression is mediated by functional E-cadherin and may play a role in density-dependent contact inhibition by regulating tyrosine phosphorylation in E-cadherin complexes.
Identification of a novel AU-rich-element-binding protein which is related to AUF1
Authors: DEAN Jonathan L.E. , SULLY Gareth , WAIT Robin , RAWLINSON Lesley , CLARK Andrew R. , SAKLATVALA Jeremy
Published: July 2002

The AU-rich element (ARE) is an important instability determinant for a large number of early-response-gene mRNAs. AREs also mediate the stabilization of certain pro-inflammatory mRNAs, such as tumour necrosis factor (TNF)-α and cyclo-oxygenase-2 (COX-2), in response to inflammatory stimuli. To understand how AREs control mRNA stability, it is necessary to identify trans-acting factors. We have purified a new ARE-binding protein and identified it as CArG box-binding factor-A (CBF-A). The amino acid sequence of CBF-A is highly similar to that of the ARE-binding protein AUF1. Recombinant CBF-A bound the COX-2 and TNF-α AREs, but not a non-specific control RNA. In contrast, in an electrophoretic-mobility-shift assay (EMSA) of crude RAW 264.7 macrophage-like cell extracts, an antiserum that recognizes both AUF1 and CBF-A failed to supershift complexes formed on the TNF-α ARE, but did supershift a complex specific for the COX-2 ARE. CBF-A exists as two isoforms, p37 and p42, that differ by a 47-amino-acid insertion close to the C-terminus. By expressing epitope-tagged isoforms of CBF-A it was shown that the p42 isoform binds the COX-2 ARE in EMSA of crude cell extracts. In a HeLa-cell tetracycline-regulated reporter system, overexpression of the p42 CBF-A isoform resulted in stabilization of a COX-2 ARE reporter mRNA. Epitope-tagged p42 CBF-A expressed in HeLa cells co-immunoprecipitated with endogenous COX-2 mRNA, but not glyceraldehyde-3-phosphate dehydrogenase mRNA, as shown by reverse-transcription PCR. The similarity between CBF-A and AUF1 suggests that CBF-A could be re-named AUF2.
Irreversible inhibition of the bacterial cysteine protease-transpeptidase sortase (SrtA) by substrate-derived affinity labels
Authors: SCOTT Christopher J. , McDOWELL Andrew , MARTIN S. Lorraine , LYNAS John F. , VANDENBROECK Koen , WALKER Brian
Published: July 2002

We report on the first synthesis, kinetic evaluation and application of novel substrate-derived inhibitors against the Staphylococcus aureus cysteine protease-transpeptidase, sortase (staphylococcal surface protein sorting A, SrtA). The peptidyl-diazomethane and peptidyl-chloromethane analogues, Cbz (benzyloxycarbonyl)-Leu-Pro-Ala-Thr-CHN2 (I) and Cbz-Leu-Pro-Ala-Thr-CH2Cl (II) respectively were found to act as time-dependent irreversible inhibitors of recombinant sortase (SrtAΔN). The peptidyl-chloromethane analogue (II) was the most powerful with an inhibitor specificity constant (ki/Ki) of 5.3×104M-1·min-1, approx. 2-fold greater than that determined for the peptidyl-diazomethane (I). Additionally, using Western-blot analysis, we have been able to demonstrate that a biotinylated version of the peptidyl-diazomethane analogue, biotin-Ahx (aminohexanoyl)-Leu-Pro-Ala-Thr-CHN2 (III), can be used as an affinity label to detect the presence of wild-type SrtA in crude cell lysates prepared from S. aureus.
Prison‐based substance abuse treatment, residential aftercare and recidivism
Authors: Hiller Matthew L. , Knight Kevin , Simpson D. Dwayne
Published: July 2002

Aims. This study examined the impact of residential aftercare on recidivism following prison‐based treatment for drug‐involved offenders. Design. A matched group quasi‐experimental design. Survival regression analyses were used to predict time until rearrest. A logistic regression model was constructed for predicting aftercare completion. Setting. A 9‐month in‐prison therapeutic community (ITC) and several communitybased transitional therapeutic communities (TTCs). Participants. Data were collected from 396 male inmates (293 treated, 103 untreated). Measurements. Background information (gender, ethnicity, age, education level, criminal history and risk for recidivism) was abstracted from the state criminal justice databases and a structured interview led by clinical staff. During treatment process measures were based on inmate self‐ratings of their counselors, program and peers. A post‐treatment interview conducted by field research staff assessed satisfaction with transitional aftercare. Post‐release recidivism was based on state‐maintained computerized criminal history records. Findings. ITC treatment, especially when followed by residential aftercare, was effective for reducing post‐release recidivism rates. Lower satisfaction with transitional aftercare treatment was associated with not completing the residential phase of community‐based aftercare. Conclusions. Corrections‐based treatment policy should emphasize a continuum of care model (from institution to community) with high quality programs and services .
Drunkenness, feeling the effects and 5 + measures
Authors: Midanik Lorraine T.
Published: July 2002

Aims. The purpose of this study is to compare changes in the definitions of drunkenness and feeling the effects of drinking between 1979 and 1995 in US surveys, and compare three measures of heavier drinking as predictors of negative consequences of heavier alcohol use. Design. A trend analysis using the 1979 (N = 1772) and 1995 (N = 2178) US National Alcohol Surveys and a cross‐sectional analysis using only the 1995 National Alcohol Survey. Participants. The analysis included only those respondents who reported consuming alcohol in the last year. The data were collected by face‐to‐face interviews. Measurements. Three measures of heavier drinking were obtained in each survey: frequency of drunkenness, frequency of feeling the effects of alcohol and frequency drinking five or more drinks on one day. The three outcome measures were derived as indices of social consequences, alcohol dependence symptoms and alcohol‐related harm. Findings. Significantly higher proportions of respondents reported being drunk or feeling the effects of alcohol in 1995, and they also reported a lower number of drinks needed to become drunk (8.2 vs. 6.3 drinks) and to feel the effects (4.7 vs. 4.0 drinks). Frequency of drunkenness was found to be the best predictor of social consequences, alcohol dependence symptoms and alcohol‐related harm. Conclusions. These results point to some of the cultural shifts that have occurred in the United States during this "drying" period and the importance of considering more subjective measures of heavier drinking to assess risk.