bluuebunny/crossref_metadata_2025 huggingface dataset:
Redistribution and unmasking of Annexin V binding sites in apoptotic Raji cells
Authors: di Certo Maria Grazia , Faggioni Alberto , Barile Giuseppe
Published: June 2003

The complement receptor type 2 (CR2) associates with other surface antigens and proteins and its redistribution and/or unmasking occurs through still unknown mechanism(s). The data presented demonstrate that high‐density cultured CR2‐positive cells undergo apoptosis and that the redistribution and unmasking of Annexin V binding sites occurs in a fashion similar to the redistribution and unmasking of CR2. Therefore, apoptotic and non apoptotic cells from the same lineage may share a similar mechanism for the exposition of neo‐surface markers
Differential gene expression of bone‐related proteins in epithelial and fibroblastic cells derived from human periodontal ligament
Authors: Mouri Yoshihiro , Shiba Hideki , Mizuno Noriyoshi , Noguchi Takuji , Ogawa Tetsuji , Kurihara Hidemi
Published: June 2003

Hertwig's epithelial root sheath (HERS) is involved in the differentiation of cementoblasts. The cells of epithelial rests of Malassez (ERM) may contribute to that process. However, little is known about the role of these epithelial cells in cementum repair. In the present study, we investigated the expression of alkaline phosphatase (ALPase), osteopontin (OPN), bone morphogenetic protein (BMP)‐2 and BMP‐4 in epithelial cells (E cells) and fibroblastic cells (F cells) derived from the same human periodontal ligament. E cells were identified by immunoblotting with anti‐cytokeratin 5 and 8 antibody. Reverse transcription—polymerase chain reaction analysis showed that E cells have lower ALPase and BMP‐4 mRNA levels than F cells. On the other hand, the expression of OPN mRNA in E cells was stronger than in F cells. No significant difference was observed in BMP‐2 expression between E and F cells. Thus, they have different expression patterns of ALPase, BMP‐4 and OPN, suggesting that ERM and mesenchymal cells in periodontal ligament may be cooperatively involved in cementum repair. Furthermore, E cell cultures will be useful in elucidating the role of ERM.
Differential effects of Matrigel and its components on functional activity of CFTR and ENaC in mouse endometrial epithelial cells
Authors: Yang Jian Zhi , Ho Alice Lok Sze , Ajonuma Louis Chukwuemeka , Lam Sun Yee , Tsang Lai Ling , Tang Ning , Rowlands Dewi Kenneth , Gou Yu Lin , Chung Yiu Wa , Chan Hsiao Chang
Published: June 2003

Our previous studies have observed an effect of Matrigel, a solubilized basement membrane preparation extracted from the Engelbreth—Holm—Swarm (EHS) mouse sarcoma, on the expression of ion channels in mouse endometrial epithelia; namely the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP‐dependent Cl−channel, and the epithelial sodium channel (ENaC). The present study further investigated the effects of Matrigel and its individual components on the functional expression of CFTR and ENaC using the short‐circuit current (Isc) technique. The results showed that different components of Matrigel, namely growth factors, laminin and collagen, had differential effects on the functional activity of the two ion channels in murine endometrial epithelium. The information obtained may be useful for designing future in vitro culture models to investigate the functional roles of these ion channels in the endometrium.
Effect of BAP and IAA on the expression of G1 and G2 control points and G1‐S and G2‐M transitions in root meristem cells of Vicia faba
Authors: Polit Justyna T. , Maszewski Janusz , Kaźmierczak Andrzej
Published: June 2003

Excised, carbohydrate‐starved root meristems of Vicia faba subsp. minor have been used to investigate the impact of the auxin indole‐3‐acetic acid (IAA) and the cytokinin benzyl‐6‐aminopurine (BAP) on (1) the expression of Principal Control Points (PCPs) during the G1‐ and G2‐phases of the cell cycle, and (2) the dynamics of sucrose‐mediated resumption of DNA replication and mitosis (G1‐to‐S and G2‐to‐M transitions). Compared with the excised root tips starved in mineral medium without hormones, stationary phase meristems induced during continuous treatment with BAP, IAA, or a mixture of BAP+IAA, increased the number of G2 cells, producing characteristic profiles of nuclear DNA content. In medium containing 2% sucrose, BAP accelerated PCP1→S and PCP2→M, whereas continuous treatment with IAA resulted in marked prolongation of both transitions. Since the PCPs regulate progression through the key events of interphase and mitosis by interacting with cyclin dependent kinases (CDKs), these results seem to correspond with current data indicating functional connections between phytohormones, nutritional signals, gene expression and the cell division cycles in plants.
Inflammation: a novel mechanism for the transport of extracellular nucleotide‐induced arachidonic acid by S100A8/A9 for transcellular metabolism
Authors: Kannan Subburaj
Published: June 2003

Extracellular nucleotides cause neutrophil degranulation by activating the purinergic receptor subtype P2Y. However, the molecular mechanism involved in the signal pathway remains unknown. A hypothetical scheme suggesting that leukotriene(s) and leukotriene receptor(s) activation is required for extracellular nucleotide‐mediated neutrophil degranulation is presented here. Subsequent to the extracellular nucleotide binding to its receptors, intracellular arachidonic acid (AA) levels are elevated. Although AA is a known substrate of the lipoxygenase pathway mediated by 5‐lipoxygenase, excess AA could form a complex with S100A8/A9 for transport to the extracellular milieu. Extracellular availability of the S100A8/A9+AA complex could potentially be used for transcellular metabolism by resting and/or activated leukocytes (PMN, MN), vascular endothelium and smooth muscle cells at the inflammatory foci. Once imported into the resting and/or activated leukocytes, AA derived from the S100A8/A9+AA complex could serve as a substrate in the 5‐lipoxygenase‐mediated leukotriene pathway. Essentially, in addition to extracellular nucleotide‐induced leukotrienes, AA derived from the S100A8/A9+AA complex could also be utilized for the synthesis of inflammatory mediators such as leukotriene B4(LTB4), which in turn could trigger leukocyte degranulation, as well as cellular damage to vascular endothelium and smooth muscle cells, thereby exacerbating inflammation.
Immortalisation of a mucopolysaccharidosis type IIIC fibroblast cell line via expression of SV40 T antigen
Authors: Nelson Kathy , Melville Elizabeth L , Meikle Peter J , Anson Donald S
Published: June 2003

Mucopolysaccharidosis type IIIC is caused by a deficiency of acetyl—CoA: α‐glucosaminidase‐N‐acetyltransferase activity. This enzyme is unique among enzymes involved in the lysosomal degradation of glycosaminoglycans in that it catalyses an anabolic reaction, the addition of an acetyl group to glucosamine at the non‐reducing terminus of heparan sulphate. We have identified a mucopolysaccharidosis type IIIC skin fibroblast cell line with undetectable levels of residual acetyl—CoA: α‐glucosaminidase‐N‐acetyltransferase activity and immortalised it via expression of simian virus 40 large T antigen. Enzymatic analysis of two immortalised cell lines demonstrated that they both retained the original mucopolysaccharidosis IIIC phenotype. Variable number tandem repeat analysis confirmed that both were derived from the parental cell line.
Slow internal dynamics of membrane proteins in mechanisms of protease‐induced aggregation of platelets
Authors: Mazhul V , Chernovets T , Zaitseva E , Shcharbin D
Published: June 2003

Room temperature tryptophan phosphorescence (RTTP) of suspensions of human platelets was studied. RTTP spectra and decay kinetics of both intact platelets and those after short‐term incubation with low concentrations of thrombin or trypsin (0.3–50 μg/ml) were investigated. Protease‐induced changes in the RTTP lifetime of platelets were observed, and interpreted in terms of the slow internal dynamics of membrane protein modification. The functional role of membrane protein internal dynamics is discussed in the context of platelet aggregation and signal transduction processes.
Cellular aspects of elastogenesis in the elastic tendon of the chicken wing
Authors: Pimentel Silvia Borges , Carvalho Hernandes F.
Published: June 2003

Morphological, immunocytochemical and ultrastructural methods were used to investigate the role of cells during elastogenesis in the elastic tendon of the chicken wing. Intimate contact of the cell processes with elastic fibers was observed in adult birds. During development there was a sequential appearance of microfibril bundles that became progressively impregnated with amorphous elastin, which eventually predominated in fully developed elastic fibers. The growing elastic fibers were usually enveloped by recesses of the cell surface. The tendon cells were polarized in their association with fibrous components of the extracellular matrix. This arrangement suggests that these cells secrete and organize elastic and collagen fibers to different extracellular compartments. These results show that cells are intimately involved in producing components of different extracellular matrix fibers, in controlling their assembly, and in defining their borders and associations during development.
Stimulation of lipocalin‐type prostaglandin D synthase by retinoic acid coincides with inhibition of cell proliferation in human 3AO ovarian cancer cells
Authors: Su Bing , Guan Ming , Xia Jing , Lu Yuan
Published: June 2003

Lipocalin‐type prostaglandin D synthase (LPGDS; PGH2D‐isomerase; EC 5.3.99.2) is a bifunctional protein first identified in the mammalian brain. It acts as a PGD2‐producing enzyme and a retinoid transporter. Recent studies have shown that LPGDS is anomalously expressed in ovarian tumors and that retinoid may have a role as an ovarian cancer chemotherapeutic agent. To determine whether there is a relationship between retinoid and LPGDS in ovarian tumors, we examined the regulation of the gene encoding LPGDS by all‐trans retinoic acid (RA). Real‐time quantitative RT—PCR analysis showed that RA strongly induced the accumulation of LPGDS mRNA in human 3AO ovarian cancer cells. Furthermore, treatment of the cells with RA induced the synthesis and secretion of LPGDS into the culture medium. This increased expression of LPGDS was accompanied by an inhibition of cell proliferation in the ovarian cancer cells. Prostaglandin D synthase, ovarian cancer, retinoic acid, real‐time quantitative RT—PCR.
Radiographic signs in cats with nasal disease
Authors: Lamb C.R. , Richbell S. , Mantis P.
Published: June 2003

Radiographic signs in 64 cats that had radiography as part of the diagnostic work-up for suspected nasal disease were reviewed in a blinded fashion. Final diagnoses in these cats were rhinitis in 27, primary nasal neoplasia in 21 and non-nasal disease in 16. The signs with highest predictive value for nasal neoplasia were displacement of midline structures (73%), unilateral generalised soft tissue opacity (70%), unilateral generalised loss of turbinate detail (69%) and evidence of bone invasion (64%). The only radiographic finding that occurred more frequently in cats with rhinitis was a nasal cavity within normal limits, and the predictive value of this sign was only 38%. Radiographic signs in cats with nasal neoplasia are similar to those reported in dogs, whereas the radiographic signs in cats with rhinitis are variable and non-specific, and may be absent.
The diagnosis of heart failure in general practice: implications for the UK National Service Framework
Authors: Sparrow Nigel , Adlam David , Cowley Alan , Hampton John R.
Published: June 2003

Background:

The UK National Service Framework recommends patients with suspected heart failure undergo echocardiography. Selection of patients for this investigation in primary care is difficult. It is not clear which clinical features best identify patients with left ventricular systolic dysfunction.

Aim:

Using echocardiography, to establish the accuracy of primary care diagnosis of left ventricular systolic dysfunction. To investigate the sensitivity, specificity and predictive values of clinical features in the diagnosis of left ventricular systolic dysfunction.

Study:

A cross‐sectional study of 621 patients from a population prescribed loop diuretics in 7 general practices.

Method:

Clinical diagnoses were extracted from general practice records. Symptoms, clinical signs, ECG features, brain natriuretic peptide levels and echocardiographic findings were studied in a research clinic.

Results:

Left ventricular systolic dysfunction (ejection fraction <40%) was present in 50% of 621 patients prescribed loop diuretics in primary care. General practice diagnoses showed high false positive rates. Individual or combinations of clinical features did not accurately predict left ventricular systolic dysfunction.

Conclusion:

These results suggest the clinical diagnosis of left ventricular systolic dysfunction is inaccurate in this population. General practitioners should have a low threshold for referring patients prescribed loop diuretics for echocardiography. Increased open access echocardiography facilities will be needed.
Difficulties of introducing the National Service Framework for heart failure into general practice in the UK
Authors: Sparrow Nigel , Adlam David , Cowley Alan , Hampton John R.
Published: June 2003

Background:

The National Service Framework (NSF) sets standards for the management of heart failure in the UK. Loop diuretics are commonly first prescribed in primary care. Some patients taking these drugs have heart failure and may benefit from other treatments including ACE inhibitors. Accurate diagnosis in primary care is essential for the aims of the NSF to be realised.

Aims:

To investigate loop diuretic prescribing in general practice, to analyse recorded clinical features, patient investigations and ACE inhibitor use in this population.

Method:

One thousand three hundred and one patients taking loop diuretics were identified from prescription records of seven general practices. Demographic details, clinical features, investigations and drug treatments were extracted from patient records.

Results:

The prevalence of loop diuretic prescribing increased with age. Twenty percent of patients were attributed a diagnosis of heart failure but relevant clinical features were recorded in less than 50% of patient records. Open access echocardiography was used in 8.9% of patients. ACE inhibitors were prescribed in 39.8% of patients considered to have heart failure. 18.2% of these were taking the recommended target dose.

Conclusion:

Loop diuretics are prescribed commonly, particularly in the elderly. There is no clear pattern of documented clinical features that leads to prescription of these drugs. Open access echocardiography is rarely used to aid diagnosis. ACE inhibitors are under‐prescribed and under‐dosed in patients diagnosed with heart failure in this study population.
The heart failure revascularisation trial (HEART): rationale, design and methodology
Authors: Cleland J.G.F. , Freemantle N. , Ball S.G. , Bonser R.S. , Camici P. , Chattopadhyay S. , Dutka D. , Eastaugh J. , Hampton J. , Large S. , Norell M.S. , Pennell D.J. , Pepper J. , Sanda S. , Senior R. , Smith D.
Published: June 2003

Background:

Most patients with heart failure due to left ventricular systolic dysfunction (LVSD) secondary to coronary artery disease (CAD) have evidence of myocardium in jeopardy (reversible ischaemia and/or stunning hibernation). It is not known whether revascularisation in such cases is safe or beneficial.

Aims

To determine whether revascularisation will improve the survival of patients with LVSD and heart failure secondary to CAD and myocardium in jeopardy.

Methods:

This is a randomised controlled trial comparing revascularisation or not, in addition to optimal medical therapy with ACE inhibitors, beta‐blockers, aldosterone antagonists and an anti‐thrombotic agent. Patients must have heart failure requiring treatment with diuretics, a left ventricular ejection fraction <35% and evidence of coronary disease. Myocardial viability and ischaemia are assessed by a broad range of techniques including stress echocardiography and nuclear imaging. All imaging tests are reviewed in core laboratories to ensure uniform reporting. Any conventional revascularisation technique is permitted. The primary outcome measure is all cause mortality. Symptoms, quality of life and health economic issues will also be explored. Assuming an annual mortality of 10% in the control group and allowing for substantial cross‐over rates, a study of 800 patients followed for 5 years has 80% power with an alpha of 0.05 (two‐sided) to show a 25% reduction in mortality with revascularisation.

Results:

At the time of writing 180 patients have been screened for inclusion, 111 have consented to participate and 70 have been randomised. The results of viability testing are awaited in 22 patients. Twenty‐six patients had been investigated for myocardial viability and/or by angiography prior to consent, as part of the routine practice in that cardiology department. Of 68 patients who have completed assessment only after consent, 47 (69%) were included. The principal reason for drop‐out between consent and randomisation was lack of evidence of myocardial ischaemia or hibernation.

Conclusion:

The HEART trial will help to determine whether investigation of myocardial ischaemia and/or viability with a view to revascularisation should become part of the routine care of patients with heart failure due to LVSD and CAD.
Conceptualizing and Assessing Heterosexism in High Schools: A Setting‐Level Approach
Authors: Chesir-Teran Daniel
Published: June 2003

Heterosexism is defined as a setting‐level process that systematically privileges heterosexuality relative to homosexuality, based on the assumption that heterosexuality, as well as heterosexual power and privilege are the norm and the ideal. The many ways heterosexism is manifest in the physical–architectural, program–policy, suprapersonal, and social features of high schools are described followed by a proposal for a comprehensive assessment strategy. Strategies used in previous research are reviewed in terms of what is assessed, how it is assessed, and how it is analyzed. The author advocates for more comprehensive assessments and for school‐level analyses to enable comparisons between schools, facilitate research on the effects of heterosexism, and provide a basis for evaluating interventions. Additional issues include reliability and validity, links between heterosexism and other forms of oppression, heterosexism in other contexts or at other levels, and implications for theory and practice in community psychology.
Community Building with Aging and Old Lesbians
Authors: Nystrom Nancy M. , Jones Teresa C.
Published: June 2003

Descriptions of innovative methods of responding to the concerns and needs of aging and old lesbians are scarce in research literature. This paper describes the creation of the Elder Initiative, a community building effort for aging and old lesbians in a major northwest city. With the aims of providing support networks for the women and ensuring their involvement in the development of the local lesbian community, the Elder Initiative adopted the Kretzmann and McKnight model of community building to guide its objectives. In the 3 years since the group formed, membership has grown to over 550 women and several successful projects have been undertaken. The importance of the Elder Initiative and similar groups for successful aging among lesbians and areas of future research are discussed.
Stressor and Resilience Factors for Lesbians, Gay Men, and Bisexuals Confronting Antigay Politics
Authors: Russell Glenda M. , Richards Jeffrey A.
Published: June 2003

When lesbian, gay, and bisexual (LGB) people encounter antigay campaigns and elections, they face explicit and implicit homophobic attacks. In order to understand the points of stress and the bases for resilience in the face of these attacks, we developed a 130‐item quantitative survey on the basis of results of an earlier qualitative study. Three hundred, sixteen Colorado LGB people endorsed items representing sources of stress and sources of resilience associated with the campaign for and passage of an antigay referendum. Factor analyses of the results suggested 5 sources of stressors and 5 sources of resilience for LGB persons and their communities.
Coming Out in Community Psychology: Personal Narrative and Disciplinary Change
Authors: D'Augelli Anthony R.
Published: June 2003

Community psychology as a discipline has not focused its attention on the lives of lesbians, gay men, and bisexual (LGB) people, despite the field's potential to collaborate with this population in dealing with the many psychosocial challenges they face. Professional and personal barriers that community psychologists must confront conducting research and developing interventions with the LGB community are described. Community psychology work with LGB communities is often a “coming out” experience for LGB community psychologists, and a questioning of sexual orientation fot heterosexual community psychologists. Conceptual analyses and empirical reports are presented that exemplify how community psychologist can contribute to decreasing the pervasive stigma LGB people face. The use of an ecological model, a focus on the development and evaluation of preventive interventions, and a recognition of the diversity within the LGB population are core perspectives that can direct future work by community psychologists. Issues ranging from testing ways to empower young LGB people to analyses of the impact of the sociopolitical climate on LGB lives demonstrate the rich range of contributions community psychologists can make.
Working Pasteur's Quadrant: Harnessing Science and Action for Community Change
Authors: Price Richard H. , Behrens Teresa
Published: June 2003

Community psychology in general and the field of prevention in particular has unquestioningly accepted the assumption that the research process should proceed in a linear fashion from a search for basic knowledge to application in the community context. This ignores the compelling insight offered by Stokes (1997) that the drive for new knowledge and the pursuit of application can be combined in a single effort. If research in community psychology pursues the drive for application without an equal commitment to the development of knowledge about underlying community processes of social cooperation and change, it will become a field less capable of innovative and enduring contributions to community well‐being and effectiveness. Opportunities abound in community psychology for the simultaneous pursuit of new knowledge and more effective practice. We offer the example of a community leadership development program to promote collective efficacy as a case in point.
An Applied Collaborative Training Program for Graduate Students in Community Psychology: A Case Study of a Community Project Working with Lesbian, Gay, Bisexual, Transgender, and Questioning Youth
Authors: Stanley Jeanne L.
Published: June 2003

The following case study offers a detailed description of a university–organization partnership, the basis for a collaborative outreach training program between community psychology graduate students and a lesbian, gay, bisexual, transgender, and questioning (LGBTQ) youth center. Students and youth collaborated over 2 semesters on the creation of a social meeting venue within an urban LGBTQ youth‐operated center as part of a community outreach project in the students' course in community psychology. Semistructured interviews with the youth and the students provide first‐person accounts and perspectives of the project as an effective learning tool for training students in community psychology and in working with LGBTQ youth.
Methodological Issues in Research on Sexual Behavior with Latino Gay and Bisexual Men
Authors: Zea María Cecilia , Reisen Carol A. , Díaz Rafael M.
Published: June 2003

Latino gay men are at high risk for HIV/AIDS, and therefore it is critical that we increase our understanding of their sexual behavior. This paper discusses theoretical and methodological issues of conducting research with Latino gay and bisexual men. The importance of culture in psychological theory addressing sexual behavior is highlighted. Cultural and socioeconomic forces that impact the social construction of sexuality and sexual risk need to be taken into account to increase research validity. Social context and internalized sociocultural experiences can affect a variety of issues, including sexual scripts and the definition and fluidity of sexual orientation identity. Moreover, Latino gay men's sexuality may be influenced by experiences of oppression, discrimination, racism, and homophobia. Level of acculturation should also be considered. Measures that are developed by taking into account the cultural context and incorporating a Latino perspective are helpful in conducting meaningful research. Triangulation of quantitative and qualitative methods will help provide a picture rich in context and at the same time generalizable. The relationship between researchers and participants is affected by Latino cultural styles, and suggestions for approaches to conduct research in the Latino gay community are offered.
Exploring Dynamics of Abusive Lesbian Relationships: Preliminary Analysis of a Multisite, Qualitative Study
Authors: Ristock Janice L.
Published: June 2003

This paper presents preliminary results from a multisite, qualitative study on violence in lesbian relationships. A framework for conducting community‐based, empowerment research that draws on theories of community psychology, feminism, and postmodernism is presented. The study was designed to understand the dynamics of abusive lesbian relationships and social service providers' responses to the abuse. Results from 80 in‐depth interviews with lesbians who have experienced relationship violence are examined with a particular focus on a pattern of first relationships being abusive and a theme of shifting power dynamics. Analysis of focus group discussions with 45 feminist service providers (e.g., counselors, shelter workers, social workers, healthcare providers) reveals the difficulties in assessing the power dynamics of abusive same‐sex relationships and in developing appropriate responses when relying on heterosexually gendered models developed to address men's violence against women. The preliminary results present implications both for how we theorize and research this form of violence, and for improving the practices and policies of social services that work with lesbian, gay, bisexual, and transgendered communities.
Endocytosis of Synaptotagmin 1 Is Mediated by a Novel, Tryptophan‐Containing Motif
Authors: Jarousse Nadine , Wilson Joshua D. , Arac Demet , Rizo Josep , Kelly Regis B.
Published: June 2003

The rate at which a membrane protein is internalized from the plasma membrane can be regulated by revealing a latent internalization signal in response to an appropriate stimulus. Internalization of the synaptic vesicle membrane protein, synaptotagmin 1, is controlled by two distinct regions of its intracytoplasmic C2B domain, an internalization signal present in the 29 carboxyterminal (CT) amino acids and a separate regulatory region. We have now characterized the internalization motif by mutagenesis and found that it involves an essential tryptophan in the last beta strand of the C2B domain, a region that is distinct from the AP2‐binding site previously described. Internalization through the tryptophan‐based motif is sensitive to eps15 and dynamin mutants and is therefore likely to be clathrin mediated. A tryptophan‐to‐phenylalanine mutation had no effect on internalization of the CT domain alone, but completely inhibited endocytosis of the folded C2B domain. This result suggests that recognition of sorting motifs can be influenced by their structural context. We conclude that endocytosis of synaptotagmin 1 requires a novel type of internalization signal that is subject to regulation by the rest of the C2B domain.
Overexpression and Mislocalization of a Tail‐Anchored GFP Redefines the Identity of Peroxisomal ER
Authors: Lisenbee Cayle S. , Karnik Sheetal K. , Trelease Richard N.
Published: June 2003

Peroxisomal ascorbate peroxidase (APX) sorts indirectly via a subdomain of the ER (peroxisomal ER) to the boundary membrane of peroxisomes in tobacco Bright Yellow 2 cells. This novel subdomain characteristically appears as fluorescent reticular/circular compartments distributed variously in the cytoplasm. Further characterizations are presented herein. A peptide possessing the membrane targeting information for peroxisomal APX was fused to GFP (GFP‐APX). Transiently expressed GFP‐APX sorted to peroxisomes and to reticular/circular compartments; in both cases, the GFP moiety faced the cytosol. Of particular interest, both homotypic and heterotypic aggregates of peroxisomes, mitochondria, and/or plastids were formed. The latter two organelles comprised the circular portion of the reticular/circular compartments, apparently as a consequence of oligomerization (zippering) of the GFP moieties after insertion into the outer membranes of the affected organelles. These results, coupled with the accumulation of endogenous peroxisomal APX in cytoplasmic, noncircular compartment(s) following treatment with brefeldin A, indicate that authentic peroxisomal ER is composed only of a reticular compartment(s). Equally important, the data show that overexpressed, membrane‐targeted GFP fusion proteins have a propensity to form organelle aggregates that may lead to misinterpretations of sorting pathways of trafficked proteins.
The Intraflagellar Transport Machinery of Chlamydomonas reinhardtii
Authors: Cole Douglas G.
Published: June 2003

First discovered in the green alga, Chlamydomonas, intraflagellar transport (IFT) is the bidirectional movement of protein particles along the length of eukaryotic cilia and flagella. Composed of ∼16 different proteins, IFT particles are moved out to the distal tip of the organelle by kinesin‐II and are brought back to the cell body by cytoplasmic dynein 1b. Mutant analysis of the IFT motor and particle proteins using diverse organisms has revealed a conserved and essential role for IFT in the assembly and maintenance of cilia and flagella. IFT is thought to mediate this assembly through the delivery of axonemal precursors out to the distal tip of the growing organelle. Consistent with this model, the IFT particle proteins are rich in protein–protein binding motifs, suggesting that the particles may act as scaffolds for the binding of multiple cargoes. With most of the IFT proteins now identified at the level of the gene, this review will briefly examine both the structure and function of the IFT machinery of Chlamydomonas reinhardtii.
Phosphoinositide 3-kinase regulates maturation of lysosomes in rat hepatocytes
Authors: MOUSAVI Seyed Ali , BRECH Andreas , BERG Trond , KJEKEN Rune
Published: June 2003

To obtain information about the role of phosphoinositide 3-kinase (PI3K) in the endocytic pathway in hepatocytes, the uptake and intracellular transport of asialo-orosomucoid (ASOR) was followed in cells treated with wortmannin or LY294002. The two inhibitors, at concentrations known to inhibit the enzyme, did not affect internalization or the number of surface asialoglycoprotein receptors, but they caused a paradoxical increase (approx. 50% above control values) in the degradation of ASOR labelled with [125I]tyramine cellobiose ([125I]TC). Wortmannin or LY204002 inhibited the autophagic sequestration of lactate dehydrogenase very effectively, and the enhanced degradation of [125I]TC-ASOR could be an indirect effect of reduced autophagy, as an amino acid mixture known to inhibit autophagy also caused increased degradation of [125I]TC-ASOR, and its effect was not additive to that of wortmannin or LY294002. Wortmannin or LY294002 had pronounced effects on the late parts of the endocytic pathway in the hepatocytes: first, dense lysosomes disappeared and were replaced by swollen vesicles; secondly, degradation of [125I]TC-ASOR took place in an organelle of lower buoyant density (in a sucrose gradient) than the bulk of lysosomes (identified in the gradient by lysosomal marker enzymes). With increasing length of incubation with wortmannin or LY294002, the density distributions of the lysosomal markers also shifted to lower density and gradually approached that of the labelled degradation products. The labelled degradation products formed from [125I]TC-labelled proteins were trapped at the site of formation, because they did not penetrate the vesicle membranes. The results obtained indicate that internalization and intracellular transport of ASOR to lysomes may take place in the absence of PI3K activity in rat hepatocytes. On the other hand, fusion of late endosomes with lysosomes seems to produce ‘hybrid organelles’ (active lysosomes) that are unable to mature into dense lysosomes.
Organization on the plasma membrane of the retinitis pigmentosa protein RP2: investigation of association with detergent-resistant membranes and polarized sorting
Authors: CHAPPLE J. Paul , GRAYSON Celene , HARDCASTLE Alison J. , BAILEY Tracey A. , MATTER Karl , ADAMSON Peter , GRAHAM Catriona H. , WILLISON Keith R. , CHEETHAM Michael E.
Published: June 2003

Mutations in the retinitis pigmentosa protein gene RP2 account for up to 15% of X-linked retinitis pigmentosa. RP2 is a novel protein of unknown function, which is targeted to the plasma membrane by dual N-terminal acyl-modification. Dual-acylated proteins are targeted to lipid rafts, and some are subject to polarized sorting. Therefore we investigated the organization of RP2 on the plasma membrane. Endogenous RP2 protein was predominantly localized at the plasma membrane, and exogenously expressed green-fluorescent-protein-tagged protein was also targeted to the membrane in a wide range of cultured cells. High levels of endogenous RP2 protein were present in HeLa cells and in the retinal pigment epithelium-derived cell line ARPE19. A significant proportion of RP2 in cultured neuroblastoma cells was associated with detergent-resistant membranes (DRMs), but much less than other dually acylated proteins (e.g. Lyn and Fyn). In contrast, the RP2-interacting protein Arl3 (ADP-ribosylation factor-like 3) was not found to be associated with DRMs. The association of RP2 with DRMs was cholesterol-dependent. In polarized epithelial cells in culture and in vivo, RP2 was present in both the apical and basolateral domains of the plasma membrane. These data show that RP2 is not specific to either domain, unlike some other dually acylated proteins. Interestingly, the level of RP2 protein increased in the epithelial cell line Caco-2 with differentiation and polarization. These data show that RP2 is present on the membrane of all cell types examined both in vitro and in vivo, and that RP2 associates with lipid rafts, suggesting a potential role for the protein in signal transduction.
Mutagenesis of the HMGB (high-mobility group B) protein Cmb1 (cytosine-mismatch binding 1) of Schizosaccharomyces pombe: effects on recognition of DNA mismatches and damage
Authors: KUNZ Christophe , ZURBRIGGEN Karin , FLECK Oliver
Published: June 2003

Cmb1 (cytosine-mismatch binding 1) is a high-mobility group (HMG) protein of Schizosaccharomyces pombe, which consists of 223 amino acids and has a single HMG domain at the C-terminal end. We have created several mutant and deletion forms of the Cmb1 protein and studied the effects on general DNA binding and specific binding to DNA mismatches and damaged DNA. Cmb1Δ41 (i.e. Cmb1 from which the 41 N-terminal amino acids have been deleted) bound specifically to cytosine-containing mismatches, to the cisplatin-induced intrastrand cross-links cis-GG and cis-AG and to an O6-methylguanine lesion. DNA binding was not affected when the 45 N-terminal amino acids were deleted, but was abolished in the absence of the 50 N-terminal amino acids, and was reduced when Cmb1 was truncated by between five and eleven C-terminal amino acids. Cmb1, both with and without the C-terminal truncations, retained its DNA binding affinity after heating at 95 °C. The cmb1 gene was induced when S. pombe cells were treated with cisplatin. Mitotic mutation rates were increased in a S. pombe cmb1 null mutant and in a cmb1-(1–212) mutant, which encodes a Cmb1 protein lacking the 11 C-terminal amino acids. We conclude that mutation avoidance by Cmb1 is distinct from Msh2-dependent mismatch repair, but related to nucleotide excision repair.
Glucoamylase starch-binding domain of Aspergillus niger B1: molecular cloning and functional characterization
Authors: PALDI Tzur , LEVY Ilan , SHOSEYOV Oded
Published: June 2003

Carbohydrate-binding modules (CBMs) are protein domains located within a carbohydrate-active enzyme, with a discrete fold that can be separated from the catalytic domain. Starch-binding domains (SBDs) are CBMs that are usually found at the C-terminus in many amylolytic enzymes. The SBD from Aspergillus niger B1 (CMI CC 324262) was cloned and expressed in Escherichia coli as an independent domain and the recombinant protein was purified on starch. The A. niger B1 SBD was found to be similar to SBD from A. kawachii, A. niger var. awamori and A. shirusami (95–96% identity) and was classified as a member of the CBM family 20. Characterization of SBD binding to starch indicated that it is essentially irreversible and that its affinity to cationic or anionic starch, as well as to potato or corn starch, does not differ significantly. These observations indicate that the fundamental binding area on these starches is essentially the same. Natural and chemically modified starches are among the most useful biopolymers employed in the industry. Our study demonstrates that SBD binds effectively to both anionic and cationic starch.
The soluble ectodomain of RetC634Y inhibits both the wild-type and the constitutively active Ret
Authors: CERCHIA Laura , LIBRI Domenico , CARLOMAGNO Maria Stella , de FRANCISCIS Vittorio
Published: June 2003

Substitution of Cys-634 in the extracellular domain of the Ret tyrosine kinase receptor causes its dimerization and activation of its transforming potential. To gain further insight into the molecular basis leading to Ret activation we purified a mutant protein consisting of the entire ectodomain of the Ret carrying a Cys-634→Tyr substitution (EC-RetC634Y). The protein is glycosylated, like the native one, and is biologically active. By using an in vitro cell system we show that EC-RetC634Y inhibits the membrane-bound receptor RetC634Y, interfering with its dimerization. Furthermore, we demonstrate that EC-RetC634Y competes with the wild-type Ret receptor for ligand binding. The results presented support the notion of the possible involvment of glial cell line-derived neurotrophic factor (GDNF) with multiple endocrine neoplasia type 2A (MEN2A) tumours, and describe a useful tool for generating molecular mimetics directed towards specific mutations of the ret oncogene.
Synergism between nuclear receptors bound to specific hormone response elements of the hepatic control region-1 and the proximal apolipoprotein C-II promoter mediate apolipoprotein C-II gene regulation by bile acids and retinoids
Authors: KARDASSIS Dimitris , ROUSSOU Anastasia , PAPAKOSTA Paraskevi , BOULIAS Konstantinos , TALIANIDIS Iannis , ZANNIS Vassilis I.
Published: June 2003

We have shown previously that the hepatic control region 1 (HCR-1) enhances the activity of the human apolipoprotein C-II (apoC-II) promoter in HepG2 cells via two hormone response elements (HREs) present in the apoC-II promoter. In the present paper, we report that the HCR-1 selectively mediates the transactivation of the apoC-II promoter by chenodeoxycholic acid (CDCA) and 9-cis-retinoic acid. CDCA, which is a natural ligand of farnesoid X receptor α (FXRα), increases the steady-state apoC-II mRNA levels in HepG2 cells. This increase in transcription requires the binding of retinoid X receptor α (RXRα)–FXRα heterodimers to a novel inverted repeat with one nucleotide spacing (IR-1) present in the HCR-1. This element also binds hepatocyte nuclear factor 4 and apoA-I regulatory protein-1. Transactivation of the HCR-1/apoC-II promoter cluster by RXRα–FXRα heterodimers in the presence of CDCA was abolished by mutations either in the IR-1 HRE of the HCR-1 or in the thyroid HRE of the proximal apoC-II promoter, which binds RXRα–thyroid hormone receptor β (T3Rβ) heterodimers. The same mutations also abolished transactivation of the HCR-1/apoC-II promoter cluster by RXRα–T3Rβ heterodimers in the presence of tri-iodothyronine. The findings establish synergism between nuclear receptors bound to specific HREs of the proximal apoC-II promoter and the HCR-1, and suggest that this synergism mediates the induction of the HCR-1/apoC-II promoter cluster by bile acids and retinoids.
Identification of regions involved in the binding of alpha-bungarotoxin to the human alpha7 neuronal nicotinic acetylcholine receptor using synthetic peptides
Authors: MARINOU Martha , TZARTOS Socrates J.
Published: June 2003

The neuronal α7 nicotinic acetylcholine receptor (AChR) binds the neurotoxin α-bungarotoxin (α-Bgt). Fine mapping of the α-Bgt-binding site on the human α7 AChR was performed using synthetic peptides covering the entire extracellular domain of the human α7 subunit (residues 1–206). Screening of these peptides for 125I-α-Bgt binding resulted in the identification of at least two toxin-binding sites, one at residues 186–197, which exhibited the best 125I-α-Bgt binding, and one at residues 159–165, with weak toxin-binding capacity; these correspond, respectively, to loops C and IV of the agonist-binding site. Toxin binding to the α7(186–197) peptide was almost completely inhibited by unlabelled α-Bgt or d-tubocurarine. Alanine substitutions within the sequence 186–198 revealed a predominant contribution of aromatic and negatively charged residues to the binding site. This sequence is homologous to the α-Bgt binding site of the α1 subunit (residues 188–200 in Torpedo AChR). In competition experiments, the soluble peptides α7(186–197) and Torpedo α1(184–200) inhibited the binding of 125I-α-Bgt to the immobilized α7(186–197) peptide, to native Torpedo AChR, and to the extracellular domain of the human α1 subunit. These results suggest that the toxin-binding sites of the neuronal α7 and muscle-type AChRs bind to identical or overlapping sites on the α-Bgt molecule. In support of this, when synthetic α-Bgt peptides were tested for binding to the recombinant extracellular domains of the human α7 and α1 subunits, and to native Torpedo and α7 AChR, the results indicated that α-Bgt interacts with both neuronal and muscle-type AChRs through its central loop II and C-terminal tail.
Encoding of progesterone stimulus intensity by intracellular [Ca2+] ([Ca2+]i) in human spermatozoa
Authors: HARPER Claire V. , KIRKMAN-BROWN Jackson C. , BARRATT Christopher L. R. , PUBLICOVER Stephen J.
Published: June 2003

Progesterone induces a biphasic Ca2+ influx and consequent acrosome reaction in human spermatozoa. We have used two procedures to vary the stimulus (dosage and prior receptor desensitization) to investigate the encoding of stimulus strength by intracellular [Ca2+] ([Ca2+]i). Acrosome reaction and amplitude (but not kinetics) of the transient [Ca2+]i response (population measurement) showed sigmoidal dose sensitivity over the range 0.3 nM–3 μM, saturating at ≈300 nM (ED50≈30 nM). The amplitude of the sustained response saturated at 3 μM. Single-cell imaging showed that the amplitudes of both transient and sustained [Ca2+]i responses were highly dose-dependent, but that their frequency of occurrence and kinetics were largely dose-independent. Fluorimetric measurements confirmed that progesterone-induced [Ca2+]i influx was subject to desensitization, with second and subsequent applications of 3 μM progesterone being ineffective. However, sequential additions of 3 nM, 30 nM and 3 μM progesterone generated transient [Ca2+]i responses at each concentration, the amplitude and duration of the response to 3 μM progesterone being reduced compared with non-pretreated cells. Single-cell imaging revealed that pretreatment had no effect on the proportion of responsive cells, but single-cell responses, similarly to population responses, were smaller and markedly reduced in duration, consistent with an effect of desensitization on a late component of the [Ca2+]i transient. We conclude that the strength of the progesterone stimulus, when varied by dosage or by desensitization, is encoded by an analogue [Ca2+]i signal. Dose dependency of the acrosome reaction is therefore determined not by the number of progesterone-responsive cells but by variation in the probability of exocytosis in a ‘constant’ responsive population.
Partial characterization of cell-type X collagen interactions
Authors: LUCKMAN Steven P. , REES Elaine , KWAN Alvin P. L.
Published: June 2003

Type X collagen is a short-chain non-fibrillar collagen that is deposited exclusively at sites of new bone formation. Although this collagen has been implicated in chondrocyte hypertrophy and endochondral ossification, its precise function remains unclear. One possible function could be to regulate the processes of chondrocyte hypertrophy through direct cell–type X collagen interactions. Adhesions of embryonic chick chondrocytes, and cell lines with known expression of collagen-binding integrins (MG63 and HOS), were assayed on chick type X collagen substrates, including the native, heat-denatured and pepsin-digested collagen, and the isolated C-terminal non-collagenous (NC1) domain. Type X collagen supported the greatest level of adhesion for all cell types tested. The involvement of the α2β1 integrin in type X collagen–cell interaction was demonstrated by adhesion studies in the presence of Mg2+ and Ca2+ ions and integrin-function-blocking antibodies. Cells expressing α2β1 integrin (chick chondrocytes and MG63 cells) also adhered to heat-denatured type X collagen and the isolated NC1 domain; however, removal of the non-collagenous domains by limited pepsinization of type X collagen resulted in very low levels of adhesion. Both focal contacts and actin stress-fibre formation were apparent in cells plated on type X collagen. The presence of α2 and β1 integrin subunits in isolated chondrocytes and epiphyseal cartilage was also confirmed by immunolocalization. Our results demonstrate, for the first time, that type X collagen is capable of interacting directly with chondrocytes and other cells, primarily via α2β1 integrin. These findings are atypical from the fibrillar collagen–cell interactions via collagen binding integrins in that: (1) the triple-helical conformation is not strictly required for cell adhesion; (2) the NC1 domain is also involved in the adhesion of α2β1-expressing cells. These data form the basis for further studies into the mechanism and biological significance of type X collagen deposition in the growth plate.