bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a transcription factor, encoded by two vaccinia virus early genes, that regulates the intermediate stage of viral gene expression
Authors: Sanz Patrick , Moss Bernard
Published: July 2002

Vaccinia virus early, intermediate, and late stage genes are sequentially transcribed by the viral RNA polymerase within the cytoplasm of infected cells. We found that the 34- and 45-kDa polypeptides encoded by vaccinia virus ORFs A8R and A23R, respectively, were necessary to reconstitute transcription of a template with an intermediate stage promoter. Coexpression of the A8R and A23R genes in
Escherichia coli
was required for
in vitro
activity. In addition, the two polypeptides copurified, indicating their association as protein subunits of a vaccinia virus intermediate transcription factor. This factor, which we named VITF-3, complemented three viral proteins—namely, the RNA polymerase, capping enzyme, and a 30-kDa protein called VITF-1 that is also a subunit of the RNA polymerase—and an unidentified cell factor called VITF-2. Expression of the A8R and A23R genes occurred between 1 and 5 h after vaccinia virus infection and was not prevented by an inhibitor of DNA replication, consistent with a role for VITF-3 in specifically regulating intermediate transcription
in vivo
. The vaccinia virus A8R and A23R genes are highly conserved among vertebrate poxviruses, but no other viral or cellular homologs were identified.
Hypermutation in derepressed operons ofEscherichia coliK12
Authors: Wright Barbara E. , Longacre Angelika , Reimers Jacqueline M.
Published: July 2002

This article presents evidence that starvation for leucine in anEscherichia coliauxotroph triggers metabolic activities that specifically target theleuoperon for derepression, increased rates of transcription, and mutation. Derepression of theleuoperon was a prerequisite for its activation by the signal nucleotide, guanosine tetraphosphate, which accumulates in response to nutritional stress (the stringent response). A quantitative correlation was established betweenleuBmRNA abundance andleuB−reversion rates. To further demonstrate that derepression increased mutation rates, the chromosomalleuoperon was placed under the control of the inducibletacpromoter. When theleuoperon was induced by isopropyl-d-thiogalactoside, bothleuBmRNA abundance andleuB−reversion rates increased. These investigations suggest that guanosine tetraphosphate may contribute as much as attenuation in regulatingleuoperon expression and that higher rates of mutation are specifically associated with the derepressedleuoperon.
Loss of imprinting of a paternally expressed transcript, with antisense orientation to K
V
LQT1, occurs frequently in Beckwith–Wiedemann syndrome and is independent of insulin-like growth factor II imprinting
Authors: Lee Maxwell P. , DeBaun Michael R. , Mitsuya Kohzoh , Galonek Heidi L. , Brandenburg Sheri , Oshimura Mitsuo , Feinberg Andrew P.
Published: July 2002

Genomic imprinting plays a fundamental role in cancer and some hereditary diseases, including Beckwith–Wiedemann syndrome (BWS), a disorder of prenatal overgrowth and predisposition to embryonal malignancies such as Wilms tumor. We have previously shown that the K
V
LQT1 gene on chromosomal band 11p15 is imprinted, with expression of the maternal allele, and that the maternal allele is disrupted in rare BWS patients with balanced germ-line chromosomal rearrangements. We now show that an antisense orientation transcript within K
V
LQT1, termed LIT1 (long QT intronic transcript 1) is expressed normally from the paternal allele, from which K
V
LQT1 transcription is silent, and that in the majority of patients with BWS, LIT1 is abnormally expressed from both the paternal and maternal alleles. Eight of sixteen informative BWS patients (50%) showed biallelic expression, i.e., loss of imprinting (LOI) of LIT1. Similarly, 21 of 36 (58%) BWS patients showed loss of maternal allele-specific methylation of a CpG island upstream of LIT1. Surprisingly, LOI of LIT1 was not linked to LOI of insulin-like growth factor II (IGF2), which was found in 2 of 10 (20%) BWS patients, even though LOI of IGF2 occurs frequently in Wilms and other tumors, and in some patients with BWS. Thus, LOI of LIT1 is the most common genetic alteration in BWS. We propose that 11p15 harbors two imprinted gene domains—a more centromeric domain including K
V
LQT1 and p57
KIP2
, alterations in which are more common in BWS, and a more telomeric domain including IGF2, alterations in which are more common in cancer.
Sequence-specific binding of counterions toB-DNA
Authors: Denisov Vladimir P. , Halle Bertil
Published: July 2002

Recent studies by x-ray crystallography, NMR, and molecular simulations have suggested that monovalent counterions can penetrate deeply into the minor groove ofBform DNA. Such groove-bound ions potentially could play an important role in AT-tract bending and groove narrowing, thereby modulating DNA functionin vivo. To address this issue, we report here23Na magnetic relaxation dispersion measurements on oligonucleotides, including difference experiments with the groove-binding drug netropsin. The exquisite sensitivity of this method to ions in long-lived and intimate association with DNA allows us to detect sequence-specific sodium ion binding in the minor groove AT tract of threeB-DNA dodecamers. The sodium ion occupancy is only a few percent, however, and therefore is not likely to contribute importantly to the ensemble ofB-DNA structures. We also report results of ion competition experiments, indicating that potassium, rubidium, and cesium ions bind to the minor groove with similarly weak affinity as sodium ions, whereas ammonium ion binding is somewhat stronger. The present findings are discussed in the light of previous NMR and diffraction studies of sequence-specific counterion binding to DNA.
International perspectives in psychological research
Authors: Ritchie Pierre L.‐J.
Published: July 2002

The following two papers by (1) Bond and Sabourin and (2) d'Ydewalle and Denis were prepared for the World Social Science Report. They illustrate some of the current scope and diversity of the discipline.
Raising School Performance in the League Tables (Part 2): Barriers to responsiveness in three disadvantaged schools
Authors: Woods Philip A. , Levaçić Rosalind
Published: July 2002

Abstract This is the second of two articles drawing from the Economic and Social Research Council‐funded Impact of Competition on Secondary Schools study. The first article reported quantitative data concerning the relationship between the rate of school improvement and degree of social disadvantage amongst school student communities. This article examines qualitative data from three case‐study schools in order to illuminate the barriers to responsiveness that face socially disadvantaged schools and impede school improvement. Attention is drawn to the cumulative impact of contextual influences, which interact with internal school factors, particularly the influence of local school hierarchies and processes of labelling that affect students and their learning experiences.
The Global Alliance for Vaccines and Immunization - A Millennial Challenge
Authors: Nossal Gustav J. V.
Published: July 2002

As the new millennium dawns, a number of factors have conspired to make the outlook for global immunisation truly promising. These include private philanthropy aiming to raise immunisation rates in developing countries; a real head of steam behind the global poliomyelitis eradication campaign; the very recent introduction of several powerful new vaccines; and a 2-year-long effort to make the various elements of the United Nations system work closely together with non-governmental organisations and the private sector in wide co-operation. The aim is to prevent 3-4 million deaths per year. Despite a great deal of heartening progress, there is still need for further research. We have no vaccine for HIV/AIDS or malaria, and the only tuberculosis vaccine, namely BCG, is poorly effective in the prevention of adult pulmonary tuberculosis. However, new resources are bringing new players into the research field as well, so the longer term outlook is heartening.
Why do People Pay for Information?
Authors: Colomb Robert M.
Published: July 2002

This paper investigates the situations where there are incentives for people to pay a premium over the channel costs for information content. It concludes that there are at least four: premium low relative to channel costs and monopoly, which are less interesting as they are not specific to information; where the information need is idiosyncratic; and where the quality of the information source is critical.
The Dutch East India Company, Christiaan Huygens and the Marine Clock, 1682-95
Authors: Van der Kraan Alfons
Published: July 2002

The story of the Dutch East India Company, Christiaan Huygens and the marine clock shows that in the seventeenth century Dutch Republic there was a tendency towards the formation of a modern partnership between business, science and technology. This emerging relationship was personified by Johannes Hudde (1628-1704) and Christiaan Huygens (1629-1695), men from entirely different walks of life except for their shared interest in science, especially mathematics. It was this shared interest in mathematics which brought them together and indirectly led to the marine clock research project. Hudde was a Director of the Dutch East India Company as well as a mathematician of international standing, whilst Huygens was both a brilliant theoretical scientist and extremely skilled innovator.Through his interest in mathematics, Hudde had come to know Huygens - he had corresponded with him and was broadly familiar with the work Huygens had been doing. So when Huygens, in 1682, returned to Holland from France, Hudde conceived the idea, which was entirely novel at the time, to enlist the support of the East India Company for one of Huygens' research projects, a project, of course, in which the Company had a direct economic interest, namely, the marine clock which it needed to find the longitude at sea.
From POTS to E-commerce: What Have the Developing Countries Learnt About Property Rights Over the Last 50 Years?
Authors: Singh J. P.
Published: July 2002

The challenge of electronic commerce is new to the developing world. Will this technology-driven initiative allow developing countries in Asia to leapfrog? Electronic commerce will trot or walk depending upon the property rights shaping its behaviour. The history of information infrastructural provision teaches us that efficient property rights can only be expected in rare circumstances, when the polity has a highly developed civil society and existing institutions produce restraint. Sequencing and the fit between domestic institutions and the types of property rights are important. Well-organized large user groups are clear winners from reforms, but universal service in countries like South Korea and Singapore resulted from state prerogatives. Three layers of an electronic commerce network along with five conditions of property rights efficiency are identified.
Are We Eating our Seed Corn?: Basic Research in the US Corporate Sector
Authors: Varma Roli
Published: July 2002

Since the mid-1980s, industrial research in the United States has gone through major organizational changes. Funding for centralized corporate research laboratories in the high technology industry, which leads in research, has shifted from corporate sources to business divisions. Research has been either transferred into individual business units or organized along product lines for well-known markets. As a result, support has shifted to low-risk, mission-oriented, and short-term research, and an extensive involvement of business elements in research activities. Basic research projects seem to be completely gone from centralized corporate research laboratories. In the long run, the shift away from the untargeted inquiry can be problematic to the company, as well as to the country.
Accelerating Technology: The Pace of Transmission Systems
Authors: Noll A. Michael
Published: July 2002

The growths in capacity of various generations of transmission technology were studied. The results show that the growth rates for different transmission technologies are themselves increasing, which means that the pace of transmission technology is accelerating. What is somewhat surprising is that long-distance rates are not decreasing more steeply. What is not surprising is the bandwidth glut in backbone networks. However, the increasing availability of transmission capacity in the bandwidth-rich world of the future could herald a return to circuit switching for data telecommunication.
The role of substitute therapy in the treatment of problem amphetamine use
Authors: KLEE HILARY , WRIGHT SAMANTHA , CARNWATH TOM , MERRILL JOHN
Published: July 2002

The aim of the project was to examine the effectiveness of treatment services for amphetamine users in the North West of England. Semi‐structured interviews were conducted by researchers in a longitudinal matched case‐control design in which each client was matched with an amphetamine user not in contact with services. Interviews yielded quantitative data which were subjected to standard statistical tests, and also rich qualitative data that provided further insights into their attitudes and behaviour. This paper reports on the subgroup of drug agency clients who were prescribed dexamphetamine (PDAs) and the changes in their use of street amphetamine. Analyses comparing prescribed clients with their controls revealed a significant reduction in amount and frequency of street amphetamine consumption over the first 3 months of treatment. Such changes in amphetamine use were most marked in the first month of treatment. Comparisons between PDAs and clients in treatment but not receiving a prescription (NPDAs) revealed that the reduction in amount was greater for the prescribed clients. There was no difference in the frequency of use between these client groups. Different patterns of change were observed among injecting clients. Retention in treatment was significantly greater for PDAs. Clients' compliance with treatment regimens and the concerns of health professionals about substitute prescribing with dexamphetamine were revealed through qualitative analyses.
Characteristics of amphetamine users seeking information, help and treatment in Adelaide, South Australia
Authors: VINCENT NIKI , SHOOBRIDGE JODIE , ASK ALEX , ALLSOP STEVE , ALI ROBERT
Published: July 2002

This study was designed as a needs assessment for interventions to respond to hazardous and harmful amphetamine use, including options for treatment. Research techniques from Rapid Assessment Methodology (RAM) were utilized to collect data. This included a survey of 100 amphetamine users, from which the data for the current paper has been drawn. This paper is focused on the characteristics of those who had sought information and help in the sample, and on those who were dependent on amphetamine and who felt they needed treatment, in comparison to those who were also dependent but did not feel the need for treatment. Findings in regard to treatment‐seekers were largely consistent with previous Australian and overseas research, and thus underscore the importance of considering psychological morbidity, both prior and subsequent to amphetamine use, in treatment settings for amphetamine users. Having previously sought general information about amphetamines, safer drug use and blood‐borne diseases was best predicted by higher sexual risk‐taking and poorer physical functioning. Having previously sought help for an amphetamine‐related concern or problem was best predicted by higher severity of dependence on amphetamines and poorer social functioning. Peers were considered to be important sources of information and help, as were GPs. In developing interventions for amphetamine users, consideration should be given to the characteristics of those who seek particular types of assistance.
Evaluation of Inflammatory Cytokine Secretion by Human Alveolar Macrophages
Authors: García J. E. Losa , Rodríguez F. M. , de Cabo M. R. Martín , Salgado M. J. García , Losada J. P. , Villarón L. G. , López A. J. , Arellano J. L. P.
Published: July 2002

The alveolar macrophage (AM) secretes interleukin 1β (IL‐1β), tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8), all of them inflammatory cytokines involved in the pathogenesis of many lung diseases. The aim of the present work was to evaluate the basal and stimulated secretion of these cytokines by human AMs. Human AMs were collected by bronchoalveolar lavage (BAL) from four healthy controls and 13 patients with diffuse interstitial lung disease (five cases of sarcoidosis, three of hypersensitivity pneumonitis and five of idiopathic pulmonary fibrosis). AMs were cultured in the presence or absence of different concentrations of lipopolysaccharide (LPS), phorbolmyristate and gammainterferon. IL‐1β, TNF‐α, IL‐6 and IL‐8 levels were measured in BAL fluid and culture supernatant using specific enzyme‐linked immunosorbent assays. The substance found to stimulate the secretion of inflammatory cytokines to the greatest extent was LPS at a concentration of 10 μg/ml. Regarding the secretion of IL‐1β, four observations were of interest: basal secretion was very low; LPS exerted a potent stimulatory effect; considerable within‐group variability was observed; and there were no significant differences in the comparisons among groups. With respect to TNF‐α secretion, the results were similar. The only striking finding was the higher basal secretion of this cytokine with respect to that of IL‐1β. Regarding the secretion of IL‐6, the same pattern followed by TNF‐α was found. However, it should be stressed that the increase induced by LPS was smaller than in the two previous cytokines. Regarding the secretion of IL‐8, three findings were patent: the strong basal secretion of this cytokine; the moderate increase induced by LPS; and the existence of significant differences among the different groups with respect to the stimulated secretion of this cytokine, which reached maximum values in patients with idiopathic pulmonary fibrosis. Finally, it should be noted that the pattern of cytokines observed in the BAL fluid was similar to that found in cultured AM supernatants. The pattern of inflammatory cytokine secretion by AMs differs from that of other cells of the mononuclear phagocyte system (MPS). In this sense. AMs secrete low amounts of IL‐1, moderate amounts of TNF‐α and IL‐6, and high quantities of IL‐8. Adherence is an important stimulus in the secretion of these molecules and LPS elicits an increased secretion inverse to the basal secretion. There is considerable individual variability in the secretion of inflammatory cytokines by the AMs of patients with interstitial lung disease and the AMs of these patients are primed in vivo for the secretion of these cytokines. The results of our study, carried out in vitro, can be extrapolated to the in vivo setting.
Book and media reviews
Authors: COOK CHRIS
Published: July 2002

Books reviewed in this article:

Book reviews in this column will primarily be of titles focusing completely, or in part, on biological aspects of addiction. However, significant titles of general relevance to the addictions field will also be included, even if they are not “biological”, as will titles of general methodological and clinical relevance, even if they are not on “addictions”. Similar considerations will apply to other media (software, audio tapes and CDs, videos, etc). However, specific “addictions” software applications seem to be relatively uncommon and, as these items are rarely reviewed elsewhere, we will endeavour to include reviews of some of the older programmes that are still useful, as well as new titles that appear. I would appreciate suggestions of any items suitable for reviews, but especially software and other media of specific relevance to the addictions. Please contact: Professor Chris Cook, Kent Institute of Medicine and Health Sciences, University of Kent at Canterbury, Kent CT2 7NR, UK. Email: c.c.h.cook@ukc.ac.uk

Cocaine: Effects on the Developing Brain J.A. HARVEY & B.E. KOSOFSKY (Eds)

Co‐morbidity in Affective Disorders MAURICIO TOHEN (Ed.)

Designer Drugs Directory KARL VALTER & PHILIPPE ARRIZABALAGA

Forbidden Drugs, 2nd edn PHILIP ROBSON

Aviation Medicine, 3rd edn J. ERNSTING, A. N. NICHOLSON & D. J. RAINFORD

Research in Medicine: Planning a Project to Writing a Thesis GEORGE MURELL, CHRISTOPHER HAUNG & HAROLD ELLIS

Multivariable Analysis: a Practical Guide for Clinicians MITCHELL H. KATZ

Statistical Genomics: Linkage, Mapping and QTL Analysis BEN HUI LIU

Spiritual Evolution J.M. TEMPLETON & K. S. GINGER

The Troubled Helix—Social and Psychological Implications of the New Human Genetics THERESA MARTEAU & MARTIN RICHARDS (Eds)
Information processing deficits in withdrawing alcoholics
Authors: Keedwell Paul A. , Kumari Veena , Poon Lucia , Marshall E. JANE , Checkley Stuart A.
Published: July 2002

Prepulse inhibition (PPI) of the acoustic startle response (a reduction in response to an intense, startling stimulus (the pulse) if preceded by 30–50 ms by a weaker, non‐startling stimulus) is an established model to index information processing deficits in thought‐disordered schizophrenic patients. The present study aimed to investigate the influence of alcohol withdrawal on the PPI effect. Eight withdrawing alcoholic patients underwent testing for PPI of the acoustic startle response (defined as percentage reduction of the response over pulse‐alone stimulus; prepulses 15 dB above the background) on three occasions (1, 3 and 7 days following the last drink). The results demonstrated remarkably low levels of PPI on days 1 and 3, with this being very robust in three patients who had a history of delirium tremens; there was a trend towards normalization of PPI on day 7. This study, although preliminary, suggests strongly that there is a deficit in the filtering of sensory information in alcohol‐dependent patients undergoing alcohol withdrawal. This was most apparent in those with a history of delirium tremens. Further studies are needed to define the cause and chronicity of these deficits.
Propofol dependency after treatment of tension headache
Authors: Schneider Udo , Rada Dominique , Rollnik Jens D. , Passie Thorsten , Emrich Hinderk M.
Published: July 2002

Propofol is a widely used general anaesthetic with growing usage outside anaesthesia. Despite its potential for abuse only five cases of propofol dependency have yet been reported. These comprise health care professionals with easy access to the drug. This is the first reported case of propofol dependency in a lay person. One should be aware of the abuse potential of propofol with regard to its growing use outside the context of general anaesthesia.
Book and Media Reviews
Authors: Ball David
Published: July 2002

Book reviewed in this article:

Book reviews in this column will primarily be of titles focusing completely, or in part, on biological aspects of addiction. However, significant titles of general relevance to the addictions field will also be included, even if they are not “biological”, as will titles of general methodological and clinical relevance, even if they are not on “addictions”. Similar considerations will apply to other media (software, audio tapes and CDs, videos, etc.). However, specific “addictions” software applications seem to be relatively uncommon and, as these items are rarely reviewed elsewhere, we will endeavour to include reviews of some of the older programmes that are still useful, as well as new titles that appear. I would appreciate suggestions of any items suitable for reviews, but especially software and other media of specific relevance to the addictions. Please contact: Dr David Ball, National Addiction Centre, 4 Windsor Walk, London SE5 8AF, UK. Email: media@addictionbiology.com

The Science of Marijuana L. L. IVERSEN

Memory Disorders in Psychiatric Practice G. E. BERRIOS & J. R. HODGES (Eds)

Drug Use and Prisons D. SHEWAN & J. B. DAVIES (Eds)

Meta‐analysis in Medicine and Health Policy D. K. STANGL & D. A. BERRY (Eds)

Brain Imaging in Clinical Psychiatry R. R. KRISHNAN & P. M. DORAISWAMY (Eds)
Carbohydrate deficient transferrin in alcoholic and non‐alcoholic liver disease: a comparison of two assay methods
Authors: KEATING JAMES , CHEUNG CONNIE , PETERS TIMOTHY J. , PRZEMIOSLO ROBERT , WILLIAMS ROGER , SHERWOOD ROY A.
Published: July 2002

Carbohydrate‐deficient transferrin (CDT) was assayed in 105 patients with non‐alcohol‐related liver diseases, 50 patients with alcohol‐induced liver disease and 40 alcohol misusers with minimal hepatic dysfunction. The patients with liver disease were hospitalized for assessment of suitability for orthotopic liver transplantation. CDT was measured by two commercially available micro anion exchange methods; CDTect (Pharmacia) and %CDT (AXIS). Additionally, total transferrin was measured to allow expression of the CDTect results as ‘relative’ measurements. Overall, 41/105 (39%) of patients with non‐alcohol‐related liver diseases had a raised CDTect result, 13/105 (12%) had a raised %CDT result and 26/105 (25%) an abnormal CDTect/total transferrin ratio. In general, patients with cholestatic liver disease, primary biliary cirrhosis or primary sclerosing cholangitis had more ‘false positive’ results by CDTect than did those with viral or autoimmune hepatitis. The sensitivity of the two methods for the detection of alcohol misuse was similar; CDTect 77%, %CDT 65% and CDTect/total transferrin ratio 70%. This was, however, lower than the sensitivity of the conventional marker GGT (82%). In patients with severe liver disease, where up to 30% of the serum total transferrin concentrations fall outside the reference range, ‘relative’ CDT methods have greater specificity than ‘absolute’ measurements.
Metabolism of drugs of abuse by cytochromes P450
Authors: CHO ARTHUR K. , NARIMATSU SHIZUO , KUMAGAI YOSHITO
Published: July 2002

Studies of most drugs of abuse utilize in vivo animal experimentation so that the responses measured reflect the pharmacokinetics of the administered drug as well as its pharmacodynamics. These drugs are generally lipid soluble chemicals and their elimination is dependent on metabolism, so an understanding of this process is critical to the interpretation of responses. This review summarizes the interaction between drugs of abuse and cytochromes P450, the oxidative enzymes that catalyze the first step of the metabolic process. Although they process their substrates by a common chemical mechanism, these enzymes differ markedly in their regulation, i.e. induction and inhibition, their substrate selectivities, the metabolites they generate and their relative concentration in different species. The activity of an enzyme catalyzing a specific metabolic reaction can be altered by prior xenobiotic exposure, by its genetics and by a co‐administered drug, so that the pharmacokinetics of the drug under study can vary with the history of the individual subject. These issues are obviously important in human studies so, when possible, the relevant human enzymes involved in the processes described have been identified.
Amino Acid Sequence of the Protease Inhibitor BWI‐4a From Buckwheat Seeds
Authors: Belozersky Mikhail A. , Dunaevsky Yakov E. , Musolyamov Alexander Kh. , Egorov Tsezi A.
Published: July 2002

The complete amino acid sequence of protease inhibitor BWI‐4a from buckwheat (Fagopyrum esculentum Moench) seeds, consisting of 67 amino acid residues with a single disulfide bond, has been established by Edman degradation in combination with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Its N terminus is blocked by a pyroglutamic acid residue. Mass spectrometric analysis revealed that inhibitor BWI‐4a is present in buckwheat seeds in two isoforms with a single amino acid substitution of Ala40 for Gly40. The reactive site of the inhibitor contains an Arg43‐Asp44 bond. Analysis of the amino acid sequence suggests that the buckwheat seed protease inhibitor is a member of the potato proteinase inhibitor I family.
Transport of Mitochondria During Axonogenesis
Authors: Dedov Vadim N. , Dedova Irina V. , Armati Patricia J.
Published: July 2002

The cellular mechanisms involved in axonogenesis are still unclear. In the present work we found that formation of neurites in cultured neonatal dorsal root ganglion neurons co‐incided with the redistribution of highly charged mitochondria. Radially distributed in subplasmalemmal space 3 h after plating, highly charged mitochondria formed clusters in the hillocks of predominant neurites during the next 24?48 h and then redistributed into the axons. These results provide evidence that accumulation of a critical mass of charged mitochondria at the site of the future axonal hillock may represent the slow initiation stage of axonogenesis, followed by a fast growth phase.
Nuclear Factor of Activated T Cells (NFAT1‐C) Represses the Enhancer II and Pregenomic Promoter (EnII/Cp) of Hepatitis B Virus (HBV) Through its Responsive Site GGAGA and Nullifies the HBx‐Driven Transcriptional Activation
Authors: Lee Joong Hyuk , Rho Hyune Mo
Published: July 2002

The immunosuppressant cyclosporin A (CsA)‐sensitive nuclear factor of activated T cells 1 (NFAT1) has been known to be a transcriptional regulator of cytokine and viral genes during the immune response. By analyses of serial deletion, mutation, and heterologous promoter assay, we report here that the CsA‐sensitive NFAT1‐C represses the transcriptional activity of enhancer II and pregenomic promoter (EnII/Cp) of HBV through the NFAT1‐C responsive site (GGAGA, nt 1603‐1618) and nullifies the HBx‐driven transcriptional activation of the EnII/Cp of HBV in a dose‐dependent manner. These results suggest that a CsA‐sensitive NFAT1‐C may control the viral activity in HBV‐infected cells by inhibiting the EnII/Cp and nullifying the HBx‐driven transcriptional activation.
Kinesin Light Chains Are Essential for Axonal Transport in Drosophila
Authors: Gindhart Joseph G. , Desai Chand J. , Beushausen Sven , Zinn Kai , Goldstein Lawrence S.B.
Published: July 2002

Kinesin is a heterotetramer composed of two 115-kD heavy chains and two 58-kD light chains. The microtubule motor activity of kinesin is performed by the heavy chains, but the functions of the light chains are poorly understood. Mutations were generated in the Drosophila gene Kinesin light chain (Klc), and the phenotypic consequences of loss of Klc function were analyzed at the behavioral and cellular levels. Loss of Klc function results in progressive lethargy, crawling defects, and paralysis followed by death at the end of the second larval instar. Klc mutant axons contain large aggregates of membranous organelles in segmental nerve axons. These aggregates, or organelle jams (Hurd, D.D., and W.M. Saxton. 1996. Genetics. 144: 1075–1085), contain synaptic vesicle precursors as well as organelles that may be transported by kinesin, kinesin-like protein 68D, and cytoplasmic dynein, thus providing evidence that the loss of Klc function blocks multiple pathways of axonal transport. The similarity of the Klc and Khc (Saxton et al. 1991. Cell 64:1093–1102; Hurd, D.D., and W.M. Saxton. 1996. Genetics 144: 1075–1085) mutant phenotypes indicates that KLC is essential for kinesin function, perhaps by tethering KHC to intracellular cargos or by activating the kinesin motor.
ARF1 Mediates Paxillin Recruitment to Focal Adhesions and Potentiates Rho-stimulated Stress Fiber Formation in Intact and Permeabilized Swiss 3T3 Fibroblasts
Authors: Norman J.C. , Jones D. , Barry S.T. , Holt M.R. , Cockcroft S. , Critchley D.R.
Published: July 2002

Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTPγS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPγS at 8 min after permeabilization still induced paxillin recruitment to focal adhesion–like structures at the ends of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two proteins are regulated by different mechanisms. Paxillin recruitment was largely rho-independent, but could be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH2-terminally truncated Δ17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPγS- induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regulate paxillin recruitment to focal adhesions was confirmed by microinjection of Q71LARF1 and Δ17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA- induced assembly of actin stress fibers was potentiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers.
GMAP-210, A Cis-Golgi Network-associated Protein, Is a Minus End Microtubule-binding Protein
Authors: Infante Carlos , Ramos-Morales Francisco , Fedriani Concepción , Bornens Michel , Rios Rosa M.
Published: July 2002

We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997–1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1,979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210–encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.
A Cytoplasmic Dynein Heavy Chain Is Required for Oscillatory Nuclear Movement of Meiotic Prophase and Efficient Meiotic Recombination in Fission Yeast
Authors: Yamamoto Ayumu , West Robert R. , McIntosh J. Richard , Hiraoka Yasushi
Published: July 2002

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination.
Matching of Calcineurin Activity to Upstream Effectors Is Critical for Skeletal Muscle Fiber Growth
Authors: Dunn Shannon E. , Chin Eva R. , Michel Robin N.
Published: July 2002

Calcineurin-dependent pathways have been implicated in the hypertrophic response of skeletal muscle to functional overload (OV) (Dunn, S.E., J.L. Burns, and R.N. Michel. 1999. J. Biol. Chem. 274:21908–21912). Here we show that skeletal muscles overexpressing an activated form of calcineurin (CnA*) exhibit a phenotype indistinguishable from wild-type counterparts under normal weightbearing conditions and respond to OV with a similar doubling in cell size and slow fiber number. These adaptations occurred despite the fact that CnA* muscles displayed threefold higher calcineurin activity and enhanced dephosphorylation of the calcineurin targets NFATc1, MEF2A, and MEF2D. Moreover, when calcineurin signaling is compromised with cyclosporin A, muscles from OV wild-type mice display a lower molecular weight form of CnA, originally detected in failing hearts, whereas CnA* muscles are spared this manifestation. We also show that OV-induced growth and type transformations are prevented in muscle fibers of transgenic mice overexpressing a peptide that inhibits calmodulin from signaling to target enzymes. Taken together, these findings provide evidence that both calcineurin and its activity-linked upstream signaling elements are crucial for muscle adaptations to OV and that, unless significantly compromised, endogenous levels of this enzyme can accommodate large fluctuations in upstream calcium-dependent signaling events.
The Telomerase/Vault-Associated Protein Tep1 Is Required for Vault RNA Stability and Its Association with the Vault Particle
Authors: Kickhoefer Valerie A. , Liu Yie , Kong Lawrence B. , Snow Bryan E. , Stewart Phoebe L. , Harrington Lea , Rome Leonard H.
Published: July 2002

Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTep1−/− mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTep1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTep1−/− mice. Although vaults purified from the livers of mTep1−/− mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTep1−/− vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.
Nucleolar Components Involved in Ribosome Biogenesis Cycle between the Nucleolus and Nucleoplasm in Interphase Cells
Authors: Chen Danyang , Huang Sui
Published: July 2002

We examined the mobilities of nucleolar components that act at various steps of the ribosome biogenesis pathway. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) analyses demonstrate that factors involved in rRNA transcription (upstream-binding factor [UBF]), processing (nucleolin, fibrillarin, and RNase MRP subunits, Rpp29), and ribosome assembly (B23) exchange rapidly between the nucleoplasm and nucleolus. In contrast, the mobilities of ribosomal subunit proteins (S5, L9) are much slower. Selective inhibition of RNA polymerase I transcription does not prevent the exchanges but influences the rates of exchange differentially for different nucleolar components. These findings suggest that the rapid exchange of nucleolar components between the nucleolus and nucleoplasm may represent a new level of regulation for rRNA synthesis. The different dynamic properties of proteins involved in different steps of ribosome biogenesis imply that the nucleolar association of these proteins is due to their specific functional roles rather than simply their specific nucleolar-targeting events.
Biogenesis of the Signal Recognition Particle (Srp) Involves Import of Srp Proteins into the Nucleolus, Assembly with the Srp-Rna, and Xpo1p-Mediated Export
Authors: Grosshans Helge , Deinert Karina , Hurt Ed , Simos George
Published: July 2002

The signal recognition particle (SRP) targets nascent secretory proteins to the ER, but how and where the SRP assembles is largely unknown. Here we analyze the biogenesis of yeast SRP, which consists of an RNA molecule (scR1) and six proteins, by localizing all its components. Although scR1 is cytoplasmic in wild-type cells, nuclear localization was observed in cells lacking any one of the four SRP “core proteins” Srp14p, Srp21p, Srp68p, or Srp72p. Consistently, a major nucleolar pool was detected for these proteins. Sec65p, on the other hand, was found in both the nucleoplasm and the nucleolus, whereas Srp54p was predominantly cytoplasmic. Import of the core proteins into the nucleolus requires the ribosomal protein import receptors Pse1p and Kap123p/Yrb4p, which might, thus, constitute a nucleolar import pathway. Nuclear export of scR1 is mediated by the nuclear export signal receptor Xpo1p, is distinct from mRNA transport, and requires, as evidenced by the nucleolar accumulation of scR1 in a dis3/rrp44 exosome component mutant, an intact scR1 3′ end. A subset of nucleoporins, including Nsp1p and Nup159p (Rat7p), are also necessary for efficient translocation of scR1 from the nucleus to the cytoplasm. We propose that assembly of the SRP requires import of all SRP core proteins into the nucleolus, where they assemble into a pre-SRP with scR1. This particle can then be targeted to the nuclear pores and is subsequently exported to the cytoplasm in an Xpo1p-dependent way.
Evidence that Golgi structure depends on a p115 activity that is independent of the vesicle tether components giantin and GM130
Authors: Puthenveedu Manojkumar A. , Linstedt Adam D.
Published: July 2002

Inhibition of the putative coatomer protein I (COPI) vesicle tethering complex, giantin–p115–GM130, may contribute to mitotic Golgi breakdown. However, neither this, nor the role of the giantin–p115–GM130 complex in the maintenance of Golgi structure has been demonstrated in vivo. Therefore, we generated antibodies directed against the mapped binding sites in each protein of the complex and injected these into mammalian tissue culture cells. Surprisingly, the injected anti-p115 and antigiantin antibodies caused proteasome-mediated degradation of the corresponding antigens. Reduction of p115 levels below detection led to COPI-dependent Golgi fragmentation and apparent accumulation of Golgi-derived vesicles. In contrast, neither reduction of giantin below detectable levels, nor inhibition of p115 binding to GM130, had any detectable effect on Golgi structure or Golgi reassembly after cell division or brefeldin A washout. These observations indicate that inhibition of p115 can induce a mitotic-like Golgi disassembly, but its essential role in Golgi structure is independent of its Golgi-localized binding partners giantin and GM130.
Dislocation and degradation from the ER are regulated by cytosolic stress
Authors: VanSlyke Judy K. , Musil Linda S.
Published: July 2002

Akey step in ER-associated degradation (ERAD) is dislocation of the substrate protein from the ER into the cytosol to gain access to the proteasome. Very little is known about how this process is regulated, especially in the case of polytopic proteins. Using pulse-chase analysis combined with subcellular fractionation, we show that connexins, the four transmembrane structural components of gap junctions, can be chased in an intact form from the ER membrane into the cytosol of proteasome inhibitor–treated cells. Dislocation of endogenously expressed connexin from the ER was reduced 50–80% when the cytosolic heat shock response was induced by mild oxidative or thermal stress, but not by treatments that instead upregulate the ER unfolded protein response. Cytosolic but not ER stresses slowed the normally rapid degradation of connexins, and led to a striking increase in gap junction formation and function in otherwise assembly-inefficient cell types. These treatments also inhibited the dislocation and turnover of a connexin-unrelated ERAD substrate, unassembled major histocompatibility complex class I heavy chain. Our findings demonstrate that dislocation is negatively regulated by physiologically relevant, nonlethal stress. They also reveal a previously unrecognized relationship between cytosolic stress and intercellular communication.
Cytotoxic T Lymphocyte–associated Antigen 4 (CTLA-4) Regulates the Unfolding of Autoimmune Diabetes
Authors: Lühder Fred , Höglund Petter , Allison James P. , Benoist Christophe , Mathis Diane
Published: July 2002

Evidence has been accumulating that shows that insulin-dependent diabetes is subject to immunoregulation. To determine whether cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is involved, we injected anti–CTLA-4 mAb into a TCR transgenic model of diabetes at different stages of disease. When injected into young mice, months before they would normally become diabetic, anti–CTLA-4 induced diabetes rapidly and essentially universally; this was not the result of a global activation of T lymphocytes, but did reflect a much more aggressive T cell infiltrate in the pancreatic islets. These effects were only observed if anti–CTLA-4 was injected during a narrow time window, before the initiation of insulitis. Thus, engagement of CTLA-4 at the time when potentially diabetogenic T cells are first activated is a pivotal event; if engagement is permitted, invasion of the islets occurs, but remains quite innocuous for months, if not, insulitis is much more aggressive, and diabetes quickly ensues.