bluuebunny/crossref_metadata_2025 huggingface dataset:
Energy-transducing thylakoid membranes remain highly impermeable to ions during protein translocation
Authors: Teter Sarah A. , Theg Steven M.
Published: July 2002

We investigated the operation of a posttranslational protein translocation pathway to determine whether ions are excluded from the translocase during protein transport. The membrane capacitance during protein translocation across chloroplast thylakoid membranes was monitored via electric-field-indicating carotenoid electrochromic bandshift measurements. Evidence is presented that shows that the membrane ion conductance is not increased during the complete cycle of binding, transport, and substrate release by the ΔpH-dependent translocase; i.e., the membrane remains ion-tight during protein translocation. We further demonstrate that a synthetic targeting peptide that directs proteins across this membrane does not gate translocation pores. We conclude that protein transport across the thylakoid membrane does not compromise its ability to maintain ion gradients and is, thus, unlikely to affect its functions in energy transduction.
The distribution of oriented contours in the real world
Authors: Coppola David M. , Purves Harriett R. , McCoy Allison N. , Purves Dale
Published: July 2002

In both humans and experimental animals, the ability to perceive contours that are vertically or horizontally oriented is superior to the perception of oblique angles. There is, however, no consensus about the developmental origins or functional basis of this phenomenon. Here, we report the analysis of a large library of digitized scenes using image processing with orientation-sensitive filters. Our results show a prevalence of vertical and horizontal orientations in indoor, outdoor, and even entirely natural settings. Because visual experience is known to influence the development of visual cortical circuitry, we suggest that this real world anisotropy is related to the enhanced ability of humans and other animals to process contours in the cardinal axes, perhaps by stimulating the development of a greater amount of visual circuitry devoted to processing vertical and horizontal contours.
An experimental solution for the Luria–Delbrück fluctuation problem in measuring hypermutation rates
Authors: Bachl Jürgen , Dessing Mark , Olsson Carina , von Borstel R. C. , Steinberg Charles
Published: July 2002

A cell line harboring all trans-acting elements necessary for hypermutation was transfected with a plasmid harboring the major cis-acting elements plus a green fluorescent protein gene containing a premature chain-termination codon. Transfected cells do not fluoresce unless the stop codon reverts. When a sizable cell population is purged of revertants by sorting, the frequency of mutants increases linearly with time, and there is no Luria–Delbrück fluctuation effect. Moreover, as mutant frequencies seemed to vary less than cell numbers in replicate cultures, it is suggested that hypermutation might not be coupled closely to cell division.
On the role of thymic epithelium vs. bone marrow-derived cells in repertoire selection of T cells
Authors: Zinkernagel Rolf M. , Althage Alana
Published: July 2002

T lymphocytes mature in the thymus to become functional T cells. Studies with chimeric mice and T cell receptor (TCR) transgenic (tg) mice have indicated that the major histocompatibility gene complex (MHC) of thymic radio-resistant (presumed to be epithelial) cells positively select the MHC-restricted T cell repertoire. Surprisingly, mice without a thymus reconstituted with an MHC-incompatible thymus generate effector T cells which are, in general, specific for the host and not for the thymic MHC. The present study reanalyzed this longstanding paradox in nude mice that were reconstituted with an MHC-incompatible thymus plus or minus immunologically defective bone marrow-derived cells or in nude mice expressing a transgenic T cell receptor. A pathway of thymus-dependent but thymic MHC-independent T cell maturation is revealed where expansion of the antiviral T cell repertoire depends on the MHC of bone marrow-derived cells. These results indicate an alternative, if not a general, pathway of T cell maturation and selection: the thymus may function essentially as an organ promoting T cell receptor expression; T cell specificity, however, reflects repertoire expansion plus cell survival and effector T cell induction driven by the MHC of bone marrow-derived cells. Therefore pure thymus defects can be efficiently reconstituted by allo- and xenogeneic thymic grafts.
The Tyr-265-to-Cys mutator mutant of DNA polymerase β induces a mutator phenotype in mouse LN12 cells
Authors: Clairmont Caroline A. , Narayanan Latha , Sun Ka-Wai , Glazer Peter M. , Sweasy Joann B.
Published: July 2002

DNA polymerase β functions in both base excision repair and meiosis. Errors committed by polymerase β during these processes could result in mutations. Using a complementation system, in which rat DNA polymerase β substitutes for DNA polymerase I of
Escherichia coli
, we previously isolated a DNA polymerase β mutant in which Tyr-265 was altered to Cys (Y265C). The Y265C mutant is dominant to wild-type DNA polymerase β and possesses an intrinsic mutator activity. We now have expressed the wild-type DNA polymerase and the Y265C mutator mutant in mouse LN12 cells, which have endogenous DNA polymerase β activity. We demonstrate that expression of the Y265C mutator mutant in the LN12 cells results in an 8-fold increase in the spontaneous mutation frequency of λ
cII
mutants compared with expression of the wild-type protein. Expression of Y265C results in at least a 40-fold increase in the frequency of deletions of three bases or more and a 7-fold increase in point mutations. Our results suggest that the mutations we observe
in vivo
result directly from the action of the mutator polymerase. To our knowledge, this is the first demonstration of a mutator phenotype resulting from expression of a DNA polymerase mutator mutant in mammalian cells. This work raises the possibility that variant polymerases may act in a dominant fashion in human cells, leading to genetic instability and carcinogenesis.
Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes
Authors: Makovets Svetlana , Doronina Victoria A. , Murray Noreen E.
Published: July 2002

ClpXP-dependent proteolysis has been implicated in the delayed detection of restriction activity after the acquisition of the genes (
hsdR
,
hsdM
, and
hsdS
) that specify
Eco
KI and
Eco
AI, representatives of two families of type I restriction and modification (R-M) systems. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However, unmodified targets may be generated in the DNA of an
hsd
+
bacterium as the result of replication errors or recombination-dependent repair. We show that ClpXP-dependent regulation of the endonuclease activity enables bacteria that acquire unmodified chromosomal target sequences to survive. In such bacteria, HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP. A mutation that blocks only the modification activity of
Eco
KI, leaving the cell with ≈600 unmodified targets, is not lethal provided that ClpXP is present. Our data support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences, but before completion of the pathway that would result in DNA breakage.
Crystal structure of triosephosphate isomerase from
Trypanosoma cruzi
in hexane
Authors: Gao Xiu-Gong , Maldonado Ernesto , Pérez-Montfort Ruy , Garza-Ramos Georgina , de Gómez-Puyou Marietta Tuena , Gómez-Puyou Armando , Rodríguez-Romero Adela
Published: July 2002

To gain insight into the mechanisms of enzyme catalysis in organic solvents, the x-ray structure of some monomeric enzymes in organic solvents was determined. However, it remained to be explored whether the structure of oligomeric proteins is also amenable to such analysis. The field acquired new perspectives when it was proposed that the x-ray structure of enzymes in nonaqueous media could reveal binding sites for organic solvents that in principle could represent the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite
Trypanosoma cruzi
was soaked and diffracted in hexane and its structure solved at 2-Å resolution. Its overall structure and the dimer interface were not altered by hexane. However, there were differences in the orientation of the side chains of several amino acids, including that of the catalytic Glu-168 in one of the monomers. No hexane molecules were detected in the active site or in the dimer interface. However, three hexane molecules were identified on the surface of the protein at sites, which in the native crystal did not have water molecules. The number of water molecules in the hexane structure was higher than in the native crystal. Two hexanes localized at <4 Å from residues that form the dimer interface; they were in close proximity to a site that has been considered a potential target for drug design.
Postexposure immunoprophylaxis of primary isolates by an antibody to HIV receptor complex
Authors: Wang Chang Yi , Sawyer Lynette S. W. , Murthy Krishna K. , Fang Xinde , Walfield Alan M. , Ye John , Wang James J. G. , Chen Pei De , Li Ming Lie , Salas Mary T. , Shen Ming , Gauduin Marie-Claire , Boyle Rosanne W. , Koup Richard A. , Montefiori David C. , Mascola John R. , Koff Wayne C. , Hanson Carl V.
Published: July 2002

mAb B4 is a monoclonal antibody directed against HIV receptor complex. The antibody had broad neutralizing activity against HIV and provided postexposure prophylaxis to hu–peripheral blood leukocyte (PBL)–severe combined immunodeficient mice and chimpanzees. B4 recognized a complex receptor site for HIV on the T cell surface that includes CD4 and also may be influenced by interaction with HIV coreceptors. mAb B4 preferentially neutralized primary HIV-1 isolates compared with T cell line-adapted strains, including syncytium-inducing and non-syncytium-inducing phenotypes, representatives from HIV-1 subtypes A-G, as well as HIV-2, simian immunodeficiency virus, and chimeric simian/human immunodeficiency virus (SHIV). Neutralization was demonstrated in both pre- and postinfection models. The administration of mAb B4 after infectious challenge totally interrupted the infection of hu-PBL–severe combined immunodeficient mice by PBL-grown HIV-1 and the infection of chimpanzees by chimp-adapted HIV-1. This mode of protection suggested that the anti-HIV receptor antibody is efficacious for prophylaxis after exposure to HIV and for prevention of maternal transmission and may be an effective antiretroviral agent for treatment.
Identification of genes controlled by quorum sensing in
Pseudomonas aeruginosa
Authors: Whiteley Marvin , Lee Kimberly M. , Greenberg E. P.
Published: July 2002

Bacteria communicate with each other to coordinate expression of specific genes in a cell density-dependent fashion, a phenomenon called quorum sensing and response. Although we know that quorum sensing via acyl-homoserine lactone (HSL) signals controls expression of several virulence genes in the human pathogen
Pseudomonas aeruginosa
, the number and types of genes controlled by quorum sensing have not been studied systematically. We have constructed a library of random insertions in the chromosome of a
P. aeruginosa
acyl-HSL synthesis mutant by using a transposon containing a promoterless
lacZ
. This library was screened for acyl-HSL induction of
lacZ
. Thirty-nine quorum sensing-regulated genes were identified. The genes were organized into classes depending on the pattern of regulation. About half of the genes appear to be in seven operons, some seem organized in large patches on the genome. Many of the quorum sensing-regulated genes code for putative virulence factors or production of secondary metabolites. Many of the genes identified showed a high level of induction by acyl-HSL signaling.
Imaging of biological macromolecules on mica in humid air by scanning electrochemical microscopy
Authors: Fan Fu-Ren F. , Bard Allen J.
Published: July 2002

Imaging of DNA, keyhole limpet hemocyanin, mouse monoclonal IgG, and glucose oxidase on a mica substrate has been accomplished by scanning electrochemical microscopy with a tungsten tip. The technique requires the use of a high relative humidity to form a thin film of water on the mica surface that allows electrochemical reactions to take place at the tip and produce a faradaic current (≈1 pA) that can be used to control tip position. The effect of relative humidity and surface pretreatment with buffer solutions on the ionic conductivity of a mica surface was investigated to find appropriate conditions for imaging. Resolution of the order of 1 nm was obtained.
Receptor-induced polymerization of coatomer
Authors: Reinhard Constanze , Harter Cordula , Bremser Martina , Brügger Britta , Sohn Kai , Helms J. Bernd , Wieland Felix
Published: July 2002

Coatomer, the coat protein complex of COPI vesicles, is involved in
the budding of these vesicles, but the underlying mechanism is unknown.
Toward a better understanding of this process, the interaction between
coatomer and the cytoplasmic domain of a major transmembrane protein of
COPI vesicles, p23, was studied. Interaction of coatomer with this
peptide domain results in a conformational change and polymerization of
the complex
in vitro
. This changed conformation also is
observed
in vivo
, i.e., on the surface of authentic,
isolated COPI vesicles. An average of four peptides was found
associated with one coatomer complex after polymerization. Based on
these results, we propose a mechanism by which the induced
conformational change of coatomer results in its polymerization, and
thus drives formation of the bud on the Golgi membrane during
biogenesis of a COPI vesicle.
Spatial attention affects brain activity in human primary visual cortex
Authors: Gandhi Sunil P. , Heeger David J. , Boynton Geoffrey M.
Published: July 2002

Functional MRI was used to test whether instructing subjects to attend to one or another location in a visual scene would affect neural activity in human primary visual cortex. Stimuli were moving gratings restricted to a pair of peripheral, circular apertures, positioned to the right and to the left of a central fixation point. Subjects were trained to perform a motion discrimination task, attending (without moving their eyes) at any moment to one of the two stimulus apertures. Functional MRI responses were recorded while subjects were cued to alternate their attention between the two apertures. Primary visual cortex responses in each hemisphere modulated with the alternation of the cue; responses were greater when the subject attended to the stimuli in the contralateral hemifield. The attentional modulation of the brain activity was about 25% of that evoked by alternating the stimulus with a uniform field.
Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium
Authors: Ziemienowicz Alicja , Görlich Dirk , Lanka Erich , Hohn Barbara , Rossi Luca
Published: July 2002

Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells.
Agrobacterium tumefaciens
, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells.
Agrobacterium
-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation.
Agrobacterium
virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA)
in vitro
. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA–protein complexes into nuclei. Import of VirD2–ssDNA–VirE2 complexes was fast and efficient, and was shown to depended on importin α, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein–DNA complex, made
in vitro
, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.
Catalytic role of enzymes: Short strong H-bond-induced partial proton shuttles and charge redistributions
Authors: Kim Kwang S. , Oh Kyung Seok , Lee Jin Yong
Published: July 2002

A two-step reaction mechanism (catalyzed alternatively by
acid and base) with partial proton shuttles and charge redistributions
promoted by short strong H bonds (SSHBs) (playing a dual role as an
amphi-acid/base catalyst) is proposed to explain the enormous rate
enhancement observed in enzymatic reactions involving carbanion
intermediates. The SSHBs in the two-step reactions are found to be
responsible for enhancing enzyme–substrate interactions in favor of
the transition state structure over that of reactant. The detailed
quantum theoretical studies of ketosteroid isomerase provide evidence
of assisting roles of SSHB in enzymatic activity. The understanding of
the two-step reaction mechanism would be a useful aid in
designing novel functional enzymes and abzymes.
The N-terminal domains of histones H3 and H4 are not necessary for chromatin assembly factor-1- mediated nucleosome assembly onto replicated DNA
in vitro
Authors: Shibahara Kei-ichi , Verreault Alain , Stillman Bruce
Published: July 2002

An
in vitro
reconstitution system for the analysis of replication-coupled nucleosome assembly is described. In this “two-step system,” nucleosome assembly is performed in a separate reaction from DNA replication, wherein purified newly replicated DNA remains noncovalently marked for subsequent chromatin assembly factor-1 (CAF-1)-dependent nucleosome assembly. Because the nucleosome assembly is performed separately from the DNA replication step, this system is more versatile and biochemically tractable when compared with nucleosome assembly during simian virus 40 (SV40) DNA replication. The N-terminal domains of histones H3 and H4 play an important but redundant function in nucleosome assembly in the budding yeast,
Saccharomyces cerevisiae
. It had been proposed that at least one tail of histone H3 or H4 is required for replication-coupled nucleosome assembly. However, we demonstrate that the N-terminal domains of both histone H3 and H4 are dispensable for CAF-1-mediated formation of nucleosome cores onto newly replicated DNA
in vitro
. CAF-1 and each of its individual subunits stably bound to recombinant (H3.H4)
2
tetramers lacking the N-terminal domains of both H3 and H4. Therefore, the N-terminal tails of histone H3 and H4 that contain the specific acetylation sites are not necessary for CAF-1-dependent nucleosome assembly onto replicated DNA. We suggest that the histone acetylation may be required for a CAF-1 independent pathway or function after deposition, by marking of newly replicated chromatin.
Striking a balance: Modulation of the actin cytoskeleton bySalmonella
Authors: Galán Jorge E. , Zhou Daoguo
Published: July 2002

Salmonellaspp. have evolved the ability to enter into cells that are normally nonphagocytic. The internalization process is the result of a remarkable interaction between the bacteria and the host cells. Immediately on contact,Salmonelladelivers a number of bacterial effector proteins into the host cell cytosol through the function of a specialized organelle termed the type III secretion system. Initially, two of the delivered proteins, SopE and SopB, stimulate the small GTP-binding proteins Cdc42 and Rac. SopE is an exchange factor for these GTPases, and SopB is an inositol polyphosphate phosphatase. Stimulation of Cdc42 and Rac leads to marked actin cytoskeleton rearrangements, which are further enhanced by SipA, aSalmonellaprotein also delivered into the host cell by the type III secretion system. SipA lowers the critical concentration of G-actin, stabilizes F-actin at the site of bacterial entry, and increases the bundling activity of the host-cell protein T-plastin (fimbrin). The cellular responses stimulated bySalmonellaare short-lived; therefore, immediately after bacterial entry, the cell regains its normal architecture. Remarkably, this process is mediated by SptP, another target of the type III secretion system. SptP exert its function by serving as a GTPase-activating protein for Cdc42 and Rac, turning these G proteins off after their stimulation by the bacterial effectors SopE and SopB. The balanced interaction ofSalmonellawith host cells constitutes a remarkable example of the sophisticated nature of a pathogen/host relationship shaped by evolution through a longstanding coexistence.
Protease-activated receptor-2 modulates myocardial ischemia-reperfusion injury in the rat heart
Authors: Napoli Claudio , Cicala Carla , Wallace John L. , de Nigris Filomena , Santagada Vincenzo , Caliendo Giuseppe , Franconi Flavia , Ignarro Louis J. , Cirino Giuseppe
Published: July 2002

Protease-activated receptor-2 (PAR-2) is a member of seven transmembrane domain G protein-coupled receptors activated by proteolytic cleavage whose better known member is the thrombin receptor. The pathophysiological role of PAR-2 remains poorly understood. Because PAR-2 is involved in inflammatory and injury response events, we investigated the role of PAR-2 in experimental myocardial ischemia-reperfusion injury. We show for the first time that PAR-2 activation protects against reperfusion-injury. After PAR-2-activating peptide (2AP) infusion, we found a significant recovery of myocardial function and decrease in oxidation at reflow. Indeed, the glutathione cycle (glutathione and oxidized glutathione) and lipid peroxidation analysis showed a reduced oxidative reperfusion-injury. Moreover, ischemic risk zone and creatine kinase release were decreased after PAR-2AP treatment. These events were coupled to elevation of PAR-2 and tumor necrosis factor α (TNFα) expression in both nuclear extracts and whole heart homogenates. The recovery of coronary flow was not reverted by L-nitroarginine methylester, indicating a NO-independent pathway for this effect. Genistein, a tyrosine kinase inhibitor, did not revert the PAR-2AP effect. During early reperfusion injury
in vivo
not only oxygen radicals are produced but also numerous proinflammatory mediators promoting neutrophil and monocyte targeting. In this context, we show that TNFα and PAR-2 are involved in signaling in pathophysiological conditions, such as myocardial ischemia-reperfusion. At the same time, because TNFα may exert pro-inflammatory actions and PAR-2 may constitute one of the first protective mechanisms that signals a primary inflammatory response, our data support the concept that this network may regulate body responses to tissue injury.
The Relations of Children’s Situational Empathy-related Emotions to Dispositional Prosocial Behaviour
Authors: Holmgren Robin A. , Eisenberg Nancy , Fabes Richard A.
Published: July 2002

Although empathy-related responding has frequently been associated with prosocial behaviour directed towards the target of an individual’s vicarious emotional responding, relations between dispositional prosocial behaviour and sympathy or personal distress have seldom been examined, particularly with a multimethod approach. Kindergarten to third-graders’ empathy-related responses to a ”lm were examined as predictors of dispositional prosocial behaviour as reported by teachers, parents, and peers. Teachers’ ratings of dispositional behaviour were related to children’s facial and skin conductance reactions to the ”lm. Parents’ and peers’ ratings of dispositional prosocial behaviour were infrequently associated with children’s empathy-related responding, although peer reports were positively related to heart rate acceleration for boys. Reasons for the differential patterns of relations for different reporters are considered.
The debate over the National Curriculum for history in England and Wales, 1989–90: the role of the press
Authors: Jones Gareth Elwyn
Published: July 2002

The construction of a history curriculum for England and for Wales under the terms of the 1988 Education Reform Act occasioned more debate in the media than any other subject in the National Curriculum. The links between the content of a history syllabus and perceptions within both nations of the part this plays in moulding national spirit made this inevitable. The disparity of view between the two groups charged with devising the syllabus and the government, particularly the Prime Minister, heightened the tension. This article explores the way in which consequent political, professional and public discussion was covered in the whole range of national newspapers. It is argued that the failure of the London press to comprehend the situation in Wales reduced significantly its appreciation of the processes involved in the making of the history curriculum.
Markers of ethnicity and their effect on measuring illicit drug use by ethnic groups
Authors: REID GARY , BEYER LORRAINE , AITKEN CAMPBELL , CROFTS NICK
Published: July 2002

Assessing the relative involvement of ethnic communities with illicit drugs relies on identification of ethnicity in collected information. Many of the indicators of ethnicity used in Victorian drug‐ and alcohol‐related databases fail to provide researchers with adequate information to accurately determine ethnic or cultural orientation. Country of birth is the variable most commonly used to identify ethnicity, but it conceals Australian‐born children of migrants who may identify primarily with their ethnic background. The self‐identified ‘ethnic/cultural background’ variable collected in the Victorian methadone registry allowed us to show that many second‐generation migrants do identify primarily with their ethnic/cultural background. Of the methadone permit forms examined, 31% identified the applicant as belonging to one of 70 different ethnic/cultural background groups. The most common ethnic groups were Vietnamese (22%), Greek (16%) and Italian (15%). The Victorian methadone registry implies that illicit drug use among a number of ethnic minority communities is substantial, unlike all other Victorian databases, which lack good ethnicity measures. Health‐related databases should reflect the multi‐cultural nature of the Victorian population; the inclusion of better ethnicity variables can allow an improved measurement about the extent of illicit drug use in ethnic minority communities and therefore illuminate a number of important education and service delivery issues.
Modernizing Böhm-Bawerk's Theory of Interest
Authors: Dorfman Robert
Published: July 2002

In 1690, John Locke proclaimed, “Whatsoever [anyone]; removes out of the State that Nature hath provided … hath mixed his Labour with, and joyned to it something that is his own, and thereby makes it his Property.” Ever since this dictum was pronounced philosophers, economists, and, especially, capital theorists have been embarrassed by the virtually universal disregard of it in practice. At the very inception of economic theorizing, Ricardo declared that explaining the division of the national income into wages, rents, and interest was “the principal problem in Political Economy.” He proposed an ingenious explanation, but was far from settling the matter. Some fifty years later, Marx, building on Ricardo's concept of value, brought the debate to a sharp focus; after him, it became largely an argument between Marxists and everyone else. Put starkly, Marx defined the value of any good or service to be the amount of labor required to produce it. Since a worker and his family can be supported for a year by the product of a good deal less than a full year's labor, this definition implies a gap between the value of a year's labor and the value of its products. Owners of land and capital are only too glad to close the gap by claiming the “surplus value.” Marx's diagnosis of the nature and origin of surplus value became the focus of discussions of the distribution of income for most of the following century.
Cocaine Dependence with and without Posttraumatic Stress Disorder: A Comparison of Substance Use, Trauma History and Psychiatric Comorbidity
Authors: Back Sudie , Dansky Bonnie S. , Coffey Scott F. , Saladin Michael E. , Sonne Susan , Brady Kathleen T.
Published: July 2002

This study examined the relationship between substance use, trauma history, post‐traumatic stress disorder (PTSD), and psychiatric comorbidity in a treatment seeking sample of cocaine dependent individuals (N = 91). Structured clinical interviews revealed that 42.9% of the sample met DSM‐III‐R criteria for lifetime PTSD. Comparisons between individuals with and without lifetime PTSD revealed that individuals with PTSD had significantly higher rates of exposure to traumatic events, earlier age of first assault, more severe symptomatology, and higher rates of Axis I and Axis II diagnoses. The results illustrate a high incidence of PTSD among cocaine dependent individuals. Routine assessment of trauma history and PTSD may assist in the identification of a subgroup of cocaine users in need of special prevention and treatment efforts.
Vanadate Reduction by Molybdenum‐Free Dissimilatory Nitrate Reductases from Vanadate‐Reducing Bacteria
Authors: Antipov A. N. , Lyalikova N. N. , Khijniak T. V. , L'vov N. P.
Published: July 2002

Molybdenum‐ and molybdenum cofactor‐free nitrate reductases recently isolated by us from vanadate‐reducing bacteria Pseudomonas isachenkovii are likely to mediate vanadate reduction. During anaerobic growth of P. isachenkovii on medium supplemented with nitrate and vanadate, vanadate dissimilation was followed by nitrate consumption, and this process was associated with some structural reorganizations of nitrate reductases. The homogeneous membrane‐bound nitrate reductase of P. isachenkovii reduced vanadate with NADH as an electron donor.
Fusion Competence of Myoblasts Rendered Genetically Null for N-Cadherin in Culture
Authors: Charlton Carol A. , Mohler William A. , Radice Glenn L. , Hynes Richard O. , Blau Helen M.
Published: July 2002

Myoblast fusion is essential to muscle tissue development yet remains poorly understood. N-cadherin, like other cell surface adhesion molecules, has been implicated by others in muscle formation based on its pattern of expression and on inhibition of myoblast aggregation and fusion by antibodies or peptide mimics. Mice rendered homozygous null for N-cadherin revealed the general importance of the molecule in early development, but did not test a role in skeletal myogenesis, since the embryos died before muscle formation. To test genetically the proposed role of N-cadherin in myoblast fusion, we successfully obtained N-cadherin null primary myoblasts in culture. Fusion of myoblasts expressing or lacking N-cadherin was found to be equivalent, both in vitro by intracistronic complementation of lacZ and in vivo by injection into the muscles of adult mice. An essential role for N-cadherin in mediating the effects of basic fibroblast growth factor was also excluded. These methods for obtaining genetically homozygous null somatic cells from adult tissues should have broad applications. Here, they demonstrate clearly that the putative fusion molecule, N-cadherin, is not essential for myoblast fusion.
Tissue-specific Expression of the L1 Cell Adhesion Molecule Is Modulated by the Neural Restrictive Silencer Element
Authors: Kallunki Pekka , Edelman Gerald M. , Jones Frederick S.
Published: July 2002

The cell adhesion molecule L1 mediates neurite outgrowth and fasciculation during embryogenesis and mutations in its gene have been linked to a number of human congenital syndromes. To identify DNA sequences that restrict expression of L1 to the nervous system, we isolated a previously unidentified segment of the mouse L1 gene containing the promoter, the first exon, and the first intron and examined its activity in vitro and in vivo. We found that a neural restrictive silencer element (NRSE) within the second intron prevented expression of L1 gene constructs in nonneural cells. For optimal silencing of L1 gene expression by the NRSE-binding factor RE-1–silencing transcription factor (REST)/NRSF, both the NRSE and sequences in the first intron were required. In transgenic mice, an L1lacZ gene construct with the NRSE generated a neurally restricted expression pattern consistent with the known pattern of L1 expression in postmitotic neurons and peripheral glia. In contrast, a similar construct lacking the NRSE produced precocious expression in the peripheral nervous system and ectopic expression in mesenchymal derivatives of the neural crest and in mesodermal and ectodermal cells. These experiments show that the NRSE and REST/NRSF are important components in restricting L1 expression to the embryonic nervous system.
Probing the Architecture of a Simple Kinetochore Using DNA–Protein Crosslinking
Authors: Espelin Christopher W. , Kaplan Kenneth B. , Sorger Peter K.
Published: July 2002

In budding yeast, accurate chromosome segregation requires that one and only one kinetochore assemble per chromosome. In this paper, we report the use of DNA–protein crosslinking and nondenaturing gel analysis to study the structure of CBF3, a four-protein complex that binds to the essential CDEIII region of Saccharomyces cerevisiae centromeres. We find that three subunits of CBF3 are in direct contact with CDEIII over a region of DNA that spans 80 bp. A highly asymmetric core complex containing p58CTF13 p64CEP3 and p110NDC10 in direct contact with DNA forms at the genetically defined center of CDEIII. This core complex spans ∼56 bp of CEN3. An extended complex comprising the core complex and additional DNA-bound p110NDC10 also forms. It spans ∼80 bp of DNA. CBF3 makes sequence-specific and -nonspecific contacts with DNA. Both contribute significantly to the energy of CBF3–DNA interaction. Moreover, important sequence-specific contacts are made with bases that are not conserved among yeast centromeres. These findings provide a foundation for understanding the organization of the CBF3–centromere complex, a structure that appears to initiate the formation of microtubule attachment sites at yeast kinetochores. These results also have implications for understanding centromere-binding proteins in higher cells.
A Putative Catenin–Cadherin System Mediates Morphogenesis of the Caenorhabditis elegans Embryo
Authors: Costa Michael , Raich William , Agbunag Cristina , Leung Ben , Hardin Jeff , Priess James R.
Published: July 2002

During morphogenesis of the Caenorhabditis elegans embryo, hypodermal (or epidermal) cells migrate to enclose the embryo in an epithelium and, subsequently, change shape coordinately to elongate the body (Priess, J.R., and D.I. Hirsh. 1986. Dev. Biol. 117:156– 173; Williams-Masson, E.M., A.N. Malik, and J. Hardin. 1997. Development [Camb.]. 124:2889–2901). We have isolated mutants defective in morphogenesis that identify three genes required for both cell migration during body enclosure and cell shape change during body elongation. Analyses of hmp-1, hmp-2, and hmr-1 mutants suggest that products of these genes anchor contractile actin filament bundles at the adherens junctions between hypodermal cells and, thereby, transmit the force of bundle contraction into cell shape change. The protein products of all three genes localize to hypodermal adherens junctions in embryos. The sequences of the predicted HMP-1, HMP-2, and HMR-1 proteins are related to the cell adhesion proteins α-catenin, β-catenin/Armadillo, and classical cadherin, respectively. This putative catenin–cadherin system is not essential for general cell adhesion in the C. elegans embryo, but rather mediates specific aspects of morphogenetic cell shape change and cytoskeletal organization.
A Complex Web of Signal-dependent Trafficking Underlies the Triorganellar Distribution of P-Selectin in Neuroendocrine PC12 Cells
Authors: Blagoveshchenskaya Anastasiya D. , Hewitt Eric W. , Cutler Daniel F.
Published: July 2002

By analyzing the trafficking of HRP–P-selectin chimeras in which the lumenal domain of P-selectin was replaced with horseradish peroxidase, we determined the sequences needed for targeting to synaptic-like microvesicles (SLMV), dense core granules (DCG), and lysosomes in neuroendocrine PC12 cells. Within the cytoplasmic domain of P-selectin, Tyr777 is needed for the appearance of P-selectin in immature and mature DCG, as well as for targeting to SLMV. The latter destination also requires additional sequences (Leu768 and 786DPSP789) which are responsible for movement through endosomes en route to the SLMV. Leu768 also mediates transfer from early transferrin (Trn)-positive endosomes to the lysosomes; i.e., operates as a lysosomal targeting signal. Furthermore, SLMV targeting of HRP–P-selectin chimeras, but not the endogenous SLMV protein synaptophysin/p38, previously shown to be delivered to SLMV directly from the plasma membrane, is a Brefeldin A–sensitive process. Together, these data are consistent with a model of SLMV biogenesis which involves an endosomal intermediate in PC12 cells. In addition, we have discovered that impairment of SLMV or DCG targeting results in a concomitant increase in lysosomal delivery, illustrating the entwined relationships between routes leading to regulated secretory organelles (RSO) and to lysosomes.
Active Caspases and Cleaved Cytokeratins Are Sequestered into Cytoplasmic Inclusions in Trail-Induced Apoptosis
Authors: MacFarlane Marion , Merrison Wendy , Dinsdale David , Cohen Gerald M.
Published: July 2002

Tumor necrosis factor–related apoptosis- inducing ligand (TRAIL) –induced apoptosis, in transformed human breast epithelial MCF-7 cells, resulted in a time-dependent activation of the initiator caspases-8 and -9 and the effector caspase-7. Cleavage of caspase-8 and its preferred substrate, Bid, preceded processing of caspases-7 and -9, indicating that caspase-8 is the apical initiator caspase in TRAIL-induced apoptosis. Using transient transfection of COOH-terminal–tagged green fluorescent protein fusion constructs, caspases-3, -7, and -8 were localized throughout the cytoplasm of MCF-7 cells. TRAIL-induced apoptosis resulted in activation of caspases-3 and -7, and the redistribution of most of their detectable catalytically active small subunits into large spheroidal cytoplasmic inclusions, which lacked a limiting membrane. These inclusions, which were also induced in untransfected cells, contained cytokeratins 8, 18, and 19, together with both a phosphorylated form and a caspase-cleavage fragment of cytokeratin 18. Similarly, in untransfected breast HBL100 and lung A549 epithelial cells, TRAIL induced the formation of cytoplasmic inclusions that contained cleaved cytokeratin 18 and colocalized with active endogenous caspase-3. We propose that effector caspase-mediated cleavage of cytokeratins, resulting in disassembly of the cytoskeleton and formation of cytoplasmic inclusions, may be a characteristic feature of epithelial cell apoptosis.
Membrane Topogenesis of a Type I Signal-Anchor Protein, Mouse Synaptotagmin Ii, on the Endoplasmic Reticulum
Authors: Kida Yuichiro , Sakaguchi Masao , Fukuda Mitsunori , Mikoshiba Katsuhiko , Mihara Katsuyoshi
Published: July 2002

Synaptotagmin II is a type I signal-anchor protein, in which the NH2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.
Plasminogen Activator Inhibitor 1 Functions as a Urokinase Response Modifier at the Level of Cell Signaling and Thereby Promotes Mcf-7 Cell Growth
Authors: Webb Donna J. , Thomas Keena S. , Gonias Steven L.
Published: July 2002

Plasminogen activator inhibitor 1 (PAI-1) is a major inhibitor of urokinase-type plasminogen activator (uPA). In this study, we explored the role of PAI-1 in cell signaling. In MCF-7 cells, PAI-1 did not directly activate the mitogen-activated protein (MAP) kinases, extracellular signal–regulated kinase (ERK) 1 and ERK2, but instead altered the response to uPA so that ERK phosphorylation was sustained. This effect required the cooperative function of uPAR and the very low density lipoprotein receptor (VLDLr). When MCF-7 cells were treated with uPA–PAI-1 complex in the presence of the VLDLr antagonist, receptor-associated protein, or with uPA–PAI-1R76E complex, which binds to the VLDLr with greatly decreased affinity, transient ERK phosphorylation (<5 min) was observed, mimicking the uPA response. ERK phosphorylation was not induced by tissue-type plasminogen activator–PAI-1 complex or by uPA–PAI-1 complex in the presence of antibodies that block uPA binding to uPAR. uPA–PAI-1 complex induced tyrosine phosphorylation of focal adhesion kinase and Shc and sustained association of Sos with Shc, whereas uPA caused transient association of Sos with Shc.

By sustaining ERK phosphorylation, PAI-1 converted uPA into an MCF-7 cell mitogen. This activity was blocked by receptor-associated protein and not observed with uPA–PAI-1R76E complex, demonstrating the importance of the VLDLr. uPA promoted the growth of other cells in which ERK phosphorylation was sustained, including β3 integrin overexpressing MCF-7 cells and HT 1080 cells. The MEK inhibitor, PD098059, blocked the growth-promoting activity of uPA and uPA–PAI-1 complex in these cells. Our results demonstrate that PAI-1 may regulate uPA-initiated cell signaling by a mechanism that requires VLDLr recruitment. The kinetics of ERK phosphorylation in response to uPAR ligation determine the function of uPA and uPA–PAI-1 complex as growth promoters.
Tom40, the Pore-Forming Component of the Protein-Conducting Tom Channel in the Outer Membrane of Mitochondria
Authors: Ahting Uwe , Thieffry Michel , Engelhardt Harald , Hegerl Reiner , Neupert Walter , Nussberger Stephan
Published: July 2002

Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex). We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex. Tom40 is organized in a high molecular mass complex of ∼350 kD. It forms a high conductance channel. Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner. The secondary structure of Tom40 comprises ∼31% β-sheet, 22% α-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of ∼2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly.
SCAR is a primary regulator of Arp2/3-dependent morphological events in Drosophila
Authors: Zallen Jennifer A. , Cohen Yehudit , Hudson Andrew M. , Cooley Lynn , Wieschaus Eric , Schejter Eyal D.
Published: July 2002

The Arp2/3 complex and its activators, Scar/WAVE and Wiskott-Aldrich Syndrome protein (WASp), promote actin polymerization in vitro and have been proposed to influence cell shape and motility in vivo. We demonstrate that the Drosophila Scar homologue, SCAR, localizes to actin-rich structures and is required for normal cell morphology in multiple cell types throughout development. In particular, SCAR function is essential for cytoplasmic organization in the blastoderm, axon development in the central nervous system, egg chamber structure during oogenesis, and adult eye morphology. Highly similar developmental requirements are found for subunits of the Arp2/3 complex. In the blastoderm, SCAR and Arp2/3 mutations result in a reduction in the amount of cortical filamentous actin and the disruption of dynamically regulated actin structures. Remarkably, the single Drosophila WASp homologue, Wasp, is largely dispensable for these numerous Arp2/3-dependent functions, whereas SCAR does not contribute to cell fate decisions in which Wasp and Arp2/3 play an essential role. These results identify SCAR as a major component of Arp2/3-dependent cell morphology during Drosophila development and demonstrate that the Arp2/3 complex can govern distinct cell biological events in response to SCAR and Wasp regulation.
The Common Cytokine Receptor γ Chain Plays an Essential Role in Regulating Lymphoid Homeostasis
Authors: Nakajima Hiroshi , Shores Elizabeth W. , Noguchi Masayuki , Leonard Warren J.
Published: July 2002

In the immune system, there is a careful regulation not only of lymphoid development and proliferation, but also of the fate of activated and proliferating cells. Although the manner in which these diverse events are coordinated is incompletely understood, cytokines are known to play major roles. Whereas IL-7 is essential for lymphoid development, IL-2 and IL-4 are vital for lymphocyte proliferation. The receptors for each of these cytokines contain the common cytokine receptor γ chain (γc), and it was previously shown that γc-deficient mice exhibit severely compromised development and responsiveness to IL-2, IL-4, and IL-7. Nevertheless, these mice exhibit an age-dependent accumulation of splenic CD4+ T cells, the majority of which have a phenotype typical of memory/activated cells. When γc-deficient mice were mated to DO11.10 T cell receptor (TCR) transgenic mice, only the T cells bearing endogenous TCRs had this phenotype, suggesting that its acquisition was TCR dependent. Not only do the CD4+ T cells from γc-deficient mice exhibit an activated phenotype and greatly enhanced incorporation of bromodeoxyuridine but, consistent with the lack of γc-dependent survival signals, they also exhibit an augmented rate of apoptosis. However, because the CD4+ T cells accumulate, it is clear that the rate of proliferation exceeds the rate of cell death. Thus, surprisingly, although γc-independent signals are sufficient to mediate expansion of CD4+ T cells in these mice, γc-dependent signals are required to regulate the fate of activated CD4+ T cells, underscoring the importance of γc-dependent signals in controlling lymphoid homeostasis.