bluuebunny/crossref_metadata_2025 huggingface dataset:
Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity
Authors: Andersen Henrik , Greenberg Daniel L. , Fujikawa Kazuo , Xu Wenfeng , Chung Dominic W. , Davie Earl W.
Published: July 2002

The activation of human platelets by thrombin is mediated primarily by protease-activated receptors (PARs). PAR1 and PAR4 are present on human platelets and are activated by the hexapeptides SFLLRN and GYPGQV, respectively. To further characterize the involvement of PAR1 and PAR4 in platelet activation, the ability of SFLLRN or GYPGQV to generate annexin V binding to newly exposed phospholipids on the platelet surface and generate procoagulant activity has been examined. Exposure of phosphatidylserine and phosphatidylethanolamine on platelets, as determined by an increase in annexin V binding, was strongly stimulated by SFLLRN, thrombin, and collagen, but only to a minor extent by GYPGQV. In a clotting assay initiated with factor VIIa, soluble tissue factor, and calcium, the clotting time in the absence of platelets was >5 min. In the presence of unstimulated platelets, the clotting time was 200 ± 20 sec. In the presence of platelets activated with SFLLRN or collagen, the clotting time decreased to 100 ± 10 sec. This shortening of the clotting time is equivalent to about a 5-fold increase in coagulant activity when stimulated platelets are compared with unstimulated platelets and activated platelets are used as a reference. These results indicate that thrombin initiates a very strong response in platelets through PAR1, leading to exposure of anionic phospholipids that support blood clotting. The response mediated by PAR4, however, was limited to platelet aggregation and similar to that triggered in platelets by weaker agonists such as ADP or epinephrine.
Prediction of protein-folding mechanisms from free-energy landscapes derived from native structures
Authors: Alm E. , Baker D.
Published: July 2002

Guided by recent experimental results suggesting that protein-folding rates and mechanisms are determined largely by native-state topology, we develop a simple model for protein folding free-energy landscapes based on native-state structures. The configurations considered by the model contain one or two contiguous stretches of residues ordered as in the native structure with all other residues completely disordered; the free energy of each configuration is the difference between the entropic cost of ordering the residues, which depends on the total number of residues ordered and the length of the loop between the two ordered segments, and the favorable attractive interactions, which are taken to be proportional to the total surface area buried by the ordered residues in the native structure. Folding kinetics are modeled by allowing only one residue to become ordered/disordered at a time, and a rigorous and exact method is used to identify free-energy maxima on the lowest free-energy paths connecting the fully disordered and fully ordered configurations. The distribution of structure in these free-energy maxima, which comprise the transition-state ensemble in the model, are reasonably consistent with experimental data on the folding transition state for five of seven proteins studied. Thus, the model appears to capture, at least in part, the basic physics underlying protein folding and the aspects of native-state topology that determine protein-folding mechanisms.
An anti-CD45RO immunotoxin eliminates T cells latently infected with HIV-1in vitro
Authors: McCoig Cynthia , Van Dyke Gregory , Chou Chin-Sheng , Picker Louis J. , Ramilo Octavio , Vitetta Ellen S.
Published: July 2002

Despite the success of highly active antiretroviral therapy (HAART) in lowering circulating HIV-1 to undetectable levels in most infected individuals, several studies have documented the presence of a small reservoir of latently infected cells in HAART patients, the majority of which are CD45RO+memory T cells. We previously have demonstrated that latently infected, replication-competent cells can be generatedin vitroafter eliminating CD25+cells with an immunotoxin (IT). The present study was designed to determine whether these latent cells could be eliminated by an anti-CD45RO IT. Our results indicate that the anti-CD45RO IT eliminates >99%, of either M-tropic or T-tropic virus produced by the latently infected cells after mitogen stimulation. This IT also appears to be as effective as the anti-CD25 IT in eliminating the activated, HIV-1-producing cells. In contrast, the anti-CD45RO IT does not kill CD45RA+naive cells. Further studies using cells from HIV-1-infected individuals on HAART will be necessary to determine the potential clinical utility of this IT.
Horizontal gene transfer and mutation: Ng
rol
genes in the genome of
Nicotiana glauca
Authors: Aoki Seishiro , Syōno Kunihiko
Published: July 2002

Ng
rol
genes (Ng
rolB
, Ng
rolC
, NgORF13, and NgORF14) that are similar in sequence to genes in the left transferred DNA (TL-DNA) of
Agrobacterium rhizogenes
have been found in the genome of untransformed plants of
Nicotiana glauca
. It has been suggested that a bacterial infection resulted in transformation of Ng
rol
genes early in the evolution of the genus
Nicotiana.
Although the corresponding four
rol
genes in TL-DNA provoked hairy-root syndrome in plants, present-day
N. glauca
and plants transformed with Ng
rol
genes did not exhibit this phenotype. Sequenced complementation analysis revealed that the Ng
rolB
gene did not induce adventitious roots because it contained two point mutations. Single-base site-directed mutagenesis at these two positions restored the capacity for root induction to the Ng
rolB
gene. When the Ng
rolB,
with these two base substitutions, was positioned under the control of the cauliflower mosaic virus 35S promoter (P35S), transgenic tobacco plants exhibited morphological abnormalities that were not observed in P35
s
-Ri
rolB
plants. In contrast, the activity of the Ng
rolC
gene may have been conserved after an ancient infection by bacteria. Discussed is the effect of the horizontal gene transfer of the Ng
rol
genes and mutations in the Ng
rolB
gene on the phenotype of ancient plants during the evolution of
N. glauca
.
Fluorescence-intensity distribution analysis and its application in biomolecular detection technology
Authors: Kask Peet , Palo Kaupo , Ullmann Dirk , Gall Karsten
Published: July 2002

A methodology, fluorescence-intensity distribution analysis, has been developed for confocal microscopy studies in which the fluorescence intensity of a sample with a heterogeneous brightness profile is monitored. An adjustable formula, modeling the spatial brightness distribution, and the technique of generating functions for calculation of theoretical photon count number distributions serve as the two cornerstones of the methodology. The method permits the simultaneous determination of concentrations and specific brightness values of a number of individual fluorescent species in solution. Accordingly, we present an extremely sensitive tool to monitor the interaction of fluorescently labeled molecules or other microparticles with their respective biological counterparts that should find a wide application in life sciences, medicine, and drug discovery. Its potential is demonstrated by studying the hybridization of 5′-(6-carboxytetramethylrhodamine)-labeled and nonlabeled complementary oligonucleotides and the subsequent cleavage of the DNA hybrids by restriction enzymes.
Regulation of cyclooxygenase-2 (COX-2) in rat renal cortex by adrenal glucocorticoids and mineralocorticoids
Authors: Zhang Ming-Zhi , Harris Raymond C. , McKanna James A.
Published: July 2002

Production of prostaglandins involved in renal salt and water homeostasis is modulated by regulated expression of the inducible form of cyclooxygenase-2 (COX-2) at restricted sites in the rat renal cortex. Because inflammatory COX-2 is suppressed by glucocorticoids, and prostaglandin levels in the kidney are sensitive to steroids, the sensitivity of COX expression to adrenalectomy (ADX) was investigated. By 2 weeks after ADX in mature rats, cortical COX-2 immunoreactivity increased 10-fold in the cortical thick ascending limb and macula densa. The constitutive isoform, COX-1, was unchanged. The magnitude of the changes and specificity of COX-2 immunoreactivity were validated by
in situ
hybridization histochemistry of COX-2 mRNA and Western blot analysis. Increased COX-2 activity (>5-fold) was documented by using a specific COX-2 inhibitor. The COX-2 up-regulation in ADX rats was reversed by replacement therapy with either corticosterone or deoxycorticosterone acetate. In normal rats, inhibition of glucocorticoid receptors with RU486 or mineralocorticoid receptors with spironolactone caused up-regulation of renal cortical COX-2. These results indicate that COX-2 expression
in situ
is tonically inhibited by adrenal steroids, and COX-2 is regulated by mineralocorticoids as well as glucocorticoids.
Reversing adipocyte differentiation: Implications for treatment of obesity
Authors: Zhou Yan-Ting , Wang Zhuo-Wei , Higa Moritake , Newgard Christopher B. , Unger Roger H.
Published: July 2002

Conventional treatment of obesity reduces fat in mature adipocytes but leaves them with lipogenic enzymes capable of rapid resynthesis of fat, a likely factor in treatment failure. Adenovirus-induced hyperleptinemia in normal rats results in rapid nonketotic fat loss that persists after hyperleptinemia disappears, whereas pair-fed controls regain their weight in 2 weeks. We report here that the hyperleptinemia depletes adipocyte fat while profoundly down-regulating lipogenic enzymes and their transcription factor, peroxisome proliferator-activated receptor (PPAR)γ in epididymal fat; enzymes of fatty acid oxidation and their transcription factor, PPARα, normally low in adipocytes, are up-regulated, as are uncoupling proteins 1 and 2. This transformation of adipocytes from cells that store triglycerides to fatty acid-oxidizing cells is accompanied by loss of the adipocyte markers, adipocyte fatty acid-binding protein 2, tumor necrosis factor α, and leptin, and by the appearance of the preadipocyte marker Pref-1. These findings suggest a strategy for the treatment of obesity by alteration of the adipocyte phenotype.
Genetic background changes the pattern of forebrain commissure defects in transgenic mice underexpressing the β-amyloid-precursor protein
Authors: Magara Fulvio , Müller Ulrike , Li Zhi-Wei , Lipp Hans-Peter , Weissmann Charles , Stagljar Marijana , Wolfer David P.
Published: July 2002

We previously have reported corpus callosum defects in transgenic mice expressing the β-amyloid precursor protein (βAPP) with a deletion of exon 2 and at only 5% of normal levels. This finding indicates a possible involvement of βAPP in the regulation or guidance of axon growth during neural development. To determine to what degree the βAPP mutation interacts with genetic background alleles that predispose for forebrain commissure defects in some mouse lines, we have assessed the size of the forebrain commissures in a sample of 298 mice. Lines with mixed genetic background were compared with congenic lines obtained by backcrossing to the parental strains C57BL/6 and 129/SvEv. Mice bearing a null mutation of the βAPP gene also were included in the analysis. We show that, independently of genetic background, both lack and underexpression of βAPP are associated with reduced brain weight and reduced size of forebrain commissures, especially of the ventral hippocampal commissure. In addition, both mutations drastically increase the frequency and severity of callosal agenesis and hippocampal commissure defects in mouse lines with 129/SvEv or 129/Ola background.
Active galactic nuclei
Authors: Fabian Andrew C.
Published: July 2002

Active galactic nuclei are the most
powerful, long-lived objects in the Universe. Recent data confirm the
theoretical idea that the power source is accretion into a massive
black hole. The common occurrence of obscuration and outflows probably
means that the contribution of active galactic nuclei to the power
density of the Universe has been generally underestimated.
Cytosolic phospholipase A2is essential for both the immediate and the delayed phases of eicosanoid generation in mouse bone marrow-derived mast cells
Authors: Fujishima Hiroshi , Sanchez Mejia René O. , Bingham Clifton O. , Lam Bing K. , Sapirstein Adam , Bonventre Joseph V. , Austen K. Frank , Arm Jonathan P.
Published: July 2002

We have used mice in which the gene for cytosolic phospholipase A2(cPLA2) has been disrupted to demonstrate the absolute requirement for cPLA2in both the immediate and the delayed phases of eicosanoid generation by bone marrow-derived mast cells. For the immediate phase, quantitative analysis of the products of the 5-lipoxygenase pathway showed that gene disruption of cPLA2prevented the provision of arachidonic acid substrate for biosynthesis of proximal intermediates. By analogy, we conclude that arachidonic acid substrate was also not available to prostaglandin endoperoxide synthase 1 in the immediate phase of prostaglandin (PG) D2generation. These defects occurred with two distinct stimuli, stem cell factor and IgE/antigen, which were, however, sufficient for signal transduction defined by exocytosis of β-hexosaminidase. Whereas cPLA2is essential for immediate eicosanoid generation by providing arachidonic acid, its role in delayed-phase PGD2generation is more complex and involves the activation-dependent induction of prostaglandin endoperoxide synthase 2 and the supply of arachidonic acid for metabolism to PGD2.
Molecular basis for a link between complement and the vascular complications of diabetes
Authors: Acosta Juan , Hettinga Judith , Flückiger Rudolf , Krumrei Nicole , Goldfine Allison , Angarita Luis , Halperin Jose
Published: July 2002

Activated terminal complement proteins C5b to C9 form the membrane attack complex (MAC) pore. Insertion of the MAC into endothelial cell membranes causes the release of growth factors that stimulate tissue growth and proliferation. The complement regulatory membrane protein CD59 restricts MAC formation. Because increased cell proliferation characterizes the major chronic vascular complications of human diabetes and because increased glucose levels in diabetes cause protein glycation and impairment of protein function, we investigated whether glycation could inhibit CD59. Glycation-inactivation of CD59 would cause increased MAC deposition and MAC-stimulated cell proliferation. Here, we report that (i) human CD59 is glycatedin vivo, (ii) glycated human CD59 loses its MAC-inhibitory function, and (iii) inactivation of CD59 increases MAC-induced growth factor release from endothelial cells. We demonstrate by site-directed mutagenesis that residues K41 and H44 form a preferential glycation motif in human CD59. The presence of this glycation motif in human CD59, but not in CD59 of other species, may help explain the distinct propensity of humans to develop vascular proliferative complications of diabetes.
Postgerminative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle
Authors: Eastmond Peter J. , Germain Veronique , Lange Peter R. , Bryce James H. , Smith Steven M. , Graham Ian A.
Published: July 2002

The glyoxylate cycle is regarded as essential for postgerminative growth and seedling establishment in oilseed plants. We have identified two allelic
Arabidopsis
mutants,
icl-1
and
icl-2,
which lack the glyoxylate cycle because of the absence of the key enzyme isocitrate lyase. These mutants demonstrate that the glyoxylate cycle is not essential for germination. Furthermore, photosynthesis can compensate for the absence of the glyoxylate cycle during postgerminative growth, and only when light intensity or day length is decreased does seedling establishment become compromised. The provision of exogenous sugars can overcome this growth deficiency. The
icl
mutants also demonstrate that the glyoxylate cycle is important for seedling survival and recovery after prolonged dark conditions that approximate growth in nature. Surprisingly, despite their inability to catalyze the net conversion of acetate to carbohydrate, mutant seedlings are able to break down storage lipids. Results suggest that lipids can be used as a source of carbon for respiration in germinating oilseeds and that products of fatty acid catabolism can pass from the peroxisome to the mitochondrion independently of the glyoxylate cycle. However, an additional anaplerotic source of carbon is required for lipid breakdown and seedling establishment. This source can be provided by the glyoxylate cycle or, in its absence, by exogenous sucrose or photosynthesis.
Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules
Authors: Garcia-Parajo M. F. , Segers-Nolten G. M. J. , Veerman J.-A. , Greve J. , van Hulst N. F.
Published: July 2002

Real-time single-molecule fluorescence detection using confocal and near-field scanning optical microscopy has been applied to elucidate the nature of the “on–off” blinking observed in the Ser-65 → Thr (S65T) mutant of the green fluorescent protein (GFP). Fluorescence time traces as a function of the excitation intensity, with a time resolution of 100 μs and observation times up to 65 s, reveal the existence of a nonemissive state responsible for the long dark intervals in the GFP. We find that excitation intensity has a dramatic effect on the blinking. Whereas the time during which the fluorescence is
on
becomes shorter as the intensity is increased, the
off-
times are independent of excitation intensity. Statistical analysis of the
on-
and
off-
times renders a characteristic
off-
time of 1.6 ± 0.2 s and allows us to calculate a transition yield of ≈0.5 × 10
−5
from the emissive to the nonemissive state. The saturation excitation intensity at which
on-
and
off-
times are equal is ≈1.5 kW/cm
2
. On the basis of the single-molecule data we calculate an absorption cross section of 6.5 × 10
−17
cm
2
for the S65T mutant. These results have important implications for the use of the GFP to follow dynamic processes in time at the single-molecular level.
Immunization against Alzheimer's β-amyloid plaques via EFRH phage administration
Authors: Frenkel Dan , Katz Odelia , Solomon Beka
Published: July 2002

The epitope EFRH, corresponding to amino acids 3–6 within the human β-amyloid peptide (AβP), acts as a regulatory site controlling both the formation and disaggregation process of the β-amyloid fibrils (Aβ). Locking of this epitope by highly specific antibodies affects the dynamics of the entire AβP molecule, preventing self-aggregation as well as enabling resolubilization of already formed aggregates. Production of such antibodies by repeated injections of toxic human Aβ fibrils into transgenic mice suggests the feasibility of vaccination against Alzheimer's disease. Here, we report the development of an immunization procedure for the production of effective anti-aggregating β-amyloid antibodies based on filamentous phages displaying the EFRH peptide as specific and nontoxic antigen. Effective autoimmune antibodies were obtained by EFRH phage administration in guinea pigs, which exhibit AβP identical to the human AβP region. Moreover, because of the high antigenicity of the phage, no adjuvant is required to obtain high affinity anti-aggregating IgG antibodies after a short immunization period of 3 weeks. Availability of such antibodies opens up possibilities for the development of an efficient and long-lasting vaccination for the prevention and treatment of Alzheimer's disease.
Structure–function analysis of the tobacco mosaic virus resistance gene
N
Authors: Dinesh-Kumar S. P. , Tham Wai-Hong , Baker Barbara J.
Published: July 2002

The tobacco
N
gene is a member of the
Toll-interleukin-1 receptor/nucleotide-binding
site/leucine-rich repeat (TIR-NBS-LRR) class of plant resistance
(
R
) genes and confers resistance to tobacco mosaic virus
(TMV). We investigated the importance of specific domains of
N
in inducing TMV resistance, by examining various
N
deletion and point mutations that introduce single
amino acid substitution mutants
in vivo
. Our deletion
analysis suggests that the TIR, NBS, and LRR domains play an
indispensable role in the induction of resistance responses against
TMV. We show that amino acids conserved among the
Toll/IL-1R/plant
R
gene TIR domain and
NBS-containing proteins play a critical role in
N
-mediated TMV resistance. Some loss-of-function
N
alleles such as the TIR deletion and point mutations
in the NBS (G216A/E/V/R, G218R, G219D,
K222E/N, and T223A/N) interfere with the wild-type
N
function and behave like dominant negative mutations.
These F
1
plants mount a hypersensitive response (HR) that
is indistinguishable from that of the wild-type
N
plants, yet TMV was able to move systemically, causing a systemic
hypersensitive response (SHR). Many amino acid substitutions in the
TIR, NBS, and LRR domains of
N
lead to a partial
loss-of-function phenotype. These mutant plants mount delayed HR
compared with the wild-type
N
plants and fail to contain
the virus to the infection site. In addition, some partial
loss-of-function alleles (W82S/A, W141S/A,
G218V/S, and G219V) interfere with the wild-type
N
function, leading to SHR. The partial loss-of-function
and dominant negative mutant alleles described in this report will be
useful in furthering our understanding of the TIR-NBS-LRR class of
R
genes.
From cell death to embryo arrest: Mathematical models of human preimplantation embryo development
Authors: Hardy K. , Spanos S. , Becker D. , Iannelli P. , Winston R. M. L. , Stark J.
Published: July 2002

Human preimplantation embryos exhibit high levels of
apoptotic cells and high rates of developmental arrest during
the first week
in vitro
. The relation between the two is
unclear and difficult to determine by conventional experimental
approaches, partly because of limited numbers of embryos. We apply a
mixture of experiment and mathematical modeling to show that observed
levels of cell death can be reconciled with the high levels of embryo
arrest seen in the human only if the developmental competence of
embryos is already established at the zygote stage, and environmental
factors merely modulate this. This suggests that research on improving
in vitro
fertilization success rates should move from
its current concentration on optimizing culture media to focus more on
the generation of a healthy zygote and on understanding the mechanisms
that cause chromosomal and other abnormalities during early cleavage
stages.
Phonological Similarity and Trace Degradation in the Serial Recall Task: When CAT helps RAT, but not MAN
Authors: Fallon Anthony B. , Groves Kim , Tehan Gerald
Published: July 2002

Phonological similarity is observed to affect serial recall detrimentally when correct‐in‐position scoring is used. Two experiments investigated the role of item and position accuracy scoring of rhyming, similar nonrhyming, and dissimilar lists under immediate recall conditions; articulatory suppression; or a filled delay. In general, rhyme lists produced the best item recall but position accuracy was highest for dissimilar lists. The results are due to a category cueing effect improving item recall for rhyme lists in conjunction with a detrimental effect of phonological similarity on position accuracy.
The Inconveniences of Nationality: German Bohemians, The Disintegration of the Habsburg Monarchy, and the Attempt to Create a “Sudeten German” Identity
Authors: Bahm Karl F.
Published: July 2002

In much of the already vast and expanding literature on nationalism there is an understandable emphasis on its political dimensions. It is generally seen as the ideological mobilization of an essentially cultural national identity—which may or may not be considered pre-existing—for the purposes of attaining sovereign state power, or in some other way influencing and affecting state power, for example attaining greater rights or autonomy within the state. Where there are no such demands directed at the state, such an understanding implies that either we are not dealing with a nation, or we are dealing with one that is still unconscious of its nationhood or that is satisfied without any political expression of that nationhood. None of these cases, in any event, would normally be considered examples of nationalism, since nationalism by definition must demand, indeed is the demand for such state expression or recognition of nationality. As John Breuilly puts it: nationalism is “above and beyond all else, about politics, and … politics is about power.”
Gender and Computing: Not the same old problem
Authors: Clegg Sue , Trayhurn Deborah
Published: July 2002

ABSTRACT The decline in numbers of women on computing courses in higher education has been the subject of much comment. However, the debate has moved from a ‘women and computing’ approach to a more sophisticated theorisation of gender. We suggest that computing also requires better theorisation. Computing is most usefully characterised as a concrete not abstract science. Women's contribution to the field is often ignored because they are seen as end‐users, not part of the academic mainstream. We report a case study of men and women on applied information technology courses. Using data from in‐depth interviews, we found that both men and women were aware of gender in computing practice. However, both men and women expressed intrinsic as well as instrumental reasons for studying and the women were confident in their own abilities. Women brought with them valuable administrative experience of using computer systems. One of the challenges is to conceptualise women's computer skills as real computing and to ask what is wrong with computing rather than what is wrong with women.
Upregulation of ICAM‐1 Expression on J774.2 Macrophages by Endotoxin Involves Activation of NF‐κB but not Protein Tyrosine Kinase: Comparison to Induction of iNOS
Authors: Ruetten Hartmut , Thiemermann Christoph , Perretti Mauro
Published: July 2002

This study compares the signal transduction pathway which leads to the upregulation of intercellular adhesion molecule‐1 (ICAM‐1) expression with that of the increase in the expression of inducible nitric oxide synthase (iNOS) protein and activity caused by endotoxin in cultured J774.2 macrophages. Treatment of J774.2 cells with lipopolysaccharide E. coli (LPS) induced a concentration‐dependent increase in the expression of ICAM‐1 on the cell surface within 4 h and an increase in iNOS protein and activity at 24 h. The upregulation of ICAM‐1 expression on J774.2 macrophages caused by LPS was significantly inhibited by pretreatment of the cells with inhibitors of the activation of the nuclear transcription factor NF‐κB, such as L‐1‐tosylamido‐2‐phenylethylchloromethyl ketone (TPCK), pyrrolidine dithiocarbamate (PDTC), rotenone or calpain inhibitor I, but not by the tyrosine kinase inhibitors, tyrphostin AG126 or genistein. In contrast, genistein or tyrphostin AG126 also prevented the induction of iNOS protein and activity in J774.2 macrophages elicited by LPS. Thus, the increase in the expression of ICAM‐1 on J774.2 macrophages by endotoxin involves the activation of NFκB, but not of protein tyrosine kinase.
Making Economic Knowledge: Reflections on Golinski's Constructivist History of Science
Authors: Weintraub E. Roy
Published: July 2002

While most scientists and philosophers of science privilege scientific knowledge, and have sought demarcations of science from non-science to justify the privilege, sociologists of science, small numbers of philosophers of science, anthropologists, and some scientists themselves have been attracted to a new way of talking about science. Prefigured by Ludwik Fleck (1935/1979) and Gaston Bachelard (1934/1984), nurtured by the controversies over Thomas Kuhn's work, and instantiated in the Edinburgh School's Strong Program, the naturalistic turn portrays science as a human activity, part of the woof and warp of culture itself. Yet curiously historians of science have been less involved in this recent reconceptualization of both science and scientific knowledge.
The oral health consequences of chewing areca nut
Authors: Trivedy C. R. , Craig G. , Warnakulasuriya S.
Published: July 2002

Abstract Deleterious effects of areca nut on oral soft tissues are published extensively in the dental literature. Its effects on dental caries and periodontal tissues, two major oral diseases, are less well researched. Areca‐induced lichenoid lesions mainly on buccal mucosa or tongue are reported at quid retained sites. In chronic chewers a condition known as betel chewer's mucosa, a discoloured areca nut‐encrusted change, is often found where the quid particles are retained. Areca nut chewing is implicated in oral leukoplakia and submucous fibrosis, both of which are potentially malignant in the oral cavity. Oral cancer often arises from such precancerous changes in Asian populations. In 1985 the International Agency for Research on Cancer concluded that there is limited evidence to conclude that areca chewing may directly lead to oral cancer. There is, however, new information linking oral cancer to pan chewing without tobacco, suggesting a strong cancer risk associated with this habit. Public health measures to quit areca use are recommended to control disabling conditions such as submucous fibrosis and oral cancer among Asian populations.
Comprehension of inferential meaning in language‐impaired and language normal children
Authors: Letts Carolyn , Leinonen Eeva
Published: July 2002

Three groups of language‐normal (LN) 6, 8 and 16/17 year olds, and a group of language‐impaired (LI) children were given a task answering questions about pictures that involved inferential meaning. A developmental progression in the types of responses given is seen, with the LI children performing like the youngest LN children. A similar progression is seen in the ability to justify the answers given to inferential questions with the young adult group giving the fewest justifications that were problematical in some way. Larger numbers of problematical justifications in the LI group can be related to some extent to non‐pragmatic aspects of their impairment, but overall this group also gave more pragmatically irrelevant responses when asked to justify answers given. It was not possible to identify any major differences between subgroups of children within the LI group, identified as pragmatically impaired (PI) and non pragmatically impaired (NPI), in terms of either answers given to inferential questions or in terms of problematical justifications for these answers. Possible reasons for these findings are discussed.
Giving the crucial information: performance on a referential communication task in Swedish children with language impairment
Authors: Wagner Christina Reuterskiöld , Nettelbladt Ulrika , Sahlén Birgitta
Published: July 2002

The study focused on the performance of a group of Swedish children with language impairment (LI) on a referential communication task as a step in the investigation of their pragmatic skills. The task entailed choosing a single card from a selection of 16 depicting a face and describing it well enough for the opponent in order for him/her to pick the correct one from his/her identical array of cards laid out behind a barrier. To give an adequate description, the player had to understand that four dimensions had to be described in order for the other person to choose the correct card. The participating children had been part of a previous study on narrative skills in children with LI. A few of them with rather poor language comprehension had shown utterances during story generation judged to be irrelevant to both the listener and the task. In the present study, language comprehension did not significantly correlate to performance on the referential communication task. The participants performed at the level of their peers without LI and there was no significant difference between the amount of relevant or irrelevant information when the children with LI interacted with an adult or with a friend. The results are discussed in relation to recent research.
Mitochondrial Regulation of Store-operated Calcium Signaling in T Lymphocytes
Authors: Hoth Markus , Fanger Christopher M. , Lewis Richard S.
Published: July 2002

Mitochondria act as potent buffers of intracellular Ca2+ in many cells, but a more active role in modulating the generation of Ca2+ signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or “capacitative” Ca2+ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca2+ store with thapsigargin (TG) activates Ca2+ release-activated Ca2+ (CRAC) channels in T cells, and the ensuing influx of Ca2+ loads a TG- insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca2+ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na+ depletion, which inhibits Na+-dependent Ca2+ export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca2+ levels that are consistent with its localization in the TG-insensitive store. Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca2+ uptake is sensitive to extracellular [Ca2+], indicating that mitochondria sense Ca2+ gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na+ depletion prevent the ability of T cells to maintain a high rate of capacitative Ca2+ entry over prolonged periods of >10 min. Under these conditions, the rate of Ca2+ influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca2+ that enters T cells via store-operated Ca2+ channels, but also play an active role in modulating the rate of capacitative Ca2+ entry.
Multiple Forms of Endocytosis In Bovine Adrenal Chromaffin Cells
Authors: Smith Corey , Neher Erwin
Published: July 2002

We studied endocytosis in chromaffin cells with both perforated patch and whole cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric catecholamine detection. We found that chromaffin cells exhibit two relatively rapid, kinetically distinct forms of stimulus-coupled endocytosis. A more prevalent “compensatory” retrieval occurs reproducibly after stimulation, recovering an approximately equivalent amount of membrane as added through the immediately preceding exocytosis. Membrane is retrieved through compensatory endocytosis at an initial rate of ∼6 fF/s. Compensatory endocytotic activity vanishes within a few minutes in the whole cell configuration. A second form of triggered membrane retrieval, termed “excess” retrieval, occurs only above a certain stimulus threshold and proceeds at a faster initial rate of ∼248 fF/s. It typically undershoots the capacitance value preceding the stimulus, and its magnitude has no clear relationship to the amount of membrane added through the immediately preceding exocytotic event. Excess endocytotic activity persists in the whole cell configuration. Thus, two kinetically distinct forms of endocytosis coexist in intact cells during perforated patch recording. Both are fast enough to retrieve membrane after exocytosis within a few seconds. We argue that the slower one, termed compensatory endocytosis, exhibits properties that make it the most likely mechanism for membrane recycling during normal secretory activity.
Mitochondrial Fusion in Yeast Requires the Transmembrane GTPase Fzo1p
Authors: Hermann Greg J. , Thatcher John W. , Mills John P. , Hales Karen G. , Fuller Margaret T. , Nunnari Jodi , Shaw Janet M.
Published: July 2002

Membrane fusion is required to establish the morphology and cellular distribution of the mitochondrial compartment. In Drosophila, mutations in the fuzzy onions (fzo) GTPase block a developmentally regulated mitochondrial fusion event during spermatogenesis. Here we report that the yeast orthologue of fuzzy onions, Fzo1p, plays a direct and conserved role in mitochondrial fusion. A conditional fzo1 mutation causes the mitochondrial reticulum to fragment and blocks mitochondrial fusion during yeast mating. Fzo1p is a mitochondrial integral membrane protein with its GTPase domain exposed to the cytoplasm. Point mutations that alter conserved residues in the GTPase domain do not affect Fzo1p localization but disrupt mitochondrial fusion. Suborganellar fractionation suggests that Fzo1p spans the outer and is tightly associated with the inner mitochondrial membrane. This topology may be required to coordinate the behavior of the two mitochondrial membranes during the fusion reaction. We propose that the fuzzy onions family of transmembrane GTPases act as molecular switches to regulate a key step in mitochondrial membrane docking and/or fusion.
Onset of Keratin 17 Expression Coincides with the Definition of Major Epithelial Lineages during Skin Development
Authors: McGowan Kevin M. , Coulombe Pierre A.
Published: July 2002

The type I keratin 17 (K17) shows a peculiar localization in human epithelial appendages including hair follicles, which undergo a growth cycle throughout adult life. Additionally K17 is induced, along with K6 and K16, early after acute injury to human skin. To gain further insights into its potential function(s), we cloned the mouse K17 gene and investigated its expression during skin development. Synthesis of K17 protein first occurs in a subset of epithelial cells within the single-layered, undifferentiated ectoderm of embryonic day 10.5 mouse fetuses. In the ensuing 48 h, K17-expressing cells give rise to placodes, the precursors of ectoderm-derived appendages (hair, glands, and tooth), and to periderm. During early development, there is a spatial correspondence in the distribution of K17 and that of lymphoid-enhancer factor (lef-1), a DNA-bending protein involved in inductive epithelial–mesenchymal interactions. We demonstrate that ectopic lef-1 expression induces K17 protein in the skin of adult transgenic mice. The pattern of K17 gene expression during development has direct implications for the morphogenesis of skin epithelia, and points to the existence of a molecular relationship between development and wound repair.
Peroxisome Synthesis in the Absence of Preexisting Peroxisomes
Authors: South Sarah T. , Gould Stephen J.
Published: July 2002

Zellweger syndrome and related diseases are caused by defective import of peroxisomal matrix proteins. In all previously reported Zellweger syndrome cell lines the defect could be assigned to the matrix protein import pathway since peroxisome membranes were present, and import of integral peroxisomal membrane proteins was normal. However, we report here a Zellweger syndrome patient (PBD061) with an unusual cellular phenotype, an inability to import peroxisomal membrane proteins. We also identified human PEX16, a novel integral peroxisomal membrane protein, and found that PBD061 had inactivating mutations in the PEX16 gene. Previous studies have suggested that peroxisomes arise from preexisting peroxisomes but we find that expression of PEX16 restores the formation of new peroxisomes in PBD061 cells. Peroxisome synthesis and peroxisomal membrane protein import could be detected within 2–3 h of PEX16 injection and was followed by matrix protein import. These results demonstrate that peroxisomes do not necessarily arise from division of preexisting peroxisomes. We propose that peroxisomes may form by either of two pathways: one that involves PEX11-mediated division of preexisting peroxisomes, and another that involves PEX16-mediated formation of peroxisomes in the absence of preexisting peroxisomes.
The Peroxin Pex19p Interacts with Multiple, Integral Membrane Proteins at the Peroxisomal Membrane
Authors: Snyder William B. , Koller Antonius , Choy A. Jobu , Subramani Suresh
Published: July 2002

Pex19p is a protein required for the early stages of peroxisome biogenesis, but its precise function and site of action are unknown. We tested the interaction between Pex19p and all known Pichia pastoris Pex proteins by the yeast two-hybrid assay. Pex19p interacted with six of seven known integral peroxisomal membrane proteins (iPMPs), and these interactions were confirmed by coimmunoprecipitation. The interactions were not reduced upon inhibition of new protein synthesis, suggesting that they occur with preexisting, and not newly synthesized, pools of iPMPs. By mapping the domains in six iPMPs that interact with Pex19p and the iPMP sequences responsible for targeting to the peroxisome membrane (mPTSs), we found the majority of these sites do not overlap. Coimmunoprecipitation of Pex19p from fractions that contain peroxisomes or cytosol revealed that the interactions between predominantly cytosolic Pex19p and the iPMPs occur in the organelle pellet that contains peroxisomes. These data, taken together, suggest that Pex19p may have a chaperone-like role at the peroxisome membrane and that it is not the receptor for targeting of iPMPs to the peroxisome.
The Plant Vacuolar Sorting Receptor Atelp Is Involved in Transport of Nh2-Terminal Propeptide-Containing Vacuolar Proteins in Arabidopsis thaliana
Authors: Ahmed Sharif U. , Rojo Enrique , Kovaleva Valentina , Venkataraman Sridhar , Dombrowski James E. , Matsuoka Ken , Raikhel Natasha V.
Published: July 2002

Many soluble plant vacuolar proteins are sorted away from secreted proteins into small vesicles at the trans-Golgi network by transmembrane cargo receptors. Cleavable vacuolar sorting signals include the NH2-terminal propeptide (NTPP) present in sweet potato sporamin (Spo) and the COOH-terminal propeptide (CTPP) present in barley lectin (BL). These two proteins have been found to be transported by different mechanisms to the vacuole. We examined the ability of the vacuolar cargo receptor AtELP to interact with the sorting signals of heterologous and endogenous plant vacuolar proteins in mediating vacuolar transport in Arabidopsis thaliana. AtELP extracted from microsomes was found to interact with the NTPPs of barley aleurain and Spo, but not with the CTPPs of BL or tobacco chitinase, in a pH-dependent and sequence-specific manner. In addition, EM studies revealed the colocalization of AtELP with NTPP-Spo at the Golgi apparatus, but not with BL-CTPP in roots of transgenic Arabidopsis plants. Further, we found that AtELP interacts in a similar manner with the NTPP of the endogenous vacuolar protein AtALEU (Arabidopsis thaliana Aleu), a protein highly homologous to barley aleurain. We hypothesize that AtELP functions as a vacuolar sorting receptor involved in the targeting of NTPP-, but not CTPP-containing proteins in Arabidopsis.
Characterization of Palladin, a Novel Protein Localized to Stress Fibers and Cell Adhesions
Authors: Parast Mana M. , Otey Carol A.
Published: July 2002

Here, we describe the identification of a novel phosphoprotein named palladin, which colocalizes with α-actinin in the stress fibers, focal adhesions, cell–cell junctions, and embryonic Z-lines. Palladin is expressed as a 90–92-kD doublet in fibroblasts and coimmunoprecipitates in a complex with α-actinin in fibroblast lysates. A cDNA encoding palladin was isolated by screening a mouse embryo library with mAbs. Palladin has a proline-rich region in the NH2-terminal half of the molecule and three tandem Ig C2 domains in the COOH-terminal half. In Northern and Western blots of chick and mouse tissues, multiple isoforms of palladin were detected. Palladin expression is ubiquitous in embryonic tissues, and is downregulated in certain adult tissues in the mouse. To probe the function of palladin in cultured cells, the Rcho-1 trophoblast model was used. Palladin expression was observed to increase in Rcho-1 cells when they began to assemble stress fibers. Antisense constructs were used to attenuate expression of palladin in Rcho-1 cells and fibroblasts, and disruption of the cytoskeleton was observed in both cell types. At longer times after antisense treatment, fibroblasts became fully rounded. These results suggest that palladin is required for the normal organization of the actin cytoskeleton and focal adhesions.
Endocytosis and Recycling of the HIV Coreceptor Ccr5
Authors: Signoret Nathalie , Pelchen-Matthews Annegret , Mack Matthias , Proudfoot Amanda E.I. , Marsh Mark
Published: July 2002

The chemokine receptor CCR5 is a cofactor for the entry of R5 tropic strains of human immunodeficiency viruses (HIV)-1 and -2 and simian immunodeficiency virus. Cells susceptible to infection by these viruses can be protected by treatment with the CCR5 ligands regulated on activation, normal T cell expressed and secreted (RANTES), MIP-1α, and MIP-1β. A major component of the mechanism through which chemokines protect cells from HIV infection is by inducing endocytosis of the chemokine receptor. Aminooxypentane (AOP)-RANTES, an NH2-terminal modified form of RANTES, is a potent inhibitor of infection by R5 HIV strains. AOP-RANTES efficiently downmodulates the cell surface expression of CCR5 and, in contrast with RANTES, appears to prevent recycling of CCR5 to the cell surface. Here, we investigate the cellular basis of this effect.

Using CHO cells expressing human CCR5, we show that both RANTES and AOP-RANTES induce rapid internalization of CCR5. In the absence of ligand, CCR5 shows constitutive turnover with a half-time of 6–9 h. Addition of RANTES or AOP-RANTES has little effect on the rate of CCR5 turnover. Immunofluorescence and immunoelectron microscopy show that most of the CCR5 internalized after RANTES or AOP-RANTES treatment accumulates in small membrane-bound vesicles and tubules clustered in the perinuclear region of the cell. Colocalization with transferrin receptors in the same clusters of vesicles indicates that CCR5 accumulates in recycling endosomes. After the removal of RANTES, internalized CCR5 recycles to the cell surface and is sensitive to further rounds of RANTES-induced endocytosis. In contrast, after the removal of AOP-RANTES, most CCR5 remains intracellular. We show that these CCR5 molecules do recycle to the cell surface, with kinetics equivalent to those of receptors in RANTES-treated cells. However, these recycled CCR5 molecules are rapidly reinternalized. Our results indicate that AOP-RANTES–induced changes in CCR5 alter the steady-state distribution of the receptor and provide the first evidence for G protein–coupled receptor trafficking through the recycling endosome compartment.