bluuebunny/crossref_metadata_2025 huggingface dataset:
MEYRIAT, FOSKETT E A COMPETÊNCIA EM INFORMAÇÃO
Authors: Jesus Joselito Manuel de , Alves Fernanda Maria Melo
Published: June 2020

O trabalho efetua uma revisão das contribuições teóricas de Jean Meyriat, que concebe e enfatiza a atuação do usuário da informação no sistema técnico social da informação, concebendo o documento numa perspectiva dialética, interativa e dinâmica, e de Foskett sobre os sistemas formais de comunicação do conhecimento, nas quais o usuário tem papel preponderante. A formação deste usuário proposto por Meyriat exige uma perspectiva pedagógica também interacionista quese associaao conceito de competência em informação,entendida de modo geral, como a capacidade de reconhecer quando a informação é necessária, ter a capacidade de localizar, avaliar e utilizar eficazmente a informação e preservá-la para reutilização futura, conceito proposto pela American Library Association e pela UNESCO, através de uma abordagem epistemológica e pedagógica de caráter interacionista e crítica. A partir desta triangulação aborda criticamente a perspectiva pedagógica do aprender a aprender naquilo que ela tem de redução do sujeito usuário da informação ao individualismo competitivo e hierarquizante, o que favorece o encrudescimento da exclusão que contraria os próprios princípios norteadores da Competência em Informação e dos conceitos de Documento e Documentação. Propõe a concepção pedagógica numa perspectiva emancipatória, fazendo avançar o ensino e a aprendizagem na direção democrática de um usuário solidário que ativa o documento utilizando suas capacidades cognitivas plenamente.
OS AVANÇOS CIENTÍFICOS E TECNOLÓGICOS E SUAS IMPLICAÇÕES NO CAMPO DA DESIGUALDADE E DA INCLUSÃO SOCIOEDUCACIONAL
Authors: Araújo Jurandir de Almeida , Alves Sobrinho Raquel , Neves Barbara Coelho
Published: June 2020

O mundo contemporâneo configura-se como um período da história de profundas e contínuas mudanças em todos os setores e aspectos da vida em sociedade. A ciência e as novas tecnologias da informação e comunicação, em articulação com as instituições de ensino, responsáveis pela sistematização e transmissão do conhecimento, gozam de grande prerrogativa no desenvolvimento científico e tecnológico. Assim, é objetivo desse artigo tecer uma análise teórico-crítica acerca das implicações dos avanços científicos e tecnológicos no campo das desigualdades sociais e educacionais.
TECNOLOGIA ASSISTIVA: produtos e serviços disponíveis na internet
Authors: Fraz Joeanne Neves , Hott Daniela Francescutti Martins , Moreira Geraldo Eustáquio , Rodrigues Georgete Medleg
Published: June 2020

A proposta deste estudo é compartilhar produtos e serviços disponíveis na Internet com foco no uso de tecnologia assistiva, buscando a promoção da igualdade e da inclusão social das pessoas com deficiência nas instituições e/ou organizações. Abordando, nesta reflexão, como ocorre, para as pessoas com deficiência, o acesso à Web e seus recursos, tendo em vista as suas dificuldades nesse ambiente, sendo a tecnologia um facilitador. Os objetivos foram: identificar problemas na questão de acesso com acessibilidade à informação em ambientes virtuais e digitais, trazendo à tona as barreiras que dificultam o acesso das pessoas com deficiência visual, auditiva, intelectual, física e/ou mobilidade reduzida; e, apresentar produtos e serviços de tecnologia interativa disponíveis na internet, conhecendo recursos e/ou metodologias de acessibilidades atitudinal e comunicacional. Trata-se de um estudo de abordagem exploratória e descritiva, que parte da vivência das autoras nas disciplinas cursadas e objetivos das pesquisas de doutorado em andamento em seus programas de Pós-Graduação (em Ciência da Informação e em Educação, Universidade de Brasília – UnB). A reflexão mostrou produtos e serviços de tecnologia assistiva que buscam minimizar os problemas de acessibilidade à informação em ambientes virtuais e digitais, e a importante divulgação da Tecnologia Assistiva existente e suas funções.
Biochemical heterogeneity of reverse transcriptase purified from the AIDS virus, HTLV‐III
Authors: Chandra Angelika , Gerber Thomas , Chandra Prakash
Published: July 2002

The reverse transcriptase from AIDS virus, HTLV‐III, was purified and characterized. The purified enzyme has a very high affinity for template primers (rC)
n
·(dG)12 and (rCm)
n
·(dG)12 compared to that for (rA)
n
·(dT)12. In addition, the HTLV‐III reverse transcriptase was able to transcribe (rAm)
n
·(dT)12 very efficiently. The ionic requirements are unique in the sense that HTLV‐III reverse transcriptase prefers Mg2 as divalent ions to transcribe (rC)
n
·(dG)12 and (rA)
n
·(dT)12. The M
r of the enzyme is 95000–98000. Unlike the HTLV‐I reverse transcriptase, the HTLV‐III enzyme is highly stable and has a much higher activity in the presence of (rC)
n
·(dG)12; the V
max for HTLV‐III reverse transcriptase is several‐fold higher than that for HTLV‐I enzyme. The enzyme activity of the purified reverse transcriptase from HTLV‐III was resolved into two peaks on a preparative isoelectric column, one at pH 5.75 and the other at pH 6.25. This leads us to conclude that the reverse transcriptase of HTLV‐III is biochemically heterogeneous.
The formation of plasma membrane blebs in hepatocytes exposed to agents that increase cytosolic Ca2+ is mediated by the activation of a non‐lysosomal proteolytic system
Authors: Nicotera Pierluigi , Hartzell Pia , Davis Gregory , Orrenius Sten
Published: July 2002

Exposure of isolated hepatocytes to extracellular ATP, cystamine or ionophore A23187 was associated with an increase in cytosolic Ca2+ concentration, a stimulation of intracellular proteolysis, and the appearance of plasma membrane blebs which preceded the loss of cell viability. Both bleb formation and cell killing were prevented when inhibitors of Ca2+‐activated neutral proteases, such as antipain or leupeptin, were included in the incubation medium, whereas inhibitors of lysosomal proteases had no effect. Thus, the activation of a Ca2+‐dependent, non‐lysosomal proteolytic system appears to be responsible for the plasma membrane blebbing and, ultimately, the cytotoxicity associated with treatment of hepatocytes with agents that disrupt intracellular Ca2+ homeostasis.
Stimulation of partially purified adenylate cyclase from bull sperm by bicarbonate
Authors: Garty Nira B. , Salomon Yoram
Published: July 2002

Solubilized and partially purified adenylate cyclase from bull sperm was found to be specifically activated (up to 6‐fold) by sodium bicarbonate (NaHCO3) and to a lesser extent by NANO3. Other sodium salts were either ineffective (e.g. NaCOOH) or inhibitory (e.g. NaHSO3, NaHSO4 and Na2B4O7). Stimulation by NaHCO3 was dose‐dependent in the range of 0–40 mM and was greater when enzyme activity was assayed in the presence of magnesium as compared with manganese ions. Bicarbonate seems to affect maximal enzyme velocity (V
max) and has no effect on the K
m of adenylate cyclase for Mn‐ATP. Stimulation of adenylate cyclase by NaHCO3 coincided with the elution pattern of the enzyme as recorded following chromatography on DEAE‐cellulose or gel filtration on BioGel P‐100. These results suggest that in the course of stimulation of sperm adenylate cyclase, bicarbonate is likely to interact directly with the enzyme. Furthermore, this intrinsic and unique property of sperm adenylate cyclase may explain results reported by others on the stimulation of cAMP production by bicarbonate in intact and broken sperm preparations and suggest a biochemical basis for enhanced sperm motility associated with high bicarbonate concentrations.
Binding of specific ligands to muscarinic receptors alters the fluidity of membrane fragments from rat brain A fluorescence polarization study with lipid‐specific probes
Authors: Manevich E.M. , Köiv A. , Järv J. , Molotkovsky J.G. , Bergelson L.D.
Published: July 2002

The previously suggested method of following ligand‐receptor interactions by measuring ligand‐induced changes in membrane fluidity [(1986) FEBS Lett. 194, 313–316] was employed to study the binding of specific ligands of the muscarinic receptor to rat brain membrane fragments containing a fluorescent analogue of phosphatidylcholine (APC) as a membrane probe. Upon addition of carbachol and atropine in low concentrations the fluorescence polarization of the APC‐labeled membranes decreased significantly demonstrating that binding of these ligands to the muscarinic receptor increases the fluidity of its lipid environment. The fluidity changes were specific, concentration‐dependent and saturable. In comparison with radioligand assays the fluorescent lipid probe method proved to be much more sensitive but the K
d values obtained by the two methods differed considerably.
Chloramphenicol‐induced stabilization of light‐harvesting complexes in thylakoids during development
Authors: Tzinas George , Argyroudi-Akoyunoglou Joan
Published: July 2002

Chloramphenicol treatment allows light‐harvesting apoprotein accumulation in thylakoids of intermittent light plants and prevents its digestion in plants transferred to darkness after brief pre‐exposure to continuous light. This suggests that under conditions where reaction center‐core protein synthesis is inhibited, the light‐harvesting apoproteins can be stabilized. The results support the competition hypothesis proposed to occur between reaction center polypeptides and light‐harvesting apoproteins for chlorophyll, whenever the rate of chlorophyll formation relative to that of the polypeptide components is limited.
Site‐directed mutagenesis of histidine 62 in the ‘basic patch’ region of yeast phosphoglycerate kinase
Authors: Fairbrother Wayne J. , Hall Len , Littlechild Jennifer A. , Walker Philip A. , Watson Herman C. , Williams Robert J.P.
Published: July 2002

Site‐directed mutagenesis has been used to produce a mutant form of yeast phosphoglycerate kinase (PGK) in which the ‘basic patch’ residue His 62 has been replaced by a glutamine residue. Using 1H‐NMR spectroscopy, it was found that 3‐phosphoglycerate (3‐PG) binding to the mutant protein induces the same conformational effects as for wild‐type PGK, although the affinity was reduced by 2‐ to 3‐fold. Kinetic studies show both K
m for 3‐PG and V
max to be increased by ~ 2‐fold relative to the wild‐type enzyme. These data are consistent with the suggestion that His 62 assists in the binding of the substrate to the enzyme.
Cosecretion of amylin and insulin from isolated rat pancreas
Authors: Fehmann H.C. , Weber V. , Göke R. , Göke B. , Arnold R.
Published: July 2002

Amylin, a 37 amino acid C‐terminal amidated peptide is an integral part of secretory granules of pancreatic β‐cells. Utilizing a specific radioimmunoassay system we demonstrate in the present study a cosecretion of amylin and insulin from the isolated rat pancreas. The secretion pattern of both peptides during glucose or glucose plus arginine stimulation is identical. The molar ratio of amylin amounts to 10% of that of insulin. The biological significance of amylin is still unknown, but a paracrine/endocrine role in glucose homeostasis is speculated.
Membrane‐associated phosphoinositidase C activity in Dictyostelium discoideum
Authors: Lundberg Gunilla A. , Newell Peter C.
Published: July 2002

by micromolar concentrations of free calcium with maximal activity at 100 μM.
The ram: A novel low molecular weight GTP‐binding protein cDNA from a rat megakaryocyte library
Authors: Nagata Koh-ichi , Satoh Takaya , Itoh Hiroshi , Kozasa Tohru , Okano Yukio , Doi Takefumi , Kaziro Yoshito , Nozawa Yoshinori
Published: July 2002

A novel low Mr
GTP‐binding protein cDNA was isolated from a rat megakaryocyte cDNA library with a synthetic oligonucleotide probe corresponding to an 8‐amino acid sequence specific for c25KG, a GTP‐binding protein previously isolated from human platelet cytosol fraction [(1989) J. Biol. Chem. 264, 17000–17005]. The cDNA has an open reading frame encoding a protein of 221 amino acids with a calculated Mr
of 25068. The protein is designated as ram (ras‐related gene from megakaryocyte) protein (ram p25). The amino acid sequence deduced from the ram cDNA contains the consensus sequences for GTP‐binding and GTPase domains. ram p25 shares about 23%, 39% and 80% amino acid homology with the H‐ras, smg25A and c25KG proteins, respectively. The 3.5‐kb ram mRNA was detected abundantly in spleen cells.
Regulation of neuronal migration and neuritogenesis by distinct surface proteases Relative contribution of plasmin and a thrombin‐like protease
Authors: Shea Thomas B. , Beermann Mary Lou
Published: July 2002

The relative contribution of two neuronal surface proteases, plasmin and a protease with thrombin‐like specificity, on NB2a/dl neuroblastoma migration and neuritogenesis were examined. Exogenous plasmin induced cell body rounding and increased cell migration, but did not prevent or reverse neurite outgrowth. Inhibition of endogenous plasmin by its specific inhibitor, aprotinin, suppressed migration but did not induce neuritogenesis, Removal or inhibition of the thrombin‐like protease by serum deprivation or hirudin addition, respectively, induced neurite outgrowth, as shown in our previous studies, but did not suppress migration. By contrast, trypsin induced simultaneous cell rounding and neurite retraction. These findings indicated that plasmin may regulate cell migration, while the thrombin‐like protease may regulate facets of neurite outgrowth. Although unable to induce de novo neuritogenesis, plasmin inhibition potentiated the otherwise transient neurites induced by simultaneous inhibition or the thrombin‐like protease. Since cultured neuronal cells migrate primarily in the direction of newly elaborated neurites, this finding is interpreted to indicate that cessation of neuronal migration by plasmin inhibition enhances net neurite outgrowth by inhibition of the putative thrombin‐like protease.
NO•, CO and •OH Endogenous soluble guanylyl cyclase‐activating factors
Authors: Schmidt Harald H.H.W.
Published: July 2002

Several low molecular weight compounds are capable of activating soluble guanylyl cyclase. Recent evidence suggests that some of these are formed under physiological conditions: the nitric oxide radical, carbon monoxide and the hydroxyl radical. Thus, multiple signal transduction pathways appear to exist that form a family of guanylyl cyclase activating factors and thereby regulate the intracellular cyclic guanosine 3′, 5′‐monophosphate level.
Secretagogue‐induced calcium wave shows higher and prolonged transients of nuclear calcium concentration in mast cells
Authors: Katagiri Satoshi , Takamatsu Tetsuro , Minamikawa Tetsuhiro , Fujita Setsuya
Published: July 2002

To clarify the mechanism of secretagogue (compound 40/80)‐induced calcium signaling in rat peritoneal mast cells, we analyzed serial confocal calcium images with high spatial and temporal resolution using different Ca2+‐probes. The Ca2+‐wave began at the periphery of the cytoplasm, and then spread to the center of the nucleus. Nuclear [Ca2+]i was clearly higher than cytoplasmic [Ca2+]i. The heterogeneity of [Ca2+]i continued until about 2 min after degranulation. These results suggest the existence of an intranuclear Ca2+‐store which possesses a Ca2+ ‐releasing mechanism similar to that in the cytoplasm.
Cantharidin, another natural toxin that inhibits the activity of serine/threonine protein phosphatases types 1 and 2A
Authors: Honkanen Richard E.
Published: July 2002

Cantharidin, a natural toxicant of blister beetles, is a strong inhibitor of protein phosphatases types 1(PP1) and 2A (PP2A). Like okadaic acid, cantharidin inhibits the activity of the purified catalytic subunit of PP2A (IC50 = 0.16 μM) at a lower concentration than that of PPI (IC50 = 1.7 μM) and only inhibits the activity of protein phosphatase type 2B (PP2B) at high concentrations. Dose‐inhibition studies conducted with whole cell homogenates indicate that cantharidin also inhibits the native forms of these enzymes. Thus, cantharidin, which is economical and readily available, may be useful as an additional probe for studying the functions of serine/threonine protein phosphatases.
Production and characterization of monoclonal antibodies specific to multi‐ubiquitin chains of polyubiquitinated proteins
Authors: Fujimuro Masahiro , Sawada Hitoshi , Yokosawa Hideyoshi
Published: July 2002

Polyubiquitinated proteins tagged with multi‐ubiquitin chains are substrates preferred by the 26 S proteasome (a ubiquitin/ATP‐dependent proteolytic complex). Here, we developed a simple method for the efficient preparation of polyubiquitinated proteins which are degraded by the 26 S proteasome in an ATP‐dependent manner. Our efficient method enabled us to produce ten monoclonal antibodies that recognized the multi‐ubiquitin chains of the polyubiquitinated proteins, but not free ubiquitin or the protein moieties. Eight of the antibodies recognized only the multi‐ubiquitin chains of the polyubiquitinated proteins, while the other two antibodies cross‐reacted with mono‐ubiquitin and methyl‐ubiquitin, both of which are linked to proteins via an isopeptide bond, as well as with the multi‐ubiquitin chains. Thus these antibodies are novel and useful tools for the identification and quantification of polyubiquitinated proteins in various cells and tissues under physiological and pathological conditions.
Vitellogenin gene of the silkworm, Bombyx mori: Structure and sex‐dependent expression
Authors: Yano Ken-ichi , Sakurai Mayumi Toriyama , Izumi Susumu , Tomino Shiro
Published: July 2002

Vitellogenin of Bombyx mori is a precursor of major yolk protein synthesized in the female fat body at larval—pupal ecdysis. The gene for B. mori vitellogenin is composed of seven exons interspersed by six introns. Developmental profile of the primary transcript of the gene indicated that the biosynthesis of B. mori vitellogenin is regulated transcriptionally in a sex‐ and stage‐dependent manner in the fat body. The Arg‐X‐Arg‐Arg sequence, which conforms to the recognition site of mammalian furin, occurs in a region just upstream of the putative proteolytic cleavage site of B. mori previtellogenin.
Elucidation of the poly‐l‐proline binding site in Acanthamoeba profilin I by NMR spectroscopy
Authors: Archer Sharon J. , Vinson Valda K. , Pollard Thomas D. , Torchia Dennis A.
Published: July 2002

The multifunctional protein profilin is one of the most abundant proteins in the cytoplasm and is thought to regulate actin assembly and the phosphoinositide signaling pathway. Profilin binds to several different ligands including actin, poly‐l‐proline, and the head groups of polyphosphoinositides. Knowledge of profilin/ligand interactions is important for understanding the physiology of profilin in the cell. As a first step in the characterization of profilin/ligand complexes, we have studied a profilin/poly‐Pl‐prohne complex in solution using high resolution NMR spectroscopy. Analysis of profilin NOE's and chemical shift data indicates that the protein secondary structure is conserved upon binding to poly‐l‐proline and that the binding site is located between the N‐ and C‐terminal helices in a region rich in highly conserved aromatic sidechains. This site is adjacent to the proposed binding site for actin. In addition, the rate constant for dissociation of the complex is found to be 1.6 ± 0.2 × 104 s−1.
Human μ opiate receptor
Authors: Wang Jia-Bei , Johnson Peter S. , Persico Antonio M. , Hawkins Anita L. , Griffin Constance A. , Uhl George R.
Published: July 2002

A human μ opiate receptor cDNA has been identified from a cerebral cortical cDNA library using sequences from the rat μ opiate receptor cDNA. The human μ opiate receptor (hμOR1) shares 95% amino acid identity with the rat sequence. The expressed μOR1 recognizes tested opiate drugs and opioid peptides in a sodium‐ and GTP‐sensitive fashion with affinities virtually identical to those displayed by the rat μ opiate receptor. Effects on cyclic AMP are similar to those noted for the rat μ opiate receptor. An 18 kb genomic clone hybridizing with the hμOR1 cDNA contains 63 and 489 bp exonic sequences flanked by splice donor/acceptor sequences. Analysis of hybridization to DNA prepared from human rodent hybrid cell lines and chromosomal in situ hybridization studies indicate localization to 6q24–25. An MspI polymorphism, producing a 3.7 kb band, may prove useful in assessing this gene's involvement in neuropsychiatric disorders involving opiatergic systems.
‘Camelising’ human antibody fragments: NMR studies on VH domains
Authors: Davies Julian , Riechmann Lutz
Published: July 2002

A human heavy chain variable domain (VH) was expressed in bacteria for structural analysis by NMR spectroscopy. NMR analysis was initially impossible due to the short transverse proton relaxation time of the VH, probably caused by aggregation through the exposed interface naturally in contact with the light chain. The relaxation time was improved to normal values when this interface was mutated to mimic heavy chains of camel antibodies naturally devoid of light chains and through the use of the detergent CHAPS. Assignment of NMR signals will now be possible after isotopic labeling. Implications for the design of VH domains as minimum size immunoreagents are outlined.
Transvaginal sonography and progesterone challenge for identifying endometrial pathology in postmenopausal women
Authors: Malinova M. , Pehlivanov B.
Published: July 2002

Objective: To evaluate the usefulness of transvaginal sonographic (TVS) measurement of endometrial thickness combined with a progesterone challenge test (PCT) for identifying endometrial pathology in postmenopausal women. Methods: Two hundred eighty‐four postmenopausal women were examined by TVS: 130 asymptomatic women (group A) and 154 with uterine bleeding (group B). Endometrial thickness >5 mm was considered pathological. All women with abnormal endometrium from group A underwent PCT. All women from group B underwent D&C. Results: Thirty women with a positive PCT underwent D&C. There was no endometrial cancer. One hundred seven patients from group B had abnormal sonographic and histologic findings: benign (hyperplasia, polyp) or malign (endometrial cancer). There was no cancer in cases with endometrial thickness ≤ 6 mm. The sensitivity, specificity and accuracy of TVS for detecting endometrial pathology were 99%, 89% and 96% if the cut‐off limit of 5 mm was used. Conclusion: TVS combined with PCT is a simple, well‐tolerated, safe and reliable method for identifying endometrial pathology in postmenopausal women.
THE EFFECTS OF EXTRACELLULAR MATRIX AND OSTEOGENIC PROTEIN‐1 ON THE MORPHOLOGICAL DIFFERENTIATION OF RAT SYMPATHETIC NEURONS
Authors: Lein Pamela , Guo Xin , Hedges Ann Marie , Rueger David , Johnson Mary , Higgins Dennis
Published: July 2002

The growth patterns of axons and dendrites differ with respect to their number, length, branching, and spatial orientation; therefore, it is likely that these processes differ in their growth requirements. To examine this hypothesis, we have been analyzing the responses of cultured rat sympathetic neurons to three types of stimuli: large structural proteins of the extracellular matrix, matrix‐associated growth factors, and neurotrophins. Purified structural proteins such as laminin and collagen IV have been found to promote only axonal growth; whereas the matrix associated growth factor, osteogenic protein‐1, selectively stimulates dendritic growth. In contrast, nerve growth factor modulates the growth of both types of processes. These data suggest that process‐specific interactions with the extracellular environment may be critical determinants of cell shape in neurons. Perinatal rat sympathetic neurons grown in culture in the absence of serum or glial cells extend a single process which is axonal in nature. Exposure to osteogenic protein‐1 causes the formation of additional processes which express the morphological, cytoskeletal, and ultrastructural characteristics of dendrites. Consistent with observations on the regulation of dendritic growth in sympathetic neurons in situ, the dendrite‐promoting activity of osteogenic protein‐1 is independent of synaptic or electrical activity, but is modulated by nerve growth factor. In the presence of optimal concentrations of osteogenic protein‐1 and nerve growth factor, the size of the dendritic arbor extended by cultured sympathetic neurons approximates that seen in situ at comparable developmental stages. Osteogenic protein‐1 does not promote dendritic growth in cultured neurons obtained from embryonic ciliary, dorsal root, trigeminal or nodose ganglia, suggesting that its morphogenetic effects are cell selective. Since mRNA for osteogenic protein‐1 is expressed in mature as well as embryonic target tissues of the sympathetic nervous system, we also examined the effects of osteogenic protein‐1 on cultures of sympathetic neurons derived from adult rats. Consistent with results obtained with perinatal neurons, osteogenic protein‐1 selectively promoted dendritic growth in adult neurons. These data suggest that this matrix‐associated growth factor could play a role not only in the morphogenesis of the developing nervous system, but also in the maintenance and remodeling of dendritic structures in the mature animal.
Dysthymia in DSM-IV and ICD-10: nosology and diagnosis
Authors: López-Ibor JJ
Published: July 2002

Since the DSM-III, the category of dysthymia includes patients that were previously considered as neurotic, reactive, characterological, temperamental, minor or atypical depressives as opposed to a category of endogenous, autonomous, morbid, major or typical depressives. Despite certain confusion, the need for a nosological category is very important for patients suffering from years of depressive mood, with a significant impact on their life-styles and adaptation, as they can benefit enormously from modern antidepressant treatments. DSM-IV and ICD-10 describe dysthymia in a very similar way although there are significant differences. The differences are the consequence of the lack of sufficient scientific data in some points and the need to take a decision, but also to indicate areas for future research. Other differences are the consequence of major discrepancies in basic perspectives of the classifications (ie, the importance given to disablement for the diagnosis). The similarities between both classifications allow the use of either of them for most purposes. Research on the different classifications should help in better defining the concept of dysthymia and mood disorders in general (ie, the cut-off points between them) and of psychiatric nosology.
The PWWP domain: a potential protein–protein interaction domain in nuclear proteins influencing differentiation?
Authors: Stec Ingrid , Nagl Sylvia B. , van Ommen Gert-Jan B. , den Dunnen Johan T.
Published: July 2002

Upon characterization of WHSC1, a gene mapping to the Wolf–Hirschhorn syndrome critical region and at its C‐terminus similar to the Drosophila ASH1/trithorax group proteins, we identified a novel protein domain designated PWWP domain. To gain insight into its structure, evolutionary conservation and its potential functional role, we performed database searches to identify other PWWP domain‐containing proteins. We retrieved 39 proteins, and a multiple alignment shows that the domain spans some 70 amino acids. It is present in proteins of nuclear origin and plays a role in cell growth and differentiation. Due to its position, the composition of amino acids close to the PWWP motif and the pattern of other domains present, we hypothesize that the domain is involved in protein–protein interactions.
Chemical inhibitors: a tool for plant cell cycle studies
Authors: Planchais Séverine , Glab Nathalie , Inzé Dirk , Bergounioux Catherine
Published: July 2002

Synchrony provides a large number of cells at defined points of the cell cycle. Highly synchronised cells are powerful and effective tools for molecular analyses and for studying the biochemical events of the cell cycle in plants. Usually, plant cell suspensions can be synchronised by chemical agents, which arrest the cell cycle by acting on the driving forces of the cell cycle engine such as cyclin‐dependent kinase activity, enzymes involved in DNA synthesis or proteolysis of cell cycle regulators or by acting on the cell cycle apparatus (mitotic spindle). The specificity, reversibility and efficiency of each type of cell cycle inhibitor are described and related to their mode of action.
Nuclear import factors importin α and importin β undergo mutually induced conformational changes upon association
Authors: Cingolani Gino , Lashuel Hilal A. , Gerace Larry , Müller Christoph W.
Published: July 2002

A heterodimer of importin α and importin β accomplishes the nuclear import of proteins carrying classical nuclear localization signals (NLS). The interaction between the two import factors is mediated by the IBB domain of importin α and involves an extended recognition surface as shown by X‐ray crystallography. Using a combination of biochemical and biophysical techniques we have investigated the formation of the importin β:IBB domain complex in solution. Our data suggest that upon binding to the IBB domain, importin β adopts a compact, proteolytically resistant conformation, while simultaneously the IBB domain folds into an α helix. We suggest a model to describe how these dual mutually induced conformational changes may orchestrate the nuclear import of NLS cargo in vivo.
Effects of MAP kinase cascade inhibitors on the MKK5/ERK5 pathway
Authors: Mody Nimesh , Leitch Jane , Armstrong Chris , Dixon Jack , Cohen Philip
Published: July 2002

Antibodies that recognise the active phosphorylated forms of mitogen‐activated protein kinase (MAPK) kinase 5 (MKK5) and extracellular signal‐regulated kinase 5 (ERK5) in untransfected cells have been exploited to show that the epidermal growth factor (EGF)‐induced activation of MKK5 and ERK5 occurs subsequent to the activation of ERK1 and ERK2 in HeLa cells. The drugs U0126 and PD184352, which prevent the activation of MKK1 (and hence the activation of ERK1/ERK2), also prevent the activation of MKK5, although higher concentrations are required. Our studies define physiological targets of the MKK5/ERK5 pathway as proteins whose phosphorylation is largely prevented by 10 μM PD184352, but unaffected by 2 μM PD184352. Surprisingly, 2 μM PD184352 prolongs the activation of MKK5 and ERK5 induced by EGF or H2O2, indicating negative control of the MKK5/ERK5 pathway by the classical MAPK cascade. Our results also indicate that ERK5 is not a significant activator of MAPK‐activated protein kinase‐1/RSK in HeLa cells.
Involvement of extracellular calcium in phosphatidylserine exposure during apoptosis
Authors: Hampton Mark B. , Vanags Daina M. , Isabella Pörn-Ares M. , Orrenius Sten
Published: July 2002

The appearance of phosphatidylserine (PS) on the outer surface of apoptotic cells is an important signal for their ingestion. In platelets, elevation of intracellular Ca2+ with thapsigargin can trigger large amounts of PS exposure within minutes. We detected PS exposure in U937 promonocytes and Jurkat T‐cells after incubation with thapsigargin, but in only 10% of the cells, and it took up to 6 h to occur. Tumor necrosis factor and anti‐Fas antibody rapidly trigger apoptosis in these cells, and chelation of extracellular Ca2+ with 5 mM EGTA inhibited PS exposure by 65% and 50%, respectively. Chelation of intracellular Ca2+ with BAPTA‐AM had no effect. Other parameters of apoptosis, including cell blebbing, shrinkage, nuclear fragmentation, activation of the ICE‐like proteases, and fodrin cleavage, were not inhibited by extracellular EGTA. We conclude that while an elevation of intracellular Ca2+ is an ineffective trigger of apoptosis in the cells investigated, extracellular Ca2+ is required for efficient PS exposure during apoptosis.
Translocation of cytosolic annexin 2 to a Triton‐insoluble membrane subdomain upon nicotine stimulation of chromaffin cultured cells
Authors: Sagot Isabelle , Regnouf Françoise , Henry Jean-Pierre , Louise-Anne Pradel
Published: July 2002

To gain a better understanding of the function of annexin 2, we have investigated the subcellular distribution of the monomeric and heterotetrameric forms of annexin 2 and their relationship to the cytoskeleton upon stimulation of chromaffin cells. Quantitative immunoblotting has revealed that in resting cells a large amount of annexin 2 is monomeric and cytosolic. Upon nicotine stimulation 80% of total annexin 2 becomes associated with a Triton‐X100‐insoluble fraction where the monomeric and the heterotetrameric forms are found. The translocation of monomeric annexin 2 is Ca2+‐dependent and complete at 1 μM free Ca2+. We have shown that about 66% of the annexin 2 associated with the Triton‐X100‐insoluble fraction is soluble in octylglucoside while the remnants are insoluble in the detergent and remain likely associated with actin filaments and associated cytoskeleton proteins. The octylglucoside‐soluble fraction contains integral proteins from the plasma membrane and from granule membrane, but does not contain caveolin. Moreover, upon nicotine stimulation, a redistribution of proteins was detected in this fraction. These dynamic processes appear concomitantly with the phosphorylation of annexin 2 in this compartment and with catecholamine release. It is suggested that the soluble octylglucoside fraction may represent a special lipidic membrane compartment where the NSF attachment proteins and the cytosolic proteins like annexin 2 and rab3a may become concentrated upon stimulation of the cell. The presence of annexin 2 is consistent with its proposed function on granule and target membrane proteins required for the close apposition of two distinct membranes and supports its functional role in the regulated exocytosis/endocytosis process.
Binding mode of benzhydroxamic acid to Arthromyces ramosus peroxidase shown by X‐ray crystallographic analysis of the complex at 1.6 Å resolution
Authors: Itakura Hiroyuki , Oda Yutaka , Fukuyama Keiichi
Published: July 2002

The crystal structure of Arthromyces ramosus peroxidase (ARP) in complex with benzhydroxamic acid (BHA) as determined by X‐ray analysis at 1.6 Å shows unambiguously how BHA binds to ARP. BHA is located in the distal heme pocket. Its functional groups are held by three hydrogen bonds to His56Nϵ, Arg52Nϵ, and Pro154O, but are too far away to interact with the heme iron. The aromatic ring of BHA is positioned at the entrance of the channel to the heme pocket, approximately parallel to the heme group. Most water molecules at the active site of the native enzyme are replaced by BHA, leaving a ligand, probably a water molecule, at the sixth position of the heme. Results are compared with spectroscopic data.
HIV‐1 capsid mutants inhibit the replication of wild‐type virus at both early and late infection phases
Authors: Furuta Rika A , Shimano Reika , Ogasawara Takashi , Inubushi Ritsuko , Amano Kazushi , Akari Hirofumi , Hatanaka Masakazu , Kawamura Meiko , Adachi Akio
Published: July 2002

In‐frame mutations were introduced into various portions of the human immunodeficiency virus type 1 (HIV‐1) gag gene, and potentials of the mutants to suppress the replication of wild‐type HIV‐1 were monitored. In contrast to results obtained with matrix and nucleocapsid mutants, almost all capsid mutants blocked HIV‐1 replication completely in single‐round replication assays. A capsid mutant designated C6b was demonstrated to be one of the most efficient inhibitors for HIV‐1 reported to date, and to be effective at both early and late viral replication phases. T‐cells, which are engineered to express the C6b Gag in response to HIV‐1 infection, were perfectly resistant to HIV‐1.
Coupling of rat somatostatin receptor subtypes to a G‐protein gated inwardly rectifying potassium channel (GIRK1)
Authors: Kreienkamp Hans-Jürgen , Hönck Hans-Hinrich , Richter Dietmar
Published: July 2002

The five different rat somatostatin receptor subtypes (SSTR1–SSTR5) were coexpressed with a subunit of G‐protein gated inwardly rectifying potassium channel (GIRK1) in Xenopus oocytes. SSTR2–SSTR5, but not SSTR1 coupled efficiently to the activation of GIRK currents when stimulated by SST14 or SST28. A comparison of the dose‐response curves and of the maximum currents obtained indicates that SSTR2 couples most efficiently to this effector, supporting the notion that SSTR2 is involved in activation of potassium conductances by SST in vivo.
Sodium channel protein expression enhances the invasiveness of rat and human prostate cancer cells
Authors: Smith Paul , Rhodes Nicholas P , Shortland Adam P , Fraser Scott P , Djamgoz Mustafa B.A , Ke Youqiang , Foster Christopher S
Published: July 2002

Expression of Na+ channel protein was analysed in established cell lines of rat and human prostatic carcinoma origin by flow cytometry using a fluorescein‐labelled polyclonal antibody. In many cell lines examined, the obtained frequency distribution profiles were bimodal and identified a subpopulation of cells which expressed high levels of Na+ channel protein. A significant positive correlation was demonstrated between the proportion of channel‐expressing cells and the functional ability of individual cell lines to invade a basement membrane matrix in vitro. In addition, two transfectant cell lines containing rat prostate cancer genomic DNA were found to express significantly elevated levels of Na+ channel protein when compared with the original benign recipient cell line. Enhanced Na+ channel expression by two metastatic derivatives of these transfectant cells directly correlated with increased invasiveness in vitro. These studies strongly support the hypothesis that expression of Na+ channel protein and the metastatic behaviour of prostatic carcinoma cells are functionally related, either by endowing the membranes of these cells with specialised electrophysiological properties (e.g. enhancing their motility and/or secretory activities) and/or by perturbing endogenous mechanisms regulating ionic homeostasis within the cells.
Cytochrome c in the apoptotic and antioxidant cascades
Authors: Skulachev Vladimir P
Published: July 2002

Recent progress in studies on apoptosis has revealed that cytochrome c is a pro‐apoptotic factor. It is released from its places on the outer surface of the inner mitochondrial membrane at early steps of apoptosis and, combining with some cytosolic proteins, activates conversion of the latent apoptosis‐promoting protease pro‐caspase‐9 to its active form. Cytochrome c release can be initiated by the pro‐apoptotic protein Bax. This process is blocked by the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL. The role of cytochrome c in apoptosis may be understood within the framework of the concept assuming that the evolutionary primary function of apoptosis was to purify tissues from ROS‐overproducing cells. In this context, the pro‐apoptosis activity of cytochrome c might represent one of the anti‐oxidant functions inherent in this cytochrome. Among other cytochrome c‐linked antioxidant mechanisms, the following systems can be indicated. (1) Cytochrome c released from the inner mitochondrial membrane to the intermembrane space can operate as an enzyme oxidizing O−⋅
2 back to O2. The reduced cytochrome c is oxidized by cytochrome oxidase (or in yeasts and bacteria, by cytochrome c peroxidase). (2) The intermembrane cytochrome c can activate the electron transport chain in the outer mitochondrial membrane. This bypasses the initial and middle parts of the main respiratory chain, which produce, as a rule, the major portion of ROS in the cell. (3) The main respiratory chain losing its cytochrome c is inhibited in such a fashion that antimycin‐like agents fail to stimulate ROS production.
X‐ray and spectrophotometric studies of the binding of proflavin to the S1 specificity pocket of human α‐thrombin
Authors: Conti Elena , Rivetti Claudio , Wonacott Alan , Brick Peter
Published: July 2002

Proflavin can be used to study the interactions of inhibitors and substrates with thrombin by monitoring the changes in the visible absorption spectrum that occur on dye displacement. We have used microspectrophotometric methods to investigate the binding of proflavin to crystals of an α‐thrombin‐hirugen complex and have determined the structure by X‐ray crystallography. The proflavin molecule binds in the S1 pocket of the enzyme with one of the amino groups hydrogen bonded to the carboxylate of Asp‐189 while the protonated ring nitrogen is hydrogen bonded to the carbonyl of Gly‐219. This result indicates that the proflavin displacement assay can be used to specifically monitor the binding of inhibitors to the S1 pocket.
Differential genome analysis applied to the species‐specific features of Helicobacter pylori
Authors: Huynen Martijn , Dandekar Thomas , Bork Peer
Published: July 2002

We introduce a simple and rapid strategy to identify genes that are responsible for species‐specific phenotypes. The genome of a species that has a specific phenotype is compared with at least one, closely related, species that lacks this phenotype. Homologous genes that are shared among the species compared are identified and discarded from the list of candidates for species‐specific genes. The process is automated and rapidly yields a small subset of the genome that likely contains genes responsible for the species‐specific features. Functions are assigned to the genes, and dubious annotations are filtered out. Information is extracted not only from the presence of genes, but also from their absence with respect to known phenotypes. We have applied the technique to identify a set of species‐specific genes in Helicobacter pylori by comparing it with its closest relatives for which complete genome sequences are available, Haemophilus influenzae and Escherichia coli. Of the genes of this set for which functional features can be obtained, a large fraction (63%, 123 proteins) is (potentially) involved in H. pylori's interaction with its host. We hypothesize that a family of outer membrane proteins is critical for the ability of H. pylori to colonize host cells in highly acidic environments.
Pharmacology of recombinant γ‐aminobutyric acidA receptors rendered diazepam‐insensitive by point‐mutated α‐subunits
Authors: Benson Jack A , Löw Karin , Keist Ruth , Mohler Hanns , Rudolph Uwe
Published: July 2002

Amino acids in the α‐ and γ‐subunits contribute to the benzodiazepine binding site of GABAA‐receptors. We show that the mutation of a conserved histidine residue in the N‐terminal extracellular segment (α1H101R, α2H101R, α3H126R, and α5H105R) results not only in diazepam‐insensitivity of the respective αxβ2,3γ2‐receptors but also in an increased potentiation of the GABA‐induced currents by the partial agonist bretazenil. Furthermore, Ro 15‐4513, an inverse agonist at wild‐type receptors, acts as an agonist at all mutant receptors. This conserved molecular switch can be exploited to identify the pharmacological significance of specific GABAA‐receptor subtypes in vivo.
The relationship between synonymous codon usage and protein structure
Authors: Tao Xie , Dafu Ding
Published: July 2002

The hypothesis that synonymous codon usage is related to protein three‐dimensional structure is examined by investigating the correlation between synonymous codon usage and protein secondary structure. All except two codons inE. colishow the same secondary structural preference for alpha‐helix, beta‐strand or coil as that of amino acids to be encoded by the respective codons, while 17 codons show secondary structural bias in mammalian proteins. The results indicate that there is no significant correlation between synonymous codon usage and protein secondary structure inE. coli, but there is a correlation in mammals. It could be deduced that synonymous codons carry much less structural information in prokaryotes than in eukaryotes due to their divergent evolutionary mechanism.
Rat liver mitochondria can hydrolyse thiamine pyrophosphate to thiamine monophosphate which can cross the mitochondrial membrane in a carrier‐mediated process
Authors: Barile Maria , Valenti Daniela , Brizio Carmen , Quagliariello Ernesto , Passarella Salvatore
Published: July 2002

We show here that TPP→TMP conversion can take place in rat liver mitochondria. This occurs via the novel, putative TPP pyrophosphatase localised in the mitochondrial matrix, as shown both by digitonin titration and by an HPLC enzyme assay carried out on the mitochondrial matrix fraction. Certain features of the reaction, including the substrate and pH dependence, are reported. Additional evidence is given that externally added TMP can cross the mitochondrial membrane in a manner consistent with the occurrence of a carrier‐mediated process. This can occur both via the TPP translocator and via a novel translocator, inhibited by CAT but different from the ADP/ATP carrier.
Two distinct classes of rat intestinal mucosal enzymes incorporating putrescine into protein
Authors: Tsai Yu-Hui , Lai Wen-Fu Thomas , Wu Yu-Wei , Johnson Leonard R
Published: July 2002

Tissue‐transglutaminase (t‐TGase) is a family of calcium‐dependent enzymes. A Ca2+‐independent soluble enzyme, in addition to t‐TGase, capable of incorporating polyamines into proteins was demonstrated in rat intestinal mucosa. The Ca2+‐independent enzyme was stimulated 2‐ to 5‐fold by Fe2+and Co2+ions but inhibited by Cu2+and Zn2+ions. The Ca2+‐stimulated t‐TGase activity was inhibited by divalent ions in the following order: Zn2+, Fe2+>Co2+>Cu2+. The opposite effects of EGTA, Fe2+and Co2+on these two enzyme activities indicate that they are two distinct classes of enzymes. Competition studies demonstrated differential preferences of the two enzymes for substrates. The Ca2+‐dependent enzyme preferred putrescine, monodansylcadaverine>cadaverine, spermidine, spermine>1,10‐diaminodecane>triethylbutylamine. On the other hand, the Ca2+‐independent enzyme preferred putrescine>cadaverine>spermine, 1,10‐diaminodecane>spermidine>monodansylcadaverine>triethylbutylamine. Further studies with divalent ions excluded the possible association of this novel Ca2+‐independent enzyme with diamine oxidase. Finally, the Ca2+‐independent enzyme had a higher affinity for putrescine (Km=0.02 mM) than did Ca2+‐dependent t‐TGase (0.2 mM). As judged by gel filtration on HiPrep Sephacryl 200 column, the Ca2+‐independent enzyme had a molecular weight of ∼48 kDa, the intestinal Ca2+‐dependent t‐TGase was about 188 kDa while that of testicular t‐TGase was about 96 kDa. In conclusion, the Ca2+‐independent enzyme is stimulated by cobalt or ferric ions, and selectively incorporates aliphatic diamines or polyamines with symmetric amino groups. The observed Ca2+‐independent enzyme activity is not related to diamine oxidase or its products. With a 10 times greater affinity for putrescine, the calcium‐independent, 48‐kDa intestinal enzyme may mediate polyamine function better than calcium dependent, 188‐kDa intestinal tissue transglutaminase in the intestinal mucosa.
Enhanced fMLP‐stimulated chemotaxis in human neutrophils from individuals carrying the G protein β3 subunit 825 T‐allele
Authors: Virchow Sebastian , Ansorge Nikolaus , Rübben Herbert , Siffert Gerlinde , Siffert Winfried
Published: July 2002

We have recently described a C825T polymorphism in the gene encoding for the Gβ3 subunit of heterotrimeric G proteins. The 825T allele is associated with a novel splice variant (Gβ3‐s) and enhanced signal transduction via pertussis toxin (PTX)‐sensitive G proteins. fMLP‐induced chemotaxis, but not O2− generation, was increased in neutrophils with the TC/TT (EC50=1.5±1.3 nM) genotypes compared to the CC genotype (EC50=5.9±1.5 nM). Maximal fMLP‐induced increase in [Ca2+]i was significantly reduced in neutrophils from individuals with TC/TT genotype vs. CC genotype (212.9±10.1 nM vs. 146.4±24.2 nM). Gβ3‐s appears to be associated with enhanced immune cell function in humans.