bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of calcium in the organization of fibrillin microfibrils
Authors: Kielty Cay M. , Shuttleworth C.Adrian
Published: July 2002

The microfibrillar glycoprotein fibrillin has a multidomain structure which contains forty‐three epidermal growth factor‐like motifs with calciumbinding consensus sequences. We have utilized intact microfibrils isolated from human dermal fibroblast cultures to investigate the putative influence of bound calcium on microfibrillar organization and integrity. Incubation with EDTA or EGTA rapidly resulted in gross disruption of microfibril morphology. The treatment induced disorganization of the interbead domains although the regular beaded arrangement was always apparent. These changes were readily reversible on replacing calcium, indicating that the treatment had not compromised microfibrillar integrity. The data localize calcium binding EGF‐like repeats to the interbead domains and indicate that lateral packing of fibrillin monomers is calcium‐dependent. This arrangement suggests how mutations in epidermal growth factor‐like domains of fibrillin might cause the disruption in microfibril organization and interactions which underlies the clinical symptoms of some Marian syndrome patients.
A ubiquityl‐calmodulin synthetase that effectively recognizes the Ca2+‐free form of calmodulin
Authors: Majetschak M. , Laub M. , Jennissen H.P.
Published: July 2002

Ubiquityl‐calmodulin synthetase (uCaM‐synthetase) activity as detected in reticulocyte lysate and the crude extracts of rabbit tissues [FEBS Lett. 294 (1991) 229‐233] has been well characterized as being essentially Ca2+‐dependent (−Ca2+/+Ca2+ activity ratio: 0.15–0.2). However, during the purification of this enzyme on ubiquitin‐Sepharose the Ca2+‐dependent activity is lost and an essentially Ca2+‐independent enzyme (−Ca2++Ca2+ activity ratio: 1.0–1.5) is obtained which was purified 90‐fold (uCaM‐Syn Fl) to a final specific activity of 0.32 pkat/mg. During the purification procedure a second protein factor (uCaM‐Syn F2) was isolated that has no catalytic activity by itself but restores Ca2+ dependence to the uCaM‐Syn Fl fraction (−Ca2++Ca2+ activity ratio: 0.1) and enhances the catalytic activity in uCaM‐Syn Fl in the presence of Ca2+ over 40‐fold. It is concluded that several (possibly interdependent) forms of uCaM‐synthetase exist which display different substrate specificities for calmodulin.
Expression of PP2A B regulatory subunit β isotype in rat testis
Authors: Hatano Yoshiaki , Shima Hiroshi , Haneji Tatsuji , Miura Akira B. , Sugimura Takashi , Nagao Minako
Published: July 2002

We isolated a rat cDNA encoding part of the β‐isotype of the B regulatory subunit (BRß) of protein phosphatase 2A (PP2A). The isolated cDNA encoded the region corresponding to amino acids positions 8(R) to 177(N) of human BRß. The identities of the nucleotide and amino acid sequences of the rat and human BRßs were 95.7% and 100%, respectively. The BRß mRNA was specifically expressed in rat brain and testis, the lengths of mRNAs in these two organs being different. In the testis, the BRß mRNA was first detected 40 days after birth, increasing gradually thereafter, and was expressed specifically in elongated spermatids, while mRNA of the α‐isotype (BRα) was expressed equally in all spermatogenic cells. After meiosis, round spermatids change morphologically to elongated spermatids. BRß may regulate the activity of the PP2A catalytic subunit in spermatids, and be involved in spermatogenic maturation, especially spermatid elongation.
A study of branched‐chain amino acid aminotransferase and isolation of mutations affecting the catabolism of branched‐chain amino acids in Saccharomyces cerevisiae
Authors: Dickinson J.Richard , Norte Valia
Published: July 2002

The specific activity of branched‐chain amino acid aminotransferase was highest when S. cerevisiae was grown in minimal medium containing a branched‐chain amino acid as nitrogen source. Growth in complex media with glycerol or ethanol gave moderately high levels, whereas with glucose and fructose the specific activity was very low. Mutagenesis defined three genes (BAA1 to BAA3) required for branched‐chain amino acid catabolism. The baal mutation reduced the specific activity of the aminotransferase, the stationary phase density in YEPD and caused gross morphological disturbance. Branched‐chain amino acid aminotransferase is essential for sporulation.
Cloning of cDNA for granzyme‐like protein III, a novel serine proteinase from rat duodenum
Authors: Grigorenko Vitaly G. , Yarovoi Serge V. , Paulauskaite Ramune , Amerik Alexander Yu.
Published: July 2002

We previously reported cloning of cDNAs which encode two granzyme‐like serine proteinases (GLP I and GLP II) from rat duodenum. In this paper we present the cDNA sequence for a novel member of the granzyme‐like protein family from rat duodenum, GLP III. The amino acid sequence deduced from the cDNA consists of 248 residues and shows 88.2% identity to GLP I and 50.6% identity to GLP II. Comparison of the amino acid sequence of GLP III with sequences of related proteinases reveals the location of the catalytic amino acid triad and enables the prediction of the substrate specificity. Despite close similarity to GLP I, GLP III is expected to demonstrate different substrate specificity due to a substitution of the Arg residue by Glu at the critical position. Northern blot analysis demonstrates that the GLP III transcript is present only in duodenum.
Cost‐benefit analysis of laparoscopic adnexectomy
Authors: Tintara H. , Leetanaporn R.
Published: July 2002

Objective: To determine the economic feasibility (from the patient's viewpoint) of laparoscopic adnexectomy (LA) in a developing country. Method: Forty‐four LA cases of benign ovarian cysts or tubal pregnancy were compared with 44 conventional open adnexectomy (OA) cases treated during the same period and matched by diagnosis, procedure and degree of difficulty. Result: The postoperative hospital stay was 1.3 ± 0.6 days (mean ± S.D.) and 5.3 ± 1.1 days (P < 0.001) for the LA and OA groups, respectively. LA cases had a shorter recovery period (5.8 ± 2.2 days vs. 27.2 ± 6.6 days, P < 0.001), but higher hospital charges (US$463.3 ± 84.9 vs. US$229.8 ± 92.2, P < 0.001), caused mainly (89.2%) by disposable laparoscopic instruments. Incremental cost‐benefit analysis revealed a possible saving with LA for higher income patients (US$9.2 per day). Conclusions: LA in Thailand may have economic benefit only to the higher income patients. Benefits to other patients in developing countries will depend on future cost reductions of disposable instruments and/or more effective use of reusable instruments.
Taurine deficiency in dissociated mouse cerebellar cultures affects neuronal migration
Authors: Maar Thomas , Morán Julio , Schousboe Arne , Pasantes‐Morales Herminia
Published: July 2002

The role of taurine in the process of neuronal migration was studied in a microwell cell culture system. Immunocytochemical analysis of the cellular composition of this culture system revealed the presence of the astrocytic marker GFAP in some structures such as the aggregates of neuronal bodies and in those cables used for migration, resembling what is described in vivo. The neuronal marker γ‐enolase stained practically all structures, including the aggregates and all cables. The intracellular taurine concentration was reduced by 60% in mouse cerebellar granule cells treated with a blocker of taurine transport, guanidinoethane sulfonate (GES). Under these conditions cell migration was markedly reduced to approximately 50% of that in untrated cultures. Both, taurine depletion and impairment of cell migration induced by GES were prevented by adding taurine to the culture medium. Taurine deficiency similarly affected different morphological parameters such as the number of cables suitable for neuronal migration as well as the number of migrating neurons. The number of aggregates of neuronal bodies was significantly increased, by about 30%, as a consequence of the reduced migration. Taurine alone did not exert any effect on the parameters evaluated. GES treatment of granule cells did not affect mitochondrial metabolism or K+‐stimulated Ca2+‐dependent [3H]‐d‐aspartate release. This suggests that the described effects of taurine deficiency were not due to an alteration of neuronal viability and that the action of GES was not simply due to unspecific and deleterious effects. These results are in agreement with those obtained in in vivo studies. This approach represents a useful model to investigate the role played by taurine in the process of neuronal migration.
Structural models for carcinoembryonic antigen and its complex with the single‐chain Fv antibody molecule MFE23
Authors: Boehm Mark K. , Perkins Stephen J.
Published: July 2002

MFE23 is a single chain Fv antibody that has a high affinity for carcinoembryonic antigen (CEA). A full homology model for CEA based on V‐type, I‐type and C2‐type immunoglobulin folds, 28 oligosaccharides and the interdomain angle of CD2 was validated using solution scattering data. The superimposition of the intermolecular contacts observed in our recent crystal structure of MFE23 with the N‐terminal domain of CEA permitted the MFE23–CEA complex to be modelled. Good surface and electrostatic complementarity and carbohydrate‐unhindered access of MFE23 with the indentation between the first two CEA domains was observed. The model is supported by biochemical data and provides insight on the high affinity of MFE23 for CEA.
Genomic Exploration of the Hemiascomycetous Yeasts: 4. The genome of Saccharomyces cerevisiae revisited
Authors: Blandin Gaëlle , Durrens Pascal , Tekaia Fredj , Aigle Michel , Bolotin-Fukuhara Monique , Bon Elisabeth , Casarégola Serge , de Montigny Jacky , Gaillardin Claude , Lépingle Andrée , Llorente Bertrand , Malpertuy Alain , Neuvéglise Cécile , Ozier-Kalogeropoulos Odile , Perrin Arnaud , Potier Serge , Souciet Jean-Luc , Talla Emmanuel , Toffano-Nioche Claire , Wésolowski-Louvel Micheline , Marck Christian , Dujon Bernard
Published: July 2002

Since its completion more than 4 years ago, the sequence of Saccharomyces cerevisiae has been extensively used and studied. The original sequence has received a few corrections, and the identification of genes has been completed, thanks in particular to transcriptome analyses and to specialized studies on introns, tRNA genes, transposons or multigene families. In order to undertake the extensive comparative sequence analysis of this program, we have entirely revisited the S. cerevisiae sequence using the same criteria for all 16 chromosomes and taking into account publicly available annotations for genes and elements that cannot be predicted. Comparison with the other yeast species of this program indicates the existence of 50 novel genes in segments previously considered as ‘intergenic’ and suggests extensions for 26 of the previously annotated genes.
Functions of WW domains in the nucleus
Authors: Sudol Marius , Sliwa Krzysztof , Russo Tommaso
Published: July 2002

The WW domain is a protein module found in a wide range of signaling proteins. It is one of the smallest protein modules that folds as a monomer without disulfide bridges or cofactors. WW domains bind proteins containing short linear peptide motifs that are proline‐rich or contain at least one proline. Although the WW domain was initially considered a ‘cytoplasmic module’, the proteins containing WW domains have also been localized in the cell nucleus. Moreover, these proteins have been documented to participate in co‐activation of transcription and modulation of RNA polymerase II activity. The carboxy‐terminal domain (CTD) of RNA polymerase II acts as an assembly platform for distinct WW domain‐containing proteins that affect the function of the RNA polymerase II. The formation of complexes between CTD and WW domain‐containing proteins is regulated by phosphorylation of the CTD. Since the CTD sequence is highly repetitive and a target of several post‐translational modifications and conformational changes, it presents a unique structure capable of enormous molecular diversity. The WW domain has been implicated in several human diseases including Alzheimer's disease. The WW domain‐containing iso‐prolyl isomerase named Pin1, a protein known to be essential for cell cycle progression, was shown to be active in restoration of the microtubule‐binding activity of Tau, a protein of neurofibrillar tangles found in the brains of Alzheimer's patients. It is the WW domain of Pin1 that interacts directly with Tau protein. In addition, the WW domain‐containing adapter protein FE65 was shown to regulate processing of Alzheimer's amyloid precursor protein. It is expected that by understanding the details of the WW domain‐mediated protein–protein interactions, we will be able to illuminate numerous signaling pathways which control certain aspects of transcription and cell cycle.
The second cysteine‐rich domain of Mac1p is a potent transactivator that modulates DNA binding efficiency and functionality of the protein
Authors: Voutsina Alexandra , Fragiadakis George S , Boutla Alexandra , Alexandraki Despina
Published: July 2002

Mac1p is a Saccharomyces cerevisiae DNA binding transcription factor that activates genes involved in copper uptake. A copper‐induced N–C‐terminal intramolecular interaction and copper‐independent homodimerization affect its function. Here, we present a functional analysis of Mac1p deletion derivatives that attributes new roles to the second cysteine‐rich (REPII) domain of the protein. This domain exhibits the copper‐responsive potent transactivation function when assayed independently and, in the context of the entire protein, modulates the efficiency of Mac1p binding to DNA. The efficiency of binding to both copper‐response promoter elements can determine the in vivo functionality of Mac1p independent of homodimerization.
Role of the terminal repeat GAGC trimer, the major Rep78 binding site, in adeno‐associated virus DNA replication
Authors: Bishop B.M. , Santin A.D. , Quirk J.G. , Hermonat P.L.
Published: July 2002

The adeno‐associated virus (AAV) terminal repeats (TR) are cis required, and the AAV encoded Rep78 protein is trans required, for AAV DNA replication. The Rep78 protein recognizes and interacts with at least three regions within the TR DNA. The major binding site, with the highest affinity for Rep78 binding, is within the TR stem (nt 36‐16) and includes the ‘core’ GAGC trimer (GAGC3, nt 33‐22; Fig. 2) sequence. In this study mutations were made within the GAGC trimer and these mutants assayed for their ability to allow for AAV double stranded (ds DNA, prepackaging DNA replication), and single stranded DNA (ss DNA, due to virion packaging) replication. Here, it is shown that when the two inside GAGC motifs are mutated, with only motif no. 1 left intact (see Fig. 2), the resulting AAV (mutA) genome was significantly defective for both ds DNA (17% of wild type) and ss DNA (9%). If the TRs contained only the two outside motifs intact (mutB), motifs no. 1 and 2, the AAV genome had a significant but reduced level of both ds (50%) and ss (34%) DNA replication. Finally, if only the middle motif no. 2 was mutated, with motifs no. 1 and 3 left intact (mutC), the resulting DNA replication for both ds and ss forms was essentially wild type (80% that of wild type). These data suggest that the GAGC trimer plays a role in AAV DNA replication, and that GAGC motif no. 3 is the most important of the three motifs for both ds and ss DNA replication.
Coexpression of a human P450 (CYP3A4) and P450 reductase generates a highly functional monooxygenase system in Escherichia coli
Authors: Blake Jennifer A.R. , Pritchard Mike , Ding Shaohong , Smith Graeme C.M. , Burchell Brian , Wolf C.Roland , Friedberg Thomas
Published: July 2002

The catalytic activities of recombinant cytochrome P450s expressed in E. coli have been impeded by the absence of endogenous P450 reductase. To solve this problem, we coexpressed P450 reductase with CYP3A4. Membranes from this strain contained 215 pmol P450/mg protein and a reductase activity of 1315 nmol cytochrome c reduced/min per mg. We detected 6β‐hydroxylation of testosterone and oxidation of nifedipine in vivo with turnover numbers of 15.2 and 17.3 min−1, respectively. These values compare favourably with those obtained using an optimally reconstituted system. Our data demonstrate that a catalytically efficient human P450 system can be generated in E. coli.
Purification of IpaC, a protein involved in entry of Shigella flexneri into epithelial cells and characterization of its interaction with lipid membranes
Authors: De Geyter Carine , Vogt Bas , Benjelloun-Touimi Zineb , Sansonetti Philippe J , Ruysschaert Jean-Marie , Parsot Claude , Cabiaux Véronique
Published: July 2002

Entry of Shigella flexneri into epithelial cells and lysis of the phagosome involve the secreted IpaA–D proteins. A complex containing IpaC and IpaB is able to promote uptake of inert particles by epithelial cells. This suggested that Ipa proteins, either individually or as a complex, might interact with the cell membrane. We have purified IpaC and demonstrated its interaction with lipid vesicles. This interaction is modulated by the pH, which might be relevant to the dual role of Ipa proteins, in induction of membrane ruffles upon entry and lysis of the endosome membrane thereafter.
Secretion and binding of HMG1 protein to the external surface of the membrane are required for murine erythroleukemia cell differentiation
Authors: Passalacqua Mario , Zicca Antonio , Sparatore Bianca , Patrone Mauro , Melloni Edon , Pontremoli Sandro
Published: July 2002

We show here that murine erythroleukemia (MEL) cells, following induction with hexamethylene bisacetamide, accumulate high mobility group (HMG)1 protein onto the external surface of the cell in a membrane‐associated form detectable by immunostaining with a specific anti‐HMG1 protein antibody. This association is maximal at a time corresponding to cell commitment. At longer times, immunostainable cells are progressively reduced and become almost completely undetectable along with the appearance of hemoglobin molecules. Binding to MEL cells does not affect the native molecular structure of HMG1 protein. The type of functional correlation between HMG1 protein and MEL cell differentiation is suggested by the observation that if an anti‐HMG1 protein antibody is added at the same time of the inducer almost complete inhibition of cell differentiation is observed, whereas if the antibody is added within the time period in which cells undergo through irreversible commitment, inhibition progressively disappears. A correlation between MEL cell commitment and the biological effect of HMG1 protein can thus be consistently suggested.
Overlapping distribution of KATP channel‐forming Kir6.2 subunit and the sulfonylurea receptor SUR1 in rodent brain
Authors: Karschin Christine , Ecke Claudia , Ashcroft Frances M , Karschin Andreas
Published: July 2002

ATP‐sensitive K+ channels comprise a complex of at least two proteins: a member of the inwardly rectifying Kir6 family (e.g. Kir6.2) and a sulphonylurea receptor (e.g. SUR1) which belongs to the ATP‐binding cassette (ABC) superfamily. Using specific radiolabeled antisense oligonucleotides, the cellular localization of both mRNAs was investigated in the rodent brain by in situ hybridization. The distribution of both transcripts was widespread throughout the brain and showed a high degree of overlap with peak expression levels in the hippocampus, neocortex, olfactory bulb, cerebellum, and several distinct nuclei of the midbrain and brainstem, indicating their important role in vital brain function.
Laccase is essential for lignin degradation by the white‐rot fungus Pycnoporus cinnabarinus
Authors: Eggert Claudia , Temp Ulrike , Eriksson Karl-Erik L.
Published: July 2002

The white‐rot fungus, Pycnoporus cinnabarinus, provides an excellent model organism to elucidate the controversial role of laccase in lignin degradation. P. cinnabarinus produces laccase in one isoform as the predominant phenoloxidase in ligninolytic cultures, and neither LiP nor MnP are secreted. Yet, P. cinnabarinus degrades lignin very efficiently. In the present work, we show that laccase‐less mutants of P. cinnabarinus were greatly reduced in their ability to metabolize 14C ring‐labeled DHP. However, 14CO2 evolution in these mutant cultures could be restored to levels comparable to those of the wild‐type cultures by addition of purified P. cinnabarinus laccase. This clearly indicates that laccase is absolutely essential for lignin degradation by P. cinnabarinus.
Autocatalytic processes in cooperative mechanisms of prion diseases
Authors: Laurent Michel
Published: July 2002

According to the leading theory, the agent responsible for prion diseases would be the conformational isomer PrPSc of a cellular protein PrPC, the pathogenic form PrPSc multiplying by converting the normal protein into a likeness of itself. The pathogenic isoform could catalyze the conformational transition so that the process, taken as a whole, is autocatalytic. However, in this simple but atypic model, unrealistic values of rate parameters are needed in order to account for the kinetics of the propagation of prion diseases. In this paper, I show that these limits can be overcome by assuming that catalysis proceeds through a multimeric assembly of the pathogenic isoform of the prion protein. Such a structure would indeed be able to provide cooperativity both at the assembly and conformational change levels, strongly reinforcing the autocatalytic character of the activated process. Moreover, such a property is a prerequisite to endow the metabolic system with dynamic bistability. Together with a good agreement regarding experimental data, this analysis is closely akin to Griffith's original idea concerning the thermodynamic conditions required for autocatalyzed modifications of any protein.
Oxygen‐dependent regulation of the respiration and growth of Escherichia coli by nitric oxide
Authors: Yu Hidenori , Sato Eisuke F , Nagata Kumiko , Nishikawa Manabu , Kashiba Misato , Arakawa Tetsuo , Kobayashi Kenzo , Tamura Toshihide , Inoue Masayasu
Published: July 2002

To elucidate the role of nitric oxide (NO) in the metabolisms of enteric bacteria, its effect on the respiration and growth of Escherichia coli was examined. Respiration of E. coli was reversibly inhibited by NO particularly under low oxygen tensions. Growth of E. coli was also inhibited by NO more strongly under low oxygen tension than at its high concentration. Because the intestinal lumen is anaerobic, even a small amount of NO might strongly inhibit the energy metabolism and growth of E. coli and other enteric bacteria in vivo than in air atmospheric conditions in which oxygen tension is unphysiologically high.
JC‐1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess ΔΨ changes in intact cells: implications for studies on mitochondrial functionality during apoptosis
Authors: Salvioli Stefano , Ardizzoni Andrea , Franceschi Claudio , Cossarizza Andrea
Published: July 2002

The sensitivity and specificity of three fluorescent probes used for cytofluorimetric analysis of mitochondrial membrane potential (ΔΨ) were studied in the U937 human cell line. First, the role of plasmamembrane in influencing the binding of the probes to mitochondria has been investigated. The depolarization of plasmamembrane with high doses of extracellular KCl had no immediate effects on the loading of JC‐1, DiOC6(3) and rhodamine 123 (R123). However, after a few hours of culture in the presence of KCl, significant changes were observed only in cells stained with DiOC6(3). Second, a comparative study was performed concerning the effects of agents capable of collapsing ΔΨ. While adding FCCP to cell cultures resulted in consistent changes in the fluorescence emission of both JC‐1 and DiOC6(3) – but not of R123 – only cells stained with JC‐1 responded to valinomycin. On the whole, our data indicate that JC‐1 is a reliable probe for analyzing ΔΨ changes with flow cytometry, while the others show a lower sensitivity (R123), or a non‐coherent behaviour, due to a high sensitivity to changes in plasmamembrane potential [DiOC6(3)]. These data cast some doubts on those studies that, using fluorescent probes that have a low sensitivity to ΔΨ, hypothesized that the fall in ΔΨ is one of the early events, if not one of the main causes, of apoptosis.
Isolation and characterization of a neomycin‐resistant mutant of Methanobacterium thermoautotrophicum with a lesion in Na+‐translocating ATPase (synthase)
Authors: Šmigáň Peter , Polák Peter , Majernı́k Alan , Greksák Miloslav
Published: July 2002

A mutant of Methanobacterium thermoautotrophicum with a lesion in membrane Na+‐translocating ATPase (synthase) was isolated. The total ATPase activity in permeabilized cells of this mutant was elevated three‐fold as compared with the wild‐type strain. In contrast to wild‐type cells, mutant ATPase was neither inhibited by DCCD nor stimulated by Na+ ions. The methane formation rate of the mutant cells at pH 7.5 under non‐growing conditions was nearly twice that of the wild‐type strain and was stimulated by sodium ions. On the other hand, the ATP synthesis driven by methanogenesis under the same conditions was lower that of the wild‐type under the same conditions, and contrary to the wild‐type was not stimulated by Na+ ions. ATP synthesis driven by a potassium diffusion potential in the presence of sodium ions was markedly diminished in the mutant cells. The membrane potential values of the wild‐type and the mutant cells in the presence of 10 mM NaCl at pH 7.0 were comparable at energized conditions (−223 mV and −230 mV respectively). The Mg2+‐dependent ATPase activity of the 105×g supernatant of broken cells from the mutant cells was 30% higher than in the wild‐type. On the other hand, two bands with Mg2+‐dependent ATPase activity were identified by native PAGE in this fraction in both wild‐type as well as in mutant. These data suggest that the binding of Na+‐translocating ATPase (synthase) to the membrane spanning part is changed in the mutant strain.
Serpin‐like properties of α1‐antitrypsin Portland towards furin convertase
Authors: Dufour Erick K , Denault Jean-Bernard , Hopkins Paul C.R , Leduc Richard
Published: July 2002

Recent studies have demonstrated that a serpin variant, α1‐antitrypsin Portland (AT‐PDX), can inhibit the mammalian convertase furin. Here, we examine the mechanism by which this inhibition takes place. We find that furin, which does not belong to the trypsin‐like serine protease family, the usual targets of serpins, forms an SDS‐heat denaturation‐resistant complex with AT‐PDX both in vitro and in vivo. AT‐PDX inhibited furin with an association rate constant (k
ass) of 1.5×106 M−1 s−1 which is similar to k
ass values reported for serpins with trypsin‐like enzymes. These results illustrate that AT can be modified to act essentially as a suicide inhibitor of furin, an enzyme of the subtilase superfamily of serine proteases.
Following G‐quartet formation by UV‐spectroscopy
Authors: Mergny Jean-Louis , Phan Anh-Tuan , Lacroix Laurent
Published: July 2002

Oligodeoxynucleotides which include stretches of guanines form a well‐known tetrameric structure. We show that the recording of reversible absorbance changes at 295 nm allows to precisely monitor intramolecular guanine (G)‐quartet formation and dissociation. Accurate T
m and thermodynamic values could be easily extracted from the data, whereas classical recordings at 260 nm led to a much larger uncertainty and in extreme cases, to completely inaccurate measurements. This inverted denaturation profile was observed for all G‐quartet‐forming oligonucleotides studied so far. This technique is very useful in all cases where intramolecular or intermolecular quadruplex formation is suspected.
Human immunodeficiency virus type 1 Vif‐derived peptides inhibit the viral protease and arrest virus production
Authors: Baraz Lea , Friedler Assaf , Blumenzweig Immanuel , Nussinuv Orna , Chen Nissim , Steinitz Michael , Gilon Chaim , Kotler Moshe
Published: July 2002

Human immunodeficiency virus type 1 (HIV‐1) Vif protein is required for productive HIV‐1 infection of peripheral blood lymphocytes and macrophages in cell culture and for pathogenesis in the SCID‐hu mouse model of HIV‐1 infection. Vif inhibits the viral protease (PR)‐dependent autoprocessing of truncated HIV‐1 Gag‐Pol precursors expressed in bacterial cells and efficiently inhibits the PR‐mediated hydrolysis of peptides in cell‐free systems. The obstructive activity of Vif has been assigned to the 92 amino acids residing at its N′‐terminus (N‐Vif). To determine the minimal Vif sequence required to inhibit PR, we synthesized overlapping peptides derived from N‐Vif. These peptides were then assessed, using two in vitro and two in vivo systems: (i) inhibition of purified PR, (ii) binding of PR, (iii) inhibition of the autoprocessing of the Gag‐Pol polyprotein expressed by a vaccinia virus vector, and (iv) inhibition of mature virus production in human cells. The peptides derived from two regions of N‐Vif encompassing residues Tyr‐30–Val‐65 and Asp‐78–Val‐98, inhibited PR activity in both the in vitro and the in vivo assays. Thus, these peptides can be used as lead compounds to design new PR inhibitors.
Discovery of a receptor related to the galanin receptors
Authors: Lee Dennis K , Nguyen Tuan , O'Neill Gary P , Cheng Regina , Liu Yang , Howard Andrew D , Coulombe Nathalie , Tan Carina P , Tang-Nguyen Anh-Thi , George Susan R , O'Dowd Brian F
Published: July 2002

We report the isolation of a cDNA clone named GPR54, which encodes a novel G protein‐coupled receptor (GPCR). A PCR search of rat brain cDNA retrieved a clone partially encoding a GPCR. In a library screening this clone was used to isolate a cDNA with an open reading frame (ORF) encoding a receptor of 396 amino acids long which shared significant identities in the transmembrane regions with rat galanin receptors GalR1 (45%), GalR3 (45%) and GalR2 (44%). Northern blot and in situ hybridization analyses revealed that GPR54 is expressed in brain regions (pons, midbrain, thalamus, hypothalamus, hippocampus, amygdala, cortex, frontal cortex, and striatum) as well as peripheral regions (liver and intestine). In COS cell expression of GPR54 no specific binding was observed for 125I‐galanin. A recent BLAST search with the rat GPR54 ORF nucleotide sequence recovered the human orthologue of GPR54 in a 3.5 Mb contig localized to chromosome 19p13.3.
A 45 amino acid residue domain necessary and sufficient for proteolytic cleavage of the MAP1B polyprotein precursor
Authors: Tögel Martin , Eichinger René , Wiche Gerhard , Propst Friedrich
Published: July 2002

The microtubule‐associated proteins 1B and 1A are synthesized as polyprotein precursors which are rapidly cleaved to give rise to heavy and light chains constituting the respective microtubule‐associated protein 1B or microtubule‐associated protein 1A complex. To identify domains necessary for precursor processing, we expressed microtubule‐associated protein 1B deletion mutants in fibroblasts and monitored proteolytic cleavage of the precursor proteins by immunoblot analysis. We found that a novel hydrophilic, proline‐rich 45 amino acid domain containing the cleavage site is necessary and sufficient for processing. This domain is conserved in microtubule‐associated protein 1A. Additional sequences in the N‐terminal half of the heavy chain contribute to the efficiency of cleavage.
Transformation of rat fibroblasts by phospholipase C‐γ1 overexpression is accompanied by tyrosine dephosphorylation of paxillin
Authors: Chang Jong-Soo , Iwashita Shintaro , Lee Young Han , Kim Myung Jong , Ryu Sung Ho , Suh Pann-Ghill
Published: July 2002

We previously have shown that the overexpression of phospholipase C‐γ1 (PLC‐γ1) in rat 3Y1 fibroblasts results in malignant transformation (Chang, J.‐S., Noh, D.Y., Park, I.A., Kim, M.J., Song, H., Ryu, S.H. and Suh, P.‐G. (1997) Cancer Res. 57, 5465–5468). The transformed cells, which initially are in an elongated and flat form after seeding in plastic dishes, become rounded during continued culture. We found that tyrosine dephosphorylation of paxillin accompanies this morphological change of the transformed cells and that PLC‐γ1 co‐immunoprecipitates together with paxillin and vice versa, but not after the cells have become round. Transformed cells growing on fibronectin‐pre‐coated dishes regain their flat morphology and this is accompanied by paxillin tyrosine phosphorylation. Furthermore, immunoprecipitation analysis showed that paxillin forms a heteromeric complex with PLC‐γ1 in cells grown on fibronectin. These results suggest that a complex formation between paxillin and PLC‐γ1 may play a role in cell‐substrate adhesion.
Identification and characterization of the RNA helicase activity of Japanese encephalitis virus NS3 protein
Authors: Utama Andi , Shimizu Hiroyuki , Morikawa Shigeru , Hasebe Futoshi , Morita Kouichi , Igarashi Akira , Hatsu Masahiro , Takamizawa Kazuhiro , Miyamura Tatsuo
Published: July 2002

The NS3 protein of Japanese encephalitis virus (JEV) contains motifs typical of RNA helicase/NTPase but no RNA helicase activity has been reported for this protein. To identify and characterize the RNA helicase activity of JEV NS3, a truncated form of the protein with a His‐tag was expressed in Escherichia coli and purified. The purified JEV NS3 protein showed an RNA helicase activity, which was dependent on divalent cations and ATP. An Asp‐285‐to‐Ala substitution in motif II of the JEV NS3 protein abolished the ATPase and RNA helicase activities. These results indicate that the C‐terminal 457 residues are sufficient to exhibit the RNA helicase activity of JEV NS3.
Computerized analysis of fetal heart rate parameters by gestational age
Authors: Park M.I , Hwang J.H , Cha K.J , Park Y.S , Koh S.K
Published: July 2002

Objective: The aim of this study is to define the reference ranges for fetal heart rate (FHR) parameters according to gestational age, by determining the relationship between the FHR and gestational age using a computerized FHR analysis system. Methods: Using our own software developed by Hanyang University Hospital in Korea, non‐stress tests were performed for 20 min. FHR parameters for 6455 subjects were analyzed for various gestational groups; <25 weeks, 25–28 weeks, 29–32 weeks, 33–36 weeks, 37–40 weeks, and >40 weeks. Results. The FHR parameters were related to gestational age. The mean baseline FHR, signal loss, and fetal movements decreased significantly with gestation (P<0.0001). The variability and accelerations of FHR were highest for the 37–40 weeks gestational group (P<0.05). Conclusions: The results showed that overall, the differences in the FHR parameters between gestational groups were statistically significant, and the gestational age of the fetus should be considered when interpreting FHR patterns.
Sexually transmitted diseases: global importance
Authors: Sciarra J.J.
Published: July 2002

The worldwide incidence of sexually transmitted diseases (STDs) including the human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) continues to increase and represents a major public health problem especially in the developing world causing infertility and ectopic pregnancy. Prevention of the spread of STDs, along with early detection and appropriate therapy has the potential to minimize the reproductive tract damage of these infections and simultaneously improve the health of women in all areas of the world.
The FIGO study group on women's sexual and reproductive rights
Authors: Cook R.J. , Dickens B.M.
Published: July 2002

In October 1998, the FIGO Executive Board established a Study Group on Women's Sexual and Reproductive Rights to develop specific details regarding observance, enforcement and advancement. At its inaugural meeting in April 1999 the Study Group reviewed international and national progress in respect of such rights. International initiatives had achieved considerable progress towards specification and monitoring of rights, particularly through the committee receiving reports of national compliance under the Convention on the Elimination of All Forms of Discrimination Against Women. National progress was more mixed. While some countries had improved women's sexual and reproductive rights, many had not, and rights in some had regressed. The Study Group considered actions available to FIGO member societies to advance rights in their countries, how advancement of rights might be pursued in countries in general, and initiatives that FIGO itself might undertake and facilitate to protect and promote women's sexual and reproductive rights.
Development of tensile strength during distraction osteogenesis in a rat model
Authors: Aronson J. , Hogue W. R. , Flahiff C. M. , Gao G. G. , Shen X. C. , Skinner R. A. , Badger T. M. , Lumpkin C. K.
Published: July 2002

These studies were designed to determine the reliability of in vitro tensile testing to measure the temporal development of regenerate bone strength in rats during limb lengthening (distraction osteogenesis, DO). External fixators were placed on the right tibiae of 36 virus‐free, 400–450 g male Sprague Dawley rats, and osteotomies (n = 33) were performed. Distraction was initiated the following morning (0 day latency) at 0.4 mm/day and continued to day 20. The 8 mm gap was allowed to consolidate for up to 50 days (day 70 postop). Contralateral unoperated and operated (fixator only) controls were included. On days 20, 30, 50 and 70 postop, the rats were anesthetized, and their tibiae were radiographed prior to undergoing sacrifice for histological or tensile analysis. On day 70, an additional group was tested by three‐point bending. Radiodensity measurements demonstrated progressive mineralization of the DO gap, and histology confirmed typical intramembranous ossification of collagen bundles oriented parallel to the distraction force. Tensile stiffness increased significantly between days 20 and 30 postop, this increase correlated with initial radiographic and histologic bridging of the DO gap. Energy to failure and ultimate tensile strength increased progressively to day 70. At day 70, the force to failure for three‐point bending was 65% of control tibiae. In conclusion, in vitro tensile testing provides a reliable method to test the development of structural integrity during the early stages of DO. Therefore, the biomechanical effects of postulated modulators of bone repair can be measured during early stages (bone formation, bridging, early consolidation) of DO in a rat model. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Cytokine regulation of embryonic rat dopamine and serotonin neuronal survival in vitro
Authors: Jarskog L.Fredrik , Xiao Hong , Wilkie Mary Beth , Lauder Jean M. , Gilmore John H.
Published: July 2002

Interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), and tumor necrosis factor‐α (TNF‐α) are cytokines with pleiotropic effects in the central nervous system (CNS), including an emerging role in neurodevelopment. This study measured the effects of cytokines on the survival of tyrosine hydroxylase (TH) immunoreactive dopamine neurons from the substantia nigra (SN), and 5‐hydroxytryptamine (5‐HT) immunoreactive serotonin neurons from the rostral raphe (RR), using cultures from embryonic day 14 (E14) rat brain. IL‐1β, IL‐6, and TNF‐α were added to cell cultures at 1, 10 and 100 U/ml. After 3 days in vitro, TH and 5‐HT neurons were counted. The survival of 5‐HT neurons was significantly reduced by 20–30% at 10 U/ml of IL‐6. IL‐1β and TNF‐α at doses of 1 and 10 U/ml appeared to have a similar effect on the survival of these neurons, but this effect was not statistically significant. Comparable non‐significant reductions of survival also occurred for TH neurons at the lower doses of IL‐6 and TNF‐α. In separate experiments, SN and RR cultures were exposed to the cytokines at a higher dose (1000 U/ml), causing a significant 30–40% decrease in the survival of TH neurons, but little or no change in 5‐HT neuronal survival. Taken together, these results show that IL‐1β, IL‐6, and TNF‐α can affect developing monoamine neurons at physiologically relevant concentrations, and that high doses differentially inhibit the survival of TH and 5‐HT neurons after short exposures.
Seasonal influences on admissions for affective disorder and schizophrenia in Ireland: a comparison of first and readmissions
Authors: Clarke M. , Moran P. , Keogh F. , Morris M. , Kinsella A. , Larkin C. , Walsh D. , O'Callaghan E.
Published: July 2002

Although genetic and environmental factors operating before or around the time of birth have been demonstrated to be relevant to the aetiology of the major psychoses, a seasonal variation in the rates of admission of such patients has long been recognised. Few studies have compared first and readmissions. This study examined for seasonal variation of admission in the major psychoses, and compared diagnostic categories by admission status. Patients admitted to Irish psychiatric inpatient facilities between 1989 and 1994 with an ICD-9/10 diagnosis of schizophrenia or affective disorder were identified from the National Psychiatric Inpatient Reporting System (NPIRS). The data were analysed using a hierarchical log linear model, the chi-square test, a Kolmogorov-Smirnov (KS) type statistic, and the method of Walter and Elwood. The hierarchical log linear model demonstrated significant interactions between the month of admission and admission order (change in scaled deviance 28.77, df = 11, P < 0.003). Both first admissions with mania, and readmissions with bipolar affective disorder exhibited significant seasonality. In contrast, only first admissions with schizophrenia showed significant seasonal effects. Although first admissions with mania and readmissions with bipolar disorder both show seasonality, seasonal influences appear to be more relevant to onset of schizophrenia than subsequent relapse.
Associations Between Smoking and Systemic Lupus Erythematosus–Related Cytokines and Chemokines Among US Female Nurses
Authors: Hahn Jill , Leatherwood Cianna , Malspeis Susan , Liu Xinyi , Lu Bing , Roberts Andrea L. , Sparks Jeffrey A. , Karlson Elizabeth W. , Feldman Candace H. , Munroe Melissa E. , James Judith A. , Kubzansky Laura D. , Costenbader Karen H.
Published: July 2020

Objective

Smoking has been associated with increased systemic lupus erythematosus (SLE) risk, but the biologic basis for this association is unknown. Our objective was to investigate whether women’s smoking was positively associated with SLE‐associated proinflammatory chemokines/cytokines (stem cell factor [SCF], B lymphocyte stimulator [BLyS], interferon‐γ–inducible 10‐kd protein [IP‐10], and interferon‐α); or negatively associated with antiinflammatory cytokine interleukin‐10 (IL‐10); and whether associations were modified by SLE‐related autoantibody status.

Methods

The Nurses’ Health Study (NHS, n = 121,700) and NHSII (n = 116,429) cohorts were begun in 1976 and 1989. In 1988–1990 (NHS) and 1996–1999 (NHSII), ~25% of participants donated blood samples. We identified 1,177 women without SLE with banked samples, and we tested by enzyme‐linked immunoassay (ELISA) for chemokines/cytokines as well as anti‐Sm, anti‐Ro/SSA, anti‐La/SSB, and anti‐RNP. Antinuclear antibodies (ANAs) were detected by HEp‐2 cell indirect immunofluorescence, and anti–double‐stranded DNA antibodies and were assayed by ELISA. Smoking was assessed until blood draw. Separate tobit and linear regression analyses, adjusted for potential confounders, modeled associations between smoking and log‐transformed chemokine/cytokine concentrations. Analyses were stratified by autoantibody status. Effect estimates were calculated as ratios of geometric means expressed as percentage differences.

Results

Among the 15% of current/recent versus 85% of past/never smokers, BLyS levels were 8.7% higher (P < 0.01) and were 24% higher (P < 0.0001) among those who were ANA positive. Current/recent smokers had IL‐10 concentrations 46% lower (P < 0.01) than past/never smokers; each 10 pack‐years of smoking was associated with a 17% decrease in IL‐10 level (P < 0.001). Smoking was not associated with IP‐10 or SCF.

Conclusion

Elevated BLyS and lower IL‐10 levels among current smokers, particularly among ANA‐positive women, may be involved in SLE pathogenesis.
Direct Asymmetric Arylation of Imines
Authors: Egorov Ilya N. , Santra Sougata , Kopchuk Dmitry S. , Kovalev Igor S. , Zyryanov Grigory V. , Majee Adinath , Ranu Brindaban C. , Rusinov Vladimir L. , Chupakhin Oleg N.
Published: July 2020

The Friedel‐Crafts reaction represents one of the most efficient tools for the formation of carbon‐carbon bonds and direct derivatization of aromatic compounds. Asymmetric arylation of imines involving Friedel‐Crafts reaction has received much interest recently. The present review summarizes the works on the asymmetric arylation of imines by different approaches including Friedel‐Crafts reaction and their development to date. The review is divided into three main sections: addition reaction of arenes to imines in the presence of chiral catalysts; addition reaction of arenes to imines containing chiral tails and asymmetric coupling reaction of aryl boronic acids and imines.

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An integrative approach to studying plasticity in growth disruption and outcomes: A bioarchaeological case study of Napoleonic soldiers
Authors: Holder Sammantha , Miliauskienė Žydrūnė , Jankauskas Rimantas , Dupras Tosha
Published: July 2020

Objectives

The aim of this study was to investigate how much variation in adult stature and body mass can be explained by growth disruption among soldiers who served in Napoleon's Grand Army during the Russian Campaign of 1812.

Methods

Linear enamel hypoplasia (LEH) were recorded as representations of early life growth disruption, while the impact on future growth was assessed using maximum femur length (n = 73) as a proxy for stature and maximum femoral head diameter (n = 25) as a proxy for body mass. LEH frequency, severity, age at first formation, and age at last formation served as explanatory variables in a multiple regression analysis to test the effect of these variables on maximum femur length and maximum femoral head diameter.

Results

The multiple regression model produced statistically significant results for maximum femur length (F‐statistic = 3.05, df = 5 and 67, P = .02), with some variation in stature (adjusted r2 = 0.13) attributable to variation in growth disruption. The multiple regression model for maximum femoral head diameter was not statistically significant (F‐statistic = 1.87, df = 5 and 19, P = .15).

Conclusions

We hypothesized stress events during early life growth and development would have significant, negative, and cumulative effects on growth outcomes in adulthood. The results did not support our hypothesis. Instead, some variables and interactions had negative effects on stature, whereas others had positive effects. This is likely due to catch‐up growth, the relationship between acute and chronic stress and growth, resilience, and plasticity in human growth over the life course.