bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclosporin synthetase is a 1.4 MDa multienzyme polypeptide Re‐evaluation of the molecular mass of various peptide synthetases
Authors: Schmidt Bettina , Riesner Detlev , Lawen Alfons , Kleinkauf Horst
Published: July 2002

The earlier determined molecular mass of 0.8 MDa for the multifunctional polypeptide, cyclosporin synthetase, was re‐evaluated by SDS‐PAGE and CsCl density gradient centrifugation. In SDS‐PAGE, new molecular mass values as standards were available from sequencing data. In the CsCl density gradient extremly low protein concentrations, such as 10–50 nM could be analysed due to the fluorescence detection system of the analytical ultracentrifuge. Both methods yielded approximately the same value of about 1.4 MDa. Using this molecular mass of cyclosporin synthetase as a reference the molecular masses of various related enzymes could be re‐evaluated in SDS‐PAGE. The sedimentation coefficient of 26.3 S for cyclosporin synthetase indicates an oblate overall shape of the enzyme.
Retinoic acid decreases retinoic acid and triiodothyronine nuclear receptor expression in the liver of hyperthyroidic rats
Authors: Higueret P. , Pallet V. , Coustaut M. , Audouin I. , Begueret J. , Garcin H.
Published: July 2002

Retinoic acid (RA) and triiodothyronine (T3) exert many of their actions by binding to specific nuclear receptors (respectively, RA receptor (RAR) and T3) receptor (TR) belonging to a ‘superfamily’ of receptors. Some heterologous regulation of these receptors has been shown, and in particular regulation of the maximum binding capacity of TR by either retinol or RA. Now, using hyperthyroidic rats as a model, the effect of RA on binding capacity and on the mRNA levels of TR and RAR was investigated. The results show that the benefit of vitamin A treatment for the hyperthyroidic state, which has been described for a long time, could be the result of a down‐heteroregulation of TR by RA, the active metabolite of retinol.
Escherichia coli DNA gyrase: Genetic analysis of gyr A and gyr B mutations responsible for thermosensitive enzyme activity
Authors: Oram Mark , Kuroda Reiko , Fisher L.Mark
Published: July 2002

Escherichia coli gyr A 43 and gyr B 203 alleles conferring temperature‐sensitive (ts) growth encoded Gly751 → Asp and Pro171 → ser substitutions in the DNA gyrase A and B subunits, respectively. A plasmid‐borne gyr A 43 allele wasg genetically dominant over a chromosomal quinolone‐resistant gyr A gene at 30°C but not at 42°C. These results and others confirm the ts phenotype of the mutation, the first to be identified in the C‐terminal DNA binding/complex stabilizing domain of gyrase A protein. By contrast, the Pro171 → Ser mutation is located near the ATP‐binding site of gyrase B protein and could interfere with energy coupling during DNA supercooling. These data are discussed in regard to recently described gyr A (ts) mutations that affect the control of chromosome segregation.
Resonance Raman studies of the carbonmonoxy form of catalase Evidence for and effects of phenolate ligation
Authors: Hu Songzhou , Kincaid James R.
Published: July 2002

Resonance Raman spectra are reported for the carbon monoxide (CO) adduct of catalase formed from the reaction of peracetic acid or hydrogen peroxide with the azide adduct or catalase in the presence of CO. The expected three normal vibrations of the Fe–CO fragment are detected at 1,908, 593 and 543 cm−1 for the ν(C–O), δ(Fe–C–O) and ν(Fe–CO), respectively. The expected coordination or the phenolate group in this adduct is confirmed by the enhancement of an internal vibration of phenolate, ν19a at 1,515 cm−1, and an extraordinary intensity enhancement or the ν(Fe–CO) mode.
Arachidonic acid abolishes the mitogen‐induced increase in cytosolic free Ca2+ and intracellular pHi in rat thymocytes
Authors: Astashkin E.I. , Khodorova A.B. , Surin A.M.
Published: July 2002

The effects of arachidonic acid (AA) and the lectin mitogens, concanavalin A (Con A) and phytohemagglutinin (PHA), on [Ca2+], and pHi in rat thymocytes have been studied by using the intracellular fluorescent probes, Fura‐2 and BCECF. It was revealed that exogenous AA (3 μM), in addition to the well‐known changes in basal [Ca2+]i and pHi, also caused a complete blockade of [Ca2+], and pH, signals induced by Con A(10μg/ml) and PHA (10 μg/ml). In contrast, exposure of thymocytes to mitogens did not prevent the AA‐induced increase in [Ca2+]i, and decrease in pHi, In experiments with sodium propionate, the similarity between AA action and EIPA (ethylisopropylamiloride), an inhibitor of Na+/H+ exchangers, was revealed. It is proposed that the inhibitory effect of AA on mitogen‐induced lymphocyte proliferation is due primarily to the blockade of transmembrane [Ca2+]i, and pHi signals, associated with a sustained cytosolic acidification.
Key active site residues in the inhibition of acetylcholinesterases by soman
Authors: Qian Naifeng , Kovach Ildiko M.
Published: July 2002

Molecular modeling (GEMM 7.3) and molecular mechanics calculations (YETI V 5.3) using the X‐ray coordinates for acetylcholinesterase (AChE) from Torpedo californica indicate electrostatic stabilization by the active site. Glu‐199, of the developing positive charge on the incipient carbonium ion in the dealkylation in the adducts of AChE with PSCR and PsCs diastereomers of 2‐(3,3‐dimethylbutyl) methylphosphonofluoridate (soman). His‐440 is indispensable as a general acid catalyst of C‐O bond breaking in the dealkylation reaction and that of bond breaking to the Ser γ‐O in reactivation. This demand for catalysis seems to be satisfied for the reactivation of enzyme from the PsCs, diastereomer of soman, but not from the P(S)C(R) diastereomer.
The C‐terminal domain of yeast cytochrome b is essential for a correct assembly of the mitochondrial cytochrome bc1 complex
Authors: di Rago Jean-Paul , Macadre Catherine , Lazowska Jaga , Slonimski Piotr P.
Published: July 2002

Yeast mutants modifying the C‐terminal region of mitochondrial cytochrome b were isolated and characterized. A nonsense mutation of the leucine codon 335 (TTA → TAA), 50 residues before the normal C‐terminus, blocks incorporation of heme into the apocytochrome b and prevents growth on non‐fermentable substrates. The same defects were observed in a frameshift mutant (after codon 348, TAT → TATT) in which the last 37 C‐terminal residues are predicted to be replaced by a novel sequence of 33 amino acids. Function was regained in the nonsense mutant only by true back mutations restoring a protein of the wild‐type sequence. The respiratory capacity was restored to wild‐type levels in the frameshift mutant by a variety of single base subtractions located within a window of 24 bases before or after the original +T addition, these pseudo‐reversions resulted in single or multiple (up to five) consecutive amino acid replacements between positions 346 and 354 and restored the wild‐type sequence from position 355 to 385. These data, combined with hydropathy calculations and sequence comparisons, suggest that the C‐terminal domain of cytochrome b forms a transmembrane segment essential for the correct assembly of the cytochrome bc
1 complex.
Inhibitors of the intracellular Ca2+ release mechanism prevent muscarinicinduced Ca2+ influx in rat sublingual mucous acini
Authors: Zhang Guo H. , Melvin James E.
Published: July 2002

The effects of inhibitors of the intracellular Ca2+ release mechanism on divalent cation fluxes were examined in acinar cells loaded with the Ca2+‐sensitive, Mn2+‐quenchable dye, fura‐2. TMB‐8 and dantrolene (DTL) dramatically inhibited the carbachol (CCh)‐stimulated increase in [Ca2+]i and Mn2+ influx. These agents do not directly inhibit divalent cation entry since addition of TMB‐8 or DTL after CCh stimulation did not block Mn2+ influx. TMB‐8 did not influence the [Ca2+]i increase or the Mn2+ influx produced by thapsigargin. These results indicate that TMB‐8 and DTL do not interfere with divalent cation influx by inhibiting a step distal to depletion of the intracellular Ca2+ pool. TMB‐8 and DTL did not significantly influence the muscarinic‐stimulated production of inositol trisphosphate (IP3) and inositol tetrakisphosphate (IP4), although TMB‐8, but not DTL, did decrease the CCh‐stimulated 1,4,5‐IP3 levels approximately 55%. The above results directly demonstrate that the filling state of the intracellular Ca2+ store primarily regulates the Ca2+ entry mechanism in sublingual mucous acinar cells.
Starvation effect on rat kidney peroxisomal and microsomal fatty acid oxidation
Authors: Orellana Myriam , Fuentes Oscar , Valdés Elena
Published: July 2002

Microsomal lauric acid 12‐hydroxy lauric acid (ω)‐hydroxylation and fatty acid peroxisomal β‐oxidation were studied in kidney tissue from starved rats. Starvation increased the microsomal ω‐hydroxylation and peroxisomal β‐oxidation of fatty acids with a high correlation between both processes. Earlier, we reported similar results in liver. Our results support the hypothesis that the role of microsomal fatty acids ω‐hydroxylation is the generation of substrate for peroxisomal β‐oxidation, with the final purpose of contributing to a catabolic or gluconeogenic pathway from fatty acids.
Pentoxifylline inhibits the expression of tissue factor mRNA in endotoxin‐activated human monocytes
Authors: Ollivier V. , Ternisien C. , Vu T. , Hakim J. , de Prost D.
Published: July 2002

Tissue factor (TF) is a transmembrane glycoprotein which, in association with factor VII(a), is the main activator of coagulation. In illnesses such as Gram‐negative endotoxemia, circulating monocytes synthesize and express substantial TF activity, resulting in extensive disseminated intravascular coagulation. We investigated the way in which TF is suppressed by pentoxifylline (PTX), and found that PTX down‐regulates immunologic TF expression and specific mRNA production in response to LPS. Since TF mRNA stability is not altered, this effect appears to take place at the transcriptional level.
Solubilisation partial characterisation of the α‐MSH receptor on primary rat Schwann cells
Authors: Dyer Jason K. , Ahmed Abdel R.H. , Oliver George W.J. , Poulton Colin W. , Haynes Laurence W.
Published: July 2002

The ACTH/MSH4–10 melanocortin core peptide sequence possesses neurotrophic properties in peripheral nerve. During functional neuroanatomical recovery after damage to peripheral nerves, Schwann cells play a significant role in facilitating regeneration. Here we employ a modified super‐potent α‐MSH analogue to solubilise α‐MSH receptor proteins from cultured primary rat Schwann cells. [125I‐Tyr2,Nle4,d‐Phe7,ATB‐Lys11]‐α‐MSH photoaffinity labelled proteins from Schwann cells were analyzed by SDS‐PAGE followed by autoradiography. The results indicate that the α‐MSH receptor proteins labelled have a molecular weight of 42–45 kDa. These data are the first to demonstrate solubilisation and characterisation of α‐MSH receptors from non‐melanoma cells.
Oligomerization of an avian thymic parvalbumin Chemical evidence for a Ca2+‐specific conformation
Authors: Hapak Raymond C. , Zhao Hongmei , Henzl Michael T.
Published: July 2002

CPV3, the third parvalbumin isoform to be identified in the chicken, is produced exclusively in the thymus gland. Although parvalbumins are typically cysteine‐deficient, CPV3 contains two cysteine residues, at positions 18 and 72. The reported three‐dimensional parvalbumin structures suggest that the side chain of cysteine‐72 should be solvent‐accessible. Accordingly, we find that CPV3 readily forms disulfide‐linked oligomers in the absence of reducing agents. The reaction, employing either O2 or ferricyanide ion as the oxidant, is apparently restricted to the Ca2+‐bound form of the protein. The differing reactivity of the Ca2+, Mg2+, and apo‐forms has significant structural implications.
Construction and in vivo analysis of new split lactose permeases
Authors: Wrubel Wolfgang , Stochaj Ursula , Ehring Ruth
Published: July 2002

The capacity of incomplete segments of Escherichia coli lactose permease to form transport‐competent complexes in vivo was further tested. Two series of mutant lacY genes were constructed. One encoded N‐terminal lactose permease segments of different length. The proteins specified by the other group contained deletions of different length and location within the N‐terminal region. Several pairs of such mutant proteins reconstituted active lactose transport. For certain combinations duplications of protein segments were compatible with the formation of an active carrier. Duplication of helices could also be tolerated, when part of a single polypeptide chain.
The detergent Triton X‐100 induces a death pattern in human carcinoma cell lines that resembles cytotoxic lymphocyte‐induced apoptosis
Authors: Borner Markus M. , Schneider Erasmus , Pirnia Farzaneh , Sartor Oliver , Trepel Jane B. , Myers Charles E.
Published: July 2002

The detergent Triton X‐100 (TX100) was used with the intention to establish a model for necrotic cell death. However TX100 was found to induce apoptotic and necrotic death in prostate and colon cancer cell lines. Apoptosis was characterized by the typical morphological features and internucleosomal DNA fragmentation. The rapid onset within 60 min and the lack of inhibition by cycloheximide indicated that apoptosis induced by TX100 was not dependent on protein synthesis. Removal of extracellular calcium blocked internucleosomal DNA fragmentation. This pattern of cell death shows a striking similarity to the effect of cytotoxic lymphocytes on their target cells.
The R‐form of DNA does exist
Authors: Shchyolkina Anna K. , Timofeev Edward N. , Borisova Olga F. , Il'icheva Irina A. , Minyat Elvira E. , Khomyakova Elena B. , Florentiev Vladimir L.
Published: July 2002

Oligonucleotide 5'‐d(CATGCTAACT)‐L‐d(AGTTAGCATG)‐L‐d(CATGCTAACT)‐3' [L = pO(CH2CH2O)3P] is shown to fold back on itself twice forming at pH 7 a sufficiently stable triplex (T
m, is about 30°C) with parallel‐orientated identical strands (the recombinant or R‐form of DNA). Experimental evidence was obtained by studying thermal denaturation, chemical modification and binding of fluorescent probes. The stability of the R‐triplex increases in the presence of divalent ions or spermidine. Its structure is characterized by a certain heterogeneity that causes the cooperativity of a triplex‐to‐duplex transition to decrease. On the basis of conformational modeling, the possible types of base tripling in all four triplets are proposed. The experimental data as well as the molecular mechanic calculations indicate that the stabilities of triplets in the R‐triplex decrease in the order: G:C‐G = A:T‐A ⪢ T:A‐T > C:G‐C.
Drosophila lebanonensis ADH: analysis of recombinant wild‐type enzyme and site‐directed mutants
Authors: Albalat Ricard , Atrian Sílvia , Gonzàlez-Duarte Roser
Published: July 2002

Unique amino acid substitutions occur in D. lebanonensis ADH. They are found within the putative NAD + ‐binding domain and affect residues that are otherwise highly conserved in all other species of the genus. To restore the consensus ainino acids, we have constructed an expression system for this enzyme in E. coli, and engineered two mutants, Ala13 Gly and Asn56 Thr. The biochemical and kinetic features of these retromutants are consistent with increased catalytic efficiency and thermal stability. Thus, results show that wild‐type D. lebanonensis ADH can be improved by site‐directed mutagenesis.
Modifications induced by acylphosphatase in the functional properties of heart sarcolemma Na+,K+ pump
Authors: Nassi Paolo , Nediani Chiara , Fiorillo Claudia , Marchetti Elena , Liguri Gianfranco , Ramponi Giampietro
Published: July 2002

Acylphosphatase purified from cardiac muscle actively hydrolyzes the phosphoenzyme intermediate of heart sarcolemma Na+,K+‐ATPase. This effect occurred with acylphosphatase amounts (up to 800 membrane protein) that fall within the physiological range and the low value of the apparent K
m (0.69 × 10−7 M) indicates a considerable affinity of the enzyme towards this specific substrate. Acylphosphatase addition to purified sarcolemmal vesicles significantly increased the rate of Na+,K+‐dependent ATP hydrolysis. Maximal stimulation, observed with 800 protein, resulted in an ATPase activity which was about 2‐fold over basal value. The same acylphosphatase amounts significantly stimulated, in a similar and to an even greater extent, the rate of ATP driven Na+ transport into sarcolemmal vesicles. These findings lead to suppose that an accelerated hydrolysis of the phosphoenzyme may result in an enhanced activity of heart sarcolemmal Na+,K+ pump, therefore suggesting a potential role of acylphosphatase in the control of this active transport system.
Specific association of phosphatidylinositol 3‐kinase with the protooncogene product Cbl in Fcγ receptor signaling
Authors: Matsuo Tsuyoshi , Hazeki Kaoru , Hazeki Osamu , Katada Toshiaki , Ui Michio
Published: July 2002

A tyrosine‐phosphorylated protein with a molecular mass of 115 kDa was reported to be tightly associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3‐kinase, when the enzyme was essentially activated upon ligand engagement of Fcγ receptors (FcγR) leading to engulfment of IgG‐coated erythrocytes by phagocytes [Ninomiya et al. (1994) J. Biol. Chem. 269, 22732‐22737]. Here, the 115‐kDa protein is identified as the product of human c‐cbl protooncogene. Cross‐linking of FcγRII on the surface of THP‐1 cells triggered (a) prominent tyrosine phosphorylation of Cbl, (b) activation of PI 3‐kinase that was immunoprecipitated with the anti‐Cbl or the anti‐phosphotyrosine antibody, and (c) specific association of Cbl with p85, Thus, Cbl functions in phagocytes as a result of its association with PI 3‐kinase in response to FcγR ligation.
2‐Nitrosofluorene and N‐hydroxy‐2‐aminofluorene react with the ubiquinone‐reduction center (center N) of the mitochondrial cytochrome bc1 complex
Authors: Klöhn Peter-Christian , Brandt Ulrich , Neumann Hans-Günter
Published: July 2002

We determined the sites of artificial electron transfer onto 2‐nitrosofluorene (NOF), a metabolite of carcinogenic 2‐acetylaminofluorene in mitochondria and isolated cytochrome bc
1 complex. NOF‐induced O2 consumption in mitochondria was sensitive to antimycin A, but insensitive to myxothiazol. In the isolated cytochrome bc
1 complex, NOF induced rapid MOA‐stilbene‐insensitive reoxidation of cytochrome b, whereas in the presence of antimycin A, reoxidation was very slow. The corresponding hydroxylamine, N‐hydroxy‐2‐aminofluorene (N‐OH‐AF), reduced cytochrome b specifically through center N of the cytochrome bc
1 complex. We conclude that NOF and N‐OH‐AF bind to center N of the cytochrome bc
1 complex and acts as electron acceptor and donor, respectively. The N‐OH‐AF/NOF interconversion is considered to be involved in the cytotoxicity of 2‐acetylaminofluorene in vivo.
Molecular cloning and bacterial expression of a cDNA encoding furostanol glycoside 26‐O‐β‐glucosidase of Costus speciosus
Authors: Inoue Kentaro , Shibuya Masaaki , Yamamoto Kazuo , Ebizuka Yutaka
Published: July 2002

Furostanol glycoside 26‐O‐β‐glucosidase (F26G) purified from Costus speciosus rhizomes was digested with endoproteinase, and several internal peptide fragments were obtained. Degenerate oligonucleotide primers based on amino acid sequences of the peptides were used for amplification of F26G cDNA fragments by applying nested polymerase chain reactions to cDNAs from in vitro cultured plantlets of C. speciosus. Using primers based on sequences of the cDNA fragments, the 5′‐ and 3′‐end clones were isolated by rapid amplification of cDNA ends (RACE) methods. Finally, the entire coding portion of F26G cDNA was cloned by using primers designed from sequences of the RACE products. The deduced amino acid sequence of CSF26G1, the protein encoded by the cloned cDNA, consists of 562 amino acids and shows high homology to a widely distributed family of β‐glucosidases (BGA family). Cell‐free homogenate of Escherichia coli expressing CSF26G1 cDNA showed β‐glucosidase activity specific for cleavage of the C‐26 glucosidic bond of furostanol glycosides.
Identification of the functional region on the superantigen Yersinia pseudotuberculosis‐derived mitogen responsible for induction of lymphocyte proliferation by using synthetic peptides
Authors: Yoshino Ken-ichi , Takao Toshifumi , Ishibashi Makiko , Samejima Yuji , Shimonishi Yasutsugu , Takeda Tae
Published: July 2002

Yersinia pseudotuberculosis‐derived mitogen (YPM) is the unique Gram‐negative bacillary superantigen known. In order to identify the regions on the YPM molecule involved in its superantigenic activity, seven overlapping peptides of the entire YPM molecule were synthesized and tested to evaluate their effects on the YPM‐induced proliferation of human peripheral blood lymphocytes. A peptide corresponding to the N‐terminal amino acid sequence (1–23) was found to inhibit YPM‐induced lymphocyte proliferation in a concentration‐dependent manner. The N‐terminal peptide was found to show no inhibition of the proliferation induced by the other superantigen (staphylococcal enterotoxin B) or the other T‐cell mitogen pertussis toxin, indicating that the inhibition is specific to YPM‐induced proliferation. Thus, we have identified the N‐terminal region (1–23) of the YPM as one of the functional regions responsible for its superantigenic activity.
Serum CA 125 and first trimester abortion
Authors: Scarpellini F. , Mastrone M. , Sbracia M. , Scarpellini L.
Published: July 2002

Objective: The study was carried out to assess the clinical value of serum CA 125 in association with serum β‐human chorionic gonadotropin (hCG) for predicting pregnancy outcome. Methods: One hundred spontaneous pregnancies, 52 non‐threatened pregnancies and 48 with threatened abortions, observed in the Department of Obstetrics and Gynecology at the University ‘La Sapienza’, Rome, Italy, were evaluated during the first trimester using ultrasound examination, CA 125 and β‐hCG titrations. Results: Threatened pregnancies had statistically significantly higher CA 125 serum values than non‐threatened pregnancies, especially those with a negative outcome (P < 0.01). The CA 125 levels in the threatened pregnancies were positively correlated with the tropho‐decidual hematoma volume (r = 0.839, P < 0.0001). The CA 125‐β‐hCG association showed a higher prognostic value (sensitivity 78.9%, specificity 96.5%) in assessing pregnancy outcome than CA 125 or β‐hCG alone (sensitivity 78.9% and 57.9%, respectively; specificity 75.8% and 86.2%, respectively). Conclusions: Our findings are in accordance with the hypothesis of a tropho‐decidual origin of this marker, suggesting its possible usefulness in the prognostic evaluation of first trimester threatened abortion.
Surgical treatment alternatives in stress incontinence
Authors: Güner H. , Ahmed S. , Nas T. , Yildirim M.
Published: July 2002

Objectives: This study was performed to evaluate the different surgical treatment alternatives for urinary stress incontinence and their clinical results. Methods: Of the patients who visited our clinic between January 1989 and June 1994 complaining of urinary stress incontinence, 408 were found to have genuine stress incontinence (GSI) after undergoing clinical and laboratory investigations. These 408 patients were treated with different surgical techniques. Their postoperative results are discussed. Results: After postoperative follow‐up, the success rates of the different surgical techniques were as follows; anterior colporrhaphy (Kelly plication) 66.2%, Marshall‐Marchetti‐Krantz 88.7%, Burch procedure 94.1%, and sling urethropexy 97.5%. Conclusions: Patients with a clear‐cut diagnosis of GSI must be surgically treated. There are several different techniques, all of which have advantages and disadvantages. If the right technique is used for the right patient then the success rate is high.
Variability of peripartum vaginal group B streptococcal colonization
Authors: Itakura A. , Kurauchi O. , Morikawa S. , Matsuzawa K. , Mizutani S. , Tomoda Y.
Published: July 2002

Objectives: Our purpose was to examine the change in peripartum vaginal group B streptococcal concentration. Methods: From 1989 to 1993, 251 pregnant women who delivered over 48 h after admission were included in this study. Group B streptococcal quantitative culture of vaginal specimens was performed within 24 h before delivery and on the previous day, and the GBS concentrations compared. Results: A statistical difference between positive rates for group B Streptococcus on the delivery day and that on the previous day was found (14 vs. 22%). In 20/58 (34%) of the cases between the two time points the vaginal group B streptococcal colonization increased. Conclusions: This study suggests the existence of major variation in peripartum vaginal group B streptococcal concentrations. We conclude that the rate and the concentration of perinatal vaginal group B streptococcal colonization even one day before delivery cannot be used to predict that at delivery.
Agreement on diagnoses of mental disorder in the primary health care of Turku, Finland
Authors: Lehtinen V , Joukamaa M , Karlsson H , Rouhe E
Published: July 2002

Primary health care is in the central position in the general sphere of mental health services. This article deals with, as a part of a joint Nordic project, the occurrence of mental disorders of patients in primary health care as based on the material (n= 1,000) collected in the health centre of Turku. The relation of the occurrence of mental disorders as diagnosed by means of a standardized psychiatric interview method (PSE) to the disorders, recognized by general practitioners (GPs) working in the health centre is also observed. It was found that one fourth of the patients had a clear mental disorder that required treatment, while studies in the general population, using the same method, reveal only 10% as having some kind of mental disorder. Three quarters of the evident mental disorders recognized by GPs were diagnosed as such also by the PSE, but the GPs were capable of recognizing only 43% of the cases identified by the PSE. On the basis of these results, the importance of the mental health issue in the basic and postgraduate training of the general practitioners must be emphasized.
Dysthymia as a temperamental variant of affective disorder
Authors: Akiskal HS
Published: July 2002

Dysthymia is estimated to afflict at least 3% of the population worldwide. Because it is a chronic disorder, its prevalence is high in psychiatric and general medical settings. The mystery of this incapacitating depressive subtype — long recognized but only recently sanctioned in the DSM-IV and ICD-10 — is that, in their habitual condition, those suffering from dysthymia lack the classical ‘objective’ or ‘major’ signs of acute clinical depression, such as profound changes in psychomotor and vegetative functions. Instead, patients consult their doctors for more fluctuating complaints consisting of gloominess, lethargy, self-doubt, malaise, and lack of joie de vivre. They typically work hard, but do not enjoy their work. If married, they are deadlocked in bitter and unhappy marriages which lead neither to reconciliation nor separation. For them, their entire existence is a burden: they are satisfied with nothing, complain of everything, and brood about the uselessness of existence. As a result, in the past they were labeled ‘existential depressives’ or ‘depressive characters’ and condemned to the couch, often on a chronic basis. Several lines of research over the past fifteen years have shed new light on the biological origins of this disorder. Sleep neurophysiologic findings have shown that many parameters of paradoxical sleep in dysthymia (such as REM percentage, REM latency, and circadian distribution of REM) are similar to those observed in major affective illness. Furthermore, family studies of dysthymia have demonstrated a significant excess of mood disorders. Indeed, dysthymia has been identified in childhood, and prospective follow-up has demonstrated major affective breakdowns including bipolar switches in up to 20%. Coupled with sleep findings, these family and follow-up data suggest that dysthymia is best considered as ‘trait depression’, a constitutional variant of major affective illness. As expected from the early onset chronic nature of the disturbance, in both clinical and epidemiological studies, the social and health burden of dysthymia has been found to be considerable and comparable to that of major medical disorders. The foregoing clinical and biological data have provided the impetus for well-designed pharmacological trials in dysthymia, and a new therapeutic optimism.
Development of a novel biomarker of free radical damage in reperfusion injury after cardiac arrest
Authors: Basu S. , Nozari A. , Liu X.L. , Rubertsson S. , Wiklund L.
Published: July 2002

In a porcine model of cardiopulmonary resuscitation (CPR), we investigated changes in the plasma levels of 8‐iso‐PGF2α, a marker for oxidative injury, and 15‐keto‐dihydro‐PGF2α, an inflammatory response indicator during the post‐resuscitation period after cardiac arrest. Twelve piglets were subjected to either 2 or 5 min (VF2 and VF5 group) of ventricular fibrillation (VF) followed by 5 min of closed‐chest CPR. Six piglets without cardiac arrest were used as controls. In VF5 group, 8‐iso‐PGF2α in the jugular bulb plasma (draining the brain) increased four‐fold. Jugular bulb 8‐iso‐PGF2α in the control group remained unchanged. The 15‐keto‐dihydro‐PGF2α also increased four‐fold in the VF5 group. Thus, 8‐iso‐PGF2α and 15‐keto‐dihydro‐PGF2α measurements in jugular bulb plasma may be used as biomarkers for quantification of free radical catalyzed oxidative brain injury and inflammatory response in reperfusion injury.
Evidence for the presence of a component of the Mn complex of the photosystem II reaction centre which is exposed to water in the S2 state of the water oxidation complex
Authors: Evans Michael C.W. , Rich Anne M. , Nugent Jonathan H.A.
Published: July 2002

The interaction of water oxidising photosystem II preparations with the aqueous environment has been investigated using electron spin echo envelope modulation spectroscopy in the presence of 2H2O. The spectra show interaction of 2H of 2H2O with the preparation in the S2 state. The component interacting with water decays during 1–4 weeks storage at 77 K. No interaction of water with the classical multiline S2 Mn signal, which is more stable on storage at 77 K, was detected. The results show that a component of the water oxidation complex, possibly involving the Mn centre, is accessible to water and may be the water binding site for photosynthetic water oxidation.
Functional genomics by mass spectrometry
Authors: Andersen Jens S , Mann Matthias
Published: July 2002

Systematic analysis of the function of genes can take place at the oligonucleotide or protein level. The latter has the advantage of being closest to function, since it is proteins that perform most of the reactions necessary for the cell. For most protein based (‘proteomic’) approaches to gene function, mass spectrometry is the method of choice. Mass spectrometry can now identify proteins with very high sensitivity and medium to high throughput. New instrumentation for the analysis of the proteome has been developed including a MALDI hybrid quadrupole time of flight instrument which combines advantages of the mass finger printing and peptide sequencing methods for protein identification. New approaches include the isotopic labeling of proteins to obtain accurate quantitative data by mass spectrometry, methods to analyze peptides derived from crude protein mixtures and approaches to analyze large numbers of intact proteins by mass spectrometry directly. Examples from this laboratory illustrate biological problem solving by modern mass spectrometric techniques. These include the analysis of the structure and function of the nucleolus and the analysis of signaling complexes.
Expression of doubly labeled Saccharomyces cerevisiaeiso‐1 ferricytochrome c and 1H, 13C and 15N chemical shift assignments by multidimensional NMR
Authors: Szabo Christina M , Sanders Lori K , Le H.Carl , Chien Ellen Y.T , Oldfield Eric
Published: July 2002

We have expressed [U‐13C,15N]‐labeled Saccharomyces cerevisiae iso‐1 cytochrome c C102T;K72A in Escherichia coli with a yield of 11 mg/l of growth medium. Nuclear magnetic resonance (NMR) studies were conducted on the Fe3+ form of the protein. We report herein chemical shift assignments for amide 1H and 15N, 13C°, 13Cα, 13Cβ, 1Hα and 1Hβ resonances based upon a series of three‐dimensional NMR experiments: HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB, HCA(CO)N, HCCH‐TOCSY and HBHA(CBCA)NH. An investigation of the chemical shifts of the threonine residues was also made by using density functional theory in order to help solve discrepancies between 15N chemical shift assignments reported in this study and those reported previously.
Kinetic mechanism of NADH‐enoyl‐ACP reductase from Brassica napus
Authors: Fawcett Tony , Copse Catherine L , Simon J.William , Slabas Antoni R
Published: July 2002

Enoyl‐ACP reductase, a component of fatty acid synthase, is a target for anti‐microbial agents and herbicides. Here we demonstrate the kinetic mechanism to be a compulsory‐order ternary complex with NADH binding before the acyl substrate. Matrix‐assisted laser desorption ionisation mass spectrometry analysis of enzymatically and synthesised crotonyl‐ACP substrate showed the former to contain a single acyl group, whereas the latter contained up to four additional crotonylations. The use of authentic crotonyl‐ACP will be important in future kinetic and crystallographic studies.
Chemical and serological characterization of the Bordetella hinzii lipopolysaccharides1
Authors: Aussel Laurent , Chaby Richard , Le Blay Karine , Kelly John , Thibault Pierre , Perry Malcolm B. , Caroff Martine
Published: July 2002

Bordetella hinzii has recently been isolated from immunocompromised human hosts. The polysaccharides isolated from its endotoxin (lipopolysaccharide, LPS) were investigated using chemical analyses, NMR, gas‐liquid chromatography/mass spectrometry and mass spectrometry by plasma desorption, matrix‐assisted laser desorption/ionization and electrospray. The following structure for the O‐chain‐free LPS was deduced from the experimental results: Mass spectrometry and serology revealed that the O‐chains were different from the homopolymer common to Bordetella bronchiseptica and Bordetella parapertussis strains and were composed of a trisaccharide repeating unit. Masses up to 8 kDa were obtained for native LPS molecular species.
Ecm10, a novel Hsp70 homolog in the mitochondrial matrix of the yeast Saccharomyces cerevisiae
Authors: Baumann Frank , Milisav Irina , Neupert Walter , Herrmann Johannes M
Published: July 2002

Members of the heat shock protein 70 (Hsp70) family are found in most of the compartments of eukaryotic cells where they play essential roles in protein metabolism. In yeast mitochondria, two Hsp70 proteins are known: Ssc1 and Ssq1. We identified Ecm10 as a third Hsp70 protein in the mitochondrial matrix. Ecm10 shares 82% amino acid identity with Ssc1 and 54% with Ssq1. Overexpression of Ecm10 mitigates protein import defects in ssc1 mutants suggesting that Ecm10 can play a role in protein translocation. Like Ssc1, Ecm10 interacts with the nucleotide exchange factor Mge1 in an ATP‐dependent manner. Deletion of ecm10 leads to synthetic growth defects with ssc1 mutations at low temperature. Our data suggest an overlapping function of Ecm10 and Ssc1.
Characterization and functional analysis of the nucleotide binding fold in human peroxisomal ATP binding cassette transporters
Authors: Roerig Peter , Mayerhofer Peter , Holzinger Andreas , Gärtner Jutta
Published: July 2002

The 70‐kDa peroxisomal membrane protein (PMP70) and the adrenoleukodystrophy protein (ALDP) are half ATP binding cassette (ABC) transporters in the peroxisome membrane. Mutations in the ALD gene encoding ALDP result in the X‐linked neurodegenerative disorder adrenoleukodystrophy. Plausible models exist to show a role for ATP hydrolysis in peroxisomal ABC transporter functions. Here, we describe the first measurements of the rate of ATP binding and hydrolysis by purified nucleotide binding fold (NBF) fusion proteins of PMP70 and ALDP. Both proteins act as an ATP specific binding subunit releasing ADP after ATP hydrolysis; they did not exhibit GTPase activity. Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Furthermore, our results indicate that these mutations do not influence homodimerization or heterodimerization of ALDP or PMP70. The study provides evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter.
Non‐destructive quantitation of spermine in human prostate tissue samples using HRMAS 1H NMR spectroscopy at 9.4 T
Authors: Cheng Leo Ling , Wu Chin-lee , Smith Matthew R. , Gonzalez R.Gilberto
Published: July 2002

We present the results of a study of human prostate specimens evaluated by high resolution magic angle spinning 1H nuclear magnetic resonance (NMR) spectroscopy at 400 MHz (9.4 T) and by quantitative histopathology. We demonstrate that NMR and pathology data can be obtained from the same intact specimens, and report for the first time a linear correlation between the NMR measured concentration of spermine, a proposed endogenous inhibitor to prostate cancer growth, and the volume percentage of normal prostatic epithelial cells as quantified by histopathology. Our results show that NMR may serve as a critical tool for the investigation of the inhibitory mechanism of spermine in human subjects.
A deeper investigation on carbohydrate cycling in Sinorhizobium meliloti
Authors: Gosselin Isabelle , Wattraint Olivier , Riboul David , Barbotin Jean-Noël , Portais Jean-Charles
Published: July 2002

Recycling of triose‐phosphate and pentose‐phosphate was previously reported on glucose in Sinorhizobium meliloti, a polysaccharide‐synthesizing bacterium, but the metabolic basis of such processes remained unclear. In this work, we have used 13C‐labelling strategies to demonstrate that carbohydrate cycling in this organism is independent of the gluconate bypass, the alternative pathway for glucose assimilation involving its periplasmic oxidation into gluconate. Furthermore, carbohydrate cycling in S. meliloti is also observed on fructose, making the situation in this bacterium significantly different from that depicted for alginate‐synthesizing species.
Biosynthetically lipid‐modified human scFv fragments from phage display libraries as targeting molecules for immunoliposomes
Authors: de Kruif John , Storm Gert , van Bloois Louis , Logtenberg Ton
Published: July 2002

A human anti‐CD22 single chain (sc) Fv antibody fragment from a synthetic phage antibody display library was biosynthetically lipid‐tagged by using Escherichia coli lipoprotein sequences. The purified anti‐CD22 scFv lipoprotein was incorporated into liposomes by detergent dilution. Anti‐CD22 immunoliposomes were shown to bind specifically in a dose‐ and time‐dependent manner to CD22+ cell lines and CD22+ B‐lymphocytes present in freshly isolated samples of blood mononuclear cells. The immunoliposomes were demonstrated to accumulate in intracellular compartments. Biosynthetically lipid‐tagged human scFv antibody fragments isolated from phage display libraries may facilitate the construction of immunoliposomes with improved properties.
Identification of the secretory vesicle membrane binding region of chromogranin A1
Authors: Kang Yong Koo , Yoo Seung Hyun
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Immuno‐purification of a dimeric subcomplex of the respiratory NAHD‐CoQ reductase of Rhodobacter capsulatus equivalent to the FP fraction of the mitochondrial complex I
Authors: Duborjal Hervé , Dupuis Alain , Chapel Agnès , Kieffer Silvie , Lunardi Joël , Issartel Jean-Paul
Published: July 2002

The Rhodobacter capsulatus genes encoding the NUOE and NUOF subunits, equivalent to the 24 kDa and 51 kDa subunits of the mammalian mitochondrial complex I, have been sequenced. According to the nucleotide sequence, the NUOE subunit is 389 amino acids long and has a molecular mass of 41.3 kDa. In comparison to the mitochondrial equivalent subunit, NUOE is extended at the C terminus by more than 150 amino acids. The NUOF subunit is 431 amino acids long and has a molecular mass of 47.1 kDa. A subcomplex containing both the NUOE and NUOF subunits was extracted by detergent treatment of R. capsulatus membranes and immuno‐purified. This subcomplex is homologous to the mitochondrial FP fragment. Mass spectrometry after trypsin treatment of the NUOE subunit validates the atypical primary structure deduced from the sequence of the gene.
c‐Ras is required for the activation of the matrix metalloproteinases by concanavalin A in 3Y1 cells
Authors: Thant Aye Aye , Serbulea Mihaela , Kikkawa Fumitaka , Liu Enbo , Tomoda Yutaka , Hamaguchi Michinari
Published: July 2002

Concanavalin A (Con A) is known to trigger augmented secretion and proteolytic activation of the matrix metalloproteinases (MMPs) in fibroblasts. To study the signaling pathway critical for the activation of MMPs in fibroblasts, we examined the effects of dominant negative ras (S17N ras) expression under the control of conditionally inducible promoter in Con A‐activated 3Y1 cells. We found that augmented secretion and proteolytic activation of MMP‐2 and MMP‐9 together with expression of MT1‐MMP in Con A‐activated 3Y1 were dramatically suppressed by S17N ras expression. These results strongly suggest that c‐Ras plays a critical role in the augmented expression and proteolytic activation of MMPs in fibroblasts.
PDK1, one of the missing links in insulin signal transduction?1
Authors: Cohen Philip , Alessi Dario R , Cross Darren A.E
Published: July 2002

The initial steps in insulin signal transduction occur at the plasma membrane and lead to the activation of phosphatidylinositide (PtdIns) 3‐kinase and the formation of PtdIns(3,4,5,)P3 in the inner leaflet of the plasma membrane which is then converted to PtdIns(3,4)P2 by a specific phosphatase. Inhibitors of PtdIns 3‐kinase suppress nearly all the metabolic actions of insulin indicating that PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2 are key ‘second messengers’ for this hormone. A major effect of insulin is its ability to stimulate the synthesis of glycogen in skeletal muscle. By ‘working backwards’ from glycogen synthesis, we have dissected an insulin‐stimulated protein kinase cascade which is triggered by the activation of PtdIns 3‐kinase. The first enzyme in this cascade is termed 3‐phosphoinositide‐dependent protein kinase (PDK1), because it is only active in the presence of PtdIns(3,4,5)P3 or PtdIns(3,4)P2. PDK1 then activates protein kinase B (PKB) which, in turn, inactivates glycogen synthase kinase‐3 (GSK3), leading to the dephosphorylation and activation of glycogen synthase and hence to an acceleration of glycogen synthesis. We review the evidence which indicates that the phosphorylation of other proteins by PKB and GSK3 is likely to mediate many of the intracellular actions of insulin.
Design of sensitive fluorogenic substrates for human cathepsin D
Authors: Gulnik Sergei V. , Suvorov Leonid I. , Majer Pavel , Collins Jack , Kane Bradley P. , Johnson Donald G. , Erickson John W.
Published: July 2002

Cathepsin D is a lysosomal aspartic proteinase that has been implicated in several pathological processes such as breast cancer and Alzheimer's disease. We designed and synthesized a number of quenched fluorogenic substrates with P2 variations in the series AcEE(EDANS)KPIXFFRLGK(DABCYL)E‐NH2, where X=cysteine, methylcysteine, ethylcysteine, tert‐butylcysteine, carboxymethylcysteine, methionine, valine or isoleucine. Most of the fluorogenic substrates exhibited greater k
cat/K
m ratios than the best cathepsin D substrates described so far. Differences in kinetic constants, which were rationalized using structure‐based modeling, might make certain substrates useful for particular applications, such as active site titrations or initial velocity determination using a fluorescent plate reader.
The Su(Ste) repeat in the Y chromosome and βCK2tes gene encode predicted isoforms of regulatory β‐subunit of protein kinase CK2 in Drosophila melanogaster
Authors: Kalmykova A.I. , Dobritsa A.A. , Gvozdev V.A.
Published: July 2002

We report an exon‐intron structure of the Su(Ste) repeat capable of encoding an isoform of the β‐subunit of protein kinase CK2. The predicted Su(Ste) gene product contains a drastically changed amino acid sequence of the N‐terminal fragment as compared to the earlier described bCK2tes gene considered to be an ancestor of the Su(Ste) repeats. The following peculiarities of molecular divergence of the Su(Ste) and βCK2tes genes are revealed: damages of the autophosphorylation site; usage of an alternative splicing site instead of a damaged one; conservation of the zinc finger domain in spite of local ORF alterations.
Oxidative stress and hypoxia/reoxygenation trigger CD95 (APO‐1/Fas) ligand expression in microglial cells
Authors: Vogt Markus , Bauer Manuel K.A , Ferrari Davide , Schulze-Osthoff Klaus
Published: July 2002

Apoptosis plays an important role in neurodegeneration, although the mechanisms and mediators in the brain are largely unknown. Because microglial cells have been suggested to contribute to apoptosis in neurological disorders, we investigated the expression of the death ligand CD95L in this cell type. We found that, compared to classical mediators of microglial activation, the most potent inducer of CD95L was oxidative stress. Exposure of microglial cells to H2O2 or paraquat rapidly triggered CD95L mRNA and protein expression, associated with the activation of transcription factor NF‐κB. Enhanced expression of CD95L was further found following exposure of cells to hypoxia and subsequent reoxygenation. Our results indicate a potential role of CD95L in oxidative stress‐mediated cell death, ischemia/reperfusion and other diseases with a disturbed redox balance.
The GalR2 galanin receptor mediates galanin‐induced jejunal contraction, but not feeding behavior, in the rat: differentiation of central and peripheral effects of receptor subtype activation
Authors: Wang Suke , Ghibaudi Lorraine , Hashemi Tanaz , He Chaogang , Strader Catherine , Bayne Marvin , Davis Harry , Hwa Joyce J
Published: July 2002

The neuropeptide galanin mediates a diverse array of physiological functions through activation of specific receptors. Roles of the three recently cloned galanin receptors (GalRs) in rat intestinal contraction and food intake were examined using GalR‐selective ligands and the results were compared with the pharmacological profiles of defined GalRs. The action profile of these ligands in jejunal contraction resembled only that of GalR2 and only a high level of GalR2 mRNA was detected in the tissue, supporting GalR2 as the receptor mediating jejunal contraction. The action profile for food intake in rats excluded GalR2, GalR3 and the putative pituitary galanin receptor as the ‘feeding receptor’, suggesting that either GalR1 or an unidentified GalR is responsible for mediating this function.