bluuebunny/crossref_metadata_2025 huggingface dataset:
The mitochondrial permeability transition pore may comprise VDAC molecules
Authors: Szabó Ildikó , Pinto Vito De , Zoratti Mario
Published: July 2002

The electrophysiological properties of isolated mitochondrial porin (VDAC), reconstituted in planar bilayers or proteoliposomes, resemble those of the mitochondrial megachannel believed to be the permeability transition pore. In particular, a correspondence was found with regard to the voltage dependence: VDAC was driven to closed states by potentials of either sign, but the effect was not symmetrical; voltages negative in the compartment to which VDAC was added were more effective. The results are consistent with the hypothesis that the FTP may consist of two cooperating VDAC channels, plus presumably an adenine nucleotide carrier dimer and a third component known to be part of the mitochondrial benzodiazepine receptor.
Alternative transcripts of the human CD23/FcϵRII
Authors: Matsui Minoru , Nunez Rafael , Sachi Yoshihumi , Lynch Richard G. , Yodoi Junji
Published: July 2002

Human CD23/FcϵRII is a 45 kDa type‐II membrane glycoprotein having two isoforms (a and b) that only differ in the structures of their intracytoplasmic tails. CD23/FcϵRII has been demonstrated to have multiple roles in the immune system such as regulation of lymphocyte growth and differentiation and IgE‐mediated immune responses. Here, we found that the human B‐cell line RPMI8866, in addition to a and b transcripts, contained shorter transcripts (a' and b') that lack the entire third exon. These alternative transcripts were also detected in peripheral blood lymphocytes as well as other hematopoietic cell lines with CD23/FcϵRII. Because exon 3 encodes all of the transmembrane segment and the anchoring region of the cytoplasmic tail, it is suggested that a' and b' transcripts encode secretory forms of CD23/FcϵRII or they may function as regulatory transcripts involved in the control of CD23/FcϵRII expression.
Enhancement by retinoid of hemin‐induced differentiation of human leukemia K562 cell line
Authors: Nakajima Osamu , Hashimoto Yuichi , Iwasaki Shigeo
Published: July 2002

The effect of retinoid on human leukemia K562 cell differentiation induced by hemin was examined. Retinoids (retinoic acid and synthetic retinoids [Am80 and Ch55]) dose‐dependently enhanced hemin‐induced erythroid differentiation of K562 cells, though these retinoids themselves did not induce the differentiation. Under optimal conditions, these retinoids caused a doubling of the population of hemin‐induced differentiated cells. In addition, co‐treatment of cells with hemin and retinoid led to longer maintenance of the differentiated state after the removal of hemin, which might imply aquisition of irreversibility of hemin‐induced differentiation. These results suggest that the combination of retinoids with other differentiation inducers might be useful for leukemia therapy in cases where the leukemic cells are poorly responsive or unresponsive to retinoids, alone.
Expression cloning of an epithelial amiloride‐sensitive Na+ channel
Authors: Lingueglia Eric , Voilley Nicolas , Waldmann Rainer , Lazdunski Michel , Barbry Pascal
Published: July 2002

A complementary DNA encoding an amiloride‐sensitive Na+ channel has been cloned and characterized from rat colon. The protein encoded by the cDNA has a sequence of 699 amino acids (79 kDa) containing several putative membrane spanning domains and potential phosphorylation sites. It forms a channel that has the electrophysiological and pharmacological properties characteristic of the epithelial Na+ channel. Homologies (including in transmembrane domains) have been found between a part of the channel sequence and the Mec4 gene product of Caenorhabditis elegans, a protein associated with mutation‐induced neuronal degeneration.
Carboxyl group hydrogen bonding in X‐ray protein structures analysed using neutron studies on amino acids
Authors: Ramanadham M. , Jakkal V.S. , Chidambaram R.
Published: July 2002

A method is proposed to make a distinction between ionized and neutral carboxyl groups in X‐ray protein structures. This is based on an analysis of the relative hydrogen bonding populations and bond‐length bond‐valence correlations in high‐precision neutron studies of amino acids and small peptides. With the help of this method, four amino acid residues containing carboxyl groups in the refined structure of triclinic hen egg‐white lysozyme have been analysed. Two of these, Glu‐35 and Asp‐52, are involved in lysozyme function, while the other two, Glu‐7 and Asp‐101, form a protein‐protein inter‐molecular contact in the triclinic structure.
Glutamate transporters from brain
Authors: Kanner Baruch I.
Published: July 2002

The removal of neurotransmitters by their transporters in presynaptic nerve terminals and glial cells plays an important role in the termination of synaptic transmission. Many neurotransmitter transporters, which are sodium‐ and chloride‐coupled, have been cloned and shown to constitute a large superfamily. Glutamate is the major excitatory neurotransmitter in the central nervous system. If not efficiently removed, it causes death of neuronal cells. Its transporter couples the flow of glutamate to that of sodium and potassium. Recently three different but related glutamate transporters have been cloned, which have no significant homology to the members of the superfamily.
X‐linked Kallmann syndrome
Authors: Lutz Beat , Rugarli Elena I. , Eichele Gregor , Ballabio Andrea
Published: July 2002

Kallmann syndrome is a human genetic disorder characterized by the association of hypogonadism with the inability to smell, and is due to defects in the olfactory system development (i.e. incomplete migration of olfactory axons and of gonadotropin‐releasing hormone producing neurons from the olfactory epithelium to the forebrain; aplasia or hypoplasia of olfactory bulbs and tracts). The human X‐linked Kallmann syndrome gene and its chicken homologue have been cloned. Their protein products contain fibronectin type III repeats and a ‘four‐disulfide‐core’ domain also found in molecules that are involved in neural development. Consistent with the human phenotype, the chicken Kallmann gene is expressed in the developing olfactory bulb. At present the molecular and cellular mechanism of action of the Kallmann syndrome gene product is unknown. Based on expression studies and the characteristic domains of the predicted protein, it is hypothesized that the protein may be involved in targeting olfactory axons to the bulb. Alternatively, the Kalunann protein could be an extracellular matrix component required for the proper formation of the multilayered structure of the olfactory bulb.
Phosphatidylglycerol dependent protein translocation across the Escherichia coli inner membrane is inhibited by the anti‐cancer drug doxorubicin
Authors: Phoenix David A. , de Wolf Frits A. , Staffhorst Rutger W.H.M. , Hikita Chinami , Mizushima Shoji , Kruijff Bende
Published: July 2002

OmpF‐Lpp, a model secretory protein, requires both a positively charged signal sequence and phosphatidylglycerol (PG) for efficient translocation across the E. coli inner membrane. Modification of the signal sequence can, however, remove both these prerequisites for translocation providing OmpF‐Lpp mutants which undergo either PG and charge dependent or PG and charge independent translocation. Here we show that positively charged membrane interactive compounds (polylysine & doxorubicin) are able to inhibit PG dependent translocation of the OmpF‐Lpp signal sequence mutants but not PG independent translocation. Doxorubicin is also shown to bind more efficiently to liposomes containing increased levels of anionic lipid indicating that in these assays it may be inhibiting translocation by preventing electrostatic interaction between the anionic lipid head group and the positively charged signal sequences.
Cloning and expression of a cDNA for the human prostacyclin receptor
Authors: Katsuyama Masato , Sugimoto Yukihiko , Namba Tsunehisa , Irie Atsushi , Negishi Manabu , Narumiya Shuh , Ichikawa Atsushi
Published: July 2002

A functional cDNA for the human prostacyclin receptor was isolated from a cDNA library of CMK cells, a human megakaryocytic leukaemia cell line. The cDNA encodes a protein consisting of 386 amino acid residues with seven putative transmembrane domains and a deduced molecular weight of 40,956. [3H]Iloprost specifically bound to the membrane of CHO cells stably expressing the cDNA with a Kd of 3.3 nM. This binding was displaced by unlabelled prostanoids in the order of iloprost = cicaprost ⪢ carbacyclin ⪢ prostaglandin E1 (PGE1) > STA2. PGE2, PGD2 and PGF2α did not inhibit it. Iloprost in a concentration‐dependent manner increased the cAMP level and generated inositol trisphosphate in these cells, indicating that this human receptor can couple to multiple signal transduction pathways.
Deletion analysis of the dystrophin‐actin binding domain
Authors: Corrado K. , Mills P.L. , Chamberlain J.S.
Published: July 2002

Three sequence motifs at the N‐terminus of dystrophin have previously been proposed to be important for binding to actin. By analyzing a series of purified bacterial fusion proteins deleted for each of these sites we have demonstrated that none of the three are critical for dystrophin‐actin interactions. Instead, our data suggest that sequences in the N‐terminal 90 amino acids of dystrophin, excluding a conserved KTFT motif, contain the major site for interaction with actin.
Independent control of transcription initiations from two sites by an initiator‐like element and TATA box in the human c‐erbB‐2 promoter
Authors: Mizuguchi Gaku , Kanei-Ishii Chie , Sawazaki Tetsuya , Horikoshi Masami , Roeder Robert G. , Yamamoto Tadashi , Ishii Shunsuke
Published: July 2002

Transcription of the human c‐erbB‐2‐proto‐oncogene starts mainly at two sites, nucleotide positions + 1 and −69. The present studies have identified an initiator‐like element that specifies the position of transcription initiation at position −69. This initiator‐like element contains six GGA repeats and is located just downstream from the transcription start site between positions −68 and −45. In addition, both in vitro and in vivo studies indicated that transcription initiation at position +1 is specified by a TATA box 25 bp upstream from the transcription startpoint. Thus, initiation at two sites in the c‐erbB‐2 promoter is controlled independently by the initiator‐like element and the TATA box.
The binding of tissue prokallikrein to isolated human neutrophils
Authors: Anders Jonas , Kemme Michael
Published: July 2002

We have investigated the binding of human tissue kallikrein and the corresponding proenzyme to isolated human lymphocytes and neutrophils, respectively, by the means of steady‐state binding assays. It was demonstrated that only prokallikrein bound to washed neutrophils in a time‐dependent, specific and saturable manner, whereas in vitro binding of activated kallikrein to neutrophils or lymphocytes could be excluded. Our data provide strong evidence for the specific interaction of prokallikrein with intact human neutrophils and indicate a neutrophil surface binding site for the tissue kallikrein precursor which could be involved in kallikrein endocytosis and selective prokallikrein‐to‐kallikrein conversion.
Bilirubin and ascorbate antioxidant activity in neonatal plasma
Authors: Gopinathan Vimala , Miller Nicholas J. , Milner Anthony D. , Rice-Evans Catherine A.
Published: July 2002

Extremely low birth weight premature infants have been known for many years to have limited antioxidant protective capacity, especially with reference to those antioxidant components which do not cross the placenta until the third trimester of gestation. In this study the total antioxidant activity and the concentrations of individual antioxidants in plasma from premature neonates (27 ± 2 weeks gestation) compared to term babies (38–41 weeks gestation) have been examined. The results show elevated levels of ascorbate at birth in the plasma of premature neonates compared with those of term babies, but the total plasma antioxidant status of the premature babies is significantly lower than that of term babies. At 5 days post‐partum the ascorbate levels are within the normal adult range and plasma bilirubin levels are considerably enhanced in both groups, while the total plasma antioxidant status of the premature neonates has increased. Analysis of the relationship between the total plasma antioxidant activity and the bilirubin concentration show a direct, highly significant correlation for the term group, r
2 = 0.774, consistent with significance of bilirubin as a plasma antioxidant.
Voltage‐dependent Ca2+ channels in Chinese hamster ovary (CHO) cells
Authors: Skryma R. , Prevarskaya N. , Vacher P. , Dufy B.
Published: July 2002

Voltage‐dependent Ca2+ channels were identified in CHO‐K1 cells, currently used in molecular biology studies. Experimental data obtained at both macroscopic and single‐channel levels using the patch‐clamp technique show that the Ca2+ current in CHO cells is similar to the high‐threshold L‐type of Ca2+ current previously observed in excitable cells. It can be carried by Ca2+ or Ba2+ ions, blocked by both inorganic (Co2+) and organic (nifedipine, isradipine) Ca2+ channel blockers. The unitary Ca2+ channel activity was characterized by a conductance of 19 pS in 60 mM Ca2+, by single exponential distribution of open times (t = 0.84 ms) and biexponential distribution of closed times (t
f, = 1.67 ms, t
s = 7.9 ms ). However, the functional role of these Ca2+ channels in CHO cells remains unclear.
Nicotinamide, a missing link in the early stress response in eukaryotic cells: A hypothesis with special reference to oxidative stress in plants
Authors: Berglund Torkel
Published: July 2002

A hypothesis is presented suggesting that nicotinamide (NIC) is an initial signal substance in the response of eukaryotic cells to conditions which cause DNA‐strand breakage, especially in connection with oxidative stress. In the stressed cell, NIC is released as a result of the activity of poly(ADP‐ribose)polymerase (PADPRP). PADPRP is known to be activated by DNA‐strand breakage, caused by e.g. oxidative stress or mutagens. NIC and its metabolite trigonelline (N‐methylnicotinic acid) can induce defensive metabolism at the gene level. Connections between NIC and DNA‐methylation are also considered. This hypothesis is discussed in the light of own observations and literature reports.
Structural analysis of recombinant von Willebrand factor: Identification of hetero‐ and homo‐dimers
Authors: Fischer Bernhard , Mitterer Artur , Schlokat Uwe , DenBouwmeester Renate , Dorner Friedrich
Published: July 2002

Wild‐type full‐length cDNA of von Willebrand factor (vWF) was expressed in CHO cells. Recombinant vWF (rvWF) was obtained and its molecular composition investigated by two‐dimensional electrophoresis. Results of first dimension electrophoresis under non‐reducing conditions showed that rvWF‐dimer represents a mixture of three different species. Second dimension electrophoresis under reducing conditions revealed, that these protein species represent (i) homo‐dimers of either two unprocessed or two fully processed, mature vWF polypeptides, and (ii) the hetero‐dimer of unprocessed and mature rvWF monomers. Compared with vWF‐dimers from human plasma, which contained predominantly proteolytically degraded polypeptides, recombinant vWF‐dimers were shown to consist of non‐proteolyzed subunits only.
Confirmation of the existence of a third family among peptidyl‐prolyl cis/trans isomerases Amino acid sequence and recombinant production of parvulin
Authors: Rahfeld Jens-U. , Rücknagel Karl Peter , Schelbert Birte , Ludwig Birgit , Hacker Jörg , Mann Karlheinz , Fischer Gunter
Published: July 2002

In addition to the major cyclophilin‐like peptidyl‐prolyl cis/trans isomerases (PPIases) of Escherichia coli an enzyme of very low relative molecular mass (10.1 kDa) was discovered in this organism which gave first indication of the existence of a novel family in this enzyme class [1994, FEBS Lett. 343, 65–69]. In the present report we describe the chemically determined amino acid sequence of four peptides derived from the 10.1 kDa protein by the treatment with either cyanogen bromide or endoproteinase Lys‐C. Together with a continuous run of 75 amino acids starting N‐terminally, the sequence of the mature enzyme, 92 residues in length, was elucidated. Cloning and determination of the primary structure of a DNA fragment encoding this enzyme were also performed. Overexpression of the enzyme by using multicopies of plasmid pSEP38 in E. coli and detecting an enhanced PPIase activity attributed to the 10.1 kDa enzyme provided additional proof that the 92 amino acid protein was a PPIase. The enzyme was called parvulin (lat.: parvulus, very small). Homology analyses indicated that several parvulin‐like proteins could be found in the database screened. To further elucidate the functional role of PPIases it might be of some importance that homologous proteins like the PrtM protein of Lactococcus lactis and the PrsA lipoprotein of Bacillus subtilis are known to be involved in the protein export and maturation machinery of the bacteria.
Synaptobrevin cleavage by the tetanus toxin light chain is linked to the inhibition of exocytosis in chromaffin cells
Authors: Höhne-Zell Barbara , Ecker Axel , Weller Ulrich , Gratzl Manfred
Published: July 2002

Exocytosis of secretory granules by adrenal chromaffin cells is blocked by the tetanus toxin light chain in a zinc specific manner. Here we show that cellular synaptobrevin is almost completely degraded by the tetanus toxin light chain within 15 min. We used highly purified adrenal secretory granules to show that synaptobrevin, which can be cleaved by the tetanus toxin light chain, is localized in the vesicular membrane. Proteolysis of synaptobrevin in cells and in secretory granules is reversibly inhibited by the zinc chelating agent dipicolinic acid. Moreover, cleavage of synaptobrevin present in secretory granules by the tetanus toxin light chain is blocked by the zinc peptidase inhibitor captopril and by synaptobrevin derived peptides. Our data indicate that the tetanus toxin light chain acts as a zinc dependent protease that cleaves synaptobrevin of secretory granules, an essential component of the exocytosis machinery in adrenal chromaffin cells.
Calcium influx and protein kinase Cα activation mediate arachidonic acid mobilization by the human NK‐2 receptor expressed in Chinese Hamster ovary cells
Authors: Arkinstall Steve , Emergy Isabelle , Church Dennis , Chollet Andre , Kawashima Eric
Published: July 2002

We have cloned a cDNA encoding the human ileal neurokinin‐2 (NK‐2) receptor which mediates powerful neurokinin A‐stimulated arachidonic acid (AA) and prostaglandin release when expressed in CHO cells. Two major signal transduction events appear to underlie this response. Firstly, AA liberation is critically dependent upon agonist‐stimulated influx of extracellular Ca2+ although not release from intracellular stores. Secondly, NK‐2 receptor‐linked AA mobilization requires concomitant PKC activation and based upon limited subtype immunodetectability as well as NKA‐stimulated translocation, PKCα could play a major role. While NKA‐stimulated Ca2+ mobilization is insensitive to preincubation with pertussis toxin, identical pretreatment inhibits AA release partially and blocks PKCa translocation completely. These observations indicate that in this cell system AA liberation reflects NK‐2 receptor‐dependent activation of two distinct but converging signal transduction pathways regulated by different G‐protein species and involving Ca2+ influx and PKCa activation.
The novel hexapeptide motif found in the acyltransferases LpxA and LpxD of lipid A biosynthesis is conserved in various bacteria
Authors: Vuorio Riitta , Härkönen Taina , Tolvanen Martti , Vaara Martti
Published: July 2002

Two bacterial acyltransferases (LpxA of Escherichia coli, LpxD of E. coli and Salmonella typhimurium) have previously been shown to consist of a very unusual tandem‐repeat structure with tens of repeating hexapeptides (24 hexapeptides in LpxA, 26 in LpxD). By sequencing LpxD of Yersinia
enterocolitica (a distant relative of E. coli and S. typhimurium within Enterobacteriaceae) as well as LpxA of S. typhimurium and Y. enterocolitica, and by analyzing the existing data on these enzymes of Ricketsia
rickettsii, it was now shown that the hexapeptide repeat pattern is a very conservative property of these enzymes. Even though the overall homology (allowing equivalent amino acids) between the four proteins was only 59% in LpxA and 58% in LpxD, the homology in the first residue of each hexapeptide was 87% in LpxA and 100% in LpxD. Secondary structure prediction by PredictProtein server suggested a very strong beta strand dominance in all the hexad regions. Accordingly, LpxA and LpxD of various bacterial origins can now be regarded as structurally very unusual enzymes, largely consisting of hexad repeats.
Trypanosoma cruzi epimastigote forms possess a Ca2+‐calmodulin dependent protein kinase
Authors: Ogueta S.B. , Solari A. , Téllez-Iñón M.T.
Published: July 2002

Trypanosoma cruzi epimastigote forms showed a tightly bound Ca2+‐calmodulin‐dependent protein kinase activity, which could be partially extracted from membranes and axonemes. The enzyme is constituted by subunits which were autophosphorylated in the absence of exogenous substrates. An antibody against CaM kinase II recognized a Ca2+‐ or Ca2+‐CaM‐dependent conformational epitope in these fractions. The detected bands were of molecular weights similar to the α and β subunits of the corresponding bovine brain enzyme (60 and 50 kDa). Studies using [125I]CaM revealed the presence of a CaM‐binding domain. These experiments confirm that the parasite possesses a paniculate CaM kinase with characteristics similar to the bovine brain enzyme.
Acylation of subtilisin with long fatty acyl residues affects its activity and thermostability in aqueous medium
Authors: Plou Francisco J. , Ballesteros Antonio
Published: July 2002

Subtilisin Carlsberg has been artificially hydrophobized by acylation with octanoyl or palmitoyl chlorides. Samples with several degrees of substitution were obtained. Hydrophobization facilitates in some cases the binding of synthetic or natural substrates. Furthermore, derivatized subtilisins show improved thermal stability (15‐fold at 45°C) in aqueous solution. As a result, octanoyl‐subtilisin exhibits enhanced thermostability without losing biological activity.
Wortmannin inhibits insulin‐stimulated but not contraction‐stimulated glucose transport activity in skeletal muscle
Authors: Lee Abraham D. , Hansen Polly A. , Holloszy John O.
Published: July 2002

In skeletal muscle, glucose transport is stimulated by insulin, contractions and hypoxia. In this study, we used the phosphatidylinositol 3‐kinase (PI 3‐kinase) inhibitor wortmannin to examine whether (i) PI 3‐kinase activity is necessary for stimulation of glucose transport by insulin in muscle, and (ii) PI 3‐kinase mediates a step in the pathway by which contractions/hypoxia stimulate glucose transport. Wortmannin completely blocked insulin‐ and insulin‐like growth factor‐1‐stimulated glucose transport in muscle. In contrast, wortmannin had no effect on the stimulation of glucose transport by contractions or hypoxia, providing evidence that PI 3‐kinase activity is not involved in the activation of glucose transport by these stimuli.
Green fluorescent protein: applications in cell biology
Authors: Gerdes Hans-Hermann , Kaether Christoph
Published: July 2002

The green fluorescent protein (GFP) of Aequorea victoria is a unique in vivo reporter for monitoring dynamic processes in cells or organisms. As a fusion tag GFP can be used to localize proteins, to follow their movement or to study the dynamics of the subcellular compartments to which these proteins are targeted. Recent studies where GFP technology has revealed new insights regarding physiological activities of living cells are discussed.
Characterization of human presenilin 1 using N‐terminal specific monoclonal antibodies: Evidence that Alzheimer mutations affect proteolytic processing
Authors: Mercken Marc , Takahashi Hiroshi , Honda Toshiyuki , Sato Kazuki , Murayama Miyuki , Nakazato Yuko , Noguchi Kaori , Imahori Kasutomo , Takashima Akihiko
Published: July 2002

The majority of cases of early‐onset familial Alzheimer disease are caused by mutations in the recently identified presenilin 1 (PS1) gene, located on chromosome 14. PS1, a 467 amino acid protein, is predicted to be an integral membrane protein containing seven putative transmembrane domains and a large hydrophilic loop between the sixth and seventh membrane‐spanning domain. We produced 7 monoclonal antibodies that react with 3 non‐overlapping epitopes on the N‐terminal hydrophilic tail of PS1. The monoclonal antibodies can detect the full‐size PS1 at M
r 47 000 and a more abundant M
r 28 000 product in membrane extracts from human brain and human cell lines. PC12 cells transiently transfected with PS1 constructs containing two different Alzheimer mutations fail to generate the 28 kDa degradation product in contrast to PC12 cells transfected with wild‐type PS1. Our results indicate that missense mutations in this form of familial Alzheimer disease may act via a mechanism of impaired proteolytic processing of PS1.
Morphological and electrophysiological properties of centrifuged stratified Xenopus oocytes
Authors: Richter Hans‐Peter , Hoock Christian , Neumcke Berthold
Published: July 2002

Summary— To separate and concentrate various cytoplasmic organelles in wild type and albino Xenopus oocytes, defolliculated cells were loaded on a Ficoll‐400 gradient and centrifuged. Optimum results were obtained with centrifugations at 10 000 g for 5 min at 20°C. The cells became pear‐shaped and appeared stratified with the white lipid yolk on top, an intermediate transparent zone of about 100–300 μm, and the greenish protein yolk at the bottom. To determine the cellular constituents, particularly of the transparent zone, electron microscopy was performed. The transparent zone was found to contain (from animal to vegetal) the various endoplasmic reticula, a layer of mitochondria, cytoplasm enriched in ribosomes and the depressed nucleus. In centrifuged stratified wild type oocytes, most of the pigment was layered on top of the protein yolk. The typical cortical aspects of the oocyte persisted. Centrifuged albino oocytes had a very pronounced transparent zone with sharp transitions to the lipid phase and to the protein yolk. The resting membrane potentials of centrifuged oocytes were between −35 and −65 mV, and the membrane resistances were in the 500 kΩ to 1 MΩ range. Under voltage clamp conditions, the oocytes exhibited Ca2+‐activated Cl− currents with biphasic kinetics and spontaneous oscillations of these currents. It is concluded that centrifuged stratified oocytes have normal electrophysiological properties, and that they are a suitable preparation to study the contribution of various cellular organelles to the propagation of second messengers in the cytosol.
Production of transforming growth factor (TGF) β by fetal lung cells
Authors: de Bortoli Christelle , Chailley‐Heu Bernadette , Bourbon Jacques R
Published: July 2002

Summary— TGF β is supposed to play an important role in the process of epithelial maturation in the developing fetal lung. Using an immunofluorescence approach, we showed that fetal rat lung fibroblasts elaborate the three TGF β isoforms known in mammals (TGF β1, β2 and β3) whereas epithelial cells appear to synthesize only TGF β1 and β3. Isolated fibroblasts secrete the three isoforms. Biological assay of TGF β activity in fibroblast‐conditioned media did not reveal significant changes according to the stage when fibroblasts were isolated.
Autoradiographic studies on the uptake of 3H‐noradrenaline and 3H‐serotonin by neurones and astrocytes in explant and primary cultures of rat CNS: Effects of antidepressants
Authors: Hösli E. , Hösli L.
Published: July 2002

Autoradiographic studies were made on the uptake of 3H‐noradrenaline and 3H‐serotonin in explant cultures and primary astrocyte cultures from various regions of rat central nervous system (cortex, cerebellum, locus coeruleus, nucleus raphé, spinal cord). In explant cultures from locus coeruleus and nucleus raphé cell bodies and processes of many neurones revealed intense labelling by 3H‐noradrenaline and 3H‐serotonin, respectively. In cultures from cortex, cerebellum and spinal cord the cell bodies of neurones did not show labelling by the monoamines but many nerve fibres in the outgrowth zone had taken up 3H‐noradrenaline and 3H‐serotonin. Astrocytes in explant cultures did not take up 3H‐noradrenaline and 3H‐serotonin whereas astrocytes in primary cultures showed heavy uptake of both monoamines. In contrast, amino acid transmitters such as 3H‐GABA and 3H‐glutamate were accumulated by astrocytes in explant as well as in primary cultures. Uptake of both 3H‐noradrenaline and 3H‐serotonin by neurones and astrocytes was considerably reduced or inhibited in Na+‐free incubation medium or at low temperature, suggesting an active uptake mechanism. Addition of the antidepressants maprotiline and (+)oxaprotiline inhibited the uptake of 3H‐noradrenaline by neuronal cell bodies and fibres in explant cultures and by astrocytes in primary cultures. The uptake of 3H‐serotonin by neurones and astrocytes was blocked by citalopram and paroxetine. Our studies demonstrate that astrocytes in primary cultures are able to actively take up 3H‐noradrenaline and 3H‐serotonin whereas there was no uptake of monoamines into astrocytes in explant cultures, suggesting that there is a difference between astrocytes in different culture systems (explant cultures vs primary cultures) with respect to the uptake of monoamine transmitters.
Increase in nitric oxide synthase and NADPH‐diaphorase in the adrenal gland of streptozotocin‐diabetic Wistar rats and its prevention by ganglioside
Authors: Afework Mekbeb , Lincoln Jill , Belai Abebech , Burnstock Geoffrey
Published: July 2002

Levels of nitric oxide synthase (NOS) and NADPH‐diaphorase in adrenal glands of streptozotocin‐diabetic rats of 8 and 12 weeks' duration compared with control rats were assessed with histochemical and biochemical techniques. Adrenal glands from streptozotocin‐diabetic rats of 8 weeks' duration treated with ganglioside were examined also. In the adrenal medulla of 8‐weeks‐ and 12‐weeks‐diabetic rats, NOS‐immunoreactive nerve fibres were increased and decreased, respectively; additional NOS‐immunoreactive and NADPH‐diaphorase stained cells, which appeared to be cortical cells, were located in medulla and cortex compared with controls. Increased intensity in NADPH‐diaphorase staining of the cortical cells of diabetic rats was observed also. Ganglioside treatment of the 8‐weeks‐diabetic rats prevented the diabetic‐induced increase in NOS‐immunoreactive nerve fibres. Also, it reduced most of the increase in the NOS‐immunoreactive and NADPH‐diaphorase stained cells and the intensity of NADPH‐diaphorase staining of cortical cells. With biochemical assay, a significant increase in NOS activity was found in the adrenal glands from 8‐weeks‐diabetic rats, and this increase was reduced by ganglioside treatment in four out of six diabetic rats.

In summary, streptozotocin‐induced diabetes causes an initial increase in the levels of NOS and NADPH‐diaphorase in the adrenal gland of rat, which was prevented by ganglioside treatment.
Six-month and lifetime prevalences of psychiatric disorders in inpatients with diabetes mellitus
Authors: de Mont-Marin F , Hardy P , Lepine JP , Halfon P , Feline A
Published: July 2002

The Composite International Diagnostic Interview (CIDI) was used to assess the prevalence of psychiatric disorders in a French population of 46 inpatients with diabetes mellitus. According to DSM-III-R criteria, 52.2% of subjects presented at least one psychiatric diagnosis in their lifetime and 41.3% did so less than six months before the study. Affective and anxiety disorders represented at least 83% of the psychiatric diagnoses. The risk for those disorders seems to be restricted to a predisposed group as only one of the 16 subjects who had suffered from an anxiety or depressive episode within the previous six months had never experienced such an episode before.
A conserved small GTP‐binding protein Alp41 is essential for the cofactor‐dependent biogenesis of microtubules in fission yeast
Authors: Radcliffe Pippa A. , Vardy Leah , Toda Takashi
Published: July 2002

The proper folding of tubulins and their incorporation into microtubules consist of a series of reactions, in which evolutionarily conserved proteins, cofactors A to E, play a vital role. We have cloned a fission yeast gene (alp41
+) which encodes a highly conserved small GTP‐binding protein homologous to budding yeast CIN4 and human ARF‐like Arl2. alp41
+ is essential, disruption of which results in microtubule dysfunction and growth polarity defects. Genetic analysis indicates that Alp41 plays a crucial role in the cofactor‐dependent pathway, in which it functions upstream of the cofactor D homologue Alp1D and possibly in concert with Alp21E.
A novel rod‐like opsin isolated from the extra‐retinal photoreceptors of teleost fish
Authors: Philp Alisdair R. , Bellingham James , Garcia-Fernandez Josè-M. , Foster Russell G.
Published: July 2002

We have isolated a novel opsin from the pineal complex of Atlantic salmon (Salmo salar) and from the brain of the puffer fish (Fugu rubripes). These extra‐retinal opsins share approximately 74% identity at the nucleotide and amino acid level with rod‐opsins from the retina of these species. By PCR, we have determined that the novel rod‐like opsin is not expressed in the salmon retina, and the retinal rod‐opsin is not expressed in the salmon pineal. Phylogenetic analysis suggests that the rod‐like opsins arose from a gene duplication event approximately 205 million years ago, a time of considerable adaptive radiation of the bony fish. In view of the large differences in the coding sequences of the pineal/brain rod‐like opsins, their extra‐retinal sites of expression, and phylogenetic position we have termed these novel opsins ‘extra‐retinal rod‐like opsins’ (ERrod‐like opsins). We speculate that the differences between retinal rod‐opsins and ERrod‐like opsins have arisen from their differing photosensory roles and/or genetic drift after the gene duplication event in the Triassic.
Induction of cell death by the lysosomotropic detergent MSDH
Authors: Li Wei , Yuan XiMing , Nordgren Gunnar , Dalen Helge , Dubowchik Gene M. , Firestone Raymond A. , Brunk Ulf T.
Published: July 2002

Controlled lysosomal rupture was initiated in lysosome‐rich, macrophage‐like cells by the synthetic lysosomotropic detergent, O‐methyl‐serine dodecylamide hydrochloride (MSDH). When MSDH was applied at low concentrations, resulting in partial lysosomal rupture, activation of pro‐caspase‐3‐like proteases and apoptosis followed after some hours. Early during apoptosis, but clearly secondary to lysosomal destabilization, the mitochondrial transmembrane potential declined. At high concentrations, MSDH caused extensive lysosomal rupture and necrosis. It is suggested that lysosomal proteases, if released to the cytosol, may cause apoptosis directly by pro‐caspase activation and/or indirectly by mitochondrial attack with ensuing discharge of pro‐apoptotic factors.
A novel SCN5A mutation associated with idiopathic ventricular fibrillation without typical ECG findings of Brugada syndrome
Authors: Akai Jun , Makita Naomasa , Sakurada Harumizu , Shirai Nobumasa , Ueda Kazuo , Kitabatake Akira , Nakazawa Kiyoshi , Kimura Akinori , Hiraoka Masayasu
Published: July 2002

Mutations in the human cardiac Na+ channel α subunit gene (SCN5A) are responsible for Brugada syndrome, an idiopathic ventricular fibrillation (IVF) subgroup characterized by right bundle branch block and ST elevation on an electrocardiogram (ECG). However, the molecular basis of IVF in subgroups lacking these ECG findings has not been elucidated. We performed genetic screenings of Japanese IVF patients and found a novel SCN5A missense mutation (S1710L) in one symptomatic IVF patient that did not exhibit the typical Brugada ECG. Heterologously expressed S1710L channels showed marked acceleration in the current decay together with a large hyperpolarizing shift of steady‐state inactivation and depolarizing shift of activation. These findings suggest that SCN5A is one of the responsible genes for IVF patients who do not show typical ECG manifestations of the Brugada syndrome.
Isolation and characterization of a higher plant ADP‐glucose pyrophosphorylase small subunit homotetramer
Authors: Salamone Peter R. , Greene Thomas W. , Kavakli Ibrahim H. , Okita Thomas W.
Published: July 2002

ADP‐glucose pyrophosphorylase (AGPase) is the allosterically regulated gateway for carbon entry into transient and storage starch in plants as well as glycogen in bacteria. This enzyme plays a key role in the modulation of photosynthetic efficiency in source tissues and directly determines the level of storage starch in sink tissues, thus influencing overall crop yield potential. AGPase is a tetrameric enzyme; in higher plants it consists of two regulatory large subunits (LS) and two catalytic small subunits (SS), while in cyanobacteria and prokaryotes the enzyme is homotetrameric. The potato SS gene in pML10 was mutated by hydroxylamine and mutants were screened for elevated homotetrameric activity by iodine vapor staining. This search strategy led to the isolation of SS mutants (SUP‐1, TG‐15) that had pyrophosphorylase activity in the absence of the LS. TG‐15 has a leucine to phenylalanine change at position 48 (L48F) that corresponds to a phenylalanine residue at the analogous position in theEscherichia colihomotetrameric AGPase as well as a valine to isoleucine change at position 59 (V59I). TG‐15 was partially purified and kinetic analysis revealed substrate and effector affinities equal to wild type heterotetrameric enzyme with the exception of ATP binding.
Trp12, a novel Trp related protein from kidney
Authors: Wissenbach Ulrich , Bödding Matthias , Freichel Marc , Flockerzi Veit
Published: July 2002

A novel member of the transient receptor potential (Trp) family of ion channels, Trp12, was identified. The Trp12 mRNA is abundantly expressed in mouse kidney and encodes a protein of 871 amino acid residues. Trp12 transfected cells reveal an elevated cytosolic Ca2+ and respond with a further increase of cytosolic Ca2+ to perfusion with hypoosmotic solutions. The human orthologue of murine Trp12 was localized on a genomic clone derived from human chromosome 12. It is composed of 15 translated exons. The intron placement within that primary structure does not correlate with the previously postulated splice sites in transcripts encoding the stretch‐inhibitable channel which shares a high degree of amino acid sequence identity with Trp12 and the vanilloid receptor type 1.
Osmotic stress activates distinct lipid and MAPK signalling pathways in plants
Authors: Munnik Teun , Meijer Harold J.G
Published: July 2002

Plants are continuously exposed to all kinds of water stress such as drought and salinity. In order to survive and adapt, they have developed survival strategies that have been well studied, but little is known about the early mechanisms by which the osmotic stress is perceived and transduced into these responses. During the last few years, however, a variety of reports suggest that specific lipid and MAPK pathways are involved. This review briefly summarises them and presents a model showing that osmotic stress is transmitted by multiple signalling pathways.
Cloning and functional analysis of the hematopoietic cell‐specific phospholipase Cγ2 promoter
Authors: Kang Jong Sun , Kohlhuber Franz , Hug Hubert , Marmé Dieter , Eick Dirk , Ueffing Marius
Published: July 2002

Phospholipase Cγ
2 (PLCγ
2) is a phospholipid‐converting enzyme which, upon receptor stimulation, is activated within membrane‐bound signalling complexes. In contrast to the highly ubiquitous PLCγ
1, PLCγ
2 is expressed predominantly in B‐lymphocytes. Associated with antigen‐coupling receptors it is activated by tyrosine phosphorylation after the triggering of B‐cell surface immunoglobulin. We have cloned and sequenced the human PLCγ
2 promoter. Primer extension analysis reveals the existence of a major transcriptional start site. The TATA‐less promoter contains G+C‐rich stretches with a cluster of contiguous SP1 consensus sites, an NF1, and an AP2 site between by −220 to −70. A construct containing the region from −189 to +78 confers full promoter activity, as shown by fusion to a luciferase reporter gene construct. The distal part of the promoter between by −662 to −293 containing an SRE, EBF and CACCC box contributed negatively to promoter activity in the B‐cell line Raji but not in three adherent cell lines. In Raji cells, PLCγ
2 mRNA is expressed at low levels with a half life greater than 4 h. After treatment with serum, TPA, retinoic acid, or with 5‐azacytidine increased levels of PLCγ
2 mRNA were induced in B‐cells.
Stabilization of cationic liposome‐plasmid DNA complexes by polyamines and poly(ethylene glycol)‐phospholipid conjugates for efficient in vivo gene delivery
Authors: Hong Keelung , Zheng Weiwen , Baker Andrew , Papahadjopoulos Demetrios
Published: July 2002

Stable complexes of cationic liposomes with plasmid DNA were prepared by (1) including a small amount of poly(ethylene glycol)‐phospholipid conjugate or (2) condensing the DNA with polyamines prior to the formation of liposome‐plasmid complexes. These preparations were stable for months at 4°C and gave reproducible high transfection activity for in vivo gene delivery after intravenous injection in mice. Under these conditions, the expression of marker gene (luciferase) was primarily in the lungs (reaching values up to 3 ng expression per mg tissue protein), but also in other tissues to a lesser extent. Non‐stabilized formulations lost all their transfection activity in 4 days. In these formulations cholesterol, not dioleoylphosphatidylethanolamine, was the helper lipid effective for sustaining high transfection activity in vivo. These new developments in formulation technology should enhance the potential for liposome‐mediated gene therapy.
Control of energy fluxes by the sarcoplasmic reticulum Ca2+‐ATPase: ATP hydrolysis, ATP synthesis and heat production
Authors: de Meis Leopoldo , Bianconi M.Lucia , Suzano Valdecir A
Published: July 2002

The experiments described indicate that heat is released when Ca2+leaks through the Ca2+‐ATPase of sarcoplasmic reticulum vesicles. In the presence of a transmembrane Ca2+concentration gradient, agents that modify the amount of ATP synthesized from ADP and Pialso modify the amount of heat produced by the hydrolysis of each ATP molecule. Thus, in the presence of heparin, less ATP is synthesized and more heat is produced. Conversely, with dimethyl sulfoxide more ATP is synthesized and less heat is produced. The data indicate that between limits (−10 to −30 kcal/mol) the Ca2+‐ATPase can regulate the interconversion of energy in such a way as to vary the fraction of energy derived from ATP hydrolysis which is converted into heat and that which is converted into other forms of energy.
Identification of a cyclic peptide inhibitor of platelet‐derived growth factor–BB receptor‐binding and mitogen‐induced DNA synthesis in human fibroblasts
Authors: Brennand David M. , Dennehy Ulla , Ellis Vincent , Scully Michael F. , Tripathi Poonam , Kakkar Vijay V. , Patel Geeta
Published: July 2002

Peptides corresponding to residues from Loops I and III of platelet‐derived growth factor–BB (PDGF‐BB) were examined for their potential to act as PDGF antagonists. We have identified two peptides which directly stimulated DNA synthesis in human dermal fibroblasts and a cyclic peptide which inhibited PDGF‐induced DNA synthesis. The inhibitory action of cyclic PDGF‐BB73‐81, on DNA synthesis was shown to be restricted to cells which express PDGF receptors. Also cyclic PDGF‐BB73‐81 specifically competed for 125I‐labelled PDGF‐BB but not for 125I‐labelled EGF binding to their respective cellular receptors. The cyclic peptide therefore provides a minimum structure to investigate PDGF/receptor interactions and our findings confirm the importance of the loop configuration of PDGF‐BB73‐81 in the native molecule. The cyclic peptide may constitute a basis for developing more potent inhibitors of PDGF action.
Apoptosis induction by inhibitors of Ser/Thr phosphatases 1 and 2A is associated with transglutaminase activation in two different human epithelial tumour lines
Authors: von Zezschwitz Caroline , Vorwerk Hilke , Tergau Frithjof , Steinfelder Hans Jürgen
Published: July 2002

Two epithelial tumour lines, HeLa and KB, were treated with okadaic acid and calyculin A, specific inhibitors of Ser/Thr phosphatases (PP), esp. PP1 and PP2A. Morphological criteria, analysis of DNA fragmentation and studies of membrane integrity revealed that both agents concentration‐ and time‐dependently induced apoptosis at nanomolar concentrations which in these cells was associated with the stimulation of a transglutaminase activity. Since a non‐functional derivative of okadaic acid did not affect cell viability apoptosis was apparently related to the inhibition of PP1 and PP2A. Membrane damage marker activity was delayed by at least 24 h when compared to nuclear alterations.
High levels of rhodopsin phosphorylation in missense mutations of C‐terminal region of rhodopsin
Authors: Ohguro Hiroshi
Published: July 2002

Rhodopsin phosphorylation was investigated using synthetic C‐terminal peptides from rhodopsin. The peptides were phosphorylated by expressed rhodopsin kinase (RK) in the presence of a photolyzed truncated rhodopsin at the C‐terminus. No peptide phosphorylation was detected under dark or in conditions in which RK was inactive. However, the phosphorylation rate was significantly higher in the following three peptides: (345M Rho, 330DDEASTTVSKTETSQMAPA; 347S Rho, 330DDEASTTVSKTETSQVASA; and 347L Rho: 330DDEASTTVSKTETSQVALA) taken from missense mutations of rhodopsin found in patients with autosomal retinitis pigmentosa (ADRP) as compared with that from wild‐type rhodopsin (330DDEASTTVSKTETSQVAPA). Distribution of the phosphorylation showed a similar ratio among three serines (334, 338 and 343) in 347L Rho mutation to wild type. However, 345M Rho and 347S Rho peptides showed higher phosphorylation at Ser334. The data obtained suggests that an abnormally high rate of phosphorylation in missense mutations around the rhodopsin C‐terminus may change the position of phosphorylation and inactivation process of the visual transduction.
Mitochondrial membrane potential and hydroethidine‐monitored superoxide generation in cultured cerebellar granule cells
Authors: Budd Samantha L. , Castilho Roger F. , Nicholls David G.
Published: July 2002

Mitochondrial depolarisation has been reported to enhance the generation of superoxide anion (O⋅−
2) in a number of cell preparations while an inhibition has been observed with isolated mitochondria. Cerebellar granule cells equilibrated with >1 μM hydroethidine (dihydroethidium) which is oxidised to the fluorescent ethidium cation by O⋅−
2 showed a large increase in fluorescence on protonophore addition. However, controls showed the fluorescent enhancement to be a consequence of release of unbound preformed ethidium from the mitochondrial matrix within the cell with resultant fluorescent enhancement. This ambiguity was removed by the use of low (1 μM) concentrations of dye in which case generated ethidium remained bound within the mitochondria. Under these conditions antimycin A, but not protonophore addition, produced an increase in fluorescence. It is concluded that excess ethidium acts as an indicator of mitochondrial membrane potential obscuring the monitoring of O⋅−
2 and that certain experiments employing this indicator in cells may require re‐evaluation.