bluuebunny/crossref_metadata_2025 huggingface dataset:
Attenuation of agonist‐induced desensitization of the rat substance P receptor by progressive truncation of the C‐terminus
Authors: Sasakawa Nobuyuki , Sharif Mohammed , Hanley Michael R.
Published: July 2002

We have investigated the C‐terminal tail of the rat substance P receptor (SPR) as a domain essential for agonist‐induced desensitization. Four progressively shorter mutants, using premature termination in the C‐terminus, were constructed and compared with the unaltered SPR using ectopic expression of wild‐type and mutant receptors in Xenopus oocytes. These mutants were designated D16, D47, D70 and D96 with 16, 47, 70 and 96 amino acids residues deleted from the tail, respectively. Wild type SPR, D16 and D47 exhibited normal current responses when challenged with substance P, but D70 and D96 had reduced maximal current responses (70% and 5% of wild type SPR, respectively). D70, however, exhibited substantial resistance to substance P‐induced desensitization in that 55%, versus 8% for wild type SPR, of the peak current of the first response was preserved on second challenge with substance P. Therefore, a domain from residues 338 to 360 of the rat SPR, though not necessary for the functional activity of the receptor, plays an essential role in agonist‐induced desensitization.
A molecular mechanism for qE‐quenching
Authors: Crofts Antony R. , Yerkes Cristine T.
Published: July 2002

We discuss energy‐dependent fluorescence lowering (qE‐quenching), and suggest a model to explain the experimental data currently available. The main elements of the model are: (a) the qE‐quenching reflects a mechanism associated with a component of the light‐harvesting antenna rather than the reaction center of photosystem (PS) II — we suggest that it occurs through formation of an efficient quencher in one of the minor chlorophyll protein (CP) complexes; (b) the minor CPs have glutamate residues instead of glutamines at positions shown in light‐harvesting complex II (LHCII) to be ligands to chlorophylls near the lumenal interface. We suggest that the quenching reflects a change in ligation of chlorophyll on protonation of these glutamate residues leading to formation of an exciton coupled dimer with a neighboring pigment, in which additional energy levels allow vibrational relaxation of the excited singlet. The model accounts for the dependence on low lumenal pH, the ligand residue changes between LHCII and the minor CPs, the preferential distribution of components of the xanthophyll cycle in the minor CPs, the inhibition of qE‐quenching by DCCD, and the specific binding of DCCD to the minor CPs.
Increased β‐amyloid release and levels of amyloid precursor protein (APP) in fibroblast cell lines from family members with the Swedish Alzheimer's disease APP670/671 mutation
Authors: Johnston Janet A. , Cowburn Richard F. , Norgren Svante , Wiehager Birgitta , Venizelos Nikolaos , Winblad Bengt , Vigo-Pelfrey Carmen , Schenk Dale , Lannfelt Lars , O'Neill Cora
Published: July 2002

Cell lines transfected with the Swedish Alzheimer's disease amyloid precursor protein APP670/671 mutation release significantly more β‐amyloid than wild‐type cells. Citron et al. [Proc. Natl. Acad. Sci. USA (1994) in press] have recently shown that fibroblasts carrying the APP670/671 mutation also release more β‐amyloid than control cells [1]. The present study confirms a ca. threefold increase in β‐amyloid release from mutation‐bearing fibroblasts. APP mRNA levels did not differ between mutation‐bearing and control cells, although mutation‐bearing fibroblasts contained significantly more APP751/770 than controls. Mild stress decreased β‐amyloid secretion and increased APP751/770 levels in all cell lines. In conclusion, the proportion of APP committed to amyloidogenic processing is increased in fibroblasts from family members with the APP670/671 mutation, and this mutation may also compromise the APP stress response.
p53 expression in nitric oxide‐induced apoptosis
Authors: Meßmer Udo K. , Ankarcrona Maria , Nicotera Pierluigi , Brüne Bernhard
Published: July 2002

Nitric oxide (NO) is a diffusible messenger involved in several patho‐physiological processes including immune‐mediated cytotoxicity and neural cell killing. NO or the products of its redox chemistry can cause DNA damage and activate subsequent lethal reactions including energy depletion and cell necrosis. However, regardless of whether it is endogenously produced in response to cytokines, or generated by chemical breakdown of donor molecules, NO can also induce apoptosis in different systems. Here, we report that NO generation in response to a cytokine induced NO‐synthase or by NO donors stimulates the expression of the tumor suppressor gene, p53, in RAW 264.7 macrophages or pancreatic RINm5F cells prior to apoptosis. NO‐synthase inhibitors such as N
G‐monomethyl‐l‐arginine prevent the inducible NO generation as well as p53 expression and apoptosis. Since p53 expression is linked to apoptosis in some cells exposed to DNA damaging agents, we suggest that NO‐induced apoptosis in these cell systems is the consequence of DNA damage and subsequent expression of this tumor suppressor gene.
Structure of the large ribosomal subunit RNA of Phytophthora megasperma, and phylogeny of the oomycetes
Authors: Van der Auwera Gert , Chapelle Sabine , De Wächter Rupert
Published: July 2002

The 5.8S and 28S rRNA sequences of the oomycete Phytophthora megasperma were determined in order to study the secondary structure of these molecules and to assess the phylogenetic position of the oomycetes among the eukaryotes. Preliminary results point to an affiliation between the oomycetes, dinoflagellates and ciliates, a cluster which seems related to the fungi. In the course of this work, we developed a set of primers which allow sequencing and PCR amplification of eukaryotic large ribosomal subunit RNA genes of a wide range of phylogenetically distant organisms.
Mitochondrial respiratory chain inhibitors induce apoptosis
Authors: Wolvetang Ernst J. , Johnson Karina L. , Krauer Kenia , Ralph Steve J. , Linnane Anthony W.
Published: July 2002

In this paper the specific mitochondrial respiratory chain inhibitors rotenone and antimycin A and the highly specific mitochondrial ATP‐synthase inhibitor oligomycin are shown to induce an apoptotic suicide response in cultured human lymphoblastoid and other mammalian cells within 12–18 h. The mitochondrial inhibitors do not induce apoptosis in cells depleted of mitochondrial DNA and thus lacking an intact mitochondrial respiratory chain. Apoptosis induced by respiratory chain inhibitors is not inhibited by the presence of Bcl‐2. We discuss the possible role of mitochondrial induced apoptosis in the ageing process and age‐associated diseases.
Na+‐dependent high affinity uptake of l‐glutamate in primary cultures of human fibroblasts isolated from three different types of tissue
Authors: Balcar Vladimir J. , Shen Jie , Bao Shisan , King Nicholas J.C.
Published: July 2002

Cultured human fibroblasts isolated from embryonic muscle, skin and peripheral nerve tissues were found to accumulate [3H]L‐glutamate by a Na+‐dependent uptake process strongly inhibited by several glutamate/aspartate analogues including d‐ and l‐aspartate, d‐ and l‐thero‐3‐hydroxyaspartate and l‐trans‐pyrrolidine‐2,4‐dicarboxylate but not d‐glutamate. It was also reduced by elevated concentrations of K+, Rb+ and Cs+. The values of K
m
's were 5–20 μM, well within the ‘high affinity’ region. Variations in the capacity (Vmax) of [3H]l‐glutamate uptake did not correlate with the origin (muscle, skin or nerve tissue) of the fibroblasts. The uptake characteristics suggest that it is mediated by a transport system similar to that commonly observed only in brain tissue.
β‐Cyclodextrin interacts with the Alzheimer amyloid β‐A4 peptide
Authors: Camilleri Patrick , Haskins Neville J. , Hewlett David R.
Published: July 2002

Electrospray ionisation mass spectrometry has been used to show that the synthetic 40 amino acid β‐amyloid peptide (β1‐40) interacts with the cyclic oligosaccharide β‐cyclodextrin. This interaction, presumably with the hydrophobic aromatic moieties on the peptide, has been shown to diminish substantially the neurotoxic effects of β1‐40 in a cell line.
Rapid validation of molecular structures of biological samples by electrospray‐mass spectrometry
Authors: Ashton D.S. , Beddell C.R. , Green B.N. , Oliver R.W.A.
Published: July 2002

A short account is presented of the method of measuring molecular masses (M
r) of pure biological samples by electrospray ionisation mass spectrometry. It is demonstrated that the technique yields M
r values with an effective accuracy equal to or better than 0.008% of the calculated M
r, provided that the correct molecular structure is employed in the calculation. It is therefore recommended that this method of measuring M
r's should be considered to form an essential part of all studies aimed at elucidating the molecular structure of purified biological macromolecules or for confirming the identity of labelled samples of such molecules.
Glycoprotein biosynthesis in Saccharomyces cerevisiae: ngd29, an N‐glycosylation mutant allelic to och1 having a defect in the initiation of outer chain formation
Authors: Lehle Ludwig , Eiden Antje , Lehnert Klaus , Haselbeck Anton , Kopetzki Erhard
Published: July 2002

Outer chain glycosylation in Saccharomyces cerevisiae leads to heterogeneous and immunogenic asparagine‐linked saccharide chains containing more than 50 mannose residues on secreted glycoproteins. Using a [3H]mannose suicide selection procedure a collection of N‐glycosylation defective mutants (designated ngd) was isolated. One mutant, ngd29, was found to have a defect in the initiation of the outer chain and displayed a temperature growth sensitivity at 37°C allowing the isolation of the corresponding gene by complementation. Cloning, sequencing and disruption of NGD29 showed that it is a non lethal gene and identical to OCH1. It complemented both the glycosylation and growth defect. Membranes isolated from an ngd29 disruptant or an ngd29mnn1 double mutant were no longer able, in contrast to membranes from wild type cells, to transfer mannose from GDPmannose to Man8GlcNAc2, the in vivo acceptor for building up the outer chain. Heterologous expression of glucose oxidase from Aspergillus niger in an ngd29mnn1 double mutant produced a secreted uniform glycoprotein with exclusively Man8GlcNAc2 structure that in wild type yeast is heavily hyperglycosylated. The data indicate that this mutant strain is a suitable host for the expression of recombinant glycoproteins from different origin in S. cerevisiae to obtain mammalian oligomannosidic type N‐linked carbohydrate chains.
Attempts to convert chymotrypsin to trypsin
Authors: Venekei István , Szilágyi László , Gráf László , Rutter William J.
Published: July 2002

Trypsin and chymotrypsin have specificity pockets of essentially the same geometry, yet trypsin is specific for basic while chymotrypsin for bulky hydrophobic residues at the PI site of the substrate. A model by Steitz, Henderson and Blow suggested the presence of a negative charge at site 189 as the major specificity determinant: Asp189 results in tryptic, while the lack of it chymotryptic specificity. However, recent mutagenesis studies have shown that a successful conversion of the specificity of trypsin to that of chymotrypsin requires the substitution of amino acids at sites 138, 172 and at thirteen other positions in two surface loops, that do not directly contact the substrate. For further testing the significance of these sites in substrate discrimination in trypsin and chymotrypsin, we tried to change the chymotrypsin specificity to Typsin‐like specificity by introducing reverse substitutions in rat chymotrypsin. We report here that the specificity conversion is poor: the Ser189Asp mutation reduced the activity but the specificity remained chymotrypsin‐like; on further substitutions the activity decreased further on both tryptic and chymotryptic substrates and the specificity was lost or became slightly Typsin‐like. Our results indicate that in addition to structural elements already studied, further (chymotrypsin) specific sites have to be mutated to accomplish a chymotrypsin → trypsin specificity conversion.
Peroxynitrite formed by simultaneous generation of nitric oxide and superoxide selectively inhibits bovine aortic prostacyclin synthase
Authors: Zou M.-H. , Ullrich V.
Published: July 2002

The effect of various oxidants on bovine aortic prostacyclin synthase was tested with 14C‐labelled prostaglandia endoperoxide as substrate. No sensitivity against hydrogen peroxide, superoxide or hydroxyl radicals was observed but hypochlorite inhibited with an IC50 value of 7 μM. Among the reactive nitrogen species nitric oxide and nitrogen dioxide radicals were ineffective, but peroxynitrite irreversibly blocked prostacyclin biosynthesis with an IC50 value of 50 nM. Peroxynitrite acted within seconds whereas hypochlorite required up to 30 min for completion. Simultaneous generation of nitric oxide and superoxide also caused inhibition which suggested that under pathological conditions like ischemia‐reperfusion not only the vasodilatory effects of nitric oxide but also those of prostacyclin could be eliminated.
Identification of the nuclear transcription factor NFκB in rat brain after in vivo ethanol administration
Authors: Ward Roberta J. , Zhang Ying , Crichton Robert R. , Piret Bernard , Piette Jacques , de Witte Phillipe
Published: July 2002

NFκB a nuclear transcription factor, was induced in the brain nuclear fraction of naive rats after an acute injection of ethanol, 2 g/kg. In contrast, rats which had been chronically alcoholised showed the constitutively active NFκB‐like complex only after a further acute dose of ethanol. Hepatic nuclear fractions did not exhibit the specific NFκB‐like complex during the first 45 min after acute ethanol injection, beyond that which was normally constitutively present. Such activation of NFκB‐like complex in the brains of the naive rats may play an important role in the cellular protective response.
Stability of positive and negative symptoms in schizophrenic patients: a 3-year follow-up study
Authors: Dollfus S , Petit M
Published: July 2002

The course of negative and positive symptoms was studied in neuroleptic-treated patients over a 3-year period, in consideration also of the initial phase of illness (post-acute or chronic). This study was carried out in a broadly defined schizophrenic sample, in order not to give preference to one diagnostic subgroup over another. Forty-six patients were evaluated every year for 3 years, 23 in the post-acute group and 23 in the chronic group. Aggravations of the Clinical Global Impression (CGI) and of the SANS total score were observed, regardless of the group (chronic or post-acute). This global aggravation confirmed Kraepelin's concept of dementia praecox; moreover, this aggravation was not due to an increase in the number of patients relapsing, or to an aggravation of akinesia. Three types of negative and positive symptom courses were observed: i) the mean sub-scores of positive symptoms, such as hallucinations, delusions, positive formal thought disorders, and of negative symptoms such as flattening affect, avolition/apathy and attentional impairment, did not vary significantly over time in either group; ii) the mean sub-scores of bizarre behavior and alogia fluctuated over time (p < 0.05) and only poverty of speech was perfectly stable among the items constituting alogia; iii) the mean subscores of anhedonia/asociality worsened significantly over time irrespective of the groups (p < 0.05), and among the items constituting anhedonia, recreational interest-activities and intimacy-closeness abilities worsened (p < 0.05 and p < 0.01, respectively). This aggravation was neither due to an increase in neuroleptic doses nor to the duration and chronicity of illness. However, negative symptoms, except anhedonia, can be reversible in some patients. The very strong stability of anhedonia, whatever the group, emphasize the importance of taking anhedonia into account in future diagnostic classifications.
Depression, perceived parental rearing and self-acceptance
Authors: Richter J , Richter G , Eisemann M , Seering B , Bartsch M
Published: July 2002

Psychoanalytical, behavioural and cognitive theories assume a continuous process in the development of self-acceptance as an important psychological variable by parent-child interaction during childhood and adolescence. The objective of the present study was to investigate the relationships between perceived parental rearing behaviour and self-acceptance in psychiatric inpatients. The results of extreme group comparisons pointed to the reciprocal discriminative power of parental rearing factors and self-acceptance scores, supporting the hypothesis of a continuous process in the development of self-acceptance and mood traits. Perceived parental rearing predicted aspects of psychopathology in adulthood. The effects of maternal and paternal behaviour appeared to be gender-specific. Alternative interpretations of these findings are discussed.
Inhibition of voltage‐dependent sodium channels by Ro 31‐8220, a ‘specific’ protein kinase C inhibitor
Authors: Lingameneni Ratnakumari , Vysotskaya Tanya N. , Duch Daniel S. , Hemmings Hugh C.
Published: July 2002

We find that several protein kinase C (PKC) inhibitors, previously considered to be specific, directly inhibit voltage‐dependent Na+ channels at their useful concentrations. Bisindolylmaleimide I (GF 1092037), IX (Ro 31‐8220) and V (an inactive analogue), but not H7 (a non‐selective isoquinolinesulfonamide protein kinase inhibitor), inhibited Na+ channels assessed by several independent criteria: Na+ channel‐dependent glutamate release and [3H]batrachotoxinin‐A 20‐α‐benzoate binding in rat cortical synaptosomes, veratridine‐stimulated 22Na+ influx in CHO cells expressing rat CNaIIa Na+ channels and Na+ currents measured in isolated rat dorsal root ganglion neurons by whole cell patch‐clamp recording. These findings limit the usefulness of the bisindolylmaleimide class PKC inhibitors in excitable cells.
Involvement of poly(ADP‐ribose) polymerase and activation of caspase‐3‐like protease in heat shock‐induced apoptosis in tobacco suspension cells
Authors: Tian Rui-Hua , Zhang Gui-You , Yan Chang-Hui , Dai Yao-Ren
Published: July 2002

The cleavage of poly(ADP‐ribose) polymerase (PARP) by caspase (casp)‐3 is an essential link in the apoptotic pathway in animal cells. In plant cells, however, there is no authentic evidence for the similar role that PARP may play during apoptosis. Using a heat shock (HS)‐induced apoptosis system of tobacco cells, we found that immediately after a 4 h heat treatment, PARP was cleaved to form an 89 kDa signature fragment, while DNA laddering appeared only after a 20 h recovery following the HS. An activation of casp‐3‐like protease was also observed. The results suggest that apoptosis in plants and animals may share common mechanisms. On the other hand, when cells were preincubated with 4 mM 3‐aminobenzamide or 2–8 mM nicotinamide, the specific inhibitors of PARP, before HS treatment, apoptotic cell death was reduced significantly. Our results thus imply that PARP may also be involved in apoptosis in a different way from the casp‐related events.
Cell‐cycle regulation of the p53‐inducible gene B99
Authors: Collavin Licio , Monte Martin , Verardo Roberto , Pfleger Cathie , Schneider Claudio
Published: July 2002

B99 is a p53‐inducible gene whose accumulation upon p53 activation is restricted to late S/G2 cells. Here we have analyzed B99 regulation during the cell cycle in murine cells with or without functional p53. We report that B99 accumulates in late S/G2 phase, is phosphorylated in mitosis, and disappears in G1 phase, regardless of the status of p53. As a complement to this observation, we show that B99 is not induced by p53 in quiescent cells. Therefore, B99 expression is modulated both by cell‐cycle regulatory mechanisms and by p53, and p53 can increase the cellular levels of B99 only during the window of the cell cycle when it is normally expressed. On the basis of these observations we rename B99 Gtse‐1 (G‐two‐ and S‐phase‐expressed).
Immunocontent and secretion of S100B in astrocyte cultures from different brain regions in relation to morphology
Authors: Pinto Silvana S. , Gottfried Carmem , Mendez Andreas , Gonçalves Daniela , Karl Juliana , Gonçalves Carlos A. , Wofchuk Susana , Rodnight Richard
Published: July 2002

Primary astrocyte cultures prepared from neonatal hippocampus, cerebral cortex and cerebellum were morphologically distinct. Cells from hippocampus and cortex were almost entirely protoplasmic, whereas cerebellar astrocytes had many processes; in the absence of serum these differences were accentuated. We compared the immunocontent and secretion of the mitogenic protein S100B in these cultures. Immunocontent was 2.5 times higher in cerebellar astrocytes than in hippocampal or cortical astrocytes. Cells from all three regions secreted S100B under basal conditions, but the secretion rate was higher in cerebellar astrocytes. Secretion depended on protein synthesis and was increased by incubation with forskolin or lysophosphatidic acid in mechanisms which were additive. The stellate morphology induced by forskolin was reversed by lysophosphatidic acid in hippocampal but not in cerebellar cultures, suggesting that S100B secretion was not associated with a process‐bearing phenotype of astrocytes.
Early signaling components in ultraviolet‐B responses: distinct roles for different reactive oxygen species and nitric oxide
Authors: A.-H.-Mackerness Soheila , John C.Fred , Jordan Brian , Thomas Brian
Published: July 2002

The nature and origin of the reactive oxygen species (ROS) involved in the early part of Ultraviolet‐B (UV‐B)‐induced signaling pathways were investigated in Arabidopsis thaliana using a range of enzyme inhibitors and free radical scavengers. The increase in PR‐1 transcript and decrease in Lhcb transcript in response to UV‐B exposure was shown to be mediated through pathways involving hydrogen peroxide (H2O2) derived from superoxide (O2

−). In contrast, the up‐regulation of PDF1.2 transcript was mediated through a pathway involving O2

− directly. The origins of the ROS were also shown to be distinct and to involve NADPH oxidase and peroxidase(s). The up‐regulation of Chs by UV‐B was not affected by ROS scavengers, but was reduced by inhibitors of nitric oxide synthase (NOS) or NO scavengers. Together these results suggest that UV‐B exposure leads to the generation of ROS, from multiple sources, and NO, through increased NOS activity, giving rise to parallel signaling pathways mediating responses of specific genes to UV‐B radiation.
Growth factor‐dependent signaling and cell cycle progression
Authors: Jones Steven M , Kazlauskas Andrius
Published: July 2002

There are three central ideas contained within this review. Firstly, growth factor‐stimulated signaling is not restricted to a 30–60 min window, but occurs at a much later time as well. Secondly, the second wave of signaling overlaps temporally with the cell cycle program and may be directly responsible for engaging it. Thirdly, the G1 to S interval appears to encompass two distinct phases of the cell cycle, during which the coordinated activation of distinct sets of signaling enzymes drives cell cycle progression. Each of these concepts is likely to initiate new investigation and hence provide additional insight into the fundamental question of how growth factors drive cell proliferation.
Analysis of N‐glycans of pathological tau: possible occurrence of aberrant processing of tau in Alzheimer's disease
Authors: Sato Yuji , Naito Yasuhide , Grundke-Iqbal Inge , Iqbal Khalid , Endo Tamao
Published: July 2002

In a previous study [Wang et al. (1996) Nat. Med. 2, 871–875], Wang et al. found (i) that abnormally hyperphosphorylated tau (AD P‐tau) isolated from Alzheimer's disease (AD) brain as paired helical filaments (PHF)‐tau and as cytosolic AD P‐tau but not tau from normal brain were stained by lectins, and (ii) that on in vitro deglycosylation the PHF untwisted into sheets of thin straight filaments, suggesting that tau only in AD brains is glycosylated. To elucidate the primary structure of N‐glycans, we comparatively analyzed the N‐glycan structures obtained from PHF‐tau and AD P‐tau. More than half of N‐glycans found in PHF‐tau and AD P‐tau were different. High mannose‐type sugar chains and truncated N‐glycans were found in both taus in addition to a small amount of sialylated bi‐ and triantennary sugar chains. More truncated glycans were richer in PHF‐tau than AD P‐tau. This enrichment of more truncated glycans in PHF might be involved in promoting the assembly and or stabilizing the pathological fibrils in AD.
GM3 ganglioside inhibits endothelin‐1‐mediated signal transduction in C6 glioma cells
Authors: Bassi Rosaria , Viani Paola , Giussani Paola , Riboni Laura , Tettamanti Guido
Published: July 2002

We found that sparse and confluent C6 glioma cells differ both in GM3 content, which increases with cell density, and in endothelin‐1 (ET‐1)‐induced phosphoinositide hydrolysis, which was markedly higher in the sparse cells than in the confluent. Also after manipulation of the cellular GM3 content through treatment with exogenous GM3 or with drugs known to affect GM3 metabolism, the ET‐1 effect was inversely related to GM3 cellular levels. Cell treatment with an anti‐GM3 mAb resulted in the enhancement of ET‐1‐induced phospholipase C activation and restored the capacity of GM3‐treated cells to respond to ET‐1. These findings suggest that the GM3 ganglioside represents a physiological modulator of ET‐1 signaling in glial cells.
Identification and characterization of a predominant isoform of human MKK3
Authors: Han Jiahuai , Wang Xinghao , Jiang Yong , Ulevitch Richard J , Lin Shengcai
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Mitochondrial acyl carrier protein is involved in lipoic acid synthesis in Saccharomyces cerevisiae
Authors: Brody Stuart , Oh Changkyu , Hoja Ursula , Schweizer Eckhart
Published: July 2002

The yeast gene, ACP1, encoding the mitochondrial acyl carrier protein, was deleted by gene replacement. The resulting acp1‐deficient mutants had only 5–10% of the wild‐type lipoic acid content remaining, and exhibited a respiratory‐deficient phenotype. Upon meiosis, the lipoate deficiency co‐segregated with the acp1 deletion. The role of ACP1 in long‐chain fatty acid synthesis was studied in fas1 and fas2 null mutants completely lacking cytoplasmic fatty acid synthase. When grown on odd‐chain (13:0 and 15:0) fatty acids, these cells showed less than 1% of C‐16 and C‐18 acids in their total lipids. Mitochondrial ACP is therefore suggested to be involved with the biosynthesis of octanoate, a precurser to lipoic acid.
The uptake of cholesterol at the small‐intestinal brush border membrane is inhibited by apolipoproteins
Authors: Boffelli Dario , Compassi Sabina , Werder Moritz , Weber Franz E , Phillips Michael C , Schulthess Georg , Hauser Helmut
Published: July 2002

The uptake of free and esterified cholesterol at the brush border membrane is protein‐mediated. Here we show that this sterol uptake is effectively inhibited by exchangeable serum apolipoproteins. Binding of the apolipoprotein to the brush border membrane mediates the inhibitory effect. Evidence is presented to show that the structural motif responsible for the inhibition is the amphipathic α‐helix.
Yeast mitochondrial F1F0‐ATPase: the novel subunit e is identical to Tim11
Authors: Arnold Isabel , Bauer Matthias F. , Brunner Michael , Neupert Walter , Stuart Rosemary A.
Published: July 2002

We report here the identification of the novel subunit of the mitochondrial F1F0‐ATPase from Saccharomyces cerevisiae, ATPase subunit e. Yeast ATPase subunit e displays significant similarities in both amino acid sequence, properties (hydropathy and predicted coiled‐coil structure) and orientation in the inner membrane, with previously identified mammalian ATPase subunit e proteins. Estimation of its native molecular mass and ability to be co‐immunoprecipitated with α subunit of the F1‐ATPase, demonstrate that subunit e is a subunit of the F1F0‐ATPase. Stable expression of subunit e requires the presence of the mitochondrially encoded subunits of the F0‐ATPase. Subunit e had been previously identified as Tim11 and was proposed to be involved in the process of sorting of proteins to the mitochondrial inner membrane.
Nitric oxide directly activates calcium‐activated potassium channels from rat brain reconstituted into planar lipid bilayer
Authors: Shin Jung Hoon , Chung Sungkwon , Park Eun Joo , Uhm Dae-Yong , Suh Chang Kook
Published: July 2002

Using the planar lipid bilayer technique, we tested whether NO directly activates calcium‐activated potassium (Maxi‐K) channels isolated from rat brain. We used streptozotocin (STZ) as NO donor, and the NO release was controlled with light. In the presence of 100–800 μM STZ, the Maxi‐K channel activity increased up to 3‐fold within several tens of seconds after the light was on, and reversed to the control level several minutes after shutting off the light. Similar activation was observed with other NO donors such as S‐nitroso‐N‐acetylpenicillamine and sodium nitroprusside. The degree of activity increase was dependent upon the initial open probability (P
init). When the P
init was lower, the activity increase was greater. These results demonstrate that NO can directly affect the Maxi‐K channel activity, and suggest that the Maxi‐K channel might be one of the physiological targets of NO in brain.
Characterization of two receptors for TRAIL1
Authors: Schneider Pascal , Bodmer Jean-Luc , Thome Margot , Hofmann Kay , Holler Nils , Tschopp Jürg
Published: July 2002

Two receptors for TRAIL, designated TRAIL‐R2 and TRAIL‐R3, have been identified. Both are members of the tumor necrosis factor receptor family. TRAIL‐R2 is structurally similar to the death‐domain‐containing receptor TRAIL‐R1 (DR‐4), and is capable of inducing apoptosis. In contrast, TRAIL‐R3 does not promote cell death. TRAIL‐R3 is highly glycosylated and is membrane bound via a putative phosphatidylinositol anchor. The extended structure of TRAIL‐R3 is due to the presence of multiple threonine‐, alanine‐, proline‐ and glutamine‐rich repeats (TAPE repeats). TRAIL‐R2 shows a broad tissue distribution, whereas the expression of TRAIL‐R3 is restricted to peripheral blood lymphocytes (PBLs) and skeletal muscle. All three TRAIL receptors bind TRAIL with similar affinity, suggesting a complex regulation of TRAIL‐mediated signals.
Human prion proteins expressed in Escherichia coli and purified by high‐affinity column refolding
Authors: Zahn Ralph , von Schroetter Christine , Wüthrich Kurt
Published: July 2002

An efficient method is presented for the production of intact mammalian prion proteins and partial sequences thereof. As an illustration we describe the production of polypeptides comprising residues 23–231, 81–231, 90–231 and 121–231 of the human prion protein (hPrP)

1
. Polypeptides were expressed as histidine tail fusion proteins into inclusion bodies in the cytoplasm of Escherichia coli and refolded and oxidized while N‐terminally immobilized on a nickel‐NTA agarose resin. This ‘high‐affinity column refolding’ facilitates the preparation of prion proteins by preventing protein aggregation and intermolecular disulfide formation. After elution from the resin the histidine tail can be removed using thrombin without cleaving the prion protein polypeptide chain. The same protocol as used here for hPrP has been successfully applied with bovine and murine prion proteins. The protein preparations are stable for weeks at room temperature in concentrated solution and are thus suitable for detailed structural studies. Preliminary biophysical characterization of hPrP(23–231) suggests that the C‐terminal half of the polypeptide chain forms a well‐structured globular domain, and that the N‐terminal half does not form extensive regular secondary structures.
Identification of rhodopsin in the pigeon deep brain
Authors: Wada Yasutaka , Okano Toshiyuki , Adachi Akihito , Ebihara Shizufumi , Fukada Yoshitaka
Published: July 2002

We detected rhodopsin gene expression in the pigeon lateral septum, a photosensitive deep brain region that is responsible for the photoperiodic gonadal response. The nucleotide sequence of the deep brain rhodopsin cDNA clone exactly matched that of the retinal one, indicating that a single rhodopsin gene is transcribed in the two tissues. Immunohistochemical analysis localized rhodopsin in the cerebrospinal fluid‐contacting neurons, which have been assumed to be photoreceptive cells in the deep brain. Pigeon rhodopsin seems to play dual important roles in the visual and non‐visual systems, the latter of which contributes to the photoperiodic response.
Cloning and characterization of MUPP1, a novel PDZ domain protein
Authors: Ullmer Christoph , Schmuck Karin , Figge Andrea , Lübbert Hermann
Published: July 2002

Using the yeast two‐hybrid system we isolated a cDNA clone encoding a novel protein interacting with the C‐terminal domain of the 5‐HT2C receptor. The protein, named MUPP1 (multi‐PDZ‐domain protein), contains thirteen PDZ domains and no obvious catalytic domain; it is related to hINADL and a putative C. elegans polypeptide referred to as C52A11.4 containing six or ten PDZ domains, respectively. Domains highly similar to those of MUPP1 are arrayed in the same order in all three proteins. The MUPP1 gene is localized on human chromosome 9p24‐p22. Transcripts encoding MUPP1 are abundant in the brain as well as in several peripheral organs.
Nitric oxide (NO) disrupts specific DNA binding of the transcription factor c‐Myb in vitro
Authors: Brendeford Elen M , Andersson Kristin B , Gabrielsen Odd S
Published: July 2002

In an attempt to elucidate signal transduction pathways which may modulate DNA binding of the transcription factor c‐Myb, we investigated whether c‐Myb could be a target for the signaling molecule nitric oxide (NO) in vitro. NO‐generating agents severely inhibited specific DNA binding of the c‐Myb minimal DNA‐binding domain R2R3. This inhibition was readily reversible upon treatment with excess DTT. A redox‐sensitive cysteine (C130) was required for this NO sensitivity. Moreover, a DNA‐binding domain carrying two of the avian myeloblastosis virus (AMV)‐specific mutations (L106H, V117D) appeared to be less sensitive to S‐nitrosylation than the wild‐type c‐Myb. This difference in NO sensitivity may influence the regulation of wild type versus AMV v‐Myb protein function.
Repulsive interparticle interactions in a denatured protein solution revealed by small angle neutron scattering
Authors: Receveur Véronique , Durand Dominique , Desmadril Michel , Calmettes Patrick
Published: July 2002

In order to investigate the effect of concentration in biological processes such as protein folding, small angle neutron scattering measurements were used to determine the second virial coefficient of solutions of both native and strongly denatured phosphoglycerate kinase and the radius of gyration of the protein at zero concentration. The value of the second virial coefficient is a good probe of the non‐ideality of a solution. The present results show that the unfolding of the protein leads to a drastic change in the repulsive intermolecular interactions. We conclude that these interactions are due mainly to the behaviour of the denatured polypeptide chain as an excluded volume polymer.
Structural analysis and proteolytic processing of recombinant G domain of mouse laminin α2 chain
Authors: Talts Jan F , Mann Karlheinz , Yamada Yoshihiko , Timpl Rupert
Published: July 2002

Four individual LG modules from the C‐terminus of the laminin α2 chain (LG1, LG2, LG4 and LG5) and combinations of these modules were prepared as recombinant products from transfected mammalian cells. This demonstrated that LG modules represent autonomously folding protein domains. Successful production depended on proper alignment of module borders and required a sequence correction at the C‐terminus which added an extra cysteine. The LG modules were glycosylated and shown by electron microscopy to have a globular shape, indicating proper folding. Evidence is provided for the splicing of a 12 bp exon in LG2, although this did not impair folding. Proteolytic cleavage at the C‐terminus of a basic sequence was observed close to the N‐terminus of LG3. A similar processing also occurs in tissue‐derived laminin‐2 and ‐4 which contain the α2 chain.
Statistical analysis of protein kinase specificity determinants
Authors: Kreegipuu Andres , Blom Nikolaj , Brunak Søren , Järv Jaak
Published: July 2002

The site and sequence specificity of protein kinases, as well as the role of the secondary structure and surface accessibility of the phosphorylation sites on substrate proteins, was statistically analyzed. The experimental data were collected from the literature and are available on the World Wide Web at http://www.cbs.dtu.dk/databases/PhosphoBase/. The set of data involved 1008 phosphorylatable sites in 406 proteins, which were phosphorylated by 58 protein kinases. It was found that there exists almost absolute Ser/Thr or Tyr specificity, with rare exceptions. The sequence specificity determinants were less strict and were located between positions −4 and +4 relative to the phosphorylation site. Secondary structure and surface accessibility predictions revealed that most of the phosphorylation sites were located on the surface of the target proteins.
Defective intracellular activity of GDP‐d‐mannose‐4,6‐dehydratase in leukocyte adhesion deficiency type II syndrome
Authors: Sturla Laura , Etzioni Amos , Bisso Angela , Zanardi Davide , De Flora Giovanni , Silengo Lorenzo , De Flora Antonio , Tonetti Michela
Published: July 2002

Leukocyte adhesion deficiency type II (LAD II) is a rare genetic disease characterized by severe immunodeficiency which is related to defective expression in leukocytes of sialyl‐Lewis X (SLeX), a fucosylated ligand for endothelial selectins. The molecular basis of LAD II is still unknown, but has been tentatively localized in the de novo pathway of GDP‐l‐fucose biosynthesis from GDP‐d‐mannose. Here, we demonstrate that in cell lysates from a LAD II patient, GDP‐d‐mannose‐4,6‐dehydratase (GMD), the first of the two enzymes of the pathway has a defective activity compared to control subjects. GMD in cell lysates from both parents showed intermediate activity levels. Cloning of GMD from patient and control lymphocytes ruled out any mutation affecting the amino acid GMD sequence and the purified recombinant proteins from both controls and the patient showed identical specific activities. Since the levels of immunoreactive GMD in cell lysates were comparable in the patient and in controls, the biochemical deficiency of intracellular GMD activity in LAD II seems to be due to mutation(s) affecting some still unidentified GMD‐regulating protein.
Mutational analysis of caveolin‐induced vesicle formation
Authors: Li Shengwen , Galbiati Ferruccio , Volonte' Daniela , Sargiacomo Massimo , Engelman Jeffrey A , Das Kallol , Scherer Philipp E , Lisanti Michael P
Published: July 2002

Caveolae are vesicular organelles with a characteristic uniform diameter in the range of 50–100 nm. Although recombinant expression of caveolin‐1 is sufficient to drive caveolae formation, it remains unknown what controls the uniform diameter of these organelles. One hypothesis is that specific caveolin‐caveolin interactions regulate the size of caveolae, as caveolin‐1 undergoes two stages of self‐oligomerization. To test this hypothesis directly, we have created two caveolin‐1 deletion mutants that lack regions of caveolin‐1 that are involved in directing the self‐assembly of caveolin‐1 oligomers. More specifically, Cav‐1 Δ61–100 lacks a region of the N‐terminal domain that directs the formation of high molecular mass caveolin‐1 homo‐oligomers, while Cav‐1 ΔC lacks a complete C‐terminal domain that is required to allow caveolin homo‐oligomers to interact with each other, forming a caveolin network. It is important to note that these two mutants retain an intact transmembrane domain. Our current results show that although Cav‐1 Δ61–100 and Cav‐1 ΔC are competent to drive vesicle formation, these vesicles vary widely in their size and shape with diameters up to 500–1000 nm. In addition, caveolin‐induced vesicle formation appears to be isoform‐specific. Recombinant expression of caveolin‐2 under the same conditions failed to drive the formation of vesicles, while caveolin‐3 expression yielded caveolae‐sized vesicles. These results are consistent with the previous observation that in transformed NIH 3T3 cells that lack caveolin‐1 expression, but continue to express caveolin‐2, no morphologically distinguishable caveolae are observed. In addition, as caveolin‐2 alone exists mainly as a monomer or homo‐dimer, while caveolins 1 and 3 exist as high molecular mass homo‐oligomers, our results are consistent with the idea that the formation of high molecular mass oligomers of caveolin are required to regulate the formation of uniform caveolae‐sized vesicles. In direct support of this notion, regulated induction of caveolin‐1 expression in transformed NIH 3T3 cells was sufficient to recruit caveolin‐2 to caveolae membranes. The ability of caveolin‐1 to recruit caveolin‐2 most likely occurs through a direct interaction between caveolins 1 and 2, as caveolins 1 and 2 are normally co‐expressed and interact with each other to form high molecular mass hetero‐oligomers containing both caveolins 1 and 2.
A new human nm23 homologue (nm23‐H5) specifically expressed in testis germinal cells1
Authors: Munier Annie , Feral Chloé , Milon Laurence , Pinon Véronique Phung-Ba , Gyapay Gabor , Capeau Jacqueline , Guellaen Georges , Lacombe Marie-Lise
Published: July 2002

We have identified a cDNA encoding a 212 amino acid protein (Nm23‐H5) with 27–31% identity to the human members of the nm23/nucleoside diphosphate (NDP) kinase gene family. The nm23‐H5 gene is located on chromosome 5q23–31 and is transcribed as one main transcript of 1.1 kb which is highly and specifically expressed in testis, in the spermatogonia and early spermatocytes. Nm23‐H5 possesses most of the residues conserved among NDP kinases plus an additional COOH‐terminus end of 55 amino acids unique to this protein. However, under usual assay conditions, Nm23‐H5 expressed in Escherichia coli did not exhibit NDP kinase activity. These results suggest that Nm23‐H5 is specifically involved in early stages of spermatogenesis.
Mutational analysis of the cysteines in the extracellular domain of the human Ca2+ receptor: effects on cell surface expression, dimerization and signal transduction
Authors: Fan Gao-Feng , Ray Kausik , Zhao Xin-mei , Goldsmith Paul K , Spiegel Allen M
Published: July 2002

Mammalian calcium receptors (CaRs) share with the metabotropic glutamate receptors (mGluRs) the relative positions of 16 cysteine residues in the amino‐terminal extracellular domain. To investigate the role of these cysteines, a series of mutants in the extracellular domain of the human CaR was prepared in which each of these 16 cysteine residues and three others not conserved in the mGluRs were replaced by serines. Wild‐type and mutant CaR cDNAs were expressed in HEK‐293 cells, and evaluated for expression and response to extracellular calcium. Mutation of three non‐conserved cysteines and of two conserved cysteines produced proteins with near wild‐type phenotype. In contrast, mutation of the other conserved cysteines gave proteins that showed drastic reduction in cell surface expression and/or failed to respond to calcium. We identified 14 cysteines essential for proper trafficking and function of the receptor, two of which may be involved in formation of a disulfide‐linked dimer.
Tau proteins with FTDP‐17 mutations have a reduced ability to promote microtubule assembly
Authors: Hasegawa Masato , Smith Michael J , Goedert Michel
Published: July 2002

Recently exonic and intronic mutations in the gene for microtubule‐associated protein tau have been discovered in cases of familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17). Intronic mutations have been shown to lead to an abnormal preponderance of four‐repeat tau isoforms. The effects of the exonic mutations are unknown. We report here that the G272V, P301L, V337M and R406W mutations lead to a marked reduction in the ability of tau to promote microtubule assembly. This partial loss‐of‐function may be the primary effect of the known missense mutations in tau.
Selective suppression of stress‐activated protein kinase pathway by protein phosphatase 2C in mammalian cells
Authors: Hanada Masahito , Kobayashi Takayasu , Ohnishi Motoko , Ikeda Shoko , Wang Hong , Katsura Koji , Yanagawa Yuchio , Hiraga Akira , Kanamaru Ryunosuke , Tamura Shinri
Published: July 2002

Protein phosphatase 2Cα (PP2Cα) or PP2Cβ‐1 expressed in COS7 cells suppressed anisomycin‐ and NaCl‐enhanced phosphorylations of p38 co‐expressed in the cells. PP2Cα or PP2Cβ‐1 expression also suppressed both basal and stress‐enhanced phosphorylations of MKK3b and MKK6b, which are upstream protein kinases of p38, and of MKK4, which is one of the major upstream protein kinases of JNK. Basal activity of MKK7, another upstream protein kinase of JNK, was also suppressed by PP2Cα or PP2Cβ‐1 expression. However, basal as well as serum‐activated phosphorylation of MKK1a, an upstream protein kinase of ERKs, was not affected by PP2Cβ or PP2Cβ‐1. A catalytically inactive mutant of PP2Cβ‐1 further enhanced the NaCl‐stimulated phosphorylations of MMK3b, MKK4 and MKK6b, suggesting that this mutant PP2Cβ‐1 works as a dominant negative form. These results suggest that PP2C selectively inhibits the SAPK pathways through suppression of MKK3b, MKK4, MKK6b and MKK7 activities in mammalian cells.
Kinetic stabilisation of a modular protein by domain interactions
Authors: Wenk Martina , Jaenicke Rainer , Mayr Eva-Maria
Published: July 2002

Protein S, a two‐domain spore coat protein from Myxococcus xanthus, is structurally related to eye‐lens βγ‐crystallins. No natural monomeric one‐domain member of this protein superfamily is known. To determine the stability of the single domains and to explain the ubiquitous domain duplication, the isolated domains of protein S were constructed. The N‐domain is thermodynamically more stable than the C‐domain. In intact protein S, domain interactions lead to an apparent decrease in stability of the N‐terminal domain, whereas the C‐terminal domain is stabilised. In contrast, unfolding kinetics of both domains are decreased 100‐fold due to interactions in the complete molecule.
Intestinal absorption of luteolin and luteolin 7‐O‐β‐glucoside in rats and humans
Authors: Shimoi Kayoko , Okada Hisae , Furugori Michiyo , Goda Toshinao , Takase Sachiko , Suzuki Masayuki , Hara Yukihiko , Yamamoto Hiroyo , Kinae Naohide
Published: July 2002

In this study, we investigated the intestinal absorption of luteolin and luteolin 7‐O‐β‐glucoside in rats by HPLC. The absorption analysis using rat everted small intestine demonstrated that luteolin was converted to glucuronides during passing through the intestinal mucosa and that luteolin 7‐O‐β‐glucoside was absorbed after hydrolysis to luteolin. Free luteolin, its conjugates and methylated conjugates were present in rat plasma after dosing. This suggests that some luteolin can escape the intestinal conjugation and the hepatic sulfation/methylation. LC/MS analysis showed that the main conjugate which circulates in the blood was a monoglucuronide of the unchanged aglycone. Luteolin in propyleneglycol was absorbed more rapidly than that in 0.5% carboxymethyl cellulose. The plasma concentration of luteolin and its conjugates reached the highest level 15 min and 30 min after dosing with luteolin in propyleneglycol, respectively. HPLC analysis also allowed us to demonstrate the presence of free luteolin and its monoglucuronide in human serum after ingestion of luteolin.