bluuebunny/crossref_metadata_2025 huggingface dataset:
A mutation causing constitutive synthesis of the pyruvate dehydrogenase complex in Escherichia coli is located within the pdhR gene
Authors: Haydon David J. , Quail Michael A. , Guest John R.
Published: July 2002

The aceE‐aceF‐lpd genes encoding the pyruvate dehydrogenase (PDH) complex of Escherichia coli are preceded by a gene encoding a putative transcriptional regulator, PdhR (formerly designated Gen A). Enzymological tests and studies with pdhR‐lacZ and aceE‐lacZ translational fusions have shown that a constitutive mutation (acec816), which increases PDH complex synthesis to the pyruvate‐induced level in the absence of inducer, is recessive to the wild‐type pdhR gene in trans. Sequence comparisons further showed that the acec816 mutation affects a single site in the pdhR gene leading to an Arg118 (CGU) → Cys (UGU) substitution in the PdhR protein. The results support the view that synthesis of the PDH complex is regulated from the pdhR promoter of a pdhR‐aceEF‐lpd operon.
No enzymatic activities are necessary for the stabilization of ascorbic acid by K‐562 cells
Authors: Schweinzer Esther , Waeg Georg , Esterbauer Hermann , Goldenberg Hans
Published: July 2002

We disprove that living cells stabilize ascorbate by the activity of a trans plasma membrane semidehydroascorbate reductase. The two processes show different specificities for both substrate and inhibitor. Not only cells but also cell‐conditioned buffers stabilize ascorbate as long as compounds with molecular weights above 10 kDa are not removed. The effect is most probably due to chelation of traces of transition metals.
Early cell cycle diacylglycerol (DAG) content and protein kinase C (PKC) activity enhancement potentiates prostaglandin F2α (PGF2α) induced mitogenesis in Swiss 3T3 cells
Authors: Goin Mercedes , Pignataro Omar , de Asua Luis Jimenez
Published: July 2002

R59022, a diacylglycerol kinase inhibitor, enhances the prostaglandin F2α (PGF2α)‐induced diacylglycerol (DAG) synthesis in Swiss 3T3 cells. It also potentiates the PGF2α‐mediated protein kinase C (PKC)‐dependent 80 kDa protein (80K) phosphorylation and initiation of DNA replication. R 59022 enhances the PGF2α mitogenic response by increasing the rate of entry into the S phase. Insulin does not cause 80K phosphorylation, and does not enhance its induction but it potentiates the PGF2α mitogenic response. These results suggest that mitogenically triggered fluctuations in DAG content and PKC activity play a pivotal role in controlling the PGF2α‐induced DNA synthesis while insulin acts via a different mechanism.
Atomic force microscope of bacteriophage T4 and its tube‐baseplate complex
Authors: Ikai Atsushi , Yoshimura Kousei , Arisaka Fumio , Ritani Akishige , Imai Kensaku
Published: July 2002

Bacteriophage T4 was imaged by atomic force microscopy with the finest resolution to date with a clear image of tail fibers of an estimated diameter of 2–3 nm. T4 phages were spread on a clean surface of silicon wafer and dried under air before observation with an atomic force microscope. The head, tail and tail fibers were routinely imaged with somewhat distorted dimensions. The ease of imaging isolated phage particles with a good resolution raised our expectation for the further use of AFM in biomedical applications.
Calcium binding to HMG1 protein induces DNA looping by the HMG‐box domains
Authors: S̆tros Michal , Reich Josef , Kolíbalová Anna
Published: July 2002

Electron microscopy has shown that non‐histone chromosomal HMG1 could induce DNA looping or compaction in the presence (but not in the absence) of Ca2+. The effect of calcium on DNA looping and compaction was interpreted as calcium binding to the acidic C‐domain of HMG1. Both individual DNA‐binding HMG1‐box domains A and B were found to be involved in DNA looping and compaction. Treatment of HMG1 with a thiol‐specific reagent, N‐ethylmaleimide, inhibited the ability of the protein to induce DNA looping and compaction but not the electrostatic interaction with DNA. These results indicated that cysteine‐sulfhydryl groups of the HMG1‐box domains A and B are specifically involved in DNA looping and compaction, and that in the absence of calcium the acidic C‐domain down‐regulates these effects by modulation of the DNA‐binding properties of the HMG1‐box domains.
Structural similarity of cytochrome c2 from Rhodopseudomonas viridis to mitochondrial cytochromes c revealed by its crystal structure at 2.7 Å resolution
Authors: Sogabe Satoshi , Ezoe Toshihide , Kasai Nobutami , Saeda Masahiko , Uno Atsushi , Miki Miyako , Miki Kunio
Published: July 2002

The crystal structure of cytochrome c
2 from Rhodopseudomonas viridis has been refined using molecular dynamics and restrained least‐squares methods to a crystallographic R‐factor of 0.216 at 2.7 Å resolution. A structural comparison between Rps. viridis cytochrome c
2 and the other bacterial cytochromes C
2 or mitochondrial cytochromes c indicates that the overall protein foldings are very similar to each other with the exception of the surface loop and terminal region of the polypeptide chain. However, the position and hydrogen‐bond pattern of the evolutionarily conserved water molecule buried within the heme binding pocket in Rps. viridis cytochrome c
2 are common to those in the mitochondrial cytochromes c. This fact indicates that Rps. viridis cytochrome c
2 is structurally more similar to mitochondrial cytochromes c than to the other bacterial cytochromes c
2.
Energy‐coupled transport through the outer membrane of Escherichia coli small deletions in the gating loop convert the FhuA transport protein into a diffusion channel
Authors: Braun Volkmar , Killmann Helmut , Benz Roland
Published: July 2002

Active transport of Fe3+ as ferrichrome complex through the outer membrane of Escherichia coli is mediated by the FhuA outer membrane protein and the TonB—ExbB—ExbD protein complex in the cytoplasmic membrane. The required energy is provided by the electrochemical potential of the cytoplasmic membrane which is assumed to induce a conformation of the TonB protein that causes a conformational change in FhuA so that bound ferrichrome is released into the periplasmic space located between the outer and the cytoplasmic membrane. Excision of segments as small as 12 amino acids in the largest surface loop of FhuA converted FhuA into an open channel through which ferrichrome and antibiotics diffused independent of TonB—ExbB—ExbD. It is proposed that FhuA forms a closed channel which is opened by movement of the gating loop through a kind of allosteric interaction with TonB. The gating loop is also involved in binding of all FhuA ligands which in addition to ferrichrome are the phages T1, T5, φ80, colicin M and the antibiotic albomycin.
Structural model of the ATP‐binding domain of the F1‐β subunit based on analogy to the RecA protein
Authors: Amano Toyoki , Yoshida Masasuke , Matsuo Yo , Nishikawa Ken
Published: July 2002

In contrast to the previous topological model of the ATP binding domain of the F1‐ATPase β subunit based on analogies to those of ras p21 and adenylate kinase, a more consistent model can be constructed with the known structure of the recA protein as a reference. The secondary structure of the F1‐ATPase β subunit predicted from the primary structure agrees well with that of the recA protein. The topology includes a repetitive βαCβαβαβαβ structure where all β strands are parallel and surround the central αC helix above which bound ATP is located. Several residues thought to be located at catalytic site as reported in genetic and chemical labeling work can be consistently positioned in this model.
Phorbol 12‐myristate 13‐acetic acid inhibits PTP1B activity in human mesangial cells A possible mechanism of enhanced tyrosine phosphorylation
Authors: Kiyomoto Hideyasu , Fouqueray Bruno , Abboud Hanna E. , Choudhury Goutam Ghosh
Published: July 2002

Activation of protein kinase C (PKC) by phorbol 12‐myristate 13‐acetic acid (PMA) stimulates DNA synthesis in human glomerular mesangial cells. Incubation of these cells with PMA stimulates the tyrosine phosphorylation of a set of proteins ranging from 110 to 39 kDa with different time kinetics. PMA inhibits total protein tyrosine phosphatase (PTPase) activity in these cells. Immunoprecipitation of PTP1B, an intracytoplasmic tyrosine phosphatase, with subsequent assay of the immunobeads for PTPase shows a significant inhibition of its activity in PMA‐treated cells. Immunoblot analysis of mesangial cell lysates using the same antibody revealed that PMA does not affect the level of this 50 kDa PTP1B protein. These data indicate that inhibition of total PTPase, and specifically PTP1B, activity may provide a mechanism for stimulation of tyrosine phosphorylation by PMA in these cells and thereby contribute to its mitogenic effect.
Interleukin‐1β dissociates β‐amyloid precursor protein and β‐amyloid peptide secretion
Authors: Vasilakos J.P. , Carroll R.T. , Emmerling M.R. , Doyle P.D. , Davis R.E. , Kim K.S. , Shivers B.D.
Published: July 2002

A heightened production of interleukin 1β(IL‐1β) has been reported in microglial‐associated amyloid deposits in Alzheimer's disease (AD) brains. These plaques are composed predominantly of β/A4 peptide derived from β‐amyloid precursor protein (βAPP). We demonstrate that short‐term (1 h) IL‐1β‐treatment increases βAPPs secretion and concomitantly decreases cell‐associated βAPP in human H4 neuroglioma cells. Long‐term (5 h) IL‐1β treatment did not alter secreted or cell‐associated βAPP content. In contrast, the secretion of β/A4‐containing epitope was not affected by short‐term IL‐1β stimulation; however, long‐term IL‐1β treatment decreased the amount of β/A4‐containing epitope secreted from the cells. These results show that IL‐1β modifies the processing and secretion of βAPP to exacerbate perhaps the neuropathology of AD.
The interleukin‐1‐stimulated protein kinase that phosphorylates heat shock protein hsp27 is activated by MAP kinase
Authors: Bird T.A. , Schule H.D. , Delaney P. , de Roos P. , Sleath P. , Dower S.K. , Virca G.D.
Published: July 2002

In KB cells, interleukin‐1 (IL‐1), epidermal growth factor and phorbol ester transiently activated both MAP kinase and a serine kinase which phosphorylated the heat shock protein hsp27. Extracts made from IL‐1‐stimulated KB cells phosphorylated recombinant hsp27, in vitro, on serine residues 78 and 82 which are contained within Arg‐X‐X‐Ser motifs similar to those phosphorylated by the ribosomal protein S6 kinases. Upon size exclusion chromatography, however, hsp27 kinase eluted as a single peak of activity at 50–60 kDa, clearly separated from ribosomal protein S6 kinases. Treatment of partially purified hsp27 kinase with protein phosphatase‐2a reduced its activity by 80%. De‐phosphorylated hsp27 kinase could be approximately 50% reactivated by a factor present in IL‐1‐treated cell extracts in the presence of ATP. This factor co‐eluted with MAP kinase after partial purification by DEAE‐cellulose, phenyl Sepharose, and size exclusion chromatography. Purified sea star p44mpk and recombinant ERK2 MAP kinases were also capable of re‐activating hsp27 kinase to a similar extent. These data suggest that hsp27 kinase is downstream from, and probably a direct target of MAP kinase.
Labeling of v‐Src and BCR‐ABL tyrosine kinases with [14C]herbimycin A and its use in the elucidation of the kinase inactivation mechanism
Authors: Fukazawa Hidesuke , Uehara Yoshimasa , Murakami Yuko , Mizuno Satoshi , Hamada Masa , Takeuchi Tomio
Published: July 2002

The ansamycin antibiotic, herbimycin A, selectively inactivates cytoplasmic tyrosine kinases, most likely by binding irreversibly to the reactive SH group(s) of kinases. To further investigate the mechanism of herbimycin A action, we attempted to label tyrosine kinases with [14C]herbimycin A. p60
v‐src
and p2 10
BCR‐ABL
in immune complexes were labeled with [14C]herbimycin A, demonstrating that the antibiotic binds directly to tyrosine kinases. Digestion of [14C]herbimycin A‐labeled p60
v‐src
with Staphylococcus taureus V8 protease revealed that the herbimycin A binding site is within the C‐terminal 26‐kDa fragment of p60
v‐src
, which contains the tyrosine kinase domain. Herbimycin A treatment inhibited labeling of p60
v‐src
by [14]C]fluorosulfonylbenzoyl adenosine, an affinity labeling reagent of nucleotide binding sites, indicating that herbimycin A‐modified p60
v‐src
cannot interact with ATP. The results suggest that herbimycin A inactivates tyrosine kinases by binding directly to the kinase domain, thereby inhibiting access to ATP.
Anaesthetic phencyclidine, blocker of the ATP‐sensitive potassium channels
Authors: Kokoz Yuri M. , Alekseev Alexey E. , Povzun Alexander A. , Korystova Antonina F. , Peres-Saad Hector
Published: July 2002

The double sucrose gap and patch‐clamp studies revealed that phencyclidine blocked the ATP‐sensitive K+ channel in isolated cardiac cells (half‐maximal inhibition at ≈20 μM; Hill coefficient ≈1). 10μM phencyclidine increased the inward Ca2+ current and blocked the outward K+ current in the frog auricle trabeculae. The phencyclidine effects on the frog auricle trabeculae and the isolated cardiac cells proved to be quite reversible.
The threonine residues in MAP kinase kinase 1 phosphorylated by MAP kinase in vitro are also phosphorylated in nerve growth factor‐stimulated rat phaeochromocytoma (PC12) cells
Authors: Saito Yuji , Gomez Nestor , Campbell David G. , Ashworth Alan , Marshall Chris J. , Cohen Philip
Published: July 2002

The residues on MAP kinase kinase‐1 (MAPKK1) phosphorylated by MAP kinase in vitro have been identified as Thr‐291 and Thr‐385. Both threonines are phosphorylated in PC12 cells and the 32P‐labelling of each residue increases after stimulation with nerve growth factor (NGF). The results establish that MAPKK1 is a physiological substrate for MAP kinase. The two active forms of MAPKK that are resolved by Mono Q chromatography of PC12 cell extracts are both phosphorylated at Thr‐291 and Thr‐385, demonstrating that neither species is the MAPKK2 isoform which lacks Thr‐291.
Induction of c‐fos gene expression by urokinase‐type plasminogen activator in human ovarian cancer cells
Authors: Dumler I. , Petri T. , Schleuning W.-D.
Published: July 2002

Binding of urokinase‐type plasminogen activator (u‐PA) to u‐PA receptor (u‐PAR) induces the rapid and transient expression of c‐fos in OC‐7 ovarian carcinoma cells. The pretreatment of the cells with protein tyrosine kinase (PTK) inhibitors, but not the inactivation of the u‐PA active site by DFP (diisopropyl fluorophosphate), abrogates this effect. A soluble u‐PAR fragment, expressed in baculovirus‐infected Sf9 cells and purified by affinity chromatography, competes for binding of u‐PA to u‐PAR and inhibits c‐fos induction. We conclude that activation of u‐PAR after interaction with u‐PA at the cell surface initiates a transmembrane signal, most likely in conjunction with other still unknown protein(s). This signal generates PTK activity feeding into a signal transduction pathway which activates nuclear transcription factors.
Identification of axial ligands of cytochrome c552 from Nitrosomonas europaea
Authors: Arciero David M. , Peng Qinyun , Peterson Jim , Hooper Alan B.
Published: July 2002

Cytochrome c
552 from Nitrosomonas europaea was analyzed by visible, EPR and MCD spectroscopies. The visible and MCD data show that histidine and methionine are the axial ligands to the heme iron of the ferric protein. The EPR spectrum of the cytochrome shows an atypical highly axial low spin (HALS) type signal with g‐values that make it difficult to identify the axial ligands. These results reinforce the value of near‐infrared MCD spectroscopy for assigning ligands in ferric heme systems and point out the difficulties in using only EPR spectroscopy for the same purpose. The description of another c‐cytochrome exhibiting a HALS‐type EPR signal will eventually be helpful in explaining the physical basis for this unusual signal.
Do voltage‐gated Kv1.1 and inward rectifier Kir2.1 potassium channels form heteromultimers?
Authors: Tytgat Jan , Buyse Gunnar , Eggermont Jan , Droogmans Guy , Nilius Bernd , Daenens Paul
Published: July 2002

Possible heteromultimer formation between Kv‐ and Kir‐type K+ channels was investigated, in connection with the known functional diversity of K+ channels in vivo. Voltage‐clamp experiments were performed on Xenopus oocytes, either injected with concatenated Kir2.1‐Kv1.1 mRNA, or co‐injected with Kv1.1 and Kir2.1 mRNA. K+ currents could be approximated by the algebraic sum of the 2 K+ current types alone. The tandem construct did not show functional expression, although it could be detected by Western blotting. We conclude that Kv1.1 and Kir2.1 α‐subunit proteins fail to assemble and do not contribute functional diversity to K+ channels.
Depression and cognitive impairment in the elderly: a multicentre study
Authors: Kielholz P , Adams C , Bech P , Bocksberger JP , Crombecque A , de Girolamo G , Fayolle K , Holsboer E , Katschnig H , Korten A , Küng A , Meyer JW , Pöldinger W , Prilipko L , Reubi I , Sartorius N
Published: July 2002

The present study, conducted in collaboration between the Departments of Psychiatry in Swiss Universities and the World Health Organization, had two main goals: to develop assessment methods which could subsequently be used in the Swiss centres in a standard manner; and to make arrangements for continuing collaboration between the centres in Switzerland and the acquisition of new knowledge about the distinctions between depression and cognitive impairment. For this aim, three different groups of elderly patients of either sex were selected during the period of November 1989 to July 1991 for inclusion in the study. The first two groups included the first ten patients of either sex over 60 years of age consecutively contacting the participating institutions and showing depression with or without clinically significant symptoms of cognitive impairment; the control group included patients showing no depression or clinically significant symptoms of cognitive impairment. A total of 125 patients were included in the initial evaluation, 69 of which were reassessed at a seven-month follow up (on average). Each patient was administered a number of clinician-rated or self-report instruments for the assessment of depression, cognitive impairment, disabilities, physical status and onset of disorders. The study has shown that a variety of instruments can be used for the reliable assessment of depression or cognitive impairment in the elderly; but the instruments for the assessment of depression differentiate only poorly between patients with or without cognitive impairment. Because of the importance of identifying both depressed and cognitively impaired patients among the elderly, different assessment instruments targeted at the different symptom clusters need to be administered simultaneously.
Increased frequency of aberrant CSF circulation in schizophrenic patients compared to healthy volunteers
Authors: Oxenstierna G , Bergstrand G , Edman G , Flyckt L , Nybäck H , Sedvall G
Published: July 2002

In a previous cisternographic study of the cerebrospinal fluid (CSF) circulation in schizophrenic patients, indications for disturbed flow dynamics were found in 10 of 30 subjects. In order to replicate and investigate the clinical and pathophysiological significance of this finding, 39 schizophrenic patients and 42 healthy subjects were examined with an improved method for measurement of CSF circulation. 99mTc-DTPA was injected intrathecally and the gamma cisternograms were evaluated blindly. Correlations between cisternography findings and age, duration of disease, previous hospitalizations, positive or negative symptomatology, exposure to neuroleptics, psychiatric family history, CT findings and CSF levels of protein, tryptophan and monoamine metabolites, were calculated. Seven of the patients showed abnormalities in the cisternograms with a slow or obstructed flow of CSF over the convexities (P < 0.01) whereas none of the healthy volunteers showed abnormalities. There were no correlations between disturbed CSF circulation in the patients and the clinical and biochemical parameters, thus the significance of the deviations, similar to other biological aberrations found in schizophrenic patients, is not known. Recent developments in magnetic resonance imaging offer new possibilities to further examine CSF circulation abnormalities in schizophrenia.
Alternative splicing within the TGF‐β type I receptor gene (ALK‐5) generates two major functional isoforms in vascular smooth muscle cells
Authors: Agrotis Alex , Condron Melanie , Bobik Alex
Published: July 2002

We have identified in rat vascular smooth muscle cells (SMCs) the simultaneous expression of two TGF‐β type I receptor (ALK‐5) cDNAs, occurring as a consequence of alternate usage of AG splice acceptor motifs separated by 12 nucleotides located at an intron‐exon junction. When translated the resultant full length proteins differ from each other only by the in‐frame presence or absence of Gly‐Pro‐Phe‐Ser residues adjacent to their transmembrane domain. Stable expression of these alternate ALK‐5 isoforms in ALK‐5‐deficient cells demonstrated that both were competent in signaling TGF‐β‐induced growth inhibition and gene transcription, but with an apparently distinct potency. Our data suggest that alternate splicing within the ALK‐5 gene is an important mechanism whereby SMCs may regulate their response to TGF‐β.
Voltage and substrate dependence of the inverse transport mode of the rabbit Na+/glucose cotransporter (SGLT1)
Authors: Sauer G.A. , Nagel G. , Koepsell H. , Bamberg E. , Hartung K.
Published: July 2002

Properties of the cytoplasmic binding sites of the rabbit Na+/glucose cotransporter, SGLT1, expressed in Xenopus oocytes were investigated using the giant excised patch clamp technique. Voltage and substrate dependence of the outward cotransport were studied using α‐methyl D‐glucopyranoside (αMDG) as a substrate. The apparent affinity for αMDG depends on the cytoplasmic Na+ concentration and voltage. At 0 mV the K
M for αMDG is 7 mM at 110 mM Na+ and 31 mM at 10 mM Na+. The apparent affinity for αMDG and Na+ is voltage dependent and increases at positive potentials. At 0 mV holding potential the outward current is half‐maximal at about 70 mM. The results show that SGLT1 can mediate sugar transport out of the cell under appropriate concentration and voltage conditions, but under physiological conditions this transport is highly improbable due to the low affinity for sugar.
Role of the histidine residues of visna virus nucleocapsid protein in metal ion and DNA binding
Authors: Morcock David R. , Sowder Raymond C. , Casas-Finet José R.
Published: July 2002

Zinc finger (ZF) domains in retroviral nucleocapsid proteins usually contain one histidine per metal ion coordination complex (Cys‐X2‐Cys‐X4‐His‐X4‐Cys). Visna virus nucleocapsid protein, p8, has two additional histidines (in the second of its two ZFs) that could potentially bind metal ions. Absorption spectra of cobalt‐bound ZF2 peptides were altered by Cys alkylation and mutation, but not by mutation of the extra histidines. Our results show that visna p8 ZFs involve three Cys and one His in the canonical spacing in metal ion coordination, and that the two additional histidines appear to interact with nucleic acid bases in p8–DNA complexes.
Do the extracellular enzymes cellobiose dehydrogenase and manganese peroxidase form a pathway in lignin biodegradation?
Authors: Hildén Lars , Johansson Gunnar , Pettersson Göran , Li Jiebing , Ljungquist Pierre , Henriksson Gunnar
Published: July 2002

The extracellular enzyme manganese peroxidase is believed to degrade lignin by a hydrogen peroxide‐dependent oxidation of Mn(II) to the reactive species Mn(III) that attacks the lignin. However, Mn(III) is not able to directly oxidise the non‐phenolic lignin structures that predominate in native lignin. We show here that pretreatment of a non‐phenolic lignin model compound with another extracellular fungal enzyme, cellobiose dehydrogenase, allows the manganese peroxidase system to oxidise this molecule. The mechanism behind this effect is demethoxylation and/or hydroxylation, i.e. conversion of a non‐phenolic structure to a phenolic one, mediated by hydroxyl radicals generated by cellobiose dehydrogenase. This suggests that cellobiose dehydrogenase and manganese peroxidase may act in an extracellular pathway in fungal lignin biodegradation. Analytical techniques used in this paper are reverse‐phase high‐pressure liquid chromatography, gas chromatography connected to mass spectroscopy and UV‐visible spectroscopy.
Intermediates in the reaction of substrate‐free cytochrome P450cam with peroxy acetic acid
Authors: Schünemann V. , Jung C. , Trautwein A.X. , Mandon D. , Weiss R.
Published: July 2002

Freeze‐quenched intermediates of substrate‐free cytochrome 57Fe‐P450cam in reaction with peroxy acetic acid as oxidizing agent have been characterized by EPR and Mössbauer spectroscopy. After 8 ms of reaction time the reaction mixture consists of ∼90% of ferric low‐spin iron with g‐factors and hyperfine parameters of the starting material; the remaining ∼10% are identified as a free radical (S′=1/2) by its EPR and as an iron(IV) (S=1) species by its Mössbauer signature. After 5 min of reaction time the intermediates have disappeared and the Mössbauer and EPR‐spectra exhibit 100% of the starting material. We note that the spin‐Hamiltonian analysis of the spectra of the 8 ms reactant clearly reveals that the two paramagnetic species, e.g. the ferryl (iron(IV)) species and the radical, are not exchanged coupled. This led to the conclusion that under the conditions used, peroxy acetic acid oxidized a tyrosine residue (probably Tyr‐96) into a tyrosine radical (Tyr‐96), and the iron(III) center of substrate‐free P450cam to iron(IV).
Tetraplex formation by the progressive myoclonus epilepsy type‐1 repeat: implications for instability in the repeat expansion diseases
Authors: Saha Tapas , Usdin Karen
Published: July 2002

The repeat expansion diseases are a group of genetic disorders resulting from an increase in size or expansion of a specific array of tandem repeats. It has been suggested that DNA secondary structures are responsible for this expansion. If this is so, we would expect that all unstable repeats should form such structures. We show here that the unstable repeat that causes progressive myoclonus epilepsy type‐1 (EPM1), like the repeats associated with other diseases in this category, forms a variety of secondary structures. However, EPM1 is unique in that tetraplexes are the only structures likely to form in long unpaired repeat tracts under physiological conditions.
Nuclear localization signals in the Xenopus FGF embryonic early response 1 protein
Authors: Post Janine N. , Gillespie Laura L. , Paterno Gary D.
Published: July 2002

Xenopus early response 1 (XER1) is a fibroblast growth factor‐inducible transcription factor whose developmentally regulated nuclear localization is thought to be important in the control of cell differentation during embryonic development [Luchman et al., Mech. Dev. 80 (1999) 111–114]. Analysis of the XER1 amino acid sequence revealed four regions which contain potential nuclear localization sequences (NLSs). Using mutant XER1 proteins and portions of XER1 fused to green fluorescent protein (GFP) transfected into NIH 3T3 cells, we have determined that only one of these, NLS4, located near the carboxy‐terminus of XER1, is necessary and sufficient for targeting exclusively to the nucleus. Of the other three predicted NLS sequences, only NLS1, consisting of the sequence 138RPRRCK143 was shown to function as a cryptic, weak NLS. NLS4 contains a core region consisting of the sequence 463RPIKRQRMD471 which is similar to the core NLS directing the human c‐MYC protein to the nucleus. The core sequence is flanked by a predicted cdc2/protein kinase A phosphorylation motif, however mutation of the serine472 to alanine or aspartic acid had no detectable effect on accumulation of GFP‐XER1 fusion proteins in the nucleus, demonstrating that this putative phosphorylation site plays no role in regulating nuclear transport.
The Wilms tumor suppressor gene WT1 induces G1 arrest and apoptosis in myeloblastic leukemia M1 cells
Authors: Murata Yoji , Kudoh Tetsuhiro , Sugiyama Haruo , Toyoshima Kumao , Akiyama Tetsu
Published: July 2002

WT1 was isolated as a tumor suppressor gene of Wilms tumor. However, high expression of WT1 correlates with poor prognosis in acute leukemia. In addition suppression of WT1 expression by WT1 anti‐sense oligonucleotide inhibits proliferation of leukemia cells, suggesting that WT1 is important for their proliferation. To further elucidate the biological significance of WT1 in leukemic cell growth, we overexpressed exogenous WT1 in murine M1 myeloblastic leukemia cells using the isopropyl‐β‐d‐thiogalactoside (IPTG)‐controlled expression system. We found that induction of one splicing variant of WT1 [WT1‐17AA(+)‐KTS(−)] in M1 cells induces cell cycle arrest and apoptotic cell death. These results suggest that the role of WT1 is different depending on the type of leukemia cell in which it is expressed.
Overexpression of hIGF‐1 exclusively in skeletal muscle increases the number of dihydropyridine receptors in adult transgenic mice
Authors: Renganathan M , Messi M.L , Schwartz R , Delbono O
Published: July 2002

The number of dihydropyridine receptors (DHPR) and sarcoplasmic reticulum (SR) Ca2+ release channels (RyR1) and their interaction determine the efficacy of the sarcolemmal excitation‐SR Ca2+ release‐contraction coupling (ECC). Both receptors play a central role in ECC as demonstrated in various animal species and muscle subtypes. In the present work we studied the effect of transgenic overexpression of human insulin‐like growth factor 1 (hIGF‐1) on the levels of these two Ca2+ channels in extensor digitorum longus (EDL) (fast‐twitch), soleus (slow‐twitch) and pool of fast‐ and slow‐twitch muscles from adult C57BL/6 mice. Muscles from hIGF‐1 transgenic mice showed a significant increase in IGF‐1 concentration (20–30‐fold) and in the number of DHPR (52% increase) whereas no significant change in RyR1 binding sites was detected. The differential effect on DHPR and RyR1 resulted in a 30% increase in DHPR/RyR1 ratio. Fast‐ and slow‐twitch muscles showed 50 and 70% increase in the number of DHPR and 30 and 80% increase in DHPR/RyR1, respectively. These results support the concept that the increased autocrine/paracrine secretion of hIGF‐1 exerts potent stimulatory effects on DHPR α1 subunit expression in adult skeletal muscle.
Association of glucose‐regulated protein (grp78) with human keratin 8
Authors: Liao Jian , Price Daniel , Omary M.Bishr
Published: July 2002

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament proteins that are expressed in ‘simple‐type’ epithelial cells. They associate with several proteins including the 70 kDa cytoplasmic heat shock proteins (hsp70). We identified the human 78 kDa glucose‐regulated protein (grp78) as a keratin‐associated protein. Keratin‐grp78 association was noted after co‐immunoprecipitation of K8/18 from HT29 detergent solubilized cell lysates, and appears to involve non‐posttranslationally modified grp78. The grp78‐K8/18 association is induced by culturing cells in the presence of tunicamycin or after glucose starvation. K8/18‐bound grp78 can be dissociated by Mg‐ATP and the association can be reconstituted in vitro using purified grp78, then redissociated again by Mg‐ATP. Binding of grp78 occurs preferentially with K8, and when reconstituted does not depend on the posttranslational modification state of K8/18. Co‐incubation of K8/18 with hsp70 and grp78 shows preferential association with hsp70. Our results demonstrate a direct association of grp78 with K8 under conditions that induce grp78 expression.
Identification of neutral and acidic sphingomyelinases in Helicobacter pylori
Authors: Lin Yuh-Ling , Liu Jai-Shin , Chen Kuei-Tian , Chen Chien-Tsu , Chan Err-Cheng
Published: July 2002

We demonstrated for the first time the presence of sphingomyelinase (SMase) in Helicobacter pylori. Activation of SMase has been implicated as the cause of elevation of cellular ceramide levels and consequently of apoptosis. The data indicate that there are two classes of SMase, defined by their optimal pHs and cellular locations, existing in H. pylori. One is an Mg2+‐dependent membrane‐bound enzyme with an optimal activity at pH 7, and the other is an Mg2+‐independent cytosolic enzyme with an optimal activity at pH 5. Bisalumin, a bismuth salt, was found to inhibit the activities of both forms of SMase regardless of the presence of Mg2+. By Western blot analysis, the membrane‐bound SMases of H. pylori and Bacillus cereus were shown to be antigenically related and to have a similar denatured molecular mass of 28 kDa.
Quantitative model for the cooperative interaction of the bacteriorhodopsin molecules in purple membranes
Authors: Tokaji Zsolt
Published: July 2002

The trimeric, asymmetric and sequential model for the cooperative interaction of the bacteriorhodopsin molecules in purple membranes [Zs. Tokaji, Biophys. J. 65 (1993) 1130–1134] is being extended in the paper. Analyses of data from absorption kinetic measurements with preexcitation and green background illumination, and photoselection measurements on oriented samples confirm the main features of this cooperative interaction and support the validity of the extended model. This model includes the observed heterogeneity of the non‐excited state of the bacteriorhodopsin molecules in purple membranes, and the agreement with the data suggests that molecules in any other state than the bacteriorhodopsin ground state can alter the photocycle of their neighbors. The presented results seem to contradict other models for the cooperation of the bacteriorhodopsin molecules.
cDNA cloning and sequencing of the human ryanodine receptor type 3 (RYR3) reveals a novel alternative splice site in the RYR3 gene
Authors: Leeb Tosso , Brenig Bertram
Published: July 2002

The human ryanodine receptor type 3 (RYR3) was cloned from a fetal brain cDNA library and its complete sequence was determined (EMBL accession number AJ001515). The sequenced cDNA spanned 15 564 bp and contained an open reading frame of 14 613 bp. The corresponding protein consisted of 4870 amino acids with a calculated molecular mass of 552 kDa. Amino acid sequence identities to the RYR3 proteins from rabbit, mink, and chicken were 96%, 95%, and 83% respectively. A previously unidentified alternative splice site was detected generating a transcript that lacked bases 11 569–11 650 and encoded a truncated protein.
Differential scanning calorimetric studies of the glycoprotein, winged bean acidic lectin, isolated from the seeds of Psophocarpus tetrogonolobus
Authors: Srinivas V.R , Singha Netai C , Schwarz Fredrick P , Surolia Avadhesa
Published: July 2002

Differential scanning calorimetry of solutions of WBAII and in presence of sugar ligands shows that WBAII dimer dissociates to its constituent monomeric subunits at the denaturation temperature. The thermal denaturation of WBAII consists of the unfolding of two independent domains of WBAII similar to that of basic winged bean lectin and ECorL and in contrast to concanavalin A (conA), pea and lentil lectin, which unfold as single entities. Apparently, the glycosylation reduces the structural integrity of WBAII as compared to conA, pea and lentil lectin. The increase in the denaturation temperature of the sugar‐lectin complexes yields binding constants close to the binding constants extrapolated from the ITC results and confirms the mechanism proposed for its thermal unfolding.
Plant profilin induces actin polymerization from actin:β‐thymosin complexes and competes directly with β‐thymosins and with negative co‐operativity with DNase I for binding to actin
Authors: Ballweber Edda , Giehl Klaudia , Hannappel Ewald , Huff Thomas , Jockusch Brigitte M , Mannherz Hans Georg
Published: July 2002

Recombinant plant (birch) profilin was analyzed for its ability to promote actin polymerization from the actin:thymosin β4and β9complex. Depending on the nature of the divalent cation, recombinant plant (birch) profilin exhibited two different modes of interaction with actin, like mammalian profilin. In the presence of magnesium ions birch profilin promoted the polymerization of actin from A:Tβ4. In contrast, in the presence of calcium but absence of magnesium ions birch profilin was unable to initiate the polymerization of actin from the complex with Tβ4. However, under these conditions profilin formed a stable stoichiometric complex with skeletal muscle α‐actin, as verified by its ability to increase the critical concentration of actin polymerization. Chemical cross‐linking indicated that birch profilin competes with Tβ4for actin binding. Ternary complex formation of birch profilin with actin:DNase I complex was suggested by chemical cross‐linking. However, the determination of the critical concentrations of actin polymerization in the simultaneous presence of birch profilin and DNase I indicated that profilin and DNase I did not form a ternary complex. These data indicated a negative co‐operativity between the profilin and DNase I binding sites on actin.
Calcium modulates conformational changes in F‐actin induced by smooth muscle heavy meromyosin
Authors: Avrova Stanyslava V , Borovikov Yurii S , Efimova Natalia N , Chacko Samuel
Published: July 2002

The effect of Ca2+ on conformational changes in rhodamine‐phalloidin‐labeled F‐actin induced by binding of smooth muscle heavy meromyosin (HMM) with either phosphorylated or dephosphorylated regulatory light chains (LC20) was studied by polarized fluorimetry. LC20 phosphorylation caused alterations in the F‐actin structure typical of the force‐producing (strong‐binding) state, while dephosphorylation of the chains led to alterations typical of the formation of non‐force‐producing (weak‐binding) state of the actomyosin complex. The presence of Ca2+ enhanced the effect of LC20 phosphorylation and weakened the effect of LC20 dephosphorylation. These data suggest that Ca2+ modulates actin‐myosin interaction in smooth muscle by promoting formation of the strong‐binding state.
Molecular cloning of a bombinin gene from Bombina orientalis: detection of NF‐κB and NF‐IL6 binding sites in its promoter
Authors: Miele Rossella , Ponti Donatella , Boman Hans G , Barra Donatella , Simmaco Maurizio
Published: July 2002

The sequence of a gene from Bombina orientalis was determined which codes for antibacterial peptides. The gene comprises two exons separated by a large intron. Exon 1 codes for the signal peptide, while exon 2 contains the genetic information for two identical bombinins and one bombinin H. The promoter region of the bombinin gene contains putative recognition sites for nuclear factors, such as NFκB and NF‐IL6. In vivo experiments on B. orientalis have shown that a short contact with bacteria is sufficient to induce a marked increase in the amount of antibacterial peptides in the skin secretion of frogs. This increase was suppressed by pretreatment with glucocorticoids. In the latter case, a significant increase of IκBα in the secretion is also detectable.
Phospholipase C cDNAs from sponge and hydra: antiquity of genes involved in the inositol phospholipid signaling pathway1
Authors: Koyanagi Mitsumasa , Ono Kanako , Suga Hiroshi , Iwabe Naoyuki , Miyata Takashi
Published: July 2002

To know whether or not the set of genes involved in the inositol phospholipid signaling pathway already existed in the early evolution of animals, we carried out cloning of cDNAs encoding phospholipase Cs (PLCs) from Ephydatia fluviatilis (freshwater sponge) and Hydra magnipapillata strain 105 (hydra). We isolated two PLC cDNAs, PLC‐βS and PLC‐γS, from sponge and three cDNAs, PLC‐βH1, PLC‐βH2, and PLC‐δH, from hydra. From the domain organization and the divergence pattern in the PLC family tree, the sponge PLC‐βS and PLC‐γS and the hydra PLC‐δH are possibly homologous to the vertebrate PLC‐β, PLC‐γ and PLC‐δ subtypes, respectively. A detailed phylogenetic analysis suggests that the hydra PLC‐βH1 and PLC‐βH2 are homologs of the vertebrate PLC‐β1/2/3/Drosophila PLC21 and the vertebrate PLC‐β4/Drosophila norpA, respectively. A phylogenetic analysis of the PLC family and the protein kinase C (PKC) family, together with that of the G protein α subunit (Gα) family, revealed that the origin of the set of genes Gαq, PLC, PKC involved in the inositol phospholipid signaling pathway is very old, going back to dates before the parazoan‐eumetazoan split, the earliest branching among extant animal phyla.
Lysophosphatidylcholine generates superoxide anions through activation of phosphatidylinositol 3‐kinase in human neutrophils
Authors: Nishioka Hiroaki , Horiuchi Hisanori , Arai Hidenori , Kita Toru
Published: July 2002

Lysophosphatidylcholine (LPC) accumulates in inflammatory tissues, where neutrophils are recruited to generate superoxide anions (O⋅−
2). Here, we show that LPC stimulates O⋅−
2 generation in human neutrophils and that the activity is inhibited with phosphatidylinositol 3‐kinase (PI3 kinase) inhibitors, but not with protein kinase C (PKC) inhibitors. Furthermore, we demonstrate that LPC activates PI3 kinase in neutrophils. Thus, LPC might contribute to host defense by generating O⋅−
2 in neutrophils through PI3 kinase activation, but not through PKC activation.
Localization of Rho GTPase in sea urchin eggs
Authors: Nishimura Yukako , Nakano Kentaro , Mabuchi Issei
Published: July 2002

We isolated the urho1 (rchin in English or ni in Japanese) gene from the sea urchin cDNA library which encodes a Rho GTPase. Anti‐URho1 antibodies specifically recognized a 22 kDa protein in the extracts of echinoderm eggs. URho1 was concentrated in the cortices from both unfertilized and fertilized eggs as judged by immunoblot analysis. URho1 may bind directly to the cell membrane but not be a component of the cortical layer. Immunofluorescence microscopy revealed that URho1 is localized to the cleavage furrow and the midbody during cytokinesis.
Helicobacter pylori lipopolysaccharide enhances the expression of NADPH oxidase components in cultured guinea pig gastric mucosal cells
Authors: Teshima Shigetada , Tsunawaki Shohko , Rokutan Kazuhito
Published: July 2002

Recently, we showed that cultured guinea pig gastric pit cells possess a phagocyte NADPH oxidase‐like activity, which was up‐regulated by Helicobacter pylori lipopolysaccharide. We demonstrate here that these cells express all of the phagocyte NADPH oxidase components (gp91‐, p22‐, p67‐, p47‐, and p40‐phoxes). Treatment with lipopolysaccharide increased the expression of gp91‐, p22‐, and p67‐phoxes, but not that of p47‐ and p40‐phoxes. Intriguingly, the p67‐phox expression consistently correlated with up‐regulation of superoxide anion‐producing ability. Thus, the gastric pit cell NADPH oxidase may play an important role in regulation of the inflammatory response associated with H. pylori infection.
Nuclear and nucleolar localization of Saccharomyces cerevisiae ribosomal proteins S22 and S25
Authors: Timmers Antonius C.J , Stuger Rogier , Schaap Peter J , van 't Riet Jan , Raué Hendrik A
Published: July 2002

Nuclear import usually relies on the presence of nuclear localization sequences (NLSs). NLSs are recognized by NLS receptors (importins), which target their substrates to the nuclear pore. We identified the NLSs of the yeast ribosomal proteins S22 and S25 and studied the former by mutational analysis. Furthermore, in S25 the nucleolar targeting information was found to overlap with its NLS. Comparison with previously published data on yeast ribosomal protein NLSs and computer analysis indicates the existence of a novel type of ribosomal protein‐specific NLS that differs from the classical Chelsky and bipartite NLSs. The existence of such a ribosomal protein‐specific NLS is in accordance with the recent identification of ribosomal protein‐specific importins.
Photoelectric response of the N intermediate of bacteriorhodopsin and its mutant T46V
Authors: Tóth-Boconádi R. , Szabó-Nagy A. , Taneva S.G. , Keszthelyi L.
Published: July 2002

Double flash experiments were performed in order to gain information about the characteristics of the N intermediates of the photocycle of bacteriorhodopsin. The N intermediates of wild‐type bacteriorhodopsin and mutant T46V were excited at different delay times after the first laser flash which induced the photocycle and the electric responses were registered. These electric signals revealed that charge motions occurred in both cases, though charge translocation, i.e. H+ pumping, could not be observed. The delay time dependence of the electric signals is characterized by two distinct processes corresponding to two substates of the N intermediates.
The protein kinase C inhibitors bisindolylmaleimide I (GF 109203x) and IX (Ro 31‐8220) are potent inhibitors of glycogen synthase kinase‐3 activity
Authors: Hers Ingeborg , Tavaré Jeremy M , Denton Richard M
Published: July 2002

Here we report that the widely used protein kinase C inhibitors, bisindolylmaleimide I and IX, are potent inhibitors of glycogen synthase kinase‐3 (GSK‐3). Bisindolylmaleimide I and IX inhibited GSK‐3 in vitro, when assayed either in cell lysates (IC50 360 nM and 6.8 nM, respectively) or in GSK‐3β immunoprecipitates (IC50 170 nM and 2.8 nM, respectively) derived from rat epididymal adipocytes. Pretreatment of adipocytes with bisindolylmaleimide I (5 μM) and IX (2 μM) reduced GSK‐3 activity in total cell lysates, to 25.1±4.3% and 12.9±3.0% of control, respectively. By contrast, bisindolylmaleimide V (5 μM), which lacks the functional groups present on bisindolylmaleimide I and IX, had little apparent effect. We propose that bisindolylmaleimide I and IX can directly inhibit GSK‐3, and that this may explain some of the previously reported insulin‐like effects on glycogen synthase activity.
Permeabilizing action of an antimicrobial lactoferricin‐derived peptide on bacterial and artificial membranes
Authors: Aguilera O. , Ostolaza H. , Quirós L.M. , Fierro J.F.
Published: July 2002

A synthetic peptide (23 residues) that includes the antibacterial and lipopolysaccharide‐binding regions of human lactoferricin, an antimicrobial sequence of lactoferrin, was used to study its action on cytoplasmic membrane of Escherichia coli 0111 and E. coli phospholipid vesicles. The peptide caused a depolarization of the bacterial cytoplasmic membrane, loss of the pH gradient, and a bactericidal effect on E. coli. Similarly, the binding of the peptide to liposomes dissipated previously created transmembrane electrical and pH gradients. The dramatic consequences of the transmembrane ion flux during the peptide exposure indicate that the adverse effect on bacterial cells occurs at the bacterial inner membrane.