bluuebunny/crossref_metadata_2025 huggingface dataset:
Recognition of direction of uniform and accelerated visual motion and EEG alpha wave phases
Authors: Shevelev Igor A. , Kamenkovich Viktorina M. , Kostelianetz Nina B. , Sharaev George A.
Published: April 2003

The perception of visual motion in seven subjects was studied comparing motion towards or away from the fixation point in the left or right hemifield. The light target was moved either at a constant velocity or positively or negatively accelerated to compensate for the magnification factor of the visual cortex. We compared probability and latency of motion recognition when it was asynchronous or synchronized to different phases of the alpha wave of the EEG recorded over the occipital cortex. If the motion accelerated away from the fixation point and was synchronized with the alpha wave it was more likely to be perceived whereas if it was towards the fixation point it was less likely to be detected. However, perception of the constant velocity motion was not changed by locking it to the alpha wave phase. These results support the hypothesis that the scanning waves of excitation spread over the visual cortex periodically and that they are locked to the alpha component of the EEG.
Interaction studies between the p21Cip1/Waf1 cyclin‐dependent kinase inhibitor and proliferating cell nuclear antigen (PCNA) by surface plasmo resonance
Authors: Knibiehler Martine , Goubin Françoise , Escalas Nathalie , Jónsson Zophonias O. , Mazarguil Honoré , Hübscher Ulrich , Ducommun Bernard
Published: April 2003

The cyclin‐dependent kinase (CDK) inhibitor p21Cip1 consists of two domains that interact with CDKs and proliferating cell nuclear antigen (PCNA), respectively. We have investigated the interaction between p21Cip1 and PCNA using surface plasmon resonance (SPR) technology and compared the results with those obtained from other sources such as the yeast two‐hybrid system. Whilst other methods are only semi‐quantitative, the SPR technique allowed us to determine the kinetic parameters of the interaction. The apparent equilibrium constant K
KD calculated for these kinetic parameters was 3.2 × 10−7 M. We further demonstrate the use of SPR to study the interaction between mutant proteins and to determine their actual KD. The interaction between p21Cip1/PCNA is shown to be dependent upon the trimeric conformation of PCNA since a point mutant that abolishes PCNA‐PCNA interaction also abolishes PCNA's interaction with p21Cip1. Finally, we demonstrate that SPR can be used to characterise the interaction of p21Cip1 and PCNA in the presence of short competitive peptides.
Impaired glutathione biosynthesis in the ischemic‐reperfused rabbit myocardium
Authors: Lapenna Domenico , de Gioia Sergio , Ciofani Giuliano , Mezzetti Andrea , Pierdomenico Sante D. , Di Ilio Carmine , Cuccurullo Franco
Published: April 2003

Non‐protein thiols (NP‐SH) and the activities of the glutathione status‐regulating enzymes γ‐glutamylcysteine synthetase (G‐GCS), γ‐glutamyl transpeptidase (G‐GT) and glutathione reductase (GR) were assessed in perfused rabbit hearts subjected to severe (60 min) or mild (7 min) total ischemia and 30 min reperfusion. Severe ischemia significantly decreased NP‐SH, which were further depressed on reperfusion together with a significant decline in G‐GCS activity; G‐GT and GR activities were unchanged. Specific analytes were unaffected by mild ischemia‐reperfusion. Thus, impaired enzymatic biosynthesis of GSH is operative in the reperfused rabbit myocardium after 60 min ischemia. This phenomenon may favour myocardial GSH depression and oxidative reperfusion injury after severe ischemia.
Differential post‐transcriptional control of ornithine decarboxylase and spermidine‐spermine N1‐acetyltransferase by polyamines
Authors: Fogel-Petrovic Mirjana , Vujcic Slavoljub , Miller John , Porter Carl W.
Published: April 2003

Ornithine decarboxylase (ODC) and spermidine/spermine N
1‐acetyltransferase (SSAT) are short‐lived polyamine enzymes with rate‐limiting roles in controlling polyamine biosynthesis and catabolism, respectively. We have found that treatment of MALME‐3M human melanoma cells for 6 h with 10 μg/ml cycloheximide (CHX) increases ODC and SSAT mRNA 6–9‐fold. When cells containing CHX‐induced SSAT mRNA were washed and post‐incubated for an additional 6 h in drug free media, enzyme activity increased only 2‐fold above that in untreated cells despite the > 6‐fold increase in accumulated mRNA. Inclusion of 10 μM spermine or spermidine in the post‐incubation medium increased SSAT activity ∼7‐fold without further elevating SSAT mRNA levels. This indicates post‐transcriptional regulation which, due to the similarity between polyamine‐mediated increases in SSAT activity and available mRNA, probably occurs at the level of mRNA translation. In contrast to the SSAT response, polyamines markedly reduced ODC activity (but not mRNA) to one sixth that in cells not exposed to polyamines. The findings illustrate how via post‐transcriptional mechanisms, shifts in intracellular polyamine pools can simultaneously and differentially regulate polyamine biosynthesis and catabolism. It is hypothesized that these post‐transcriptional responses enable cells to rapidly and sensitively control intracellular spermidine and spermine pools.
Co‐expression of mRNA for type I and type II Interleukin‐1 receptors and the IL‐1 receptor accessory protein correlates to IL‐1 responsiveness
Authors: Wesche H. , Neumann D. , Resch K. , Martin M.U.
Published: April 2003

Three cell surface molecules participate in Interleukin‐1 (IL‐1) binding and signal generation, the two distinct types of receptors (type I IL‐1R and type II IL‐1R) and the IL‐1 receptor accessory protein (IL‐1RAcP). Low surface expression hampers the detection of all three components on a protein level in most cell types, thus the highly sensitive RT‐PCR was used to analyse the mRNA expression in a panel of 18 murine cell types of different hemopoietic lineages and fibroblasts. The transcription of both types of IL‐1 receptors was detected in all cell lines tested. In most cell lines the IL‐1RAcP was co‐expressed with the IL‐1 receptors, and only these lines responded to IL‐1. However, in three cell lines no mRNA for the IL‐1RAcP could be detected, and these cells did not respond to IL‐1. These results suggest that the expression of the IL‐1RAcP correlates with IL‐1 responsiveness and they point to a pivotal role for the IL‐1RAcP in IL‐1 signal generation.
Action of bovine serum albumin on cytochrome c oxidase activity and proton pumping: a role for fatty acids in enzyme function?
Authors: Sharpe Martyn , Perin Ivano , Nicholls Peter
Published: April 2003

Bovine serum albumin (BSA) at micromolar concentrations causes a red shift of the Soret band of bovine cytochrome c oxidase with a slow biphasic time course. It also inhibits the turnover of detergent‐isolated enzyme in a similarly slow manner; the progress of this inhibition is halted by palmitate and other fatty acids. The inhibitory bovine serum albumin effect may involve fatty acid depletion from the enzyme. Respiration by cytochrome c oxidase vesicles (proteoliposomes) in the presence of ionophores (uncontrolled) shows only a small inhibition by BSA but preincubation of such vesicles with BSA induces a loss of proton pumping activity. After incubation of BSA‐depleted proteoliposomes in the presence of reductant with combinations of fatty acids, pumping activity can be fully restored, suggesting a supportive or even essential role of endogenous fatty acids in H+ translocation by this membranous enzyme.
Interaction between the left‐handed Z‐DNA and polyamine‐2
Authors: Ohishi Hirofumi , Nakanishi Isao , Inubushi Kouichi , van der Marel Gijs , van Boom Jacques H. , Rich Alexander , Wang Andrew H.J. , Hakoshima Toshio , Tomita Ken-ichi
Published: April 2003

This paper deals with the crystal structure of d(CG)3‐spermidine complex. The DNA fragment, d(CG)3, was crystallized with N‐(2‐amino‐propyl)‐1,4‐diamino‐butane, PA(34), spermidine. The results of its X‐ray crystallographic analysis showed many intermolecular contacts between d(CG)3 and spermidine, but the binding mode of spermidine to the d(CG)3 molecule is different from that of the d(CG)3 and N‐(2‐amino‐ethyl)‐1,4‐diamino‐butane [PA(24)]complex: a spermidine molecule bound to the d(CG)3 and its symmetrically related neighboring d(CG)3 molecules through the water molecules with hydrogen bonds, while one PA(24) molecule connected directly to one d(CG)3 molecule, but not to its neighboring d(CD)3 molecule. In the crystal, the d(CG)3 molecule was the left‐handed Z‐form, and three magnesium cations and a sodium cation were observed around the d(CG)3 moiety with different binding modes from the case of the d(CG)3‐PA(24) complex.
The three‐dimensional structure of capsule‐specific CMP: 2‐keto‐3‐deoxy‐manno‐octonic acid synthetase from Escherichia coli
Authors: Jelakovic Stefan , Jann Klaus , Schulz Georg E.
Published: April 2003

CMP‐Kdo synthetases from Gram‐negative bacteria activate Kdo for incorporation into lipo‐ and capsule‐polysaccharides. Here we report the crystal structure of the capsule‐specific synthetase from E. coli at 2.3 Å resolution. The enzyme is a dimer of 2 × 245 amino acid residues assuming C2 symmetry. It contains a central predominantly parallel β‐sheet with surrounding helices. The chain fold is novel; it is remotely related to a double Rossmann fold. A large pocket at the carboxyl terminal ends of the central β‐strands most likely accommodates the catalytic center. A putative phosphate binding site at the loop between the first β‐strand and the following helix is indicated by a bound iridium hexachloride anion.
Presence of ε‐adenosine tetraphosphate in chromaffin granules after transport of ε‐ATP
Authors: Gualix Javier , Abal Miguel , Pintor Jesús , Miras-Portugal Maria Teresa
Published: April 2003

Adenosine 5′‐tetraphosphate (Ap4) is a natural constituent of chromaffin granules with concentration values of 2.2±0.1 nmol/mg of protein and a ratio 245±40 times lower with respect to ATP (n=4). The granular transport of ε‐ATP resulted in a time‐ and concentration‐dependent production of ε‐adenosine tetraphosphate (ε‐Ap4) at the intragranular level. The ε‐Ap4 formation followed a hyperbolic saturation kinetic at low ε‐ATP concentrations with K
m value of 0.4 μM ε‐ATP intragranular (1.15 pmol/mg of granular protein). Intragranular concentrations of ε‐ATP higher than 500 pmol/mg of protein (≈ 175 μM intragranular) resulted in a non‐saturable production of ε‐Ap4.
Localisation of an ATP‐binding site on adenylyl cyclase type I after chemical and enzymatic fragmentation
Authors: Droste Martin , Mollner Stefan , Pfeuffer Thomas
Published: April 2003

Photolabeling of partially purified bovine brain adenylyl cyclase (AC I) with [γ
32P8‐N‐ATP led to incorporation of 32P into the 115 kDa catalyst. Further treatment with N‐chlorosuccinimide, which cleaves proteins at tryptophan residues, yielded a 14 kDA 32P‐labeled fragment. The latter was immunoprecipitated by antibody BBC1, recognizing the extreme C‐terminus of AC I, but not by antibody BBC2, recognizing a more remote epitope. Further fragmentation of photolabeled AC I by the proteases Glu‐C and Asp‐N yielded 32P‐labeled peptides corresponding to 2.9 kDa and 5.6 kDa fragments, which were not recognized by any of these antibodies. This narrows the ATP binding site down to a 25 amino acid sequence containing a general motif G(X0–7)KG(X0–4)L/M(X5–7)S/T present in all eukaryotic adenylyl cyclases so far cloned, but also in a variety of bacterial adenylyl cyclases.
Conformational change in DNA induced by cationic bilayer membranes
Authors: Akao Tetsuyuki , Fukumoto Takanori , Ihara Hirotaka , Ito Akio
Published: April 2003

The effect of synthetic cationic lipids on the structure of DNA was studied. The fluorescence enhancement of ethidium bromide on intercalation into DNA was suppressed by the addition of bilayer‐forming lipids, but not by micellar ones. Results on the fluorescence depolarization index suggest that ethidium bromide is not released from DNA by lipids intercalated into DNA. CD spectra of the DNA‐lipid complexes revealed that the structure of DNA was changed only by bilayer‐forming lipids at temperatures lower than their T

c
values. Thus, the conformation of DNA is forced to change by cationic lipids forming the rigid bilayer membrane so that ethidium bromide fluorescence might be reduced, and the conformation can be controlled by selection of the appropriate lipid and temperature.
The role of the T‐loop of the signal transducing protein PII from Escherichia coli
Authors: Jaggi Rene , Ybarlucea Wendy , Cheah Eong , Carr Paul D. , Edwards Karen J. , Ollis David L. , Vasudevan Subhash G.
Published: April 2003

The 3D structure of PII, the central protein that controls the level of transcription and the enzymatic activity of glutamine synthetase in enteric bacteria revealed that residues 37–55 form the ‘T’ loop, part of which protrudes from the core of the protein. Within this loop are the only two tyrosine residues that occur in the polypeptide, and one of them, Tyr‐51, has been shown by chemical modification studies to be the site of uridylylation. Since tyrosine at position 46 is conserved in all known PII, proteins, oligonucleotide directed mutagenesis was used to investigate the role of the two residues. Changing Tyr‐51 to phenylalanine or serine abolished uridylylation. Altering tyrosine at position 46 to phenylalanine affected the rate of uridylylation of the protein. This latter mutation does not alter the structure of PII but the reduction in the uridylylation efficiency suggests a role for this residue in recognition and binding of the sensor enzyme uridylyl transferase.
Effect of D42N substitution in Escherichia coli inorganic pyrophosphatase on catalytic activity and Mg2+ binding
Authors: Avaeva Svetlana M. , Rodina Elena V. , Kurilova Svetlana A. , Nazarova Tatjana I. , Vorobyeva Natalya N.
Published: April 2003

Asp‐42 located in the active site of E. coli inorganic pyrophosphatase (PPase) has been substituted by Asn by site‐directed mutagenesis. This resulted in a 3‐fold increase in hydrolytic activity measured under optimal conditions, a 15.5‐fold increase in the K
m value and retention of the pK values of groups for enzyme and enzyme‐substrate complex. The active site of the enzyme contains 4 metal binding centers (I–IV) [Harutyunyan et al. (1996) Eur. J. Biochem., in press]. Asp‐42 is located near centers II and IV. The D42N replacement had no effect on Mg2+ binding with center II. At the same time, occupation of center IV eliminates the inhibition of inorganic pyrophosphate hydrolysis by high Mg2+ concentrations typical of wild‐type PPase. It is proposed that the increase in activity and decrease in affinity for substrate of the D42N PPase results from changes in Mg2+ binding to center IV. The Mg2+ binding centers of E. coli PPase are lined up in filling order.
Distinct effects of heat shock and ATP depletion on distribution and isoform patterns of human Hsp27 in endothelial cells
Authors: Loktionova Svetlana A. , Ilyinskaya Olga P. , Gabai Vladimir L. , Kabakov Alexander E.
Published: April 2003

To study the cytoprotective capacity of Hsp27 under various cellular stresses, we compared the effects of heating and energy deprivation on its distribution and isoform composition. Cultured endothelial cells from human aorta or umbilical vein were subjected to heat shock (45°C) and ATP‐depleting metabolic stress (CCCP or rotenone in a glucose‐free medium). Both exposures led to the translocation of Hsp27 into the Triton X‐100‐insoluble cellular fraction, whereas the immunofluorescent Hsp27 pattern was characteristic for each stress employed. Heating (5–30 min) caused unexpected association of Hsp27 with thick bundles of actin microfilaments (stress fibers). ATP depletion within 30–120 min resulted in the appearance of Hsp27‐containing compact granules in the nucleus. The insolubilization and relocalization of Hsp27 were reversible in both cases. The stress‐induced shifts in the Hsp27 isoform spectrum indicate an increase in phosphorylation of Hsp27 in heat‐shocked cells and its dephosphorylation in ATP‐depleted cells. We suggest that these stresses diversely affect the phosphorylation status of endothelial Hsp27, thus altering its localization, supramolecular organization and functional activity toward actin.
Structural characterization of extracellular ribonuclease of Bacillus polymyxa: amino acid sequence determination and spatial structure prediction
Authors: Lebedev A.A. , Shlyapnikov S.V. , Pustobaev V.N. , Kirpichnikov M.P. , Dementiev A.A.
Published: April 2003

The primary structure of extracellular Bacillus polymyxa ribonuclease (RNase Bpo) was established by mass spectroscopy analysis and automatic Edman degradation of the individual peptides obtained from protein digestion with Glu‐specific protease V8. RNase Bpo consists of 111 amino acid residues, with a relative molecular weight of 12 607. RNase Bpo is a close structural homolog of RNases of B. amyloliquefaciens (RNase Ba) and B. intermedius (RNase Bi), the similarity of their primary structures being 68%. Molecular modelling of the structure of the complex of RNase Bpo with substrate analog d(CGAC) was performed and a spatial model based on the known crystal structure of RNase Ba complex with the corresponding nucleotide was constructed using the methods of interactive computer graphics and energy minimization. The differences in the primary and tertiary structures of the enzymes were analyzed in order to understand the substrate specificity of Bacillus RNases.
Alkynyl phosphates are potent inhibitors of serine enzyme
Authors: Segal Dina , Shalitin Channa , Shalitin Yechiel , Fischer David R. , Stang Peter J.
Published: April 2003

Propynyl, hexynyl and t‐butylethynyl diethyl phosphates were found to be very powerful covalent inhibitors of serine enzymes. Esterases were inhibited with second‐order rate constants of 107–108 M−1 min−1. Most proteases were inhibited with a rate constant of 104–105 M−1 min−1. By inhibiting chymotrypsin with (3‐14C)‐1‐propynyl diethyl phosphate, it was established that inhibition was caused by binding of the phosphate group to the enzyme active site.
Mitogen crosstalk accompanying urokinase receptor expression in stimulated vascular smooth muscle cells
Authors: Reuning Ute , Dixon Eric P. , Little Sheila P. , Bang Nils U.
Published: April 2003

Thrombin and other mitogens regulate the expression of the urokinase‐type plasminogen activator receptor (uPAR) protein and mRNA levels in bovine vascular smooth muscle cells (SMC). We investigated interactions between mitogens capable of increasing uPAR mRNA levels in SMC. Up‐regulation of uPAR mRNA upon thrombin and basic fibroblast growth factor (bFGF) stimulation was preceded by a 2–3‐fold transient increase in bFGF mRNA within 1 h. TGF‐β
1 did not result in a significant change in bFGF mRNA levels. Platelet‐derived growth factor (PDGF) while substantially enhancing uPAR mRNA levels, diminished bFGF mRNA levels by 3–4‐fold. Both thrombin and bFGF induced the message for bFGF‐R 2–3‐fold. Thrombin also provoked a 3–4‐fold rise in TGF‐β
1 mRNA levels within 30 min. In summary, on the mRNA level, we demonstrated both positive as well as negative feed‐back mechanisms between different mitogens, among them bFGF revealing in addition to autoinduction also up‐regulation of the transcript concentration of its own receptor. Thus, cooperation and possible amplification of mitogenic effects might be implicated in the fine‐tuned regulation of uPAR mRNA in stimulated bovine aorta SMC.
Neurotoxic glutamate treatment of cultured cerebellar granule cells induces Ca2+‐dependent collapse of mitochondrial membrane potential and ultrastructural alterations of mitochondria
Authors: Isaev Nikolaj K. , Zorov Dmitry B. , Stelmashook Elena V. , Uzbekov Rustem E. , Kozhemyakin Maxim B. , Victorov Ilya V.
Published: April 2003

Rhodamine 123 staining and electron microscopy were used to reveal a correlation between the ultrastructural and functional state of cultured cerebellar granule cells after short glutamate treatment. Glutamate exposure (15 min, 100 μM) in Mg2+‐free solution caused considerable ultrastructural alterations in a granule cell: clumping of the chromatin, swelling of the endoplasmic reticulum and mitochondria, and disruption of the mitochondrial cristae. After glutamate treatment, the mitochondria of the neurons lost their ability to sequester rhodamine 123. Both the N‐methyl‐d‐aspartate receptor channel blocker MK‐801 (30 μM) and cobalt chloride (2 mM) prevented the deteriorative effects of glutamate. These data suggest that glutamate‐induced Ca2+ overload of the neurons can lead to non‐specific permeability of the inner mitochondrial membrane, resulting in neuronal death.
The monofunctional glycosyltransferase of Escherichia coli is a member of a new class of peptidoglycan‐synthesising enzymes
Authors: Di Berardino Marco , Dijkstra Arnoud , Stüber Dietrich , Keck Wolfgang , Gubler Marcel
Published: April 2003

Using conserved fingerprints in the glycosyltransferase (GTase) domain of high‐molecular‐weight penicillin‐binding proteins (PBP), a gene (mgt) encoding a putative monofunctional glycosyltransferase has been identified in Haemophilus influenzae and in other bacterial species. Here we report the cloning of the homologous Escherichia coli gene and show that the solubilised membrane fraction of E. coli cells overexpressing the mgt gene contain a significantly increased peptidoglycan synthesis activity. In contrast to the high‐molecular‐weight PBPs, this activity is not inhibited by Flavomycin.
ROCK‐I and ROCK‐II, two isoforms of Rho‐associated coiled‐coil forming protein serine/threonine kinase in mice
Authors: Nakagawa Osamu , Fujisawa Kazuko , Ishizaki Toshimasa , Saito Yuji , Nakao Kazuwa , Narumiya Shuh
Published: April 2003

We recently identified a novel human protein kinase, p160 ROCK, as a putative downstream target of the small GTPase Rho. Using the human ROCK cDNA as a probe, we isolated cDNA of two distinct, highly related sequences from mouse libraries. One encoded a mouse counterpart of human ROCK (ROCK‐I), and the other encoded a novel ROCK‐related kinase (ROCK‐II). Like ROCK/ROCK‐I, ROCK‐II also bound to GTP‐Rho selectively. ROCK‐I mRNA was ubiquitously expressed except in the brain and muscle, whereas ROCK‐II mRNA was expressed abundantly in the brain, muscle, heart, lung and placenta. These results suggest that at least two ROCK isoforms are present in a single species and play distinct roles in Rho‐mediated signalling pathways.
Novel harmful effects of [60]fullerene on mouse embryos in vitro and in vivo
Authors: Tsuchiya Toshie , Oguri Ikuko , Yamakoshi Yoko Nakajima , Miyata Naoki
Published: April 2003

[60]Fullerene (C60) was solubilized with poly(vinylpyrrolidone) (PVP) in water, and the aqueous solution was applied to a mouse midbrain cell differentiation system. On incubation of C60 with various concentrations of PVP, cell differentiation and proliferation were potently inhibited, although weaker than the vehicle controls. C60 was clearly distributed into the yolk sac and embryos by intraperitoneal administration to pregnant mice at 50 mg/kg and had a harmful effect on both conceptuses by microscopical evaluation. This in vivo and in vitro action on embryogenesis is a novel and seriously harmful activity of C60.
Distribution of bending propensity in DNA sequences
Authors: Gabrielian Andrei , Simoncsits András , Pongor Sándor
Published: April 2003

Local binding propensity and curvature of DNA can be characterized using a vector description of DNA bendability, based on a set of parameters derived from deoxyribonuclease I (DNase I) cleavage experiments. Two characteristics — arithmetic and vector averages of bendability — were successfully used to predict experimentally known bendable, rigid and curved segments in DNA. A characteristic distribution of bendability is conserved in evolutionarily related kinetoplast sequences. An analysis of the M. genitalium and H. influenza genomes as well as fragments of human and yeast genomes shows, on the other hand, that highly curved segments — similar to artificially designed curved oligonucleotides — are extremely rare in natural DNA.
Alzheimer's presenilin 1 gene expression in platelets and megakaryocytes
Authors: Vidal Ruben , Ghiso Jorge , Wisniewski Thomas , Frangione Blas
Published: April 2003

The presenilin 1 (PSI) gene located on chromosome 14 has been linked with the majority of early‐onset FAD. The normal biological role of PS1 as well as the mechanism by which mutations in PS1 cause FAD remains unknown. PS1 expression in platelets and the Dami megakaryocytic cell line was examined by Western blot analysis and RT‐PCR. Using an anti‐N‐terminus PS1 antibody we detected PS1 immunoreactive bands of 44, 32 and 27 kDa in both cell types. After RT‐PCR we observed that platelets and megakaryocytes carry at least four different PS1 transcripts. One of them is a novel PS1 splice variant that lacks the coding sequence for exon 10 resulting in a shorter 409 amino acid protein.
Differential translocation of protein kinase C isozymes in rats characterized by a chronic lack of LTP induction and cognitive impairment
Authors: Caputi Antonio , Rurale Sabrina , Pastorino Lucia , Cimino Mauro , Cattabeni Flaminio N. , Luca Monica Di
Published: April 2003

The translocation of protein kinase C isozymes was investigated in an animal model of cognitive deficit and lack of induction of long‐term potentiation (LTP). In MAM rats, presynaptic α, β, ϵ PKC showed enhanced translocation, while postsynaptic γ PKC displayed decreased translocation when compared to control levels. This imbalance of PKC isozyme translocation between the pre‐ and post‐synaptic compartment might therefore represent a possible molecular cause for the lack of synaptic plasticity observed in these animals.
NF‐κB‐mediated regulation of urokinase gene expression by PMA and TNF‐α in human A549 cells
Authors: Guerrini Luisa , Casalino Laura , Corti Angelo , Blasi Francesco
Published: April 2003

Binding sites for different transcription factors have been identified in the regulatory region of the human uPA gene. We investigated the role of NF‐κB and AP‐1 families of transcription factors in the induction of uPA mRNA by TNF‐α and PMA in the A549 cell line constitutively expressing uPA mRNA and protein. Using the protein synthesis inhibitor cycloheximide and the antioxidant PDTC, that have an opposite effect on NF‐κB and AP‐1 activation, we showed that uPA mRNA induction by TNF‐α and PMA in the A549 cell line is mainly due to NF‐κB activation.
Insulin markedly potentiates the capacity of parathyroid hormone to increase expression of 25‐hydroxyvitamin D3‐24‐hydroxylase in rat osteoblastic cells in the presence of 1,25‐dihydroxyvitamin D3
Authors: Armbrecht H.J. , Wongsurawat V.J. , Hodam T.L. , Wongsurawat N.
Published: April 2003

We have previously shown that insulin alters the renal metabolism of 25‐hydroxyvitamin D. To examine the effect of insulin on vitamin D metabolism in bone, we have used UMR‐106 osteoblast‐like cells to study the regulation of 25(OH)D3‐24‐hydroxylase (24‐hydroxylase) expression by insulin. The 24‐hydroxylase is an important enzyme in degrading 1,25‐dihydroxyvitamin D3 (1,25(OH)2D) in target tissues. Insulin alone had no effect on mRNA levels of the cytochrome P450 component (CYP24) of the 24‐hydroxylase or on 24‐hydroxylase activity itself in UMR cells. However, insulin increased the capacity of parathyroid hormone (PTH) to elevate CYP24 mRNA levels by 3–4 fold and to increase 24‐hydroxylase activity by 2‐fold in the presence of 1,25(OH)2D. Insulin increased the maximal responsiveness of UMR cells to PTH without altering their sensitivity. The action of insulin required the presence of 1,25(OH)2D and was partly dependent on new protein synthesis. Insulin‐like growth factor 1 also potentiated the effects of PTH. This marked stimulation of the 24‐hydroxylase by PTH and insulin may serve to regulate 1,25(OH)2D action and/or to produce 24,25‐dihydroxyvitamin D in bone cells.
Activation of a recombinant membrane type 1‐matrix metalloproteinase (MT1‐MMP) by furin and its interaction with tissue inhibitor of metalloproteinases (TIMP)‐2
Authors: Sato Hiroshi , Kinoshita Takeshi , Takino Takahisa , Nakayama Kazuo , Seiki Motoharu
Published: April 2003

Membrane type 1‐matrix metalloproteinase (MT1‐MMP) initiates the activation of the zymogen progelatinase A/72‐kDa type IV collagenase by cleavage of the Asn66‐Leu peptide bond. We previously pointed out that MT1‐MMP possesses a unique amino acid sequence Arg‐Arg‐Lys‐Arg111 which is a potential recognition sequence for furin‐like proteases (Nature, 370 (1994) 61–65). Here, using a recombinant MT1‐MMP expressed in Escherichia coli we demonstrated that furin specifically cleaves MT1‐MMP between Arg111‐Tyr in vitro, which resulted in a stimulation of progelatinase A‐activation function. Tissue inhibitor of metalloproteinases (TIMP)‐2 inhibited activation of progelatinase A by forming a stable complex with activated MT1‐MMP.
Fatty acids stimulate activity and restore respiratory control in a proton channel mutant of cytochrome c oxidase
Authors: Fetter John , Sharpe Martyn , Qian Jie , Mills Denise , Ferguson-Miller Shelagh , Nicholls Peter
Published: April 2003

(1) Removal of a carboxyl at residue 132 of subunit I of Rhodobacter sphaeroides cytochrome c oxidase significantly inhibits electron transfer and makes proton pumping undetectable [Fetter et al. (1995) Proc. Natl. Acad. Sci. USA 92, 1604–1608]. When reconstituted into phospholipid vesicles (COV), wild‐type oxidase shows respiratory control that is partially released by either valinomycin or nigericin and fully released by the two ionophores combined. Under the same conditions, the D132A mutant COV show anomalous ionophore responses, including inhibition by valinomycin or by CCCP. Nevertheless, oxidase activity results in development of a similar membrane potential in COV containing either wild‐type or D132A oxidase, and the ionophore responses of the membrane potential are similar for both enzymes. (2) Long chain fatty acids such as arachidonic acid, but not fatty alcohols, stimulate steady‐state electron transfer activity 3–7‐fold, with either detergent‐solubilized (purified) D132A oxidase or the reconstituted form. The effect is specific for this mutant and is not seen with wild‐type or other mutants of similar overall activity. Arachidonate‐treated D132A COV show normal ionophore responses to valinomycin and nigericin and full release of respiration in presence of both ionophores or of CCCP. Thus, arachidonate and some other fatty acids abolish the ionophore anomalies seen when the D132A enzyme is reconstituted in their absence. (3) Fatty acid addition does not restore proton pumping, likely because fatty acids also induce proton permeability and some degree of uncoupling. A model of D132A function is presented and possible roles for the fatty acids in ‘chemical rescue’ of the mutant are discussed.
Assignment of protein disulphides by a computer method using mass spectrometric data
Authors: Caporale Carlo , Sepe Ciro , Caruso Carla , Pucci Piero , Buonocore Vincenzo
Published: April 2003

We designed a computer program for the assignment of protein disulphides using mass spectrometric data. All the theoretical linear peptides containing from one to three cysteines are generated on the basis of the protein sequence. Their combination ways are determined in order to create all the possible disulphide‐bridged fragments containing from two to six cysteines and to calculate their molecular weight. One, two and three S‐S bridges per linked fragment were considered, taking into account the possibility that the fragments contain unabridged residues. The mass data obtained from the spectral analysis of peptide mixtures of the digested protein are then associated to the fitting structures of disulphide‐bridged peptides, giving rise to the primary output. This output can then be screened by using information on the specificity of the proteolytic agent(s) used and/or any further mass data provided by Edman degradation and/or carboxypeptidase treatment of the peptide mixtures. The need for such a computer aid is discussed and examples of application are given.
Analysis of the recognition mechanism of the alternative pathway of complement by monoclonal anti‐factor H antibodies: evidence for multiple interactions between H and surface bound C3b
Authors: Sakari Jokiranta T. , Zipfel Peter F. , Hakulinen Juha , Kühn Sabine , Pangburn Michael K. , Tamerius John D. , Meri Seppo
Published: April 2003

The ability of the alternative pathway of complement to discriminate targets as either activators or non‐activators is mediated by different binding properties of factor H to surface‐associated C3b molecules. In the present study we have probed the interaction between H and C3b using five anti‐H mAb. The binding sites of the mAb were mapped by Western blotting using both recombinant and trypsin‐generated H fragments. Two mAb bound to CCP1 (90X, 196X), two to CCP5 (MRC OX24, 86X) and one to CCP8‐15a (131X). At a molar ratio 2:1 of 125I‐H:mAb all tested mAb enhanced binding of H to both activator‐ and non‐activator‐bound C3b. At higher concentrations two mAb had an inhibitory effect on H binding to surface‐associated C3b (OX24, 131X). Thus the mAb 131X inhibits H binding to surface‐bound C3b but unlike OX24 it does not bind to the previously described C3b binding site within or near CCP4‐5. These results indicate that there is an additional interaction site on factor H for surface‐bound C3b.
Removal of proteasomes from the nucleus and their accumulation in apoptotic blebs during programmed cell death
Authors: Pitzer Frank , Dantes Ada , Fuchs Tania , Baumeister Wolfgang , Amsterdam Abraham
Published: April 2003

Apoptosis can be initiated in immortalized cAMP‐stimulated rat ovarian granulosa cells by induction of wild‐type p53 activity. Immunocytochemical studies using confocal microscopy reveal that in apoptotic, unlike in normal growing cells, the proteasomes are removed from the nucleus and accumulate within the apoptotic blebs at the periphery of the cell. In parallel, a striking reorganization of the actin cytoskeleton is observed which forms a spherical network separating the apoptotic blebs from the cytoplasmic organelles, such as mitochondria and lipid droplets which remain in the perinuclear region. The reorganization of the actin cytoskeleton as well as disappearance of proteasomes from the nucleus suggest possible function of proteasomes in apoptotic regulation.
Phenoxymethylpenicillin amidohydrolases from Penicillium chrysogenum
Authors: Whiteman P.A. , Abraham E.P.
Published: April 2003

A phenoxymethylpenicillin amidohydrolase which hydrolyses phenoxymethylpenicillin to 6‐aminopenicillanic acid (6‐APA) has been isolated from two species of Penicillium chrysogenum. The amidohydrolase had a molecular mass of approx. 42 kDa. Its activity with benzylpenicillin as substrate was only 1.5% of that with phenoxymethylpenicillin and it was inhibited by its products. No penicillin formation from 6‐APA and phenoxyacetyl or phenylacetyl coenzyme A was observed. The enzyme is thus distinct from the phenylacetyl coenzyme A: 6‐APA acyltransferase, which also has amidohydrolase activity and is involved in the final stages of the biosynthesis of penicillins.
Purification and characterization of nuclear alkaline phospholipase A2 in rat ascites hepatoma cells
Authors: Oishi Takashi , Tamiya-Koizumi Keiko , Kudo Ichiro , Iino Satoshi , Takagi Kenzo , Yoshida Shonen
Published: April 2003

The alkaline phospholipase A2 (PLA2) was purified from nuclei of rat ascites hepatoma cells (AH7974) by column chromatography with a Sephacryl S‐300 column and an immunoadsorbent using anti‐group II PLA2 monoclonal antibody. From these two columns, the alkaline PLA2 was eluted in parallel with a 17‐kDa protein which is reactive to another antigroup II PLA2 polyclonal antibody. Approximately 80% of nuclear PLA2 was inhibited by this antibody. The alkaline PLA2 was found in association with the chromatin fraction among subnuclear fractions. By an immunocytochemical staining, the nuclei of AH7974 were stained more strongly than other parts of cells with anti‐group II PLA2 antiserum.
CO2 response for expression of ribulose‐1,5‐bisphosphate carboxylase/oxygenase genes is inhibited by AT‐rich decoy in the cyanobacterium
Authors: Onizuka Takuo , Akiyama Hideo , Endo Sumiyo , Kanai Shozo , Hirano Masahiko , Tanaka Satoshi , Miyasaka Hitoshi
Published: April 2003

The CO2‐regulatory function of the AT‐rich element in the promoter for ribulose‐1,5‐bisphosphate carboxylase/oxygenase (rbc) genes in the cyanobacterium Synechococcus sp. PCC7002 was analyzed using the transcription factor decoy approach. Double‐stranded phosphorothioate AT‐rich oligonucleotides with high affinity for a sequence‐specific DNA‐binding protein were successfully introduced into cyanobacterial cells in culture without any transfection reagent. The AT‐rich decoy oligonucleotides interfered with CO2 regulation of rbc expression by blocking the binding of the sequence‐specific DNA‐binding protein, indicating that the AT‐rich element plays a critical role in CO2 regulation for rbc genes. The decoy oligonucleotide approach to cyanobacteria provides a simple and excellent tool for investigating transcriptional regulation in vivo.
Isoproterenol produces a rapid increase in sialidase activity in rat heart tissue and cardiomyocyte‐derived H9c2 cells in culture
Authors: Saito Megumi , Sakiyama Katsuhiro , Shiota Tomoko , Ito Masaki
Published: April 2003

The effects of isoproterenol on sialidase activity in rat cardiomyocytes were examined. Administration of isoproterenol to rats (0.2 or 2 mg/kg body weight) produced an increase in sialidase activity in total membrane fraction of heart tissue within 120 min (121±13% of the control at 120 min after administration of 0.2 mg isoproterenol/kg, n=5, P<0.05). Sialidase activity in cardiomyocyte‐derived H9c2 cells was also increased by treatment with isoproterenol (10 μM) for 60 min. The effect of isoproterenol on sialidase activity was amplified by the addition of 3‐isobutyl‐1‐methylxanthine (IBMX). Sialidase activity in H9c2 cells was elevated by treatment with dibutyryl cAMP plus IBMX without isoproterenol. The content of N‐acetylneuraminic acid in cells decreased by 22% after treatment with isoproterenol plus IBMX. These results suggest that sialidase activity in rat cardiomyocytes is regulated by β‐adrenergic stimulators via a cAMP‐dependent process. The increased activity of sialidase may account for the reduction of sialic acid content of cells.
Molecular evolution of the modulator of chloroplast ATP synthase: origin of the conformational change dependent regulation
Authors: Hisabori Toru , Ueoka-Nakanishi Hanayo , Konno Hiroki , Koyama Fumie
Published: April 2003

Chloroplast ATP synthase synthesizes ATP by utilizing a proton gradient as an energy supply, which is generated by photosynthetic electron transport. The activity of the chloroplast ATP synthase is regulated in several specific ways to avoid futile hydrolysis of ATP under various physiological conditions. Several regulatory signals such as ΔμH+, tight binding of ADP and its release, thiol modulation, and inhibition by the intrinsic inhibitory subunit ϵ are sensed by this complex. In this review, we describe the function of two regulatory subunits, γ and ϵ, of ATP synthase based on their possible conformational changes and discuss the evolutionary origin of these regulation systems.
Proton translocation driven by ATP hydrolysis in V‐ATPases
Authors: Kawasaki-Nishi Shoko , Nishi Tsuyoshi , Forgac Michael
Published: April 2003

The vacuolar H+‐ATPases (or V‐ATPases) are a family of ATP‐dependent proton pumps responsible for acidification of intracellular compartments and, in certain cases, proton transport across the plasma membrane of eukaryotic cells. They are multisubunit complexes composed of a peripheral domain (V1) responsible for ATP hydrolysis and an integral domain (V0) responsible for proton translocation. Based upon their structural similarity to the F1F0 ATP synthases, the V‐ATPases are thought to operate by a rotary mechanism in which ATP hydrolysis in V1 drives rotation of a ring of proteolipid subunits in V0. This review is focused on the current structural knowledge of the V‐ATPases as it relates to the mechanism of ATP‐driven proton translocation.
Drug‐use initiation and conduct disorder among adolescents in drug treatment
Authors: Hser Yih‐Ing , Grella Christine E. , Collins Cyleste , Teruya Cheryl
Published: April 2003

This study investigated effects of drug‐use initiation and conduct disorder (CD) among 1031 adolescents who participated in the Drug Abuse Treatment Outcomes Studies for Adolescents (DATOS‐A) sponsored by the National Institute on Drug Abuse (NIDA). The mean age of first drug use was 12.7 (s.d.=2.2), 57% met DSM‐III‐R criteria for CD, and earlier initiators were more likely to have CD. About 78% of the adolescents with CD reported that their first CD symptom occurred prior to drug‐use initiation. The proportions of adolescents who had prior treatment were similar (about 28%) across all groups, but earlier initiators reported a greater number of treatment episodes and younger ages at their first treatment. Conduct disordered adolescents revealed greater problems prior to DATOS‐A treatment, but they appeared to be more motivated and ready for treatment. Although adolescents with CD still showed worse outcomes after treatment, the impact of CD appeared to lessen when pretreatment differences were controlled. To a lesser extent, adolescents who began using drugs at earlier ages had greater alcohol and drug use and other problems at intake, but their treatment outcomes appeared to be similar to later initiators. There were few significant interaction effects of initiation and CD. Findings from this study highlight the importance of better understanding the progression of drug use, treatment utilization, and psychiatric comorbidity among adolescents with substance abuse problems.
Ventrally emigrating neural tube cells migrate into the developing vestibulocochlear nerve and otic vesicle
Authors: Ali M.M. , Jayabalan S. , Machnicki M. , Sohal G.S.
Published: April 2003

Virtually all cell types in the inner ear develop from the cells of the otic vesicle. The otic vesicle is formed by the invagination of non‐neural ectodermal cells known as the otic placode. We investigated whether a recently described cell population, originating from the ventral part of the hindbrain neural tube known as the ventrally emigrating neural tube (VENT) cells, also contributes cells to the otic vesicle. The ventral hindbrain neural tube cells were labeled with the fluorescent vital dye DiI or replication‐deficient retroviruses containing the LacZ gene in chick embryos on embryonic day 2, after the emigration of neural crest from this region. One day later, the labeled cells were detected only in the hindbrain neural tube. Shortly thereafter, the labeled cells began to appear in the eighth (vestibulocochlear) cranial nerve and otic vesicle. From embryonic day 3.5–5, the labeled cells were detected in the major derivatives of the otic vesicle, i.e. the endolymphatic duct, semicircular canals, utricle, saccule, cochlea, and vestibulocochlear ganglion. That the emigrated cells originated from the ventral part of the hindbrain neural tube was confirmed by focal application of DiI impregnated filter paper and with quail chimeras. It is concluded that, in addition to the otic placode cells, the otic vesicle also contains the ventrally emigrating neural tube cells, and that both cell populations contribute to the structures and cell types in the inner ear. It is well known that inductive signals from the hindbrain are required for the morphogenesis of the inner ear. The migration of the hindbrain neural tube cells into the otic vesicle raises the possibility that the inductive effect of the hindbrain might be mediated, at least in part, by the ventrally emigrating neural tube cells and that, therefore, a mechanism exists that involves cells rather than diffusible molecules only.
Antinociceptive effects of RB101(S), a complete inhibitor of enkephalin‐catabolizing enzymes, are enhanced by (+)‐HA966, a functional NMDA receptor antagonist: a c‐Fos study in the rat spinal cord
Authors: Buritova Jaroslava , Le Guen Stéphanie , Fournié‐Zaluski Marie‐Claude , Roques Bernard P , Besson Jean‐Marie
Published: April 2003

The effects of the S enantiomer of RB101, a complete inhibitor of enkephalin‐catabolizing enzymes, alone or in combination with a functional NMDA receptor antagonist, (+)‐HA966 were studied on the spinal c‐Fos protein expression in the carrageenan model of inflammatory nociception. One hour 30 min after intraplantar carrageenan in awake rats, c‐Fos immunoreactive (c‐Fos‐IR) nuclei were preferentially located in the laminae I—II and V—VI of the spinal dorsal horn, i.e., spinal areas containing numerous neurons responding exclusively, or not, to peripheral nociceptive stimuli. RB101(S) (5, 10, 20 and 40 mg/kg i.v.) dose‐dependently reduced the total number of carrageenan‐evoked c‐Fos‐IR nuclei (r=0.63, P<0.01), with 49±3% reduction (P<0.001) for the highest dose. Two highest doses of RB101(S) (20 and 40 mg/kg) significantly reduced the number of carrageenan‐evoked c‐Fos‐IR nuclei in both superficial I—II (32±7% and 36±5% reduction, respectively, P<0.05 for both) and deep V—VI (42±6% and 61±2% reduction, respectively, P<0.001 for both) laminae. The effects of RB101(S) were naloxone‐reversible. Combination of low doses of RB101(S) (2.5 or 10 mg/kg i.v.) and an inactive dose of (+)‐HA966 (2.5 mg/kg s.c.) produced supra‐additive effects (39±4% and 51±5% reduction of the total number of c‐Fos‐IR nuclei, respectively, P<0.001 for both). These effects were partially reversed by naloxone. These results provide evidence for the potent effects of combination of RB101(S) and (+)‐HA966. Considering the absence of major opioid side effects of RB101(S) and the marked increase of its antinociceptive effects by NMDA receptor antagonist, this type of drug combination could have beneficial therapeutical application.