bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of the catalytic subunit of type‐1 protein phosphatase by the v‐abl tyrosine kinase
Authors: Villa-Moruzzi Emma , Zonca Paolo Dalla , Crabb John W.
Published: July 2002

The catalytic subunit of type‐1 protein phosphatase (PP1) was phosphorylated by the tyrosine kinase v‐abl as follows: (i) cytosolic PP1 was phosphorylated more (0.73 mol/mol) than PP1 obtained from the glycogen particles (0.076 mol/mol), while free catalytic subunit isolated in the active or inactive form from cytosolic PP1 was phosphorylated even less and catalytic subunit complexed with inhibitory was not phosphorylated; (ii) phosphorylation stoichiometry was dependent on the concentration of PP1 and 3 h incubation at 30°C was required for maximal phosphorylation; (iii) phosphorylation was on a tyrosine residue located in the C‐terminal region of PP1 which is lost during proteolysis; (iv) phosphorylation did not affect enzyme activity but allowed conversion from the active to the inactive form upon incubation with inhibitory of a PP1 form that in its dephospho‐form did not convert.
Do submaximal InsP3 concentrations only induce the partial discharge of permeabilized hepatocyte calcium pools because of the concomitant reduction of intraluminal Ca2+ concentration?
Authors: Combettes Laurent , Claret Michel , Champeil Philippe
Published: July 2002

In several types of cells whose cytoplasmic Ca2+ is regulated by inositol phosphate derivatives, low concentrations of InsP3 added to permeabilized cell suspensions induce the rapid discharge or part of the InsP3‐sensitive Ca2+ pool instead of slow monophasic release of Ca2+ from the entire pool. As a tentative explanation for this puzzling observation, sometimes called ‘quantal release’, it was suggested that the reduced intraluminal Ca2+ concentration remaining in the Ca2+ pool after a certain amount of Ca2+ had been released might allosterically reduce the channels' affinity for InsP3 and the corresponding InsP3‐dependent Ca2+ efflux, and thus result in partial pool discharge (Irvine, R.F. (1990) FEBS Lett. 263, 5–9). We have tested this hypothesis by manipulating the Ca2+ pool contents with ionophore, and found that the rate of InsP3‐dependent Ca2+ efflux after ionophore‐induced partial discharge of the Ca2+ pools was much faster than what was predicted on the basis of this hypothesis. Heterogeneity of the Ca2+ pools appears to be a more likely reason for the ‘quantal release’ behavior.
A novel ETA antagonist (BQ‐123) inhibits endothelin‐1‐induced phosphoinositide breakdown and DNA synthesis in rat vascular smooth muscle cells
Authors: Eguchi Satoru , Hirata Yukio , Ihara Masaki , Yano Mitsuo , Marumo Fumiaki
Published: July 2002

The effects of a novel cyclic pentapeptide (BQ‐123), an endothelin (ET) antagonist selective for the ETA receptor subtype, on phosphoinositide breakdown and DNA synthesis stimulated by ET‐1 were studied in cultured rat vascular smooth muscle cells (VSMC). BQ‐123 competitively inhibited the binding of [125I]ET‐1 to VSMC with the apparent K
i of 4 × 10−9 M. BQ‐123 dose‐dependently inhibited formation of inositol‐1,4,5‐trisphosphate and [3H]thymidine uptake stimulated by ET‐1. These data suggest that the ET‐1‐induced DNA synthesis in VSMC is mainly mediated by ETA receptor subtype.
Structure/function analyses of human sex hormone‐binding globulin by site‐directed mutagenesis
Authors: Bocchinfuso W.P. , Warmels-Rodenhiser S. , Hammond G.L.
Published: July 2002

Human sex hormone‐binding globulin (hSHBG) and rat androgen‐binding protein (rABP) exhibit distinct affinities for sex‐steroids. We therefore constructed and expressed a hSHBG/rABP hybrid cDNA encoding the N‐terminal portion of hSHBG (205 residues) and the C‐terminal portion of rABP (168 residues). The resulting chimera displayed similar steroid‐binding characteristics as hSHBG and was recognised by a monoclonal antibody (S1B5) for hSHBG. We then created substitutions at Ser‐133, His‐136 and Met‐139. The Asp‐133 and Gln‐136 mutants bound steroids in the same way as normal hSHBG while the steroid‐binding affinity of Trp‐139 was reduced. All three mutants cross‐reacted similarly in a hSHBG radioimmunoassay, but Gln‐136 was recognised poorly by the S1B5 antibody. These data imply that residues involved in steroid‐binding are located within the N‐terminal half of hSHBG and include Met‐139, and that the S1B5 epitope is located in this region.
Developmental expression of voltage‐sensitive K+ channels in mouse skeletal muscle and C2C12 cells
Authors: Lesage Florian , Attali Bernard , Lazdunski Michel , Barhanin Jacques
Published: July 2002

The developmental expression of voltage‐sensitive K+ channels was analyzed by Northern blot in mouse skeletal muscle. Of nine Shaker‐like genes studied, eight are expressed in this mammalian muscle. Their expression is differentially regulated during development. The mouse cell line C2C12 has been used to study expression of voltage‐sensitive K+ channels during in vitro myotube differentiation. Different voltage‐sensitive K+ channel messages are also expressed in these cells which display a pattern of expression depending upon the differentiation stage. The message for the very peculiar K+ channel or IsK type could only be detected by polymerase chain reaction on skeletal muscle mRNA.
Modulation of rat liver peroxisomal and microsomal fatty acid oxidation by starvation
Authors: Orellana Myriam , Fuentes Oscar , Rosenbluth Helen , Lara Mauricio , Valdés Elena
Published: July 2002

In this work the microsomal lauric acid ω‐hydroxylation, fatty acid peroxisomal β‐oxidation, and the levels of cytochrome P‐450 IVAI were studied in liver tissue from starved rats. Starvation increased the peroxisomal β‐oxidation and the microsomal hydroxylation of fatty acids. The correlation between these activities would support the proposal that both processes are linked, contributing in part to catabolism of fatty acids in liver of starved rats.
Human guanylin: cDNA isolation, structure, and activity
Authors: Wiegand Roger C. , Kato Johji , Huang Margaret D. , Fok Kam F. , Kachur James F. , Currie Mark G.
Published: July 2002

Guanylin is a mammalian peptide homologue of heat‐stable enterotoxins that acts on intestinal guanylate cyclase to elicit an increase in cyclic GMP. We have isolated a cDNA encoding an apparent precursor of guanylin from a human intestinal cDNA library. The mRNA is expressed at high levels in human ileum and colon. Human guanylin stimulated increases in T84 cell cyclic GMP levels, displaced 125I‐labelled heat‐stable enterotoxin (STa) binding to this cell line, and stimulated increases in short‐circuit current (Isc) or isolated rat proximal colonic mucosa. This peptide may play a role in regulating fluid and electrolyte absorption in human intestines.
Affinities of bovine photoreceptor cGMP phosphodiesterases for rod and cone inhibitory subunits
Authors: Hamilton Susan E. , Prusti Rabi K. , Bentley J.Kelley , Beavo Joseph A. , Hurley James B.
Published: July 2002

Rods and cones have analogous phototransduction components and cycles, but differ from each other in their physiological response to light. Differences between the affinities of rod and cone phosphodiesterase (PDE) catalytic subunits for their respective inhibitory subunits could potentially contribute to these physiological differences. To test this idea, we expressed both the 13 kDa PDE subunit, unique to a subset of bovine retinal cones [(1990) J. Biol. Chem. 265, 11259‐11264], and the rod PDE 11 kDa inhibitory subunit in E. coll, purified them, and compared their abilities to inhibit rod and cone PDE catalytic subunits. Rod PDE has similar K
i values (~80 pM) for both the rod and cone recombinant inhibitory subunits. Activated cone PDE has K
i values of 200 pM for the cone 13 kDa subunit and 600 pM for rod PDEγ.
Functional expression of gastric H,K‐ATPase using the baculovirus expression system
Authors: Klaassen Corné H.W. , Van Uem Tom J.F. , De Moel Mariëlle P. , De Caluwé Godelieve L.J. , Swarts Herman G.P. , De Pont Jan Joep H.H.M.
Published: July 2002

A novel approach to construct a single recombinant baculovirus expressing two protein subunits simultaneously by replacing polyhedrin as well as p10 coding sequences is described. The recombinant baculovirus expressed the α‐ as well as the β‐subunit of the gastric H.K‐ATPase. Sf9 cells infected with this virus exhibited a K+‐ and SCH 28080‐sensitive ATP‐dependent phosphorylation capacity in purified Sf9 membranes similar to Natlve H,K‐ATPase. This activity was not present in control membranes containing only one of the two H,K‐ATPase subunits. We therefore conclude that both subunits are essential for the phosphorylation capacity of H.K‐ATPase.
Peroxynitrite modification of low‐density lipoprotein leads to recognition by the macrophage scavenger receptor
Authors: Graham Annette , Hogg Neil , Kalyanaraman Balaraman , O'Leary Vanessa , Darley-Usmar Victor , Moncada Salvador
Published: July 2002

Peroxynitrite is an oxidant which could be formed in the vasculature by the reaction of superoxide with nitric oxide. It is capable of modifying amino acid residues and of initiating lipid peroxidation. In the present study we have shown that peroxynitrite converts low density lipoprotein to a form recognized by the macrophage scavenger receptor and that this process is associated with modification of the protein and lipid, and with the oxidation of α‐tocopherol to α‐tocopherol quinone.
Cloning, characterization and overexpression of a Streptococcus pyogenes gene encoding a new type of mitogenic factor
Authors: Iwasaki Makoto , Igarashi Hisanaga , Hinuma Yorio , Yutsudo Takashi
Published: July 2002

A new type of mitogenic factor, termed MF, has been found in the culture supernatant of Streptococcus pyogenes and its N‐terminal amino acid sequence has been determined. On the basis of this sequence, an S. pyogenes gene encoding MF was cloned and its nucleotide sequence was determined. The MF gene includes a long, open reading frame with 813 nucleotides capable of encoding the MF precursor protein with 271 amino acids. Removal of the putative 43 residues as a signal peptide results in the mature MF protein with 228 amino acids. The molecular mass of the mature MF is calculated as 25,363 which is consistent with the previously determined value of 25,370 for MF secreted from S. pyogenes. Neither nucleotide nor amino acid sequence homology was found between the mature MF and other streptococcal pyrogenic exotoxins, such as SPE A, SPE B and SPE C. The mature MF was recombinantly overexpressed as a fusion protein with glutathione S‐transferase in Escherichia coli. The recombinant protein showed mitogenic activity in rabbit peripheral blood lymphocytes and immunoreactivity with the rabbit antiserum raised against the secreted MF from S. pyogenes. These data indicate that a unique gene encoding MF was cloned from S. pyogenes.
Sarcoplasmic reticulum release channels from frog skeletal muscle display two types of calcium dependence
Authors: Bull Ricardo , Marengo Juan José
Published: July 2002

Calcium channels derived from sarcoplasmic reticulum of frog skeletal muscle were fused with planar lipid bilayers. Fractional open times displayed two types of calcium dependence: (i) blockable channels showed a bell‐shaped calcium dependence with an activation constant of 4.5 μM, a Hill coefficient for activation of 1.46 and a blocking constant of 226 μM, and (ii) non‐blockable channels displayed a sigmoidal calcium dependence with an activation constant of 1.1 μM and a Hill coefficient of 1.42; no blocking effect was seen with calcium up to 0.5 mM. These two types of calcium dependence may underlie the coexistence of two different pathways for calcium release in frog skeletal muscle.
Molecular cloning and gene organization of the mouse mitochondrial 3,2‐trans‐enoyl‐CoA isomerase
Authors: Stoffel Wilhelm , Düker Maria , Hofmann Kay
Published: July 2002

3,2‐trans‐enoyl‐CoA isomerase (mECI, E.C. 5.3.3.8) is the key enzyme of mitochondrial β‐oxidation of unsaturated fatty acids. A mouse cDNA clone spanning the entire coding region of mECI was isolated and sequenced. Subsequently, two overlapping genomic clones containing the complete mECI gene were isolated and characterized. The mouse mECI cDNA comprises an open reading frame of 867 bp, encoding a protein of 32 kDa. The mECI gene, spanning about 15 kb, consists of seven exons. Multiple transcription starts were determined by primer extension experiments. Knowledge of the gene organization and availability of genomic clones for mouse mECI will facilitate the study of unsaturated fatty acid metabolism in normal and pathological states.
Verapamil and diltiazem inhibit receptor‐operated calcium channels and intracellular calcium oscillations in rat hepatocytes
Authors: Striggow Frank , Bohnensack Ralf
Published: July 2002

Fura‐2 loaded rat hepatocytes were used to determine whether the L‐type channel blockers, verapamil and diltiazem, affect receptor‐operated calcium channels (ROCCs). The flux through ROCCs was followed by quenching of fura‐2 fluorescence due to the influx of extracellular Mn2+ induced by vasopressin. Verapamil as well as diltiazem inhibited vasopressin‐stimulated Mn2+ influx in a dose‐dependent manner up to 60% at concentrations of 200–400 μM. Furthermore, both inhibitors decreased significantly the frequency of phenylephrine‐induced oscillation of [Ca2+]i. The experimental findings indicate that L‐type channel blockers inhibit ROCCs in rat hepatocytes.
Production of the Alzheimer's β‐amyloid peptide by C6 glioma cells
Authors: Morato Esperanza , Mayor Federico
Published: July 2002

The β‐amyloid peptide (Aß) is a 4 kDa proteolytic fragment derived from the β‐amyloid precursor protein (βAPP) which is deposited as amyloid fibrils in the brains of patients with Alzheimer's disease. βAPP processing was investigated in C6 glioma cells using several affinity‐purified anti‐peptide antibodies raised against different domains of the protein. Both direct immunoblot analysis of C6 glioma conditioned medium and metabolic labeling of cells followed by immunoprecipitation of extracellular medium with specific antibodies revealed that these glial cells normally produce and release a soluble 4 kDa peptide which co‐migrates with synthetic Aß (1–40) and is specifically recognized by antibodies raised against N‐ or C‐terminal domains of the β‐amyloid peptide. Our results further suggest that glial cells may prove a major source of β‐amyloid production in the nervous tissue.
PDI and glutathione‐mediated reduction of the glutathionylated variant of human lysozyme
Authors: Hayano Toshiya , Inaka Koji , Otsu Mieko , Taniyama Yoshio , Miki Kunio , Matsushima Masaaki , Kikuchi Masakazu
Published: July 2002

A mutant human lysozyme, designated as C77A‐a, in which glutathione is bound to Cys95, has been shown to mimic an intermediate in the formation of a disulfide bond during folding of human (h)‐lysozyme. Protein disulfide isomerase (PDI), which is believed to catalyze disulfide bond formation and associated protein folding in the endoplasmic reticulum, attacked the glutathionylated h‐lysozyme C77A‐a to dissociate the glutathione molecule. Structural analyses showed that the protein is folded and that the structure around the disulfide bond, buried in a hydrophobic core, between the protein and the bound glutathione is fairly rigid. Thioredoxin, which has higher reducing activity of protein disulfides than PDI, catalyzed the reduction with lower efficiency. These results strongly suggest that PDI can catalyze the disulfide formation in intermediates with compact structure like the native states in the later step of in vivo protein folding.
The intriguing link between modulation of both multidrug resistance and ligand‐toxin conjugate cytotoxicity
Authors: Jaffrézou Jean-Pierre , Laurent Guy
Published: July 2002

Pharmacological agents which possess a chemosensitizing activity (i.e. the ability to modulate the multidrug resistance phenotype) can equally enhance ligand‐toxin conjugate cytotoxicity. By confronting results obtained in both fields of research it appears that quite a number of agents, which are structurally unrelated, possess this bilateral effect. We have therefore attempted to provide a brief review of the literature and to discuss a hypothesis by which a common mechanism such as modifications in intracellular vesicle sorting and/or lipid metabolism may be implicated. We believe that these observations may provide clues for future research.
Selective coupling of the human anaphylatoxin C5a receptor and α16 in human kidney 293 cells
Authors: Buhl Anne Mette , Eisfelder Bartholomew J. , Worthen G.Scott , Johnson Gary L. , Russell Marijane
Published: July 2002

The peptide C5a which is generated during the complement cascade is an important chemotactic factor involved in the inflammatory response. The C5a receptor (C5aR) primary sequence suggests that it has a serpentine structure of seven transmembrane domains which is typical of classical G‐protein‐coupled receptors. To investigate the signal transduction mechanism of C5a we transiently expressed the C5aR in combination with different G‐protein a subunits in human kidney 293 cells and measured the PLC activity induced upon C5a stimulation. Cotransfection of C5aR and α16 stimulated PLC while cotransfection of C5aR with either αq or α12 did not.
Endothelin‐1 stimulated phospholipase D in A10 vascular smooth muscle derived cells is dependent on tyrosine kinase Evidence for involvement in stimulation of mitogenesis
Authors: Wilkes Lesley C. , Patel Viral , Purkiss John R. , Boarder Michael R.
Published: July 2002

The mechanism whereby endothelin stimulates motogenesis of vascular smooth muscle cells is not understood. Here we show that endothelin‐1 stimulates phospholipase D by a protein kinase C and tyrosine kinase dependent mechanism, and present evidence that implicate the phosphatidic acid formed by phospholipase D in the mitogenic response.
Proprotein processing activity and cleavage site selectivity of the Kex2‐like endoprotease PACE4
Authors: Creemers John W.M. , Kormelink Paul J.Groot , Roebroek Anton J.M. , Nakayama Kazuhisa , Van de Ven Wim J.M.
Published: July 2002

Proprotein processing activity of the Kex2‐like mammalian endoprotease PACE4 and its cleavage selectivity for sites with basic amino acid residues were determined. Using a recombinant vaccinia virus‐based expression system, PACE4 was expressed in pig kidney PK(15) cells and, like two other Kex2‐like endoproteases furin and PC6A, shown to correctly process the precursor of von Willebrand factor (pro‐vWF). Furthermore, characteristics of the cleavage site selectivity of PACE4 were compared to those of furin and PC6A using the vWF cleavage site mutants vWFR‐lG, vWFK‐2A, and vWFR‐4A as substrates. Cleavage site selectivity of PACE4 and PC6A appeared to be similar but they differed from that of furin.
Role of protein kinase C in cholinergic stimulation of lacrimal gland protein secretion
Authors: Zoukhri Driss , Hodges Robin R. , Dicker Deanna M. , Dartt Darlene A.
Published: July 2002

The purpose of this study was to determine the role of protein kinase C (PKC) isozymes in carbachol‐induced protein secretion in the lacrimal gland. Three isoforms of PKC are present in rat lacrimal gland: PKC‐α, ‐δ and ‐g3. Carbachol translocated PKC‐ε during 5 s incubation. Pretreatment with PdBu for 0 to 4 h down‐regulated PKC‐α by 31% at 20 min, PKC‐ε by 36% at 2 h, and PKC‐δ by 37% at 4 h. A 2 h phorbol ester treatment inhibited carbachol‐induced secretion completely at 1 min and partially at 5, and 20 min, but did not alter the carbachol‐induced increase in the intracellular [Ca2+]. We conclude that PKC‐α and ‐g3, but not PKC‐δ, are implicated in cholinergic agonist‐induced protein secretion in rat lacrimal gland.
A high‐conductance voltage‐dependent multistate Ca2+ channel found in sea urchin and mouse spermatozoa
Authors: Beltrán C. , Darszon A. , Labarca P. , Liévano A.
Published: July 2002

Ion fluxes through poorly understood channel‐mediated mechanisms participate in the interaction between spermatozoa and egg. Previously, we reported the characterization in planar bilayers of a high conductance Ca2+‐selective, voltage‐dependent multistate channel from S. purpuratus sea urchin sperm plasma membranes [14]. Here we show that this ion channel can be directly transferred to planar lipid bilayers upon sperm addition, from sea urchin [S. purpuratus and L. pictus) and from mouse. We found that spermatozoa from these species posses a conspicuous Ca2+‐selective, high conductance, multi‐state, voltage‐dependent channel, which displays similar voltage dependence and equal PBa2+/PK
+ ~4 in the three species. The presence of this Ca2+ channel in such diverse species suggests it plays a relevant role in sperm physiology. The high sensitivity of planar bilayers to detect single ion channels can now be used to study ion channel regulation and gamete interaction.
The N‐terminal half of a mitochondrial presequence peptide inserts into cardiolipin‐containing membranes Consequences for the action of a transmembrane potential
Authors: Leenhouts J.M. , Török Z. , Mandieau V. , Goormaghtigh E. , de Kruijff B.
Published: July 2002

The orientation of a mitochondrial presequence peptide, associated with anionic lipid‐containing model membranes, was investigated. The peptide inserts with its N‐terminal α‐helical part into cardiolipin (CL) monolayers so that the N‐terminal 14 residues are protected from proteinase K. In phosphatidylglycerol (PG) monolayers the inserted peptide was fully accessible to the protease. A consequence of the different orientations of the peptide was that membrane potential‐dependent protection from trypsin was much faster for the peptide bound to PG‐containing vesicles compared to CL‐containing membranes, suggesting that in the mitochondrial protein import process other components of the import apparatus are involved in the efficient potential‐driven translocation of presequences across the inner mitochondrial membrane.
Interleukin‐1 receptor antagonist expression in epithelial cells of human endometrium
Authors: Fukuda M. , Azuma C. , Kanai T. , Koyama M. , Kimura T. , Saji F.
Published: July 2002

Objective: To examine the expression of interleukin‐1 (IL‐1) and IL‐1 receptor antagonist (IL‐1ra) in the human endometrium in the follicular and luteal phases. Methods: The concentrations of IL‐1α and IL‐1β in the culture supernatants of endometrial cells were determined by enzyme‐linked immunosorbent assay. Transcription of the IL‐1ra gene in the endometrium was investigated by reverse polymerase chain reaction (PCR). Human endometrium was immunohistochemically stained using a monoclonal antibody specific to IL‐1ra. Results: The concentrations of IL‐1α and IL‐1β in the culture supernatants were 11 and 55 pg/ml, respectively, in the follicular phase, and 10 and 40 pg/ml, respectively, in the luteal phase. The concentration of IL‐1ra was 465 pg/ml in the follicular phase and 1710 pg/ml in the luteal phase. Densitometric analysis of the reverse PCR products showed that the expression of IL‐1ra mRNA was increased in endometrial cells in the luteal phase. Immunohistochemical staining revealed that epithelial cells were the main source of IL‐1ra in human endometrium. Conclusions: Human endometrial cells produce IL‐1 (mainly IL‐1β) and IL‐1ra. The level of IL‐1ra production in human endometrial epithelial cells was greater in the luteal phase than in the follicular phase due to the increased transcription of the IL‐1ra gene.
Free alpha‐subunits of human chorionic gonadotropin in preeclampsia
Authors: Moodley D. , Moodley J. , Buck R. , Haneef R. , Payne A.
Published: July 2002

Objective: To investigate free α‐human chorionic gonadotropin (hCG) as a marker of preeclampsia. Methods: Four groups of patients were studied: normal pregnancies, preeclampsia, eclampsia and normal pregnant women < 20 weeks' gestation. Patients were further divided according to parity and gestational age (≤20, 21–30, 31–40 weeks). An immunoradiometric assay employing monoclonal antibodies specific for free α‐hCG was used. Results: A total of 313 patients were analyzed. Thirty‐four patients ≤20 weeks' gestation were followed until delivery: five (14.7%) developed preeclampsia; none had abnormal α‐hCG levels before onset of preeclampsia. Patients with preeclampsia (21–30 weeks' gestation) demonstrated a mean α‐hCG level greater than that of normotensive controls but this was not statistically significant. Between 31 and 40 weeks' gestation, mean α‐hCG levels in the hypertensive and control groups were 210.8 ng/ml and 115.8 ng/ml, respectively (P < 0.001). A stronger association was observed between α‐hCG and preeclampsia with increasing gestational age (relative risk [RR]2.07, 21–30 weeks; RR 3.02, 31–40 weeks) and severity (RR 4.51, mild; RR 12.15, severe; RR 16.88, eclampsia). Conclusion: There is a strong association between α‐hCG and preeclampsia, nevertheless this test is unsuitable for predicting preeclampsia.
Removal of levonorgestrel capsules from the biceps muscle
Authors: Creinin M.D. , Klaisle C.M.
Published: July 2002

Multiple levonorgestrel implants were identified and removed from below the biceps fascia using radiography to assist in localization, axillary block for anesthesia, careful dissection, and palpation of the implants within the deep muscle belly. This difficult implant removal demonstrates the importance of both a clinician experienced in levonorgestrel implant removal and a general surgeon experienced in arm surgery when performing such procedures.
SNS/PN3 and SNS2/NaN sodium channel‐like immunoreactivity in human adult and neonate injured sensory nerves
Authors: Yiangou Y. , Birch R. , Sangameswaran L. , Eglen R. , Anand P.
Published: July 2002

Two tetrodotoxin‐resistant voltage‐gated sodium channels, SNS/PN3 and SNS2/NaN, have been described recently in small‐diameter sensory neurones of the rat, and play a key role in neuropathic pain. Using region‐specific antibodies raised against different peptide sequences of their α subunits, we show by Western blot evidence for the presence of these channels in human nerves and sensory ganglia. The expected fully mature 260 kDa component of SNS/PN3 was noted in all injured nerve tissues obtained from adults; however, for SNS2/NaN, smaller bands were found, most likely arising from protein degradation. There was increased intensity of the SNS/PN3 260 kDa band in nerves proximal to the site of injury, whereas it was decreased distally, suggesting accumulation at sites of injury; all adult patients had a positive Tinel's sign at the site of nerve injury, indicating mechanical hypersensitivity. Injured nerves from human neonates showed similar results for both channels, but neonate neuromas lacked the SNS2/NaN 180 kDa molecular form, which was strongly present in adult neuromas. The distribution of SNS/PN3 and SNS2/NaN sodium channels in injured human nerves indicates that they represent targets for novel analgesics, and could account for some differences in the development of neuropathic pain in infants.
Structure of the 5th transmembrane segment of the Na,K‐ATPase α subunit: a cysteine‐scanning mutagenesis study
Authors: Guennoun Saı̈da , Horisberger Jean-Daniel
Published: July 2002

To study the structure of the pathway of cations across the Na,K‐ATPase, we applied the substituted cysteine accessibility method to the putative 5th transmembrane segment of the α subunit of the Na,K‐ATPase of the toad Bufo marinus. Only the most extracellular amino acid position (A796) was accessible from the extracellular side in the native Na,K‐pump. After treatment with palytoxin, six other positions (Y778, L780, S782, P785, E786 and L791), distributed along the whole length of the segment, became readily accessible to a small‐size methanethiosulfonate compound (2‐aminoethyl methanethiosulfonate). The accessible residues are not located on the same side of an α‐helical model but the pattern of reactivity would rather suggest a β‐sheet structure for the inner half of the putative transmembrane segment. These results demonstrate the contribution of the 5th transmembrane segment to the palytoxin‐induced channel and indicate which amino acid positions are exposed to the pore of this channel.
Spontaneous subunit exchange in porcine liver fructose‐1,6‐bisphosphatase
Authors: Nelson Scott W , Honzatko Richard B , Fromm Herbert J
Published: July 2002

No evidence to date suggests the possibility of subunit exchange between tetramers of mammalian fructose‐1,6‐bisphosphatase. An engineered fructose‐1,6‐bisphosphatase, with subunits of altered electrostatic charge, exhibits spontaneous subunit exchange with wild‐type enzyme in the absence of ligands. The exchange process reaches equilibrium in approximately 5 h at 4°C, as monitored by non‐denaturing gel electrophoresis and anion exchange chromatography. Active site ligands, such as fructose 6‐phosphate, abolish subunit exchange at the level of the monomer, but permit dimer–dimer exchanges. AMP, alone or in the presence of active site ligands, abolishes all exchange processes. Exchange phenomena may play a role in the kinetic mechanism of allosteric regulation of fructose‐1,6‐bisphosphatase.
Gene therapy for streptozotocin‐induced diabetic mice by electroporational transfer of naked human insulin precursor DNA into skeletal muscle in vivo
Authors: Yin Dong , Tang Jian-Guo
Published: July 2002

Transfer of naked plasmid with insulin precursor DNA into skeletal muscle of streptozotocin (STZ)‐induced diabetic mice through electroporation and detection of gene expression is described. Four different human insulin precursor DNA fragments were inserted into pcDNA3.1(−), downstream of a CMV promoter. Three of them, with a secretion signal sequence, succeeded in lowering blood glucose at a range of 30–50% in STZ diabetic mice. The other, with a synthetic DNA fragment encoding human proinsulin, failed. The mortality rate of very seriously STZ diabetic mice was reduced significantly by the treatment. The circulating insulin‐like protein (mouse insulin, human proinsulin, or intermediates during conversion of proinsulin to insulin) level in the blood of less seriously STZ diabetic mice treated with the human preproinsulin gene with an intron was about 15–23 μU/ml, while that of STZ diabetic mice treated with empty vector was only about 6 μU/ml and that of normal mice was about 18 μU/ml. Transcription of the three human insulin precursor DNAs in mouse skeletal muscle was also detected by RT‐PCR. The human preproinsulin gene with the intron showed a slightly higher potency in reducing blood glucose of mildly diabetic mice. These studies indicate that the skeletal muscle transferred with appropriate preproinsulin DNA by electroporation in vivo can secrete insulin‐like protein resulting in reduction of blood glucose, and a basal blood insulin level can be achieved for at least 1 month.
Calcium currents and transients of native and heterologously expressed mutant skeletal muscle DHP receptor α1 subunits (R528H)
Authors: Jurkat-Rott Karin , Uetz Ulrich , Pika-Hartlaub Ursula , Powell Jeanne , Fontaine Bertrand , Melzer Werner , Lehmann-Horn Frank
Published: July 2002

Rabbit cDNA of the α1 subunit of the skeletal muscle dihydropyridine (DHP) receptor was functionally expressed in a muscular dysgenesis mouse (mdg) cell line, GLT. L‐type calcium currents and transients were recorded for the wild type and a mutant α1 subunit carrying an R528H substitution in the supposed voltage sensor of the second channel domain that is linked to a human disease, hypokalemic periodic paralysis. L‐type channels expressed in GLT myotubes exhibited currents similar to those described for primary cultured mdg cells injected with rabbit wild type cDNA, indicating this system to be useful for functional studies of heterologous DHP receptors. Voltage dependence and kinetics of activation and inactivation of L‐type calcium currents from mutant and wild type channels did not differ significantly. Intracellular calcium release activation measured by fura‐2 microfluorimetry was not grossly altered by the mutation either. Analogous measurements on myotubes of three human R528H carriers revealed calcium transients comparable to controls while the voltage dependence of both activation and inactivation of the L‐type current showed a shift to more negative potentials of approximately 6 mV. Similar effects on the voltage dependence of the fast T‐type current and changes in the expression level of the third‐type calcium current point to factors not primarily associated with the mutation perhaps participating in disease pathogenesis.
Intragenic recombinations in rotaviruses
Authors: Suzuki Yoshiyuki , Gojobori Takashi , Nakagomi Osamu
Published: July 2002

In this paper, evidence for intragenic recombination in the VP7 gene between rotavirus strains bearing different serotypes is demonstrated for the first time. Intragenic recombination may be one of the escaping mechanisms from the host immune system for rotavirus. This process involves exchanging antigenic regions, thus questioning the use of multivalent vaccines for the prevention of rotavirus infection.
Ganglioside GM2 is substrate for a sialidase in MDCK cells
Authors: Marchesini Sergio , Benaglia Giuliana , Piccinotti Alberto , Bresciani Roberto , Preti Augusto
Published: July 2002

GM1 ganglioside carrying a fluorescent fatty acid in substitution of the natural one, has been administered to cultured Madin‐Darby canine kidney (MDCK) cells for different pulse times (0.5–24 h), and its metabolic fate was followed. The fluorescent GM2, asialo‐GM2, asialo‐GM1 and ceramide were the only detectable metabolites. The complete absence of fluorescent GM3 is consistent with the presence in these cells of a sialidase working on GM1 and GM2 gangliosides. After treatment of the cells with chloroquine the fluorescent GM1 remained essentially undegraded, indicating a catabolic processing at lysosomal level.
The Pzh1 protein phosphatase and the Spm1 protein kinase are involved in the regulation of the plasma membrane H+‐ATPase in fission yeast
Authors: Balcells Lluı́s , Martı́n Rocı́o , Ruiz M.Carmen , Gómez Néstor , Ramos José , Ariño Joaquı́n
Published: July 2002

We have previously shown that the mutation of the Schizosaccharomyces pombe PPZ‐like protein phosphatase encoded by the gene pzh1+
results in increased tolerance to sodium and in hypersensitivity to potassium ions. A similar phenotype has also been reported for deletants in the spm1/pmk1 gene, encoding a mitogen‐activated protein (MAP) kinase. We have found that the sodium tolerance phenotype of pzh1 deletants is stronger than that of spm1 mutants, and both effects are additive. Therefore, most probably both gene products mediate different pathways on sodium tolerance. In our hands, mutation of the kinase does not alter the tolerance to potassium, but it yields cells more tolerant to magnesium ions. While in budding yeast the mutations are synthetically lethal, fission yeast cells lacking both the phosphatase and the kinase genes are viable. Interestingly, their ability to export H+ to the medium is greatly impaired (although not that of pzh1 or spm1 single mutants). We have observed that, although the amount of the H+‐ATPase in the plasma membrane is not altered, the activity of the enzyme is lower than normal and cannot be induced by glucose. These observations suggest that the activity of the H+‐ATPase in fission yeast might be regulated by phospho‐dephosphorylation mechanisms that might involve the pzh1+
and spm1+
gene products.
Functional expression and genomic structure of human chondroitin 6‐sulfotransferase1
Authors: Tsutsumi Kae , Shimakawa Hiromi , Kitagawa Hiroshi , Sugahara Kazuyuki
Published: July 2002

The cDNA and gene encoding human chondroitin 6‐sulfotransferase (C6ST) have been cloned. The expression of a soluble recombinant form of the protein in COS‐1 cells produced an active sulfotransferase, which used as acceptor substrates polymer chondroitin, various chondroitin sulfate isoforms and chondroitin sulfate tetrasaccharides. The identification of the reaction products demonstrated that the enzyme transferred sulfate to position 6 of GalNAc in the GlcAβ1‐3GalNAc but not the IdoAα1‐3GalNAc nor the GlcAβ1‐3GalNAc(4‐O‐sulfate) sequences. The human C6ST gene spans more than 20 kb and consists of three exons. The protein‐coding domain of the C6ST gene is divided into two discrete exons.
A matrix metalloproteinase inhibitor which prevents fibroblast‐mediated collagen lattice contraction
Authors: Scott Kate A , Wood Edward J , Karran Eric H
Published: July 2002

Matrix metalloproteinases (MMPs) and the specific tissue inhibitors of metalloproteinases (TIMPs) are involved in tissue turnover in normal and pathological processes including wound healing. Marimastat, a potent inhibitor of MMPs, was used to investigate the role of MMPs in an in vitro wound contraction model, the dermal equivalent, in which fibroblasts are grown in a collagen matrix. Marimastat inhibited fibroblast‐mediated lattice contraction and this inhibition was reversible upon removal of the inhibitor, indicating that MMPs play an important role in fibroblast‐mediated collagen lattice contraction, modelling what may happen when granulation tissue contracts in a healing wound.
Li+ protects nerve cells against destabilization of Ca2+ homeostasis and delayed death caused by removal of external Na+
Authors: Khodorov B. , Pinelis V. , Vinskaya N. , Sorokina E. , Grigortsevich N. , Storozhevykh T.
Published: July 2002

In experiments with fura‐2 loaded cultured rat cerebellar granule cells we have compared the changes in [Ca2+]i homeostasis produced by replacement of external Na+ with the organic cation N‐methyl‐d‐glucamine (NMDG) or Li+. The Na+/NMDG replacement caused an increase in baseline [Ca2+]i and a considerable delay in [Ca2+]i recovery following a glutamate (Glu) pulse in almost all the cells. In contrast Na+/Li+ replacement usually did not change baseline [Ca2+]i and produced only a small (if any) delay in the post‐glutamate [Ca2+]i recovery. Previously [Storozhevykh et al. (1998) FEBS Lett. 431, 215–218] we revealed that perturbation of [Ca2+]i homeostasis caused by Na+/NMDG replacement cannot be explained by a reversal of the Na+/Ca2+ exchange but is mainly due to Ca2+ influx through NMDA channels activated by Na+ dependent release of endogenous excitatory amino acids (‘reversed Glu uptake'). In the present work we confirmed this conclusion and obtained evidence suggesting that in contrast to NMDG Li+ interferes with the ‘reversed Glu uptake' triggered by removal of external Na+. Thus it has been shown that the addition of Li+ (20 mM) to a Na+‐free NMDGcontaining solution suppressed both the perturbation of [Ca2+]i homeostasis and delayed neuronal death caused by Na+/NMDG replacement. Li+ is also able to abolish the [Ca2+]i response induced by PDC which at high concentrations (>200 μM) is shown to stimulate the release of endogenous Glu. In contrast to Na+/Li+, Na+/NMDG replacement greatly enhances [Ca2+]i increase caused by PDC. Control experiments showed that Na+/Li+ replacement does not decrease the [Ca2+]i response to the Glu pulse. Therefore we concluded that a considerable quantitative difference between the effects of Na+/NMDG and Na+/Li+ replacements on both [Ca2+]i homeostasis and cell viability resulted mainly from the ability of Li+ to attenuate the release of endogenous Glu in response to the removal of external Na+.
Induction of uncoupling protein (UCP) 2 in primary cultured hepatocytes
Authors: Kimura Kazuhiro , Jung Bae Dong , Kanehira Katsushi , Irie Yukiko , Cañas Xavier , Saito Masayuki
Published: July 2002

Uncoupling protein 2 (UCP2) mRNA expression and function was examined in rat primary cultured hepatocytes. UCP2 mRNA was not expressed in freshly isolated hepatocytes, but appeared during a 24–144 h primary culture period. Isolated mitochondria from 144 h cultured hepatocytes showed a lower oxygen consumption rate in the presence of succinate and ADP. However, the ratio of the oxygen consumption rate when media contained succinate alone to that with succinate and ADP was increased by 166% versus control mitochondria. Moreover, the mitochondrial potential in the presence of succinate was decreased by 60%, indicating the potential role of UCP2 in hepatocyte mitochondria as an active uncoupler.
A low content in zeatin type cytokinins is not restrictive for the occurrence of G1/S transition in tobacco BY‐2 cells
Authors: Laureys Françoise , Smets Rafaël , Lenjou Marc , Van Bockstaele Dirk , Inzé Dirk , Van Onckelen Harry
Published: July 2002

Theories on the importance of cytokinins in G1/S transition control are manifold and contradictory. By establishing a double Aϕ‐PZ block, maximal synchronization of a BY‐2 suspension culture was obtained to investigate the effect of cytokinin depletion on G1/S transition. Lovastatin was used as a specific inhibitor of cytokinin biosynthesis. Flow cytometry showed that the G1/S transition occurred regardless of the cytokinin drop. This observation indicates an extremely low dose requiry for that stage of the cell cycle. It is very likely that precisely the downregulation of zeatin type cytokinins matters for the G1/S transition to occur, since cytokinin addition at early G1 blocked the cycle at G1/S.
The function of the microtubule‐associated protein tau is variably modulated by graded changes in glycogen synthase kinase‐3β activity
Authors: Leroy K. , Menu R. , Conreur J.L. , Dayanandan R. , Lovestone S. , Anderton B.H. , Brion J.P.
Published: July 2002

The microtubule‐associated protein tau favors microtubule nucleation and stabilization and plays a role in the elongation of axons. We have investigated the ability of glycogen synthase kinase‐3β (GSK‐3β) to control tau‐induced processes outgrowth. Tau‐transfected Chinese hamster ovary (CHO) cells developed processes containing microtubule bundles after cytochalasin treatment, but a significant reduction in the number of cells harboring processes was observed in tau/GSK‐3β‐co‐transfected cells. Lithium, an inhibitor of GSK‐3β, counteracted in a dose‐dependent manner this inhibitory effect of GSK‐3β. These findings suggest that GSK‐3β modulates in a graded manner the ability of tau to control the microtubule‐dependent induction of cell processes.
Non‐closure of visceral peritoneum at abdominal hysterectomy
Authors: Kucuk M , Okman T.K
Published: July 2002

Our purpose was to determine whether non‐closure of the visceral peritoneum alters post‐operative outcome at abdominal hysterectomy. A prospective, randomized trial was performed. Of the 100 evaluable subjects, 50 had the visceral peritoneum left open whereas 50 were closed. Patients were observed for evidence of morbidity after the operation. Analysis of data was performed with use of the unpaired t‐test for continuous variables. The visceral peritonization process at abdominal hysterectomy does not provide immediate post‐operative benefits while unnecessarily lengthening surgical time and anesthesia exposure. We suggest that visceral peritonization process can be abolished at abdominal hysterectomy.
Bacterial adherence and immune response in recurrent urinary tract infection
Authors: Suman E. , Gopalkrishna Bhat K. , Hegde B.M.
Published: July 2002

Objective: To study the significance of bacterial adherence and humoral immune response in the pathogenesis of recurrent urinary tract infection in adult women. Methods: The study population consisted of 30 women with recurrent urinary tract infection (UTI) and 30 healthy women as controls. Bacterial adherence to vaginal epithelial cells was studied. The concentration of serum and urine antibodies against mixed coliform antigen and clinical isolate was determined by enzyme‐linked immunosorbant assay (ELISA). The surface hydrophobicity of the organisms was determined. Student's unpaired t‐test and Pearson's correlation coefficient test were used in the statistical analysis. Results: There was an increased adherence of bacteria to vaginal epithelial cells of women with recurrent UTI. The SIgA level in urine was low compared with other antibodies. There was good correlation between the level of free urinary IgG antibody and bacterial adherence. Conclusions: This study showed that increased receptivity of vaginal epithelial cells to pathogens and lower local immunity may play an important role in the pathogenesis of recurrent UTI in females.
Tap test, shake test and phosphatidylglycerol in the assessment of fetal pulmonary maturity
Authors: Kucuk M.
Published: July 2002

Objective: No single test of amniotic fluid has yet been found completely reliable, easily performed and universally applicable in the prediction of fetal pulmonary maturity. Setting: Department of obstetrics and gynecology of a university hospital. Method: The tap test and shake test were compared to the phosphatidylglycerol (PG) on amniotic fluid samples in 180 fetuses. Results: We found that the tap test results were correlated to the absence or presence of the respiratory distress syndrome in the newborn. When the tap test was evaluated at 5 min, the sensitivity (80.00%), specificity (91.03%) and predictive value for maturity (94.96%) and immaturity (68.29%) were comparable to those of the PG, which has a sensitivity of 82.85%, specificity of 92.41% and predictive value for maturity of 95.71% and immaturity of 72.50%. Conclusion: Our findings suggest that the tap test can be a valuable clinical procedure.
Trends and causes of maternal mortality in Kazakhstan
Authors: Kaupova N , Nukusheva S , Biktasheva H , Goyaux N , Thonneau P
Published: July 2002

Objective: The aim of this study was to determine trends in the rate and causes of maternal mortality in Kazakhstan. Methods: Maternal deaths occurring between 1960 and 1996 in the 19 oblast (administrative regions) of Kazakhstan were documented and analyzed. Results: After an initial phase of decrease in the period 1960–1980, the level of maternal mortality in Kazakhstan has been stable at 70–80 maternal deaths per 100 000 live births. Maternal deaths due to hypertension and abortion complications have not substantially decreased. Over the last 10 years, illegal abortions have accounted for most Kazakh maternal deaths resulting from abortion complications. Conclusion: To reduce the maternal death rate, the Kazakh health authorities and health workers (obstetricians, midwives, health educators) must develop and rapidly implement maternal health and family planning policies.
On the applicability of two different stimulation techniques for intra‐operative peroneal nerve conduction testing
Authors: Nebelung W. , Wissel H. , Awiszus F.
Published: July 2002

Dysfunction of the peroneal nerve is an important complication of knee surgery. We compared two monitoring procedures of peroneal nerve function during a standardized operation, a closing wedge high tibial osteotomy. For two types of stimulation the evoked compound motor unit action potentials (CMAPs) were recorded on the tibialis anterior muscle. We used direct perineural electrical stimulation of the common peroneal nerve distal to the cuff (dCMAPs) after nerve identification in the surgical field. Additionally, magnetic stimulation of the sacral plexus proximal to the cuff (pCMAPs) was performed. It was found that dCMAPs were recorded during almost one hour of tourniquet time whereas the pCMAPs were blocked after 25–30 min in 9 out of 11 cases. On the other hand, the CMAP obtained after proximal stimulation exhibited a latency shift with tourniquet yielding an indicator of ischaemic changes present beneath and distal to the tourniquet cuff. In conclusion, different applicabilities of both stimulation techniques under tourniquet conditions were demonstrated. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.