bluuebunny/crossref_metadata_2025 huggingface dataset:
The sequence of the hemoregulatory peptide is present in Giα proteins
Authors: Laerum Ole Didrik , Frostad Stein , Tøn Hege I. , Kamp Dietmar
Published: July 2002

The hemoregulatory peptide PyroGlu‐Glu‐Asp‐Cys‐Lys (HP5b), which inhibits myelopoietic colony formation in vitro, is shown to be a sequence motif which is also part of the effector domain of Giα proteins. Out of 8 synthetic peptides with sequence variations of HP5b, those with the closest similarity to the Giα sequence are biologically active. The inhibitory effect appears to be dependent on the blocked N‐tenninus. It is postulated that these peptides may interfere with signal transduction mediated by Gα proteins.
Revised structure of the peptide lactone antibiotic, TL‐119 and/or A‐3302‐B
Authors: Kitajima Yasuo , Waki Michinori , Shoji Jun'ichi , Ueno Tamio , Izumiya Nobuo
Published: July 2002

The peptide lactone antibiotic TL‐119 and/or A‐3302‐B was chemically synthesized in order to confirm the proposed structure. The synthetic compound was different from both natural TL‐119 and A‐3302‐B in their physicochemical properties and in biological activity. Re‐examination of the configuration of the constituent amino acid residues in natural TL‐119 and/or A‐3302‐B indicated that natural TL‐119 and A‐3302‐B contains D‐aThr instead of the original L‐Thr. We tentatively propose a revised structure for TL‐119 and/or A‐3302‐B.
Evidence for receptor‐mediated inhibition of intrinsic activity of GTP‐binding protein, Gi1 and Gi2, but not G0 in reconstitution experiments
Authors: Ueda Hiroshi , Uno Shouichi , Harada Jun , Kobayashi Ichiro , Katada Toshiaki , Ui Michio , Satoh Masamichi
Published: July 2002

The receptor‐mediated inhibition of intrinsic activities of GTP‐binding proteins (G‐proteins) was studied. Pertussis toxin (IAP)‐substrate G‐protein, Gi1, Gi2 or G0, was prelabeled with [α‐32P]GDP and reconstituted with synaptic membranes of the guinea pig cerebellum in the presence of 0.02% of Chaps. Intrinsic activities of G‐proteins were evaluated by the release of [α‐32P]GDP in exchange for added GppNHp or GDP in reconstituted preparations. U‐50,488H (1 nM‐10 μM), a specific ϰ‐subtype of opioid receptor agonist, inhibited the [α‐32P]GDP release in exchange for added 1 μM GppNHp in Gi1‐reconstituted preparations in a concentration‐dependent manner. On the other hand, the ϰ‐opioid agonist at 10 μM increases the K
m values of GppNHp, but not GDP in exchange for [α‐32P]GDP release in preparations reconstituted with Gi1 or Gi2, but not with G0. These findings indicate that ϰ‐opioid receptor is coupled to inhibition of intrinsic activities of Gi1 and Gi2, but not G0, in guinea pig cerebellar membranes. In addition, it was revealed that the mode of action is mediated by a decrease in affinity of GTP (or its analog) for G‐proteins, but not by a change in affinity of GDP.
Multicatalytic and 26 S ubiquitin/ATP‐stimulated proteases in maturing rabbit red blood cells
Authors: Di Cola Domenico , Pratt Greg , Rechsteiner Martin
Published: July 2002

Rabbit red blood cells of various ages were separated on Percoll gradients and the activities of two large cytosolic proteases were measured. Both the multicatalytic protease (MCP), assayed by hydrolysis of fluorigenic peptides, and the 26 S ubiquitin/ATP‐stimulated protease, assayed by degradation of ubiquitin—lysozyme conjugates, declined 3‐fold or less during maturation of rabbit reticulocytes to erythrocytes. The ability of MCP to hydrolyze three classes of peptides decreased in parallel indicating that the 20 S protease is not significantly remodeled during red blood cell maturation.
The vacuolar H+‐translocating ATPase of renal tubules contains a 115‐kDa glycosylated subunit
Authors: Gillespie John , Ozanne Susan , Percy Judith , Warren Mark , Haywood Jeff , Apps David
Published: July 2002

Kidney microsomes were fractionated with Triton X‐114, to give a fraction enriched in the renal tubule H+‐translocating ATPase, as judged by the sensitivity of its ATPase activity to bafilomycin A1, and its content of two polypeptides recognized by antibodies directed against subunits of plant tonoplast ATPases. This fraction contained a polypeptide of apparent molecular mass of 115 kDa, that was recognized by an antibody to the largest (120 kDa) subunit chromaffin‐granule membrane H+‐ATPase, and, like this subunit, was reduced in molecular weight on treatment with glycopeptidase F. We conclude that, like other mammalian vacuolar H+‐ATPases, the kidney H+‐ATPase contains a large, glycosylated subunit.
Characterization of cDNA encoding the mouse hepatic triglyceride lipase and expression by in vitro translation
Authors: Chang Shau-Feng , Netter Hans Jürgen , Will Hans
Published: July 2002

A cDNA coding for the mouse hepatic triglyceride lipase has been isolated from a mouse liver cDNA library. The nucleotide sequence of the cDNA shows an open reading frame encoding a polypeptide of 510 amino acids that is 91.5% and 86% homologous to rat and human hepatic lipase, respectively. The most drastic protein sequence divergence is found at the carboxyterminal end which was speculated to harbour one heparin‐binding site. By in vitro translation of cRNA in the presence of pancreatic membranes the hepatic lipase was shown to be glycosylated and to have an electrophoretic mobility of 53 kDa.
Regulation of interleukin‐1 receptors on AtT‐20 mouse pituitary tumour cells
Authors: Kobayashi Hiromasa , Fukata Junichi , Tominaga Tomoko , Murakami Norihiko , Fukushima Mitsuo , Ebisui Osamu , Segawa Hajime , Nakai Yoshikatsu , Imura Hiroo
Published: July 2002

To study the cellular mechanisms of interleukin‐I (IL‐1) in the pituitary corticotroph, we studied the properties of IL‐1 receptors on a mouse pituitary ACTH‐producing cell line, AtT‐20. Scatchard plot analysis revealed a single type of receptor with a K
d (dissociation constant) of 93 pM, and 482 binding sites/cell. [125I]IL‐1α binding competed with IL‐1α and IL‐1β in an equimolar fashion. A 24 h pre‐incubation with either CRH, epinephrine or nor‐epinephrine increased the [125I]IL‐1α binding sites in the AtT‐20 cells and conversely, a similar pre‐incubation with either dexamethasone or tumour necrosis factor‐α (TNFα) decreased them without affecting the affinity of the receptors in either case.
Glucose binding enhances the papain susceptibility of the intracellular loop of the GLUT1 glucose transporter
Authors: Asano Tomoichiro , Katagiri Hideki , Tsukuda Katsunori , Lin Jiann-Liang , Ishihara Hisamitsu , Inukai Kouichi , Yazaki Yoshio , Oka Yoshitomo
Published: July 2002

Digestion of human GLUT1 protein in erythrocytes with 5 μg/ml papain for 5 min yielded several fragments. By using several site‐specific antibodies, two of these fragments containing the intracellular loop domain between M6 and M7 were demonstrated to be further digested by a prolonged incubation with papain. The addition of 0.2 M d‐glucose enhanced this digestion between M6 and M7 by approximately 3.5‐fold, while the addition of 0.2 M d‐sorbitol exhibited no effects. These results strongly suggest that d‐glucose binding induces the conformational change of the intracellular loop domain between M6 and M7 of GLUT1 protein. Since the homology of the amino acid sequence was low in this intracellular domain among the five facilitative glucose transporter isoforms, this intracellular loop might contribute to the difference in their K
m and V
max values for glucose uptake.
Increase of poly(ADP‐ribose) polymerase mRNA levels during TPA‐induced differentiation of human lymphocytes
Authors: Menegazzi Marta , Suzuki Hisanori , de Prati Alessandra Carecreri , Tommasi Marina , Miwa Masanao , Gandini Giorgio , Gerosa Franca
Published: July 2002

The non‐mitogenic stimulation of human peripheral blood mononuclear cells (PBMC) with low concentrations of the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acatate (TPA) caused a progressive increase in the percent fraction of the cells that were positive for the early activating antigen CD69. At the same time, it caused a progressive increase in the steady‐state levels of poly(ADP‐ribose) polymerase (pADPRP) transcripts. A further increase in TPA concentration, while inducing the maximal expression of the levels of CD69 activating surface antigen, both in the presence or in the absence of proliferative activity, did not evoke any additional hightening of pADPRP mRNA levels. Time course of PBMC stimulation with a non‐mitogenic dose or TPA showed an early increase in the accumulation of pADPRP mRNA, which changed at 8‐16 h. and remained high for several days thereafter. On the basis of these data, we suggest flat the increase in pADPRP mRNA may be associated with the commitment of human lymphocytes from a quiescent (G0) to an activated (G1) state.
Cloning and characterization of the cDNA encoding human adenylosuccinate synthetase
Authors: Powell Steven M. , Zalkin Howard , Dixon Jack E.
Published: July 2002

Adenylosuccinate synthetase (AS) catalyzes the first committed step in the conversion of IMP to AMP. A cDNA was isolated from a human liver library which encodes a protein of 455 amino acids (M
r of 49.925). Alignments of human, mouse, Dictyostelium discoideum and E. coli AS sequences identify a number of invariant residues which are likely to be important for structure and/or catalysis. The human AS sequence was also 19% identical to the human urea cycle enzyme, arginosuccinate synthetase (ASS), which catalyses a chemically similar reaction. Both human liver and HcLa AS mRNA showed signals of 2.3 and 2.8 kb. An unmodified N‐terminus is required for function of the human AS enzyme in E. coli mutants lacking the bacterial enzyme. The human cDNA provides a means to assess the possible role of AS abnormalities in unclassified, idiopathic cases of gout.
Protein structural effects of agonist binding to the nicotinic acetylcholine receptor
Authors: Castresana Jose , Fernandez-Ballester Gregorio , Fernandez Asia M. , Laynez Jose L. , Arrondo Jose-Luis R. , Ferragut Jose A. , Gonzalez-Ros Jose M.
Published: July 2002

The effects on the protein structure produced by binding of cholinergic agonists to purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles, has been studied by Fourier‐transform infrared spectroscopy and differential scanning calorimetry. Spectral changes in the conformationally sensitive amide 1 infrared band indicates that the exposure of the AcChR to the agonist carbamylcholine, under conditions which drive the AcChR into the desensitized state, produces alterations in the protein secondary structure. Quantitative estimation of these agonist‐induced alterations by band‐fitting analysis of the amide 1 spectral band reveals no appreciable changes in the percent of α‐helix, but a decrease in β‐sheet structure, concomitant with an increase in less ordered structures. Additionally, agonist binding results in a concentration‐dependent increase in the protein thermal stability, as indicated by the temperature dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization induced by the cholinerpic agonist implies significant rearrangements in the protein structure.
Age and sex influence expression of plasmid DNA directly injected into mouse skeletal muscle
Authors: Wells Dominic J. , Goldspink Geoffrey
Published: July 2002

Direct injection of plasmid DNA into the skeletal muscle has been proposed as a means of effecting somatic gene therapy. We examined the influence of age and sex on the level of expression of an SV40‐CAT construct injected into mouse muscle. Age markedly affected expression, with peak values in the 4–6 week age class which were significantly higher than in animals older than 10 weeks. Sex also altered expression, with higher levels of CAT activity seen in males compared to females. We conclude that the rate of growth is important in determining leves of expression of directly injected DNA.
Receptor‐binding capability of pancreatic phospholipase A2 is separable from its enzymatic activity
Authors: Ishizaki Jun , Kishino Junji , Teraoka Hiroshi , Ohara Osamu , Arita Hitoshi
Published: July 2002

Mammalian pancreatic phospholipase A2 (PLA2‐I) has its specific receptor through which PLA2‐I induces a variety of biological responses. In this study, a fundamental relationship between the enzymatic and the receptor‐binding activities of PLA2‐I was investigated. The specific binding of PLA2‐I to the receptor was found to be independent of Ca2+ which is requisite for the PLA2 activity. On the basis of this observation, we designed and produced mutant PLA2‐Is without Ca2+‐binding abilities in order to demonstrate that the structural requirement for the enzymatic activity of PLA2‐I is not identical with that for its receptor‐binding reaction. These mutant PLA2‐Is lost almost all enzymatic activity through a disturbance at the Ca2+‐binding site, as expected, but still retained a substantial affinity to the receptor, allowing us to conclude that the receptor‐binding reaction of PLA2‐I is separable from its catalytic action.
Use of bimanyl actin derivative (TMB‐actin) for studying complexation of β‐thymosins
Authors: Heintz Daniela , Reichert Andreas , Mihelic Mirna , Voelter Wolfgang , Faulstich Heinz
Published: July 2002

By reacting trimethylammoniobromobimane bromide (TMB bromide) with rabbit muscle actin, a fluorescent reporter group was linked to cysteine at position 374. Fluorescence of TMB‐actin decreased significantly on addition of thymosin β4 (Tβ4), a peptide of 43 amino acid residues reported to bind to monomeric actin and to prevent filament formation. Based on this effect, we determined the K

d
value of the thymosin β4 complex as 0.8 μM, a value that is in agreement with previous determinations. In addition to the main compound thymosin β4, bovine tissue contains a related peptide, thymosin β9 (Tβ9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that Tβ9, similar to Tβ4, forms a 1:1 complex with monomeric actin, and hereby inhibits actin polymerization. With a K

d
value of 1.1 μM the affinity of Tβ9 is in the same range as that of Tβ4, suggesting that Tβ9, like Tβ4, contributes to maintaining the pool of monomeric actin in bovine non‐muscle cells. Further proof of the interaction of Tβ9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslinking experiments. Using different crosslinking reagents, like water‐soluble carbodiimide (EDC), m‐maleimidobenzoyl‐N‐hydroxysuccinimidate (MBS), and disuccinimidylsuberate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and Tβ9 could be identified by immunoblotting. When, in the MBS crosslinking experiments, native actin was replaced with (374‐NEM)actin, the 47 kDa band was not seen, indicating that Cys‐374 takes part in the thiol‐specific crosslinking reaction. This suggests that part of the binding site of Tβ9 must be located close to the carboxy‐tenninus.
Selective proteolysis of the wheat Em polypeptide
Authors: Taylor Richard M. , Cuming Andrew C.
Published: July 2002

The ‘Em’ polypeptide is the most abundant cytosolic polypeptide in mature wheat embryos. It is selectively and completely degraded within the first 24 h of germination. Extracts from germinated embryos contain endopeptidase activities which degrade the Em polypeptide. These are separable into a major and minor component by ion‐exchange chromatography and the use of inhibitors shows the major component to be a cysteine proteinase. This activity shows a strong preference for the Em polypeptide as a substrate, being inactive against polypeptides which are not developmentally regulated and showing only low activity towards developmentally related, but otherwise nonhomologous ‘dehydrin’ polypeptides.
Identification of 1,4‐dihydropyridine binding domains within the primary structure of the α1 subunit of the skeletal muscle L‐type calcium channel
Authors: Kalasz Huba , Watanabe Toshiro , Yabana Hideo , Itagaki Kiyoshi , Naito Kazuaki , Nakayama Hitoshi , Schwartz Arnold , Vaghy Pal L.
Published: July 2002

Calcium channel blockers are drugs that bind to the α1 subunit of L‐type calcium channels and selectively inhibit ion movements through these channels. Determination of the mechanism of channel blockade requires localization of drug‐binding sites within the primary structure of the receptor. In this study the 1,4‐dihydropyridine‐binding site of the membrane bound receptor has been identified. The covalently labeled receptor was purified and digested with trypsin. The labeled peptide fragments were immunoprecipitated with sequence‐directed antibodies. The data indicate the existence of at least three distinct dihydropyridine‐binding domains within the primary structure of the α1 subunit.
Glibenclamide and meglitinide block the transport of low molecular weight solutes into malaria‐infected erythrocytes
Authors: Kirk Kiaran , Horner Heather A. , Spillett Dominique J. , Elford Barry C.
Published: July 2002

Following infection by the malaria parasite, human erythrocytes show increased uptake of a wide variety of low molecular weight solutes via pathways with functional characteristics different from those of the transporters of normal erythrocytes. In this study glibenclamide and meglitinide were shown to inhibit the induced transport of a sugar alcohol (sorbitol), an amino acid (threonine), an inorganic anion (Cl−) and an organic cation (choline) into human erythrocytes infected in vitro with Plasmodium falciparum. The results are consistent with the hypothesis that a diverse range of substrates enter malaria‐infected cells via common pathways which have features in common with Cl‐ channels in other cell types. Glibenclamide and meglitinide were also shown to inhibit the in vitro growth of the intracellular parasite which would suggest that these pathways may be a viable chemotherapeutic target.
Biochemical and immunological evidence that an acidic domain of hsp 90 is involved in the stabilization of untransformed glucocorticoid receptor complexes
Authors: Tbarka N. , Richard-Méreau C. , Formstecher P. , Dautrevaux M.
Published: July 2002

Polyclonal antibodies (AS 232‐266) have been raised against the 232–266 amino acid sequence of the mouse hsp 84. This sequence posseses 54% acidic residues. AS 232–266 react with both the denatured and the free native murine hsp 84, but not with the bound hsp 84 present in the untransformed glucocorticoid receptor complexes (GR). Both AS 232–266 and peptide 232–266 were shown to decrease [3H]dexamethasone binding by GR. Moreover synthetic peptide 232–266, when added to 7 nm untransformed GR, convert them into 5 nm hsp 84‐free GR. Taken together these data suggest that the acidic 232–266 sequence of hsp 84 is involved in the stabilization of the hsp 84—GR interaction, which is known to result in 7 nm complex formation and in GR ligand binding activity improvement. Both peptide 232–266 and AS 232–266 destabilize this interaction.
Differentiation‐associated expression of prostaglandin G/H synthase in monocytic cells
Authors: Hoff Torsten , DeWitt David , Kaever Volkhard , Resch Klaus , Goppelt-Struebe Margarete
Published: July 2002

Significant progress in the investigation of the regulation of prostanoid formation has recently been made by cloning a second gene coding for prostaglandin G/H synthase (PGHS; EC 1.14.99.1). In this study we examined the expression of the two PGHS isoforms during phorbol ester induced monocytic differentiation of human myeloid leukemia cells (U937). Murine and ovine PGHS‐1 probes hybridized to 2.8‐ and 5.5‐kb mRNA species, whereas the murine PGHS‐2 probe hybridized to a 5.3‐kb species. Western blot analysis using antisera to mouse PGHS‐1 and to a synthetic peptide derived from a mouse PGHS‐2‐specific region revealed a band of 70 kDa for PGHS‐1 and a doublet of about 85 kDa for PGHS‐2. Unlike PGHS‐2, which was not expressed in U937 control cells, both PGHS‐1 protein and mRNA were detected in untreated U937 cells. TPA strongly induced PGHS‐2 protein and also increased the amount of PGHS‐1 protein. Correspondingly, a marked induction of PGHS‐2 mRNA was found, but virtually no change in the expression of the PGHS‐1 2.8‐kb mRNA occurred. The induction of both PGHS isoforms turned out to be dexamethasone‐sensitive. The suppression of PGHS‐2 induction was more pronounced. These results suggest that both PGHS‐1 and to a larger extent PGHS‐2 contribute to the upregulation of prostanoid synthesis during monocytic differentiation.
Tamoxifen decreases drug efflux from liposomes: Relevance to its ability to reverse multidrug resistance in cancer cells?
Authors: Kayyali Reem , Marriott Christopher , Wiseman Helen
Published: July 2002

Tamoxifen decreased the efflux of the fluorescent marker drug, chloroquine, from phosphatidylcholine liposomes. Tamoxifen is a known structural‐mimic of cholesterol, which were both found to be similarly effective in preventing drug release from liposomes. This ability of tamoxifen and cholesterol to decrease drug efflux in a concentration‐dependent manner is likely to arise from their known ability to decrease membrane fluidity both in liposomes and also in cancer cells. The possible importance of the ability of tamoxifen to inhibit drug efflux from liposomes in relation to its ability to reverse multidrug resistance in cancer patients caused by the efflux of cytotoxic therapeutic agents, is discussed.
Oxygen exchange reactions catalyzed by vacuolar H+‐translocating pyrophosphatase Evidence for reversible formation of enzyme‐bound pyrophosphate
Authors: Baykov Alexander A. , Kasho Vladimir N. , Bakuleva Natalia P. , Rea Philip A.
Published: July 2002

Vacuolar membrane‐derived vesicles isolated from Vigna radiata catalyze oxygen exchange between medium phosphate and water. On the basis of the inhibitor sensitivity and cation requirements of the exchange activity, it is almost exclusively attributable to the vacuolar H+‐pyrophosphatase (V‐PPase). The invariance of the partition coefficient and the results of kinetic modeling indicate that exchange proceeds via a single reaction pathway and results from the reversal of enzyme‐bound pyrophosphate synthesis. Comparison of the exchange reactions catalyzed by V‐PPase and soluble PPases suggests that the two classes of enzyme mediate Pi–HOH exchange by the same mechanism and that the intrinsic reversibility of the V‐PPase is no greater than that of soluble PPases.
Sphingosine‐1‐phosphate inhibits extracellular matrix protein‐induced haptotactic motility but not adhesion of B16 mouse melanoma cells
Authors: Sadahira Yoshito , Zheng Mingzhe , Ruan Fuqiang , Hakomori Sen-itiroh , Igarashi Yasuyuki
Published: July 2002

Sphingosine‐1‐phosphate (Sph‐1‐P), the initial product of Sph catabolism, inhibited chemotactic motility of a few lines of tumor cells [(1992) Proc. Natl. Acad. Sci. USA 89, 9686]. We now report that Sph‐1‐P even at very low concentration (10–100 nM) inhibits integrin‐dependent motility of melanoma cells induced by extracellular matrix (ECM), although it did not affect integrin‐dependent adhesion to ECM. Other Sph‐related compounds tested (including sphinganine‐1‐P) were much less effective than Sph‐1‐P at inhibiting motility, and also had no effect on integrin‐dependent adhesion of tumor cells to ECM. Our findings suggest that Sph‐1‐P inhibits actin filament reorganization by affecting cytoplasmic connection to integrin in ECM‐stimulated motility of melanoma cells.
A novel myosin I from bovine adrenal gland
Authors: Zhu Tong , Ikebe Mitsuo
Published: July 2002

A 3.5 kb cDNA clone was isolated from bovine adrenal gland cDNA library. The clone contained a full‐length 3.1 kb open reading frame, encoding a novel myosin I. The deduced amino acid sequence was highly homologous to other known myosin Is in the N‐terminal 2 kb region which corresponds to the myosin head domain, while no strong homology was detected in the tail region. The headtail junction contained the Ca2+‐independent calmodulin binding consensus sequence, suggesting that the novel myosin I binds calmodulin. This was confirmed by calmodulin overlay which showed the binding of 125I‐calmodulin to the recombinant myosin I expressed in E. coli. Northern blots with probes from head and tail regions of this myosin I revealed that this novel myosin I is widely distributed among various tissues.
Aromatic hydroxylation and nitration of phenylalanine and tyrosine by peroxynitrite
Authors: van der Vliet Albert , O'Neill Charles A. , Halliwell Barry , Cross Carroll E. , Kaur Harparkash
Published: July 2002

Peroxynitrite is a highly reactive species, generated from Superoxide and nitric oxide. Some effects of peroxynitrite are ascribed to the molecule itself, but decomposition products of the protonated form, peroxynitrous acid, may account for much of its reactivity in biological systems. Suggested products include highly‐reactive hydroxyl radicals, but thermodynamic calculations have been used to claim that free hydroxyl radicals cannot be formed from peroxynitrite. We utilized aromatic hydroxylation of phenylalanine as a specific detector of hydroxyl radicals, and found that incubation of phenylalanine with peroxynitrite leads to a small amount of p‐, m‐ and o‐tyrosine, specific products of attack by this radical. Products of nitration of phenylalanine and tyrosine were also detected, as was dityrosine. Peroxynitrite decomposition generates several reactive species, including some that can nitrate aromatic rings. Formation of nitro‐aromatic compounds may be a useful marker of peroxynitrite generation in biological systems.
Functional analysis of the cytoplastic domain of the human Mpl receptor for tyrosine‐phosphorylation of the signaling molecules, proliferation and differentiation
Authors: Morita Haruhiko , Tahara Tomoyuki , Matsumoto Atsushi , Kato Takashi , Miyazaki Hiroshi , Ohashi Hideya
Published: July 2002

To investigate the functional domains for signal transduction of human Mpl, we constructed a series of human c‐mpl cDNAs with various deletions in the cytoplasmic domain, and then introduced each cDNA into murine IL‐3‐dependent myeloid leukemia FDC/P2 cells to establish stable transformants. We examined the growth and differentiation responses and tyrosine phosphorylation of the intracellular signaling proteins including Jak2, Tyk2, Stat3, Stat5, Vav, SHPTP2, Cbl, Shc and Shc‐associated p145 when receptor stimulation occurred after thrombopoietin (TPO) binding. TPO stimulated cell proliferation and induced the expression of megakaryocyte lineage‐specific AP‐51 and CD61 cell surface antigens and tyrosine phosphorylation of the signaling proteins in transformants expressing full length human Mpl. These results suggested that Mpl not only induced proliferation but also transduced megakaryocyte‐specific differentiation signals into FDC/P2 cells. Mutational analysis of human Mpl indicated that the N‐terminal region of its cytoplasmic domain is necessary and sufficient to transduce proliferation and differentiation signals into cells, while the C‐terminal region may also play important roles in transducing the differentiation signals.
Trends in maternal mortality in Saudi Arabia
Authors: Al‐Meshari A. , Chattopadhyay S.K. , Younes B. , Hassonah M.
Published: July 2002

Objectives: To identify trends and factors related to maternal death, and areas that need improvement. Methods: A prospective national survey, a multistage sample of the hospitals of Saudi Arabia. The setting was a maternal mortality survey committee in Riyadh with field/area coordinators in different areas of the Kingdom. The subjects were women who died or who were dead on arrival in hospital during pregnancy or within 6 weeks of the end of pregnancy. All the data were coded and analyzed. EPINFO software was used to calculate the maternal mortality ratio (MMR) and the relative risk for the necessary variables. Results: The MMR was 18 per 100 000 births (155/880 248). Mortality was higher in older multiparous women of low income and no education. Hemorrhage, both antepartum and postpartum, was the leading cause of maternal death, together with rupture of the uterus and abortive bleeding, constituting 43% of direct and 29% of total maternal deaths. Substandard care was identified in 73% of direct maternal deaths. Conclusion: The MMR in Saudi Arabia compares favorably with that of developed countries and the oil‐producing Gulf states. Improving the number of booked patients, especially older grand multiparas, increasing the availability of banked blood and adopting a positive approach towards life‐saving surgery are likely to reduce maternal deaths.
A simulation analysis of sequencing alternatives for JIT lines using kanbans
Authors: Lummus Rhonda R.
Published: July 2002

Some companies use JIT techniques to increase flexibility for meeting changing customer demand. However, producing more than one product may require longer processing times or more setup time as production shifts between models. Using a simulated JIT process, this research investigates the effects of sequencing production given various product mix combinations and various setup or processing time additions.

The results indicate sequencing does improve output of the JIT process when a second product is added. However, given large increases in setup or processing times, the sequencing logic may break down and throughput time may still increase. The size of the added processing/setup time may result in a bottleneck and control the output of the entire process. Large increases in setup time have a more adverse effect on the process than equivalent increases in processing time.
An acidic amino acid cluster regulates the nucleolar localization and ribosome assembly of human ribosomal protein L22
Authors: Shu-Nu Chang , Lin Chi-Hung , Lin Alan
Published: July 2002

The control of human ribosomal protein L22 (rpL22) to enter into the nucleolus and its ability to be assembled into the ribosome is regulated by its sequence. The nuclear import of rpL22 depends on a classical nuclear localization signal of four lysines at positions 13–16. RpL22 normally enters the nucleolus via a compulsory sequence of KKYLKK (I‐domain, positions 88–93). An acidic residue cluster at the C‐terminal end (C‐domain) plays a nuclear retention role. The retention is concealed by the N‐domain (positions 1–9) which weakly interacts with the C‐domain as demonstrated in the yeast two‐hybrid system. Once it reaches the nucleolus, the question of whether rpL22 is assembled into the ribosome depends upon the presence of the N‐domain. This suggests that the N‐domain, on dissociation from its interaction with the C‐domain, binds to a specific region of the 28S rRNA for ribosome assembly.
Hydroxyl radical‐induced apoptosis in human tumor cells is associated with telomere shortening but not telomerase inhibition and caspase activation
Authors: Ren Jian-Guo , Xia Hui-Li , Just Tom , Dai Yao-Ren
Published: July 2002

Reactive oxygen species (ROS) have been found to trigger apoptosis in tumor cells. At the same time, telomerase is found to be associated with malignancy and reduced apoptosis. However little is known about the linkage between ROS such as OH and telomerase/telomere. To address the interrelations between OH and telomerase/telomere in tumor cell killing, HeLa, 293 and MW451 cells were induced to undergo apoptosis with OH radicals generated via Fe2+‐mediated Fenton reactions (0.1 mM FeSO4 plus 0.3–0.9 mM H2O2) and telomerase activity, telomere length were measured during apoptosis. We found that during OH‐induced apoptosis, telomere shortening took place while no changes in telomerase activity were observed. Our results suggest that OH‐induced telomere shortening is not through telomerase inhibition but possibly a direct effect of OH on telomeres themselves indicating that telomere shortening but not telomerase inhibition is the primary event during OH‐induced apoptosis. Strikingly, we also found that OH‐induced apoptosis in HeLa cells is caspase‐3‐independent but is associated with reduction of mitochondrial transmembrane potential. Our results indicate that OH triggers apoptotic tumor cell death through a telomere‐related, caspase‐independent pathway.
The rules and roles of nucleocytoplasmic shuttling proteins
Authors: Gama-Carvalho Margarida , Carmo-Fonseca Maria
Published: July 2002

The spatial separation of mRNA synthesis from translation, while providing eukaryotes with the possibility to achieve higher complexity through a more elaborate regulation of gene expression, has set the need for transport mechanisms through the nuclear envelope. In a simplistic view of nucleocytoplasmic transport, nuclear proteins are imported into the nucleus while RNAs are exported to the cytoplasm. The reality is, however, that transport of either proteins or RNAs across the nuclear envelope can be bi‐directional. During the past years, an increasing number of proteins have been identified that shuttle continuously back and forth between the nucleus and the cytoplasm. The emerging picture is that shuttling proteins are key factors in conveying information on nuclear and cytoplasmic activities within the cell.
Purification of ADAM 10 from bovine spleen as a TNFα convertase
Authors: Lunn Charles A , Fan Xuedong , Dalie Barbara , Miller Kenneth , Zavodny Paul J , Narula Satwant K , Lundell Daniel
Published: July 2002

We have purified a protease with characteristics of TNFα convertase from bovine spleen membranes. Peptide sequencing of the purified protein identified it as ADAM 10 (Genbank accession no. Z21961). This metalloprotease cleaves a recombinant proTNFα substrate to mature TNFα, and can cleave a synthetic peptide substrate to yield the mature TNFα amino terminus in vitro. The enzyme is sensitive to a hydroxamate inhibitor of MMPs, but insensitive to phosphoramidon. In addition, cloned ADAM 10 mediates proTNFα processing in a processing‐incompetent cell line.
The identification of flavonoids as glycosides in human plasma
Authors: Paganga George , Rice-Evans Catherine A
Published: July 2002

This study describes evidence for the absorption of flavonoids and their presence in human plasma in the glycosylated form by HPLC analysis with photodiode array detection. Rutin and other quercetin glycosides, phloridzin, as well as an anthocyanin are detected simultaneously. In addition, a compound eluting with the spectral properties of the aurone family is identified. The results reveal that phloretin and quercetin are absorbed from the diet as glycosides. The polyphenols are detected in plasma from non‐supplemented humans at individual levels in the range 0.5–1.6 μM.
Isoform‐dependent activation of adenylyl cyclase by proteolysis
Authors: Ebina Toshiaki , Toya Yoshiyuki , Oka Naoki , Kawabe Jun-ichi , Schwencke Carsten , Ishikawa Yoshihiro
Published: July 2002

Recent findings have suggested that the cellular proteolytic system plays a major role in the regulation of various intra‐ and extra‐cellular signaling. It was previously shown that proteolytic treatment of adenylyl cyclase leads to the activation of this enzyme. We demonstrate that this activation occurs in an adenylyl cyclase isoform‐dependent manner. The type II isoform was strongly activated (∼500%), the type III isoform was modestly activated (∼30%), and the type V isoform was inhibited by trypsin. Activation of type II adenylyl cyclase occurred in trypsin dose‐ and time‐dependent manners and was blocked by a trypsin inhibitor in a dose‐dependent manner. Other proteases, such as thrombin and plasminogen, similarly activated the type II isoform, but not the others. Our data suggest that proteolytic activation is an isoform‐ and thus cell type‐dependent mechanism of altering adenylyl cyclase catalytic activity.
Partial coverage of phospholipid model membranes with annexin V may completely inhibit their degradation by phospholipase A2
Authors: Speijer Han , Jans Sylvia W.S , Reutelingsperger Chris P.M , Hack C.Erik , van der Vusse Ger J , Hermens Wim Th
Published: July 2002

Phospholipase A2 (PLA2)‐mediated hydrolysis of membrane phospholipids was measured by ellipsometry, and the inhibition of this process by annexin V was studied. Planar membranes, consisting of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (PC/PE/PS; 54:33:13, on molar basis), were degraded by pancreatic PLA2, and the rate of hydrolysis was limited to about 0.7%/min. The influence of graded coverage of the membrane with annexin V was studied. The degree of PLA2 inhibition was nonlinearly related to the amount of membrane‐bound annexin V, and binding of only 12% and 54% of full membrane coverage resulted in, respectively, 50% and 93% inhibition. These findings indicate that the inhibition of PLA2‐mediated hydrolysis by annexin V cannot be simply explained by shielding of phospholipid substrates from the enzyme. Moreover, the present results leave room for a role of endogenous annexin V in regulating phospholipid turnover in the plasma membrane of parenchymal cells such as cardiomyocytes.
Transcriptional and translational control over sodium‐glucose‐linked transporter (SGLT1) gene expression in adult rat small intestine
Authors: Dong Rong , Srai S.Kaila , Debnam Edward , Smith Michael
Published: July 2002

We have measured SGLT1 mRNA content and SGLT1‐mediated glucose transport at different positions along the small intestine of control and streptozotocin diabetic rats and shown both parameters to be similar but higher in jejunal compared with ileal tissue. No such correlation was seen when comparing measurements of SGLT1 mRNA along jejunal villi with previous estimates of SGLT1 protein and SGLT1‐mediated glucose transport [Debnam et al., Eur. J. Physiol. 430 (1995) 151–159]. This is the first time it has been possible to directly relate these three aspects of SGLT1 gene expression in a single species. Results are discussed in terms of a possible time rather than positional control over translation of SGLT1 mRNA. © 1997 Federation of European Biochemical Societies.
A novel system that reports the G‐proteins linked to a given receptor: a study of type 3 somatostatin receptor
Authors: Komatsuzaki Katsumi , Murayama Yoshitake , Giambarella Ugo , Ogata Etsuro , Seino Susumu , Nishimoto Ikuo
Published: July 2002

SSTR3, a somatostatin (SST) receptor, is an adenylyl cyclase (AC)‐inhibiting receptor. To assign the G‐protein α‐subunit (Gα) linked to this receptor, we created a novel reporter system which utilizes the well‐established facts that the C‐terminal 5 residues of Gα are the receptor contact site and Gαs stimulates all subtypes of AC. We constructed chimeric Gαs the C‐terminal 5 residues of which were replaced with the corresponding C‐terminus of each known Gα, and examined which chimera confers SSTR3‐induced activation of AC. Cellular transfection of SSTR3 and measurement of SST‐dependent AC activity through co‐transfected chimeric Gαs revealed that SSTR3 recognizes the C‐termini of Gαi1/2 but not of Gαo or Gαz, and those of Gα14 and Gα16, but not of Gαq or Gα11. As predicted by the chimeric Gαs, SST‐bound SSTR3 stimulated polyphosphoinositide turnover only when Gα16 or Gα14 was co‐transfected. We conclude that the chimeric Gαs system provides a new approach towards the assignment of G‐proteins linked to a given receptor.
Bacterial aspartic proteinases
Authors: Hill Jeffrey , Phylip Lowri H
Published: July 2002

Regions of genomic DNA encoding putative aspartic proteinase domains were amplified by PCR from the bacterial species, Escherichia coli and Haemophilus influenzae. Expression of each of these DNA fragments resulted in the accumulation of the corresponding recombinant proteins in insoluble aggregates. Each recombinant protein was solubilised, refolded and shown to be able to cleave synthetic peptides that have been extensively used previously as substrates for aspartic proteinases of vertebrate, fungal and retroviral origin. Each activity was completely blocked by the diagnostic aspartic proteinase inhibitor, acetyl‐pepstatin. This is thus the first report demonstrating unequivocally that aspartic proteinases may be present in bacteria.
Defective cell migration in an ovarian cancer cell line is associated with impaired urokinase‐induced tyrosine phosphorylation
Authors: Mirshahi Shah Sultan , Lounes Karim C , Lu He , Pujade-Lauraine Eric , Mishal Zohar , Bénard Jean , Bernadou Alain , Soria Claudine , Soria Jeannette
Published: July 2002

The urokinase receptor (u‐PAR), a protein anchored to cell membrane by a glycosyl phosphatidylinositol, plays a central role in cancer cell invasion and metastasis by binding urokinase plasminogen activator (u‐PA), thereby facilitating plasminogen activation. Plasmin can promote cell migration either directly or by activating metalloproteinases that degrade some of the components of the extra cellular matrix. However, the IGR‐OV1‐Adria cell line contains the u‐PAR but does not migrate even in the presence of exogenous u‐PA, although the parental IGR‐OV1 cell line migrates normally in the presence of u‐PA. We therefore investigated the role of cell signalling for u‐PA induced cell locomotion. We show that cell migration induced by u‐PA–u‐PAR complex is always associated with tyrosine kinase activation for the following reasons: (1) the blockade of the u‐PAR by a chimeric molecule (albumin‐ATF) inhibits not only the u‐PA‐induced cell migration, but also the signalling in IGR‐OV1 line; (2) the binding of u‐PA to u‐PAR on non‐migrating IGR‐OV1‐Adria cells was not associated with tyrosine kinase activation; (3) the inhibition of tyrosine kinase also blocked cell migration of IGR‐OV1. Therefore tyrosine kinase activation seems to be essential for the u‐PA‐induced cell locomotion possibly by the formation of a complex u‐PAR–u‐PA with a protein whose transmembrane domain can ensure cell signalling. Thus, IGR‐OV1 and IGR‐OV1‐Adria cell lines represent a good model for the analysis of the mechanism of u‐PA–u‐PAR‐induced cell locomotion.
MDR‐1 gene expression is a minor factor in determining the multidrug resistance phenotype of MCF7/ADR and KB‐V1 cells
Authors: Kim Hwan Mook , Oh Goo Taeg , Hong Dong Ho , Kim Moon Soon , Kang Jong Seong , Park Sun Mi , Han Sang Bae
Published: July 2002

The relevance of MDR‐1 gene expression to the multidrug resistance phenotype was investigated. Drug‐resistant cells, KB‐V1 and MCF7/ADR, constantly expressed mRNA of the MDR‐1 gene and were more resistant to vinblastine and adriamycin than drug‐sensitive cells, KB‐3–1 and MCF7. The drug efflux rate of KB‐V1 was the same as KB‐3–1 although the MDR‐1 gene was expressed in only the resistant cell. The higher intracellular drug concentration of KB‐3–1 than KB‐V1 was due to the large drug influx. In the case of MCF7 and MCF7/ADR, the influx and efflux of the drug had nearly the same pattern and drug efflux was not affected by verapamil. The amount of ATP, cofactor of drug pumping activity of P‐glycoprotein, was not changed by the resistance. These observations suggested that drug efflux mediated by MDR‐1 gene expression was not a major determining factor of drug resistance in the present cell systems, and that the drug resistance could be derived from the change in drug uptake and other mechanisms.
Aromatic amino acid methyl ester analogs form quinonoidal species with Dopa decarboxylase
Authors: Moore Patrick S , Bertoldi Mariarita , Dominici Paola , Borri Voltattorni Carla
Published: July 2002

This study reports for the first time that binding of aromatic methyl ester analogs to Dopa decarboxylase in the native and inactive nicked forms causes the appearance of a dead‐end quinonoidal species absorbing at 500 nm, in addition to an external aldimine absorbing at 398 nm. The equilibrium mixture of these species varies depending on both the analog structure and the enzyme form. The above mentioned intermediates are also characterized with respect to their CD properties and the equilibria for their formation are determined as a function of pH. The results have provided evidence that the establishment of proper contacts between the active site and hydroxyl groups of the ligand are indispensable in order to limit unwanted side reactions.
Trans‐activation by the Drosophila myb gene product requires a Drosophila homologue of CBP
Authors: Hou De-Xing , Akimaru Hiroshi , Ishii Shunsuke
Published: July 2002

Attempts to demonstrate trans‐activation activity by the Drosophila myb gene product (D‐Myb) have been unsuccessful so far. We demonstrate that co‐transfection of Schneider cells with a plasmid expressing the Drosophila homologue of transcriptional co‐activator CBP (dCBP) results in trans‐activation by D‐Myb. Using this assay system, the functional domains of D‐Myb were analyzed. Two domains located in the N‐proximal region, one of which is required for DNA binding and the other for dCBP binding, are both necessary and sufficient for trans‐activation. In this respect, D‐Myb is similar to c‐Myb and A‐Myb, but different from mammalian B‐Myb. These results shed light on how the myb gene diverged during the course of evolution.
Heregulin β1 induces the down regulation and the ubiquitin‐proteasome degradation pathway of p185HER2 oncoprotein
Authors: Magnifico A , Tagliabue E , Ardini E , Casalini P , Colnaghi M.I , Ménard S
Published: July 2002

Analysis of the fate of the p185HER2 oncoprotein following activation by heregulin β1 revealed the induction of the tyrosine‐phosphorylation, down‐modulation, and polyubiquitination of p185HER2. Receptor ubiquitination was suppressed in cells treated with heregulin β1 in the presence of sodium azide, an inhibitor of ATP‐dependent reactions, or genistein, a tyrosine kinase protein inhibitor, indicating the requirement for kinase activity and ATP in p185HER2 polyubiquitination. Ubiquitinated p185HER2 was degradated by the 26S proteasome proteolytic pathway. Kinetics and inhibition experiments indicated that endocytosis of the receptor occurs downstream of the initiation of the degradation process.
Involvement of protein‐tyrosine phosphorylation and dephosphorylation in sperm‐induced Xenopus egg activation
Authors: Sato Ken-ichi , Iwasaki Tetsushi , Tamaki Ikuo , Aoto Mamoru , Tokmakov Alexander A , Fukami Yasuo
Published: July 2002

We have analyzed tyrosine‐phosphorylated proteins in Xenopus laevis eggs before and after fertilization by immunoblotting with anti‐phosphotyrosine antibody. A number of egg proteins with different subcellular distribution became tyrosine‐phosphorylated or dephosphorylated within 30 min after insemination. Tyrosine kinase‐specific inhibitors genistein and herbimycin A were found to inhibit sperm‐induced egg activation judged by the egg cortical contraction. Surprisingly, sodium orthovanadate, a tyrosine phosphatase inhibitor, also inhibited the egg activation. Moreover, we found that fertilization‐dependent tyrosine dephosphorylation of 42‐kDa mitogen‐activated protein kinase was inhibited in genistein‐treated eggs. These results suggest that both protein‐tyrosine phosphorylation and dephosphorylation pathways play an important role in the sperm‐induced Xenopus egg activation.
Identification of lysine‐238 of Escherichia coli biotin carboxylase as an ATP‐binding residue
Authors: Kazuta Yasuaki , Tokunaga Eiko , Aramaki Eiji , Kondo Hiroki
Published: July 2002

Escherichia coli biotin carboxylase was affinity labeled with adenosine diphosphopyridoxal to identify its ATP binding site. Lysyl endopeptidase digestion of the modified protein, followed by high performance liquid chromatography separation and amino acid sequencing allowed to identify lysine‐238 to be the site of modification. Site‐directed mutagenesis of this residue into alanine, arginine or glutamine resulted in mutants with much decreased activity. Lysine‐238 seems to interact with the γ‐phosphate group of ATP but is not involved in catalysis.