bluuebunny/crossref_metadata_2025 huggingface dataset:
High Incidence of Reflux Esophagitis Observed by Routine Endoscopic Examination after Gastric Pull‐up Esophagectomy
Authors: Shibuya Shunsuke , Fukudo Shin , Shineha Ryuzaburo , Miyazaki Shukichi , Miyata Go , Sugawara Koh , Mori Takahiro , Tanabe Shuichi , Tonotsuka Norio , Satomi Susumu
Published: May 2003

Abstract

A gastric tube has been widely used for reconstruction of the esophagus after esophagectomy for esophageal cancer. Reflux esophagitis after esophagectomy is frequently observed. Therefore we retrospectively investigated the risk factors for reflux esophagitis after gastric pull‐up esophagectomy in 74 outpatients with thoracic esophageal cancer. Reflux esophagitis was diagnosed endoscopically. Esophagitis was classified according to the Los Angeles classification. Reflux symptoms, medications, and the surgical procedure were reviewed. The relation between reflux symptoms and reflux esophagitis and the influence of the anastomotic site were evaluated. Reflux esophagitis was observed in 53 patients. Severe esophagitis (grade C or D) was found in 75.6% of these patients. Although all patients with esophagitis took antacid agents, histamine receptor‐2 blocker was effective in only 35% of them. The correlation between reflux symptoms and reflux esophagitis was not significant. Reflux esophagitis was present in 56.4% of patients with neck anastomosis and in 88.6% of patients with intrathoracic anastomosis (p = 0.0039). We concluded that routine endoscopic examination is necessary after gastric pull‐up esophagectomy because reflux esophagitis is not diagnosed based on reflux symptoms. When a gastric tube is used for reconstruction after esophagectomy, neck anastomosis is recommended to lower the risk of reflux esophagitis.
Review of the “Chirurgia” of Giovanni de Vigo: Estimate of His Position in the History of Surgery
Authors: Gurunluoglu Raffi , Gurunluoglu Aslin , Piza‐Katzer Hildegunde
Published: May 2003

Abstract

Giovanni de Vigo, who was born in at Rapallo, Italy, lived in the early Renaissance period (1450–1525). In 1503, De Vigo became the personal surgeon to Pope Julius II. He wrote a surgical book, “Practica Copiosa in Arte Chirurgia,” which was completed in 1514 and published in Latin. It was translated into English by Richard Traheron and printed by Edward Whytchurch in 1543. Vigo’s “Chirurgia” consists of nine books ranging from a consideration of anatomy necessary for a surgeon, to sections on abscesses, wounds, ulcers, benign and malignant tumors, fractures and dislocations, pharmaceuticals, ointments and plasters, as well as sections on dentistry, exercise, diet, syphilis, among others. De Vigo introduces a novel approach for treating mandible dislocations and describes a trephine he invented, as well as a number of new instruments. Examination of his surgical piece demonstrates that he had a broad spectrum of knowledge in surgery based in part on the ancient Greek and Arabic medical literature but mainly on his personal experience. Giovanni de Vigo contributed significantly to the revival of medicine in the sixteenth century, and he can be considered as a bridge between Greek medicine of antiquity, Arabic medicine, and the Renaissance.
Expression of Vascular Endothelial Growth Factor as a Prognostic Factor in Node‐positive Squamous Cell Carcinoma in the Thoracic Esophagus: Long‐term Follow‐up Study
Authors: Ogata Yutaka , Fujita Hiromasa , Yamana Hideaki , Sueyoshi Susumu , Shirouzu Kazuo
Published: May 2003

Abstract

To clarify the clinical significance of the expression of vascular endothelial growth factor (VEGF) in squamous cell carcinoma in the thoracic esophagus, particularly as a prognostic factor, we have investigated the correlation between VEGF expression in tumor cells and microvessel density (MVD), pathologic factors, and survival. A total of 92 specimens, each from a thoracic esophageal squamous cell cancer patient who underwent transthoracic curative R0 esophagectomy between 1991 and 1994, were examined immunohistochemically using anti‐human VEGF and anti‐human von Willebrand factor antibodies. The incidence of VEGF expression in the tumor cells was relatively low, at 23.9% of all specimens. There was no significant correlation between VEGF expression and histopathologic factors. The MVD at the tumor margin in patients with VEGF‐positive tumor cells was significantly greater than that in VEGF‐negative cases (35.2 ± 8.9 vs. 22.7 ± 8.2). The survival rate for patients with VEGF expression was significantly lower than that of those without VEGF expression; the same situation was found in node‐positive patients but not in node‐negative patients. In addition, multivariate analysis revealed that VEGF expression was an independent prognostic factor in node‐positive patients. VEGF may be implicated in the definition of the malignant phenotype of squamous cell esophageal cancer via tumor angiogenesis. Accordingly, VEGF expression in the tumor cells could be a useful prognostic factor for esophageal cancer, particularly in node‐positive patients.
Cloning and functional expression of the cDNA encoding an inwardly‐rectifying potassium channel expressed in pancreatic β‐cells and in the brain
Authors: Bond C.T. , Ämmälä C. , Ashfield R. , Blair T.A. , Gribble F. , Khan R.N. , Lee K. , Proks P. , Rowe I.C.M. , Sakura H. , Ashford M.J. , Adelman J.P. , Ashcroft F.M.
Published: May 2003

A cDNA clone encoding an inwardly‐rectifying K‐channel (BIR1) was isolated from insulinoma cells. The predicted amino acid sequence shares 72% identity with the cardiac ATP‐sensitive K‐channel rcKATP(KATP‐1; [6]). The mRNA is expressed in the brain and insulinoma cells. Heterologous expression inXenopusoocytes produced currents which were K+‐selective, time‐independent and showed inward rectification. The currents were blocked by external barium and caesium, but insensitive to tolbutamide and diazoxide. In inside‐out patches, channel activity was not blocked by 1 mM internal ATP. The sequence homology with KATP‐1 suggests that BIR1 is a subunit of a brain and β‐cell KATPchannel. However, pharmacological differences and the lack of ATP‐sensitivity, suggest that if, this is the case, heterologous subunits must exert strong modulatory influences on the native channel.
Basic properties of a novel ryanodine‐sensitive, caffeine‐insensitive calcium‐induced calcium release mechanism in permeabilised human vascular smooth muscle cells
Authors: Lynn Stephen , Gillespie James I.
Published: May 2003

The efflux of 45Ca2+ from preloaded intracellular stores of saponin‐permeabilised human uterine artery smooth muscle cultured cells was used to study the mechanisms underlying Ca2+ release from the sarcoplasmic reticulum (SR). The present paper demonstrates directly a functional Ca2+ release mechanism that is dependent on an increase in free Ca2+ (100 nM‐30 μM) and is completely inhibited by 20 μM Ruthenium red. The amount of Ca2+ released at 30 μM free Ca2+ was reduced by approximately 50% compared to the release at 10 μM. This Ca2+‐induced Ca2+ release (CICR) mechanism was not sensitive to caffeine. Exposure of cells to low free Ca2+‐containing solutions (10 nM) indicated that a component of the CICR mechanism may be functional at basal free Ca2+ levels of 100 nM. Application of ryanodine (0.1–100 μM) induced 45Ca2+ efflux from the sarcoplasmic reticulum and this release was also inhibited by 20 μM Ruthenium red.
Changes in the state of water, induced by radiofrequency electromagnetic fields
Authors: Fesenko Evgenii E. , Gluvstein Alexander Ya.
Published: May 2003

Microwave irradiation (f = 36 GHz) changes the properties of distilled water within the first 1–10 min. The new state is retained for at least tens of minutes and manifests itself as changes in power density spectrum of periodic fading voltage fluctuations that are generated during discharge of a capacitor in which water is used as a dielectric. It is assumed that long‐term changes in water properties mediate the effect of electromagnetic fields on biological systems.
Parallel purine‐pyrimidine‐purine triplex: experimental evidence for existence
Authors: Shchyolkina Anna K. , Borisova Olga F. , Minyat Elvira E. , Timofeev Edward N. , Il'icheva Irina A. , Khomyakova Elena B. , Florentiev Vladimir L.
Published: May 2003

Oligonucleotides 5′‐d(CT)5‐L‐d(AG)5‐L‐d(GA)5‐3′ and 5′‐d(GA)5‐L‐d(TC)5‐L‐d(GA)5‐3′ [L = pO(CH2CH2O)3p] were studied by thermal denaturation, chemical modification and binding of fluorescent dyes. Both oligonucleotides are shown to fold back on itself twice forming at pH 7 a sufficiently stable triplex ether with antiparallel‐oriented oligopurine strands (the first compound) or parallel‐oriented oligopurine strands (the second compounds). The parallel triplex is significantly less stable than the antiparallel one. On the basis of conformational modeling, possible types of base tripling in the triplets are proposed. Thus our data provide the first convincingly evidence for the existence of a purine‐pyrimidine‐purine triplex with parallel orientation of identical strands.
Apoptosis without decrease of cell DNA content
Authors: Fournel Sylvie , Genestier Laurent , Rouault Jean-Pierre , Lizard Gerard , Flacher Monique , Assossou Olga , Revillard Jean-Pierre
Published: May 2003

Apoptosis of human B cells and murine T and B cells was analyzed by DNA agarose gel electrophoresis, clamped homogeneous electric field, measurement of cell DNA content by flow cytometry, transmission electron microscopy and by UV microscopy. Apoptosis was induced by etoposide (an inhibitor of topoisomerase II), by the calcium ionophore ionomycin or by cross‐linking of membrane immunoglobulins (Ig) with anti‐Ig‐antibodies. Two types of apoptosis could be defined. Apoptosis resulting in small DNA fragments (180–200 base pairs and multiples thereof) was associated with a typical ‘ladder’ in agarose gel electrophoresis and a decrease in cell DNA content assessed by flow cytometry. Conversely apoptosis with large DNA fragments (100–150 kilobase pairs) was only demonstrated by clamped homogeneous electric field but was not associated with decreased cell DNA content or the observation of DNA ladders. Nuclear condensation without fragmentation was more frequent when apoptosis generated large DNA fragments. The type of apoptosis appears to be an intrinsic property of each cell type.
Remarkable activity enhancement of thermolysin mutants
Authors: Kidokoro Shun-ichi , Miki Yoichiro , Endo Kimiko , Wada Akiyoshi , Nagao Hiromasa , Miyake Toshio , Aoyama Atsuo , Yoneya Takashi , Kai Kenichi , Ooe Seigo
Published: May 2003

Most attempts to modify the properties of enzymes by amino acid substitution around the active sites have resulted in suppression of the biological activity, suggesting that the structure of natural enzymes should be almost optimized evolutionally to show the highest activity. In contrast, we found an interesting site of a well‐known metalloendopeptidase, thermolysin (EC. 3.4.24.4), where almost all the amino acid replacement causes a remarkable increase in the hydrolytic activity. Negative correlation between the activity and the thermal stability was observed. The flexibility around the substrate binding site is suggested to be a key to the correlation. Nature may have selected the amino acid at this site, which suppresses the flexibility of the molecule, to get the highest thermal stability at the expense of the activity.
Cloning and expression of cDNA encoding a new type of ascorbate peroxidase from spinach
Authors: Ishikawa Takahiro , Sakai Kosuke , Takeda Toru , Shigeoka Shigeru
Published: May 2003

A cDNA clone (SAP1) encoding a peroxidase was isolated from a spinach cDNA library using monoclonal antibodies raised against Euglena ascorbate peroxidase. The deduced amino acid sequence of SAP1 had higher homology with the cytosolic ascorbate peroxidases from plant sources than with bacterial peroxidases and classical plant peroxidases. The peroxidase activity of recombinant SAP1 protein expressed in E. coli was 1.6‐fold higher with ascorbate than with guaiacol, which was similar to those of endogenous cytosolic ascorbate peroxidases. Here we conclude that SAP1 belongs to new type of ascorbate peroxidase from spinach.
Proteolysis of protein kinase C: mM and μM calcium‐requiring calpains have different abilities to generate, and degrade the free catalytic subunit, protein kinase M
Authors: Cressman Corinne M. , Mohan Panaiyur S. , Nixon Ralph A. , Shea Thomas B.
Published: May 2003

Limited proteolysis of protein kinase C (PKC) by calpain under cell free conditions cleaves the regulatory and catalytic PKC subunits, generating a free, co‐factor independent catalytic subunit, termed PKM. In the present study, we demonstrate distinct differences in the rate, nature, and lipid‐sensitivity of PKC and PKM proteolysis by μM and mM calcium‐requiring calpain isozymes (μ calpain or m calpain, respectively). PKC is a preferred substrate for m calpain; not even a 100‐fold increase in μ calpain was capable of degrading PKC as fast as in calpain. PKM was generated by both m and μ calpains, but was itself rapidly degraded by m calpain and therefore was only transiently detectable. By contrast, PKM was formed but not degraded by μ calpain, and persisted in the presence of μ calpain long after all PKC had been degraded. Phosphatidyl serine (PS) inhibited PKC hydrolysis by m calpain yet enhanced PKC hydrolysis by μ calpain. The ability of either calpain isoenzyme to degrade [14C]azocasein was unaffected by PS, suggesting that the influence of PS was on PKC conformation. These findings point towards distinct roles for μ and m calpain in PKC regulation.
Reverse transcriptase‐polymerase chain reaction detects induction of cardiac‐like α myosin heavy chain mRNA in low frequency stimulated rabbit fast‐twitch muscle
Authors: Peuker Heidemarie , Pette Dirk
Published: May 2003

Using reverse transcriptase‐polymerase chain reaction we quantified in rabbit skeletal muscles expression levels of the highly homologous cardiac α and β myosin heavy chain (αMHC, βMHC) mRNA isoforms. Masseter muscle displayed highest levels of a cardiac‐like αMHC mRNA. This isoform was present at 20‐fold lower amounts in slow soleus and at 200‐fold lower levels in several fast‐twitch muscles. Low‐frequency stimulation periods exceeding 20 days drastically induced the αMHC mRNA in fast tibialis anterior. The αMHC mRNA was 140‐fold elevated after 60 days when βMHC mRNA had increased 50‐fold. Our results demonstrate the wide distribution of a cardiac‐like αMHC mRNA in skeletal muscle and its marked induction during fast‐to‐slow transition as induced by low‐frequency stimulation.
Alcohol dehydrogenase of class I: kiwi liver enzyme, parallel evolution in separate vertebrate lines, and correlation with 12S rRNA patterns
Authors: Hjelmqvist Lars , Metsis Madis , Persson Håkan , Höög Jan-Olov , McLennan John , Jörnvall Hans
Published: May 2003

Alcohol dehydrogenase class I from kiwi liver has been purified, analyzed, and compared with that of other alcohol dehydrogenases. The results show that several avian and mammalian forms of the enzyme exhibit parallel evolutionary patterns in two independent lineages of a single protein, establishing a pattern in common. Furthermore, the data correlate the enzyme evolutionary pattern with that of 12S rRNA. Biologically, the patterns complement those on ratite and other avian relationships. Functionally, the enzyme has a low K
m with ethanol and a branched‐chain residue at position 141, like the mammalian enzymes but in contrast to the other characterized ratite enzyme (with Ala‐141 and a higher K
m). This pattern of natural variability suggests a frequent but not fully complete correlation between a large residue size at position 141 and tight ethanol binding.
Site specificity of p72syk protein tyrosine kinase: efficient phosphorylation of motifs recognized by Src homology 2 domains of the Src family
Authors: Brunati Anna Maria , Donella-Deana Arianna , Ruzzene Maria , Marin Oriano , Pinna Lorenzo A.
Published: May 2003

Protein tyrosine kinase p72
syk
purified from rat spleen has been assayed for its ability to phosphorylate a number of peptide substrates derived from naturally occurring phospho‐acceptor sites. The phosphorylation efficiency is extremely variable, depending on the peptide sequence, with K
m values in the 3–1500 μM range. The by far best peptide substrates, with K
m values of 3 and 4 μM are those reproducing the phospho‐acceptor sites of Vav and HS1 proteins, respectively. These sites include multiple acidic residues flanking tyrosine on both sides and they also display the consensus sequences (YEDL and YEEV) preferred by the SH2 domains of the Src family. Alteration of this consensus in the HS1 peptide, by replacing either the glutamic acid or valine, also reduces the phosphorylation efficiency by p72
syk
. Also the replacement of acidic residues at position −1 and, to a lesser extent at positions −3 and −4 (but not at positions +3 and +5) are detrimental. These observations may suggest a role of p72
syk
in the recruitment of ligands/substrates for the Src family enzymes. We also show that the HS1 peptide can be used for the specific monitoring of p72
syk
since neither the two Src‐related c‐Fgr and Lyn kinases (needing a hydrophobic instead of acidic residue at position −1) nor CSK appreciably phosphorylate it.
Behavior of Rhizobium meliloti in oxygen gradients
Authors: Zhulin Igor B. , Lois Augusto F. , Taylor Barry L.
Published: May 2003

Rhizobium meliloti cells responded to an abrupt change in oxygen concentration by changing the cell speed (chemokinesis), but they did not alter the frequency at which swimming cells stopped briefly (aerotaxis). Changes in cell speed upon stimulation with oxygen coincided with changes in membrane potential. The cells did not form an aerotactic band in a spatial gradient of oxygen as do the cells of other bacterial species. The fixL and fixJ genes which encode a heme‐containing protein kinase that senses oxygen and a response regulator, respectively, were not involved in the behavior of R. meliloti in oxygen gradients.
Alternative system of succinate oxidation in glyoxysomes of higher plants
Authors: Igamberdiev A.U , Popov V.N , Falaleeva M.I
Published: May 2003

Succinate oxidation in scutella of germinating seeds of wheat and maize was investigated. Besides oxidation via succinate dehydrogenase (SDH; EC 1.3.99.1), an alternative path of succinate oxidation insensitive to SDH inhibitors — malonate and thenoyltrifluoroacetone (TTFA) — was revealed. Using isopicnic sucrose gradient it was shown that this path is localized in glyoxysomal membranes. Glyoxysomal succinate oxidase (GSO) converts succinate directly into malate with the production of hydrogen peroxide identified using auxilary enzymes malate dehydrogenase and peroxidase. GSO is most active during the intensive operation of the glyoxylate cycle (3–5 days of germination). Quinacrine, the inhibitor of flavine‐containing oxidases, strongly suppressed the activity of GSO. K
m for succinate is 18 mM for GSO from maize scutellum. It is concluded that in scutella of cereal seeds the glyoxysomal succinate oxidation non‐linked with ATP synthesis operates.
Intracellular transport of inositol‐containing sphingolipids in the yeast, Saccharomyces cerevisiae
Authors: Hechtberger Petra , Daum Günther
Published: May 2003

Organelles of the early protein secretion pathway (ER, Golgi) are involved in biosynthesis and intracellular migration of the yeast sphingolipids, inositolphosphorylceramide (IPC), mannosylinositolphosphorylceramide (MIPC), and mannosyldiinositolphosphorylceramide (M(IP)2C). Cycloheximide and nocodazole neither block biosynthesis of sphingolipids, nor ER to Golgi transport of IPC. In contrast, treatment of yeast cells with brefeldin A, which affects integrity of the Golgi, decreases formation of IPC and MIPC. Interruption of late steps of protein secretion (Golgi to plasma membrane transport) in temperature‐sensitive secretory mutants prevents sphingolipids from being transported to the cell periphery.
Rapid agonist mediated phosphorylation of the metabotropic glutamate receptor 1α by protein kinase C in permanently transfected BHK cells
Authors: Alaluf Simon , Mulvihill Eileen R. , McIlhinney Robert A.J.
Published: May 2003

Clonal BHK cells permanently transfected with the metabotropic glutamate receptor 1α (mGluR1α), which is coupled to phospholipase C, were used to study the phosphorylation state of the receptor. Cells were labelled with 32PO4
3−, lysed, the receptor immunoprecipitated with specific anti‐peptide antibodies and the immunoprecipitates analysed by SDS‐PAGE followed by autoradiography. A significant basal level of receptor phosphorylation was observed which was rapidly and transiently increased in response to agonist activation of the receptor. This agonist effect was found to be dose dependent with a rapid time course and could be abolished by the specific PKC inhibitor Ro318220, suggesting that PKC was responsible for the agonist mediated phosphorylation of the receptor.
An interface point‐mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations
Authors: Borchert T.V. , Zeelen J.Ph. , Schliebs W. , Callens M. , Minke W. , Jaenicke R. , Wierenga R.K.
Published: May 2003

Wild‐type trypanosomal triosephosphate isomerase (wtTIM) is a very tight dimer. The interface residue His‐47 of wtTIM has been mutated into an asparagine. Ultracentrifugation data show that this variant (H47N) only dimerises at protein concentrations above 3 mg/ml. H47N has been characterised at a protein concentration where it is predominantly a monomer. Circular dichroism measurements in the near‐UV and far‐UV show that this monomer is a compactly folded protein with secondary structure similar as in wtTIM. The thermal stability of the monomeric H47N is decreased compared to wtTIM: temperature gradient gel electrophoresis (TGGE) measurements give T
m‐values of 41°C for wtTIM, whereas the T
m‐value for the monomeric form of H47N is approximately 7°C lower.
Horseradish peroxidase inhibition by thiouracils
Authors: Zatón Ana María L. , Ochoa de Aspuru Eduardo
Published: May 2003

In this paper, the activity of horseradish peroxidase was further determined in the presence of several uracil derivatives. The rate of guaiacol peroxidation decreases in presence of 2‐thiouracil and of 6‐n‐propyl‐2‐thiouracil, but is not changed by 6‐n‐propyluracil nor uracil. Thus, thiouracils inhibit horseradish peroxidase in a noncompetitive form. The binding of 6‐n‐propyl‐2‐thiouracil, 2‐thiouracil, 6‐n‐propyluracil and uracil with horseradish peroxidase shows difference spectra due to changes in the environment of heme group in peroxidase. Then, the binding sites for these uracil derivatives are in an hydrophobic pocket at the heme periphery of peroxidase. The lesser binding rates were for uracil and propyluracil, which did not inhibit the peroxidase activity. These results point to the thiol group in uracils as responsible for the inhibition of peroxidase activity through interaction with an allosteric binding site, in peroxidase heme environment.
Reconstitution photoactive yellow protein from apoprotein and p‐coumaric acid derivatives
Authors: Imamoto Yasushi , Ito Takeshi , Kataoka Mikio , Tokunaga Fumio
Published: May 2003

We report reconstitution of photoactive yellow protein (PYP) from apoPYP and p‐coumaric acid derivatives. The addition of p‐coumaric acid to the apoPYP sample did not result in the recovery of PYP. In contrast, yellow products were obtained by the addition of p‐coumaryl thiophenyl ester or p‐coumaric anhydride to the apoPYP sample, the absorption spectra of which were indistinguishable from the spectrum of intact PYP. Our findings provide strong evidence that PYP has the p‐coumaryl chromophore. This reconstitution technique opens the way for further biophysical studies of PYP using artificial chromophore analogs.
Transfer of rps10 from the mitochondrion to the nucleus in Arabidopsis thaliana: evidence for RNA‐mediated transfer and exon shuffling at the integration site
Authors: Wischmann Christine , Schuster Wolfgang
Published: May 2003

Rps10, a gene coding for ribosomal protein S10 of Arabidopsis mitochondria has been transferred to the nuclear compartment, while in pea and potato the active rps10 is mitochondrially located. The nuclear rps10 gene contains an intron at the junction of the target signal sequence and the mitochondrial‐derived sequence, indicating that exon shuffling may have been involved in the addition of the transit peptide signal. Sequence comparison of Arabidopsis rps10 to the plant mitochondrial‐counterparts shows that the edited version is present in the nucleus of Arabidopsis. This finding corroborates RNA as an intermediate of a functional gene transfer between mitochondria and the nucleus. In vitro‐translated RPS10 protein is efficiently imported into potato mitochondria and a presequence of about 7 kDa is removed resulting in a mature protein that is larger compared to organellar and bacterial RPS10 proteins.
Involvement of the three inter‐α‐trypsin inhibitor (ITI) heavy chains in each member of the serum ITI family
Authors: Héron Antoine , Bourguignon Jeannette , Diarra-Mehrpour Maryam , Dautréaux Brigitte , Martin Jean-Pierre , Sesboüé Richard
Published: May 2003

Partial cDNAs coding for each of the three human inter‐α‐trypsin inhibitor (ITI) heavy chains were expressed in a bacterial plasmid system and rabbits were immunised with the fusion peptides obtained. Despite the strong sequence homology of these chains, the antisera turned out to be highly specific in the analysis of corresponding mRNA translation products or partially digested serum ITI. Besides classical serum ITI members, their use in Western blotting made it possible to evidence an H3‐related ITI form and a low‐amount H1‐related HC/bikunin component. The relative levels of ITI family members was further studied in baboon and foetal calf sera.
Tissue‐specific regulation of the expression of rat intestinal bile acid‐binding protein
Authors: Sato Eiju , Fujii Hiroshi , Fujita Michiyo , Kanda Tatsuo , Iseki Shoichi , Hatakeyama Katsuyoshi , Tanaka Toshinobu , Ono Teruo
Published: May 2003

A lipid‐binding protein identical to the rat intestinal bile acid‐binding protein, termed I‐15P, was expressed in steroid hormone‐producing tissues such as ovary and adrenal gland, but not testis. In immature rats, I‐15P was expressed in intestine but not in ovaries. The expression of I‐15P in the ovaries of immature rats was induced to the level in immature rats by gonadotropin treatment. This suggests that the expression of I‐15P in the ovaries is controlled by the ovarian cycle. The present results indicate that the expression of I‐15P is developmentally and hormonally controlled in a tissue‐specific manner.
Isolation of an active and heat‐stable monomeric form of Cu,Zn superoxide dismutase from the periplasmic space of Escherichia coli
Authors: Battistoni Andrea , Rotilio Giuseppe
Published: May 2003

We have purified the Cu,Zn superoxide dismutase (CuZnSOD) from the periplasmic space of an Escherichia coli strain unable to synthesize MnSOD and FeSOD. Gel filtration chromatography evidenced that under all the experimental conditions tested the enzyme was monomeric. The catalytic activity of this CuZnSOD was comparable to that of other well characterized dimeric eukaryotic isoenzymes, indicating that a dimeric structure is not essential to ensure enzymatic efficiency. Furthermore the purified enzyme proved to be highly heat‐stable and, uniquely among CuZnSODs, protease‐sensitive. The latter property may explain the previously described lability of this protein in cell extracts.
Stabilization of a semiquinone radical at the high‐affinity quinone‐binding site (QH) of the Escherichia coli bo‐type ubiquinol oxidase
Authors: Sato-Watanabe Mariko , Itoh Sigeru , Mogi Tatsushi , Matsuura Katsumi , Miyoshi Hideto , Anraku Yasuhiro
Published: May 2003

Reaction of ubiquinone in the high‐affinity quinone‐binding site (QH) in bo‐type ubiquinol oxidase from Escherichia coli was revealed by EPR and optical studies. In the QH site, ubiquinol was shown to be oxidized to ubisemiquinone and to ubiquinone, while no semiquinone signal was detected in the oxidase isolated from mutant cells that cannot synthesize ubiquinone. The QH site highly stabilized ubisemiquinone radical with a stability constant of 1–4 at pH 8.5 and the stability became lower at the lower pH. Midpoint potential of QH2/Q couple was −2 mV at pH 8.5 and showed −60 mV/pH dependence indicative of 2H+/2e− reaction. The E
m was more negative than that of low‐spin heme b above pH 7.0. We conclude that the QH mediates intramolecular electron transfer from ubiquinol in the low‐affinity quinol oxidation site (QL) to low‐spin heme b. Unique roles of the quinone‐binding sites in the bacterial ubiquinol oxidase are discussed.
Crystallization and preliminary diffraction studies of the structural domain E of Thermus flavus 2S rRNA
Authors: Nolte Alexis , Kluβman Sven , Lorenz Siegfried , Bald Rolf , Betzel Christian , Dauter Zbigniev , Wilson Keith , Fürste Jens Peter , Erdmann Volker A.
Published: May 2003

The ribosomal 5S RNA is an essential constituent of the large ribosomal subunit. To overcome the difficulties of crystallizing large RNA molecules such as 5S rRNAs, we decided to divide the 5S rRNA in five domains A through E to determine their structure. Recently we determined the crystal structural of the helical domain A. Here we report the crystallization of the chemically synthesized domain E of the Thermus flavus 5S rRNA. The crystal form is trigonal with unit cell dimensions: and . Diffraction‐data to 2.8 Å have been recorded and the structure solution is currently underway by means of MIR and MAD techniques.
Human vascular smooth muscle cells express an estrogen receptor isoform
Authors: Karas Richard H. , Baur Wendy E. , van Eickles Martin , Mendelsohn Michael E.
Published: May 2003

In women, estrogen (E2) exerts a clinically relevant anti‐atherogenic effect. The atheroprotective effects of E2 are mediated both by E2‐induced changes in systemic factors and by direct effects of E2 on the blood vessel wall. In studies to characterize E2 signaling pathways in vascular smooth muscle cells (VSMC), we recently demonstrated that human VSMC express a functional estrogen receptor [1]. In the present study, we applied a reverse transcription/PCR‐based strategy to identify isoforms of the E2 receptor in human VSMC. We now report that in addition to the classical E2 receptor, human VSMC derived from both mammary artery and saphenous vein express an estrogen receptor isoform containing an in‐frame deletion of Exon 4 (ERΔ4). RNase protection assays confirm the presence of ERΔ4 message in VSMC and demonstrate it is nearly as abundant as the classical E2 receptor. Transient transfection experiments in VSMC and HeLa cells demonstrate that, in contrast to the classical 67 kDa nuclear‐localized E2 receptor, ERΔ4: (a) is a 55 kDa protein that is widely distributed throughout the cell; (b) does not transactivate an E2 response element‐driven reporter plasmid in response to E2; and (c) does not modulate transactivation of the ERE‐reporter by the classical (wild type) estrogen receptor. Thus, human VSMC express an E2 receptor isoform that does not appear to alter gene transcription. The presence of a novel isoform of the E2 receptor may have important implications for studies of E2‐mediated signaling in VSMC.
Molecular cloning and functional expression of a recombinant 72.5 kDa fragment of the 110 kDa regulatory subunit of smooth muscle protein phosphatase 1M
Authors: Haystead Clare M.M. , Gailly Philippe , Somlyo Andrew P. , Somlyo Avril V. , Haystead Timothy A.J.
Published: May 2003

We have cloned a partial rat kidney cDNA that encodes a 72.5 kDa N terminal fragment of a third isoform of the M110 subunit of phosphatase 1. This new isoform contains an insert in the 542–597 position not present in the M110 previously cloned (Chen et al. (1994) FEBS Lett. 356, 51–55) from the same species. The encoded cDNA was expressed as a soluble GST‐fusion protein in E. coli, and its ability to interact with native PP‐1C was measured both in vitro and in permeabilized smooth muscle. In vitro, the fusion protein was capable of selectively binding PP‐1C and increasing the substrate specificity of the phosphatase towards myosin 13.2 ± 3.5‐fold (S.E. of the mean, n = 3). In permeabilized smooth muscle pretreated with microcystin, the recombinant protein alone (1.0 μM) did not cause relaxation, but did significantly enhance the ability of PP‐1C (0.3 μM) to relax the muscle. These findings show that the N terminal domain of the M110 subunit is the primary site for both PP‐1C and myosin binding, and thereby determines myosin specificity. The presence of isoformic variation within this sequence may permit organ/cell specific regulation of phosphorylation sites.
Characterization of functionally independent domains in the human ubiquitin conjugating enzyme UbcH2
Authors: Kaiser Peter , Mandl Sonja , Schweiger Manfred , Schneider Rainer
Published: May 2003

UbcH2 encodes a human ubiquitin conjugating enzyme (E2) able to conjugate ubiquitin to histone H2A in an E3 independent manner in vitro, which indicates that UbcH2 directly interacts with its substrates. To identify parts of the enzyme that are capable of binding H2A, we expressed several deletion mutants of UbcH2 in E. coli and tested the ability of the affinity purified mutant proteins to ubiquitinate H2A in the presence of bacterial expressed E1 and ubiquitin. With this in vitro assay we identified a C‐terminal part of UbcH2 to be important for the interaction with H2A. Transfer of this C‐terminal domain to another human E2, which is unable to catalyze ubiquitination of histones, leads to a fully active hybrid human ubiquitin conjugating enzyme capable of H2A ubiquitination. These results demonstrate that UbcH2 consists of two functionally independent domains. A N‐terminal core domain with ubiquitin conjugating activity, and a C‐terminal domain which interacts with substrate proteins.
Site‐directed mutagenesis of Thermus thermophilus EF‐Tu: the substitution of threonine‐62 by serine or alanine
Authors: Reza Ahmadian M. , Kreutzer Roland , Blechschmidt Bernd , Sprinzl Mathias
Published: May 2003

The invariant threonine‐62, which occurs in the effector region of all GTP/GDP‐binding regulatory proteins, was substituted via site‐directed mutagenesis by alanine and serine in the elongation factor Tu from Thermus thermophilus. The altered proteins were overproduced in Escherichia coli, purified and characterized. The EF‐Tu T62S variant had similar properties with respect to thermostability, aminoacyl‐tRNA binding, GTPase activity and in vitro translation as the wild‐type EF‐Tu. In contrast, EF‐Tu T62A is severely impaired in its ability to sustain polypeptide synthesis and has only very low intrinsic and ribosome‐induced GTPase activity. The affinity of aminoacyl‐tRNA to the EF‐Tu T62A·GTP complex is almost 40 times lower as compared to the native EF‐Tu·GTP. These observations are in agreement with the tertiary structure of EF‐Tu·GTP, in which threonine‐62 is interacting with the Mg2+ ion, γ‐phosphate of GTP and a water molecule, which is presumably involved in the GTP hydrolysis.
Phospholipid‐synthesizing enzymes in Golgi membranes of the yeast, Saccharomyces cerevisiae
Authors: Leber Andrea , Hrastnik Claudia , Daum Günther
Published: May 2003

Golgi membranes of the yeast, Saccharomyces cerevisiae, were isolated by a method similar to the procedure described by Cleves et al. [Cell 64 (1991) 789–800]. Marker proteins of the Golgi, such as Kex2 protease and GDPase, are highly enriched in these preparations. The phospholipid and ergosterol content of Golgi membranes is low. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol are the major phospholipids of this compartment. The amount of phosphatidylserine in the Golgi is significantly higher than in yeast bulk membranes. Inositol‐containing sphingolipids, especially inositolphosphorylceramide, are highly enriched in Golgi membranes. Two phospholipid‐synthesizing enzymes, namely phosphatidylinositol synthase and sn‐1,2‐diacylglycerol cholinephosphotransferase, are detected in the Golgi at a specific activity which exceeds that of the endoplasmic reticulum.
Apoptosis is triggered by the cyclic AMP signalling pathway in renal mesangial cells
Authors: Mühl Heiko , Nitsch Dorothea , Sandau Katrin , Brüne Bernhard , Varga Zoltan , Pfeilschifter Josef
Published: May 2003

Glomerular mesangial cells are regarded as specialized smooth muscle cells located within the renal glomeruli and fulfilling important functions in glomerular physiology and pathophysiology. Here, we report that activation of the cyclic AMP signalling pathway by dibutyryl cyclic AMP, forskolin, or the β
2‐adrenergic receptor agonist salbutamol results in induction of apoptosis in mesangial cells. Activation of the apoptotic programme results in DNA fragmentation which is visible for most forms of apoptosis and is paralleled by enrichment of cytosolic DNA/histone complexes, an increasing number of cellular 3′‐OH‐fragmented DNA ends and typical nuclear chromatin condensation. Induction of apoptosis was found to be dependent on translation and independent of nitric oxide synthase activity.
Mitochondrial alterations induced by aspirin in rat hepatocytes expressing mitochondrially targeted green fluorescent protein (mtGFP)
Authors: Venerando R. , Miotto G. , Pizzo P. , Rizzuto R. , Siliprandi N.
Published: May 2003

Mitochondria in primary living hepatocytes were visualized in cells transfected with a chimeric plasmid encoding for the green fluorescent protein (GFP) of Aequorea victoria engineered to be specifically targeted to mitochondria, as described recently (Rizzuto et al. (1995) Curr. Biol. 5, 635–642). The identification of the fluorescent organelles as authentic mitochondria was confirmed by double labeling with rhodamine 123. Acetylsalicylate treatment of hepatocytes induced in mitochondria typical morphological alterations closely analogous to the swelling promoted by acetylsalicylate in isolated mitochondria. Cyclosporin A, which in isolated mitochondria prevents the changes induced by acetylsalicylate, had no protective action but induced per se specific alterations in the morphology of mitochondria. Moreover, exposure of hepatocytes to cyclosporin A followed by acetylsalicylate caused the same mitochondrial changes induced by each of the two compounds separately. The structural alterations caused by acetylsalicylate were constantly associated with a decrease in mitochondrial urea synthesis and cell viability.
Human testis specifically expresses a homologue of the rodent T lymphocytes RT6 mRNA
Authors: Lévy Isabelle , Wu Yuan-Qing , Roeckel Nathalie , Bulle Frédérique , Pawlak André , Siegrist Sylvie , Mattéi Marie Geneviève , Guellaën Georges
Published: May 2003

A human homologue of the rodent T cell mono ADP‐ribosyl transferase RT6 mRNA was identified by a systematic analysis of human testis transcripts. This messenger encodes for a precursor protein of 367 aa (MW: 41.5 kDa) which exhibits a peptide signal, consensus domains for mono ADP‐ribosyl transferase and a C‐terminal part characteristic of glycophosphatidyl inositol anchored protein. This mRNA, transcribed from a gene localized in 4q13‐q21, is not expressed in white blood cells but is specific for human testis in which it is likely to correspond to a new ADP‐ribosyl transferase.
Vigilin contains a functional nuclear localisation sequence and is present in both the cytoplasm and the nucleus
Authors: Kügler S. , Grünweller A. , Probst C. , Klinger M. , Müller P.K. , Kruse C.
Published: May 2003

Vigilin is a member of the KH protein family and contains 14 tandemly arranged potential RNA‐binding domains. Between KH domains 2 and 3 we have identified a nuclear localization sequence by cloning this sequence into the NH2‐terminal region of phage T7 RNA polymerase as a reporter protein and by showing its transfer into the nucleus. Furthermore we provide experimental evidence that Vigilin is present both in the nucleus and in the cytoplasm in similar concentrations. These observations support the notion that Vigilin may shuttle between nucleus and cytoplasm presumably in contact with RNA molecules.
The impact of just‐in‐time manufacturing on firm performance in the US
Authors: Huson Mark , Nanda Dhananjay
Published: May 2003

Just‐In‐Time manufacturing has been subjected to numerous studies both empirical and methodological. This work attempts to measure the impact of JIT on accounting measures of performance. Most technologies and investments are justified on the basis of their impact on financial and accounting measures which are not easily quantified. Our empirical methodology, simultaneous equation estimation, allows us to isolate the partial effects of JIT on various accounting measures thus gauging the true impact of this method on firm performance. Our results show that after JIT adoption firms reduced the labor content in facilities, increased inventory turnover and enhanced earnings. There was no significant impact on prices charged by the firm. These results support the anecdotal evidence on JIT and the theoretical work done by various authors. Even though the firms studied experienced a downturn in their performance our empirical methodology could identify positive benefits resulting from JIT adoption.
Economics of operations management: A research perspective
Authors: Banker Rajiv D. , Khosla Inder S.
Published: May 2003

In response to intense competition from overseas in the last two decades, U.S. companies have instituted a variety of management practices in their quest to improve their competitiveness. Among the many practices that have been adopted by a large number of firms are just‐in‐time, total quality management, worker empowerment, and design for manufacturability. However, there has been little systematic analysis of the organizational factors that drive the adoption of these practices as well as their successful implementation. This paper posits that the theories and methods from economics can provide a useful aid in such an analysis. Using the basic tenets of economics, a structural framework is proposed to highlight critical issues in operations management that deserve attention. Examples from industry are used to illustrate how the framework may provide answers to questions that have received little attention in the research literature.
Machine dedication and process flexibility in a group technology environment
Authors: Jensen John B. , Malhotra Manoj K. , Philipoom Patrick R.
Published: May 2003

This study investigates changes in inventory and customer service performance of a job‐shop that desires to adopt a Group Technology (GT) philosophy in its shop floor operations. Simulation methodology is pursued to explore tradeoffs in shop performance between the routing flexibility of non‐dedicated machines in a functional job‐shop and the setup efficiency of dedicated machines in shops that have machine cell layouts. Further, traditional and GT‐based scheduling procedures are investigated in these different shops to determine the conditions under which a GT philosophy may be profitably employed only in layout decisions, only in scheduling decisions, or in both layout and scheduling decisions. Results from this study show that shop layout choice is not a simple decision that can capitalize either on the high routing flexibility of the functional job‐shop or on the setup efficiency advantages of a cell shop. The tradeoffs between routing flexibility and setup efficiency must be made carefully. The impact of demand variability on performance is also dependent on the type of layout. In general, product volume variability more adversely affects the performance of functional job‐shops, while product mix variability has greater impact on the performance of cell shops. Finally, sensitivity analysis is performed to show that maintaining balance between the utilization of machines is a major determinant of performance, and consequently the best layout.
Critical regions for the sweetness of brazzein1
Authors: Jin Zheyuan , Danilova Vicktoria , Assadi-Porter Fariba M , Aceti David J , Markley John L , Hellekant Göran
Published: May 2003

Brazzein is a small, heat‐stable, intensely sweet protein consisting of 54 amino acid residues. Based on the wild‐type brazzein, 25 brazzein mutants have been produced to identify critical regions important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29–33 and 39–43, plus residue 36 between these stretches, as well as the C‐terminus are involved in the sweetness of brazzein. Second, charge plays an important role in the interaction between brazzein and the sweet taste receptor.
Structural insights into the mechanism of proton pumping by bacteriorhodopsin
Authors: Hirai Teruhisa , Subramaniam Sriram
Published: May 2003

For over three decades, bacteriorhodopsin has served as a paradigm for the study of the mechanisms underlying ion pumping across biological membranes. It is perhaps among the simplest known ion pumps, which functions by converting light energy into an electrochemical gradient by pumping protons out of the cytoplasm. The combination of spectroscopic, biochemical and crystallographic studies on bacteriorhodopsin provides a unique opportunity to dissect the principal elements underlying the mechanism of transmembrane proton transport. Here, we provide a brief review of recent developments related to the determination of the structural changes during proton transport using crystallographic approaches. Taken together with previous spectroscopic and biochemical investigations, these studies allow the description of a detailed molecular mechanism of the main steps in vectorial proton transport by bacteriorhodopsin.
Variation in proton donor/acceptor pathways in succinate:quinone oxidoreductases
Authors: Cecchini Gary , Maklashina Elena , Yankovskaya Victoria , Iverson Tina M , Iwata So
Published: May 2003

The anaerobically expressed fumarate reductase and aerobically expressed succinate dehydrogenase from Escherichia coli comprise two different classes of succinate:quinone oxidoreductases (SQR), often termed respiratory complex II. The X‐ray structures of both membrane‐bound complexes have revealed that while the catalytic/soluble domains are structurally similar the quinone binding domains of the enzyme complexes are significantly different. These results suggest that the anaerobic and aerobic forms of complex II have evolved different mechanisms for electron and proton transfer in their respective membrane domains.
Short interfering RNA‐directed inhibition of hepatitis B virus replication
Authors: Hamasaki Keisuke , Nakao Kazuhiko , Matsumoto Kojiro , Ichikawa Tatsuki , Ishikawa Hiroki , Eguchi Katsumi
Published: May 2003

RNA interference (RNAi) is the process by which double‐stranded RNA directs sequence‐specific degradation of mRNA. In mammalian cells, RNAi can be triggered by 21‐nucleotide duplexes of short interfering RNA (siRNA). We examined effects of siRNA on hepatitis B virus (HBV) replication. Human hepatoma cells were transfected with HBV DNA and siRNA against HBV‐pregenome RNA. Transfection experiments demonstrated that the siRNA reduced the amount of HBV‐pregenome RNA and resulted in reduction of the levels of replicative intermediates and viral protein. Our results indicate that siRNA‐mediated gene silencing inhibits HBV replication through suppression of viral RNA, which may be useful as a potential therapeutic modality.