bluuebunny/crossref_metadata_2025 huggingface dataset:
β‐Hydroxyaspartic acid in vitamin K‐dependent plasma proteins from scorbutic and warfarin‐treated guinea pigs
Authors: Stenflo Johan , Fernlund Per
Published: July 2002

β‐Hydroxyaspartic acid is a rare amino acid, present in all vitamin K‐dependent plasma proteins except prothrombin, and is formed by a post‐translational hydroxylation of aspartic acid. We have now investigated whether this hydroxylation, like that of proline in collagen, is vitamin C‐dependent. The vitamin K‐dependent plasma proteins were isolated from normal and scorbutic guinea pig plasma by barium citrate adsorption and the β‐hydroxyaspartic acid content was determined. Compared with normal animals, scorbutic animals showed no significant reduction of β‐hydroxyaspartic acid content. In warfarin‐treated animals there was a decreased content of both β‐hydroxyaspartic acid and γ‐carboxyglutamic acid in the barium citrate adsorbed fraction. It was concluded that the post‐translational hydroxylation of aspartic acid is unlikely to be vitamin C‐dependent.
Effects of VIP and forskolin on alanine metabolism in isolated hepatocytes
Authors: Leiser Jeffrey , Blum J.J.
Published: July 2002

Vasoactive intestinal polypeptide
Forskolin
Gluconeogenesis
Alanine Glycogen metabolism
Proteins from the prokaryotic nucleoid
Authors: Lammi Matilde , Paci Maurizio , Gualerzi Claudio O.
Published: July 2002

The interaction of the prokaryotic DNA‐scaffolding proteins NS (from Escherichia coli and BS‐NS (from Bacillus stearothermophilus) with DNA has been investigated. Upon binding of NS to DNA, the resolution of its 400 MHz 1H‐NMR spectrum is lost, due to the broadening of all resonance lines. These effects are reversed by increasing the ionic strength. Since with increasing amounts of DNA added all resonances broaden progressively and simultaneously without prior selective loss of the spectral features due to the tertiary and quarternary structure of the protein, it is suggested that NS binds to DNA in the aggregated form (octameric?) and without gross alteration of its tertiary structure. By selective chemical modification of BS‐NS it was found that at least one Arg residue, located in the major hydrophilic, positively charged, conserved peptide of the protein (positions 51–70) is necessary for the interaction of BS‐NS with DNA.
Interaction of methionine‐specific tRNAs from Escherichia coli with immobilized elongation factor Tu
Authors: Fischer W. , Doi T. , Ikehara M. , Ohtsuka E. , Sprinzl M.
Published: July 2002

The interaction of three different Met‐tRNAsMet from E. coli with bacterial elongation factor (EF) Tu · GTP was investigated by affinity chromatography. Met‐tRNAfMet which lacks the base pair at the end of the acceptor stem binds only weakly to EF‐Tu·GTP, while Met‐tRNAmMet has a high affinity for the elongation factor. A modified Met‐tRNAfMet which has a C1‐G72 base pair binds much more strongly to immobilized EF‐Tu·GTP than the native aminoacyl(aa)‐tRNA with non‐base‐paired C1A72 at this position, demonstrating that the base pair including the first nucleotide in the tRNA is one of the essential structural requirements for the aa‐tRNA·EF‐Tu·GTP ternary complex formation.
Biochemical characterization of plasma membrane isolated from human skeletal muscle
Authors: Desnuelle C. , Liot D. , Serratrice G. , Lombet A.
Published: July 2002

Specific components of ion translocation systems were studied in excitable plasma membranes isolated from normal human muscle. Na+ ‐ K+ ATPase and ouabain‐sensitive K+ phosphatase activities were 8.9 ± 1 per mg protein and 96 ± 9 per mg protein, respectively. Scatchard analysis of equilibrium binding assays with [3H]ouabain showed non‐linear curves consistent with high‐ and low‐affinity sites (estimated K
d 3 nM and 0.22 μM). Two families of receptors with different affinities for a tritiated TTX derivative (estimated K
d 0.4 and 4 nM) were also identified suggesting the existence in human muscle of at least two classes of voltage‐dependent Na+ channels. In addition (+)‐[methyl‐3H]PN200‐110, a potent Ca2+ antagonist used for labeling voltage‐dependent Ca2+ channels, was observed to bind to a homogeneous population of receptors in the plasma membrane (K
d = 0.2 nM).
Evidence that human prostatic 5α‐reductase is located exclusively in the nucleus
Authors: Houston B. , Chisholm G.D. , Habib F.K.
Published: July 2002

The 5α‐reductase activities in human prostatic nuclei and microsomes were compared. The activities in both subcellular fractions were identical with respect to pH dependence, heat inactivation and Michaelis constants for NADPH and testosterone. Subcellular distribution studies using DNA and nicotinamide mononucleotide adenyl transferase as nuclear markers showed that the amount of 5α‐reductase present in the microsomes was directly proportional to the amount of nuclear contamination. These results indicate that human prostatic tissue contains only one form of 5α‐reductase, which is located exclusively in the nucleus. This finding has important implications for the mechanism of steroid action in the prostate.
Identification of the D‐glucose binding polypeptide of the renal Na+‐D‐glucose cotransporter with a covalently binding D‐glucose analog
Authors: Neeb Martin , Fasold Hugo , Koepsell Hermann
Published: July 2002

Affinity labeling
D‐Glucose analog
D‐Glucose binding site
Na±D‐glucose cotransporter
Brush‐border membrane
Kidney
Rat pancreas contains the proglucagon(64–69) fragment and arginine stimulates its release
Authors: Yanaihara Chizuko , Matsumoto Tomoaki , Kadowaki Makoto , Iguchi Kazuaki , Yanaihara Noboru
Published: July 2002

Rat proglucagon(64–69) corresponding to the C‐terminal hexapeptide of putative rat glicentin sequence in the precursor was synthesized. A glicentin C‐terminal hexapeptide specific radioimmunoassay, using the synthetic hexapeptide as standard, demonstrated the presence in rat pancreas of a peptide identified with the synthetic rat proglucagon(64–69): H‐Asn‐Arg‐Asn‐Asn‐Ile‐Ala‐OH. The hexapeptide was released concomitantly with glucagon by arginine stimulation from the isolated perfused rat pancreas. The results indicate that the pancreas co‐stores and possibly co‐releases the hexapeptide with glucagon as one of the processing products of proglucagon.
Regulation of pantothenate kinase from various tissues of the rat
Authors: Fisher Mark N. , Neely James R.
Published: July 2002

Pantothenic acid
Coenzyme synthesis
Carnitine kinase
Pantothenate kinase
Carnitine synthesis
CoA synthesis
A significant increase of lysophosphatidylinositol 4‐phosphate with insulin in isolated rat fat cells
Authors: Kuroda Yoshihiko , Nakayama Hidetaka , Ishibashi Teruo , Aoki Shin , Tushima Satoshi , Nakagawa Shoichi
Published: July 2002

We studied the effects of insulin on the incorporation of 32Pi into phospholipids in rat fat cells. When the cells were treated with insulin, a new radioactive phospholipid was detected on thin layer chromatography. The substance migrated slower than phosphatidylinositol 4,5‐bisphosphate and was hardly detectable in the absence of insulin. This effect of insulin was both time‐ and dose‐dependent with half‐maximal stimulation at 120 μU/ml. Pretreatment of insulin with anti‐insulin antibody or the cells with anti‐insulin receptor anti‐body inhibited the effect of insulin. The product of phosphatidylinositol 4‐phosphate hydrolysed by phospholipase A2 was coincided with the substance on thin layer chromatography. Quinacrine inhibited the formation of the substance in a dose‐dependent manner. These results suggested that insulin stimulates the generation of lysophosphatidylinositol 4‐phosphate through the insulin‐receptor interaction.
Correlation between molecular clock ticking, codon usage, fidelity of DNA repair, chromosome banding and chromatin compactness in germline cells
Authors: Filipski J.
Published: July 2002

The vertebrate genome is built of long DNA regions, relatively homogeneous in GC content, which likely correspond to bands on stained chromosomes. Large differences in composition have been found among DNA regions belonging to the same genome. They are paralleled by differences in codon usage in genes differently localized. The hypothesis presented here asserts that these differences in composition are caused by different mutational bias of α and β DNA polymerases, these polymerases being involved to different extents in the repair of DNA lesions in compact and relaxed chromatin, respectively, in germline cells.
Phorbol ester stimulates the synthesis of sphingomyelin in NIH 3T3 cells A diminished response in cells transformed with human A‐raf carrying retrovirus
Authors: Kiss Zoltan , Rapp Ulf R. , Anderson Wayne B.
Published: July 2002

The tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) stimulated the synthesis of sphingomyelin (CerPCho) from a [14C]choline‐labelled phosphatidylcholine (PtdCho) pool in NIH 3T3 cells. Maximal stimulation (68%) of CerP‐Cho synthesis, accompanied by an increase (38%) in its cellular content, required only 2 nM TPA. Higher concentrations of TPA (2–100 nM) had progressively less effect on CerPCho synthesis which correlated with increased hydrolysis of precursor PtdCho. In cells transformed with human or mouse A‐raf carrying retroviruses TPA‐stimulated PtdCho hydrolysis, but not CerPCho synthesis, suggesting independent regulation of these processes by the TPA‐stimulated signal transduction system.
Formation of the 680 nm‐absorbing form of the cytochrome bd oxidase complex of Escherichia coli by reaction of hydrogen peroxide with the ferric form
Authors: Poole Robert K. , Williams Huw D.
Published: July 2002

Reduced minus aerated difference spectra of membranes from Escherichia coli (grown under oxygen‐limited conditions) show, in addition to the 650 nm trough attributed to the oxygenated form of cytochrome d, a smaller trough centred at about 680 nm of unknown origin. When the reference spectrum is that of a sample oxidized with ferricyanide and to which hydrogen peroxide was added, the trough proportions changed, the 680 nm species being more dominant. Similarly, when 8.8 mM hydrogen peroxide is added to a persulphate‐oxidized sample, a peak at 680 nm is immediately formed. No such compound is observed when peroxide is added to persulphate‐oxidized membranes from a cytochrome d‐deficient mutant. It is concluded that the 680 nm species represents a peroxy form of haem d, which is stable at room temperature and is probably an intermediate in the reaction mechanism of this oxidase.
Protein chemical characterization of Mucor pusillus aspartic proteinase Amino acid sequence homology with the other aspartic proteinases, disulfide bond arrangement and site of carbohydrate attachment
Authors: Baudys̆ Miroslav , Foundling Stephen , Pavlík Manfred , Blundell Tom , Kostka Vladimír
Published: July 2002

The amino acid sequence of Mucor pusillus aspartic protenaise was determined by analysis of fragments obtained from cleavage of the enzyme by CNBr and limited tryptic digestion. The protenaise is a single polypeptide chain protein containing 361 amino acid residues, cross‐linked by two disulfide bonds. A sugar moiety composed of two GlcNAc residues and four neutral sugar residues is asparagine‐linked to the chain. The sequence of M. pusillus proteinase is highly homologous with the M. miehei protonaise (83% identity). The homology with other aspartic proteinases is low (22–24%) and indicates that the Mucor proteinases diverged at an early evolutionary phase. The most conservative regions of the molecule are those involved in catalysis and forming the binding cleft and the core region of the molecule.
Pyruvate decarboxylase is like acetolactate synthase (ILV2) and not like the pyruvate dehydrogenase E1 subunit
Authors: Green Jeremy B.A.
Published: July 2002

Protein sequences of pyruvate decarboxylase (PDC) derived from cloned yeast (Saccharomyces cerevisiae) and bacterial (Zymomonas mobilis) genes were compared with each other and with sequence databases. Extensive sequence similarities were found between them and with two others: cytochrome‐linked pyruvate oxidase from Escherichia coli and acetolactate synthase (ilvI in E. coli; ILV2 gene in S. cerevisiae). All catalyse decarboxylation of pyruvate using thiamine pyrophosphate (TPP) as cofactor. General overall similarity suggests common ancestry for these enzymes. None of the sequences was similar to the E1 component of pyruvate dehydrogenase from E. coli which also decarboxylates pyruvate with the help of TPP.
Location of phosphorylation site and DNA‐binding site of a positive regulator, OmpR, involved in activation of the osmoregulatory genes of Escherichia coli
Authors: Kato Masashi , Aiba Hirofumi , Tate Shin-ichi , Nishimura Yoshifumi , Mizuno Takeshi
Published: July 2002

The OmpR protein of Escherichia coli is a positive regulator involved in activation of the ompF and ompC genes which encode the major outer membrane proteins OmpF and OmpC, respectively. By employing recombinant DNA techniques, we isolated the N‐ and C‐terminal halves of the OmpR molecule. From the results of biochemical analyses of these fragments, it was concluded that the N‐terminal portion contains a site involved in phosphorylation by an OmpR‐specific protein kinase EnvZ, whereas the C‐terminal part possesses a DNA‐binding site for the ompC and ompF promoters.
Antisera against an acetylcholine receptor α3 fusion protein bind to ganglionic but not to brain nicotinic acetylcholine receptors
Authors: Schoepfer Ralf , Halvorsen Stanley W. , Conroy William G. , Whiting Paul , Lindstrom Jon
Published: July 2002

Neuronal nicotinic acetylcholine receptor (AChR) subtypes have been defined pharmacologically, immunologically, and by DNA cloning, but the correlations between these approaches are incomplete. Vertebrate neuronal AChRs that have been isolated are composed of structural subunits and ACh‐binding subunits. A single kind of subunit can be used in more than one AChR subtype. Monoclonal antibody (mAb) 35 binds to structural subunits of subtypes of AChRs from both chicken brain and ganglia. By using antisera to a unique sequence of α3 ACh‐binding subunits expressed in bacteria, we show that ganglionic AChRs contain α3 ACh‐binding subunits, whereas the brain AChR subtype that binds mAb 35 does not. Subunit‐specific antisera raised against recombinant proteins should be a valuable approach for identifying the subunit composition of receptors in multigene, multisubunit families.
Effect of divalent and monovalent cations on calf thymus PCNA‐independent DNA polymerase δ and its 3' → 5' exonuclease
Authors: Focher Federico , Verri Annalisa , Maga Giovanni , Spadari Silvio , Hübscher Ulrich
Published: July 2002

Recent data suggest that DNA polymerases α and δ might have a coordinate functional role at the replication fork. In this communication we show that Mg2+ is likely the natural metal activator for both enzymes. Mn2+, a known mutagenic agent, is a competitive inhibitor of Mg2+ for DNA polymerase δ and acompetitive for DNA polymerase α. The 3'→ 5' exonuclease activity associated with DNA polymerase δ is not affected upon addition of Mn2+, Be2+, another mutagenic agent, on the other hand, has an inhibitory effect on the 3' → 5' exonuclease, but not on the DNA polymerase δ. The data presented might explain the mutagenic and carcinogenic potential of these two divalent cations.
Reduction of HSV‐1 binding to BHK cells after treatment with phosphatidylinositol‐specific phospholipase C
Authors: Langeland N. , Moore L.J.
Published: July 2002

Baby‐hamster kidney cells were treated with unspecific and phosphatidylinositol‐specific phospholipase C (PI‐PLC) prior to infection with herpes simplex virus type 1 or 2. Subsequent to PI‐PLC treatment, a 30% reduction of infectivity and receptor binding was observed for type 1 virus, while type 2 was unaffected. Treating the cells with unspecific phospholipase C did not affect subsequent infection with either virus. Treatment of the virus particles with the unspecific phospholipase reduced its infectivity, probably due to loss of the viral envelope. PI‐PLC treatment of virus particles did not have any such effect on virus infectivity.
In vivo immediate early gene expression induced in intestinal and colonic mucosa by feeding
Authors: Holt Peter R. , DuBois Raymond N.
Published: July 2002

Since the gut responds rapidly to food intake, the levels of expression of several immediate early genes were measured in mucosa from small and large intestine of rats starved for 3 days or refed. Within 1 h of refeeding, jejunal and ileal c‐fos, jun B and zif/268 mRNA and colonil zif/268 dramatically increased. The zif/268 gene in jejunum corresponded in size to the full‐length cDNA but. in ileum, an RNA band of about 1.2 kb in size increased greatly after feeding. This represents a physiologic in vivo model for the study of gene regulation associated with intestinal epithelial cellular responses to feeding.
Porcine pancreas extract decreases blood‐ionized calcium in mice and inhibits osteoclast formation and bone resorption in culture
Authors: Yoneda Toshiyuki , Takaoka Yoshito , Alsina Maria M. , Garcia Joyce , Mundy Gregory R.
Published: July 2002

Patients with acute pancreatitis commonly manifest hypocalcemia for reasons which are unknown. We found that porcine pancreas extracts (PX) significantly decreased blood‐ionized calcium in Balb/e mice. Partially‐purified PX with a molecular mass of approximately 27 kDa decreased blood‐ionized calcium in the mice. Partially‐purified PX suppressed not only 41Ca release from fetal rat long bones which had been stimulated by parathyroid hormone, interleukin‐1 α, tumor necrosis factor, transforming growth factor‐α, 1.25‐dihydroxyvitamin D3 and prostaglandin E2, but tartrate‐resistant acid phosphatase‐positive multinucleated cell formation in the presence of 1,25‐dihydroxyvitamin D3 in mouse marrow cultures. The results suggest that there is an as yet‐unidentified bone metabolism‐regulating substance in porcine pancreas which might be responsible for the hypocalcemia associated with acute pancreatitis.
Cloning and sequencing of the gene encoding the large (α‐) subunit of the proteasome from Thermoplasma acidophilum
Authors: Zwickl Peter , Lottspeich Friedrich , Dahlmann Burkhardt , Baumeister Wolfgang
Published: July 2002

The gene encoding the α‐subunit of the proteasome from Thermoplasma acidophilum was cloned and sequenced. The gene encodes for a polypeptide with 233 amino acid and reduces a calculated molecular weight of 25870. Sequence similarity of the α‐subunit with the Saccharomyces cerevislae wild‐type suppressor gene sell* encoded polypeptide, which is probably identical with the subunit YC7‐α of the yeast proteasome, lends support to a putative role of proteasomes in the regulation of gene expression. The significant sequence similarity to the various subunits of eukaryotic proteasomes make it likely that proteasomal proteins are encoded by one gene family of ancient origin.
Structural dynamics in F1ATPase during the first reaction cycle of ATP hydrolysis
Authors: Neidhurdt Axel , Nawroth Thomas , Hütsch Matthias , Dose Klaus
Published: July 2002

The velocity of ATP hydrolysis, catalyzed by purified F1ATPase from Micrococcus luteus, was decelerated on decreasing the temperature. At 13′C one reaction cycle is completed after 20 s. Hydrolysis was triggered upon rapid mixing of the enzyme with ATP. During the first reaction cycle, succeeding structural alterations of the F1ATPase were traced by time resolved X‐ray scattering. The scattering spectra obtained from consecutive intervals of 1 s, revealed the F1ATPase to pass a conformational state exhibiting an expanded (6%) molecular shape. The expanded state was observed between 45% and 65% of the time required to complete the reaction cycle. This pointx out a conformational pulsation during ATP hydrolysis.
Diazipine, a novel photoaffinity probe for dihydropyridine receptors of calcium channels
Authors: Taki Motohiko , Nakayama Hitoshi , Kanaoka Yuichi
Published: July 2002

A new 1,4‐dihydropyridine photoaffinity ligand, [3H]diazipine, has been assessed by binding and photolabeling, and compared with a currently used [3H]Diazipine reversibly binds to skeletal muscle Ca2+ channels with a similar affinity of [3H]azidopine, but [3H]diazipine labels the channels two times more efficiently and no release of the incorporated amount is observed after dithiothreitol treatment.
A chemically cross‐linked nonlinear proOmpA molecule can be translocated into everted membrane vesicles of Escherichia coli in the presence of the proton motive force
Authors: Tani Katsuko , Mizushima Shoji
Published: July 2002

The chemical cross‐linking between the two cysteine residues at positions +290 and +302 of proOmpA was performed with N,N′‐bis(3‐maleimido‐propionyl)‐2‐hydroxy‐1,3‐propanediamine. In the absence of the proton motive force (ΔgmH+), the cross‐linked proOmpA was only partially translocated into everted membrane vesicles, leading to accumulation of translocation intermediates. In the presence of ΔgmH+, the cross‐linked proOmpA was completely translocated. The translocated OmpA still possessed the cross‐linked loop composed of 13 amino acid residues and the cross‐linker. It is concluded that polypeptide chains need not be necessarily linear and fully expanded to be translocated.
A small basic ribosomal protein from the extreme thermophilic archaebacterium Sulfolobus solfataricus that has no equivalent in Escherichia coli
Authors: Ramirez Celia , Louie K.Andrea , Matheson Alastair T.
Published: July 2002

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene has been confirmed by partial amino acid sequencing. The protein shows no sequence similarity to any of the ribosomal proteins from eubacteria (Escherichia coli) or to those that have been reported from eukaryotes.
Transduction of enzyme‐ligand binding energy into catalytic driving force
Authors: Fisher Harvey F. , Singh Narinder
Published: July 2002

We propose a testable general mechanism by which ligand binding energy can be used to drive a catalytic step in an enzyme catalyzed reaction or to do other forms of work involving protein molecules. This energy transduction theory is based on our finding of the widespread occurrence of ligand binding‐induced protein macrostate interconversions each having a large invariant ΔH° accompanied by a small but highly variable ΔG°. This phenomenon, which can be recognized by the large ΔCp°'s it generates, can provide the necessary energy input step but is not in itself sufficient to constitute a workable transduction mechanism. A viable mechanism requires the additional presence of an ‘energy transmission step’ which is terminated to trigger the ‘power’ stroke at a precise location on the reaction coordinate, followed by an energetically inexpensive ‘return’ step to restore the machine to its initial conditions. In the model we propose here, these additional steps are provided by the existence of ligand inducible 2‐state transitions in the free enzyme and in each of the enzyme complexes that occur along the reaction coordinate, and by the selective blocking of certain of these interconversions by high energetic barriers. We provide direct experimental evidence supporting the facts that these additional mechanistic components do exist and that the liver glutamate dehydrogenase reaction is indeed driven by just such machinery. We describe some aspects of the chemical nature of these transitions, and evidence for their occurrence in other systems.
Phorbol ester‐induced redistribution of the ASGP receptor is independent of receptor phosphorylation
Authors: Geffen Iris , Spiess Martin
Published: July 2002

Like virtually all endocytic receptors, the human asialoglycoprotein (ASGP) receptor is phosphorylated by protein kinase C at serine residues within the cytoplasmic domains of its two subunits H1 and H2. Activation of protein kinase C by phorbol esters results in hyperphosphorylation and in a concomitant net redistribution of receptors to intracellular compartments (down‐regulation) in HepG2 cells. To test whether there is a causal relationship between receptor hyperphosphorylation and redistribution, we examined the effect of phorbol ester treatment on the ASGP receptor composed of either wild‐type subunits or of mutant subunits lacking any cytoplasmic serine residues in transfected NIH3T3 fibroblast and COS‐7 cells. Although the wild‐type subunits were hyperphosphorylated in fibroblast cells, the distribution of neither the wild‐type nor the mutant receptors was affected. In contrast, phorbol ester treatment of transfected COS‐7 cells induced down‐regulation of both wild‐type and mutant receptors. These findings indicate that redistribution of the receptor is independent of its cytoplasmic serines and is not caused by receptor phosphorylation.
Tandem translation of Bacillus subtilis initiation factor IF2 in E. coli Over‐expression of infBB.su in E. coli and purification of α‐ and β‐forms of IF2B.su
Authors: Hubert Michel , Nyengaard Niels Randel , Shazand Kamaran , Mortensen Kim Kusk , Lassen Søren Flensted , Grunberg-Manago Marianne , Sperling-Petersen Hans Uffe
Published: July 2002

The protein synthesis initiation factor, IF2, in Bacillus subtilis has previously been characterized as being present in two forms, α and β, of molecular mass 79 and 68 kDa, respectively, on the basis of their cross‐reaction with anti‐E. coli IF2 antibodies and by the DNA sequence of the gene for IF2, infB
B.su. In this work we have cloned infB
B.su in E. coli cells. Two proteins of molecular mass identical to the B. subtilis IF2α and ‐β were over‐expressed and purified using a new three‐step ion‐exchange chromatography procedure. The N‐terminal amino acid sequence of the two proteins was determined and the results confirmed that the two forms were IF2α and ‐β, both encoded by the infB gene. The N‐terminal amino acid sequence determined for IF2β is Met94‐Gln‐Asn‐Asn‐Gln‐Phe. The presence of methionine at position 94 shows that this form is, in fact, the result of a second translational initiation in infB
B.su mRNA, since the codon at amino acid position 94 is GUG, which is the normal codon for valine, but also known to be an initiator codon. This is a new example of the unusual tandem translation in E. coli of an open mRNA reading frame.
Dynamics of intermediate filaments
Authors: Georgatos Spyros D.
Published: July 2002

Intermediate filaments (IFs) have always been considered as the most static and ‘skeletal’ cellular elements. This view is now changing: new information reveals that IFs exchange subunits at steady‐state, that IF networks can be assembled de novo, and that IF proteins are subject to elaborate chemical modification and de‐modification during mitosis. I describe below some of the key observations which have made us realize that IFs are dynamic structures. I also discuss some of the remaining questions pertinent to the pathways of IF assembly under in vivo conditions.
Regulation of CCK mRNA in the human neuroepithelioma cell line SK‐N‐MCIXC in response to second messenger activators
Authors: Mania-Farnell Barbara L. , Merrill Bradley J. , Konings Pierre N.M. , Davis Thomas P.
Published: July 2002

Regulation of cholecystokinin (CCK) expression was studied in the human neuroepithelioma cell line SK‐N‐MCIXC. The cells were treated with the phosphodiesterase inhibitor isobutyl‐methylxanthine and the tumor promoting phorbol ester, phorbol‐12‐myristate 13‐acetate; activators of the cyclic AMP (cAMP) and protein kinase C (PKC) second messenger pathways, respectively. Levels of CCK mRNA were determined after 6, 12 and 24 hour drug treatments, with Northern blot analysis using human CCK cDNA hybridization probes. Activation of both cAMP and PKC second messenger pathways increased CCK mRNA levels in SK‐N‐MCIXC cells. These results indicate that the levels of CCK mRNA in SK‐N‐MCIXC cells are regulated by cAMP and PKC dependent mechanisms.
Calvasculin, as a factor affecting the microfilament assemblies in rat fibroblasts transfected by src gene
Authors: Watanabe Yasuo , Usada Nobuteru , Minami Hiroyuki , Morita Takashi , Tsugane Shinichiro , Ishikawa Ryoki , Kohama Kazuhiro , Tomida Yasuhiro , Hidaka Hiroyoshi
Published: July 2002

Cell transformations accompany alterations in cell morphology and microfilament patterns. Calvasculin encodes mRNA termed pEL‐98, 18A2, 42A, p9Ka, or mtsl, found to be elevated in several metastatic cell lines. We report the elevation of calvasculin expression in SR‐3Y1 cells, which show disappearance of ordered microfilaments, compared to that in 3Y1 cells and that the similar distribution of calvasculin to that of actin filaments. Interestingly, calvasculin co‐sediments with F‐actin and bundles actin filaments in a Ca2+‐dependent manner. This activity, along with the elevation of calvasculin following transformation, suggests that the disorganization of filaments in SR‐3Y1 cell is due to the cross‐linking activity of calvasculin.
Synthetic peptides of the rab3 effector domain stimulate a membrane fusion event involved in regulated exocytosis
Authors: Edwardson J.Michael , MacLean Carol M. , Law Greg J.
Published: July 2002

We have developed a system in which the fusion of pancreatic zymogen granules with plasma membranes can be studied in vitro. Here we show that this membrane fusion event is stimulated specifically by peptides of the effector domain of rab3, a small, monomeric GTP‐binding protein. In addition, we demonstrate that the stimulatory effect of the peptides involves their binding to a target on the plasma membrane, and is both qualitatively and quantitatively different from the effect of GTPγS, which also enhances membrane fusion. We suggest that regulated exocytosis in the pancreatic acinar cell may be under the control of more than one type of GTP‐binding protein.
Insulin‐like growth factor‐I (IGF‐I) mRNA in the small intestine of suckling and adult rats
Authors: Dvořák Bohuslav , Stephana Audrey L. , Holubec Hana , Williams Catherine S. , Philipps Anthony F. , Koldovskoý Otakar
Published: July 2002

The presence and cellular localization of insulin‐like growth factor‐I (IGF‐I) mRNA in the small intestine of suckling and adult rats was studied. A sensitive reverse transcription (RT) competitive‐polymerase chain reaction (PCR) revealed IGF‐I gene expression in both age groups. Adult tissue contained 3‐fold higher levels of IGF‐I mRNA in comparison with sucklings. Using an in situ hybridization technique, IGF‐I transcripts were localized mainly in enterocytes and goblet cells in the intestinal crypts of adult rats. By using this technique, IGF‐I mRNA was not detected in jejunum of 12‐day‐old rats.
Mutational analysis of the highly conserved C‐terminal residues of the XylS protein, a member of the AraC family of transcriptional regulators
Authors: Manzanera Maximino , Marqués Silvia , Ramos Juan L
Published: July 2002

The XylS protein of the TOL plasmid of Pseudomonas putida belongs to the so‐called AraC/XylS family of regulators, that includes more than 100 different bacterial proteins. A conserved stretch of about 100 amino acids is present at the C‐terminal end. This conserved region is believed to contain seven α‐helices, including two helix‐turn‐helix (HTH) DNA binding motifs (α2‐T‐α3 and α5‐Tα‐6), connected by a linker α‐helix (α4), and two flanking α‐helices (α1 and α7). The second HTH motif is the region with the highest homology in the proteins of the family, with certain residues showing almost 90% identity. We have constructed XylS single mutants in the most conserved residues and have analysed their ability to stimulate transcription from its cognate promoter, Pm, fused to ′lacZ. The analysis revealed that mutations in the α5‐helix conserved residues had little effect on the XylS transcriptional activity, whereas the distribution of polarity in the α6‐helix was important for the activity. The strongest effect of the mutations was observed in conserved residues located outside the DNA binding domain, namely, Gly‐290 in the turn between the two helices, Pro‐309 located downstream of α6, and Leu‐313, in the small last helix α7, that seems to play an important role in the activation of RNA‐polymerase. Our analysis shows that conservation of amino acids in the family reflects structural requirements rather than functionality in specific DNA interactions.
Thrombin–thrombomodulin activation of protein C facilitates the activation of progelatinase A
Authors: Pekovich Stevan R , Bock Paul E , Hoover Richard L
Published: July 2002

The activation of the matrix metalloproteinase progelatinase A (MMP‐2) has been of keen interest because an increase in MMP‐2 activity has been implicated in disease states such as cancer and atherosclerosis. Activation of MMP‐2 occurs on the surface of specific cell types in two steps. In the first step, primary cleavage of MMP‐2 by a membrane‐type matrix metalloproteinase generates an intermediate. A secondary cleavage and activation of the intermediate is thought to occur autocatalytically. Previous studies have shown that thrombin can also activate progelatinase A in the presence of endothelial cells. We show that this cell‐dependent mechanism of MMP‐2 activation also occurs with THP‐1 cells and involves binding of thrombin to thrombomodulin present on the cell surface and generation of the anti‐coagulant protein, activated protein C. We demonstrate that activated protein C is directly responsible for activation and cleavage of the gelatinase A intermediate. This work contributes new mechanistic insights into the activation of MMP‐2 and provides a novel link between matrix metalloproteinase activation and anti‐coagulation.
On the target of a novel class of antibiotics, oxazolidinones, active against multidrug‐resistant Gram‐positive bacteria
Authors: Burghardt Helena , Schimz Karl-Ludwig , Müller Matthias
Published: July 2002

Oxazolidinones are a promising new class of synthetic antibiotics active against multidrug‐resistant Gram‐positive bacteria. To elucidate their mode of action, the effect of DuP 721 on individual steps of protein translation was studied. The drug does not interfere with translation initiation at the stage of mRNA binding or formation of 30S pre‐initiation complexes. However, it inhibits the puromycin‐mediated release of [35S]formyl‐methionine from 70S initiation complexes in a dose‐dependent manner. Inhibition involves binding of the oxazolidinone to the large ribosomal subunit and is twice as high with 50S subunits from Gram‐positive as with those from Gram‐negative bacteria.
Characterisation of a new rubredoxin isolated from Desulfovibrio desulfuricans 27774: definition of a new family of rubredoxins
Authors: LeGall Jean , Liu Ming Y. , Gomes Cláudio M. , Braga Vera , Pacheco Isabel , Regalla Manuela , Xavier António V. , Teixeira Miguel
Published: July 2002

A new rubredoxin from the sulphate‐reducing bacterium Desulfovibrio desulfuricans ATCC 27774, grown with nitrate as terminal electron acceptor, was isolated and characterised. The protein is an 8.5 kDa monomer containing one iron atom per molecule, with a reduction potential of 25±5 mV at pH 7.6. Like the recombinant Rdl protein from D. vulgaris, expressed in Escherichia coli [Lumpio, H.L., Shenvi, N.V., Garg, R.P., Summers, A.O. and Kurtz, D.M., J. Bacteriol. 179 (1997) 4607–4615], it contains an unusual spacing of four amino acids between the first two of the iron coordinating cysteinyl residues. This difference is reflected in the structure of the iron centre, as observed by visible and EPR spectroscopies. All together, these features make these proteins the first members of a new family of rubredoxins.
Contribution of a PS1‐like element to the tissue‐ and cell‐specific expression of the human GLP‐1 receptor gene
Authors: Galehshahi Firouzeh Shoghi , Göke Burkhard , Lankat-Buttgereit Brigitte
Published: July 2002

The GLP‐1 receptor (GLP‐1R) mediates the insulinotropic effects of the incretion hormone glucagon‐like peptide 1 (7–36) amide (GLP‐1). Recently, we cloned the 5′‐flanking region of the human GLP‐1R gene. To characterize tissue‐ and cell‐specific cis‐regulatory elements, we constructed a series of 5′‐deletions of the promoter. The activity of these constructs was tested in different cell lines. An element with high homology to PS1 was found to repress GLP‐1R promoter activity in fibroblasts and pancreatic D‐cells, but was not active in pancreatic A‐ and B‐cells. PS1 was described to inhibit activation of a D‐cell‐specific enhancer. Cloning the PS1‐like element upstream a heterologous promoter (SV40) revealed that it is functionally active independently from this enhancer. Our data suggest that basal activity of the GLP‐1R promoter is silenced in a tissue‐ and cell‐specific manner by negatively acting cis‐regulatory elements, including a PS1‐like element.
The structure of the most studied DNA fragment changes under the influence of ions: a new packing of d(CGCGAATTCGCG)
Authors: Liu Jie , Malinina Lucy , Huynh-Dinh Tam , Subirana Juan A
Published: July 2002

The title oligonucleotide and many related dodecamers have been extensively studied alone and as DNA‐drug complexes. In practically all cases they were found to crystallize in the same space group, stabilized by interactions among the terminal guanine bases. Here we report new packing interactions (R3) in the presence of Ca2+. The oligonucleotides interact by placing their terminal guanines in the narrow groove of a neighbor molecule, an interaction which had never been found in dodecamers.
Contributions of the ionization states of acidic residues to the stability of the coiled coil domain of matrilin‐1
Authors: Dames Sonja A. , Kammerer Richard A. , Moskau Detlef , Engel Jürgen , Alexandrescu Andrei T.
Published: July 2002

The pK
a values of eight glutamic acid residues in the homotrimeric coiled coil domain of chicken matrilin‐1 have been determined from 2D H(CA)CO NMR spectra recorded as a function of the solution pH. The pK
a values span a range between 4.0 and 4.7, close to or above those for glutamic acid residues in unstructured polypeptides. These results suggest only small favorable contributions to the stability of the coiled coil from the ionization of its acidic residues.
Demonstration of the pathogenic effect of point mutated keratin 9 in vivo
Authors: Kobayashi Setsu , Kore-eda Satoshi , Tanaka Toshihiro
Published: July 2002

A wild type keratin 9 (K9) cDNA and a point mutated keratin 9 cDNA were injected subcutaneously into mouse skin. The hemagglutinin tag staining of the wild type K9 cDNA injected specimens mainly showed a homogeneous pattern, whereas the point mutated K9 cDNA injected specimens mainly showed a granular pattern in the suprabasal cells. Double staining of K9 and the endogenous keratin revealed the incorporation of de novo synthesized K9 into the keratin network. These results demonstrate that (1) a naked DNA transfection into mouse skin can detect the pathogenic changes of point mutated keratin in vivo and (2) the keratin 9 mutation disrupts the keratin network formation in the suprabasal cells in vivo.
The extensin multigene family responds differentially to superoxide or hydrogen peroxide in tomato cell cultures
Authors: Wisniewski Jean-Pierre , Cornille Patrick , Agnel Jean-Pierre , Montillet Jean-Luc
Published: July 2002

Changes in extensin gene expression were examined in cultured tomato cells following treatments leading to the production of activated oxygen species. Digitonin, a steroid glycoalkaloid compound, has been shown to trigger a rapid and transient production of superoxide anion, O−⋅
2. 6 h after application of 50 or 100 μM of digitonin, the accumulation of four extensin transcripts (1.5, 2.6, 4.0 and 6.1 kb) was observed. Superoxide dismutase strongly inhibited the digitonin‐mediated response, suggesting a key role of O−⋅
2 in the signalling cascade. Furthermore, cells treated with enzymatically produced O−⋅
2 generated by xanthine oxidase (0.015 U/ml) gave a similar extensin response and again, SOD exerted a strong inhibitory effect on the response. On the other hand, H2O2 (2 mM) or the enzymatic H2O2 generator, glucose oxidase (0.34 U/ml), elicited the accumulation of only three of the four transcripts (1.5, 2.6 and 4.0 kb), indicating that the corresponding genes could be regulated either by H2O2 or O−⋅
2 but that the gene encoding the 6.1 kb transcript was exclusively expressed in response to O−⋅
2. Finally, it was shown that lipid peroxidation, which was only induced when cells were exposed to H2O2, did not participate in the AOS‐mediated gene expression for extensin. It can be concluded from these results that tomato cells are able to discriminate H2O2 from O−⋅
2 and they probably sense the latter by the specific oxidation of an extracellular component.
Isolation of reductase for SoxR that governs an oxidative response regulon from Escherichia coli
Authors: Kobayashi Kazuo , Tagawa Seiichi
Published: July 2002

SoxR is a transcription factor triggered by oxidative stress in Escherichia coli. Recent evidence suggests that novel redox regulation couples oxidation state to promoter activation. We have isolated the reductase for SoxR in E. coli using an assay of NADPH‐ and SoxR‐dependent cytochrome c reductase activity. When the purified protein was incubated in an anaerobic reaction mixture containing SoxR and NADPH, the reduction of [2Fe‐2S] cluster of SoxR was observed by optical and EPR spectroscopy. Our results indicate that the purified protein serves as an NADPH‐dependent reduction system for SoxR.
A pure S=3/2 [Fe4S4]+ cluster in the A33Y variant of Pyrococcus furiosus ferredoxin
Authors: Duderstadt Randall E , Brereton Phillip S , Adams Michael W.W , Johnson Michael K
Published: July 2002

The properties of the [4Fe‐4S]2+/+ cluster in wild‐type and the A33Y variant of Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR, variable‐temperature magnetic circular dichroism (VTMCD) and resonance Raman (RR) spectroscopies. The A33Y variant involves the replacement of an alanine whose α‐C is less than 4 Å from one of the cluster iron atoms by a tyrosine residue. Although the spectroscopic results give no indication of tyrosyl cluster ligation, the presence of a tyrosine residue in close proximity to the cluster results in a 38‐mV decrease in the midpoint potential of the [4Fe‐4S]2+,+ couple and has a marked effect on the ground state properties of the reduced cluster. The mixed spin [4Fe‐4S]+ cluster in the wild‐type protein, 80% S=3/2 (E/D=0.22, D=+3.3 cm−1) and 20% S=1/2 (g=2.10, 1.87, 1.80), is converted into a homogeneous S=3/2 (E/D=0.30, D=−0.7 cm−1) form in the A33Y variant. As the first example of a pure S=3/2 [4Fe‐4S]+ cluster in a ferredoxin, this variant affords the opportunity for detailed characterization of the excited electronic properties via VTMCD studies and demonstrates that the protein environment can play a crucial role in determining the ground state properties of [4Fe‐4S]+ clusters.
Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin‐like domain
Authors: Laitinen Olli H. , Airenne Kari J. , Marttila Ari T. , Kulik Tikva , Porkka Eevaleena , Bayer Edward A. , Wilchek Meir , Kulomaa Markku S.
Published: July 2002

Sea urchin fibropellins are epidermal growth factor homologues that harbor a C‐terminal domain, similar in sequence to hen egg‐white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin‐binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp‐110‐Lys, Trp‐70‐Arg and the double mutant, were expressed in a baculovirus‐infected insect cell system. A mutant of streptavidin, Trp‐120‐Lys, was similarly expressed. The homologous tryptophan to lysine (W→K) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K
d∼10−15 M of the wild‐type tetramer down to K
d∼10−8 M for both W→K mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin. The Trp‐70‐Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast‐protein liquid chromatography assay, both W→K mutants were found to form stable dimers in solution. These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin‐binding, but may serve to secure an appropriate structure for facilitating dimerization.
Interdomain interactions within the gene 3 protein of filamentous phage
Authors: Chatellier Jean , Hartley Oliver , Griffiths Andrew D. , Fersht Alan R. , Winter Greg , Riechmann Lutz
Published: July 2002

Infection of Escherichia coli by filamentous phage fd is mediated by the phage gene 3 protein (g3p). The g3p consists of three domains (g3p‐D1, D2 and D3) linked by flexible glycine‐rich linkers. All three domains are indispensable for phage infectivity; the g3p‐D1 domain binds to the TolA receptor presumably at the inner face of the outer membrane, the g3p‐D2 domain to the F‐pilus and the g3p‐D3 domain anchors g3p to the phage coat. The N‐terminal domains g3p‐D1 and D2 interact with each other; this interaction is abrogated by binding of g3p‐D2 to the F‐pilus leading to the release of g3p‐D1 to bind to TolA. Here, using phages with deletions in g3p, we have discovered a specific interaction between the two N‐terminal domains and g3p‐D3, the C‐terminal domain of g3p. We propose that these interdomain interactions within g3p lead to a compact and stable organisation when displayed on the phage tip, but that during infection, this compact state must be unraveled.
Trophoblastic disease: Twenty years' experience at Niigata University
Authors: Kanazawa K.
Published: July 2002

The author engaged in the registration work and the clinical studies of trophoblastic disease over 20 years at Niigata University in Niigata prefecture, before transferring to University of the Ryukyus in Okinawa prefecture in 1992.

In this paper, the following four topics are reviewed and discussed. 1) the registration data of trophoblastic disease in Niigata prefecture, 2) the treatment results of gestational chorio‐carcinoma at the Niigata University Hospital, 3) a trial administration of Gn‐RH analogue in patients with gestational choriocarcinoma to suppress secretion of hCG‐like substance (hLS) which might cross‐react anti‐hCG antibodies in hCG immunoassay system, 4) the two rare cases of extrauterine gestational choriocarcinoma who were presented to the Ryukyu University Hospistal.