bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative measurement of fusion between human immunodeficiency virus and cultured cells using membrane fluorescence dequenching
Authors: Sinangil Faruk , Loyter Abraham , Volsky D.J.
Published: July 2002

Human immunodeficiency virus (HIV) was purified by sucrose gradient centrifugation and labeled with octadecylrhodamine B‐chloride (R‐18) under conditions resulting in 90% quenching of the fluorescence label. Incubation of R‐18‐labeled HIV (R‐18/HIV) with CD4‐positive CEM and HUT‐102 cells, but not with CD4‐negative MLA‐144 cells, resulted in fluorescence dequenching (DQ, increase in fluorescence) of 20–25%. Similar level of DQ was observed upon incubation of CEM cells with R‐18‐labeled Sendai virus. DQ was observed when R‐18/HIV was incubated with CD4+ cells at 37°C, but not at 4°C. Most of the increase in fluorescence occurred within 5 min of incubation at 37°C and was independent of medium pH over the range of pH 5–8. Preincubation of cells with the lysosomotropic agent NH4Cl had no inhibitory effect on DQ. Complete inhibition was observed when target cells were fixed with glutaraldehyde prior to R‐18/HIV addition. Our results demonstrate application of membrane fluorescence dequenching method to a quantitative measurement of fusion between HIV and target cell membranes. As determined by DQ, HIV penetrates into target cells by a rapid, pH‐independent, receptor‐mediated and specific process of fusion between viral envelope and cell plasma membrane, quite similar to that observed with Sendai virus.
Methyllycaconitine and (+)‐anatoxin‐a differentiate between nicotinic receptors in vertebrate and invertebrate nervous systems
Authors: Macallan David R.E. , Lunt George G. , Wonnacott Susan , Swanson Karen L. , Rapoport Henry , Albuquerque Edson X.
Published: July 2002

Specific high‐affinity binding sites for 125I‐α‐bungarotoxin and (−)‐[3H]nicotine have been measured in rat brain and locust (Schistocerca gregaria) ganglia. The binding sites for 125I‐α‐bungarotoxin had similar K
d values of 1.5 × 10−9 and 0.8 × 10−9 M for rat and locust preparations, respectively; the corresponding values for the (−)‐[3H]nicotine‐binding site were 9.3 × 10−9 and 1.7 × 10−7 M. Methyllycaconitine (MLA) potently inhibited 125I‐α‐bungarotoxin binding in both rat and locust. MLA was a less effective inhibitor of (−)‐[3H]nicotine binding whereas (+)‐anatoxin‐a was a very potent inhibitor at this site in the rat but not in the locust. These data suggest that (+)‐anatoxin‐a is a useful probe for the high‐affinity nicotine‐binding receptor in vertebrate brain, whereas MLA is a preferential probe for the subclass of receptor that binds α‐bungarotoxin.
Supercoil‐stabilized left‐handed DNA in the plasmid (dA‐dT)16 insert formed in the presence of Ni2+
Authors: Nejedlý K. , Kłysik J. , Paleček E.
Published: July 2002

The (dA‐dT)16 insert of the plasmid pAT32 was probed with diethyl pyrocarbonate (DEPC) and nuclease Bal31 in the presence of Ni2+ known to be able to induce transition to left‐handed conformation in the synthetic poly(dA‐dT)·poly (dA‐T). It has been shown that this insert in a supercoiled plasmid displays a DEPC modification pattern characteristic of left‐handed DNA under conditions not sufficient to induce a left‐handed structure in the linear plasmid and poly(dA‐dT)·poly(dA‐T).
Calcitonin gene‐related peptide‐1 (CGRP‐1) is a potent regulator of glycogen metabolism in rat skeletal muscle
Authors: Leighton Brendan , Foot Elizabeth A. , Cooper Garth G.J.S. , King Jayne M.
Published: July 2002

We investigated the effects of CGRP on glucose metabolism in intact rat skeletal muscle preparations that are largely composed of either type I (soleus) or II fibres (e.g. extensor digitorum longus (EDL) or epitrochlearis muscles). CGRP‐1 inhibited insulin‐stimulated glycogen synthesis in both soleus and EDL muscle preparations. Rat CGRP‐1 was a potent stimulator of glycogenolysis only in muscles composed of type II fibres, which depend on high rates of glycogenolysis to produce high power outputs. These results may provide the basis for understanding how CGRP regulate glycogenolysis in type II fibres in vivo.
Polyphosphates as a source of high energy phosphates in yeast mitochondria: A 31P NMR study
Authors: Beauvoit Bertrand , Rigoulet Michel , Guerin Bernard , Canioni Paul
Published: July 2002

Suspensions of purified yeast mitochondria were analyzed under bubbling oxygen by 31P NMR at 161.9 MHz. The recorded spectra indicate that polyphosphates (poly(P)) are present in mitochondrial preparations. These poly(P) further characterized by the NMR study of mitochondrial perchloric acid extracts have an average chain length of 14 ± 1 residues per chain and correspond to 10% of the total content of cellular poly(P) detected by NMR. The stability of mitochondrial poly(P) was increased by the presence of oligomycin, suggesting that this compound may play a role in the energetic metabolism of yeast mitochondria.
Isolation and expression in Saccharomyces cerevisiae of a gene encoding an α‐amylase from Schwanniomyces castellii
Authors: Abarca Dolores , Fernández-Lobato María , Claros Manuel Gonzalo , Jiménez Antonio
Published: July 2002

A gene (SWA1) encoding an α‐amylase activity from Schwanniomyces castellii has been cloned and expressed, via yeast cloning vector YEp13, in Saccharomyces cerevisiae. By using a riboprobe which is internal to the SWA1 gene, a 1.55 kb transcript was detected in the poly(A)+ RNA from both Sw. castellii and a S. cerevisiae clone harboring the SWA1 gene. This transcript should, therefore, correspond to the SWA1 gene. In addition, the DNA strand determining the α‐amylase activity has been defined. Transcription of the SWA1 gene appears to be highly regulated in Sw. castellii, whereas it is constitutive in the S. cerevisiae harboring this gene.
Synthesis and evaluation of optically pure [3H]‐(+)‐pentazocine, a highly potent and selective radioligand for σ receptors
Authors: de Costa Brian R. , Bowen Wayne D. , Hellewell Susan B. , Walker J.Michael , Thurkauf Andrew , Jacobson Arthur E. , Rice Kenner C.
Published: July 2002

Tritium‐labeled (+)‐pentazocine ([3H]‐1b) of specific activity 26.6 Ci/mmol was synthesized in 3 steps starting with (+)‐normetazocine (2) of defined optical purity. [3H]‐1b has been characterized as a highly selective ligand for labeling of σ receptors. Competition data revealed that [3H]‐1b could be displaced from guinea pig brain membrane preparations with a number of commonly used σ receptor ligands. [3H]‐1b exhibited saturable, enantioselective binding with a K
d of 5.13±0.97 nM and a B
max of 1146±122 fmol/mg protein. Phencyclidine (PCP) displaced [3H]‐1b with low affinity while MK‐801 was inactive, thus indicating insignificant activity at the PCP‐binding site; apomorphine failed to displace [3H]‐1b indicating lack of dopamine receptor cross‐reactivity. Since the affinity of [3H]‐1b is about 6 times that of the two commonly employed σ ligands ((+)‐3‐[3H]PPP and [3H]DTG) and since it is more selective for σ receptors than the benzomorphan [3H]SKF‐10,047, it represents the first example of a highly selective benzomorphan based σ receptor ligand. [3H]‐1b should prove useful for further study of the structure and function of σ receptors.
Identification of a specific receptor for interleukin‐1 on rat bone marrow cells
Authors: Mizuno Keiko , Nakai Satoru , Ohmoto Yasukazu , Hirai Yoshikatu
Published: July 2002

Interleukin‐1 receptor; Interleukin‐1α; Interleukin‐1β (Rat bone marrow cell. Mouse BALB/3T3 cell)
Molecular cloning of the mature E1b‐β subunit of human branched‐chain α‐keto acid dehydrogenase complex
Authors: Chuang Jacinta L. , Cox Rody P. , Chuang David T.
Published: July 2002

We have isolated a cDNA encoding the E1b‐β subunit of the human branched‐chain α‐keto acid dehydrogenase complex. The human E1b‐β cDNA is 1401 base pairs in length. It encodes the entire mature E1b‐β subunit consisting of 342 amino acid residues, and a mitochondrial targeting presequence of 31 residues. The calculated molecular mass of the mature human E1b‐β subunit is 37 851 Da, and the calculated isoelectric point is pH 5.18. A hydropathy plot shows that the human E1b‐β subunit is highly hydrophobic. Northern blot analysis shows that the human E1b‐β mRNA is approximately 1.4 kb in size. It is present at the normal level in fibroblasts from two unrelated maple syrup urine disease patients.
Stimulatory effect of ammonium sulfate at high concentrations on the aminoacylation of tRNA and tRNA‐like molecules
Authors: Florentz Catherine , Kern Daniel , Giege Richard
Published: July 2002

The influence of various salts on the aminoacylation of tRNAVal and the tRNA‐like structure from turnip yellow mosaic virus RNA by yeast valyl‐tRNA synthetase has been studied. As expected, increasing the concentration of salts inhibits the enzymatic reaction. However, in the presence of high concentration of ammonium sulfate, and only this salt, the inhibitory effect is suppressed. Under such conditions, the aminoacylation becomes comparable to that measured in the absence of salt. It was shown that ammonium sulfate affects both the catalytic rate of the reaction and the affinity between valyl‐tRNA synthetase and the RNAs. Because the affinity between the partners in the complex is increased when the concentration of the salt is high, it is suggested that hydrophobic effects are involved in tRNA/synthetase interactions.
Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells
Authors: Nakano Tohru , Ohara Osamu , Teraoka Hiroshi , Arita Hitoshi
Published: July 2002

Two potent inflammatory mediators, interleukin 1 (IL‐1) and tumor necrosis factor (TNF) as well as lipopolysaccharide (LPS) increased group II phospholipase A2 (PLA2) mRNA levels, which resulted in enhanced secretion of the PLA2 enzyme from rat smooth muscle cells. cAMP‐elevating agents also stimulated the release of PLA2 and increased the mRNA, but IL‐1, TNF and LPS did not affect cAMP levels. Furthermore, the effects of TNF and cAMP‐elevating agents were not additive but synergistic. Therefore, we concluded that the level of rat group II PLA2 mRNA is controlled at least by two distinct mechanisms, one involves cAMP and the other is mediated by TNF, IL‐1 and LPS. This study also suggests important roles of group II PLA2 in pathogenesis of vascular inflammation.
Identification of monoclonal antibody binding domains of Na+,K+‐ATPase by immunoelectron microscopy
Authors: Ting-Beall H.Ping , Beall Harry C. , Hastings David F. , Friedman Mark L. , Ball William J.
Published: July 2002

Treatment of purified preparations of porcine Na+,K+‐ATPase with phospholipase A2, MgCl2 and NaVO3 leads to the formation of two‐dimensional crystals exclusively in a dimeric configuration. Two‐dimensional computer‐averaged projections of the electron microscopy images of the crystalline enzyme with bound Fab fragments of monoclonal antibody M10‐P5‐C11 were accomplished using image enhancement software and showed that the antibody fragments caused only a modest increase in the unit cell size, while reducing the extent of asymmetry of the two promoters in each unit cell. The digital imaging also showed that the antibody's epitope on the α subunit resides on the ‘lobe’ or ‘hook’ region of the intracellular portion of the enzyme. Since functional studies indicate that M10‐P5‐C11 binds near or between the ATP binding site and the phosphorylation site, this visualized ‘lobe’ region of α may comprise the catalytic site. In addition, the binding of another inhibitory antibody, 9‐A5, has been found to prevent crystal formation and the presence of the carbohydrate sugars on the enzyme's β subunit shown to be required for crystal formation.
The biological activity of retinoids in melanoma cells
Authors: Redfern Christopher F.F. , Daly Ann K. , Latham Julie A.E. , Todd Carole
Published: July 2002

The expression of mRNA for retinoic acid receptor beta (RAR‐β was induced by all trans‐retinoic acid in murine S91 melanoma cells. The induction of RAR‐β was dose‐dependent, rapid and insensitive to cycloheximide. Both 13‐cis‐retinoic acid and 3,4‐didehydro‐all trans‐retinoic acid also induced expression of RAR‐β but were only effective at concentrations 100‐fold greater than all trans‐retinoic acid. The expression of RAR‐α and RAR‐γ was unaffected by retinoic acid.
Carnitine palmitoyltransferase (CPT2) from liver mitochondrial inner membrane becomes inhibitable by malonyl‐CoA if reconstituted with outer membrane malonyl‐CoA binding protein
Authors: Ghadiminejad Iraj , Saggerson E.David
Published: July 2002

A soluble extract was obtained on treatment of rat liver mitochondrial outer membranes with cholate which bound (14C]malonyl‐CoA but was essentially free of carnitine palmitoyltransferase (CPT) activity. Extraction of mitochondrial inner membranes with cholate readily solubilized a CPT activity which was insensitive to malonyl‐CoA. Combination of these two extracts caused the CPT derived from inner membranes to become inhibitable by malonyl‐CoA.
Construction and characterization of a recombinant tripartite enzyme, galactose dehydrogenase/β‐galactosidase/galactokinase
Authors: Ljungcrantz Peter , Bülow Leif , Mosbach Klaus
Published: July 2002

The in‐frame gene fusion between 3 enzymes, galactose dehydrogenase, β‐galactosidase and galactokinase, is described. The purified artificial tripartite enzyme displayed all three enzymic activities. Two major forms of the hybrid protein were found, consisting of 4 and 8 subunits respectively, but other forms could also be identified. Each subunit was made up of one monomer each of galactose dehydrogenase, β‐galactosidase and galactokinase. Proximity effects exhibited by the hybrid enzyme could be demonstrated using [14
C]galactose as a reporter molecule.
Site‐specific mutagenesis of human interleukin‐6 and its biological activity
Authors: Nishimura Chiaki , Futatsugi Kensuke , Yasukawa Kiyoshi , Kishimoto Tadamitsu , Arata Yoji
Published: July 2002

Amino acid substitutions of human interleukin‐6 (IL‐6) were performed. Single substitution Met162 → Ala and double substitutions Leu159, 166 → Val resulted in a significant decrease of IL‐6 activity in the production of immunoglobulin (lg) from B‐cells. Single substitution Leu166→Val or Leu159→Val gave a slight or no significant decrease in the Ig‐induction activity, respectively. The receptor‐binding activity of each IL‐6 mutant was also examined. It was observed that the decrease of the receptor‐binding activity was generally in parallel with that of the Ig‐induction activity. We therefore suggest that hydrophobic side‐chains existing in Met162, Leu159, and Leu164 are significantly involved in the receptor‐binding of IL‐6.
Isolation of high‐molecular‐weight C‐type natriuretic peptide from the heart of a cartilaginous fish (European dogfish, Scyliorhinus canicula)
Authors: Suzuki Reiko , Takahashi Akiyoshi , Hazon Neil , Takei Yoshio
Published: July 2002

A high‐molecular‐weight form of C‐type natriuretic peptide (CNP) was isolated from both cardiac atria and ventrieles of European dogfish, Scyliorhinus canicula, and its primary structure was determined. The peptide consists of 115 amino acid residues, in which the C‐terminal 22 residues show high homology to CNPs identified to date. This is the first direct evidence for the presence of natriuretic peptide in the cartilaginous fish, and for the presence of CNP in an organ other than the brain.
Okadaic acid inhibits amylase exocytosis from parotid acini stimulated by cyclic AMP
Authors: Takuma Taishin , Ichida Tokuro
Published: July 2002

To evaluate the role of protein phosphorylation in amylase exocytosis, we studied the effects of okadaic acid, a potent inhibitor of protein phosphatase types I and 2A, on amylase release and protein phosphorylation in rat parotid acini. Although okadaic acid by itself weakly stimulated amylase release, it did not potentiate amylase release stimulated by half‐maximum doses of isoproterenol or cAMP, and markedly inhibited their maximum effects. Okadaic acid dose‐dependently increased cAMP‐independent phosphorylation of some proteins and enhanced cAMP‐dependent phosphorylation of 21‐ and 26‐kDa proteins. These results indicate that increase in protein phosphorylation does not necessarily enhance the exocytosis of amylase from parotid acini.
Suppression by staurosporine of Ca2+‐mobilization triggered by ligation of antigen‐specific receptors on T and B lymphocytes An essential role of protein tyrosine kinase in the signal transduction
Authors: Yamashita Yumi , Hasegawa-Sasaki Hiroko , Sasaki Terukatsu
Published: July 2002

It is known that the receptor for platelet‐derived growth factor (PDGF) activates phospholipase C (PLC) by phosphorylating the γl isoform of PLC with the receptor protein‐tyrosine kinase (PTK), whereas a guanine nucleotide‐binding protein participates as a transducer in the PLC activation through the receptors for vasopressin, bombesin and prostaglandin F2α (PGF2α). We have shown in a rat fibroblast line that staurosporine is a potent PTK inhibitor capable of clearly discriminating the two types of receptor‐stimulated Ca2+ mobilization and, by inference, PLC activation: the response triggered by PDGF was completely inhibited, whereas the responses triggered by vasopressin, bombesin and PGF2α were not affected at all. The Ca2+ mobilization in human T and B cell lines induced by anti‐CD3 and anti‐immunoglobulins (Ig) was completely suppressed by staurosporine. The results indicate that the PTK activity plays an essential role in the PLC activation through the T cell receptor/CD3 complex and through membrane Ig.
Acidic pentapeptide phosphorylated in vitro by calf thymus protein kinase NII binds to DNA in the presence of Mg2+ cations
Authors: Chillemi F. , Lugaro G. , Boari D. , Cardellini E. , Bramucci M. , Miano A. , Amici D. , Gianfranceschi G.L. , Durban E.
Published: July 2002

The pentapeptide pyroGlu‐Ala‐Glu‐Ser‐Asn has been synthetized and phosphorylated in vitro at level of serine by protein kinase NII isolated from calf thymus chromatin. It is noteworthy that the calf thymus kinase NII shows a remarkable affinity for this peptide. The [32P]peptide is able to bind to several DNAs in the presence of Mg2+ (λ phage, calf thymus, pBR540 plasmid). This binding appears not specific with regard to the type of DNA and its base sequence. These data support the hypothesis that phosphorylated acidic domains of nuclear nonhistone proteins could bind directly to DNA in the presence of Mg2+ cations
An hsp70 homolog is encoded on the plastid genome of the red alga, Porphyra umbilicalis
Authors: Reith Michael , Munholland Janet
Published: July 2002

A PCR experiment using Porphyra umbilicalis DNA as the template and degenerate oligonucleotides representing conserved regions of hsp70 amino acid sequences generated a 1 kb product that hybridized exclusively to the plastid DNA of this red alga. DNA sequencing of two continguous EcoRI plastid DNA clones revealed a 620 amino acid open reading frame with 71% identity to the dnaK gene of the cyanobacterium. Synechocystis 6803. Northern hybridization experiments detected a 2.3 kb transcript that is present in control (15°C) cultures and increases approximately 7‐fold upon heat shock (75 minutes at 30°C).
Genomic organization and analysis of the 5′ end of the porcine ryanodine receptor gene (ryr1)
Authors: Brenig Bertram , Brem Gottfried
Published: July 2002

In this study we describe the isolation of genomic clones of the 5′ region of the porcine ryanodine receptor gene, a candidate for malignant hyperthermia in pigs and humans. The recombinants were isolated from a porcine liver, genomic DNA library in phage EMBL3A after screening with PCR amplified DNA fragments. The exon/intron structure of the ryanodine receptor gene was determined by DNA sequencing. Based on the sequence data it was possible to develop a simple test for the detection of malignant hyperthermia susceptible and normal pigs.
Serum calcium‐decreasing factor (caldecrin) from porcine pancreas has proteolytic activity which has no clear connection with the calcium decrease
Authors: Tomomura Akito , Fukushige Tomoko , Noda Takashi , Noikura Takenori , Saheki Takeyori
Published: July 2002

We purified a serum calcium‐decreasing factor, which showed chymotrypsin‐like protease activity, from porcine pancreas to homogeneity, The factor administered to mice intravenously at a dose of 20 μg/kg b.w. decreased serum calcium by 15%. Treatment of the factor with the serine protease inhibitor, PMSF, caused a leftward shift in the dose—response curve, showing strengthened activity. It also caused a decrease in serum calcium and hydroxyproline levels in rats. At a dose of 10 ng/ml, the factor inhibited 45Ca release from cultured fetal long bone stimulated by parathyroid hormone (PTH) and PTH‐related protein, but not by interleukin‐1α, prostaglandin E1 and 1,25‐dihydroxy vitamin D3. No other well‐known pancreatic proteases had these effects. In view of the results of experiments using protease inhibitor and pancreatic proteases, and in view of the specificity of this factor in vitro, we propose that the factor exerts its serum calcium‐decreasing activity most probably not through proteolytic degradation of PTH, but through an inhibition of PTH action on bones by a yet undefined mechanism.
A complex between replication factor A (SSB) and DNA helicase stimulates DNA synthesis of DNA polymerase α on double‐stranded DNA
Authors: Zhang Suisheng , Grosse Frank
Published: July 2002

A helicase‐like DNA unwinding activity was found in highly purified fractions of the calf thymus single‐stranded DNA binding protein (ctSSB). also known as replication protein A (RP‐A) or replication factor A (RF‐A). This activity depended on the hydrolysis of ATP or dATP, and used CTP with a lower efficiency. ctSSB promoted the homologous DNA polymerase α to perform DNA synthesis on double‐stranded templates containing replication fork‐like structures. The rate and amount of DNA synthesis was found to be dependent on the concentration of ctSSB. At a 10‐fold mass excess of ctSSB over double‐stranded DNA, products of 200–600 nucleotides in length were obtained. This comprises or even exceeds the length of a eukaryotic Okazaki fragment. The ctSSB‐associated DNA helicase activity is most likely a distinct protein rather than an inherent property of SSB, as inferred from titration experiments between SSB and DNA. The association of a helicase with SSB and the stimulatory action of this complex to the DNA polymerase α‐catalyzed synthesis of double‐stranded DNA suggests a cooperative function of the three enzymatic activities in the process of eukaryotic DNA replication.
Activation of protein kinase C attenuates the cyclic GMP responses to C‐type natriuretic peptide in cultured mouse astrocytes
Authors: Yeung V.T.F. , Ho S.K.S. , Cockram C.S. , Lee C.M. , Nicholls M.G.
Published: July 2002

C‐type natriuretic peptide (CNP), a recently discovered natriuretic peptide, has a potent stimulatory effect on cyclic GMP (cGMP) formation in cultured mouse astrocytes. Pretreatment of astrocytes with phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C (PKC). attenuated CNP‐induced cGMP responses in a dose‐dependent manner, with a half‐maximal inhibitory concentration of 6 nM, whereas the inactive phorbol ester analog, 4α‐phorbol 12,13‐didecanoate, was without effect. In the presence of staurosporine, a PKC inhibitor, the inhibitory effect of PMA on CNP‐stimulated cGMP production was reversed. These results suggest that PKC is an inhibitory modulator of CNP‐stimulated cGMP responses in astrocytes and that CNP may interact with neuropeptides which stimulate PKC.
Receptor phage
Authors: Scarselli Elisa , Esposito Gloria , Traboni Cinzia
Published: July 2002

In this paper we demonstrate that phage display technology is a suitable system for studying the interaction between the high‐affinity receptor for IgE (FcϵRI) and IgE. The α subunit extracellular domains of the human receptor were expressed on the surface of filamentous phage M 13 fused to the carboxyl‐terminal part of the gene III protein (pIII). Two constructs were made, the first with both the Ig‐like domains of the receptor α chain and the second with only the C‐terminal domain. The fusion genes were cloned in a phagemid vector to display monovalently the receptor on the phage surface. Our results indicate that the α receptor expressed on the phage is able to interact with IgE as demonstrated by an ELISA assay. In addition, by using the same system, we show that a single domain of the α receptor is sufficient for the interaction with IgE although with a binding affinity lower than that of the two‐domain receptor.
Unusual kinetic behavior predicted for α‐keto acid dehydrogenase complexes
Authors: Goldstein B.N. , Selivanov V.A.
Published: July 2002

A novel regulation type, which may be observed as an unusual kinetic ‘cooperativity’, is predicted for the α‐keto acid dehydrogenase complexes. The inter‐relationship of this regulation with the well‐known regulatory effect of enzyme phosphorylation is discussed.
Expression in mammalian cells, purification and characterization of recombinant human pancreatic ribonuclease
Authors: Russo Nello , de Nigris Mena , Ciardiello Antonietta , Di Donato Alberto , D'Alessio Giuseppe
Published: July 2002

A synthetic cDNA coding for human pancreatic RNase, equipped with a secretion signal sequence, was cloned and stably expressed in Chinese hamster ovary cells. The recombinant RNase, secreted into the culture medium, was purified and characterized. It was found to be indistinguishable, by structural and catalytic parameters, from the enzyme isolated from human pancreas. Furthermore, the glycosylated forms were separated from the non‐glycosylated form. Up until now, human RNases have been isolated only in small amounts from autoptic specimens. This has hindered the exploitation of a human RNase for the construction of immunotolerated immunotoxins. On the other hand, the availability of an effective system for the expression of a human RNase may render feasible the transfer, by protein engineering, of the interesting pharmacological actions of non‐human RNase [1993 Trends Cell Biol. 3, 106‐109] to an immunotolerated, human RNase.
Mechanism of Na+/H+ exchange by Escherichia coli NhaA in reconstituted proteoliposomes
Authors: Dibrov P.A. , Taglicht D.
Published: July 2002

Purified NhaA, a Na+/H+ antiporter from Escherichia coli, reconstituted into proteoliposomes was used to study partial reactions catalyzed by this protein. Homologous Na+/Na+ exchange as well as Na+/Li+ exchange via NhaA were detected by monitoring the effects of external Li+ and Na+ ions on the ΔpH‐driven sodium uptake into NH4 Cl‐loaded vesicles. Furthermore, a sodium counterflow reaction was demonstrated in proteoliposomes preloaded with non‐radioactive Na+ and placed into the experimental buffer containing low amounts of 22Na+ under experimental conditions when both components of protonmotive force generated by the antiporter. ΔΨ and ΔpH, were dissipated by corresponding ionophores. The apparent K
m for sodium counterflow is 1.1 mM, and V
max is 80 of protein. External Na+ accelerates the downhill efflux of 22Na+ suggesting that the translocation of the Na+loaded form of the carrier is faster than the rest of the catalytic cycle.
Identification of MIM23, a putative component of the protein import machinery of the mitochondrial inner membrane
Authors: Dekker Peter J.T. , Keil Petra , Rassow Joachim , Maarse Ammy C. , Pfanner Nikolaus , Meijer Michiel
Published: July 2002

A screening for yeast mutants impaired in mitochondrial protein import led to the identification of two genes (MPI1 and MPI2) encoding the essential components MIM44 and MIM17 of the inner membrane import machinery. We analyzed twelve additional mutants obtained in the screening and found two further complementation groups. One group represents mutants of SSC1, the gene encoding mitochondrial hsp70, an essential matrix protein required for protein import across the inner membrane. The second complementation group represents mutants of a new gene (MPI3) encoding a 23 kDa integral inner membrane protein (MIM23). MIM23 is synthesized without a presequence, and its import to the inner membrane requires a membrane potential. MIM23 contains a domain homologous to half of MIM17. We speculate that MIM23 is a new member of the protein import machinery of the mitochondrial inner membrane.
Calcium‐induced degradation of the Inositol (1,4,5)‐trisphosphate receptor/Ca2+‐channel
Authors: Magnusson Andres , Haug Lise Sofie , Walaas S.Ivar , Østvold Anne Carme
Published: July 2002

Ca2+‐induced degradation of the neuronal inositol (1,4,5)‐trisphosphate receptor, a protein which regulates Ca2+‐release from intracellular stores, has been examined. The IP3‐receptor, immunopurified from rat cerebellum, appeared to be an excellent substrate for purified Ca2+‐activated neutral protease (calpain). Incubation of membranes or immunopurified IP3‐receptor with Ca2+ and cerebellar cytosol also resulted in degradation of the receptor. Two main fragments with approximate molecular masses of 130 and 95 kDa were generated, both of which appeared to derive from the carboxyterminal Ca2+‐channel‐containing part of the protein. These data suggest that activation of the IP3‐receptor, by causing increases in intracellular [Ca2+], might result in degradation of the N‐terminal, IP3‐binding part of the receptor.
Use of reference libraries and hybridisation fingerprinting for relational genome analysis
Authors: Hoheisel Jörg D. , Lehrach Hans
Published: July 2002

The concept of relational genome analysis by hybridisation has been developed into a working system. Various genomic and cDNA libraries have been generated and are distributed via a reference system. Analysis procedures have been tested successfully in the mapping of the entire Schizosaccharomyces pombe genome. In another test‐case for their refinement, analyses on the Drosophila genome are well under way. Human and mouse libraries are being studied on all levels, from generating YAC maps to partially sequencing representative cDNA libraries. The automation of the involved processes and the development of improved image detection and analysis are well advanced.
Crystallization and preliminary crystallographic analysis of trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease
Authors: Krauth-Siegel R.Luise , Sticherling Christian , Jöst Ingrid , Walsh Christopher T. , Pai Emil F. , Kabsch Wolfgang , Lantwin Christina B.
Published: July 2002

Trypanothione reductase from Trypanosoma cruzi is the most promising target molecule for the rational design of a specific drug against Chagas' disease. The recombinant protein was purified in a single Chromatographie step and crystallized. Two crystal forms suitable for X‐ray diffraction analysis were obtained. Tetragonal crystals (a = b = 87.4 Å, c = 152.3 Å) were grown from 30% polyethylene glycol (average M
r = 8,000) in the presence of 0.2% β‐n‐octylglucoside (space group either P42 with one dimer or P4222 with one monomer in the asymmetric unit). Monoclinic crystals (space group P2, a = 136.3 Å, b = 91.1 Å, c = 126.0 Å, β = 94°) were grown from 1.2 M sodium citrate in the presence of 2% octanoyl‐N‐methyl‐glucamide. They contain two dimers of the enzyme in the asymmetric unit; both crystal forms diffract to 3 Å resolution.
Mosaic expression of membrane peptidases by confluent cultures of Caco‐2 cells
Authors: Howell Steven , Brewis Ian A. , Hooper Nigel M. , Kenny A.John , Turner Anthony J.
Published: July 2002

The cell‐surface expression of endopeptidase‐24.11 (EC 3.4.24.11) on Caco‐2 cells cultured to confluency is markedly heterogeneous unlike that of dipeptidylpeptidase IV (EC 3.4.14.5). Here we have investigated the cell‐surface expression of three other ectopeptidases: angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase N (EC 3.4.11.2) and aminopeptidase W (EC 3.4.11.16). We show by indirect immunofluorescent staining that these three enzymes are present on the surface of some cells but not on others. However, these enzymes were detected in the majority of detergent‐permeabilised Caco‐2 cells indicating the presence of intracellular pools of these enzymes. This suggests that there may either be differential regulation of apical transport for these peptidases or that they recycle at different rates.
The aromatic nature of residue 66 of the 11‐kDa subunit of ubiquinol‐cytochrome c oxidoreductase of the yeast Saccharomyces cerevisiae is important for the assembly of a functional enzyme
Authors: Hemrika Wieger , Lobo-Hajdu Gisele , Berden Jan A. , Grivell Leslie A.
Published: July 2002

Transformation of multi‐ and single‐copy plasmids carrying a mutated version (LTN2, region 66‐YWYWW‐70 replaced by SASAA) of QCR8, the gene encoding the 11‐kDa subunit of ubiquinol‐cytochrome c oxidoreductase of Saccharomyces cerevisiae, to a QCR80 strain indicated the importance of this aromatic region for the assembly of a functional enzyme. Sequencing of plasmids giving spontaneous restoration of growth to some colonies among the single‐copy LTN2 transformants showed that changing the sequence SASAA into the sequence FASAA could, to a large extent, overcome the observed assembly defect, indicating the importance of the aromatic nature of residue 66.
Activation and inactivation of thyroid hormone by type I iodothyronine deiodinase
Authors: Moreno Maria , Berry Marla J. , Horst Claus , Thoma Rudy , Goglia Fernando , Harney John W. , Larsen P.Reed , Visser Theo J.
Published: July 2002

The prohormone thyroxine (T4) is activated by outer ring deiodination (ORD) to 3,3′,5‐triiodothyronine (T3) and both hormones are degraded by inner ring deiodination (IRD) to 3,3′,5′‐triiodothyronine (rT3) and 3,3′‐diiodothyronine, respectively. Indirect evidence suggests that the type I iodothyronine deiodinase (ID‐I) in liver has both ORD and IRD activities, with preference for rT3 and sulfated iodothyronines as substrates. To establish this, we have compared the ORD of rT3 and IRD of T3 and T3 sulfate by homogenates of cells transfected with rat ID‐I cDNA and by rat liver microsomes. In both preparations rT3 is the preferred substrate, while deiodination of T3 is markedly accelerated by its sulfation. Kinetic analysis provided similar K
m and V
max values in cell homogenates and liver microsomes. These data demonstrate unequivocally that ID‐I is capable of both activating and inactivating thyroid hormone by ORD and IRD, respectively.
Certain phosphoramidate derivatives of dideoxy uridine (ddU) are active against HIV and successfully by‐pass thymidine kinase
Authors: McGuigan Christopher , Bellevergue Patrice , Sheeka Hendrika , Mahmood Naheed , Hay Alan J.
Published: July 2002

As part of our effort to deliver masked phosphates inside living cells we have discovered that certain phosphate triester derivatives of the inactive nucleoside analogue, dideoxy uridine (ddU) are inhibitors of HIV replication at μM levels. Moreover, we note that certain phosphoramidate derivatives retain their activity in thymidine kinase‐deficient cells, which indicates that they do indeed act by intracellular release of the free nucleotide, and that they successfully by‐pass the nucleoside kinase. The increased structural freedom in drug design which this allows may have implications for dealing with the emergence of resistance and may stimulate the discovery of improved therapeutic agents.
Activation of the alternative pathway of complement by apoptotic Jurkat cells
Authors: Matsui Hiroyuki , Tsuji Shoutaro , Nishimura Hitosi , Nagasawa Shigeharu
Published: July 2002

Jurkat T cells die of apoptosis upon exposure to anti‐Fas mAb. Here we show that although the alternative complement pathway generally does not attack homologous cells, anti‐Fas‐induced apoptotic Jurkat T cells were attacked antibody‐independently by the alternative pathway of human complement and opsonized with iC3b, which is a ligand of the complement receptor type 3 (CR3) of phagocytes. These results suggest that apoptotic cells become the targets of the homologous alternative complement pathway, which facilitates the clearance of apoptotic cells by phagocytes.
Molecular cloning of the second major allergen, Cry j II, from Japanese cedar pollen
Authors: Namba Motoshi , Kurose Mayumi , Torigoe Kakuji , Hino Katsuhiko , Taniguchi Yoshifumi , Fukuda Shigeharu , Usui Mitsuko , Kurimoto Masashi
Published: July 2002

Cloning of a cDNA from Cry j II, the second major allergen from Japanese cedar (Cryptomeria japonica) pollen, is described. An isolated Cry j II cDNA contained an open reading frame coding for 514 amino acid residues. The mature Cry j II protein consisted of 388 amino acid residues (R46—S433). According to a homology analysis, no amino acid sequence homology was observed between Cry j II and Cry j I, another major allergen. But Cry j II showed homology with polygalacturonase (PG) derived from tomato (40% identity) at the amino acid level. The sequence information can potentially be used to devise an effective course of immunotherapy for Japanese cedar pollinosis.
A dual effect of arachidonic acid on Ca2+ transport systems in lymphocytes
Authors: Khodorova A.B. , Astashkin E.I.
Published: July 2002

A sustained increase of cytoplasmic Ca2+ concentration ([Ca2+]i) in lymphocytes was induced by a potent inhibitor of the intracellular Ca2+‐pump 2,5‐di‐(tert‐butyl)‐1,4‐hydroquinone (BHQ) that effectively depletes Ca2+ stores and produces Ca2+ influx. The addition of low concentrations of arachidonic acid (AA) (less than 10 μM) in the presence of a BHQ‐induced Ca2+ response produced a [Ca2+]i decrease. At the same time at higher concentrations, AA induced an additional increase of [Ca2+]i. The unidirectional uptake of Mn2+ into fura‐2 loaded thymocytes was used to examine the effects of AA and BHQ on Ca2+ permeability of the plasma membrane. Judging by the rate of Mn2+‐induced fura‐2 fluorescence quenching, AA causes a concentration‐dependent increase of the divalent cations' permeability both in the control solution and in the presence of BHQ. These results allowed us to suggest that the decrease of BHQ‐induced [Ca2+]i stationary levels caused by low AA concentrations is due to the enhancement of Ca2+ extrusion from the cell, whereas the additional [Ca2+]i increase at high AA concentrations results from an elevation of cell membrane Ca2+ permeability.
Hydrogen peroxide preferentially enhances the tyrosine phosphorylation of epidermal growth factor receptor
Authors: Gamou Shinobu , Shimizu Nobuyoshi
Published: July 2002

We found that hydrogen peroxide (H2O2) enhances EGF receptor tyrosine phosphorylation in intact cells as well as solubilized membrane of an EGF receptor hyperproducing cell line NA. An antioxidant MnCl2 effectively inhibited this enhancement. Interestingly, overall phosphorylation of the EGF receptor enhanced by H2O2 was half that of the EGF‐enhanced phosphorylation when the receptor immunoprecipitated from [32P]ortho‐phosphate‐labeled cells was examined. Tryptic phospho‐peptide mapping of these receptors revealed that EGF enhanced the phosphorylation on five specific residues including serine 671, 1,046 and 1,047, threonine 669 and tyrosine 1,173, whereas H2O2 enhanced the phosphorylation remarkably on tyrosine 1,173 and three other residues and only moderately on serine 1,046 and 1,047 and threonine 669. Thus, H2O2 preferentially enhances the tyrosine phosphorylation of EGF receptor through oxidant stress.
Abundant expression of thromboxane synthase in rat macrophages
Authors: Tone Yoshinori , Miyata Atsuro , Hara Shuntaro , Yukawa Susumu , Tanabe Tadashi
Published: July 2002

The cloned cDNA for rat thromboxane (TX) synthase with a size of 1851 bp contained a 1599‐bp open reading frame which encoded a 533‐amino acid protein sharing 79.7% identity with human TX synthase. RNA blot analysis was carried out with rat cells and tissues. Rat peritoneal macrophages most abundantly expressed mRNA for TX synthase, and its level was not changed by in vivo stimulation of casein. Bone marrow, spleen, lung and thymus also expressed the TX synthase gene. These findings suggest the possibility that TXA2 plays a role in the immune system.
On the tertiary structure of the Citrus ichangensis satellite DNA
Authors: Beridze T. , Tsirekidze N. , Turishcheva M.S.
Published: July 2002

A hexamer of the repeating unit of Citrus ichangensis satellite DNA was cloned. Polyacrylamide gel electrophoresis demonstrated that the shape of cloned hexamer is other than linear. As the computing of tertiary coordinates made by Eckdahl and Anderson's BEN program proved, the hexamer is a solenoid consisting of two turns termed coiled double helix (CDH)‐form. An electron microscopic analysis revealed small diameter circles in the hexamer under investigation. Unlike the hexamer control molecules are s‐shaped. It is concluded that the CDH‐form is a characteristic of the satellite DNA of Citrus ichangensis.
Spectroscopic characterization of PS I core complexes from thermophilic Synechococcus sp
Authors: Lüneberg Jürgen , Fromme Petra , Jekow Petra , Schlodder Eberhard
Published: July 2002

Monomeric and trimeric PS I complexes missing the three stromal subunits E,C and D (termed PS I core complexes) were prepared from the thermophilic cyanobacterium Synechococcus sp. by incubation with urea. The subunits E,C and D are sequentially removed. In the monomeric PS I the subunit C is removed with a half life of approx. 5 min. This is about eight times faster than in the trimeric PS I complex. In parallel with the removal of the FAB
containing subunit C the reduction kinetics of P700+ changed from a half life of about 25 ms to about 750 μs. The partner of P700+ in the 750 μs charge recombination was identified to be FX by the difference spectrum of this phase. There are some minor differences in the spectra of trimeric and monomeric PS I core complexes. At 77K the forward electron transfer from A −
1 to FX is blocked in the major fraction of the PS I core complexes and P700+A−
1 recombines with a half life of about 220 μs. In the remaining fraction P700+FX
− is formed and decays with a half life of approx. 10 ms at 77 K. The kinetics of the forward electron transfer from A−
1 to the iron‐sulfur‐clusters was measured in the native PS I and the corresponding core complexes. The reoxidation kinetics of A−
1 are identical in both cases (t
12 = 180 ns). We conclude that Fx is an obligatory intermediate in the normal forward electron transfer.
Identification and characterization of a primary antibacterial domain in CAP18, a lipopolysaccharide binding protein from rabbit leukocytes
Authors: Tossi Alessandro , Scocchi Marco , Skerlavaj Barbara , Gennaro Renato
Published: July 2002

Secondary structure prediction studies on CAP18, a lipopolysaccharide binding protein from rabbit granulocytes, identified a highly cationic, 21‐residue sequence with the tendency to adopt an amphipathic α‐helical conformation, as observed in many antimicrobial peptides. The corresponding peptide was chemically synthesized and shown to exert a potent bacterieidal activity against both Gram‐negative and Gram‐positive bacteria, and a rapid permeabilization of the inner membrane of Escherichia coli. Five analogues were synthesized to elucidate structure/activity relationships. It was found that helix disruption virtually eliminates antibacterial activity, while the degree of amphipathicity and the presence of an aromatic residue greatly affect the kinetics of bacterial inner membrane permeabilization.
YGE1 is a yeast homologue of Escherichia coli grpE and is required for maintenance of mitochondrial functions
Authors: Ikeda Eri , Yoshida Satoshi , Mitsuzawa Hiroshi , Uno Isao , Toh-e Akio
Published: July 2002

The grpE gene is a heat shock gene of Escherichia coli whose product functions as a chaperone to (re)fold proteins. We found a yeast homologue of grpE and designated it YGE1. YGE1 can replace grpE in E coli, indicating that YGE1 is a functional homologue of grpE. Deletion of YGE1 is lethal. During depletion of the Ygel product, mitochondria are sequestered in mother cells thereby accumulating cells without mitochondria, suggesting that Ygel protein plays a pivotal role in maintaining mitochondrial functions.
The membrane proteins TRAMp and sec61αp may be involved in post‐translational transport of presecretory proteins into mammalian microsomes
Authors: Klappa Peter , Zimmermann Maria , Zimmermann Richard
Published: July 2002

The presecretory protein ppcecDHFR, a hybrid between preprocecropinA and dihydrofolate reductase, is transported into mammalian microsomes post‐translationally, i.e. independent of ribosome and signal recognition particle. Here, the involvement of microsomal proteins in ribonucleoparticle‐independent transport of ppcecDHFR was analyzed by transport into trypsin‐pretreated microsomes and by transport of a truncated version of ppcecDHFR and subsequent chemical cross‐linking. We observed that post‐translational transport of ppcecDHFR can occur into microsomes which had been pretreated with trypsin (final concentration, 100μg/ml) and that of the known transport components only TRAMp and sec61αp are still present under these conditions. Furthermore, we found that the truncated ppcecDHFR, ppcecDHFR‐98mer′, can be cross‐linked to 36 kDa microsomal membrane proteins during post‐translational transport. Therefore, the two microsomal membrane proteins with molecular masses of about 36 kDa, TRAMp and secolαp, appear to be involved in the post‐translational transport of ppcecDHFR and ppcecDHFR‐98mer.
Purification and characterisation of dRP‐A: a single‐stranded DNA binding protein from Drosophila melanogaster
Authors: Marton Richard F. , Thömmes Pia , Cotterill Sue
Published: July 2002

Replication protein A (RP‐A) is an essential single‐stranded DNA binding protein (SSB) involved in the initiation and elongation phases of eukaryotic DNA replication. It has the ability to bind single‐stranded DNA extremely tightly and possesses a characteristic hetero‐trimeric structure. Here we present a method for the purification of RP‐A from Drosophila melanogaster embryos. Drosophila RP‐A (dRP‐A) has subunits of about 66, 31 and 8 kDa, in line with analogues from other species. It binds single‐stranded DNA very tightly via the large subunit. The complete protein has at least a 10‐ to 20‐fold preference for single‐stranded DNA over double‐stranded DNA and it appears that binding is only weakly co‐operative. Band shift experiments suggest that it has an approximate site covering the size of 16 nucleotides or less, however, it shows a greater affinity for long oligonucleotides than for short ones. We also demonstrate that dRP‐A can stimulate the activity of its homologous DNA polymerase α in excess of 20 fold. Analysis of the protein's abundance during embryo development indicates that it varies in a manner akin to other replication proteins.
Regulation of gene expression at a distance: the hypothetical role of regulatory protein‐mediated topological changes of DNA
Authors: Marincs Ferenc , White Derek W.R
Published: July 2002

A theoretical model is presented that a regulatory protein may activate the transcription of a promoter by interacting with a single remote operator. In response to an inducer molecule the regulatory protein bound to the operator undergoes a conformational change, and might mediate a B to Z‐DNA conversion of the operator. This transition would remove both helical turns and supercoils from the intervening region between the operator and the promoter, resulting in the correct spatial arrangement of the −10 and −35 hexamers of the promoter, which therefore can be efficiently transcribed.