bluuebunny/crossref_metadata_2025 huggingface dataset:
Wedeloside, a powerful inhibitor and ligand of the mitochondrial ADP/ATP carrier
Authors: Klingenberg Martin , Appel Maria , Oelrichs Peter B.
Published: July 2002

The effect of wedeloside, an atractyloside analogue occurring in the Australian weed Wedelia asperrima, on the ADP/ATP carrier is investigated on 3 levels; the ADP stimulated respiration, the ADP/ATP exchange, and the binding in competition with atractylate (ATR) and carboxyatractylate (CAT). The inhibition of respiration and of ADP/ATP exchange by wedeloside is nearly uncompetitive with ADP. The competitive binding with [3H]cat and [3H]ATR reveals a high binding affinity of wedeloside similar to that of CAT. The titration of the ADP/ATP exchange and of the binding with wedeloside gives a titer of 0.35 protein for rat liver protein and the titer of the binding gives 2.7 protein for beef heart mitochondria. The 1.8‐times higher titers than with CAT may indicate that two molecules of wedeloside bind to one ADP/ATP carrier dimer in contrast to the half site reactivity known for the binding of the other ligands.
Guanine nucleotides and Ca‐dependent exocytosis
Authors: Knight D.E. , Baker P.F.
Published: July 2002

Exposure of ‘leaky’ adrenal medullary cells to GTP‐γ‐S inhibits Ca‐dependent exocytosis in bovine cells, but stimulates exocytosis in chicken cells. The inhibitory action on bovine cells persists in the presence of TPA suggesting that in this tissue an inhibitory GTP‐binding protein may modulate the action of protein kinase C on exocytosis.
Qualitative difference in mitochondria of endothermic and ectothermic animals
Authors: Akhmerov R.N.
Published: July 2002

A significantly higher rate of respiration in the absence of added ADP has been revealed in mitochondria of endotherms as compared with that of the ectotherms with similar rates of respiration during phosphorylation. A high rate of ADP‐independent (non‐coupled with ATP synthesis) respiration is observed during oxidation of succinate, NADH and ascorbate + cytochrome c, but not with NAD‐dependent substrates. It increases in the presence of cytochrome c during oxidation of succinate and NADH and is also revealed during oxidation of NAD‐dependent substrates in the presence ofNAD+ and cytochrome c. ADP‐independent respiration is not affected by oligomycin, however, it is essentially inhibited in the presence of GDP. It is suggested that the significant difference in the value of ADP‐independent respiration of endo‐ and ectotherms is due to the existence in endotherms of a non‐coupled population of mitochondria which generates heat without preliminary synthesis of ATP.
Interleukin 2 induces rapid phosphorylation of cellular proteins in murine T‐lymphocytes
Authors: Kohno Michiaki , Kuwata Shigeki , Namba Yuziro , Hanaoka Masao
Published: July 2002

When quiescent murine T‐lymphocyte cells were stimulated by the addition of interleukin 2 (IL‐2), they reinitiated DNA synthesis after a lag period of 5 h. Under these conditions, rapid but transient phosphorylation of two cellular proteins with M
r, values of 27000 and 26000 was detected; maximal phosphorylation occurred within 10–15 min after the addition of IL‐2. The protein of M
r, 27 000 contained phosphoserine, while the protein of M
r 26000 contained phosphothreonine.
Solubilization of protein BM‐40 from a basement membrane tumor with cheating agents and evidence for its identity with osteonectin and SPARC
Authors: Mann Karlheinz , Deutzmann Rainer , Paulsson Mats , Timpl Rupert
Published: July 2002

Up to 80% of the calcium‐binding protein BM‐40 could be extracted from a tumor basement membrane with a physiological buffer containing 10 mM EDTA. About half of its amino acid sequence was determined by Edman degradation demonstrating identity with cDNA deduced sequences of bone osteonectin and SPARC.
Isolation of the neuropeptide pGlu‐Gly‐Arg‐Phe‐amide from the pennatulid Renilla köllikeri
Authors: Grimmelikhuijzen C.J.P. , Groeger A.
Published: July 2002

By using a radioimmunoassay for the sequence Arg‐Phe‐amide, the peptide pGlu‐Gly‐Arg‐Phe‐amide was isolated from the pennatulid Renilla köllikeri and sequenced. This peptide is a neuropeptide and is produced in high amounts in Renilla tissue (10 wet wt).
Simplified in vitro synthesis of mutated RNA molecules
Authors: Krupp Guido , Söll Dieter
Published: July 2002

We describe a simplified method for the in vitro synthesis of mutated RNA molecules. The method makes use of an oligodeoxyribonucleotide (T7‐oligo) which contains the T7 RNA polymerase promoter sequence. In combination with a second oligonucleotide, a series of transcripts initiating and terminating at any chosen position on a cloned ss DNA (e.g. M 13 phage DNA) can be generated. The phage DNA represents the non‐coding DNA strand for the desired transcript; the T7‐oligo determines the transcription start site, whereas the second oligonucleotide permits the choice of the transcription termination site. The synthesis of the required template DNA is achieved by hybridizing the two oligonucleotides to the phage DNA and subsequently synthesizing the coding DNA strand by a fill‐in reaction with Klenow enzyme. The reaction product is used directly as a template for T7 RNA polymerase; cloning of mutants is not required.
A new GTP‐binding protein in brain tissues serving as the specific substrate of islet‐activating protein, pertussis toxin
Authors: Katada Toshiaki , Oinuma Masayuki , Kusakabe Kayoko , Ui Michio
Published: July 2002

A new GTP‐binding protein serving as the specific substrate of islet‐activating protein (IAP), pertussis toxin, was purified from porcine brain membranes as an αβγ‐heterotrimeric structure. The α‐subunit of the purified protein (α40βγ) had a molecular mass of 40 kDa and differed from that of Gi(α41βγ) or Go(α39βγ) previously purified from brain tissues. The fragmentation patterns of limited tryptic digestion and immunological cross‐reactivities among the three α were different from one another. However, the βγ‐subunit resolved from the three IAP substrates similarly inhibited a membrane‐bound adenylate cyclase and their β‐subunits were immunologically indistinguishable from one another. Thus, the α40βγ is a new IAP substrate protein different from Gi or Go, in the α‐subunit only.
Molecular cloning and expression in E. coli of a Salmonella typhi porin gene
Authors: Zaror Isabel , Gómez Isabel , Castillo Gonzalo , Yudelevich Arturo , Venegas Alejandro
Published: July 2002

Immunoscreening of a Salmonella typhi cosmid library in E. coli allowed the detection of clones producing a 36 kDa porin from S. typhi. The gene is efficiently expressed in an E. coli porin‐less mutant and the protein is exported to the outer membrane envelope. Two clones which markedly differ in their level of expression have been isolated.
Kinetic limitations in the overall reaction of mitochondrial oxidative phosphorylation accounting for flux‐dependent changes in the apparent ΔGexP/ΔH+ ratio
Authors: Kunz Wolfgang , Gellerich Frank Norbert , Schild Lorenz , Schönfeld Peter
Published: July 2002

Changes in J
0, ΔH+ and ΔG
ex
P were investigated as a function of load. The flux control coefficients, particularly those of the adenine nucleotide translocator and H+‐ATPase at the maximum rate of oxidative phosphorylation were seen to strongly depend on the phosphate concentration accounting in common for the highest share in flux control. There was no unique relationship observed between J
P and ΔH+ in load‐controlled, well coupled systems, but J
P was found to depend on ΔH+ at excessive load and increasing proton leakage. All the results presented can be elucidated on the grounds of delocalized chemiosmotic coupling.
Apoptotic hepatocytes become insoluble in detergents and chaotropic agents as a result of transglutaminase action
Authors: Fesus Laszlo , Thomazy Vilmos , Autuori Francesco , Ceru Maria P. , Tarcsa Edit , Piacentini Mauro
Published: July 2002

Physiological deletion of cells ensues programmed death which involves formation of apoptotic bodies with fragmented DNA. Here we report that apoptotic hepatocytes are insoluble in detergents, urea, guanidine hydrochloride, reducing agents and thereby can be isolated from rat liver following collagenase treatment. They are wrinkled, spherical structures similar to cornified envelopes of epidermis by phase‐contrast microscopy and show irregular, globular morphology by scanning‐electron microscopy. Part of their DNA content is cleaved into nueleosomal and oligonucleosomal fragments. Their insolubility, like that of the cornified envelope, is evoked by ε‐(γ‐glutamyl)lysine and N
1,N
8‐bis(γ‐glutamyl)spermidine protein cross‐linking bonds formed by transglutaminase.
The mechanism of action of the cytotoxic lectin from Phoradendron californicum: The RNA N‐glycosidase activity of the protein
Authors: Endo Yaeta , Oka Tatsuzo , Tsurugi Kunio , Franz Hartmut
Published: July 2002

A toxic lectin from Phoradendron californicum (PCL) was found to inactivate catalytically 60 S ribosomal subunits of rabbit reticulocytes, resulting in the inhibition of protein synthesis. To study the mechanism of action of PCL, rat liver ribosomes were treated with the toxin and the extracted rRNA was treated with aniline. A fragment containing about 450 nucleotides was released from the 28 S rRNA. Analysis of the nucleotide sequence of the fragment revealed that the aniline‐sensitive phosphodiester bond was between A4324 and G4325 of the 28 S rRNA. These results indicate that PCL inactivates the ribosomes by cleaving an N‐glycosidic bond at A4324 of 28 S rRNA in the ribosomes as does ricin A‐chain.
Identification of calcium‐dependent phospholipid‐binding proteins in higher plant cells
Authors: Boustead Catherine M. , Smallwood Margaret , Small Hazel , Bowles Dianna J. , Walker John H.
Published: July 2002

Calcium‐dependent phospholipid‐binding proteins of apparent M
r 33 000 and 35 000 were isolated from suspension cultures of tomato cells. Two‐dimensional gel electrophoresis showed the proteins to have isoelectric points of approx. 5.7 and 5.6, respectively. In the presence of calcium, both proteins bound to liposomes formed from a mixture of phosphatidylserine and phosphatidylcholine, but not to liposomes of phosphatidylcholine alone. Both proteins showed immunological similarities to previously characterized calcium‐dependent phospholipid‐binding proteins (annexins) from Torpedo marmorata and mammalian species. The protein of M
r 33 000 cross‐reacted with three separate antisera raised to the annexin Torpedo calelectrin, whereas that of M
r 35 000 cross‐reacted with antisera to the bovine annexins p68 and p32/34. We suggest that the two proteins may represent the first identification in higher plants of the annexin family of calcium‐dependent phospholipid‐binding proteins.
Methylation of repetitive DNA sequences in the brain during aging of the rat
Authors: Rath P.C. , Kanungo M.S.
Published: July 2002

Methylation of repetitive DNA sequences (RDS) of the genomic DNA of the brain of 15‐ and 88‐week old rats was analysed by digestion with HpaII, MspI, EcoRI + HpaII and EcoRI + MspI followed by end‐labelling. doublets are present in two RDS of ∼ 5 and 0.4 kb, and are also randomly distributed throughout the genome. Hemimethylated doublets also occur. Both and doublets are found more in the old than in the young. This age‐related increase in DNA methylation occurs both at CCGG sites of the RDS and in the entire genome. Such increase in DNA methylation may alter chromatin conformation and gene expression in the brain as the rat ages.
Inhibition of HIV replication in cell culture by the specific aspartic protease inhibitor pepstatin A
Authors: von der Helm Klaus , Gürtler Lutz , Eberle Josef , Deinhardt Friedrich
Published: July 2002

After incubation of H9 cells infected with human immunodeficiency virus (HIV) with pepstatin A at 10−4 M for 2, 4, or 11 days, the culture medium contained significantly less HIV core antigen (p24) than controls without pepstatin A and no or only borderline activity of reverse transcriptase was detected. In addition, after pepstatin A treatment no infectious HIV at 2 or 4 days and only minimal amounts at 11 days were detectable in the culture medium.
Parallel DNA: generation of a duplex between two Drosophila sequences in vitro
Authors: Tchurikov Nickolai A. , Chernov Boris K. , Golova Yulia B. , Nechipurenko Yurii D.
Published: July 2002

We have observed the existence of a parallel complementary region between two Drosophila DNA sequences, fragments of the suffix [(1986) EMBO J, 5, 2341‐2347] and a 5'‐non‐coding sequence of the alcohol dehydrogenase gene [(1983) Cell 33, 125‐133]. The region includes ~40 bp, 76% of which are complementary in the same polarity. Synthetic complementary 16 bp oligonucleotides corresponding to this region which were bound by the 5'‐ends through a 1.6‐hexanediol bridge form a duplex which displays both melting and annealing as judged by UV absorbance. Anti parallel complementary 16 bp long oligonucleotides bound by the 5'‐3' ends through the same bridge and a single‐strand sequence were used as controls. The Hoechst 33 258 drug binds to this parallel duplex of DNA; however, the properties of such a complex testify against the B‐form of the duplex.
Does the function of adenine nucleotide translocase in fatty acid uncoupling depend on the type of mitochondria?
Authors: Schönfeld Peter
Published: July 2002

The stimulation of respiration by long‐chain fatty adds and FCCP was studied with oligomycin‐inhibited mitochondria from rat liver, heart and kidney tissue. By addition of equal amounts of palmitate and oleate, mitochondrial respiration was increased in the order RLM < RKM < RHM. Using the classical protonophore FCCP, this difference could not be observed. Inhibition of oleate‐stimulated respiration by carboxyatractyloside decreased in the order RHM > RKM > RLM. As CAT sensitivity of oleate‐stimulated respiration and the mitochondrial ANT content were found to be correlated, it is suggested that the weak CAT sensitivity of oleate‐stimulated respiration of RLM [(1989) Biochim. Biophys. Acta 977, 266‐272] is due to the low content of ANT.
Function of the conserved triad residues in the class C β‐lactamase from Citrobacter freundii GN346
Authors: Tsukamoto Kikuo , Nishida Naoko , Tsuruoka Miyuki , Sawai Tetsuo
Published: July 2002

The conserved KTG triad in the class C β‐lactamase from Citrobacter freundii GN346 was examined as to its function by means of site‐directed mutagenesis. The following conversions were performed; Lys‐315 to arginine, alanine or glutamic acid, Thr‐316 to valine, and Gly‐317 to alanine, proline or isoleucine. The resultant mutant enzymes revealed that a basic amino acid at position 315 and a small uncharged residue at position 317 are essential for the enzyme activity, but a hydroxyl group at residue 316 is not required for the enzymatic catalysis. The kinetic properties of the purified Arg‐315 and Val‐316 enzymes provided information on the function of these residues.
Cloning and sequence analysis of a cDNA encoding a precursor for rat C‐type natriuretic peptide (CNP)
Authors: Kojima Masayasu , Minamino Naoto , Kangawa Kenji , Matsuo Hisayuki
Published: July 2002

Recent identification of a C‐type natriuretic peptide (CNP) in porcine brain strongly suggested that a third member of the natriuretic peptide family still remains to be identified in other species of mammals. A cDNA encoding a precursor for rat CNP was cloned from a rat brain cDNA library and sequenced. The precursor was a 126‐residue peptide, carrying a 23‐residue signal sequence at the N‐terminus and the known porcine CNP‐53 sequence at the C‐terminus. By RNA blot analysis, rat CNP mRNA was found to be expressed exclusively in the brain, implying that CNP may function in the central nervous system as a neuropeptide.
Chromosomal localization of the mouse genes coding for α2, α3, α4 and β2 subunits of neuronal nicotinic acetylcholine receptor
Authors: Bessis Alain , Simon-Chazottes Dominique , Devillers-Thiéry Anne , Guénet Jean-Louis , Changeux Jean-Pierre
Published: July 2002

The chromosomal localization of four neuronal nicotinic acetylcholine receptor subunit genes was performed by following the mendelian segregation of their corresponding alleles in backcrosses involving the mouse species Mus spretus and the laboratory strains C57BL/6 or BALB/c. A similar analysis previously performed with muscle nicotinic acetylcholine receptor subunits revealed that the genes coding for the α and β subunits are respectively located on chromosome 2 and 11, whereas the γ and δ subunit coding genes are linked and located on mouse chromosome 1. In this study, we show that the genes coding for the neuronal nicotinic acetylcholine receptor α2, α3 and β2 subunits are dispersed on three different mouse chromosomes, viz. 14, 9 and 3 respectively. Moreover, the α4 subunit gene is located on chromosome 2 but is not genetically linked to the α1 subunit gene.
Characterization by FTIR spectroscopy of the photoreduction of the primary quinone acceptor QA in photosystem II
Authors: Berthomieu C. , Nabedryk E. , Mäntele W. , Breton J.
Published: July 2002

Molecular changes associated with the photoreduction of the primary quinone acceptor Qa of photosystem II have been characterized by Fourier transform infrared spectroscopy. This reaction was light‐induced at room temperature on photosystem II membranes in the presence of hydroxylamine and diuron. A positive signal at 1478 cm−1 is assigned to the C⋯O stretching mode of the semiquinone anion, and can be correlated to the negative C=O mode(s) of the neutral QA at 1645 cm−1 and/or 16 cm−1. Analogies with bacterial reaction center are found in the amide I absorption range at 1672 cm−1, 1653 cm−1 and 1630 cm−1. The stabilization of QA
− does not result from a large protein conformation change, but involves perturbations of several amino acid vibrations. At 1658 cm−1, a negative feature sensitive to 1H–2H exchange is tentatively assigned to a δNH2 histidine mode, while tryptophan D2252 could contribute to the signal at cm−1.
Hepatotropin mRNA expression in human foetal liver development and in liver regeneration
Authors: Selden Clare , Jones Michael , Wade David , Hodgson Humphrey
Published: July 2002

A 569 bp probe against the β‐chain of hepatotropin was used to examine expression of RNA for this growth factor in human adult and foetal liver, foetal kidney and pancreas, and rat liver after partial hepatectomy. Low level expression of a 6kb RNA occurred in human adult and normal rat liver. 70% hepatectomy increased expression, peaking at 10 h and returning to near normal levels 24 h after resection. The 6 kb band was strongly expressed in human foetal liver, as compared with adult, but not in foetal kidney or pancreas, suggesting a major role for hepatotropin in both foetal development and regeneration of the liver.
Post‐translational processing of prepro‐urotensin II
Authors: Conlon J.M. , Arnold-Reed D. , Balment R.J.
Published: July 2002

The primary structure of a teleost prepro‐urotensin II may be deduced from the nucleotide sequence of cloned DNA complementary to carp prepro‐urotensin II mRNA but the pathway of post‐translational processing of the precursor is unknown. In this study, we have isolated four peptides from an extract of flounder urophysis that are derived from prepro‐urotensin II by proteolytic cleavage. The amino acid sequences of the peptides demonstrate that flounder prepro‐urotensin II is cleaved at two monobasic processing sites (single arginine residues) to generate peptides with limited homology to carp prepro‐urotensin II‐(22–4l)‐, ‐(42–87)‐ and ‐(88–110)‐peptides. Cleavage at a tribasic residue processing site generates a urotensin II with the primary structure: Ala‐Gly‐Thr‐Thr‐Glu‐Cys‐Phe‐Trp‐Lys‐Tyr‐Cys‐Val. Urotensin II‐(4–12)‐peptide represented a minor component in the extract.
Inhibition of electrical activity in mouse pancreatic β‐cells by the ATP/ADP translocase inhibitor, bongkrekic acid
Authors: Kiranadi B. , Bangham J.A. , Smith P.A.
Published: July 2002

Bongkrekic acid causes fatal food poisoning which is associated with hyperglycaemia. Here we demonstrate that bongkrekic acid, a potent inhibitor of the mitochondrial ATP/ADP translocase, inhibits glucose‐induced electrical activity in the pancreatic β‐cell through the simulation of ATP‐sensitive potassium channel (K‐ATP‐channel) activity. By comparison of its effects with those of oligomycin, we suggest that bongkrekic acid acts by the inhibition of glucose metabolism and may induce hyperglycaemia by impairing β‐cel function.
The functional role of the β‐subunit in the maturation and intracellular transport of Na,K‐ATPase
Authors: Geering Käthi
Published: July 2002

The minimal functional enzyme unit of Na,K‐ATPase consists of an α—β complex. The α‐subunit bears all functional domains of the enzyme and so far a regulatory role for the β‐subunit in the catalytic cycle has not been established. On the other hand, increasing experimental evidence suggests that the β‐subunit is an indispensable element for the structural and functional maturation of the enzyme as well as its intracellular transport to the plasma membrane. This brief review summarizes the experimental data supporting the hypothesis that assembly of the β‐subunit is needed for the α‐subunit to acquire the correct, stable configuration necessary for the acquisition of functional properties and its exit from the ER.
Chemical detection of natural peptides by specific structures Isolation of chicken galanin by monitoring for its N‐terminal dipeptide, and determination of the amino acid sequence
Authors: Norberg Åke , Sillard Rannar , Carlquist Mats , Jörnvall Hans , Mutt Viktor
Published: July 2002

We have isolated galanin from chicken intestine by monitoring for the N‐terminal glycyltryptophan, which constitutes a conserved part characteristic of the peptide. This monitoring method complements that previously used for C‐terminal amide detection and proves chemical monitoring of specific structures to be useful. The isolation allowed determination of the structure, which was found to be unidentical to any of the known galanins. However, N‐terminal pentadecapeptide parts are identical, showing this segment to be of special importance. In addition to common substitutions at positions 16, 18, 23, 26 and 29, chicken galanin has phenylalanine at position 28, where all known mammalian galanins have leucine.
Effects of bradykinin on cell volume and intracellular pH in NIH 3T3 fibroblasts expressing the ras oncogene
Authors: Ritter M. , Wöll E. , Häussinger D. , Lang F.
Published: July 2002

BCECF fluorescence has been applied to determine intracellular pH (pHi) in NIH 3T3 fibroblasts expressing the Ha‐ras oncogene (+ras) and otherwise identical cells not expressing the oncogene (−ras). In +ras cells, pHi is significantly more alkaline (6.79 ± 0.03 n = 12) than in −ras cells (6.64 ± 0.02, n = 8). Bradykinin (100 nmol/1) leads to intracellular alkalinization in both +ras (to 6.96 ± 0.04, n = 12) and −ras cells (to 6.85 ± 0.02, n = 8). The effect or bradykinin is completely abolished in the presence of dimelhylamiloride (100μmol/1), which does not modify pHi in the absence or bradykinin. Similar to bradykinin, cell shrinkage by addition of 15 mmol/1 NaCl to the extracellular fluid leads to intracellular alkalinization (by 0.08 ± 0.01, n = 15). Cell volume is significantly greater in +ras cells (2.7 ± 0.4 pl, n = 15) than in −ras calls (2.2 ± 0.4 pl, n = 15), Bradykinin leads to cell shrinkage in both +ras cells (by 7 ± 1%, n = 17) and −ras cells (by 5 ± 1%, n = 15). The effect of bradykinin on cell volume can be reversed by the reduction or extracellular NaCl concentration by 15 mmol/1 NaCl in +ras cells and by 7 mmol/1 NaCl in −rus cells. This maneuver completely abolishes (in −ras cells) or blunts (in +ras cells) the alkalinizing effect of bradykinin. In conclusion, +ras cells are more alkaline than −ras cells. Bradykinin leads to further intracellular alkalinization by activation of the Na+/H+‐exchanger, at least in part secondary to hormone‐induced cell shrinkage.
Stable expression of human D3 dopamine receptors in GH4C1 pituitary cells
Authors: Seabrook G.R. , Patel S. , Marwood R. , Emms F. , Knowles M.R. , Freedman S.B. , McAllister G.
Published: July 2002

Human D3 dopamine receptor DNA was stably transfected into GH4C1 pituitary cells. Displacement of iodosulpiride binding in hD3 transfected cells (Kd = 0.3 nM, B
max = 89 fmol/mg protein) by dopaminergic ligands was indistinguishable from that of hD3 receptors in CHO cells. Only two clonal cell lines exhibited weak GppNHp‐dependent shifts in [3H]N‐0437 binding, and these were used for functional assays. Neither arachidonic acid metabolism, cAMP levels, inositol phosphate turnover, intracellular calcium, or potassium currents were consistently affected by dopamine (1–10 μM). The paucity of responses indicates that human D3 receptors do not couple efficiently to these second messengers in GH4C1 cells.
The amphicrine pancreatic cell line, AR42J, secretes GABA and amylase by separate regulated pathways
Authors: Ahnert-Hilger Gudrun , Wiedenmann Bertram
Published: July 2002

Treatment of AR42J cells with dexamethasone leads to an enhanced formation of amylase‐containing granules and facilitates their regulated secretion. Besides the exocrine properties, AR42J cells possess a specific uptake system for [3H]GABA. The stored GABA can be released upon potassium depolarisation in a Ca2+‐dependent manner. After treatment with dexamethasone, potassium depolarisation fails to release GABA, but instead causes a Ca2+‐dependent secretion of amylase. Since vesicles similar to small synaptic vesicles of neurons have been identified in AR42J cells, we suggest that the regulated GABA release is mediated by this vesicle type. It is tentatively speculated that other epithelial cells, which also contain small synaptic vesicles and amino acid neurotransmitters, may release them in a similar fashion.
The structure of neutrophil defensin genes
Authors: Linzmeier Rose , Michaelson David , Liu Lide , Ganz Tomas
Published: July 2002

Defensins are a family of microbicidal peptides abundant in the granules of mammalian neutrophils, in rabbit alveolar macrophages, and in human and murine intestinal Paneth cells. We cloned and sequenced the genes of three neutrophil‐specific defensins. Human HNP‐1 and HNP‐3 are nearly identical and rabbit NP‐3a is closely related. The four known neutrophil‐specific defensin genes are strikingly similar in the structure and organization of their three exons and two introns, but the three defensin genes expressed in macrophages (MCP‐1 and ‐2) or Paneth cells (HD‐5) are organized differently: HD‐5 has only two exons, and MCP‐1 and ‐2 have a comparatively short first intron. The diverse genomic organization of defensin genes may contribute to their cell‐specific expression.
Electrophoretically defined myosin heavy chain patterns of single human muscle spindles
Authors: Pedrosa-Domellöf Fatima , Gohlsch Bärbel , Thornell Lars-Eric , Pette Dirk
Published: July 2002

At least four myosin heavy chain (MHC) isoforms were separated by SDS‐PAGE in extracts of intrafusal fibers isolated by microdissection from human lumbrical muscles. The fastest migrating MHC represents a slow isoform. The slowest migrating MHC was identified as the embryonic MHCemb. A faint band, moving slightly faster than MHCemb, most likely represents a neonatal/fetal MHC isoform. A prominent band, moving between the latter and the slow isoform is suggested to represent a hitherto unidentified, spindle‐specific MHC isoform, MHCif.
The presence of a calcium‐sensitive p26‐containing complex in bovine retina rod cells
Authors: Gorodovikova Elena N. , Philippov Pavel P.
Published: July 2002

A protein with apparent M
r, 26 kDa (p26 [(1991) Biokhnya 56, 225‐228] or recoverin [(1991) Science 251, 915‐918]) was suggested to activate GC in a calcium dependent manner in bovine retina rod cells [(1991) Science 251, 915‐918; (1991) EMBO J, 10, 793‐798]. However, according to our present data homogeneous p26 preparations do not activate the enzyme. At the same time we have revealed a complex of p26 with an unidentified protein, presumably RK, in bovine ROS. Calcium favours formation of the complex whereas EGTA addition (which corresponds to a low free Ca2+ concentration) leads to its dissociation.
Importance of Arg‐219 for correct biogenesis of α1 homooligomeric glycine receptors
Authors: Langosch Dieter , Herbold Astrid , Schmieden Volker , Borman Joachim , Kirsch Joachim
Published: July 2002

The inhibitory glycine receptor is characterized by a pentameric arrangement ofsubunits with four predicted transmembrane segments (M1‐M4) each. Here, we have mutagenized arginine residues located at both termini of the α1 subunit segment, M2, which lines the receptor's anion channel. No glycine‐gated channel formation could be detected in the plasma membrane of expressing cells for any of the mutants. In addition, mutating the arginine at the cytoplasmic terminus of M2 (R219) generated proteins which were only core‐glycosylated, retained within intracellular compartments, and aggregated to high molecular weight complexes. Thus, residue R219, which corresponds to an arginine/lysine conserved in other ligand‐gated ion channel polypeptides, is essential for correct biogenesis of the receptor.
Mutation‐spectrum of a true abasic site in codon 12 of a c‐Ha‐ras gene in mammalian cells
Authors: Kamiya Hiroyuki , Suzuki Masayuki , Ohtsuka Eiko
Published: July 2002

An abasic site is postulated to be a premutagenic lesion. We previously reported that a c‐Ha‐ras gene with an abasic site analogue at either the first or the second position of codon 12 induced the formation of foci by point‐mutational activation of the gene [Kamiya et al. (1992) Nucleic Acids Res. 20, 4409‐4415]. In this study we constructed c‐Ha‐ras genes with a true abasic site in codon 12. The ras genes were found to be activated in NIH3T3 cells by a mutation to A at the modified and flanking positions, and the flanking mutations were detected more frequently.
Latency is the major determinant of UDP‐glucuronosyltransferase activity in isolated hepatocytes
Authors: Bánhegyi Gábor , Garzó Tamás , Fulceri Rosella , Benedetti Angelo , Mandl József
Published: July 2002

The glucuronidation of p‐nitrophenol was measured in intact, saponin‐ and alamethicin‐treated isolated mouse hepatocytes. In saponin‐permeabilized cells the elevation of extrareticular UDP‐glucuronic acid concentration enhanced the rate of glucuronidation threefold. When intracellular membranes were also permeabilized by alamethicin, a further tenfold increase in the glucuronidation of p‐nitrophenol was present. Parallel measurements of the ER mannose 6‐phosphatase activity revealed that saponin selectively permeabilized the plasma membrane, whereas alamethicin permeabilized both plasma membrane and ER membranes. The inhibition of p‐nitrophenol glucuronidation by dbcAMP in intact hepatocytes was still present in saponin‐treated cells and disappeared in alamethicin‐permeabilized hepatocytes. It is suggested that the permeability of the endoplasmic reticulum membrane is a major determinant of glucuronidation not only in microsomes but in isolated hepatocytes as well.
Induction of GTP cyclohydrolase I by bacterial lipopolysaccharide in the rat
Authors: Werner-Felmayer Gabriele , Prast Helmut , Werner Ernst R. , Philippu Athineos , Wachter Helmut
Published: July 2002

A 2‐ to 3‐fold increase of GTP cyclohydrolase I (E.C. 3.5.4.16), the key enzyme of tetrahydrobiopterin biosynthesis from GTP, was observed in cerebellum, remaining brain, liver, spleen, and adrenal gland of rats treated with a single dose of lipopolysaccharide (LPS). This led to increased biopterin levels in tissues but not in plasma. Parallel induction of nitric oxide (NO) synthase was indicated by a 10‐ to 100‐fold increase of plasma nitrate levels 6 and 12 hours after injection of LPS. Furthermore, systolic blood pressure was reduced significantly by 23%. Our results demonstrate induction of tetrahydrobiopterin biosynthesis after LPS treatment in vivo.
Prevention of phospholipase‐C induced aggregation of low density lipoprotein by amphipathic apolipoproteins
Authors: Liu Hu , Scraba Douglas G. , Ryan Robert O.
Published: July 2002

Phospholipase C (PL‐C) digestion of human low density lipoprotein (LDL) results in hydrolytic cleavage of the phosphocholine head group of phosphatidylcholine, thereby generating diacylglycerol. Loss of amphiphillic surface lipids and/or accumulation of diacylglycerol causes LDL samples to develop turbidity. Examination of PL‐C treated LDL by electron microscopy revealed a progressive aggregation of LDL as a function of phosphatidylcholine hydrolysis: fused particles, clusters, and multiple stacked aggregates were observed. Lipid analysis of untreated and aggregated LDL confirmed that the phosphatidylcholine content of the latter had decreased with a corresponding increase in diacylglycerol. It is likely that phospholipolysis created hydrophobic gaps within the surface monolayer of LDL, thereby inducing LDL fusion and aggregation. When amphipathic α‐helix‐containing apolipoproteins, such as human apoA‐I or Manduca sexta apolipophorin III (apoLp‐III) were present, PL‐C treated LDL did not aggregate. Compositional analysis of apolipoprotein‐containing PL‐C LDL showed that phospholipolysis was not affected by the presence of apolipoproteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of lipoproteins re‐isolated following incubation with PL‐C revealed an association of apoA‐I or apoLp‐III with PL‐C digested LDL. Electron microscopy showed no major morphological differences between native LDL and apoprotein stabilized PL‐C treated LDL and the average particle diameter of apoA‐I stabilized PL‐C LDL was 22.5 ± 0 2.2 nm versus 22.8 ± 1.6 nm for control LDL. Incubation of tritium‐labeled apoLp‐III with LDL and PL‐C demonstrated that association of apoLp‐III with PL‐C LDL correlated with the extent of phospholipid hydrolysis, the apolipoproteins apparently being recruited to compensate for the increased hydrophobic surface created by conversion of phosphatidylcholine into diacylglycerol. The results suggest that transient association of amphipathic apolipoproteins with damaged or unstable LDL may provide a mechanism to obviate formation of atherogenic LDL aggregates in vivo.
In vivo incorporation of cobalt into Propionibacterium shermanii superoxide dismutase
Authors: Meier Beate , Sehn Anja P. , Sette Marco , Paci Maurizio , Desideri Alessandro , Rotilio Giuseppe
Published: July 2002

Propionibacterium shermanii, an aerotolerant anaerobic bacterium, has already been shown to incorporate, depending on the metal supplementation to the medium, either iron or manganese or copper into the same superoxide dismutase protein. The in vivo incorporation of cobalt in the same superoxide dismutase was obtained in an iron‐, manganese‐ and copper‐depleted medium. The protein was isolated and characterized by NMR which offers the possibility to identify the amino acid residues at the active site exploiting isotropically shifted proton resonance.
Identification of metabolic pathways of the lipid peroxidation product 4‐hydroxynonenal by mitochondria isolated from rat kidney cortex
Authors: Ullrich Oliver , Grune Tilman , Henke Wolfgang , Esterbauer Herrmann , Siems Werner G.
Published: July 2002

The cytosolic lipid peroxidation product 4‐hydroxynonenal (HNE) is rapidly metabolized in mitochondria isolated from rat kidney cortex. About 80% of HNE was degraded within 3 min of incubation. Main products of HNE which were identified in mitochondria were the hydroxynonenoic acid, the 1,4‐dihydroxynonene and the glutathione‐HNE‐conjugate. Furthermore, formation of metabolites of the tricarboxylic acid cycle from HNE is suggested. The quantitative share of HNE binding to proteins was high with about 8% of total HNE consumption after 3 min of incubation. Therefore, rapid degradation of HNE by mitochondria might be involved in an intracellular antioxidative defense system.
Expression of the human UDP‐glucuronosyltransferase UGT116 in Escherichia coli
Authors: Ouzzine Mohamed , Fournel-Gigleux Sylvie , Pillot Thierry , Burchell Brian , Siest Gérard , Magdalou Jacques
Published: July 2002

The membrane‐bound human liver UDP‐glucuronosyltransferase UGT1∗6 was expressed in Escherichia coli. Exchange of the natural signal peptide by the bacterial signal peptides of pelB or OmpT proteins considerably increased the level of expression and, as the natural signal peptide, targeted the protein to the membranes. The extent of maturation of spelB‐UGTl*6 precursor was about 30%. No processing of sOmpT‐UGTl∗6 occurred but the processing rate of this precursor could be significantly increased by mutagenesis of the first two amino acid residues of the mature sequence. These expression vectors allowed us to produce high levels of recombinant mature UGT1∗6 required for further structural studies.
New era of calpain research
Authors: Sorimachi Hiroyuki , Saido Takaomi C. , Suzuki Koichi
Published: July 2002

The recent discovery of several new calpain species other than the two species thus far studied reveals that calpain, especially the calpain large subunit, constitutes a family comprising at least six members that can be classified into ubiquitous (μ‐, m‐ and ‐types) and tissue‐specific (p94 or nCL‐1 specific for skeletal muscle, and nCL‐2 and ‐2' specific for stomach) calpains. The newly identified tissue‐specific calpains have various characteristics distinct from conventional calpains in structure, manner of expression, and enzyme activity. Unique features of tissuespecific calpains are discussed together with the evolutionary view of the calpain large subunit.
The extracellular matrix produced by bovine corneal endothelial cells contains progelatinase A
Authors: Menashi Suzanne , Vlodavsky Israel , Ishai-Michaeli Rivka , Legrand Yves , Fridman Rafael
Published: July 2002

Progelatinase A is a matrix metalloproteinase involved in the turnover of extracellular matrix (ECM). We report that the ECM produced by bovine corneal endothelial (BCE) cells contains progelatinase A free of tissue inhibitor of metalloproteinase (TIMP2). The matrix‐bound progelatinase A can be activated by APMA to generate a 62 kDa and a 45 kDa species with enzymatic activity and is inhibited by TIMP2. The bound progelatinase can be released after treatment of the ECM with gelatinase B. These studies suggest that the ECM can function as a reservoir for progelatinase A which may be readily available for cells in processes such as metastasis, angiogenesis, inflammation and wound healing.
The o‐diphenol oxidase activity of arthropod hemocyanin
Authors: Zlateva T. , Di Muro P. , Salvato B. , Beltramini M.
Published: July 2002

Arthropod hemocyanin (isolated from the crab Carcinus maenas and the lobster Homarus americanus) is usually referred to as an oxygen transport‐storage protein. The protein, however, also catalyses with low efficiency the oxidation of o‐diphenol to quinone, similarly to tyrosinase (monophenol,o‐diphenol: oxygen oxidoreductase). The enzymatic parameters of hemocyanin are affected by the aggregation state of the protein; namely V

max
exhibited by a dissociated subunit is one order of magnitude greater than that of aggregated species. The reaction velocity is increased by the presence of perchlorate, an anion of the Hofmeister series. The results are also discussed on the basis of active site accessibility in comparison with tyrosinase.
Intercellular transfer of shed tumor cell gangliosides
Authors: Olshefski Randal , Ladisch Stephan
Published: July 2002

Three distinct steps underlie immunosuppression by tumor gangliosides: (i) their shedding by the tumor cell, (ii) binding to target leukocytes in the tumor microenvironment, and (iii) action upon the target cell. While shedding is well documented, cell to cell transfer of shed gangliosides is not. To address this, we employed a dual chamber culture system. In this system, metabolically radiolabeled lymphoma cells shed gangliosides into the conditioned medium of the contralateral chamber, which contained normal fibroblasts as the target cell. The shed lymphoma cell gangliosides bound avidly to the target fibroblasts in a trypsin‐resistant manner (1−2×106 and 7×106 molecules/fibroblast in 24 and 48 h). Significantly higher than binding rates of purified lymphoma gangliosides added exogenously, these binding rates in a system which models the in vivo microenvironment suggest that cell to cell ganglioside transfer is a highly efficient process.
Dilapan tent‐gemeprost regimen vs. combinations of extra‐amniotic Rivanol‐Laminaria/Lamicel and oxytocin for second trimester abortion
Authors: Bokström H. , Bryman I. , Norström A. , Platz‐Christensen J.‐J.
Published: July 2002

Objective: To compare a new regimen for second trimester abortion using Dilapan and vaginal gemeprost suppositories with extra‐amniotic Rivanol instillation and oxytocin i.v. immediately or 16 h after instillation. Methods: A prospective study was performed in 153 women to analyze the induction‐abortion interval, the use of analgesics and the complication rate. Wilcoxon's rank sum test was used for statistical evaluation. Results: The mean induction‐abortion interval was significantly shorter in the Dilapan‐gemeprost‐treated women than in the immediate or 16‐h Rivanol‐oxytocin‐treated women, 12.5 vs. 23.3 and 26.8 h, respectively. The 24‐h cumulative abortion rate was 91% in the former group vs. 49% and 61%, respectively. The use of analgesics was less frequent among the Dilapan‐gemeprost‐treated women, whereas the complication rate did not differ. Conclusions: The Dilapan‐gemeprost treatment was advantageous with respect to a shorter induction‐abortion interval and ease of handling. However a minority of women do not respond to this treatment and it is therefore necessary to employ alternative methods to complete the abortion in these cases.
The multi‐product multi‐constraint newsboy problem: Applications, formulation and solution
Authors: Lau Hon‐Shiang , Hing‐Ling Lau Amy
Published: July 2002

Practically all of the many earlier papers on the newsboy problem consider a single newsboy product with no capacity constraint. This paper points out the real‐world prevalence of the “multiple‐product multiple‐constraint newsboy problem”, i.e., the “newstand problem”. We present a formulation and a solution procedure for this newsstand problem. Our solutions procedure has been designed to efficiently handle the common situation where a large number of products are involved; this solution procedure is shown to be necessary for the practical solution of realistic newsstand problems.
Neuronal and non‐neuronal catechol‐O‐methyltransferase in primary cultures of rat brain cells
Authors: Karhunen T. , Tilgmann C. , Ulmanen I. , Panula P.
Published: July 2002

Previous biochemical and histochemical studies have suggested that catechol‐O‐methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers. COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18‐day‐old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double‐staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, flat cells displaying type‐1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type‐2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane‐bound 28‐kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.
Secondary carnitine deficiency and impaired docosahexaenoic (22:6n‐3) acid synthesis: a common denominator in the pathophysiology of diseases of oxidative phosphorylation and β‐oxidation
Authors: Infante Juan P , Huszagh Virginia A
Published: July 2002

A critical analysis of the literature of mitochondrial disorders reveals that genetic diseases of oxidative phosphorylation are often associated with impaired β‐oxidation, and vice versa, and preferentially affect brain, retina, heart and skeletal muscle, tissues which depend on docosahexaenoic (22:6n‐3)‐containing phospholipids for functionality. Evidence suggests that an increased NADH/NAD+ ratio generated by reduced flux through the respiratory chain inhibits β‐oxidation, producing secondary carnitine deficiency while increasing reactive oxygen species and depleting α‐tocopherol (α‐TOC). These events result in impairment of the recently elucidated mitochondrial pathway for synthesis of 22:6n‐3‐containing phospholipids, since carnitine and α‐TOC are involved in their biosynthesis. Therapeutic supplementation with 22:6n‐3 and α‐TOC is suggested.
Tumor necrosis factor‐induced lethal hepatitis: pharmacological intervention with verapamil, tannic acid, picotamide and K76COOH
Authors: Van Molle Wim , Vanden Berghe Joke , Brouckaert Peter , Libert Claude
Published: July 2002

Tumor necrosis factor (TNF) induces hepatitis when injected in human beings or in rodents. The molecular mechanism by which TNF induces hepatic distress remains largely unknown, although induction of apoptosis of hepatocytes appears to be an essential step. In order to increase the therapeutic value of TNF, we have studied the protective activity of several molecules and found that four chemically totally different substances confer significant protection in the model of TNF‐induced lethal hepatitis in mice sensitized with D‐(+)‐galactosamine (GalN), but not in mice sensitized with actinomycin‐D (ActD) or against anti‐Fas‐induced lethal hepatitis. Verapamil, a calcium‐channel blocker, tannic acid, picotamide, a thromboxane A2 receptor antagonist, and K76COOH, an inhibitor, amongst others, of complement, protected significantly against induction of lethality, release of the liver‐specific enzyme alanine aminotransferase (ALT) and induction of apoptosis in the liver after TNF/GalN, except for K76COOH, which paradoxically increased ALT values after challenge, and which also protected against TNF/GalN in complement‐deficient mice. The data suggest that activation of platelets and neutrophils, as well as induction of inflammation occur in the TNF/GalN model, but not in the TNF/ActD or anti‐Fas models, in which direct induction of apoptosis of hepatocytes may be more relevant. The protective activity of the drugs may lead to an increase in therapeutic value of TNF.
Sorting of the vasopressin prohormone into the regulated secretory pathway
Authors: de Bree F.M , Knight D , Howell L , Murphy D
Published: July 2002

The sorting of soluble proteins into the regulated secretory pathway (RSP) involves aggregation, but whether an additional sorting domain is also required is not clear. By fusing vasopressin prohormone (proVP) fragments to green fluorescent protein (eGFP) we have determined whether a sorting domain can function independently of the aggregative neurophysin domain. Although eGFP itself can be immunolocalised in the RSP of Neuro2A and AtT20 cells, most of the protein enters the constitutive pathway, and is found in the culture medium. In contrast, the N‐terminal 27 residues of proVP promote residence in the RSP. Furthermore, only the processed form of this fusion was secreted when stimulated. We suggest a sorting mechanism based on the recognition of a sorting motif, the efficiency of which is enhanced by neurophysin aggregation.