bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolism of acetaldehyde in human and baboon renal cortex
Authors: Michoudet C. , Baverel G.
Published: July 2002

Acetaldehyde (1–20 mM) was metabolized at high rates and in a dose‐dependent manner in isolated human and baboon kidney‐cortex tubules. Acetaldehyde removal was accompanied by a large accumulation of acetate in both human and baboon tubules. By contrast, a large synthesis of ethanol was observed only in baboon tubules. Consistent with the latter finding, ethanol was found to be metabolized at significant rates in baboon but not human tubules. In the tubules from both species, a significant fraction of the acetaldehyde removed was also completely oxidized to CO2 and H2O. These results suggest that, in both man and baboon, the kidneys participate in the in vivo metabolism of acetaldehyde; they also suggest that, in contrast with the human kidneys, the baboon kidneys contribute to the detoxication of circulating ethanol.
Recombinant type I regulatory subunit of the cAMP‐dependent protein kinase is biologically active
Authors: Oehen Stephan , Parsons JoAnne , Jans David A. , Hemmings Brian A.
Published: July 2002

The cDNA for the porcine type I regulatory subunit (RI) of the cAMP‐dependent protein kinase (cAMP‐PK) was cloned into two different bacterial expression vectors: pKK223 and pUC18. Recombinant RI was produced by bacteria transformed with either construct, and purified by affinity chromatography. Both the native RI from the pKK223 construct and the RI with an amino terminal extension of eight amino acids from the pUC18 construct were found to be completely native with regard to inhibition of the catalytic subunit activity and cAMP binding.
Unusual 5 S ribosomal RNAs
Authors: Delihas Nicolas
Published: July 2002

Sequence comparisons of 5 S and other ribosomal RNAs by segments can be useful in understanding anomalous primary and secondary structures and in assessing phylogenetic relationships. In a segmented analysis, the 5′‐half of the Chlamydomonas reinhardii chloroplast 5 S ribosomal RNA is found to have a very close sequence homology to the green plant chloroplast and cyanobacterial 5 S RNAs; however, the 3′‐half has a highly unusual sequence. Further comparisons of homologies between regions of the 5 S RNAs from C. reinhardii and the green plant chloroplasts suggest that genetic rearrangements within the 5 S DNA may have produced the unusual sequence at the 3′‐half. Segmented analyses of the C. reinhardii and green plant chloroplast 5 S RNAs suggest a close relationship which is not revealed by overall sequence comparisons.
Effects of Ca2+, theophylline and promethazine on protein phosphorylation in intact cells of rabbit ileum Correlation with active Na and Cl absorption
Authors: Sharp Geoffrey W.G. , Hannah-White Cynthia , El-Sabban Marwan , Cohen Michael E. , Donowitz Mark
Published: July 2002

The effects of Ca2+, theophylline and promethazine on the phosphorylation of microvillus membrane proteins have been studied in rabbit ileal epithelial cells, using intact cell phosphorylation techniques followed by purification of microvillus membranes, separation of peptides by two‐dimensional polyacrylamide gel electrophoresis, and quantitation of phosphorylation by computerized densitometry of autoradiograms. The Ca2+ ionophore A23187 caused increased phosphorylation of four and possibly five polypeptides; theophylline increased phosphorylation of three peptides, two of which had the same M
r and pI values as the peptides altered by the Ca2+ ionophore; promethazine decreased the phosphorylation of one of the peptides increased by Ca2+ ionophore. The phosphorylated peptides, which respond similarly to more than one agent which affect ileal Na and Cl absorption, could be involved in the regulation of NaCl absorption either as transport proteins or regulators of transport proteins.
Properties of B‐ring analogues of colchicine
Authors: Maity Sankar N. , Bhattacharyya B.
Published: July 2002

Absorption spectra of colchicine and its analogues are affected by the presence of the B‐ring, although it is not part of the chromophore (C‐ring). Thus, 2‐methoxy‐5‐(2′,3′4′‐trimethoxyphenyl)tropone has absorption maxima at 341 nm, whereas that of desacetamidocolchicine is at 353 nm. A similar red shift in the λmax of colchicine, desacetamidocolchicine and 2‐methoxy‐(2′,3′,4′‐trimethoxyphenyl)tropone also occurs when they are immobilized in the binding site to tubulin or in pure glycerol. We also observed that the B‐ring of colchicine alone or with substituent does not affect the UV‐induced rearrangement of colchicine to lumicolchicine. However, in the absence of the B‐ring, as in the case of 2‐methoxy‐5‐(2′,3′4′‐trimethoxyphenyl)tropone, the rearrangement reaction of the C‐ring slows down significantly.
A 31P MAS NMR study of cytidine 2′‐phosphate bound to ribonuclease A in the crystalline state
Authors: Dobson Christopher M. , Lian Lu-Yun
Published: July 2002

31P CP/MAS spectra have been obtained from 2′‐CMP bound to ribonuclease A in the crystalline state. The chemical shift value is closely similar to that found in solution NMR studies under similar conditions, and corresponds to that of the dianionic state of the free compound. It is suggested that the NMR approach may be of general applicability for the comparison of the binding properties of small molecules to proteins in crystals and solution.
trp operon induction during the expression in E. coli of two IFN‐γ sequences
Authors: Sverdlov E.D. , Tsarev S.A. , Krykbaev R.A. , Chernov I.P. , Rostapshov V.M.
Published: July 2002

Two nucleotide sequences coding for mature human immune interferon (IFN‐γ) and differing from each other by nine N‐terminal nucleotides were expressed in E. coli under the control of a trp promoter. The longer gene variant after the ATG initiatory codon contained a TGT TAC TGC sequence, which was absent in the shorter gene. When expressed in E. coli under the direction of identical transcription and translation regulatory elements, these genes showed different susceptibility to induction.
CpA containing oligoribonucleotides specifically inhibit protein synthesis in rabbit reticulocytes
Authors: Wagner Thomas , Gross Martin , Sigler Paul B.
Published: July 2002

The diribonucleoside monophosphate CpA (and no others) inhibits polypeptide chain elongation in rabbit reticulocyte lysates at 10–50 μM. Furthermore, all the trinucleotides containing CpA, i.e., XpCpA and CpApX (X = U, C, A or G) block polypeptide chain elongation as well. At 10 μM the inhibition by XpCpA and not CpApX is transient because a 3'‐exonucleolytic activity destroys the critical CpA moiety. The inhibitors do not appear to interfere with the aminoacylation of tRNAs or disrupt the interaction of aminoacyl‐tRNAs with the protein synthetic machinery. High levels (200 μM) of CpA or the trinucleotides containing CpA have no effect on translation in a wheat germ cell‐free system.
Inhibition of terminal deoxynucleotidyl transferase by adenine dinucleotides
Authors: Pandey V.N. , Amrute S.B. , Satav J.G. , Modak M.J.
Published: July 2002

Terminal deoxynucleotidyltransferase (TdT) exhibits strong sensitivity to ATP and its dinucleotide analogues, Ap2A, Ap3A, Ap4A, Ap5A and Ap6A. Similar to ATP, all of the dinucleotides appear to be competitive inhibitors of TdT catalysis with respect to substrate deoxynucleoside triphosphates and effectively block the UV‐mediated substrate cross‐linking to TdT. Among the various dinucleotides, Ap5A and Ap6A (diadenosine 5'‐5' penta‐ and hexaphosphate, respectively) are significantly more effective than dinucleotides containing 2, 3 or 4 phosphate backbones. Furthermore, Ap5A is found to be the only dinucleotide which has reactivity at both substrate‐ and primer‐binding domains in TdT.
Resonance Raman study of the cytochrome P‐450 LM2‐halothane intermediate complex
Authors: Hildebrandt Peter , Garda Horacio , Stier Anton , Stockburger Manfred , Van Dyke Russel A.
Published: July 2002

Resonance Raman (RR) and absorption spectroscopic studies of purified rabbit liver cytochromes P‐450 show that the form 2 isomer (LM2) but not the form 4 isomer (LM4) forms a long‐lived complex with halothane after dithionite reduction, absorbing light at 470 nm, in which ferric 6‐coordinated heme iron in the low‐spin configuration is liganded to 2‐chloro‐1,1‐difluoroethylene. The RR data exclude the possibility that the CF3CHCl− carbanion is a ligand and are consistent with the involvement of an active‐site pocket in the cytochrome P‐450 polypeptide.
Direct and indirect electron transfer from cytochromes c and c2 to the photosynthetic reaction center in pigment‐protein complexes isolated from Rhodocyclus gelatinosus
Authors: Matsuura Katsumi , Fukushima Atsushi , Shimada Keizo , Satoh Toshio
Published: July 2002

Electron transfer from soluble cytochromes c and c
2 to the photo‐oxidized bacteriochlorophyll dimer was studied in photosynthetic reaction center (RC) complexes and RC‐B870 complexes isolated from Rhodocyclus gelatinosus. In the RC‐B870 complex which consists of 6 polypeptides including a bound cytochrome subunit, cytochrome c from horse heart or cytochrome c
2, from R. gelatinosus was oxidized by photo‐oxidized bacteriochlorophyll dimer indirectly via cytochrome c‐555 in the complex. In the RC complex which is composed of two polypeptides, the oxidized bacteriochlorophyll dimer was reduced directly by the soluble cytochromes. Although direct electron transfer from soluble cytochrome does not function in R. gelatinosus in vivo, it would replace indirect electron donation if the bound cytochrome were lost mutationally.
Selective extraction of haemagglutinin and matrix protein from Sendai virions by employing trifluoperazine as a detergent
Authors: Baiocchi Marta , Pescarmona Marina , Bruschi Maria Letizia , Montecucco Cesare , Squatriti Teodoro , Tomasi Maurizio
Published: July 2002

Incubation of trifluoperazine, a local anaesthetic, at concentrations higher than the cmc with Sendai virus particles produces the selective solubilization of the haemagglutinin neuraminidase (HN) and matrix (M) proteins. This phenomenon involves aggregation of the Sendai virions and therefore the separation of HN and M from the rest of the particle can be performed by bench centrifugation. The supernatant contains the HN and M proteins and HN, once inserted into liposomes, elicits its own biological activities. Therefore, the method seems suitable for purifying large amounts of HN.
Membrane excitability expressed in human neuroblastoma cell hybrids
Authors: Gilbert Fred , Giovanni Maria Y. , Silver Ian A. , Glick Mary Catherine
Published: July 2002

The voltage‐sensitive Na+ channel is responsible for the action potential of membrane electrical excitability in neuronal tissue. Three methods were used to demonstrate the presence of neurotoxin‐responsive Na+ channels in two hybrid cell lines resulting from the fusion of excitable human neuroblastoma cells with mouse fibroblasts. Only one of the two electrically active hybrid cell lines maintained the sensitivity of the neuroblastoma parent to tetrodotoxin (TTX). The other hybrid, although electrically active, was not responsive to TTX or scorpion venom. Comparisons of the patterns of expression of membrane excitability and of chromosome complements in these human neuroblastoma cell hybrids suggest that the phenotype of membrane excitability is composed of genetically distinct elements.
Activation energy of sulfate ion transport across methylated human erythrocyte membranes
Authors: Janas Teresa , Janas Tadeusz , Kilian Mieczysław , Przestalski Stanisław
Published: July 2002

Activation energy E
A of the sulfate ions transport process across human erythrocyte membranes modified by reductive methylation has been measured. It has been found that exhaustive reductive methylation (3 times) with formaldehyde and borohydride inhibits the sulfate‐equilibrium exchange, by a maximum of about 40%. However, methylation has no measurable effect on activation energy, since the evaluated E
A values for control and methylated cells remain the same within the experimental error range.
Complete amino acid sequence of a human platelet proteoglycan
Authors: Alliel Patrick M. , Périn Jean-Pierre , Maillet Philippe , Bonnet François , Rosa Jean-Philippe , Jollès Pierre
Published: July 2002

The primary structure of a human platelet proteoglycan (P.PG) core was established by a combination of amino acid sequence analysis and cDNA cloning. The deduced 131 amino acid long protein contains eight Ser‐Gly repeats. The significance of homologies observed between P.PG and promyelocytic leukemia cell line proteoglycans is discussed.
Proteoliposomes with right‐side‐out oriented purple membrane/bacteriorhodopsin require cations inside for proton pumping
Authors: Ihara Kunio , Mukohata Yasuo
Published: July 2002

Proteoliposomes were prepared by sonication of phospholipids and blue membranes (cation‐free purple membranes carrying little activity of light‐driven proton pumping) in an acidic medium of very low ionic strength. The majority of the bacteriorhodopsin population in these proteoliposomes was in the right‐side‐out (as in living cells) orientation as judged from the resultant polypeptides after papain digestion. By raising the pH of sonication, the population of right‐side‐out oriented bacteriorhodopsin decreased, and consequently that of the inversely oriented one increased. In KCl and NaCl up to certain concentrations or in choline chloride even at high concentrations, in the light, the proteoliposomes with right‐side‐out bacteriorhodopsin did not pump protons, whereas those with inversely oriented bacteriorhodopsin did. The former began to pump only after cations were likely incorporated/permeated into the proteoliposome and reached the carboxyl terminal (cytosol) side of bacteriorhodopsin/purple membrane.
Variable expression of SV40 large T antigen in CV1 cell clones
Authors: Matthopoulos Demetrios P. , Pagoulatos Gerassimos N.
Published: July 2002

Using immunofluorescence and immunoadsorption, CV1 cell clones MA2, V4, USA3, TR7 and P3 infected with SV40 were found to express variably SV40 large T antigen. The monoclonal antibody used was Pab 419. The results indicate that P3 cells express T antigen to a considerable level as early as 10 h post‐infection, while that of TR7 and USA3 cells is minute as judged from their positive nuclei. MA2 and V4 cells did not show any positive nuclei over this period of infection. At 20 h post‐infection MA2, V4 and USA3 cells developed a considerable amount of fluorescence in their nuclei while TR7 and P3 cells produced high values. By immunoadsorption of cell extracts for the same periods of infection, similar results were obtained on the electrophoretograms. We also relate these findings with those from induction of heat‐shock proteins by SV40 infection.
Sensitivity of intergenic regions of yeast mitochondrial DNA to single‐strand‐specific nucleases
Authors: Piškur Jure
Published: July 2002

The reactivity of mitochondrial DNA (mtDNA) sequences from Torulopsis glabrata and Saccharomyces cerevisiae towards single‐strand‐specific nucleases has been examined. AT‐rich stretches located in intergenic sequences from both yeasts were cleaved by nucleases when the sequences were contained in supercoiled plasmid DNA. In particular ori/rep sequences from the mtDNA of S. cerevisiae were shown to be sensitive to the single‐strand‐specific nucleases. The locations of the sensitive sites were related to the organisation of the sequence domains of ori/rep and the superhelicity of the DNA, as well as the presence of particular sequences. It is proposed that distortions of the DNA duplex could be generated in mtDNA molecules in vivo and that these distortions may provide a substrate for enzymes involved in transmission, recombination and/or transcription of mtDNA.
Biosynthesis of intestinal microvillar proteins Effect of castanospermine on cell‐free synthesis of aminopeptidase N
Authors: Danielsen E.Michael , Olsen Jørgen
Published: July 2002

Pig small intestinal mRNA was translated in a rabbit reticulocyte lysate system supplemented with microsomal membranes. Castanospermine, an inhibitor of glucosidase I, induced a high mannose‐glycosylated form of microvillar aminopeptidase N(EC 3.4.11.2) of increased molecular mass, indicating the blocked removal of glucose residues. In contrast to its reduced expression in a mucosal explant system [(1986) Biochem. J. 240, 777–782], this molecular form of aminopeptidase N was at least as abundant in cell‐free translation as its normal high mannose‐glycosylated counterpart, ruling out degradation taking place in the rough endoplasmic reticulum. Degradation of newly produced malprocessed enzyme must therefore occur at a later stage during intracellular transport, presumably in the sarcoplasmic reticulum or in transitional elements between this organelle the Golgi complex.
The influence of a double‐stranded hindrance on DNA synthesis performed by DNA polymerase α, T4 DNA polymerase, DNA polymerase I (Klenow fragment) and AMV reverse transcriptase
Authors: Scamrov Andrew V. , Beabealashvilli Robert S.
Published: July 2002

The influence of a double‐stranded region on DNA synthesis performed by a series of DNA polymerases on a single‐stranded template was studied. Two types of double‐stranded hindrances were employed: a stable hairpin formed by the template alone and a region formed by the template and an extraneous oligonucleotide complementary to the template. While T4 and calf thymus α DNA polymerases are strongly arrested at the beginning of either of the two double‐stranded hindrances, the Klenow fragment of E. coli DNA polymerase I and avian myeloblastosis virus reverse transcriptase can pass through the double‐stranded regions. The DNA chain‐elongation rate seems to be undisturbed in the case of reverse transcriptase but greatly reduced for DNA polymerase I.
The primary organization of nucleosomal core particles from actively dividing cells of lily
Authors: Bavykin S.G. , Undritsov I.M. , Usachenko S.I. , Strokov A.A. , Bogdanov Yu.F. , Mirzabekov A.D.
Published: July 2002

Nucleosomal core particles containing different variants of histones H2A and H2B were isolated from actively growing sepals of lily buds (Lilium candida, L.). The particles had decreased electrophoretic mobility relative to those from other sources. Comparison of the arrangement of histones along DNA in lily nucleosomes with previously obtained data for nucleosomes from yeast and animal cells, either active or repressed with regard to transcription and replication, has revealed no significant differences. The results suggest that the structure of nucleosomal core particles is highly conserved in all eukaryotes, whatever the functional state of the cell and the structure of histones H2A and H2B.
Phospholipid peroxidation as a factor in gallstone pathogenesis
Authors: Lichtenberg D. , Ragimova S. , Peled Y. , Halpern Z.
Published: July 2002

Phospholipid peroxidation markedly reduces the stability of mixed micellar systems composed of cholate, phosphatidylcholine and supersaturating levels of cholesterol. This suggests that lipid peroxidation is likely to play a significant role in the precipitation of cholesterol from gallbladder bile, thus in the pathogenesis of cholesterol gallstones. This conclusion is supported by studies of the nucleation time of cholesterol in gallbladder biles, which was significantly reduced by exposure to a stream of oxygen. This effect of phospholipid peroxidation on cholesterol solubility may occur in other biological fluids as well. In view of the increased lipid peroxidation in the elderly, it may explain the effect of age on the frequency of various diseases related to cholesterol precipitation.
Processing of chromogranin A within chromaffin granules starts at C‐ and N‐terminal cleavage sites
Authors: Wohlfarter Thomas , Fischer-Colbrie Reiner , Hogue-Angeletti Ruth , Eiden Lee E. , Winkler Hans
Published: July 2002

Specific antisera were raised against synthetic peptide fragments of bovine chromogranin A. The soluble proteins of bovine chromaffin granules were subjected to two‐dimensional immunoblotting with these antisera. The endogenous breakdown products of chromogranin A gave distinct patterns of immunostaining which enabled us to correlate these peptides with defined regions of the chromogranin A molecule. The results establish that within chromaffin granules degradation of chromogranin A by the endogenous proteases can start either at the C‐ or the N‐terminal site.
Inhibition of cholesterol side‐chain cleavage by intermediates of an alternative steroid biosynthetic pathway
Authors: van Haren E.J.M. , Tait A.D.
Published: July 2002

Mitochondrial preparations from endocrine tissues were incubated with radioactive cholesterol and the effect of hydroxylated metabolites of 23,24‐dinor‐5‐cholen‐3β‐ol (23,24‐dinor‐5‐cholene‐3β,20‐diol and 23,24‐dinor‐5‐cholene‐3β,21‐diol) on the production of pregnenolone was measured. These compounds are intermediates in an alternative, sesterterpene pathway for steroid hormone biosynthesis. It was found that these materials, like the analogous side‐chain‐hydroxylated derivatives of cholesterol (20α‐hydroxycholesterol and 22S‐hydroxycholesterol), inhibit cholesterol side‐chain cleavage. The possibility that there could be a control mechanism whereby metabolites of 23,24‐dinor‐5‐cholen‐3β‐ol inhibit steroidogenesis occurring by the cholesterol pathway is discussed.
The interaction of nuclear proteins with essential promoter element of the chicken cardiac myosin light chain 2 gene is involved in muscle‐specific transcription
Authors: Braun Thomas , Buschhausen-Denker Gregor , Tannich Egbert , Arnold Hans H.
Published: July 2002

A quantitative microinjection procedure has been developed to demonstrate muscle‐specific transcription of the myosin light chain 2‐A (MLC2‐A) promoter in differentiated chicken primary breast muscle cells. Nuclear protein binds to the distal region of the required promoter sequence but not to a mutated version of this sequence. The functional significance of this specific DNA‐protein interaction for the promoter activity is demonstrated by ‘in vivo’ competition of microinjected MLC‐CAT reporter construct together with excess of synthetic oligonucleotides encompassing the protein binding sites.
Transcellular ionic currents studied by intracellular potential recordings in Neurospora crassa hyphae Transfer of energy from proximal to apical cells
Authors: Potapova T.V. , Aslanidi K.B. , Belozerskaya T.A. , Levina N.N.
Published: July 2002

Membrane potentials, input resistances, and electric coupling in the apical parts of N.crassa growing hyphae were recorded with the aid of intracellular microelectrodes. It was revealed that the apical cells were always depolarized by 10 to 30 mV as compared to the adjacent proximal cells. The septal pore maintained an electrical resistance of 4 to 6 MΩ. The calculated values of the endogenous electrical current passing through the septal pore varied between 0.5 and 1 nA. Electrical isolation of the apical cells resulted in their depolarization from 120–150 mV to 40–60 mV, characteristics of the membrane potential value of N.crassa adult hyphae with completely blocked electrogenic pumps. A simultaneous increase in the input resistance value from 15–20 MΩ to 40–80 MΩ was observed. The above data can be explained assuming that H+‐ATPase activity was greatly lowered in the apical cells. Thus in the intact hyphae with electrically coupled cells energy is transferred from the proximal hyphal compartments to the apical ones.
Cyclic nucleotide phosphodiesterase activity in Neurospora crassa Purification by immunoaffinity chromatography and characterization
Authors: Ulloa Rita M. , Rubinstein Clara P. , Molina y Vedia Luis , Torres Héctor N. , Téllez-Iñón María T.
Published: July 2002

Monoclonal antibodies to Neurospora crassa cyclic nucleotide phosphodiesterase (PDE I) were selected by their capacity to inhibit the enzyme activity. The monoclonal immunoglobulin, coupled to Sepharose 4B, was used for the affinity purification of PDE I activity. After SDS‐polyacrylamide gel electrophoresis the affinity purified PDE I fractions showed a single polypeptide band of about 41 kDa. This band reacted in Western blots with the above mentioned monoclonal immunoglobulin.
Tyrosine sulfation is not the last modification of entactin before its secretion from 3T3‐L1 adipocytes
Authors: Aratani Yasuaki , Kitagawa Yasuo
Published: July 2002

Tyrosine sulfation of entactin was studied by labeling of 3T3‐L1 adipocytes with [35S]methionine or H2
35SO4 in the presence or absence of tunicamycin or monensin. Four precursors (EN1–4) at different steps of modification were detected in addition to mature entactin. Under normal conditions, EN2 and mature entactin were intracellular species, and the latter was sulfated and secreted. Inhibition of co‐translational transfer of N‐linked oligosaccharides by tunicamycin produced EN1 and EN3 as intracellular species, and EN3 was sulfated and secreted. Interruption of protein transport from medial to trans (distal) Golgi cisternae by monensin, and consequent blockage of terminal glycosylation caused intracellular accumulation of EN4. EN4 was sulfated and of different size compared to mature entactin. These facts suggested that tyrosine sulfation of entactin occurs in medial Golgi cisternae and is not the last modification before its secretion. Our results appeared inconsistent with recent observations by Baeuerle and Huttner [(1987) J. Cell Biol. 105, 2655–2664] that tyrosine sulfation of IgM occurred within the trans (distal) Golgi cisternae as the last modification before its exit from the Golgi complex.
Ongoing protein synthesis needed for 1,25‐(OH)2D3‐mediated rapid increase of cyclic GMP in human skin fibroblasts
Authors: Barsony Julia , Marx Stephen J.
Published: July 2002

Recently we reported that 1,25‐dihydroxyvitamin D3 (1,25‐(OH)2D3) through interaction with its specific receptor rapidly (within 1 min) stimulated intracellular cGMP production in cultured human skin fibroblasts. Here we show that this effect of 100 nM 1,25‐(OH)2D3 is prevented by brief (30 min) inhibition of RNA synthesis (with actinomycin D or α‐amanitin) or by brief inhibition of protein synthesis (with cycloheximide or diphtheria toxin). The protein synthesis inhibitors also blocked stimulation of cGMP by other steroids (testosterone or dexamethasone at 100 nM) but did not block cGMP stimulation by sodium nitroprusside. Since the time for the 1,25‐(OH)2D3 receptor to increase cGMP seems too short to require de novo protein synthesis, we conclude that the 1,25‐(OH)2D3 receptor acts together with rapidly turning over protein(s) to stimulate cGMP synthesis.
In vitro translation of cytosolic and peroxisomal epoxide hydrolase and catalase on liver polyribosomes from untreated and clofibrate‐treated C57B1/6 mice
Authors: Joste Vigg , Meijer Johan
Published: July 2002

Free and membrane‐bound polyribosomes were isolated from the livers of untreated and clofibrate‐treated male C57B1/6 mice. The in vitro translation products were investigated in a rabbit reticulocyte cell‐free system by immunoprecipitation of cytosolic epoxide hydrolase, catalase and albumin. The soluble forms of epoxide hydrolase present in cytosol and in peroxisomes were found to be synthesized on free polyribosomes and could not be distinguished from each other, since only one primary translation product was found with the methods used. Clofibrate treatment was found to increase total protein synthesis, synthesis of soluble epoxide hydrolase and translational efficiency of the isolated polyribosomes.
Similarities between the regulatory sequences of the unrelated tetracycline genes of pBR322 and TN1O
Authors: Hung A. , Pictet R.
Published: July 2002

The regulatory regions of the tetracycline genes present in pBR322 (pSC101) and in the transposon Tn10 are compared. They show a low degree of nucleotide sequence similarity but a high level of structure similarity. Furthermore, analyses of RNAs transcribed in the opposite direction of the pBR322 tet gene show that there are two mRNA initiation sites separated by 29 nucleotides. This suggests the existence of two promoters for the tet repressor gene in Tn10. These features reveal a strong resemblance of the mode of regulation between the tet operons of Tn10 and pSC101.
Modelling basic features of specificity in DNA‐aureolic acid‐derived antibiotic interactions
Authors: Chen K.-X. , Gresh N. , Hui X. , Pullman B. , Zakrzewska K.
Published: July 2002

The nonintercalative groove binding of a simplified model of olivomycin, to sequences d(CGCGCGC)2, d(TATATAT)2, and d(CICICIC)2, is investigated. A significant preference is displayed for the minor groove of the d(CG) sequence. This is due predominantly to the formation of H‐bonds between the hydroxyl groups on the aglycone of the drug and the 2‐amino group of the central guanine of the oligonucleotide.
The cysteine proteinase inhibitor chicken cystatin is a phosphoprotein
Authors: Laber Bernd , Krieglstein Kerstin , Henschen Agnes , Kos Janko , Turk Vito , Huber Robert , Bode Wolfram
Published: July 2002

Peptide maps obtained by reversed‐phase HPLC of tryptic digests of isoelectric form 1 (pI=6.5) and 2 (pI=5.6) of chicken egg white cystatin revealed that the difference was located only in a single peptide (residues Ser‐74—Lys‐91). Ser‐80 of cystatin 2 was subsequently identified as being modified by phosphorylation. Moreover, alkaline phosphatase treatment of a mixture of native cystatin forms 1 and 2 was shown by ion‐exchange chromatography to cause the disappearance of isoelectric form 2 with a concomitant increase in form 1. Thus, the existence of two isoelectric forms of chicken cystatin is due to the phosphorylated form 2 and non‐phosphorylated form 1.
Inhibitors of arachidonic acid metabolism eliminate the increase in cytosolic free calcium induced by the mitogen concanavalin A in rat thymocytes
Authors: Gukovskaya A.S. , Pulido H.Arias , Zinchenko V.P. , Evtodienko Yu.V.
Published: July 2002

Using inhibitors of arachidonic acid (AA) metabolism, the possible involvement of AA products in the generation of [Ca2+]i and the pHi rise induced by the mitogen concanavalin A (Con A) in rat thymocytes has been studied. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 μM) and the phospholipase A2 inhibitor bromophenacyl bromide (10 μM) eliminated the [Ca2+]i signal induced by Con A; the cyclooxygenase blocker indomethacin also inhibited it. However, neither NDGA nor indomethacin suppressed the pHi rise stimulated by Con A. Exogenous AA induced an increase in [Ca2+]i but not in the pHi. These results indicate that AA metabolites, probably of the lipoxygenase pathway, take part in the generation of the [Ca2+]i response to the mitogen. In contrast, they appear not to be involved in the pHi rise evoked by Con A.
ATP‐dependent peptide release from mitochondria of reticulocytes
Authors: Dubiel W. , Drung I. , Müller M. , Rapoport S.M.
Published: July 2002

ATP‐dependent release of TCA‐precipitable peptides from mitochondria‐containing stroma (MCS) is described. The process is independent of ubiquitin, but is sensitive to hemin and to heat treatment. Neither chloramphenicol nor EGTA inhibit. 50% of the activity is dependent on charged tRNA. The peptides released from MCS possess a molecular mass of about 1–5 kDa and are degraded to TCA‐soluble compounds by a cytosolic protease system (fraction II) without ubiquitin.
Methylation of elongation factor EF‐Tu affects the rate of trypsin degradation and tRNA‐dependent GTP hydrolysis
Authors: Toledo Héctor , Jerez Carlos A.
Published: July 2002

The role of methylation of elongation factor EF‐Tu from Escherichia coli was investigated. The methylated factor was obtained from cells harvested at the late stationary growth phase. Submethylated forms of the factor were obtained from either bacteria grown to the mid‐logarithmic phase or cells cultured in the presence of ethionine. When fully methylated EF‐Tu was treated in the presence of trypsin, it showed a greatly reduced rate of degradation compared with both types of undermethylated EF‐Tu factors. The methylation of EF‐Tu appeared not to affect the rate of GDP and GTP exchange. However, aminoacyl‐tRNA‐stimulated GTPase activity determined in the presence of kirromycin was greatly increased when the EF‐Tu was methylated.
Conformational changes occurring in N‐ras p21 in response to binding of guanine nucleotide and metal ions probed by proteolysis performed under controlled conditions
Authors: Grand Roger J.A. , Grant Michael L.
Published: July 2002

Variations in susceptibility to proteolysis by trypsin and chymotrypsin have been used as indicators of conformational changes taking place in N‐ras p21 in response to ligand binding. It has been observed that changes occur in undenatured protein, rendering it more resistant to degradation, in the presence of divalent cations such as Mg2+ and Ca2+ (suggesting direct binding of metals to the polypeptide) and even more markedly in the presence of GDP and/or Mg2+ GD. Monovalent cations (Na+ or K+) cannot substitute for Mg2+ or Ca2+. Some capacity to bind guanine nucleotide is also retained by p21 treated with 7 M urea, as evidenced by increased resistance to proteolytic degradation, but the ability to bind divalent cations is irreversibly lost following denaturation. Protein prepared under denaturing conditions from a eukaryotic source, however, never regains the resistance to proteolysis shown by the bacterial p21 indicating irreversible changes in secondary and tertiary structure produced under these conditions.
AVP receptors of mouse Leydig cells are regulated by LH and E2 and influenced by experimental cryptorchidism
Authors: Tahri-Joutei Abderrafih , Pointis Georges
Published: July 2002

Exposure of pubertal mouse Leydig cells for 24 h to increasing concentrations (1–100 ng/ml) of LH elicited a dose‐dependent decrease in AVP receptor content. Maximal reduction (50%) was obtained at a dose of 10 ng/ml LH. A similar treatment applied to adult Leydig cells did not influence AVP receptor density. Treatment of adult Leydig cells for 24 h by E2 (5–500 ng/ml) resulted in a dose‐dependent increase in AVP receptor content. About 50% increase was achieved with 500 ng/ml E2. AVP receptor content in pubertal Leydig cells was not modified irrespective of the concentration of E2 tested. These changes in AVP receptor number were well correlated with the response of Leydig cells to AVP (10−6M) in terms of testosterone production. 2 weeks bilateral cryptorchidism resulted in reduction of testicular weight, circulating testosterone levels associated with a marked rise in Leydig cell AVP receptor density with no change of affinity. Testosterone production by Leydig cells from cryptorchid testes in response to AVP (10−6 M) or hCG (100 ng/ml) stimulation was reduced compared to that of control Leydig cells. This study provides new arguments supporting the concept that AVP could be involved in local regulation of testicular steroidogenesis.
Primary structures of new ‘iso‐hirudins’
Authors: Scharf M. , Engels J. , Tripier D.
Published: July 2002

Crude hirudin (12.7 U/μg), a complex mixture of polypeptides obtained from the leech, could be separated by microbore HPLC. A combination of amino acid analysis, N‐terminal microsequencing and chemical as well as enzymatic fragmentation made the primary sequence of the new isohirudins Ia‐IIIb' accessible. The biological activity determined in the thrombin inhibition test showed a comparable value for all of these compounds. The results presented address the question as to whether these isohirudins are true mutations from a family of genes or a family of leeches.
Inhibitory domain‐specific antibodies to cytochrome P‐450scc
Authors: Usanov Sergey A. , Chemogolov Alexey A. , Chashchin Vadim L.
Published: July 2002

Highly specific antibodies to cytochrome P‐450scc and its F1 and F2 fragments, representing N‐ and C‐terminal sequences of the hemeprotein respectively, were raised in rabbits. These antibodies were found to be inhibitory (up to 50–90%) for the cholesterol transformation into pregnenolone in the reconstituted system, indicating the involvement of both F1 and F2 domains formed by the respective fragments in monooxygenase catalysis. Cytochrome P‐450scc in mitoplasts is not accessible for trypsin as revealed by immunological techniques. However, the treatment of submitochondrial particles with trypsin results in two main fragments identified by immunoblotting in the presence of the monospecific antibodies as F1 and F2 fragments. This indicates that the trypsin sensitive 250–257 region in cytochrome P‐450scc molecule connecting both domains is exposed to the matrix side of the inner mitochondrial membrane.
Phosphorylation of tyrosine prevents dityrosine formation in vitro
Authors: Gmeiner Bernhard , Seelos Christian
Published: July 2002

Treatment of L‐tyrosine in a peroxidase/H2O2, system results in the formation of dityrosine. However, the phosphoester derivative of tyrosine, O‐phospho‐L‐tyrosine, was unable to form dityrosine even in mixtures with free L‐tyrosine. Dephosphorylation of O‐phospho‐L‐tyrosine by alkaline phosphatase followed by horseradish peroxidase/H2O2, treatment resulted in the formation of dityrosine. Our in vitro results indicate that phosphorylation/dephosphorylation of L‐tyrosine may regulate dityrosine formation, and is supposed to play an important role in protein‐protein interactions, i.e. cross‐linking.
Monovalent cations (Na+, K+, Cs+) inhibit calcium activation of photosynthetic oxygen evolution
Authors: Waggoner Charlene M. , Pecoraro Vincent , Yocum Charles F.
Published: July 2002

In the absence of the 17 and 23 kDa water‐soluble polypeptides, the oxygen‐evolving complex of photosystem II requires addition of Cl− and Ca2+ to maximize rates of oxygen evolution activity. We have found that monovalent cations (Na+, K+, Cs+) inhibit the calcium‐binding site responsible for activation of the oxygen‐evolving complex. K+ inhibits in a purely competitive manner, while Na+, with an ionic radius much closer to that of calcium, displays mixed‐type inhibition kinetics. The K
i values for Na+ and K+ have been estimated to be in the range of 8–10 mM. These values are well below the concentrations of these ions commonly used in buffer titrations and Cl− additions to standard oxygen evolution assay media and as such could alter the interpretation of data regarding calcium activation of oxygen evolution activity. An organic cation, tetramethylammonium, does not inhibit calcium activation and can be substituted for the inorganic cation as a Cl− counterion in oxygen evolution assays.
Further characterization of monoclonal antibody 6,F‐8 reacting with Lathyrus, Lens and Pisum lectins
Authors: Kolberg Jan , Rougé Pierre
Published: July 2002

The murine monoclonal antibody (MoAB) 6,F‐8 made against the glucose/mannose‐specific Lathyrus odoratus mitogen has previously been shown to react with Lens culinaris and Pisum sativum lectins, but not with the lectin from Vicia faba [(1988) Biol. Chem. Hoppe‐Seyler 369, 365–370]. The reactivity against seven other completely sequenced Lathyrus lectins has now been tested after separation of the subunits by SDS‐polyacrylamide gel electrophoresis and electroblotting to nitrocellulose filters. Two of these lectins reacted with the antibody. Comparison of the amino acid sequences of the examined lectins and the predicted hydrophilic, flexible and accessible regions of Pisum sativum suggest that valine‐147 is involved in antibody binding.
Enhanced transcription of c‐myc proto‐oncogene in spleen lymphocytes from lupus‐prone mice during the growing process
Authors: Deguchi Yasuhiro , Kishimoto Susumu
Published: July 2002

The expression of c‐myc proto‐oncogene in spleen lymphocytes has been studied in lupus‐prone mice (MRL/Mp‐lpr/lpr), an animal model for the human autoimmune disease systemic lupus erythematosus, during the growing process, in comparison to control mice (MRL/Mp‐+/+). By Northern blot assay and nuclear run on transcription assay, we demonstrated the enhancement of c‐myc proto‐oncogene expression in spleen lymphocytes from lupus‐prone mice in comparison to control mice and the level of expression of c‐myc proto‐oncogene increased during the growing process and deterioration of lupus symptoms, such as production of autoantibodies and lymphoproliferation, in this study.
Evidence for the convergence of β‐adrenergic and muscarinic signalling systems at a post‐receptor site
Authors: Horn Valerie J. , Baum Bruce J. , Ambudkar Indu S.
Published: July 2002

The β‐adrenergic agonist isoproterenol stimulates inositol trisphosphate (IP3) formation and cytosolic Ca2+ ([Ca2+]i) mobilization in rat parotid acini via a cAMP‐dependent process. Atropine, a muscarinic antagonist, inhibited these isoproterenol responses without affecting isoproterenol‐induced amylase secretion or peak [Ca2+]i and IP3 responses elicited by α1‐adrenergic stimulation with epinephrine. Atropine had no effect on isoproterenol‐induced [Ca2+]i responses in a cell line which lacked muscarinic receptors and did not alter β‐adrenoreceptor ligand binding. These results suggest that the inhibition by atropine results from a post‐receptor effect on cAMP‐mediated stimulation of phosphatidylinositol 4,5 bisphosphate (PIP2) hydrolysis.
Characterization and N‐terminal sequence of a 5 kDa polypeptide in the photosystem I core complex from spinach
Authors: Hoshina Satoshi , Sue Shichijyo , Kunishima Naoki , Kamide Kaeko , Wada Keishiro , Itoh Shigeru
Published: July 2002

Photosystem I core complexes were isolated from spinach photosystem I particles after heat treatment in the presence of 50% (v/v) ethylene glycol (heat/EG treatment). The core complex from 58°C/EG‐treated particles was composed of polypeptides with apparent molecular masses of 63, 60 and 5 kDA; this complex contained the iron sulfur center Fx but lacked center FA and FB. The core complex obtained from the 70°C/EG‐treated preparation lacked Fx and contained a lesser amount of the 5 kDa polypeptide. The N‐terminal amino acid sequence of the 5 kDa polypeptide did not correspond to the sequence derived from any possible reading frame in the chloroplast DNA of liverwort or tobacco. Twelve of the first 29 N‐terminal amino acids were hydrophobic, suggesting that this protein is intrinsic to the photosystem I reaction center.
Prolonged ischemia differently affects phospholipase C acting against phosphatidylinositol and phosphatidylinositol 4,5‐bisphosphate in brain subsynaptosomal fraction
Authors: Strosznajder Joanna
Published: July 2002

Ischemia; Subsynaptosomal fraction; Phospholipase C
Potassium chemotaxis in Rhodobacter sphaeroides
Authors: Poole Philip S. , Brown Simon , Armitage Judith P.
Published: July 2002

The addition of potassium to Rhodobacter sphaeroides in the absence of magnesium increased the membrane potential, as measured by the carotenoid bandshift. In the presence of magnesium, potassium decreased the membrane potential. Potassium stimulated the mean population swimming speed by 33% and 48% in the presence or absence of magnesium, respectively. Chemotactic responses of R. sphaeroides to potassium were also unaffected by the presence or absence of magnesium. These results suggest that the membrane potential and by inference the ΔpH and ΔμH + are not involved in chemotactic signalling towards potassium.
Role of sialic acid residues in crossed immuno‐affinoelectrophoresis of α1‐proteinase inhibitor
Authors: Mallet B. , Franc J.L. , Zattara M.C.
Published: July 2002

We have investigated the importance of the degree of sialylation when an acute phase glycoprotein, α1‐proteinase inhibitor (α1‐Pi), was analysed both by affinity chromatography on concanavalin A (Con A)‐Sepharose and by crossed immuno‐affinoelectrophoresis (CIAE) using Con A in the first dimension. Human α1‐Pi was isolated by immunosorption chromatography and then more or less desialylated. On Con A‐Sepharose chromatography no significant difference was observed in the percentage of the two fractions (retained or not retained) whatever the degree of desialylation. In contrast by CIAE this degree was largely involved in the separation of the different isoforms obtained in the first dimension.
Changes in chromatin chain flexibility during condensation induced by sodium chloride, as evidenced by electric dichroism
Authors: Marquet Roland , Favazza Marisa , Koch Michel H.J. , Houssier Claude
Published: July 2002

The electric linear dichroism of chicken erythrocyte chromatin has been measured as a function of NaCl concentration in the 1–28 mM ionic strength range, using a specially designed Kerr cell with reduced pathlength, and thus, smaller electrode surface. This allowed the determination of the dichroism of compact chromatin in conditions where artifacts due to possible contribution from turbidity are avoided, which was not the case for previous studies in the presence of di‐ or multivalent cations. The linear dichroism of compact chromatin was found to be positive, as expected from models of the 30‐nm fibre in which the linker DNA runs perpendicular to the fibre axis. The dependence of the relaxation times on ionic strength reveals that the process of compaction is first accompanied by an increase in flexibility of the chain followed by a decrease, in the range of 5–10 mM NaCl, and a further decrease above 10 mM NaCl, corresponding to the compaction of the 30 nm fibre.
Stereoselective carbonyl reductases from rat skin and leukocyte microsomes converting 12‐ketoeicosatetraenoic acid to 12(S)‐HETE
Authors: Falgueyret Jean-Pierre , Leblanc Yves , Riendeau Denis
Published: July 2002

Cell‐free preparations from rat polymorphonuclear leukocytes and skin were found to catalyze the reduction of 12‐keto‐5,8,10,14‐eicosatetraenoic acid (12‐KETE) to 12‐hydroxyeicosatetraenoic acid (12‐HETE). The reductase activity was associated with the microsomal fraction and showed a marked preference for NADH over NADPH as reducing cofactor. Characterization of the reaction product by chiral phase HPLC of the methyl ester derivative indicated that 12‐KETE reduction generated almost exclusively 12(S)‐HETE. The results demonstrate that rat skin and leukocyte microsomes possess an NADH‐dependent 12‐KETE reductase activity that forms 12(S)‐HETE as a major product. The identification of Stereoselective 12‐KETE reductases provides a basis for further defining the role these enzymes may play in the regulation of 12‐KETE levels and in the protection against degradation of 12‐KETE to the pro‐inflammatory 12(R)‐HETE by selectively generating 12‐HETE of the S configuration.
Novel GTP‐binding proteins in plasma membranes and zymogen granule membranes from rat pancreas and in pancreatic AR 4‐2J cell membranes
Authors: Lambert Monique , Bui Ngoc-Diem , Christophe J.
Published: July 2002

Photoaffinity labelling with [α‐32P]GTP allowed to detect a 54 kDa GTP‐binding protein in rat pancreatic plasma membranes and in pancreatic AR 4‐2J cell membranes. Like the 42 and 48 kDa Gsα subunits and the 41 kDa Giα subunit, this protein was absent from zymogen granule membranes. Contrastingly, a new 28 kDa GTP‐binding protein (detected by [α‐32P]GTP binding on immobilized proteins) and a 25 kDa protein (ADP‐ribosylated by botulinum toxin D) were found in all three membrane preparations. This is to our knowledge the first report on GTP‐binding proteins in zymogen granule membranes.
Packing forces in ribonuclease crystals
Authors: Crosio Marie-Pierre , Rodier Francis , Jullien Magali
Published: July 2002

Packing in Ribonuclease A and Ribonuclease S crystals have been compared in order to determine the possible role of the precipitant on lattice contacts. Both proteins have similar tertiary structures, buth they crystallize in different space groups depending on the precipitating agent. It is found that packing differs either by the number of nearest neighbours or by the size of surface areas buried in individual contacts. Ammonium sulfate seems to promote hydrophobic interactions with interfaces similar to those found in oligomeric proteins. Organic precipitants favour electrostatic interactions.