bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of glycogenolysis in hepatocytes by angiotensin II may involve both calcium release and calcium influx
Authors: DeWitt Linda M. , Putney James W.
Published: July 2002

The stimulation of [3H]glucose release (a measure of glycogenolysis) from isolated guinea pig hepatocytes by Ca‐mobilizing agonists can be resolved into two phases. The initial transient phase is independent of extracellular Ca, and is probably a result of Ca released from an intracellular pool. The second phase occurs only in the presence of extracellular Ca, which suggests that Ca‐influx is also involved in the mechanism of Ca‐mobilization by these agents in the guinea pig hepatocyte.
Characterization of a polypeptide from human seminal plasma with inhibin (inhibition of FSH secretion)‐like activity
Authors: Sheth Anil R. , Arabatti Nandita , Carlquist Mats , Jörnvall Hans
Published: July 2002

A polypeptide in preparations of ‘large’ form (M
r ∼14 000) material with inhibin‐like activity (inhibiting FSH secretion) has been isolated from human seminal plasma. Its amino acid composition, cleavage pattern with CNBr, N‐terminal sequence, and properties on reverse‐phase high‐performance liquid chromatography establish this inhibin‐like preparation to be homogeneous. The polypeptide contains close to 130 residues, has a free N‐terminal serine residue, a methionine residue in position 19, and a dibasic structure (Arg—Lys) in positions 16–17.
Two protons are pumped from the mitochondrial matrix per electron transferred between NADH and ubiquinone
Authors: Wikström Mårten
Published: July 2002

The spectrophotometric indicators neutral red and safranine were used to determine the relative H+/e− ratios of proton uptake from the mitochondrial matrix, and the q+/e− ratios of electrical charge translocation, during oxidation of β‐OH‐butyrate and succinate by ferricyanide in rat liver mitochondria. With β‐OH‐butyrate both ratios were higher than with succinate by a factor close to 3.0. Since there is full agreement that H+/e− of proton uptake and q+/e− of charge translocation are both equal to unity for oxidation of succinate (or ubiquinol) by ferricytochrome c, the corresponding ratios for oxidation of NADH by ubiquinone and cytochrome c are near 2.0 and 3.0, respectively.
Increased translational fidelity caused by the antibiotic kasugamycin and ribosomal ambiguity in mutants harbouring the ksgA gene
Authors: van Buul C.P.J.J. , Visser W. , van Knippenberg P.H.
Published: July 2002

The aminoglycoside kasugamycin, which has previously been shown to inhibit initiation of protein biosynthesis in vitro, also affects translational accuracy in vitro. This is deduced from the observation that the drug decreases the incorporation of histidine relative to alanine into the coat protein of phage MS2, the gene of which is devoid of histidine codons. The read‐through of the MS2 coat cistron, due to frameshifts in vitro, is also suppressed by the antibiotic. In contrast, streptomycin enhances histidine incorporation and read‐through in this system. The effects of kasugamycin take place at concentrations that do not inhibit coat protein biosynthesis. Kasugamycin‐resistant mutants (ksgA) lacking dimethylation of two adjacent adenosines in 16 S ribosomal RNA, show an increased leakiness of nonsense and frameshift mutants (in the absence of antibiotic). They are therefore phenotypically similar to previously described ribosomal ambiguity mutants (ram).
Hydroxyl radical production and DNA damage induced by anthracycline‐iron complex
Authors: Muindi Josephia R.F. , Sinha Birandra K. , Gianni Luca , Myers Charles E.
Published: July 2002

Adriamycin‐Fe3+ complex catalyzes the formation of hydroxyl radical from hydrogen peroxide but the DNA‐adriamycin‐iron ternary complex is much more effective. 11‐Deoxyadriamycin, which shows no spectral evidence of complex formation with iron, was ineffective. The generation of hydroxyl radical by adriamycin‐Fe3+ complex in the presence of DNA correlates with its ability to cleave DNA. Hydroxyl radicals are thus implicated as the reactive oxygen species involved in the DNA damage caused by the adriamycin‐Fe3+ complex.
SDS—PAGE strongly overestimates the molecular masses of the neurofilament proteins
Authors: Kaufmann Eckhard , Geisler Norbert , Weber Klaus
Published: July 2002

Direct molecular mass determination of the three porcine neurofilament proteins (H, M and L) was performed in 6 M guanidine—HCl using analytical gel filtration and sedimentation equilibrium centrifugation. The results show that SDS—PAGE strongly overestimates the values of the ‘higher molecular mass’ components H and M. This discrepancy stems from the carboxyterminal extensions known to have unusual amino acid composition.
Effects of GTP,GDP[βS] and glucose on adenylate cyclase activity of E. coli B
Authors: Stein Janet M. , Kornberg H.L. , Martin B.R.
Published: July 2002

Adenylate cyclase activity was measured in suspensions of E. coli B, rendered permeable with toluene. The enzyme was activated in a dose‐dependent manner by GTP and by its non‐hydrolysable analogue, GTP[γS]. In contrast, incubation with GDP[βS], a non‐phosphorylatable analogue of GDP, caused a dose‐related inhibition of adenylate cyclase; this was partially overcome by addition of GTP. GTP did not relieve, and GDP[βS] augmented, the non‐competitive and dose‐related inhibition of E.coli adenylate cyclase by glucose.
Uptake of rat and human α2‐macroglobulin‐trypsin complexes into rat and human cells
Authors: Gliemann Jørgen , Davidsen Ole , Sottrup-Jensen Lars , Sonne Ole
Published: July 2002

Uptake of rat and human α2‐macroglobulin‐trypsin complexes was measured in rat hepatocytes, rat and human adipocytes and human fibroblasts. Uptake and degradation of 125I‐labelled rat complex were about one‐third of that of the human complex in the various isolated cell types. In rat hepatocytes, the apparent K
m for cell association of the rat complex was about 16 nM as compared to about 6 nM for the human complex. The V
max values were similar, about 1 × 104 molecules · cell−1 · min−1. Thus, rat α2‐macroglobulin (an acute‐phase protein) complexed with trypsin follows the same pathways of uptake as the human homologue, although with a somewhat lower affinity for the uptake system.
Involvement of arginine residues in inhibition of protein synthesis by ricin A‐chain
Authors: Watanabe Keiichi , Funatsu Gunki
Published: July 2002

Modifications of arginine residues in ricin A‐chain with phenylglyoxal (PGO) and 1,2‐cyclohexanedione (CHD) caused a marked loss in its inhibitory activity on cell‐free protein synthesis. The loss of activity caused by modification with PGO was much faster than the loss of total arginine residues. More than 90% activity was lost with PGO modification of about three arginine residues. Regeneration of arginine residues from the CHD‐modified residues resulted in complete recovery of the activity. These results strongly suggest the involvement of definite arginine residue(s) in A‐chain activity. Analysis of the peptides, produced by peptic digestion of the [14C]PGO‐modified A‐chain, showed that some of the six arginine residues in the N‐terminal region of A‐chain react with PGO faster than other arginine residues.
Formation of formaldehyde from S‐adenosyl‐L[methyl‐3H]methionme during enzymic transmethylation of histamine
Authors: Huszti Z. , Tyihák E.
Published: July 2002

The experiments described here show that radiolabelled formaldemethone is formed from the radiolabelled methyl group of S‐adenosyl‐L‐methionine in the course of the enzymic conversion of histamine to N
τ‐methylhistamine in the presence of dimedone suggesting that the formation of HCHO is probably linked to the enzymic transmethylation of histamine.
Cytosolic Ca2+ and the regulation of secretion in parathyroid cells
Authors: Nemeth E.F. , Scarpa Antonio
Published: July 2002

(Parathyroid cell)
Secretion
cytosolic Ca2+

Quin‐2
Fura‐2
Receptor
A new method for the selective isolation of phosphoserine‐containing peptides
Authors: Holmes Charles F.B.
Published: July 2002

Meyer et al. [(1986) FEBS Lett 204, 61–66] have shown that the phosperine can be converted to S‐ethylcysteine by β‐elimination and addition of ethanethiol. I have utilised this modification to develop a rapid method for the selective purification of phosphoserine‐containing peptides from complex mixtures. Changing phosphoserine to S‐ethylcysteine increases the hydrophobicity of a peptide, altering its mobility during reverse‐phase chromatography. The number of S‐ethylcysteine residues in a peptide can be qualified at the picomolar level, following acid hydrolysis and conversion to the phenylthiocarbamyl derivative. The procedure may be particularly powerful for the analysis of peptides that are phosphorylated at multiple sites in vivo.
Polycation‐dependent, Ca2+‐antagonized phosphorylation of calmodulin by casein kinase‐2 and a spleen tyrosine protein kinase
Authors: Meggio Flavio , Brunati Anna Maria , Pinna Lorenzo A.
Published: July 2002

Ten distinct protein kinases have been tested for their ability to phosphorylate calmodulin. Only casein kinase‐2 and a spleen tyrosine protein kinase (TPK‐III) proved effective, their phosphorylation efficiency being dramatically enhanced by histones and other polybasic peptides while being depressed by 50 μM Ca2+. Phosphorylation by CK‐2 takes place with a Km of 12 μM calmodulin, leading to the incorporation of more than 1.5 mol P/mol substrate. Ser81 and Thr79 are among the residues affected. On the other hand, the two tyrosyl residues of calmodulin are both phosphorylated by TPK‐III, Tyr99 being preferred over Tyr138.
1H NMR characterization of two crambin species
Authors: Vermeulen J.A.W.H. , Lamerichs R.M.J.N. , Berliner L.J. , De Marco A. , Llinás M. , Boelens R. , Alleman J. , Kaptein R.
Published: July 2002

Crambin displays amino acid heterogeneity at positions 22 (Pro or Ser) and 25 (Leu or Ile). Using reversed phase HPLC the crambin mixture can be resolved into two protein fractions. It is shown by amino acid analysis and NMR spectroscopy that these fractions represent single proteins (Ser‐22/Ile‐25 and Pro‐22/ Leu‐25 species). A first characterization of the 1H‐NMR spectra of these species is presented.
Expression of β‐nerve growth factor mRNA in rat glioma cells and astrocytes from rat brain
Authors: Yamakuni Tohru , Ozawa Fumiko , Hishinuma Fumio , Kuwano Ryozo , Takahashi Yasuo , Amano Takehiko
Published: July 2002

A 50‐base synthetic oligodeoxynucleotide complementary to a portion of mouse nerve growth factor (NGF) mRNA was used as a probe for analysis of the expression of NGF gene. Northern blot analysis showed the presence of a major 1.3 kb transcript, which was identical in size to mouse NGF mRNA, in both C6Bu1 cells and rat astrocytes cultured from newborn rat brain. Further, the rearrangement of DNA sequence in and around the NGF gene locus of C6Bu1 cells was not detected by Southern blot analysis. These results indicate the expression of NGF mRNA in both C6Bu1 cells and astrocytes from rat brain, suggesting that astrocytes may produce NGF protein in the rat brain, especially in developing rat brain.
The 8 kDa polypeptide in photosystem I is a probable candidate of an iron‐sulfur center protein coded by the chloroplast gene frxA
Authors: Oh-oka Hirozo , Takahashi Yasuhiro , Wada Keishiro , Matsubara Hiroshi , Ohyama Kanji , Ozeki Haruo
Published: July 2002

The N‐terminal sequence of the 8 kDa polypeptide isolated from spinach photosystem I (PS I) particles was determined by a gas‐phase sequencer. The sequence showed the characteristic distribution of cysteine residues in the bacterial‐type ferredoxins and was highly homologous to that deduced from the chloroplast gene frxA of liverwort, Marchantia polymorpha. It is strongly suggested that the 8 kDa polypeptide has to be an apoprotein of one of the iron‐sulfur center proteins in PS I particles.
Rat liver membranes contain a 120 kDa glycoprotein which serves as a substrate for the tyrosine kinases of the receptors for insulin and epidermal growth factor
Authors: Phillips Susan A. , Perrotti Nicola , Taylor Simeon I.
Published: July 2002

The receptors for insulin and epidermal growth factor possess tyrosine‐specific protein kinase activity which may play a role in mediating the biological actions of these two peptides. We have identified a 120 kDa glycoprotein (pp120) in rat liver plasma membranes which can be phosphorylated by the insulin receptor in a cell‐free system and in intact cultured hepatoma cells. In the present report, we have demonstrated in a cell‐free system that solubilized epidermal growth factor receptors can phosphorylate tyrosine residues inpp120.
Two ionic forms of exoglucanase in yeast secretory mutants
Authors: Larriba Germán , Ramírez Manuel , Hernández Luis M. , Olivero Isabel , Basco Ricardo D.
Published: July 2002

Analysis of exoglucanase activity accumulated by sec mutants from Saccharomyces cerevisiae revealed the presence of two ionic forms of the major exoglucanase (exo II) secreted into the culture medium. From the accumulation pattern of representative sec mutants and the carbohydrate composition it appears that the less acidic form is converted into the more acidic one by addition of one phosphate to one of the oligosaccharide cores as the enzyme progresses through the secretory pathway. Exoglucanase I, the heavier isoenzyme, was not accumulated by the mutants. Accordingly, it should arise from exoglucanase II after the execution point of sec1 mutation.
Chicken liver basic fatty acid‐binding protein (pI = 9.0) Purification, crystallization and preliminary X‐ray data
Authors: Scapin Giovanna , Spadon Paola , Pengo Licia , Mammi Mario , Zanotti Giuseppe , Monaco Hugo L.
Published: July 2002

Chicken liver basic fatty acid‐binding protein (pI = 9.0) has been purified with a high yield by a modification of a method originally applied to rat liver. The final product is highly homogeneous and can be used to grow crystals that belong to two different space groups. The crystals are either tetragonal, space group P42212 with a = b = 60.2 Å and c = 138.1 Å or orthorhombic, space group P212121 with a = 60.7 Å, b = 40.1 Å and c = 66.7 Å. The second form appears to be more suitable for X‐ray diffraction studies, it diffracts to at least 2.8 Å resolution and it is believed to contain one protein molecule in the crystallographic asymmetric unit.
Brain natriuretic peptide interacts with atrial natriuretic peptide receptor in cultured rat vascular smooth muscle cells
Authors: Hirata Yukio , Shichiri Masayoshi , Emori Toshiaki , Marumo Fumiaki , Kangawa Kenji , Matsuo Hisayuki
Published: July 2002

The effect of synthetic porcine brain natriuretic peptide (pBNP), a novel brain peptide with sequence homology to α‐human atrial natriuretic peptide (hANP), on receptor binding and cGMP generation, was studied in cultured rat vascular smooth muscle cells (VSMC) and compared with that of α‐hANP. 125I‐pBNP bound to the cells in a time‐dependent manner similar to that of 125I‐α‐hANP. Scatchard analysis indicated a single class of binding sites for pBNP with affinity and capacity identical to those of α‐hANP. pBNP and α‐hANP were almost equipotent in inhibiting the binding of either radioligand and stimulating intracellular cGMP generation. These data indicate that BNP and ANP interact with the same receptor sites to activate guanylate cyclase in rat VSMC.
Active center differences between cathepsins L and B: The S1 binding region
Authors: Kirschke Heidrun , Wikstrom Peter , Shaw Elliott
Published: July 2002

The substrate peptide bond cleaved by cathepsins B and L is determined not by the amino acid contributing the carboxyl group to this bond as in the case of serine proteases but rather by the presence of a neighboring amino acid with a large hydrophobic side chain. From a study of the inhibitory potency in a series, Cbz‐Phe‐X‐CHN2, in which Phe promotes binding at S2 (terminology of [(1968) Biochem. Biophys. Res. Commun. 32, 898–902]) while the amino acid X probes S1, it is shown that this region of cathepsin L also has the ability to accommodate large hydrophobic side chains. In this respect cathepsin L differs from cathepsin B. Thus Cbz‐Phe‐Tyr(O‐t‐Bu)CHN2 inactivates cathepsin L with a rate 2.5 × 104 greater than that for cathepsin B.
Interaction between the δ‐endotoxin produced by Bacillus thuringiensis ssp. entomocidus and liposomes
Authors: Yunovitz Hermona , Yawetz Aminadav
Published: July 2002

The δ‐endotoxin produced by Bacillus thuringiensis ssp. entomocidus induced the release of encapsulated [14C]sucrose from reverse‐phase vesicles composed of phosphatidylcholine and cholesterol. No such release was detected when the phospholipid component of the vesicles was either phosphatidylethanolamine, phosphatidylglycerol, or sphingomyelin. The toxin‐induced release was competitively inhibited by negatively charged organic ions while positively charged organic ions, apart from choline chloride, had no such effect. The existence of a polar head group in the phospholipid as well as intermolecular hydrogen bonding at the membrane surface, was found to be of major importance in the toxin‐liposome interaction.
Increased calmodulin synthesis in the pre‐replicative phase of rat liver regeneration
Authors: Piñol M.Rosa , Berchtold Martin W. , Bachs Oriol , Heizmann Claus W.
Published: July 2002

A cytosolic calmodulin wave has been described in the pre‐replicative phase of rat liver regeneration. Here we demonstrate that this calmodulin increase is reduced by injection of cycloheximide (translation inhibitor) and actinomycin D (transcription inhibitor). In addition, we found that calmodulin mRNA levels are increased during the early pre‐replicative period of liver regeneration, and this increase is affected by actinomycin D. Together, these results strongly indicate that the calmodulin wave observed in the pre‐replicative phase of liver regeneration is due to de novo synthesis of this protein.
A model for the δ‐receptor‐bound conformation of enkephalin
Authors: Nikiforovich G.V. , Balodis J.
Published: July 2002

Sets of low‐energy structures were determined by energy calculations for two cyclic analogues of enkephalin (Ek), [D‐Pn5]‐Ek and [D‐Pn5]‐Ek, possessing the highest specificity towards δ‐opioid receptors. Comparison of mutual spatial orientations of the α‐amino group and aromatic moieties of the Tyr and Phe residues permitted one to suggest a model for the δ‐receptor‐bound conformation of enkephalin‐related peptides. The model involves a pronounced γ‐like turn of the peptide backbone centred on the Gly3 residue.
Inhibition by staurosporine of mitogen‐induced calcium mobilisation in human T lymphoblasts
Authors: Ward Stephen G. , Cantrell Doreen , Westwick John
Published: July 2002

Addition of monoclonal antibodies (UCHT1) directed against the antigen receptor/CD3 complex or the addition of the mitogens concanavalin A or phytohemagglutinin to human T lymphoblasts induced an elevation of the cytosolic free calcium concentration ([Ca2+]i) as monitored by the fluorescence of the intracellular fura‐2. The rise in [Ca2+]i induced by these agents was effectively inhibited by staurosporine, an agent which inhibits protein kinase (PK)C and PKA. Conversely the PKC activator 12‐O‐tetradecanoyl phorbol‐13‐acetate decreased the delay and accelerated the rate of elevation of [Ca2+]i induced by the mitogens and UCHT1. Increasing the concentration of cAMP or cGMP in the T lymphoblasts had no effect on agonist‐induced elevation of [Ca2+]i. Our data indicate that PKC can exert a positive feedback on the level of cytosolic Ca2+ in T lymphocytes, in contrast to what has been observed with other cells.
Negative interactions between phosphorylation of acetyl‐CoA carboxylase by the cyclic AMP‐dependent and AMP‐activated protein kinases
Authors: Munday Michael R. , Carling David , Hardie D.Grahame
Published: July 2002

We have reported previously that cyclic AMP‐dependent protein kinase phosphorylates two sites on acetyl‐CoA carboxylase (site 1: Arg‐Met‐Ser(P)‐Phe, and site 2: Ser‐Ser(P)‐Met‐Ser‐Gly‐Leu), while the AMP‐activated protein kinase also phosphorylates site 1, plus site 3 (Ser‐Ser‐Met‐Ser(P)‐Gly‐Leu), the latter being two residues C‐terminal to site 2. We now report that prior phosphorylation of site 2 by cyclic AMP‐dependent protein kinase prevents the subsequent phosphorylation of site 3 and the consequent large decrease in V
max produced by the AMP‐activated protein kinase. Similarly, prior phosphorylation of site 3 by the AMP‐activated protein kinase prevents subsequent phosphorylation of site 2 by cyclic AMP‐dependent protein kinase.
Δψ‐dependent gating of Na+/H+ exchange in Halobacterium halobium: a Δg̃mH+‐driven Na+ pump
Authors: Konishi Tetsuya , Murakami Naoyuki
Published: July 2002

Na+/H+ antiporter‐mediated 22Na+ transport was studied in envelope vesicles from Halobacterium halobium by manipulating the size of each Δg̃m+ component, ΔpH and Δψ, in the dark. Neither inside alkaline ΔpH nor outwardly directed ΔpNa+, nor a combination could facilitate 22Na+ extrusion from the vesicles. Likewise, Δψ up to 144 mV (inside negative) was not capable of initiating 22Na+ extrusion unless ΔpH existed. This extrusion was facilitated only when approx. 100 mV Δψ (gating potential) was superimposed on ΔpH (either 1 or 2). On the other hand, no uptake of 22Na+ took place even when both inside acidic ΔpH and inwardly directed Na+ gradient were imposed with or without Δψ. Under these conditions, monensin mediated the rapid uptake of 22Na+. The present results indicate that halobacterial Na+/H+ exchange is regulated not only by a Δψ‐dependent gate but also by a certain mechanism to restrict the back flux of Na+, this making the antiporter capable of functioning as an efficient Δg̃mH+‐driven pump for Na+ in a high saline environment.
Efficiency of excitation trapping by reaction centres of complex B890 from Chromatium minutissimum
Authors: Abdourakhmanov I.A. , Danielius R.V. , Razjivin A.P.
Published: July 2002

The formation and decay of excited states of light‐harvesting bacteriochlorophyll (BChl) molecules and primary charge separation in reaction centres (RCs) of pigment‐protein complex B890 of the purple bacterium Chromatium minutissimum were studied as a function of excitation wavelength by means of picosecond absorbance difference spectroscopy. Selective excitation in the RC absorption band (at 800 nm) induced rapid bleaching of the absorption band of the RC BChl dimer (τ ∼ 1 ps) without absorption changes due to excited light‐harvesting BChl. It is concluded that excitation energy transfer from the BChl dimer of the RC to light‐harvesting BChl molecules is considerably slower than RC charge separation. Therefore, energy transfer from the light‐harvesting antenna to the RC in bacterial photosynthesis may be described as a ‘migration‐limited model’.
Electron microscopy study of non‐precipitating anti‐dinitrophenyl antibodies
Authors: Ryazantsev S.N. , Vasiliev V.D. , Abramov V.M. , Franék F. , Zav'yalov V.P.
Published: July 2002

Non‐precipitating anti‐dinitrophenyl pig immunoglobulins G have been studied by negative staining, freeze‐drying and high‐resolution shadow casting. The general morphology of the molecules is described. The predominant conformation of antibody molecules is a tripod‐like one.
Homologous sequences in cholera toxin A and B subunits to peptide domains in myelin basic protein
Authors: Caamaño Claudio A. , Zand Robert
Published: July 2002

Recent reports that myelin basic protein (MBP) can be ADP‐ribosylated and contains specific sites that bind GTP and GM1 ganglioside, have suggested an analogy to the properties of cholera toxin. Comparisons of pairs of sequences between these two proteins yielded two regions of homology between MBP and the cholera toxin B (chol B) subunit, and one region of homology with the cholera toxin A (chol A) subunit. The matching sites within chol B consisted of a 17 amino acid residue sequence (residues 30–46 in chol B and residues 102–118 in human‐MBP, hMBP, p<0.0007) and an 11 residue span (residues 31–41 in chol B and sequence 29–39 in hMBP, p<0.0004). The homologous site within chol A corresponded to an 11 residue span (residues 130–140 in chol A and 67–77 in hMBP sequence, p<0.00007). Since portions of the cholera toxin sequence are virtually identical to sections of the sequence in E. coli toxin, the homology is also valid for the same sequences in this toxin. The highly antigenic behavior of MBP that is related to the induction of experimental allergic encephalomyelitis may be paralleled by comparable neural pathology from the homologous regions of cholera toxin.
Isolation of dermenkephalin from amphibian skin, a high‐affinity (δ‐selective opioid heptapeptide containing a D‐amino acid residue
Authors: Mor Amram , Delfour Antoine , Sagan Sandrine , Amiche Mohamed , Pradelles Philippe , Rossier Jean , Nicolas Pierre
Published: July 2002

The predicted amino acid sequence of the biosynthetic precursor of dermorphin, a highly potent and nearly specific μ‐opioid peptide from amphibian skin, contains four repeats of the dermorphin progenitor sequence and one single copy of a different heptapeptide sequence. We have developed a specific enzyme immunoassay and used synthetic peptides to detect and purify the new predicted heptapeptide 2.4 μg/g dry skin) from the skin of the Phyllomedusa sauvagei frog from which dermorphin was originally isolated. The identity of the novel pro‐dermorphin related peptide, Tyr‐D‐Met‐Phe‐His‐Leu‐Met‐Asp‐NH2, was established by co‐chromatography with synthetic peptides on reverse‐phase HPLC, immunological analysis, gas‐phase sequencing, mass spectrometry and by pharmacological assays. Opioid‐binding assays in vitro demonstrated that both the natural and synthetic heptapeptides displayed exceptionally high selectivity and affinity towards the δ‐opioid receptors. Because of its origin and its δ‐opioid (enkephalin) activity and specificity, this novel D‐amino acid containing peptide is named dermenkephalin.
Halobacterial flagellins are encoded by a multigene family Identification of all five gene products
Authors: Gerl Lydia , Deutzmann Rainer , Sumper Manfred
Published: July 2002

Flagellins of Halobacterium halobium are encoded in five different but homologous genes. Flagellins isolated from purified flagella were digested and the resulting peptides sequenced. The amino acid sequence data obtained prove that all five gene products are expressed and integrated into the flagellar bundle.
Two forms of the GABAA receptor distinguished by anion‐exchange chromatography
Authors: Moffett John R. , Namboodiri Aryan , Neale Joseph H.
Published: July 2002

The GABAA receptor complex was solubilized from rat brain membranes in Triton X‐100, enriched by 1012‐S affinity chromatography, and subjected to DEAE anion‐exchange chromatography. Two forms were distinguished by their differential elution during this HPLC with a KCl gradient. They displayed similar [3H]muscimol‐ and [3H]flunitrazepam‐binding characteristics, as well as [3H]flunitrazepam‐binding inhibition by CL 218872. Rechromatography of these distinct ionic forms indicated that they were not in dynamic equilibrium during chromatography. Resolution of these two pharmacologically similar populations of GABAA receptor by anion‐exchange HPLC suggests that they differ in charge densities, a condition which may reflect differing glycosylation or phosporylation states of the complex.
Zintrons in rat α‐lactalbumin gene
Authors: Meera G. , Ramesh N. , Brahmachari Samir K.
Published: July 2002

The eukaryotic genome is characterised by the occurrence of a large amount of repetitive DNA which exists within and around coding sequences. Random repeats of (TG)
n
sequences have been observed in several introns. In this paper we show that (TG)14 and (TG)24 sequences present in the third intron of rat α‐lactalbumin gene adopt left‐handed Z‐helices under varying superhelical densities. The overall sequence of the parent plasmid further influenced the level of supercoiling at which the B to Z transition could be induced in (TG)
n
sequences. Such Z‐potential intervening sequences (zintrons) could act as buffers to maintain desired levels of supercoiling near the transcribed sequences.
Multiple gabaa receptor α subunit mRNAs revealed by developmental and regional expression in rat, chicken and human brain
Authors: Montpied Pascale , Ginns Edward I. , Martin Brian M. , Steuer Deborah , O'Carroll Anne-Marie , Lolait Stephen J. , Mahan Lawrence C. , Paul Steven M.
Published: July 2002

GABAA receptor α subunit transcripts were detected by Northern analysis of rat, chicken and human brain mRNA using a series of 32 P‐labelled antisense RNA probes derived from human α1 subunit cDNAs. These α subunit mRNAs differ in their distribution among various brain regions in the rat and at least one species is detected primarily in fetal brain. GABAA receptor αa subunit probes encoding the putative extracellular domain detect at least five α subunit transcripts in rat brain, whereas probes encoding the putative intracellular domain detect only two mRNAs. These data suggest the presence in brain of multiple GABAA receptor α subunits having homologous extracellular domains and whose expression is regionally and developmentally regulated. These α subunit transcripts may encode proteins that comprise GABAA isoreceptors differing in their pharmacological and physiological properties.
Identification of an interleukin‐1 beta binding protein in human plasma
Authors: Eastgate J.A. , Symons J.A. , Duff G.W.
Published: July 2002

A covalent cross‐linking technique was used to bind iodinated interleukin‐1 (IL1) alpha and beta to plasma proteins. One specific IL1 beta binding protein was observed, that when cross‐linked to 125I‐ILl beta migrated to approximately 60 kDa on SDS‐PAGE. The protein did not bind IL1 alpha. The 43 ‐kDa protein was partially purified using a wheat germ agglutinin affinity column. The isolated factor again specifically bound IL1 beta, and appeared to consist of single chain glycoprotein. The protein was heat stable and had a rapid association time with IL1 beta. This protein may be an important carrier molecule for IL1 beta in vivo.
Correlation between the DNA supercoiling and the initiation of transcription by Escherichia coli RNA polymerase in vitro: role of the sequences upstream of the promoter region
Authors: Mishra Rakesh K. , Gopal Vijaya , Chatterji Dipankar
Published: July 2002

Binding of Escherichia coli RNA polymerase and the abortive initiation of transcription at the A2 promoter ofbacteriophage T7, separately cloned in pBR322, was found to be strongly dependent on the degree of supercoiling of the plasmid. Supercoiling does not seem to play any role in the initiation of transcription at the T7A1 promoter under identical conditions. Plasmid containing T7A2 promoter was found to be less amenable to S1 nuclease in comparison to that having T7A1. Sequence comparison reveals a high G/C content upstream to the −35 region of T7A2 which by extra duplex stability probably renders the initiation of transcription more dependent on the state of supercoiling of the template.
Functional expression of Ca2+ ‐mobilizing α‐thrombin receptors in mRNA‐injected Xenopus oocytes
Authors: Van Obberghen-Schilling Ellen , Chambard Jean Claude , Lory Philippe , Nargeot Joel , Pouysségur Jacques
Published: July 2002

α‐Thrombin (TH) initiates a program of intracellular events that lead to DNA replication in quiescent CCL39 Chinese hamster lung fibroblasts via membrane receptors that have yet to be characterized at a molecular level. Functional TH receptors were expressed in Xenopus laevis oocytes following injection of poly(A)+ RNA from TH‐responsive CCL39 cells; their presence was demonstrated by TH‐stimulated 45Ca2+ efflux or Ca2+dependent Cl− channel activation. In voltage clamp experiments on microinjected oocytes a Ca2+ ‐activated Cl− current was detected in response to TH (0.2–10 ). The TH response was blocked by a specific TH inhibitor, and potentiated by addition of FGF or intracellular injection of GTP‐γ‐S.
Carbonic anhydrase inhibition and calcium transients in soleus fibers
Authors: Wetzel P. , Liebner T. , Gros G.
Published: July 2002

We simultaneously measured cytoplasmic Ca2+ transients using Fura‐2 and isometric force in rat soleus fiber bundles. In the presence of the carbonic anhydrase inhibitor, chlorzolamide, we observed a decreased amplitude and retarded decay of the Ca2+ signal. This corresponded with a decreased isometric force and a retarded muscle relaxation. We conclude that muscle carbonic anhydrase participates in excitation‐contraction coupling, possibly by rapidly providing protons that are exchanged for Ca2+ across the sarcoplasmic reticulum membrane.
A stereospecific enzyme of the putative biosynthetic pathway of cardenolides
Authors: Gärtner Dorothea Elisabeth , Wendroth Susanne , Seitz Hanns Ulrich
Published: July 2002

Leaves of Digitalis
purpurea contain an enzyme activity which catalyzes the conversion of progesterone to 5β‐pregnane‐3,20‐dione. Since cardenolides without exception possess a 5β‐configuration, 5β‐pregnane‐3,20‐dione can serve as a precursor for this class of secondary metabolites. It is assumed that the enzyme is part of the putative biosynthetic pathway of cardenolides. This enzyme activity was spotted in the soluble fraction of a crude homogenate. Product formation was detected by gas chromatography and by gas chromatography/mass spectroscopy (g.c./m.s.). The enzyme had a pH optimum at 8.0 and an apparent K
m value of 6 μM for progesterone. It required NADPH as a co‐substrate with an apparent K
m value of 22 μM. The optimum temperature in vitro was 30°C. The activity was not dependent on monovalent and bivalent cations.
Hydrophobie cluster analysis reveals duplication in the external structure of human α‐interferon receptor and homology with γ‐interferon receptor external domain
Authors: Gaboriaud Christine , Uzé Gilles , Lutfalla Georges , Mogensen Knud
Published: July 2002

Evidence is presented, based on sequence comparison according to Hydrophobic Cluster Analysis, of a structural and evolutionary relationship between the human α/β‐interferon receptor and the human and mouse γ‐interferon receptor. These results predict that the human α/β‐interferon receptor extracellular part is organised in two homologous subdomains connected by a proline linker. They also predict that both subdomains present some homologies to the external domain of mouse and human γ interferon receptor.
Mast cell degranulating (MCD) peptide and its optical isomer activate GTP binding protein in rat mast cells
Authors: Fujimoto Ichiro , Ikenaka Kazuhiro , Kondo Tetsuro , Aimoto Saburo , Kuno Miyuki , Mikoshiba Katsuhiko
Published: July 2002

The MCD peptide in bee venom induces degranulation in mast cells. The internal calcium concentration of mast cells increased and remained high following MCD stimulation. This calcium increase was blocked by pertussis toxin (Ptx) treatment, suggesting that MCD peptide activates Ptx‐sensitive G‐protein. Even in the absence of external calcium in the incubation medium, the calcium concentration increased by MCD treatment. but soon returned to the original level. D‐MCD, the optical isomer of the MCD peptide, also increased the internal calcium concentration through a Ptx‐sensitive pathway. We suggest that cationic clusters at one side of the surface are more important in activating the G‐protein than the α‐helix conformation.
Mutant analysis of interaction of the Bacillus subtilis transcription regulator AbrB with the antibiotic biosynthesis gene tycA
Authors: Fürbaβ Rainer , Marahiel Mohamed A.
Published: July 2002

The AbrB protein of B. subtilis represses the transcription of various postexponentially expressed genes, such as the antibiotic biosynthesis gene tycA. Recently, we have shown that AbrB binds to the tycA promoter region at two A+T‐rich sites; the ‘promoter site’ (−60 to −35) and the ‘leader site’ (+169 to +231). In this study we demonstrate that a P

tyc
—lacZ fusion missing the leader region is constitutively expressed in wild‐type B. subtilis cells and in B. subtilis cells carrying spoOA or abrB mutations. We also show that substitution mutations within the recently reported potential helix‐turn‐helix DNA binding motif of AbrB did not affect its specific DNA binding ability.
Highly conserved sequences in the 3′‐untranslated region of the COL1A1 gene bind cell‐specific nuclear proteins
Authors: Määttå Arto , Bornstein Paul , Penttinen Risto P.K.
Published: July 2002

Sequencing of the 3′ untranslated region (3′‐UTR) of the human COL1A1 gene revealed numerous putative regulatory motifs and two highly conserved regions flanking the two polyadenylation sites. The conserved regions were separated by about 700 bp of less conserved sequences. The first region consists of almost all the 3′‐UTR of the shorter (4.8 kbp) COL1A1 transcript. The second conserved domain includes a motif shared with several collagen genes. Both conserved domains bind cell‐specific nuclear proteins suggesting that the 3′‐UTR is important for cell specific expression of the COL1A1 gene.
Effect of hypothyroidism and thyroid hormone replacement on the level of protein kinase C and protein kinase A in rat liver
Authors: Meier Christoph A. , Fabbro Doriano , Meyhack Irene , Hemmings Brian , Olbrecht Ursula , Jakob Andrea , Walter Paul
Published: July 2002

We investigated the influence of the thyroid hormone status on the levels of protein kinase C (PKC) and A (PKA) in the soluble fraction of rat liver. The immunodetectable PKC level in hypothyroid liver was elevated 7.7‐fold, whereas the phorbol‐ester binding capacity and the immunodetectable α‐PKC level were increased 2.4‐ and 2.6‐fold, respectively. Conversely, in hypothyroid livers the abundance of the regulatory type I and the catalytic subunits of PKA were lowered to 42% of the euthyroid level as determined by immonoblotting and by measuring the substrate specific phosphorylation rate of PKA. These changes in the PKC and PKA levels were reversible upon treatment with 0.5 μg T4/100 g body weight for 2–21 days. The thyroid state dependent alterations in hepatic PKC and PKA levels may be responsible for the Known changes in the response of hepatocytes to other hormonal stimuli in hypothyroidism.
Change of intracellular calcium of neural cells induced by extracellular ATP
Authors: Hirano Yukiko , Okajima Fumikazu , Tomura Hideaki , Majid Mohammed Abdul , Takeuchi Toshiyuki , Kondo Yoichi
Published: July 2002

Exposure of various neural cells to ATP increased intracellular Ca2+ and the production of inositol trisphosphate. The Ca2+ responses were also observed in the absence of extracellular Ca2+, suggesting that a part of Ca2+ mobilization took place from cytosolic storage. Since adenosine had no effect on intracellular Ca2+ increment, ATP appears to act through a P2‐purinergic receptor. Islet‐activating protein or pertussis toxin pretreatment hardly influenced the increase in intracellular Ca2+ and inositol trisphosphate production induced by ATP, suggesting that IAP‐sensitive GTP‐binding proteins do not play a practical role in this reaction.
Apolipoprotein B gene expression in rat intestine The effect of dietary fiber
Authors: Mazur Andrzej , Felgines Catherine , Nassir Fatiha , Bayle Dominique , Gueux Elyett , Rémésy Christian , Rayssiguier Yves , Cardot Philippe
Published: July 2002

The effect of the dietary fiber on apo B mRNA level was studied in the intestine of rats that were fed either fiber‐free or high‐fiber (30% sugar‐beet fiber) low‐fat diets for 3 weeks. The fiber diet studied does not affect jejunal apo B mRNA levels but decreases the level of ileal apo B mRNA. In the rat cecum, in both fiber‐free and fiber groups, we failed to detect the apo B mRNA. The test fiber diet feeding markedly increased fecal bile salt and cholesterol excretions. We suggest that dietary fiber can modify apo B expression in the intestine. The increased fecal bile salt excretion might be involved in such a modification.
On the origins of genetic variants
Authors: Wintersberger Ulrike
Published: July 2002

Two contrasting mechanisms responsible for the creation of genetic variants are described: one is the manifestation of the limited accuracy of the cellular machinery for DNA replication, the other results from the ability of cells to repair damaged DNA. Replication‐dependent variants and those caused by episodical DNA damage enhance the probability that a small fraction of a cell population may survive a sudden (physical or biological) change of environmental conditions. Replication‐independent variants arise during persistent but not immediately lethal stress (e.g. starvation) of a non‐dividing population. The variants observed under these conditions are of selective advantage because they are able to cope with the particular stress situation. The molecular basis of their creation is a matter of intensive debate.
The γ3‐subunit of the GABAA‐receptor confers sensitivity to benzodiazepine receptor ligands
Authors: Knoflach F. , Rhyner Th. , Villa M. , Kellenberger S. , Drescher U. , Malherbe P. , Sigel E. , Möhler H.
Published: July 2002

The γ3‐subunit of the GABAA‐receptor in rat brain has been identified by molecular cloning. When co‐expressed with the α5‐ and β2‐subunits in transfected cells a high potency for GABA (K
a = 4.9 ± 1.2 μM; and a strong cooperativity in gating the channel (H = 1.9 ± 0.2) was observed. The GABA response was potentiated in the presence of flunitrazepam reduced by βCCM. An analogous bi‐directional modulation of the GABA response was observed with diazepam and DMCM as tested with the subunit combinations α1β2γ3 and α3β2γ3 expressed in Xenopus oocytes. Since the benzodiazepine receptor ligands were virtually inactive in the absence of the γ3‐subunit, as tested with the α3β2‐ and α5β2‐subunit combinations, the γ3‐subunit is a prerequisite for the benzodiazepine receptor sensitivity of the expressed GABAA‐receptors. The γ3‐subunit could functionally replace the γ2‐subunit with regard to the bi‐directional allosteric drug modulation.
Cloning and tissue‐specific expression of the brain calcium channel β‐subunit
Authors: Pragnell Marlon , Sakamoto Junshi , Jay Scott D. , Campbell Kevin P.
Published: July 2002

A cDNA clone encoding a protein with high homology to the β‐subunit of the rabbit skeletal muscle dihydropyridine‐sensitive calcium channel was isolated from a rat brain cDNA library. This rat brain β‐subunit cDNA hybridizes to a 3.4 kb message that is expressed in high levels in the cerebral hemispheres and hippocampus but is significantly reduced in cerebellum. The open reading frame encodes 597 amino acids with a predicted mass of 65 679 Da which is 82% homologous with the skeletal muscle β‐subunit. The brain cDNA encodes a unique 153 amino acid C‐terminus and predicts the absence of a muscle‐specific 50 amino acid internal segment. It also encodes numerous consensus phosphorylation sites suggesting a role in calcium channel regulation. The corresponding human β‐subunit gene was localized to chromosome 17. Hence the encoded brain β‐Subunit. which has a primary structure highly similar to its isoform in skeletal muscle, may have a comparable role as an integral regulatory component of a neuronal calcium channel.
The pro‐region of the Kex2 endoprotease of Saccharomyces cerevisiae is removed by self‐processing
Authors: Germain Doris , Dumas France , Vernet Thierry , Bourbonnais Yves , Thomas David Y. , Boileau Guy
Published: July 2002

We have produced in the baculovirus/insect cells expression system a soluble secreted form of the Saccharomyces cerevisiae Kex2 endoprotease. This secreted enzyme was purified and its NH2‐terminal sequence determined. The NH2‐terminal sequence started at residue Leu109 of the sequence deduced from the KEX2 gene nucleotide sequence, showing that the Kex2 enzyme is produced as a proenzyme. Residue Leu109 is preceded by a pair of basic amino acid residues (Lys107‐Arg108) which is a potential processing site for the Kex2 endopeptidase. Futhermore, expression of an inactive form of this truncated enzyme resulted in the production of a protein with a higher molecular weight. These observations suggest that the pro‐region of Kex2 endoprotease is removed by a self‐processing event.
Involvement of a non‐proton pump factor (possibly Donnan‐type equilibrium) in maintenance of an acidic pH in lysosomes
Authors: Moriyama Yoshinori , Maeda Masatomo , Futai Masamitsu
Published: July 2002

Change of the internal pH of isolated lysosomes was measured with fluorescein isothiocyanate‐dextran. In buffer of pH 7.0, isolated lysosomes had an acidic pH of about 5.5, which decreased to pH 5.2 on addition of ATP. Addition of bafilomycin inhibited the acidification by H+‐ATPase and resulted in an increase of the internal pH to 5.5 due to passive diffusion of protons across the lysosomal membrane. However, no further alkalization was observed. The acidic pH (pH 5.5) of isolated lysosomes could be maintained for at least 48 h in the absence of ATP, but increased gradually to pH 5.9–6.4 upon incubation with monovalent cations (K+ or Na+), amines, or ionophores. These results suggest that a non‐proton pump factor (possibly Donnan equilibrium) is involved in maintaining the acidic pH of isolated lysosomes.
Both membrane stretch and fatty acids directly activate large conductance Ca2+‐activated K+ channels in vascular smooth muscle cells
Authors: Kirber Michael T. , Ordway Richard W. , Clapp Lucie H. , Walsh John V. , Singer Joshua J.
Published: July 2002

Large conductance Ca2+‐activated K+ channels in rabbit pulmonary artery smooth muscle cells are activated by membrane stretch and by arachidonic acid and other fatty acids. Activation by stretch appears to occur by a direct effect of stretch on the channel itself or a closely associated component. In excised inside‐out patches stretch activation was seen under conditions which precluded possible mechanisms involving cytosolic factors, release of Ca2+ from intracellular stores, or stretch induced transmembrane flux of Ca2+ or other ions potentially capable or activating the channel, Fatty acids also directly activate this channel. Like stretch activation, fatty acid activation occurs in excised inside‐out patches in the absence of cytosolic constituents. Moreover, the channel is activated by fatty acids which, unlike arachidonic acid, are not substrates for the cyclo‐oxygenase or lypoxygenase pathways, indicating that oxygenated metabolites do not mediate the response. Thus, four distinct types of stimuli (cytosolic Ca2+, membrane potential, membrane stretch, and fatty acids) can directly affect the activity of this channel.
Structural changes in profilin accompany its binding to phosphatidylinositol 4,5‐bisphosphate
Authors: Raghunathan Vidya , Mowery Patrick , Rozycki Michael , Lindberg Uno , Schutt Clarence
Published: July 2002

The effect on the structure of profilin of phosphatidylinositol 4,5‐bisphosphate (PIP2) binding was probed by fluorescence and circular dichroism (CD) spectroscopy, Fluorescence of Trp3 and Trp31 of profilin at 292 nm showed a linear decrease in solution emission at 340 nm as PIP2/profilin was increased from 0 to 80:1, apparently due to a static quenching mechanism involving formation of a nonfluorescent PIP2/profilin complex. CD spectra revealed an increase of up to 3.3‐fold in the molar ellipticity at 222 nm for profilin as it binds PIP2, as well as changes in the Cotton effect between 250 and 310 nm. These results are consistent with a possible increase in the α‐helix content or profilin triggered by the binding or PIP2.