bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation of cGMP‐dependent conductance of rod outer segment plasma membrane induced bij cGMP
Authors: Zhainazarov A.B. , Kolesnikov S.S.
Published: July 2002

Integral cGMP‐dependent currents as well as activity of single cGMP‐activated channels in plasma membrane of rod outer segment (ROS) were recorded using the patch‐clamp method. The dependence of integral currents on cGMP concentration is shown to be bell‐shaped. Decrease in cGMP‐dependent conductance at high cGMP concentration results from a decrease in channel opening probability. Thus, cGMP in high concentrations inactivates cGMP‐dependent conductance.
A proportion of photosystem II core complexes are decoupled from the phycobilisome in light‐state 2 in the cyanobacterium Synechococcus 6301
Authors: Mullineaux Conrad W. , Holzwarth Alfred R.
Published: July 2002

Cells of Synechococcus 6301 were briefly exposed to a phycocyanin‐absorbed light in the presence of DCMU. PS II trap closure was then estimated from fluorescence induction measurements with excitation light absorbed predominantly either by chlorophyll or by phycocyanin. In cells adapted to light‐state 2, the exposure to light absorbed by phycocyanin closed only a proportion of the PS II centres that could be closed by exposure to light absorbed by chlorophyll. This distinction was reduced in cells adapted to light‐state 1. We conclude that a proportion of PS II core complexes become decoupled from the phycobilisomes during the transition to light‐state 2.
The Photosystem I 5.5 kDa subunit (the psaK gene product)
Authors: Wynn R.Max , Malkin Richard
Published: July 2002

Previous studies of the Photosystem I reaction center complex, the chlorophyll protein 1 complex (CPI), have shown it is a heterodimer of two high‐molecular‐mass subunits of 83000 and 82000, the psaA and psaB gene products. The present work has identified another intrinsic low‐molecular‐mass subunit of 5 500 (as determined by SDS‐PAGE) that is tightly associated with the CPI complex. This subunit can be separated from CPI after treatment with thiol agents. CPI complexes prepared either in the presence or absence of thiols show no differences in photochemical activity under steady‐state actinic illumination. A comparison of the N‐terminal amino acid sequence of the 5.5 kDa subunit with the sequence of a Chlamydomonas PSI subunit, P37, identifies this low‐molecular‐mass subunit as the PSI psaK gene product.
Immunological determination of the oligomeric form of mitochondrial creatine kinase in situ
Authors: Quemeneur Eric , Eichenberger Denise , Vial Christian
Published: July 2002

Whereas factors governing the interconversion of the two oligomeric forms of mitochondrial creatine kinase are relatively well known, few informations are yet available on the actual form in situ. Antibodies against purified pig and rabbit heart mitochondrial creatine kinase were obtained. The former exhibits a marked specificity for the dimer while the second reacts with both dimer and octamer. They allowed to demonstrate that no dimer can be detected in mitochondria and that CKm occurs naturally exclusively as an octamer. We present arguments that the larger part, if not the totality, of the octamer is membrane‐bound rather than soluble in the intennembrane space. However, these findings do not refute the previously proposed models for the regulation of CKm activity in the mitochondrion but urge to envisage a more complex one.
Molecular cloning of a cDNA encoding the bombesin precursor in skin of Bombina variegata
Authors: Richter Klaus , Egger Richard , Kreil Günther
Published: July 2002

A cDNA library was constructed using poly(A)‐rich RNA isolated from skin of the frog Bombina variegata. This library was screened with two oligo‐nucleotides complementary to parts of the sequence of bombesin. The nucleotide sequence of one of the cloned cDNAs encoding a bombesin precursor is presented. The predicted polypeptide contains a single copy of the end‐product. The bombesin sequence is preceded by a leucine residue suggesting an unusual type of precursor processing.
A low‐molecular weight acid phosphatase present in crystalline preparations of rabbit skeletal muscle glycogen phosphorylase b
Authors: Sotiroudis Theodore G. , Geladopoulos Taxiarchis P.
Published: July 2002

Crystalline preparations of glycogen phosphorylase b contain traces of acid phosphatase activity. Non‐denaturing gel electrophoresis of phosphorylase b reveals a single band of 1‐naphthyl phosphate phosphohydrolase activity which co‐migrates with phosphorylase. The two enzymes can be separated by Sephadex G‐200 column chromatography, where the phosphatase exhibits an apparent M
r, of 17000. The contaminant enzyme hydrolyzes effectively the phenolic ester of monoorthophosphate with optimal activity for p‐nitrophenyl phosphate and L‐phosphotyrosine between pH 5.5 and 6.0. The phosphatase is insensitive to inhibition by L(+)‐tartrate but strongly inhibited by μM vanadate and Zn2+.
The RNA component of RNase P in Schizosaccharomyces species
Authors: Zimmerly Steven , Gamulin Vera , Burkard Ulrike , Söll Dieter
Published: July 2002

In the fission yeast Schizosaccharomyces pombe, the enzyme RNAse P copurifies with two RNAs, K1‐ and K2‐RNA, which are identical except for their termini [1] and which are encoded by a single gene [2]. We have undertaken the cloning of the K‐RNA genes in related organisms in order to gain comparative structural information. Because the K‐RNA sequence is poorly conserved across species, we have cloned the K‐RNA genes in the Schizosaccharomyces species S. malidevorans, S. japonicus, S. versatilis, and S. octosporus. Of the 4 species, only S. octosporus contains a K‐RNA gene different from that in S. pombe; the gene diverges by 20%. Based on the two sequences, nuclease protection data and computer analysis, we have proposed a secondary structure model for the K‐RNA. Northern analysis shows the K‐RNA genes in all four Schizosaccharomyces species to be expressed as two RNAs, as in S. pombe.
Rearrangement and overexpression of the gene coding for tumor antigen p53 in a rat histiocytoma AK‐5
Authors: Deshpande Girish , Kumari A.Leela , Khar Ashok
Published: July 2002

The gene coding for the cellular tumor antigen p53 is rearranged and overexpressed in a rat histiocytoma, AK‐5. The protein coded by the gene was detected by immunofluorescence and its full size was confirmed by immunoprecipitation using monoclonal antibodies against p53. Southern hybridizations with a full length cDNA probe specific for p53 indicated rearrangement of the gene. Alterations in the upstream region, which probably disrupt the normal regulatory control are suggested by the pattern obtained using a 5'‐specific p53 probe in Southern hybridization.
Protein TAU variants present in paired helical filaments (PHFs) of alzheimer brains
Authors: Gache Y. , Ricolfi F. , Guilleminot J. , Theiss G. , Nunez J.
Published: July 2002

Polyclonal anti‐TAU antisera directed against native Tau protein and the NH2‐terminal side of the mouse TAU sequence were used to determine the nature of the TAU variants present in Alzheimer brains and in PHFs. These antibodies labelled specifically neuroflbrillary tangles and plaque neuntes in Alzheimer brains. On immunoblots of PHF extracts, two entities of 69 and 130 kDa were identified. These TAU‐related species were absent from control brains. Protein immunoblot of total Alzheimer and control supernatants were shown to contain the same 4–5 TAU variants but none of the 69 and 130 TAU‐related entities found in PHFs. These data suggest that specific TAU species are present in PHFs.
Synthesis of a trihexacontapeptide corresponding to the sequence 8–70 of eglin c and studies on the relationship between the structure and the inhibitory activity against human leukocyte elastase, cathepsin G and α‐chymotrypsin
Authors: Okada Yoshio , Tsuboi Satoshi , Tsuda Yuko , Nagamatsu Yoko , Yamamoto Junichiro
Published: July 2002

A trihexacontapeptide corresponding to the sequence 8–70 of eglin c and its related peptides were synthesized by the conventional solution method and their inhibitory activity against human leukocyte elastase, cathepsin G and α‐chymotrypsin was examined. Although synthetic eglin c (41–49) inhibited cathepsin G and α‐chymotrypsin (K
i = 4.0 × 10−5 M and 2.0 × 10−5 M, respectively) but not leukocyte elastase, the synthetic trihexaconta‐peptide potently inhibited cathepsin G, α‐chymotrypsin and leukocyte elastase (K
i = 1.8 × 10−9 M, 1.4 × 10−9 M and 2.2 × 10−9 M, respectively). The relationship between the stucture of eglin c and the inhibitory activity against the above enzymes is also described.
AT repeats in barley genome
Authors: Ermak G.Z. , Prosnyak M.I. , Vecher A.A. , Kartel N.A.
Published: July 2002

The DNA sequence (AT)26 of barley genome has been cloned. This sequence is arranged in intraspecific locus and is repeated 1500 times per haploid genome. This fragment is not translated and can form cruciform structures in the AT region.
Analysis of U1snRNP‐specific A protein cross‐linked complexes
Authors: Boix J. , Habets W.J. , van Venrooij W.J. , Smith H.C.
Published: July 2002

The organization of the UlsnRNP‐specific A protein (34 kDa) has been analyzed by 12 and 16 Å thiol‐reversible chemical cross‐linking and Western blotting. A‐containing cross‐linked complexes had molecular masses of 43, 47, 56, 62, 67, 105 and 125 kDa. None of these complexes could be cross‐linked following ribonuclease digestion, suggesting that UsnRNA may play important roles in the spatial organization of A and other proteins. Moreover, the data suggest that A is proximal to, and may have interactions with, UsnRNP‐specific proteins C and 70 kDa as well as with UsnRNP‐common proteins B, E and G.
Mapping the copper ligands of Cu,Zn superoxide dismutase by nuclear overhauser enhancement of the isotropically shifted 1H‐NMR lines of the Cu,Co derivative
Authors: Paci Maurizio , Desideri Alessandro , Sete Marco , Falconi Mattia , Rotilio Giuseppe
Published: July 2002

Transient nuclear Overhauser effect (NOE) experiments were made with bovine Cu,Co Superoxide dismutase on the hyperfine shifted resonances of protons of the imidazole groups bound to the Cu.Zn cluster of the native enzyme. Assignments of lines based on the observed magnetization transfers were satisfactorily obtained only by altering the arrangement of the ligands around the catalytically active copper shown by X‐ray crystallography.
Acidic phospholipids directly inhibit DNA binding of mammalian DNA topoisomerase I
Authors: Tamura Hiro-omi , Ikegami Yoji , Ono Katsuhiro , Sekimizu Kazuhisa , Andoh Toshiwo
Published: July 2002

Inhibition of mammalian DNA topoisomerase I by phospholipids was investigated using purified enzyme. Acidic phospholipids inhibited the DNA relaxation activity of topoisomerase I whereas neutral phospholipid, phosphatidylethanolamine, did not. Accumulation of a protein‐DNA cleavable complex, an intermediate which is known to accumulate upon inhibition by a specific inhibitor camptothecin, did not occur. The filter binding assay revealed that the DNA binding activity of the enzyme was inhibited by acidic phospholipids. Moreoever, direct binding of phosphatidylglycerol to topoisomerase I was demonstrated. These results indicated that the inhibitory effect of acidic phospholipids on topoisomerase I was due to the loss of the DNA binding of the enzyme as a result of direct interaction between phospholipids and the enzyme.
Class IV mammalian alcohol dehydrogenase
Authors: Parés Xavier , Moreno Alberto , Cederlund Ella , Höög Jan-Olov , Jörnvall Jans
Published: July 2002

The stomach form of alcohol dehydrogenase has been structurally evaluated by peptide analysis covering six separate regions of the rat enzyme. Overall, this new structure diners widely (32–40% residue differences) from the structures of three classes of alcohol dehydrogenase characterized before from the same species. Consequently, this novel enzyme constitutes a true fourth class of mammalian alcohol dehydrogenase. In particular, differences are extensive also towards class II, although enzymatic and physicochemical properties initially suggested overall similarities with class II. The new structure establishes the presence of one further alcohol dehydrogenase mammalian gene, extends the enzyme family derived from repeated gene duplications, and confirms tissue‐specific expressions.
Immunological similarities between cytosolic and particulate tissue transglutaminase
Authors: Knight C.R.L. , Rees R.C. , Elliott B.M. , Griffin M.
Published: July 2002

At the present time it is uncertain whether or not the cytosolic and particulate forms of tissue transglutaminase are distinct and discrete enzymes. In this study a number of physical and immunological similarities between the two forms are demonstrated, indicating that they share some common epitopes, although their native confirmations may differ.
Biophysical studies on the lipid transfer particle from the hemolymph of the tobacco hornworm, Manduca sexta
Authors: Ryan Robert O. , Hicks Leslie D. , Kay Cyril M.
Published: July 2002

Hydrodynamic studies conducted in the analytical ultracentrifuge provided evidence for two populations of lipid transfer particle (LTP) when centrifuged in a buffer solution containing 10 mM Tris, pH mM KC1. The apparent sedimentation coefficients of the two species was 23.3 S and 15.3 S. Upon changing the buffer pH to 7.0 or 5.7, two species of LTP were still present but the ratio of their relative abundance was altered. When the KC1 concentration in the buffer was lowered to 50 mM the sample sedimented as a single species with an apparent s
20,w of 22.9 S. In higher ionic strength buffers (10 mM succinate, pH mM KC1) LTP sedimented with an apparent s
20,w of 14.8 S. Further experiments revealed that these two forms are interconvertable as a function of buffer ionic strength. Given previous estimates of the molecular size of LTP we concluded that the slower sedimenting peak observed at high ionic strength represents monomeric LTP while the faster sedimenting material observed at low ionic strength is likely to be an aggregated state of LTP. This interpretation is supported by molecular weight determinations made by sedimentation equilibrium experiments conducted in 10 mM succinate, pH mM KCl which yielded a particle M
r = 887000. Circular dichroism spectra of monomeric LTP sample revealed 6% α‐helix, 49% β‐sheet, 7% β‐turn and 35% random coil while aggregated LTP contained 13% α‐helix, 66% β‐sheet and 21% random coil. The transfer activity of the two LTP forms was assayed and found to be the same indicating that either the state of LTP aggregation did not affect transfer activity or that upon exposure to a large excess of lipoprotein substrate disaggregation, without loss of activity, occurs.
ATP binding site of mitochondrial creatine kinase
Authors: James Peter , Wyss Markus , Lutsenko Svetlana , Wallimann Theo , Carafoli Ernesto
Published: July 2002

The ATP binding site of mitochondrial creatine kinase from chicken heart has been studied by modifying the purified enzyme with a 14C‐labelled ATP analogue, CIRATP, in which the reactive label was covalently bound to the γ‐phosphote group of ATP. The modified enzyme was digested by pepsin, and a single radioactive nonapeptide was isolated by HPLC. Amino acid analysis and direct sequence determination revealed that the isolated peptide corresponds to amino acids 335–343 within the C‐terminal region of Mi‐CK, this peptide being highly preserved throughout evolution. Asp‐335 is very likely the site of modification by CIRATP. The specificity of the ATP analogue for the active site of creatine kinase was demonstrated by the inhibition of the enzymatic activity of Mi‐CK by C1RATP and by the prevention of this inhibition bij ADP.
A comparative study of ribosomal and DNA binding protein II from two thermophilic bacteria, Bacillus caldolyticus strain EP 00275 and Bacillus stearothermophilus
Authors: Zierer Rainer , Yasuda Gen
Published: July 2002

Ribosomal and DNA binding proteins (DNA bp II) from an extreme thermophilic bacterium, B.caldolyticus strain EP 00275, were investigated for stability and crystallization and compared to the homologous proteins from B.stearothermophilus. Two‐dimensional gel electrophoresis of both types of proteins, the amino acid composition and the sequences of some of the peptides of DNA bp II revealed a close relationship between each other. The physico‐chemical characteristics of DNA bp II were similar but different from homologous proteins from T. thermophilus and C. pasteurineum. From our results we conclude: B. stearothermophilus and B. caldolyticus strain EP 00275 are similar organisms with regard to their ribosomal and DNA binding proteins.
New model of tertiary structure of plant 5S rRNA is confirmed by digestions with α‐sarcin
Authors: Barciszewska Miroslawa Z. , Lorenz Siegfried , Joachimiak Andrzej , Ulbrich Norbert , Erdmann Volker A. , Barciszewski Jan
Published: July 2002

The cytotoxin α‐sarcin was employed to test the model of secondary and tertiary structures of plant 5S rRNAs, which we recently proposed [(1990) Int. J. Biol. Macromol. (in press)]. α‐Sarcin is a novel ribonuclease that hydrolyzes phosphodiester bonds adjacent to purines in nucleic acids [2–4]. The digestion pattern obtained for lupin and wheat germ 5S rRNAs strongly suggests the existence of tertiary interactions between residues C34, C35, C36, A37 and G85, G86, 087, U88 as previously proposed. The results on the secondary structure of plant 5S rRNA are in line with a previously proposed model [5]
Pyruvate uptake induced by a pH jump in mesophyll chloroplasts of maize and sorghum, NADP‐malic enzyme type C4 species
Authors: Ohnishi Jun-ichi , Kanai Ryuzi
Published: July 2002

A sudden pH decrease (pH jump) of the medium enhanced pyruvate uptake in the dark in mesophyll chloroplasts (MCp) of Zea mays and Sorghum bicolor, NADP‐malic enzyme type C4 plants, while it was reported that a Na+ jump enhanced pyruvate uptake in MCp of P. miliaceum, a NAD‐malic enzyme type [(1987) FEBS Lett. 219, 347]. The enhancement effect of the pH jump decayed completely in 5 min and the decay was accelerated by proton gradient‐collapsing reagents. The results suggest that active pyruvate uptake into MCp of NADP‐malic enzyme type C4 species is primarily driven by the proton gradient across the envelope.
Calcium ion activation of the anion‐conducting channel in the rat liver mitochondrial inner membrane
Authors: Selwyn Michael J. , Ng Catherine L.T. , Choo Hui Lim
Published: July 2002

Stimulation of the rat liver mitochondrial inner‐membrane anion‐conducting channel by aeration is dependent on the concentration of Ca2+ ions in the assay medium. Ca2+ activates anion conduction in both aerated and non‐energised mitochondria but acts over a wider concentration range and produces a greater increase in anion‐conductivity in aerated mitochondria. EGTA reverses Ca2+ stimulation but takes several seconds to act, indicating slow release of Ca2+ from the activation site possibly on the matrix side of the inner mitochondrial membrane. It is suggested that this channel may respond to hormone‐induced changes in cytosolic Ca2+ concentration.
Crystallization and preliminary x‐ray diffraction study of neurotoxin‐I from Naja naja oxiana VENOM
Authors: Mikhailov A.M. , Nickitenko A.V. , Trakhanov S.D. , Vainshtein B.K. , Chetverina E.V.
Published: July 2002

Crystals of the neurotoxin‐I (NTX‐I) from the venom of the middle Asian cobra Naja
naja
oxiana have been grown by vapour diffusion and dialysis methods. The crystals belong to space group P21212 with dimension of a = 25.19 Å, b = 75.59 Å, c = 36.09 Å and diffract to 1.9 Å resolution. The asymmetric unit contains one molecule (V
m = 2.2 Å/Da). Using the molecule of α‐cobratoxin (CTX) as a starting model for NTX‐I structure determination coordinates of Cα atoms of the NTX‐I molecule were obtained and the position of NTX‐I in the unit cell was derived.
Secretion and intracellular degradation of collagen in cultures of normal and SV‐40‐transformed human fibroblasts
Authors: Berman A.E. , Morozevich G.E.
Published: July 2002

With a pulse‐chase technique, secretion and intracellular degradation of collagen were investigated in human cultured normal and SV‐40‐transformed fibroblasts. Normal cells at a proliferative phase of growth secreted collagen more actively than the stationary phase (resting) cells. Transformed fibroblasts secreted protein at a lower rate than both normal cell types. Resting and dividing normal cells displayed no differences in the rates of intracellular collagen degradation at the various stages of the chase period. Transformed cells did not differ from the normal ones in collagen degradation rates at the first hour of the chase period while at later times in the chase period, the total amount of degraded collagen was reduced by 20–30% in the transformed vs normal cultures. The data are discussed in the view of possible relations between the various mechanisms of intracellular transport and degradation of collagen.
Characterization of four GO‐type proteins purified from bovine brain membranes
Authors: Inanobe Atsushi , Shibasaki Hisayuki , Takahashi Katsunobu , Kobayashi Ichiro , Tomita Urara , Ui Michio , Katada Toshiaki
Published: July 2002

Recently we reported there were at least four types of GO or GO‐like proteins in bovine brain membranes based on their elution profiles from Mono Q columns and their immunological reactivities; one of the proteins was purified as an α‐monomeric form, and the others as αβγ‐trimers. The four proteins, of which α‐subunits were confirmed to be a family of GO‐type by an immunoblot analysis, were thus referred toas αO
1, GO
2, GO
3 and GO
4, respectively, in order of their elutions from the column. Immunostained peptide mappings arising from proteolytic digestions of the four α‐subunits, together with their fragmentation patterns containing radiolabeled ADP‐ribose that had been incorporated by pertussis toxin‐catalyzed ADP‐ribosylation, suggested that the four GO‐α were classified into either of two groups such as α1 and GO
2‐α or GO
3‐α and GO
4‐α. The kinetic parameters of their GTPase activities, however, revealed that there were different properties between αO
1 and GO
2‐α or GO
3‐α and GO
4‐α. Thus, the four GO‐type proteins appeared to be different entities from one another.
Sulphation of hirudin in BHK cells
Authors: Skern Tim , Bischoff Rainer , Jallat Sophie , Dott Karin , Ali-Hadji Dalila , Clesse Daniel , Kieny Marie Paule , Courtney Michael
Published: July 2002

Hirudin, a thrombin inhibitor of the leech, was expressed in BHK cells; the α1‐antitrypsin signal peptide was used to direct secretion into the culture medium. The recombinant hirudin so produced inhibited thrombin and was shown by labelling experiments with [35S]sulphate to have been post‐translationally modified.
CD4‐affinity purification of recombinant and native HIV gp 120 and comparison of the affinity constants for the receptor
Authors: Moritz D. , Direkx L. , Mous J. , Schneider J.
Published: July 2002

Soluble CD4‐immunoglobulin chimeric proteins were covalently attached to CNBr‐activated Sepharose. This allinity matrix was used to establish a powerful new method to isolate different species of the HIV external glycoprotein gp 120 from cell‐free culture supernatants. Recombinant gp 120 was expressed in Baculovirus‐infected insect cells and isolated from cell‐free culture supernatants. The recombinant protein has an apparent molecular mass of 95 kDa whereas the native gp 120 purified from a persistently HIV‐1 infected 119 cell culture has an apparent molecular mass of 130 kDa. These two gp 120 species where shown to be of identical molecular size after complete deglycosylation achieved by endoglycosidase treatment, and they bound to CD4‐Hyt with the same binding constant, that was reported for native forms of gp 120 and CD4. Thus the different glycosylation of gp 120 does not influence its allinity to CD4 and the gp 120‐CD4 complex can be reversibly dissociated.
Transforming growth factor‐β and epidermal growth factor modulate basal and interleukin‐6‐induced amino acid uptake and acute phase protein synthesis in cultured rat hepatocytes
Authors: Bereta J. , Szuba K. , Fiers W. , Gauldie J. , Koj A.
Published: July 2002

Rat hepatocytes cultured for 2 days with interleukin‐6 show increased synthesis of acute phase proteins and enhanced accumulation of 14C‐labelled α‐aminoisobutyric acid. Transforming growth factor‐β1 (0.1–10 ng/ml) inhibits whereas epidermal growth factor (1–100 ng/ml) enhances both basal and interleukin‐6‐induced amino acid uptake by rat hepatocytes with only a slight alteration of acute phase protein synthesis.
Solubilization of rat kidney lysosomes in reversed micelles of aerosol OT in octane
Authors: Pshezhetsky Alexey V. , Buneeva Olga A. , Wiederschain German Ya.
Published: July 2002

Intact lysosomes from rat kidneys were solubilized in the ternary system: surfactant (Aerosol OT)‐buffer‐organic solvent. According to data of laser light‐scattering analysis and kinetic experiments with the lysosomal marker enzyme, N‐acetyl‐β‐D‐hexosaminidase (EC 3.2.1.30), the solubilization of lysosomes in this system resulted in the destruction of the lysosomes and the entrapping of their components in reversed micelles.
Interaction of synthetic D‐6‐deoxy‐myo‐inositol 1,4,5‐trisphosphate with the Ca2+‐releasing D‐myo‐inositol 1,4,5‐trisphosphate receptor, and the metabolic enzymes 5‐phosphatase and 3‐kinase
Authors: Safrany S.T. , Wojcikiewicz R.J.H. , Strupish J. , Nahorski S.R. , Dubreuil D. , Cleophax J. , Gero S.D. , Potter B.V.L.
Published: July 2002

The ability of D‐6‐deoxy‐myo‐inositol 1,4,5‐trisphosphate [6‐deoxy‐Ins(1,4,5)P3], a synthetic analogue of the second messenger D‐myo‐inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3], to mobilise intracellular Ca2+ stores in permeabilised SH‐SY5Y neuroblastoma cells was investigated. 6‐Deoxy‐Ins(1,4,5)P3 was a full agonist (EC30 = 6.4 μM), but was some 70‐fold less potent than Ins (1,4,5)P3 (EC30 = 0.09 μM), indicating that the 6‐hydroxyl group of Ins(1,4,5)P3 is most important for receptor binding and stimulation of Ca2+ release, but is not an essential structural feature. 6‐Deoxy‐Ins(1,4,5)P3 was not a substrate for Ins (1,4,5)P3 5‐Phosphatase, but inhibited both the hydrolysis of 5‐[22P] + Ins (1,4,5)P3 (K
1 76 μM) and the phosphorylation of [3H]Ins(1,4,5)P3 (apparent K
1 5.7 μM) 6‐Deoxy‐Ins (1,4,5)P3 mobilized Ca2+ with different kinetics to Ins(1,4,5)P3, indicating that it is probably a substrate for Ins(1,4,5)P3 3‐kinase.
Gibberellic acid stimulates acid invertase secretion in pea ovary protoplasts
Authors: Estruch Juan José , Beltrán José Pio
Published: July 2002

Protoplasts purified from mesocarp of nonpollinated pea (Pisum sativum L.) ovaries released acid invertase to the incubation. The association of the acid invertase with microsomal fractions, and the sensitivity to energy‐metabolism inhibitors and to lunicamycin, indicated the secretory nature of the release process. In the presence of GA3 (10 μM), the protoplasts increased their invertase secretion at about 60 min. this effect being counteracted by lunicamycin but not cycloheximide. Subcellular fractionation of GA3‐treated protoplasts showed that higher invertase secretion was the result of a promotion of invertase transfer from endoplasmic reticulum (ER) to Golgi apparatus.
Differentiation associated modulation of K‐FGF expression in a human teratocarcinoma cell line and in primary germ cell tumours
Authors: Schofield Paul N. , Ekström Tomas J. , Granerus Marika , Engström Wilhelm
Published: July 2002

The human teratocarcinoma cell line Tera 2 can be induced to differentiate in vitro after exposure to retinoic acid. We show in this paper that whereas the K‐FGF oncogene is expressed in undifferentiated cells, addition of retinoic acid rapidly (< 60 min) downregulates the expression of this gene. However, when cells are cultured in RA for an extended period of time (> 15 days) K‐FGF transcripts reappear. We also report that K‐FGF is expressed in approximately one‐third of primary human germ cell tumours but not in the corresponding normal testicular tissue.
Chloroplast phosphoribulokinase associates with yeast phosphoriboisomerase in the presence of substrate
Authors: Skrukrud Cynthia L. , Anderson Louise E.
Published: July 2002

Pea chloroplastic phosphoribulokinase and yeast phosphoriboisomerase partition independently of one another in a two‐phase polyethyleneglycol, dextran system, but apparent interaction is seen when ribose‐5‐phosphate is added to the two‐phase system. It appears that the pea leaf of kinase recognizes yeast isomerase when it is carrying metabolite.
Direct cleavage of a DNA fragment by a bleomycin‐oligonucleotide derivative
Authors: Sergeyev D.S. , Godovikova T.S. , Zarytova V.F.
Published: July 2002

Bicomycin A1 oligonucleotide derivative was used for direct cleavage of a DNA target. In the presence of Fe2+ ions and 2‐mercaptoethanol, Blm‐R‐pd(CCAAACA) (I) damaged the target, pd(TGTTTGGCGAAGGA), with the yield of 80%, without affecting its own oligonucleotide tail. The sites of the cleavage were T?‐T? and G?‐G?. Unbound bleomycin A5, damaged the G6‐G7‐G8 site. Reagent I formed more stable complementary complexes with the target than parent oligonucleotide (ΔT
m=11°C)
Occurrence of chloroplast ribosome recognition sites within conserved elements of the RNA genomes of carlaviruses
Authors: Foster Gary D. , Mills Peter R.
Published: July 2002

The nucleotide sequences upstream from the carlavirus open reading frames were examined for direct sequence homology. Blocks of homology were evident upstream from the 25 K ORFs of potato virus S (PVS), potato virus M (PVM) and lily symptomless virus (LSV), and upstream from the coat protein initiation codons of PVS, PVM, LSV, carnation latent virus and Helenium virus S. These blocks, which correspond to the 5′‐terminal regions of the subgenomic RNAs, were shown to contain potential ribosome recognition sequences. The distances between the binding sites and initiation codons ranged from 20 to 40 nucleotides on the viral RNAs. Whilst the majority of chloroplasts mRNAs have a distance of 8 nucleotides between binding site and initiation codon, the remaining have a distance of 23 nucleotides which is similar to that reported here for the carlaviruses.
Molecular recognition in the binding of vitamin B12 by the cobalamin‐specific Intrinsic Factor
Authors: Andrews Elizabeth R. , Pratt John M. , Brown Kenneth L.
Published: July 2002

Equilibrium constants (given as log K/M
−1) have been determined at pH 7.4 and 4*C for binding by porcine Intrinsic Factor (B12‐binding protein from the gut, specific for the ‘cobalamin’ series of Co corrinoids) of vitamin B12 or cyanocobalamin (10.5), cyanocobinamide, α‐ribazole and α‐ribazole‐phosphate (main fragments produced by cleaving off the ‘cobalamin’ side‐chain, all ≤ 3), and cyanocobinamide in the presence of ≥ 10−9 M ribazole (5.6 and independent of ribazole concentration), i.e. ribazole catalyses the binding of the cobinamide. It is proposed that the specificity of Intrinsic Factor for the cobalamins depends on the presence of the ribazole fragment in the cobalamin side‐chain to promote an essential change in conformation before the corrinoid fragments can be bound.
5′‐Derivatives of oligonucleotides as primers of DNA polymerization catalyzed by AMV reverse transcriptase and Klenow fragment of DNA polymerase 1
Authors: Lokhova I.A. , Nevinsky G.A. , Godovikova T.S. , Ivanova E.M. , Koshkin A.A. , Sergeev D.S. , Frolova E.I. , Rudenko N.K. , Khomov V.V. , Kavsan V.M. , Zarytova V.F. , Lavrik O.I.
Published: July 2002

The K
m and V
max values for d(pT)8 and its derivatives containing various 5′‐end groups were estimated in the reaction of polymerization catalyzed with AMV‐RT and FK. The change in affinity of modified primers was more pronounced in the case of AMV‐RT than in the case of FK. Introducing in d(pT)8 of intercalators such as phenazinium, ethidium and daunomycin residues in 2.7‐, 8.7‐ and 11‐fold increases in the primer affinity to AMV‐RT, respectively. However, in the case of hemin and cholesterol derivatives the K
m values were 3 and 5 times higher than those for d(pT)8. Compared to d(pT)8, the affinity of FK to all the above analogs was 2.3—3.6 times higher with the exception of cholesterol derivative to which it was 2.4‐fold lower. The effect of the 5′‐end residues on the V
max values of d(pT)8 was small and ranged from 44% to 120% of that for d(pT)8. Therefore such reactive derivatives of oligonucleotides can be used as effective primers of AMV‐RT and FK. Possible reasons for various effects of the 5′‐end residues of the primer on its interaction with FK or AMV‐RT in the presence of poly(A) are discussed.
Penetration enhancement across a model membrane by liposomally entrapped drugs using N,N′‐diacylcystine as a bilayer lipid
Authors: Kamagami Saburo , Okabe Hideaki , Kainose Hisayasu , Kikkawa Masayoshi , Ishigami Yutaka
Published: July 2002

A liposomal system for percutaneous absorption and transdermal drug delivery was developed and evaluated using a model apparatus connecting two chambers by a membrane. When N,N′‐long chain diacyl L‐cystine was incorporated into the liposomal bilayer as well lecithin and cholesterol, liposomally entrapped calccin as the model drug transferred well across the keratin membrane functioning as the skin model.
Pancreatic secretory trypsin inhibitor (PSTI) isolated from pig intestine Influence on insulin and somatostatin release
Authors: Agerberth Birgitta , Östenson Claes-Göran , Efendic Suad , Jörnvall Hans
Published: July 2002

Upon investigation of pig intestinal peptides for effects on the relcuse of endocrine hormones from the isolated perfused rat pancreas, we reported earlier that glucose‐stimulated insulin release was inhibited by PEC‐60, a peptide with marked sequence similarity to PSTI (pancreatic secretory trypsin inhibitor). Continuing this study we found a polypeptide, which inhibited glucose‐induced insulin release but enhanced glucose‐induced somatostatin secretion. Determination of the amino acid sequence of this polypeptide revealed that it is identical to that of PSTI. Thus, PSTI modulates islet hormone release.
ATP‐citrate lyase is another enzyme the histidine phosphorylation of which is inhibited by vanadate
Authors: Krivanek Jiri , Novakova Ludmila
Published: July 2002

We have recently shown that phosphorylation of histidine residue of the α‐subunit of the succinyl‐CoA synthetase is inhibited by both vanadate and vanadyl. To assess the universality of this inhibition, we have estimated the effect of vanadate on the phosphorylation of another enzyme ATP‐citrate lyase, prepared from rat liver. This enzyme contains histidine as the only amino acid with an acid‐labile (P—N) phosphate bond. The 67% inhibition of endogenous phosphorylation by 1 mM vanadate disappeared after cleavage of the acidic P—N bond of histidine with acidic sample solution. The remaining 33 per cent radioactivity was due to labelling of the acid‐stable phosphoamino acids (P‐serine and P‐threonine), the phosphorylation of which was not affected by vanadate. The dose—response curve for vanadate inhibition closely resembles that shown previously for inhibition of phosphorylation of histidine in the succinyl‐CoA synthetase. The results suggest that the action of vanadate on histidinyl phosphorylation is a more general effect (like its influence on phosphorylation of the protein‐bound tyrosine).
Protein kinase C(19–31) pseudosubstrate inhibition of insulin action in rat adipocytes
Authors: Standaert Mary L. , Sasse Joachim , Cooper Denise R. , Farese Robert V.
Published: July 2002

Treatment of intact adipocytes with the autoregulatory PKC pseudosubstrate PKC(19–31) inhibited insulin‐stimulated hexose uptake and lipogenesis, with no effect on basal values. The effect was dose‐dependent with respective IC10 values of 30 μM and 600 μM for insulin‐stimulated hexose uptake in electroporated and intact adipocytes. These studies indicate that PKC may play a role in the mediation of insulin action in adipocytes.
A rapid purification method for neurogranin, a brain specific calmodulin‐binding protein kinase C substrate
Authors: Deloulme Jean Christophe , Sensenbrenner Monique , Baudier Jacques
Published: July 2002

A rapid purifiacation method is reported for bovine brain neurogranin, a calmodulin‐binding protein kinase C (PKC) substrate. This method takes advantage of the fact that the protein remains soluble in 2.5% perchlorie acid (PCA) and that it binds to a calmodulin‐Sepharose column in the absence of calcium. Other PKC substrate proteins that remain to be to be identified were also found to share these two properties, suggesting that a class of calmodulin‐binding PKC substrates may exist in the brain.
The α subunit of human chorionic gonadotropin hormone synthesized in insect cells using a baculovirus vector is biologically active
Authors: Nakhai Bita , Pal Rahul , Sridhar Padma , Talwar G.P. , Hasnain Seyed E.
Published: July 2002

A recombinant baculovirus, vAcαhCG, having a replacement of the viral polyhedrin gene with the cDNA encoding the α subunit of hCG was used to express αhCH, an extensively glycosylated hormone in insect cells. Virus‐infected cells, 72 h pi, secreted ∼ 11.3 μg αhCG/2 × 104 cells/ml which was identical to the native hormonal peptide in terms of electrophoretic mobility, immunoreactivity and bioactivity on association with β subunit, as evident by its binding to rat testicular cells and induction of steroidogenesis in a mouse Leydig cell bioassay system. The αhCG secreted into the medium represented ∼20–30% of the total hCG synthesized by vAcαCG infected insect cells. The implications of using a very late promoter, in a baculovirus expression system, for directing the transcription of a gene whose gene product requires extensive post‐translation modifications are discussed.
Nucleotide sequence of the replication region of the marine Rhodobacter plasmid pRD31
Authors: Matsunaga Tadashi , Mihashita Hideaki , Miyake Masato , Burgess J.Grant
Published: July 2002

The minimum region required for the replication of the marine Rhodabacter endogenous plasmid pRD31 has been sequenced. This region is located on a 1367‐bp HincII‐PstI restriction fragment and is 62% rich in G‐C base pairs. A region homologous to the bacteriophage P1 repA promoter, which overlaps two inverted repeats, has been identified. Plasmids with mutations in this 1367‐bp region could not replicate in marine Rhodobacter hosts. This is the first identified replication origin of a photosynthetic bacterium.
Differential synthesis by cultured atrial and ventricular rat cardiac myocytes of myosin light chain isoforms
Authors: Brand Thomas , Milting Hendrik , Zippel Monika , Jockusch Harald
Published: July 2002

Dissociated cells from neonatal rat atria and ventricles were cultured in monolayers for 3 days. Newly synthesized 35S‐methionine labeled myosin light chain isoforms ALC‐1, ALC‐2 (atrial) and VLC‐1, VLC‐2 (ventricular) were identified on 2D gels, and their pattern of synthesis was compared to that of myocard fragments immediately after explantation. ALCs were synthesized in 5‐ to 10‐fold excess over VLCs by atrial cultures, whereas the converse was true for ventricular cultures, with two exceptions: one third of the LC‐1 synthesized by ventricular fragments was ALC‐1, and dissociated atrial cells synthesized very little LC‐2 of either isoform. The former finding corresponds to the relatively high proportion of ALC‐1 in neonatal ventricular tissue. We conclude that the regional programme of LC isoform expression is basically retained after tissue explantation and even after dissociation and culturing of cardiac myocytes.
Modulation of type‐1 protein phosphatase by synthetic peptides corresponding to the carboxyl terminus
Authors: Martin Bruce L. , Shriner Carol L. , Brautigan David L.
Published: July 2002

Protein phosphatase type‐I (PP‐1) has a protease resistant catalytic core M
r = 35000 (PP‐35K) and a carboxyl terminal segment which affects activity with various substrates. We found that micromolar concentration of a synthetic peptide, corresponding to residues 312–326 of the PP‐1 carboxyl terminus (P312‐326) that is missing from PP‐35K, increased the phosphatase activity of PP‐35K with phosphorylase and myosin light chains as substrates by decreasing the apparent K
m without a change in V
m. Purified PP‐1 and PP‐35K were inhibited identically by okadaic acid, but peptide P312‐326 only stimulated the activity of PP‐35K, not full‐length PP‐1. Other peptides corresponding to the carboxyl terminus of phosphatase‐2A or to the amino terminus of PP‐1 did not affect the activity of PP‐35K. A sequence conserved in PP‐1 from different species, Pro‐He‐Thr‐Pro‐Pro was implicated as the active region because a derivative peptide, Ala‐Pro‐Ile‐Thr‐Pro‐Pro‐Ala, stimulated the activity of PP‐35K to the same extent as peptide P312‐326 although at higher concentrations. These results indicate that the carboxyl terminus of PP‐1 interacts with the catalytic core to modulate its activity, and suggest that the physiological regulation of PP‐1 may involve this segment.
Beneficial effect of dexamethasone on attenuated hormone‐induced uptake of calcium and glycogenolysis by perfused liver of rats infected with Fasciola hepatica
Authors: Hanisch Maureen J.E. , Behm Carolyn A. , Bygrave Fyfe L.
Published: July 2002

Infection of rats with the liver fluke, Fasciola hepatica, impaired the responses of the perfused liver to calcium uptake and glucose release induced by the synergistic action of glucagon and vasopressin. Treatment of infected rats with dexamethasone prevented the impairment of each of these two responses.
Full assignment of heme redox potentials of cytochrome c3 of D. vulgaris Miyazaki F by 1H‐NMR
Authors: Park Jang-Su , Kano Katsuhiro , Niki Katsumi , Akutsu Hideo
Published: July 2002

Site‐specific heme assignment of the 1H‐NMR spectrum of cytochrome c
3 of D. vulgaris Miyazaki F, a tetraheme protein, was established. The major reduction of the heme turned out to take place in the order of hemes I, III, IV and II (numbering in the crystal structure). The hemes with the smallest and greatest solvent accessibility were reduced at the highest and lowest potentials on average, respectively. A cooperative interheme interaction was attributed to a pair of the closest hemes, namely, hemes III and IV. This assignment can provide the physicochemical basis for the elucidation of electron transfer of this protein.
Calcium mobilization in human platelets by receptor agonists and calcium‐ATPase inhibitors
Authors: Brüne Bernhard , Ullrich Volker
Published: July 2002

Inhibitors of the endoplasmic reticulum Ca2+‐ATPase like thapsigargin (TG) and 2,5‐di (tert‐butyl)‐1,4‐benzohydroquinone (tBuBHQ) cause increases in cytosolic calcium in intact human platelets resulting from prevention of reuptake. A maximal concentration of TG (0.2 μM) mobilized 100% of sequestered Ca2+ compared to the action of a receptor agonist like thrombin (0.1 U/ml). A maximal dose of tBuBHQ (50 μM) stimulated release of about 40% of intracellular calcium compared to thrombin and TG. The reduced ability of tBuBHQ to release calcium can be explained with an inhibitory effect on the cyclooxygenase pathway (K
i ≈ 7 μM). Thererore tBuBHQ is not able to cause platelet aggregation compared to TG. In the presence of a cyclooxygenase inhibitor or a thromboxane A2 receptor antagonist the action of TG is identical to that observed with tBuBHQ. Generally, inhibition of calcium sequestration does not automatically result in platelet activation. In contrast to a receptor mediated activation Ca2+‐ATPase inhibitors require the self‐amplification mechanism of endogenously formed thromboxane A2 to cause a similar response. We conclude that the calcium store sensitive to Ca2+‐ATPase inhibitors is a subset of the receptor sensitive calcium pool.
Effects of a Ca2+ gradient and water activity on the phosphorylation of Ca2+‐ATPase by Pi
Authors: Caldeira Maria Teresa , de Meis Leopoldo
Published: July 2002

The effects of dimethyl sulfoxide (20% v/v) on the phosphorylation of Ca2+‐ATPase of the sarcoplasmic reticulum by Pi vary depending on whether or not a Ca2+ gradient is formed across the vesicle membranes. In the absence of a Ca2+ gradient the solvent promotes a large increase in the affinity for Pi. This increase is no longer observed after the formation of a Ca2+ gradient. The enzyme affinity for Mg2+ is practically the same in the presence and absence of a gradient. Addition of dimethyl sulfoxide leads to an increase of the enzyme affinity for Mg2+ both in the presence and in the absence of a gradient.
Guanosine nuceleotides modulate the inhibitory effect of Brefeldin A on protein secretion
Authors: Zeuzem Stefan , Zimmermann Petra , Schulz Irene
Published: July 2002

Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the endoplasmic reticulum (ER), leaving no definable Golgi‐apparatus, and blocks transport or proteins from the ER to distal secretory compartments of the cell. Using pulse‐chase experiments the present study shows that BFA (1 μ/ml) inhibits basal and CCK‐stimulated protein secretion in isolated pancreatic acinar cells by 65 ± 6% and 84 ± 5%, respectively. In isolated permeabilized cells higher concentrations of BFA (30 μg/ml) were necessary to obtain inhibition of protein secretion. In parallel experiments protein secretion was stimulated by GTP (1 mM). BFA had no inhibitory effect on protein secretion in the presence of GTP, indicating that BFA might act on a GTP‐binding protein. Investigating the effect of BFA on small molecular weight GTP‐binding proteins we observed that [α‐32P]GTP binding to a 21 kDa protein in a subcellular fraction enriched in ER was increased in the presence of BFA. We conclude that this 21 kDa and possibly also other GTP‐binding proteins may be the molecular target of Brefeldin A in pancreatic acinar cells.
Inhibition of 5S RNA transcription in vitro by nucleosome cores with low or high levels of histone acetylation
Authors: Roberge Michel , O'Neill Timothy E. , Bradbury E.Morton
Published: July 2002

Nucleosomes exert strong inhibitory effects on gene transcription in vitro and in vivo. Since most DNA is packaged in nucleosomes, there must exist mechanisms to alleviate this inhibition during gene activation. Nucleosomes could be destabilized by histone acetylation which is strongly correlated with gene expression. We have compared the effects of nucleosomes cores with low or high levels of histone acetylation on 5S RNA transcription with Xenopus nuclear extracts in vitro. Little or no difference was observed over a range of 1 to 15 nucleosome cores per plasmid template. This result suggests that nucleosomal DNA is not more accessible to transcription factors and to the transcription machinery in acetylated nucleosomes.
Nitrite activates the transcription of the Pseudomonas aeruginosa nitrite reductase and cytochrome c‐551 operon under anaerobic conditions
Authors: Arai Hiroyuki , Igarashi Yasuo , Kodama Tohru
Published: July 2002

The transcription of the Pseudomonas aeruginosa denAB operon, which consists of the nitrite reductase and cytochrome c‐551 genes, is induced under anaerobic conditions. However, under anaerobic non‐denitrifying conditions (anaerobic growth on arginine), the promoter activity of the operon was approximately one‐fifth of that under anaerobic denitrifying conditions (anaerobic growth in the presence of nitrite or nitrate). This result clearly demonstrates that the presence of nitrite or nitrate activates the transcription of P. aeruginosa denAB operon under anaerobic conditions.