bluuebunny/crossref_metadata_2025 huggingface dataset:
NMR solution structure of gramicidin A complex with caesium cations
Authors: Arseniev Alexander S. , Barsukov Igor L. , Bystrov Vladimir F.
Published: July 2002

The conformation of the 1:1 complex of [Val1] gramicidin A with caesium cations has been determined in methanol/chloroform (1:1) solution by 2‐dimensional1H‐NMR spectroscopy. The molecular structure was found to be a right‐handed antiparallel double helical dimer ↑↓ππLD7.2with 7.2 residues per turn, which incorporates two caesium cations.
Cytoplasmic pH, a key determinant of growth factor‐induced DNA synthesis in quiescent fibroblasts
Authors: Pouysségur J. , Franchi A. , L'Allemain G. , Paris S.
Published: July 2002

In response to growth factors, quiescent fibroblast mutants lacking Na+/H+ exchange activity fail to elevate their cytoplasmic pH (pHi) and to reinitiate DNA synthesis at neutral and acidic pHo. A pHi threshold of ~7.2 exists, below which growth factors cannot set in motion the G0 to S phase transition. Restoration of the pHi defect in mutant cells restores the wild‐type phenotype. These findings, combined with the properties of another class of mutants able to grow at very low pHo, demonstrate that pHi modulated by growth factor activation of the Na+/H+ antiporter, plays a determinant role in growth control.
Persistent activity modification of phosphatidate phosphohydrolase and fatty acyl‐CoA synthetase on incubation of adipocytes with the tumour promoter 12‐O‐tetradecanoylphorbol 13‐acetate
Authors: Hall Michael , Taylor Stephen J. , Saggerson E.David
Published: July 2002

Incubation of rat adipocytes with the phorbol ester TPA caused a dose‐dependent, persistent increase in phosphatidate phosphohydrolase activity and decrease in fatty acyl‐CoA synthetase activity. Half‐maximal effects were obtained with 1–2 × 10−10 M TPA. TPA did not alter basal or noradrenaline‐stimulated lipolysis. It is suggested that these two enzymes might be modified by protein kinase C.
Irreversible inhibition of pancreatic lipase by bis‐p‐nitrophenyl methylphosphonate
Authors: Sikk P. , Osa A. , Aaviksaar A.
Published: July 2002

The reaction of porcine pancreatic lipase with an organophosphorus compound bis‐p‐nitrophenyl methylphosphonate (BNMP) resulted in the complete and irreversible inhibition of lipase activity on tributyrin emulsion (25°C, pH 7.5, 40 mM Na‐veronal‐HCl buffer) whereas the activity of the enzyme on p‐nitrophenyl acetate solution remained unchanged. The BNMP‐modified enzyme did not bind on hydrophobic interfaces (siliconized glass beads). Tyr 49 was presumed to be the modification site, and the conclusion has been made that this residue is implicated in the interface recognition site of pancreatic lipase.
Degradation of peptides and proteins of different sizes by homogenates of human MRC5 lung fibroblasts
Authors: Wharton S.A. , Hipkiss A.R.
Published: July 2002

The degradation of haemoglobin and haemoglobin‐derived peptide fragments by homogenates of MRC5 fibroblasts has been investigated. Results show that the smaller fragments were degraded more rapidly than larger substrates at both pH 5.5 and pH 7.5. Only the smallest of the soluble cyanogen bromide peptides (Mr
3500) was degraded at pH 7.5. Degradation at pH 5.5 proceeded more rapidly than that at pH 7.5 for all substrates tested but was more marked with the larger substrates. Homogenates prepared from aged cells degraded puromycyl peptides and, to a lesser extent, cyanogen bromide peptides at a slower rate, at pH 7.5, than those prepared from younger cells. We suggest that cytosolic degradation is less selective and at least one cytosolic proteolytic activity decreases as cells age.
A low‐Mr chemoattractant for vascular endothelial cells
Authors: Büki Kálmán G. , Seppä Heikki
Published: July 2002

The formation of new blood vessels occurs by sprouting from previously existing microvasculature. The process involved directed migration of the vascular endothelial cells towards chemical signals released from the target tissue. We have used the Boyden chemotaxis chamber method to identify chemotactic signals for fetal bovine vascular endothelial cells. Human placenta organ cultures produce a high‐M
r chemoattractant for the endothelial cells from which a low‐M
r factor can be liberated with trichloroacetic acid treatment and ethanol extraction. This activity was isolated from extracts of human placenta using Sephadex LH‐20, Amberlite XAD‐2, and silica gel thin‐layer chromatography. The M
r of the factor is less than 400, it is lipophilic and resistant to proteolytic enzymes. The factor induces chemotactic migration of both aortic endothelial cells and capillary endothelial cells from the retina, but has no effect on fibroblasts or leukocytes suggesting a specific function of the compound for the vascular endothelial cells.
Isolation of two forms of the high‐molecular‐mass serine protease, ingensin, from porcine skeletal muscle
Authors: Ishiura Shoichi , Sano Motoki , Kamakura Keiko , Sugita Hideo
Published: July 2002

Porcine muscle
Protease
SDS‐activation
Difference in pathway of Escherichia coli outer membrane permeation between penicillins and cephalosporins
Authors: Yamaguchi Akihito , Tomiyama Noribumi , Hiruma Ryoichi , Sawai Tetsuo
Published: July 2002

The differences in the outer membrane permeation between two major subgroups of β‐lactam antibiotics were studied. The permeation of cephalosporins was closely related to porin channels in the outer membrane. In contrast, the outer membrane permeation of penicillins did not decrease in porin‐deficient mutants and, in Rd‐type mutants, their permeability became proportional to the hydrophobicity of the molecules. The activation enthalpy of the penicillin permeation was significantly larger than that of cephalosporins. These observations indicate that penicillins can use the hydrophobic region for the major route of outer membrane passage whereas the cephalosporin permeation is restricted to the pathway via the porin pore.
Partial sequence homology of human myc oncogene protein to beta and gamma crystallins
Authors: Crabbe M.James C.
Published: July 2002

The human cellular myc gene is one of about 20 cellular oncogenes which code for a variety of proteins including protein kinases and growth factors [1]. The human gene is related to the avian myelocytomatosis leukaemia virus MC29 [2] and produces a binding protein which may be involved in regulation of gene expression [3] and cellular differentiation and proliferation [4]. The crystallins are proteins in the eye lens synthesised at different stages of cell differentiation and proliferation, and whose short range order is necessary for lens transparency [5,6]. Computer‐based sequence comparisons show that beta Bp and gamma II crystallins, which show partial sequence homology and conservation of ‘Greek Key’ motives [7,8] are also partially homologous to two regions on the human myc protein, though this protein probably does not conserve the ‘Greek Key’ structural motives.
Effect of ascorbic acid on the production of singlet oxygen by purified human myeloperoxidase
Authors: Kanofsky Jeffrey R. , Wright Jonathan , Tauber Alfred I.
Published: July 2002

We have previously studied purified human myeloperoxidase‐hydrogen peroxide‐halide ion systems as models of possible singlet oxygen production by granulocytes. While myeloperoxidase could efficiently produce singlet oxygen, the yield of singlet oxygen at a physiological pH with Cl− was very small due to enzyme inactivation. In that Bolscher et al. [(1984) Biochim. Biophys. Acta 784, 189‐191] observed that micromolar concentrations of ascorbic acid prevented inactivation of myeloperoxidase and increased the production of hypochlorous acid, we examined whether ascorbic acid would augment singlet oxygen production by the myeloperoxidase‐hydrogen peroxide‐halide ion systems. Ascorbic acid, however, fails to increase the singlet oxygen yield, suggesting that it does not augment singlet oxygen production in the intact granulocyte by a myeloperoxidase‐dependent mechanism.
Sequences in the 3'‐terminal coding regions of 5 S and 18 S rRNA genes may contribute to co‐ordinated expression of 5 S rRNA and pre(18 S/5.8 S/26 S) rRNA genes
Authors: Lane Byron G.
Published: July 2002

It has long been known that there is remarkable affinity between 5 S and 18 S rRNA from wheat embryos. Experimental evidence has failed to support the proposition that the extensive complementarity which exists between 3'‐terminal regions of the large‐subunit (5 S) and small‐subunit (18 S) rRNA is involved in uniting the subunits of the ribosome. Alternatively, it is suggested that the degree of complementarity between the 3'‐terminal regions of wheat‐embryo 5 S and 18 S rRNA may have significance at the level of the genome. It is suggested that common sequences in the 3'‐terminal coding regions of 5 S and 18 S rRNA genes may serve to co‐ordinate the expression of 5 S rRNA genes with the expression of the remainder of the (18 S/ 5.8 S/26 S) rRNA genes.
Purification of epidermal plasminogen activator inhibitor
Authors: Hibino Toshihiko , Izaki Seiichi , Izaki Masakatsu
Published: July 2002

A plasminogen activator inhibitor was purified from human cornified cell extract by DEAE‐Sepharose, Sephacryl S‐200, and high‐performance liquid chromatographies on hydroxyapatite HPHT and anion‐exchanger Mono Q at pH 7.2 and 8.0. The purified inhibitor showed M
r 43000 and pI 5.2. 50% inhibition of fibrinolytic activity (1.5 IU) of urokinase and tissue‐type plasminogen activator was attained by 0.60 ng and 11.0 ng purified inhibitor respectively. Synthetic substrate assay demonstrated slow tight‐binding inhibition to both urokinase and tissue‐type plasminogen activator. The inhibitor did not inactivate plasmin, thrombin, glandular kallikrein or trypsin.
Protein phosphorylation of lysosomal arylsulfatase B in normal and leukemic leukocytes
Authors: Uehara Yoshio , Gasa Shinsei , Makita Akira , Sakurada Keisuke , Miyazaki Tamotsu
Published: July 2002

An acidic variant form of arylsulfatase B from normal leukocytes and chronic myelogenous leukemia (CML) leukocytes was found to be phosphorylated at its serine and threonine residues through in vivo phosphorylation with 32Pi. However, the predominant phosphorylation site was serine in normal cells, in contrast to threonine in CML cells. A cyclic AMP‐dependent protein kinase was responsible for phosphorylation of the sulfatase of CML cells.
Synthesis, processing, and secretion of rat immunoglobulin E made in Xenopus oocytes
Authors: Lund Torben , Bravo Rodrigo , Johansen Hanne R. , Zeuthen Jesper , Vuust Jens
Published: July 2002

Rat immunoglobulin E (IgE) synthesized in Xenopus laevis oocytes, injected with rat plasmacytoma mRNA, was analysed by specific immunoprecipitation and SDS‐polyacrylamide gel electrophoresis under reducing as well as non‐reducing conditions. The results indicate that the oocytes will translate and correctly process the rat IgE heavy and light chains, resulting in secretion of a correctly assembled, normal immunoglobulin molecule. The normal, extensive glycosylation of the IgE heavy chain (ε‐chain) is faithfully carried out by the oocytes; therefore, this posttranslational modification is apparently of an unspecific nature, and does not depend upon a mechanism specific for plasma cells.
Oxidation‐reduction potential dependence of reaction centre triplet formation in the isolated D1/D2/cytochrome b‐559 photosystem II complex
Authors: Telfer A. , Barber J. , Evans M.C.W.
Published: July 2002

Formation of a spin‐polarized triplet on illumination at low temperature is thought to be characteristic of charge separation in photosynthetic reaction centres. The formation of a spin‐polarized triplet was confirmed in the D1/D2/cytochrome b‐559 photosystem II reaction centre preparation. The oxidation‐reduction potential range in which triplet formation occurs was determined. Triplet formation was observed at potentials between 400 and −530 mV. Loss of triplet formation, at more negative potentials, is suggested to reflect chemical reduction of the pheophytin acceptor. At potentials more oxidised than 400 mV, illumination results in formation of a radical at g = 2.0025 with a 0.75 mT linewidth. We propose that this is the result of oxidation of P680 coupled to reduction of an electron acceptor, possibly non‐haem iron. The results confirm that none of the other electron acceptors normally found in photosystem II are functional in this preparation.
Base‐catalyzed reactivation of glucogen phosphorylase reconstituted with a coenzyme‐substrate conjugate and its analogues
Authors: Horinishi Nobutaka , Tagaya Mitsuo , Fukui Toshio
Published: July 2002

Glycogen phosphorylase reconstituted with pyridoxal (5′)diphospho(1)‐α‐D‐glucose (PLDP‐Glc) is catalytically inactive but slowly converted to the active enzyme through the cleavage of the pyrophosphate linkage. A similar reaction occurs more rapidly on PLDP‐Gal and ‐Xyl but not on PLDP‐Man. Values of pK
a for all the reactions are about 8.3, suggesting the participation of a common basic residue in these reactions. Based on the present and other results, it is presumed that Tyr‐573 or Lys‐574 acts as the base abstracting the proton from 2‐hydroxyl group of the glucosyl moiety of PLDP‐Glc.
Adenosine di‐, tri‐ and tetraphosphopyridoxals modify the same lysyl residue at the ATP‐binding site in adenylate kinase
Authors: Yagami Tatsurou , Tagaya Mitsuo , Fukui Toshio
Published: July 2002

Adenosine diphosphopyridoxal modifies Lys‐21 in adenylate kinase which is located in a glycine‐rich loop [(1987) J. Biol. Chem. 262, 8257–8261]. We presently report that adenosine tri‐ and tetraphosphopyridoxals modify the same lysyl residue more rapidly than the diphospho compound does. However, susceptibilities of the Schiff bases between the labels and the lysyl residue to sodium borohydride considerably differ in the modifications with the three reagents. These observations seem to be ascribable to the mobility of the ε‐amino group of Lys‐21 in the active‐site region of the enzyme.
Bisfunction of propionic acid on purified rat liver β‐ureidopropionase
Authors: Kikugawa Mariko , Fujimoto Shigeko , Mizota Chizuru , Tamaki Nanaya
Published: July 2002

Propionic acid and isobutyric acid, which are structural analogues of N‐carbamoyl‐β‐alanine and N‐carbamoyl‐β‐aminoisobutyric acid, respectively, acted as an allosteric activator as well as a competitive inhibitor of purified rat liver β‐ureidopropionase. Propionic acid and isobutyric acid had a K
i value of approx. 0.3 mM at pH 7.0. The Hill coefficient for N‐carbamoyl‐β‐alanine was 2.0, but the cooperativity decreased to 1.0 in the presence of 1 mM propionic acid. The K
value towards N‐carbamoyl‐β‐alanine was calculated to be 0.17 mM from Hill plots and the K
m value was determined to be 0.06 mM from replots of the apparent K
m vs propionic acid.
A novel lectin‐independent interaction of P fimbriae of Escherichia coli with immobilized fibronectin
Authors: Westerlund Benita , Kuusela Pentti , Vartio Tapio , van Die Irma , Korhonen Timo K.
Published: July 2002

Binding of P fimbriae of uropathogenic Escherichia coli to purified human plasma fibronectin and human placental type IV collagen was studied. In an enzyme immunoassay, purified P fimbriae bound strongly to immobilized intact fibronectin and to the aminoterminal 30‐kDa fragment and the 120–140‐kDa carboxyterminal fragments of fibronectin. Binding to the gelatin‐binding 40‐kDa fragment of fibronectin was considerably weaker. No binding to immobilized type IV collagen was seen. The interaction between P fimbriae and immobilized fibronectin was not inhibited by α‐D‐Gal‐(1‐4)‐β‐D‐Gal‐1‐O‐Me, a receptor analog of P fimbriae. Moreover, a mutated P fimbria lacking the lectin activity behaved similarly in the adherence assays. Recombinant strains expressing the corresponding cloned fimbriae genes bound to immobilized fibronectin, but no binding to soluble 125I‐labelled fibronectin was found. The results suggest that P fimbriae interact with immobilized fibronectin and that the binding mechanism does not involve the lectin activity of the fimbriae.
Identification of single‐strand initiation signals in the terC region of the Escherichia coli chromosome
Authors: Hiasa Hiroshi , Sakai Hiroshi , Komano Tohru
Published: July 2002

On the basis of clear‐plaque formation, we detected initiation signals in the terC region of the Escherichia coli chromosome. At least two single‐strand initiation signals were identified from the terC region. The nucleotide sequences of these two signals were determined. Sequence homologies, variations of the consensus sequence of n′ protein recognition sites, 5′‐GAAGCGG‐3′, were found within these signals. A novel conserved sequence was also found within these signals. Their initiation activities were measured both by the infection growth assay and by the ability to convert the single‐stranded DNA to the duplex replicative form DNA in vivo.
Glyoxysomal malate synthetase is specifically degraded in microbodies during greening of pumpkin cotyledons
Authors: Mori Hitoshi , Nishimura Mikio
Published: July 2002

A cDNA clone (pKMS: insert, 1.9 kbp) for glyoxysomal malate synthase was prepared from a cDNA library constructed by poly(A)+ RNA from etiolated pumpkin cotyledons and was recombined into the in vitro transcription plasmid, Bluescript. Malate synthase synthesized in an in vitro transcription‐translation system was translocated into isolated glyoxysomes. The translocated malate synthase was protected from digestion by added proteinase K. Malate synthase, a glyoxysome‐specific enzyme, could also be translocated into leaf peroxisomes prepared from green pumpkin cotyledons, indicating that the machinery for protein translocation into leaf peroxisomes is similar to that into glyoxysomes. When microbodies prepared from greening pumpkin cotyledons were used in the translocation experiments, the translocated malate synthase was observed to be degraded. This result shows that the glyoxysome‐specific enzyme, malate synthase, is degraded in microbodies during the microbody transition from glyoxysomes to leaf peroxisomes.
A powerful method of sequential proton resonance assignment in proteins using relayed 15N‐1H multiple quantum coherence spectroscopy
Authors: Gronenborn Angela M. , Bax Ad , Wingfield Paul T. , Clore G.Marius
Published: July 2002

A powerful method of sequential resonance assignment of protein 1H‐NMR spectra is presented and illustrated with respect to the DNA‐binding protein ner from phage Mu. It is based on correlating proton‐proton through‐space and through‐bond connectivities with the chemical shift of the directly bonded 15N atom. By this means, ambiguities arising from chemical shift degeneracy of amide proton resonances can be resolved. The experiments described involve combining the 1H‐detected heteronuclear multiple quantum coherence correlation experiment with homonuclear nuclear Overhauser enhancement, J‐correlated or Hartmann‐Hahn experiments.
Establishment of mouse and rat hepatoma cell clones showing stable expression of rabbit cytochrome P450 IA2
Authors: Corcos L. , Rechenmann C. , Weiss M.C. , Pompon D.
Published: July 2002

Cytochrome P450 IA2, a liver‐specific member of the 3‐methylcholanthrene‐inducible family, is never detected in established cell lines. With the aim of isolating cells stably producing this protein, we have used rat and mouse hepatoma cells as recipients in transfection experiments involving rabbit cytochrome P450 IA2 cDNA. We report here the isolation of five hepatoma cell clones expressing functional P450 IA2. The level of expression is comparable to that found in COS cells transiently transformed by other P450 cDNAs. It ranges between 0.4 and 1.6 pmol P450 IA2/mg total cell protein.
Multiple charge isomers of human recombinant interleukin‐1β
Authors: Kabir Shahjahan
Published: July 2002

Human interleukin‐1β (rhuIL‐1β), obtained by DNA recombinant technology, was radiolabelled. Its isoelectric properties were determined by various analytical techniques such as high‐voltage ultrathin layer isoelectric focusing (IEF) and chromatofocusing. The rhuIL‐ 1β molecule had a molecular mass of 18 kDa, as determined by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. When examined by IEF on a polyacrylamide gel of 1 mm thickness in the pH range of 3.5–9.5, it was resolved into two broad bands appearing in the pH range of 6.2–5.8 and 5.5–5.2. Each of the two bands was further resolved into multiple bands when electrofocused on (i) a thinner gel of 0.5 mm thickness and (ii) a narrower pH range of 5–8. Upon chromatofocusing in a liquid column, it was possible to isolate various charged components of rhuIL‐1β. However, all these components reacted to the antiserum to rhuIL‐1β and displayed a molecular mass of 18 kDa suggesting the charge heterogeneity of rhuIL‐1β.
Expression of the E. coli Lac Z gene from a defective HSV‐1 vector in various human normal, cancer‐prone and tumor cells
Authors: Boothman David A. , Geller Alfred I. , Pardee Arthur B.
Published: July 2002

Introducing foreign genetic material into human cells is essential for the elucidation of the function of various human genes and has potential use in the treatment of human diseases by gene therapy. In this study we demonstrate that a defective herpes simplex virus type 1 vector, pHSVlac, can effectively transfer and express the Escherichia coli Lac Z gene in a variety of exponential and quiescent human cells. The human cells tested included representative cells derived from cancer‐prone patients that presumably have various DNA repair deficiencies.
Unwinding of duplex DNA during transcription initiation at the Escherichia coli galactose operon overlapping promoters
Authors: Chan Bernard , Minchin Stephen , Busby Stephen
Published: July 2002

We have used potassium permanganate as a probe to detect DNA duplex unwinding in vitro, in open complexes between E. coli RNA polymerase and DNA fragments carrying the E. coli galactose operon regulatory region. This zone contains 3 overlapping promoters which specify transcription initiation at 3 distinct startpoints. We have used mutant gal derivatives carrying different single point mutations, each of which allows initiation from only one of the 3 start sites. This has allowed us to compare duplex unwinding in open complexes at the 3 different promoters, and to show that the extent of the unwinding is similar in each case. Further, the pattern of DNA modification by potassium permanganate suggests a model for discrimination between the upper and lower strands. Finally, we show that DNA modification by potassium permanganate at the gal promoters is the same in vivo as in vitro.
Evidence for posttranscriptional regulation of synthesis of the Bacillus subtilis Gnt repressor
Authors: Fujita Yasutaro , Fujita Tamie , Miwa Yasuhiko
Published: July 2002

Transcription of the Bacillus subtilis gnt operon results in a polycistronic mRNA that encodes from the 5' end the Gnt repressor, gluconate kinase and permease. The RNA is drastically induced through inactivation of the Gnt repressor by gluconate. The results of deletion analysis of the gnt promoter region upstream of the repressor gene indicated that no other promoter except the gnt promoter was present for expression of the gluconate kinase gene. In contrast to the synthesis of gluconate kinase and permease, which was markedly induced by gluconate, the results of a radioimmunoassay for the Gnt repressor indicated that synthesis of the Gnt repressor from the induced mRNA was posttranscriptionally repressed.
Phosphorylation of magainin‐2 by protein kinase C and inhibition of protein kinase C isozymes by a synthetic analogue of magainin‐2‐amide
Authors: Nakabayashi Hiroki , Brown Judith H. , Morell John L. , Chen Hao-Chia , Huang Kuo-Ping
Published: July 2002

Magainins are a family of antimicrobial peptides present in the skin extracts of Xenopus laevis. Both magainin‐1 and ‐2 do not have any significant effect on the activity of protein kinase C (PKC). Magainin‐2 was found to be readily phosphorylated by PKC to 0.5 mol‐32P/mol of peptide. Neither magainin‐1, which has a sequence of S8AGK and not S8AKK as in the case of magainin‐2, nor the magainin‐2 analogue with substitution of Ala for Ser8 was phosphorylated by the kinase, suggesting that Ser8 is the phosphorylation site of magainin‐2. One synthetic analogue of magainin, designated magainin B, which has a greater tendency for α‐helix formation in non‐aqueous environment than the parent peptide resulting from substitution of Ser8, Gly13, and Gly18 with Ala in magainin‐2‐amide, is a potent inhibitor of PKC. This peptide inhibits all three PKC isozymes with IC50 less than 20 μM. Magainin B also inhibits the binding of [3H]phorbol 12,13‐dibutyrate to the kinase. These results suggest that magainin‐2 may be modified by PKC through phosphorylation and that certain synthetic analogues of magainins may be used as inhibitors of PKC.
Reconstitution of the 9 S estrogen receptor with heat shock protein 90
Authors: Inano Kouichi , Haino Makoto , Iwasaki Masaomi , Ono Naomi , Horigome Tsuneyoshi , Sugano Hiroshi
Published: July 2002

As a first step in the investigation of the reconstitution of steroid hormone receptor systems, we studied the reconstitution of 9 S estrogen receptor (ER) from purified vero ER, which is the estradiol binding subunit, and heat shock protein 90 (hsp 90). By using a phosphate buffer containing molybdate, thiocyanate, dimethylformamide, glycerol, etc., vero ER could be converted to 9 S ER with hsp 90, but not with the control protein, ovalbumin. Inactivation of ER during the reconstitution was suppressed partially by hsp 90, but not by ovalbumin. Like native 8 S ER, the reconstituted ER was sedimented at about 8.9 S and 4.6 S on glycerol gradient centrifugation in low and high salt buffers, respectively.
Synthetic sialyl glycolipids (sialo‐cholesterol and sialo‐diglyceride) induce granulocytic differentiation of human myelogenous leukemia cell line HL‐60
Authors: Saito Masaki , Nojiri Hisao , Ogino Hidetoshi , Yuo Akira , Ogura Haruo , Itoh Masayoshi , Tornita Kenkichi , Ogawa Tomoya , Nagai Yoshitaka , Kitagawa Seiichi
Published: July 2002

When HL‐60 cells were cultivated with synthetic sialyl glycolipids, sialo‐cholesterol and sialo‐diglyceride, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was accompanied by inhibition of cell proliferation. The differentiation‐inducing activity of sialo‐cholesterol was greater than that of sialo‐diglyceride on a molar basis, and the α‐anomer of each compound was more potent than the β‐anomer, suggesting that the stereospecific structure of the compounds is important for the differentiation‐inducing activity.
Alternative splicing gives rise to two forms of the p68 Ca2+ ‐binding protein
Authors: Moss Stephen E. , Crumpton Michael J.
Published: July 2002

The p68 Ca2+ and phospholipid binding protein of the lipocortin/calpactin family appears to exist as two forms. These may be resolved into a closely‐spaced polypeptide doublet by SDS‐PAGE. The cloning and sequencing of p68 revealed an apparent 18 nucleotide alternative splice sequence, which could account for this observation. We show here that an antiserum directed against a synthetic peptide corresponding to the region containing the splice sequence, recognises only the upper band of the p68 doublet by both immunoprecipitation and Western blotting., These results are consistent with alternative splicing being responsible for the generation of the two forms of p68.
Heme methyl hyperfine shift pattern as a probe for determining the orientation of the functionally relevant proximal histidyl imidazole with respect to the heme in hemoproteins
Authors: Yamamoto Yasuhiko , Nanai Norishige , Chûjô Riichirô , Suzuki Tomohiko
Published: July 2002

Heme methyl 1H and 13C resonances of met‐cyano form of myoglobin from the shark, Geleorhinus japonicus (GJMbCN), have been assigned via 1H‐13C heteronuclear shift correlated spectroscopy (COSY) connectivities and their hyperfine shifts were compared with those of the corresponding resonances of some hemoproteins. Variation of the heme methyl 1H hyperfine shift pattern correlates well with the angle (Φ) between the projection of the proximal histidyl imidazole plane onto the heme plane and the NII‐Fe‐Niv vector. The alteration of the interaction of the heme peripheral side‐chains and/or the iron‐bound ligand with the surrounding amino acid residues cannot account for large differences in the shifts of the corresponding heme methyl resonances between GJMbCN and sperm whale MbCN. Since the heme methyl 1H shifts for GJMbCN fall in between those of the corresponding resonances for sperm whale Mb and Aplysia limacma Mb in which the Φ values have been reported to be 19° and 29°, respectively, the Φ value in GJMb is estimated to be slightly larger than 19°.
The role of introns in evolution
Authors: Rogers John H.
Published: July 2002

What are the roles of ‘classical’ introns in the evolution of nuclear genes, and what was the origin of these introns? Exon shuffling has been important in the evolution of cell surface and extracellular proteins, but the evidence for it in respect of intracellular proteins is weak. Intron distributions imply that some introns have been removed while others have been inserted in the course of evolution; ancestral patterns of introns may thus have been obscured. Recent evidence on the self‐splicing and reverse‐splicing abilities of Group II introns supports the hypothesis that these could have been the ancestors of classical introns.
Immunogold localisation of ubiquitin‐protein conjugates in primary (azurophilic) granules of polymorphonuclear neutrophils
Authors: László Lajos , Doherty Fergus J. , Watson Adrian , Self Tim , Landon Michael , Lowe James , Mayer R.John
Published: July 2002

Ubiquitin—protein conjugates are found in the primary (azurophilic) lysome‐related granules but not in the secondary (specific) granules in mature polymorphonuclear neutrophils prepared from bone marrow. This is the first reported demonstration of ubiquitin—protein conjugates in lysosome‐related membrane‐bound vesicles in granulocytes and complements our previous findings of ubiquitinated proteins in lysosomes of fibroblasts. The significance of the selective presence of conjugates in only one of the two main types of neutrophil granules remains to be elucidated but may relate to the presence of the complement of acid hydrolases, including proteases, in the azurophilic granules compared to the specific granules. Ubiquitin—protein conjugates may enter the primary granules during neutrophil maturation by an autophagic process or by a heterophagic process during the fusion of phagosomes with primary granules. Alternatively protein ubiquitination may be involved in granule biogenesis.
Quinone methide as a reactive intermediate formed during the biosynthesis of papiliochrome II, a yellow wing pigment of papilionid butterflies
Authors: Saul Steven J. , Sugumaran Manickam
Published: July 2002

Mushroom tyrosinase and the recently identified, 4‐alkyl‐a‐benzoquinone: 2‐hydroxy‐p‐quinone methide isomerase were used to investigate the mechanism of biosynthesis of papiliochrome II pigment found in the yellow scales of the papilionid butterflies. Incubation of N‐β‐alanyldopamine (NBAD) and L‐kynurenine with mushroom tyrosinase resulted in the formation of adducts tentatively characterized as NBAD quinone ‐ L‐kynurenine adducts. If quinone isomerase was included in this reaction mixture, the formation of two new products could be witnessed. These two products exhibited the same retention time and the same UV and visible spectral properties as those of papiliochrome II diastereoisomers. Since quinone isomerase catalyzes the conversion of quinones to quinone methides, the above studies indicate that papiliochrome II biosynthesis involves non‐enzymatic and hence non‐stereoselective condensation of enzymatically generated NBAD quinone methide with L‐kynurenine.
Amino acid derivatization and analysis in five minutes
Authors: Hušek Petr
Published: July 2002

A convenient method permits acid separation by capillary gas chromatography in minutes — the suitable analytic forms are prepared in seconds.
Both D factor/LIF and IL‐6 inhibit the differentation of mouse teratocarcinoma F9 cells
Authors: Hirayoshi Kazunori , Tsuru Akio , Yamashita Michiaki , Tomida Mikio , Yamamoto-Yamaguchi Yuri , Yasukawa Kiyoshi , Hozumi Motoo , Goeddel D.V. , Nagata Kazuhiro
Published: July 2002

Differentation‐stimulating factor (D factor)/leukemia inhibitory factor (LIF) and IL‐6 are reported to be cytokines having multifaced functions including the induction of differentation in mouse myeloid leukemia MI cells. We here report that both D factor/LIF and IL‐6 inhibit the differentation of mouse teratocarcinoma F9 cells induced by retinoic acid alone or combined with dibutyryl cAMP. From the microscopic observation as well as Northern blot analysis using cDNA probes encoding several marker proteins for differentiation of F9 cells, we conclude that D factor/ LIF and IL‐6 are functionaly closely related in the induction of differentation in MI cells in the inhibition of F9 differentiation.
Characterization of the pcp gene encoding the pyrrolidone carboxyl peptidase of Bacillus subtilis
Authors: Awadé A. , Cleuziat Ph. , Gonzalès Th. , Robert-Baudouy J.
Published: July 2002

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp) is an enzyme that catalyzes the removal of the N‐terminal pyroglutamyl group from some poptides or proteins. Its value in protein chemistry and bacterial diagnosis makes this enzyme an interesting subject of study. The present paper reports for the first time the cloning and characterization of a pyrrolidone carboxyl peptidase gene (pcp). This gene is present in a single copy in the genome of Bacillus subtilis as indicated by Southern blot hybridization analysis. The pcp transcripts were analyzed in Escherichia coli by Northern blot hybridization and S1 nuclease mapping. The deduced amino acid sequence predicts a protein of 215 amino acids with a calculated molecular weight of 23,777 Da. The pcp gene has been over‐expressed in E. coli, allowing the identification and partial characterization of Pcp protein.
The modular architecture of vertebrate collagens
Authors: Bork Peer
Published: July 2002

Collagens are typical mosaic proteins containing a number of shuffled domains. These domains have been classified by sequence similarity in order to characterize their structural and functional relationships to other proteins. This analysis provides an overview of homologies of collagen domains. It also reveals two new relationships: (i) a module common to type V, IX, XI, and XII collagens was found to be homologous to the heparin binding domain of thrombospondin; (ii) the modular architecture of a human type VII collagen fragment was identified. Its N‐terminal globular domain contains fibronectin type III repeats located adjacent to a Von Willebrand factor type A module. The proposed structural similarities point to analogous subfunctions of the respective domains in otherwise distinct proteins.
Relocalization of αB‐crystallin by heat shock in ovarian carcinoma cells
Authors: Voorter Christina E.M. , Wintjes Liesbeth , Bloemendal Hans , de Jong Wilfried W.
Published: July 2002

αB‐Crystallin, a major lens protein, is present in clearly detectable amounts in cultured ovarian carcinoma cells. After heat‐shock treatment of these cells at 45°C αB‐crystallin relocalizes from the detergent‐soluble, cytosolic fraction to the non‐ionic detergent‐insoluble nuclear/cytoskeletal fraction. Colchicine treatment of the cells, alhough giving rise to a vimentin collapse on the nucleus, does not result in redistribution of ß‐cyrstallin. When this colchicine treatment is followed by heat shock, αB‐crystallin relocalizes again to the insoluble fraction, indicating that this relocalization is independent of the collapse of the vimentin network.
A cell free system reveals that capacitation is a prerequisite for membrane fusion during the acrosome reaction
Authors: Spungin Ben , Levinshal Tova , Rubinstein Sara , Breitbart Haim
Published: July 2002

Plasma and outer acrosomal membranes were extracted from bovine spermatozoa and used in an in vitro fusion assay. Fusion was revealed by monitoring the merging of lipids using the chlorophyll a‐N,N′‐dioctadecyloxacarbocyanine‐p‐toluene sulfonate (DCY) method [(1984) Biochim. Biophys. Acta 769. 531–542]. The requirement for capacitation, as well as the effects or pH, calcium and spermine, on membrane fusion in our cell‐free system were similar to those observed in vivo on the acrosomal reaction. This demonstrates for the first time that capacitation and alterations in intracellular pH and calcium concentration, which must precede the acrosomal reaction, are required for the membrane fusion event.
The reaction of GroEL (cpn 60) with the ATP analogue 2',3' dialdehyde ATP
Authors: Thomson Graeme J. , Coggins John R. , Price Nicholas C.
Published: July 2002

The reaction of the E. coli chaperonin GroEL (cpn 60) with the ATP analogue 2',3' oxidised ATP (oATP) has been studied. Treatment with the reagent leads to loss of the ATPase activity of GroEL in a pseudo‐first‐order fashion; this can be prevented by inclusion of ATP in the reaction mixture. Measurements of the stoichiometry of the reaction indicate that the loss of activity corresponds to the incorporation of about one oATP per subunit of GroEL. From analysis of the sequences of modified peptides it is proposed that the reaction probably occurs with one or both of the two cysteines Cys‐457 and Cys‐518, although the instability of the adduct(s) makes a definite identification of the site(s) of reaction difficult. The involvement of Cys side chains in the reaction with oATP was confirmed by using Nbs2 (5,5'‐dithiobis(2‐nitrobenzoate)) to estimate thiol groups in both modified and unmodified GroEL.
Production of disulfide‐linked hirudin dimer by in vitro folding
Authors: Chang Jui-Yoa , Grossenbacher Hugo , Meyhack Bernd , Maerki Walter
Published: July 2002

A simple process of in vitro folding has been developed for the preparation of hirudin dimer. A variant of recombinant hirudin with Asp33 replaced by Cys was expressed in yeast and isolated by HPLC. Crude Cys33‐hirudin contains heterogeneous products that are made of one species of primary sequence. They were together reduced/denatured, and allowed to re‐fold in the sodium bicarbonate buffer (pH 8.3) alone. Active, homogeneous Cys33‐hirudin monomer folded spontaneously with a first order rate constant of 0.05 ± 0.01 min−1, followed by the oxidation of two Cys33 to produce the pure dimer. The folding yield was 90%. On an equal weight basis, both Cys33‐hirudin monomer and the dimer exhibit thrombin inhibitory activity comparable to that of wild‐type hirudin. Due to the presence of an extra cysteine, the folding of active hirudin monomer (formation of three native disulfides) was accelerated by at least 12‐fold.
Inhibition of PDGF BB stimulated DNA synthesis in rat aortic vascular smooth muscle cells by the expression of a truncated PDGFβ receptor
Authors: Kemp Paul R. , Shachar-Hill Yair , Weissberg Peter L. , Metcalfe James C.
Published: July 2002

Rat aortic vascular smooth muscle cells (VSMCs) were transfected with a vector encoding a truncated human PDGFβ receptor or the full‐length human PDGFβ receptor. Cells stably expressing the truncated human PDGFβ receptor or the full‐length human PDGFβ receptor were selected and the effect of PDGF BB on DNA synthesis in these cells was studied. Cells expressing the full‐length PDGFβ receptor entered DNA synthesis normally, however, cells expressing the truncated PDGFβ receptor failed to enter DNA synthesis in response to PDGF BB. These data show that the mitogenic response of rat aortic vascular smooth muscle cells to PDGF BB can be inhibited by the expression of a truncated PDGFβ receptor.
Structural and functional characterisation of recombinant human erythropoietin analogues
Authors: Bittorf Thomas , Jaster Robert , Brock Josef
Published: July 2002

Deletion mutants of a synthetic human erythropoietin‐cDNA were transiently expressed in COS‐7 cells and products analyzed using Western blots and a cell proliferation assay. Only two mutants with deletions affecting the N‐terminal portion and the amino acid sequence 115‐121 displayed biological activity. The exchange of hydrophilic, charged amino acids by alanine in two potential α‐helical regions, the internal amino acid sequence 102–106 and the C‐terminal sequence 154–159, causes a 2–11‐fold loss of activity. The results suggest that both regions are involved in either maintaining the active structure of the hormone or interacting with the receptor.
Regulatory squelching
Authors: Cahill Michael A. , Ernst Wolfram H. , Janknecht Ralf , Nordheim Alfred
Published: July 2002

An important function of transcription factors may be to sequester coactivators or corepressors of transcription. In this manner transcription factors could regulate in trans the activity of promoters to which they do not bind. This may be of widespread significance as a mechanism to control cell cycle‐dependent and differentiation‐specific transcriptional activity within eukaryotic cells. Therefore squelching in vivo may be more important than hitherto appreciated.
Characterization of cytosolic malic enzyme in human tumor cells
Authors: Loeber Gerhard , Dworkin Mark B. , Infante Anthony , Ahorn Horst
Published: July 2002

Cytosolic NADP+‐dependent malic enzyme (ME) from human tumor cells was characterized in detail and compared to ME from normal human tissues produced in recombinant E. coli. Kinetic properties, size as seen in SDS gels, and HPLC elution profiles of tryptic digests of human ‘normal cell’ ME and NADP+‐ME from tumor cells were identical. Thus, NADP+‐ME found in tumor cells does not constitute a tumor‐specific isoform as suggested by other studies but is identical to the ‘housekeeping protein’ predominantly expressed in human liver and white adipose tissue.
Functional analysis of individual brain myosin II isoforms through hybrid formation
Authors: Wang Yulai , Chantler Peter D.
Published: July 2002

We have used a scallop hybrid myosin test system in an attempt to determine the regulatory properties of an individual myosin II isoform from rat brain. The complete coding region of cDNA corresponding to a regulatory light chain isoform previously shown to be expressed in brain [Feinstein, Durand and Milner (1991) Mol. Brain Res. 10, 97‐105] was ligated within the prokaryotic expression vector, pAED4, overexpressed in bacteria, and the purified light chain incorporated within a scallop hybrid myosin. Actin activation was calcium insensitive for all hybrids tested, irrespective of whether light chain phosphorylation had taken place before, or subsequent to, hybrid formation. We discuss the implications of these results, including the possibility that these results constitute evidence for a myosin II isoform within brain that is regulated at the level of the thin filament. In addition, evidence is presented for the presence of an additional, novel isoform of regulatory light chain expressed in rat brain.
Inhibition of the respiratory burst in mouse macrophages by ultra‐low doses of an opioid peptide is consistent with a possible adaptation mechanism
Authors: Efanov A.M. , Koshkin A.A. , Sazanov L.A. , Borodulina O.I. , Varfolomeev S.D. , Zaitsev S.V.
Published: July 2002

The respiratory burst induced by phorbol myristate acetate in mouse macrophages was inhibited by ultra‐low doses (10−15 −10−13 M) of an opioid peptide [d‐Ala2] methionine enkephalinamide. The effect disappeared at concentrations above and below this range. The inhibition approached 50% and was statistically significant (P < 0.001). Increasing the time of the opioid incubation with cells brought about a shift in the maximal effect to lower concentrations of the opioid (from 10−13 to 5 · 10−15 M) and led to a decrease in the value of the effect, fully in accord with the previously proposed adaptation mechanism of the action of ultra‐low doses.
Identification of the axial heme ligands of cytochrome b556 in succinate: Ubiquinone oxidoreductase from Escherichia coli
Authors: Peterson Jim , Vibat Cecile , Gennis Robert B.
Published: July 2002

Electron paramagnetic resonance (EPR) and near‐infrared magnetic circular dichroism (MCD) have been used to identify the ligands to the cytochrome b
556 component of succinate: ubiquinone oxidoreductase (succinate dehydrogenase) from Escherichia coli. The ‘highly axial low spin’ (HALS) EPR spectrum suggests bis(histidine) ligation of the heme with the histidines in a staggered configuration. The near‐infrared MCD spectrum exhibits a low energy maximum at 1600 nm which is also clearly indicative of bis(histidine) ligation of the heme iron. The data unambiguously demonstrate that the heme b
556 is ligated to E. coli succinate dehydrogenase via two histidines.
pH‐dependent formation of ethylenediaminetetraacetic acid supramolecular aggregates
Authors: Müller Michele , Haeberli André
Published: July 2002

Pure ethylenediaminetetraacetic acid tetrasodium salt (EDTA · Na4) in water showed a pH‐dependent change of optical density detected in the ultraviolet region. The most important change was found between pH 4 and 8, corresponding to the deprotonation of the third ionizable group having a pK of 6.2. The absorbance change was reversible and depended on the addition/removal of only 1 proton equivalent. The presence of supramolecular aggregates was suggested by size‐exclusion chromatography, dialysis and cryo‐electronmicroscopy. The molecular weight of EDTA supramolecular aggregates was 10–15,000 and 8–14,000, as determined by gel chromatography and dialysis, respectively. Cryo‐electronmicroscopy revealed structures of 2–4 nm in diameter.
The nucleotide sequence of the bacteriophage T5 ltf gene
Authors: Kaliman Alexander V. , Kulshin Vladimir E. , Shlyapnikov Michael G. , Ksenzenko Vladimir N. , Kryukov Valentine M.
Published: July 2002

The nucleotide sequence of the bacteriophage T5 Bg/II‐BamHI fragment (4,835 bp in length) known to carry a gene encoding the LTF protein which forms the phage L‐shaped tail fibers was determined. It was shown to contain an open reading frame for 1,396 amino acid residues that corresponds to a protein of 147.8 kDa. The coding region of ltf gene is preceded by a typical Shine‐Dalgarno sequence. Downstream from the ltf gene there is a strong transcription terminator. Data bank analysis of the LTF protein sequence reveals 55.1% identity to the hypothetical protein ORF 401 of bacteriophage λ in a segment of 118 amino acids overlap.
Modulation of obese gene expression in rat brown and white adipose tissues
Authors: Moinat Madelaine , Deng Chengjun , Muzzin Patrick , Assimacopoulos-Jeannet Françoise , Seydoux Josiane , Dulloo Abdul G. , Giacobino Jean-Paul
Published: July 2002

The ob gene mRNA expression in rat brown adipose tissue (BAT) and epididymal white adipose tissue (WAT) was measured on Northern blots hybridized with a rat ob gene probe. The level of ob gene mRNA in BAT was about 40% of that in WAT. Fasting (36 h) or semi‐starvation (10 days) decreased the ob gene mRNA level in both tissues by 62–68%, and cold exposure at 6°C (24 h) decreased it in BAT (−84%)_but not in WAT. Acute administration of the β
3‐adrenergic agonist Ro 16‐8714 decreased the ob gene mRNA level in BAT (−51%) and WAT (−28%) of lean Zucker rats and only in BAT (−74%) of obese falfa rats. This study demonstrates that, in the rat, the ob gene is not only expressed in WAT but also in BAT, and suggests that in these two tissues, the modulation of the ob gene expression might be more closely associated with known alterations in cell lipid content than with changes in sympathetic activity.