bluuebunny/crossref_metadata_2025 huggingface dataset:
Human monocyte chemoattractant protein‐1 (MCP‐1) Full‐length cDNA cloning, expression in mitogen‐stimulated blood mononuclear leukocytes, and sequence similarity to mouse competence gene JE
Authors: Yoshimura Teizo , Yuhki Naoya , Moore Stephen K. , Appella Ettore , Lerman Michael I. , Leonard Edward J.
Published: July 2002

The purpose of this work was to analyze cDNA encoding human monocyte chemoattractant protein‐1 (MCP‐1), previously isolated from glioma cell line culture fluid. Screening of a cDNA library from total poly(A) RNA of glioma cell line U‐105MG yielded a clone that coded for the entire MCP‐1. Nucleotide sequence analysis and comparison with the amino acid sequence of purified MCP‐1 showed that the cDNA clone comprises a 53‐nucleotide 5′‐non‐coding region, an open reading frame coding for a 99‐residue protein of which the last 76 residues correspond exactly to pure MCP‐1, and a 389‐nucleotide 3′‐untranslated region. The hydrophobicity of the first 23 residues is typical of a signal peptide. Southern blot analysis of human and animal genomic DNA showed that there is a single MCP‐1 gene, which is conserved in several primates. MCP‐1 mRNA was induced in human peripheral blood mononuclear leukocytes (PBMNLs) by PHA, LPS and IL‐1, but not by IL‐2, TNF, or IFN‐γ. Among proteins with similar sequences, the coding regions of MCP‐1 and mouse JE show 68% identity. This suggests that MCP‐1 is the human homologue of the mouse competence gene JE.
A new family of growth factor‐like peptides ‘Trefoil’ disulphide loop structures as a common feature in breast cancer associated peptide (pS2), pancreatic spasmolytic polypeptide (PSP), and frog skin peptides (spasmolysins)
Authors: Thim Lars
Published: July 2002

Four peptides present in completely different biological sources have been shown to exhibit a large degree of structural similarity. The peptides include: (i) a 60 amino acid residue breast cancer associated pS2 peptide isolated from human gastric juice and the culture media of the human breast cancer cell line MCF‐7; (ii) a 106 amino acid residue pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas and pancreatic juice; and (iii) a 49 and 50 amino acid residue peptide predicted from a cDNA isolated from the skin of the frog, Xenopus laevis. These peptides are characterized by having one (pS2 and the frog peptides) or two (PSP) domains of a highly conserved 38–39 amino acid residue consensus sequence not found in any other known peptides or proteins. The domain sequences contain 6 cysteine residues in nearly the same positions and it is suggested that these 6 residues are linked by 3 disulphide bonds to form a characteristic ‘trefoil’ disulphide loop structure common in all four peptides. From the sources of which the peptides have been isolated and from experiments showing that PSP has a growth factor stimulatory effect on MCF‐7 cells, it is further suggested that these peptides may represent members of a new family of growth factors.
Specific transport of inorganic phosphate, glucose 6‐phosphate, dihydroxyacetone phosphate and 3‐phosphoglycerate into amyloplasts from pea roots
Authors: Borchert S. , Große H. , Heldt H.W.
Published: July 2002

Evidence is provided that amyloplasts from pea roots contain a translocator which transports, in a counter exchange mode, phosphate, glucose 6‐phosphate, dihydroxyacetone phosphate and 3‐phosphoglycerate. The translocator has a low affinity for 2‐phosphoglycerate and glucose 1‐phosphate. Metabolite transport was measured by silicone oil filtering centrifugation either directly by uptake of radioactive labelled compounds or indirectly by back exchange.
Electron‐transfer restoration by vitamin K3 in a complex III‐deficient mutant of S. cerevisiae and sequence of the corresponding cytochrome b mutation
Authors: Brivet-Chevillotte Paule , di Rago Jean-Paul
Published: July 2002

The yeast box‐mutant W7 exhibits deficiencies in cytochrome b and in nuclear coded complex III subunits, a phenotype observed previously in a patient with mitochondrial myopathy. DNA sequence analysis of mutant W7 revealed a single base transition in the cytochrome b gene; the mutated residue Gly 131 is perfectly conserved in all known cytochromes b and belongs to the Q0 domain. Mutant W7 provides a model system for evaluating the action of therapeutic agents, such as vitamin K3 which restored NADH‐oxidase activity in the mutant as well as in the antimycin‐inhibited wild type. However, with the mutant, a greater quantity of menadione was necessary due to a decrease in other complex activities, and a much lower electron‐flow fraction passed through cytochrome oxidase.
Cloning and DNA sequence analysis of the glucose oxidase gene from Aspergillus niger NRRL‐3
Authors: Kriechbaum Marion , Heilmann Hans J. , Wientjes Franz J. , Hahn Marina , Jany Klaus-D. , Gassen Hans G. , Sharif Fadel , Alaeddinoglu Gürdal
Published: July 2002

A cDNA library from Aspergillus niger strain NRRL‐3 enriched in sequences encoding the glucose oxidase was constructed. An 800 bp cDNA clone isolated from this library was used to screen 12 000 recombinant phages from an EMBL3 genomic library. A 15 kbp DNA segment isolated from this library contained the 1815 bp structural gene for glucose oxidase as well as a short 5′‐ and a longer 3′‐noncoding region. The deduced protein sequence was verified by partial peptide sequencing.
Effects of mastoparan on catecholamine release from chromaffin cells
Authors: Wilson Steven P.
Published: July 2002

Release of catecholamines from bovine adrenal chromaffin cells exposed to mastoparan, a wasp venom peptide which activates GTP‐binding proteins and phospholipase A2, was evaluated. Release of catecholamines was dependent on mastoparan concentration and time of exposure. This release was, however, independent of extracellular calcium and accompanied by release of the cytoplasmic marker lactate dehydrogenase. Mastoparan also inhibited catecholamine secretion evoked by nicotine, but the peptide had little or no effect on release induced by other socretagogues. These findings suggest that in chromaffin cells mastoparan is not a secretagogue but rather causes cell lysis and blocks nicotinic receptor function.
Na+/H+ exchange in mitochondria as monitored by BCECF fluorescence
Authors: Kapus András , Ligeti Erzsébet , Fonyó Attila
Published: July 2002

The recently developed method of loading isolated heart mitochondria with the fluorescent pH indicator, BCECF, was applied to monitor the Na+
0/H+
i exchange process from the matrix side of the membrane. The Na+‐induced changes in the pH of the matrix (pHm) showed that: (i) the Na+
0/H+
i exchange followed Michaelis‐Menten kinetics with respect to external Na+ with a K
m of approx. 20 mM; (ii) in contrast to this, the dependence of the exchange rate on the matrix [H+] did not obey the Michaelian model. No Na+‐induced alkalinization occurred above a pHm of 7.45±0.09 (n=4). Below this value the reciprocal of the transport rate and that of the matrix [H+] deviated upwardly from the straight line. The results suggest that internal H+ might exert allosteric control on the mitochondrial Na+/H+ exchange process.
Surfactant enhancement of polyethyleneglycol‐induced cell fusion
Authors: Prado Adelina , Partearroyo M.Aránzazu , Mencía Mario , Goñi Félix M. , Barberá-Guillem Emilio
Published: July 2002

B 16 mouse melanoma cells in monolayers may be satisfactorily fused with 50% PEG 1500. However, pre‐treatment with detergents in solution at low concentrations significantly increases PEG fusion, up to 8‐fold in some instances, without impairing cell viability. The practical and mechanistical implications of this finding are discussed.
An antiserum to the extracellular domain of the Alzheimer amyloid precursor recognizes 70 and 88 kDa brain proteins
Authors: Simpson James , Bladon Christine M. , Yates Celia M. , Harmar Anthony J.
Published: July 2002

An antiserum raised to the extracellular domain (residues 556–566) of the Alzheimer amyloid precursor protein recognized 70 and 88 kDa proteins in Western blots of rat, Alzheimer, Down's syndrome and control human brain separated by SDS‐PAGE. The 70 kDa protein band was resolved into 5 spots by two‐dimensional electrophoresis. The findings provide further evidence that a 70 kDa protein is a metabolite of the amyloid precursor protein and reveal an 88 kDa protein which was reduced in 3 out of 6 brains with Alzheimer pathology.
The specific incorporation of labelled aromatic amino acids into proteins through growth of bacteria in the presence of glyphosate
Authors: Kim Hyun-Won , Perez Juan A. , Ferguson Stuart J. , Campbell Iain D.
Published: July 2002

Growth of Escherichia coli in the presence of glyphosate, an inhibitor of aromatic amino acid biosynthesis, has permitted the production of proton translocating ATPase that is specifically labelled with 5‐fluorotryptophan. Five sets of 19F nuclear magnetic resonances are resolved. The use of glyphosphate should be of wide applicability in the preparation of proteins labelled in aromatic amino acid residues for NMR studies.
Escherichia coli of human origin binds to carcinoembryonic antigen (CEA) and non‐specific crossreacting antigen (NCA)
Authors: Leusch Hans-Georg , Hefta Stanley A. , Drzeniek Zofia , Hummel Klaus , Markos-Pusztai Zsuzanna , Wagener Christoph
Published: July 2002

Immobilized carcinoembryonic antigen (CEA) and non‐specific crossreacting antigen (NCA) bound 3 strains of E. coli of human origin. The binding was dose dependent, saturable, and of high avidity. Binding of the bacteria to CEA and NCA was completely abolished in the presence of 10 mM α‐methyl D‐mannopyranoside. Bacteria did not bind to concanavalin A. In addition, binding to deglycosylated CEA was either absent or significantly reduced. These findings indicate that the E. coli strains bind to D‐mannosyl residues in CEA and NCA. Considering the tissue distribution of CEA (brush border of colonic epithelium) and NCA (granulocytes), these glycoproteins may be involved in the recognition of bacteria.
Slipping pumps or proton leaks in oxidative phosphorylation
Authors: van Dam Karel , Shinohara Yasuo , Unami Akira , Yoshida Kenji , Terada Hiroshi
Published: July 2002

Evidence is presented to show that the local anesthetic bupivacaine causes slip in the mitochondrial proton pump cytochrome c oxidase.
The influence of proline residues on α‐helical structure
Authors: Woolfson Derek N. , Williams Dudley H.
Published: July 2002

Proline lacks an amide proton when found within proteins. This precludes hydrogen bonding between it and hydrogen bond acceptors, and thus often restricts the residue to the first four positions of an α‐helix. Helices with proline after position four have a pronounced kink [(1988) J. Mol. Biol. 203, 601‐619]. In these cases, we find that the proline residue almost always occurs on the solvent exposed face of each helix. This positioning facilitates the compensatory hydrogen bonding between solvent and residues P‐3 and P‐4 (relative to proline, P), through the formation of the kink. Further, it aids in the packing of long helical structures around globular protein structures.
G Protein control of inositol lipids in intact vascular smooth muscle
Authors: LaBelle Edward F. , Murray Bonnie M.
Published: July 2002

Agonist‐induced PIP2, breakdown has been demonstrated in penneabilized vascular smooth muscle and shown to depend on a G protein. Segments of rat tail artery were permeabilized with ATP and EGTA after prelabeling with [3H]inositol. Norepinephrine and GTPγS were both able to increase levels of IP, IP2, and IP3 in the segments. The effects of both norepinephrine and GTPγS on the segments was non‐additive. Aluminum fluoride also increased inositol phosphates in intact segments and norepinephrine‐stimulated increases in IP, IP2, and IP3 were insensitive to pertussis toxin.
High‐affinity inositol 1,3,4,5‐tetrakisphosphate receptor from cerebellum: solubilization, partial purification and characterization
Authors: Donié Frédéric , Hülser Eckehard , Reiser Georg
Published: July 2002

Proteins which bind with high affinity Ins 1,3,4,5‐P4 or Ins 1,4,5‐P3 were solubilized from porcine cerebellar membranes. Both binding activities were separated by heparin‐agarose chromatography. The Ins 1,3,4,5‐P4 receptor was partially purified with an approximately 1000‐fold enrichment as compared to the membrane preparation. In the receptor‐enriched preparation the Ins 1,3,4,5‐P413 binding protein had an affinity (K
d) for Ins 1,3,4,5‐P4 of 4.6 nM. Ins 1,3,4,5,6‐P5 displaced [3H]Ins 1,3,4,5‐P4binding with a comparable affinity. The Ins 1,3,4,5‐P4binding protein displayed high selectivity for Ins 1,3,4,5‐P4 over other inositolphosphates (IC50 for Ins 1,4,5,6‐P4 150 nM, for Ins‐P6 1 μM and for Ins 1,3,4‐P3 5 μM). Most importantly. Ins 1,4,5‐P3 did not displace [3H]Ins 1,3,4,5‐P4binding at concentrations up to 10μM. Binding of Ins 1,3,4,5‐P4 was maximal in the pH range between 4.5 and 6, was stable with Ca2+ concentration varied from 1 nM to 1 mM, and was suppressed by heparin (IC50 about 2 nM). The high affinity receptor for Ins 1,3,4,5‐P4 reported here, which is distinct from the Ins 1,4,5‐P3 receptor might allow to evaluate the possible functional role of Ins 1,3,4,5‐P4 in the cellular signal transduction.
Excitotoxin lesion of nucleus basalis causes a specific decrease in Go mRNA in cerebral cortex
Authors: Wood Helen , de Belleroche Jacquline
Published: July 2002

Lesions of the ascending cholinergic pathway from nucleus basalis are known to have profound effects on cortical function. In particular, a substantial potentiation of carbachol‐stimulated polyphosphoinositide turnover is detected from 1 day after lesion and is maintained for several days before returning to normal by 1 month. In this study the effect of this lesion was investigated on levels of three G‐protein α‐subunit mRNAs. Excitotoxin lesion of the nucleus basalis caused a selective reduction in the levels of Goα mRNA in cerebral cortex ipsilateral to the lesion, G5α and Giα mRNA being unaffected. The maximal effect was obtained at 3 days after lesion where levels of Goα mRNA were decreased by 40% compared to sham‐operated animals. Levels of Goα mRNA returned to normal values by 28 days. Treatment with MK‐801 caused a significant attenuation of the decrease in Goα mRNA, indicating the involvement of NMDA receptors in this response.
Penicillin acylase‐catalyzed protection and deprotection of amino groups as a promising approach in enzymatic peptide synthesis
Authors: Didžiapetris Remigijus , Drabnig Barbara , Schellenberger Volker , Jakubke Hans-Dieter , S̆vedas Vytas
Published: July 2002

Penicillin acylase from E. coli is able to catalyze both the introduction and the removal of the phenylacetyl group. We have established that phenylacetyl derivatives of amino acids and peptides can be used in protease‐catalyzed peptide synthesis. Here the synthesis of leucine‐enkephalin using enzymes for N‐terminal amino group protection, peptide bond formation and deprotection is described.
Induction of programmed cell death (apoptosis) in mature lymphocytes
Authors: Lucas Miguel , Solano Francisca , Sanz Aureo
Published: July 2002

The apoptosis of human peripheral blood lymphocytes was analyzed by the breakdown of DNA into oligonucleosome‐sized fragments. The mature lymphocytes were rendered sensitive to apoptosis by either the omission of fetal bovine serum in the culture medium or the addition of polymyxin B. In the first case it was counteracted by phorbol myristate acetate. The possible involvement of protein kinase C in cell survival is pointed out.
Isolation of a new gene (SW A2) encoding an α‐amylase from Schwanniomyces occidentalis and its expression in Saccharomyces cerevisiae
Authors: Abarca D. , Fernàndez-Lubato M. , del Pozo L. , Jimènez A.
Published: July 2002

A new gene (SW A2) encoding a secretory α‐amylase activity from Schwanniomyces occidentalis has been cloned from this year and then expressed in Saccharomyces cerevisiae. Both Sw. occidentalis and a transformant of S. cerevisiae incorporating SW A2 contain a transcript of 2.1 kb which hybridizes to DNA carrying the SW A2 gene. This indicates that the transcript is a product of the SW A2 gene. Transcription of the SW A2 gene seems to be regulated in both Sw. occidentalis and S. cerevisiae. Furthermore, the secretion of α‐amylase activity is drastically expressed by glucose in both Sw. occidentalis and a transformant of S. cerevisiae containing SW A2.
Heparin inhibits A431 cell growth independently of serum and EGF mitogenic signalling
Authors: Vannucchi Simonetta , Pasquali Franca , Chiarugi Vincenzo P. , Ruggiero Marco
Published: July 2002

In several cell types heparin exerts an antiproliferative action; here we report that heparin inhibited the growth of human epidermoid carcinoma A431 cells. Heparin binding to the cell surface was necessary for growth inhibition; binding was influenced by the molecular weight of heparin. Inhibition of A431 cell proliferation was evident in the presence and in the absence of serum, thus indicating that heparin did not act by binding and ‘subtracting’ nutrients or other serum factors. In confluent A431 cells, EGF induced DNA synthesis, but heparin did not inhibit this effect; consistently, it did not affect inositol lipid turnover triggered by EGF or bradykinin.
Bacteriochlorophyll c formation via the C5 pathway of 5‐aminolevulinic acid synthesis in Chloroflexus aurantiacus
Authors: Oh-hama Tamiko , Santander Patricio J. , Stolowich Neal J. , Scott A.Ian
Published: July 2002

Biosynthesis of 5‐aminolevulinic acid (ALA) in Chloroflexus aurantiacus, a thermophilic bacterium forming bacterioclorophyll c, is shown to proceed via the C5 pathway by demonstrating (1) the specific labeling of its chlorin ring with [1‐13C] glutamate and (2) the enzyme activity to produce ALA from glutamate in a cell‐free extract. From the phylogenetic distribution it is suggested that ALA synthase distributed in some aerobic eubacteria could be monophyletic in origin.
Evidence for ATP‐ase activity of arrestin from bovine photoreceptors
Authors: Glitscher W. , Rüppel H.
Published: July 2002

In vertebrate photoreceptors the soluble protein arrestin (45 kDa) is involved in controlling the light dependence activity of receptor proteins such as transducin or the cGMP‐phosphodiesterase. Arrestin has further been identified as the retinal‐S‐antigen which is assumed to cause the autoimmune discase uveitis. In a first communication a binding of the nucleotide ATP to arrestin was described. In this subsequent study it is shown that arrestin is also able to hydrolyse ATP at a rate (5.1 ±0.3)·10−3 U/mg min with C

= 93±5 nM and a Hill coefficient n = 1.8±0.1 at pH 7.2 and 20°C. These findings suggest a new insight into the process of regulating photoreceptor activity.
Human interleukin‐5 expressed in Escherichia coli: assignment of the disulfide bridges of the purified unglycosylated protein
Authors: Proudfoot Amanda E.I. , Davies J.Gwynfor , Turcatti Gerardo , Wingfield Paul T.
Published: July 2002

Human interleukin‐5 is a homodimer: each subunit contains two cysteine residues that form two inter‐subunit disulfide bonds. The topology of the disulfides in recombinant human interleukin‐5 produced in Escherichia coli was studied by proteolytic digestion and peptide mapping. Disulfide linked peptides containing cysteine 42 linked to cysteine 84 were isolated. This indicated that cysteine 42 and 84 of one subunit were linked in an antiparallel manner to cysteines 84 and 42 of the other subunit.
Effects of human low‐density lipoproteins on the nucleotide patterns of cultured pig aortic endothelial cells
Authors: Felbel Jochen , Siess Elmar , Zöllner Nepomuk
Published: July 2002

Cultured pig aortic endothelial cells display significant changes in their nucleotide patterns after incubation with LDL‐cholesterol purified from normal human plasma as determined by HPLC. Incubation at 70 mg/dl LDL‐cholesterol for 24 h at 37°C caused a significant decrease (P<0.001) in ATP from a control value of 14.0±0.4 nmol/mg protein to 6.6±0.9 nmol/mg protein (n=4) with a concomitant increase in ADP and AMP. At higher LDL concentrations these effects were even more pronounced but still reversible. Akin to adenine nucleotides, the guanosine and uridine phosphates as determined by HPLC were changed. In contrast to LDL, HDL and VLDL were ineffectual.
Functioning of quinone acceptors in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus
Authors: Kutuzov Michael A. , Mamedov Mahir D. , Semenov Alexey Yu. , Shinkarev Vladimir P. , Verkhovsky Michael I. , Abdulaev Nazhmutdin G. , Drachev Lel A.
Published: July 2002

The photosynthetic reaction centers (RC) of the green bacterium Chloroflexus aurantiacus have been investigated by spectral and electrometrical methods. In these reaction centers, the secondary quinone was found to be reconstituted by the addition of ubiquinone‐10. The equilibrium constant of electron transfer between primary (QA) and secondary (QB) quinones was much higher than that in RC of purple bacteria. The QB binding to the protein decreased under alkalinization with apparent pK 8.8. The single flash‐induced electric responses were about 200 mV. An additional electrogenic phase due to the QB protonation was observed after the second flash in the presence of exogenous electron donors. The magnitude of this phase was 18% of that related to the primary dipole (P+Q−
A) formation. Since the C aurantiacus RC lacks H‐subunit, this subunit was not an obligatory component for electrogenic QB protonation.
D1 protein degradation during photoinhibition of intact leaves a modification of the D1 protein precedes degradation
Authors: Kettunen Reetta , Tyystjärvi Esa , Aro Eva-Mari
Published: July 2002

Illumination of intact pumpkin leaves with high light led to severe photoinhibition of photosystem II with no net degradation of the D1 protein. Instead, however, a modified form of D1 protein with slightly slower electrophoretic mobility was induced with corresponding loss in the original form of the D1 protein. When the leaves were illuminated in the presence of chloramphenicol the modified form was degraded, which led to a decrease in the total amount of the D1 protein. Subfractionation of the thylakoid membranes further supported the conclusion that the novel form of the D1 protein was not a precursor but a high‐light modified form that was subsequently degraded.
Cloning and sequence analysis of a human type A/B hnRNP protein
Authors: Khan Farhat A. , Jaiswal Anil K. , Szer Wlodzimierz
Published: July 2002

A cDNA encoding a 284 residue long type A/B hnRNP protein has been cloned. This protein, previously referred to as type C[(1987) J. Biol. Chem. 262, 17126–17137], is an RNA unwinding protein from HeLa 40S hnRNP with a high affinity for G‐ followed by U‐rich sequences. The N‐terminal part of the protein contains two consensus RNA binding domains present in a number of other RNA binding proteins. The C‐terminal part is glycine‐rich and contains a potential ATP/GTP binding loop. The distribution of charged amino acids is highly uneven and there are multiple potential phosphorylation sites.
Mitogenic signal transduction in normal and transformed 32D hematopoietic cells
Authors: Ruggiero Marco , Wang Ling M. , Pierce Jacalyn H.
Published: July 2002

We studied mitogenic signal transduction in normal and oncogene‐transformed 32D cells, a murine hematopoietic cell line that is normally dependent on interleukin‐3 (IL3) for proliferation and survival. The formation of second messengers was measured in normal cells stimulated with IL3, and in cells transfected with foreign growth factor receptor genes and stimulated with appropriate growth factors. We also measured the steady‐state level of second messengers in 32D cells transformed by erbB, abl, and src oncogenes which abrogate growth factor requirement. We found that IL3 stimulated the formation of diacylglycerol independently of inositol lipid turnover, but concomitantly with increased turnover of phosphatidylcholine. Epidermal growth factor (EGF), and platelet‐derived growth factor (PDGF) stimulated the ‘classical’ turnover of inositol lipids with formation of diacylglycerol and calcium‐mobilizing inositol phosphates. Colony stimulating factor‐1 trigged inositol lipid turnover, although to a much lower extent than EGF and PDGF. Transformed cells showed elevated levels of diacylglycerol together with increased turnover of phosphoinositides and phosphatidylcholine. Taken together these results indicate that different growth factors and oncoproteins associate with multiple signalling pathways in 32D cells.
Insertional inactivation of the psbO gene encoding the manganese stabilizing protein of photosystem II in the cyanobacterium Synechococcus PCC7942
Authors: Bockholt Rainer , Masepohl Bernd , Pistorius Elfriede K.
Published: July 2002

A Synechococcus PCC7942 mutant in which the psbO gene was inactivated by insertion of a chloramphenicol interposon and which did not contain any detectable manganese stabilizing protein in immunoblot experiments, was constructed. Such a Synechococcus mutant was able to grow under photoautotrophic conditions. Isolated thylakoid membranes from the mutant required addition of CaCl2 and MnCl2 for photosynthetic O2 evolution, and the detectable L‐amino acid oxidase activity in the isolated thylakoid membranes from the mutant was approximately four times higher than in wild‐type thylakoids. The results are discussed with respect to our model suggesting that the water‐oxidizing enzyme may have evolved from a flavoprotein with L‐amino acid dehydrogenase/oxidase activity.
Biphasic transition curve on denaturation of chicken cystatin by guanidinium chloride Evidence for an independently unfolding structural region
Authors: Björk Ingemar , Pol Ewa
Published: July 2002

Far‐ultraviolet circular dichroism and tryptophan fluorescence measurements showed that the reversible unfolding of the cysteine proteinase inhibitor, chicken cystatin, by guanidinium chloride is a two‐step process with transition midpoints at ≈3.4 and ≈5.4 M denaturant. The partially unfolded intermediate had both far‐ and near‐ultraviolet circular dichroism and fluorescence emission spectra comparable to those of the native protein. The largely retained tertiary structure suggests that the intermediate represents a species in which a separate region of lower stability has been unfolded, rather than an intermediate of the ‘molten globule’ type. Such a structurally independent region is apparent in the three‐dimensional structure of the inhibitor.
Receptor‐mediated endocytosis of the intrinsic factor—cobalamin complex in HT 29, a human colon carcinoma cell line
Authors: Guéant J.L. , Masson C. , Schohn H. , Girr M. , Saunier M. , Nicolas J.P.
Published: July 2002

A HT 29 cell line derived from human colonic carcinoma was shown to express the intrinsic factor receptor, with about 5000 binding sites per cell and an association constant of 20 × 109 1/mol at pH 7.4 and 4°C. The number of binding sites increased dramatically between 7 and 10 days of culture time. Endocytosis of the intrinsic factor‐cobalamin‐receptor complex was inhibited by two ways: at 4°C and at 37°C by incubating the cells with vinblastine, monensin and chloroquine. The plasma membrane receptor was cross‐linked to [57Co]cobalamin‐intrinsic factor and solubilized with Triton X‐100. The cross‐linked complex had a relative molecular mass of 330 kDa in native PAGE.
Fluorescence studies on the interaction of adenine with ricin A‐chain
Authors: Watanabe Keiichi , Honjo Eijiro , Tsukamoto Takuji , Funatsu Gunki
Published: July 2002

Ricin A‐chain, an N‐glycosidase that attacks 28S rRNA at a highly conserved adenine residue, has a unique tryptophan (Trp‐211) in the putative active site cleft. Fluorescence spectroscopy revealed that specific binding of adenine to the A‐chain caused a large enhancement of Trp‐211 fluorescence (70%) and a concomitant red shift of the emission spectrum (8 nm). A Scatchard plot of the fluorescence enhancement data was not linear, indicating that the environment of Trp‐211 was altered by heterogeneous binding of adenines. These results, taken together with the protective effect of adenine on the ribosome‐inactivation by ricin A‐chain, suggest that at least two adenines bind to the active site cleft.
Spontaneous conversion of PrPC to PrPSc
Authors: Sulkowski Eugene
Published: July 2002

Octa‐repeats of prion proteins (PrP) contain histidine and tryptophan residues which are known to function as ligands for transition metals. It is proposed that the spontaneous conversion of the PrPC (cellular) isoform into PrPSc (scrapie) isoform may be triggered by the coordination of these metals.
Ca2+‐mediated interaction between negatively charged and neutral liposomes
Authors: Budker Vladimir G. , Kiseleva Helena V. , Serebryakova Marina V. , Sokoloff Alexander V.
Published: July 2002

In the present work it is shown that large unilamellar lecithin/cholesterol liposomes are able to sequester small negatively charged liposomes in the presence of divalent cations. Evidence is presented suggesting that the sequestration occurs via the formation of membrane invaginations transformed further into intraliposomal vesicles.
Amidating processing enzyme complex for bioactive peptides (PAM) shows differences in specific activity and form in secretory granules isolated from the proximal and distal parts of the hypothalamo‐neurohypophyseal tract in rats
Authors: Hu H.-M. , Thorn N.A.
Published: July 2002

In rats the PAM specific activity in hypothalamic and neurohypophyseal extracts was 0.58 ± 0.8, respectively 1.78 ± 0.6 nmol · mg prot.−1 · h−1 (n = 5). PHM specific activity in the soluble part of the granules was higher in the neurohypophyseal than in the hypothalamic granules, and the fraction of total PHM and PAL present in the soluble part increased with the distance from the hypothalamus from some 45% to approx. 85%. Western blots of membrane and soluble granule fractions showed prevalence of higher mol. wt. forms in hypothalamic granules. It would appear that higher mol. wt. forms of PAM are processed by proteolytic enzymes during transport in the neuron and that non‐neural cells in the neurohypophysis have a considerable PAM activity.
The modular architecture of a new family of growth regulators related to connective tissue growth factor
Authors: Bork Peer
Published: July 2002

Recently, several groups have characterized and sequenced members of a new family of growth regulators (originally called cef10, connective tissue growth factor, fisp‐12, cyr61, or, alternatively, βIG‐M1 and βG‐M2), all of which belong to immediate‐early genes expressed after induction by growth factors or certain oncogenes. Sequence analysis of this family revealed the presence of four distinct modules. Each module has homologues in other extracellular mosaic proteins such as Von Willebrand factor, slit, thrombospondins, fibrillar collagens, IGF‐binding proteins and mucins. Classification and analysis of these modules suggests the location of binding regions and, by analogy to better characterized modules in other proteins, sheds some light onto the structure of this new family.
Nucleotide and negatively charged lipid‐dependent vesicle aggregation caused by SecA
Authors: Breukink Eefjan , Keller Rob C.A. , Kruijff Ben de
Published: July 2002

SecA which is an overall acidic protein was found to induce an increase in the turbidity of a solution of vesicles consisting of negatively charged phospholipids. This increase was found to be due to an aggregation of the vesicles mediated by SecA. The SecA‐mediated vesicle aggregation was not found for zwitterionic 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine and showed a large dependence on both temperature and ionic strength. Furthermore it was shown that ATP and to a lesser extent ADP+Pi were able to reduce the SecA‐mediated vesicle aggregation, while no effect could be seen for a non‐hydrolysable ATP analog AMP‐PNP. Using the steady state fluorescence anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene present in 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoglycerol vesicles we could show that SecA inserts in the bilayer. Monolayer studies confirmed that SecA is able to cause close contact between two membranes and gave a direct insight into the different types of lipid‐protein interactions involved. From our results we propose that the SecA monomer possesses two lipid‐binding sites which in the functional dimer conformation are responsible for the SecA‐mediated vesicle aggregation.
Expression of mouse rod photoreceptor cGMP phosphodiesterase γ subunit in bacteria
Authors: Qin Ning , Baehr Wolfgang
Published: July 2002

We expressed the γ subunit of mouse rod photoreceptor cGMP phosphodiesterase (PDE) in the bacterial pGEX‐2TK expression vector which produces a cleavable 40 kDa fusion protein. The fusion protein can be isolated in a one step procedure by affinity chromatography on glutathione beads. The yield of purified fusion protein is approximately 10 mg from 1 liter of bacterial culture, or about 3 mg of PDEγ, equivalent to the PDEγ content of approximately 200,000 mouse retinas. Both the fusion protein and the cleaved PDEγ, to which a short kinase domain remains attached, are biologically active, inhibiting activated PDE in a manner comparable to native PDEγ. Immobilized PDEγ binds transducin α subunit charged with GTP, PDE α and β subunits, and, unexpectedly, arrestin (S‐antigen).
Chelatable iron and copper can be released from extracorporeally circulated blood during cardiopulmonary bypass
Authors: Moat Neil E. , Evans Timothy E. , Quinlan Gregory J. , Gutteridge John M.C.
Published: July 2002

During cardiopulmonary bypass surgery blood is extracorporeally oxygenated and circulated before returning to the systemic arterial circulation. Blood undergoing extracorporeal dilution and circulation is exposed to non‐physiological surfaces, which cause the activation of several regulatory cascades. Cells are also subjected to damaging shear stresses. Under such conditions neutrophils can be ‘activated’ to release reactive oxygen intermediates such as O2
− and H2O2, and other cells can release proteolytic enzymes and metalloproteins. Collectively, these events can result in the release of micromolar quantities of redox active iron and copper. Bleomycin‐detectable iron and phenanthroline‐detectable copper were found in two out of four mock bypass experiments. However, there was no correlation between the presence of chelatable iron and copper and the activation of neutrophils measured as elastase.
A structural model for the glutamate‐specific endopeptidase from Streptomyces griseus that explains substrate specificity
Authors: Barbosa João Alexandre R.G. , Garratt Richard C. , Saldanha José W.
Published: July 2002

We present a model for the three‐dimensional structure of the glutamate‐specific endopeptidase from Streptomyces griseus based on the crystal structures of other bacterial proteases of the trypsin family. For the first time a structural model is described which attempts to explain the basis of P1 glutamate specificity in serine proteases. Several important changes to the S1 pocket with respect to other members of the family of different specificity are described. Of particular interest is the presence of a histidine at position 213 and the substitution of Arg‐138 by lysine. Other biochemical evidence concerning substrate preferences can be rationalized on the basis of the model.
Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide
Authors: Cleeter M.W.J. , Cooper J.M. , Darley-Usmar V.M. , Moncada S. , Schapira A.H.V.
Published: July 2002

Incubation of rat skeletal muscle mitochondria with the nitric oxide generator, S‐nitrosoglutathione (GSNO) reversibly inhibited oxygen utilisation with all substrates tested. The visible absorption spectra of the inhibited mitochondria showed that cytochromes c+c
1, b and a+a
3 were reduced, indicating a block at the distal end of the respiratory chain. Analysis of the respiratory chain enzyme activities in the presence of GSNO localised the site of inhibition to cytochrome c oxidase alone. These results indicate that nitric oxide is capable of rapidly and reversibly inhibiting the mitochondrial respiratory chain and may be implicated in the cytotoxic effects of nitric oxide in the CNS and other tissues.
Molecular cloning, tissue distribution and chromosomal localization of a novel member of the opioid receptor gene family
Authors: Chen Yan , Fan Yi , Liu Jian , Mestek Anton , Tian Mingting , Kozak Christine A. , Yu Lei
Published: July 2002

A cDNA was isolated from rat brain by low stringency hybridization with the rat μ opioid receptor cDNA. Sequence analysis of this clone indicated that it contains an open reading frame capable of encoding a 367 amino acid protein. The deduced amino acid sequence of this protein shows high degrees of homology to all three opioid receptors, μ, κ, and δ, suggesting that it is a member of the opioid receptor gene family. RNA blot analysis detected high level expression of the receptor mRNA in the brain. Southern blot analysis suggests that it is a single‐copy gene, and mapping studies localized the gene on mouse chromosome 2. Despite the high sequence homologies between this protein and the other opioid receptors, expression studies of this clone in COS‐7 cells did not show binding to [3H]diprenorphine, a ligand that binds to the other three opioid receptors. Furthermore, co‐expression of this receptor with a G protein‐activated potassium channel in Xenopus oocytes did not show functional coupling upon stimulation with μ, κ and δ agonists. Given the similar degrees of high homology to the μ, κ and δ opioid receptors and the lack of apparent affinity for their ligands, this receptor does not appear to belong to any of the three known classes of opioid receptors. Rather, it represents a novel member of the opioid receptor gene family, not identified from previous pharmacological studies.
Saposin C induces pH‐dependent destabilization and fusion of phosphatidylserine‐containing vesicles
Authors: Vaccaro Anna Maria , Tatti Massimo , Ciaffoni Fiorella , Salvioli Rosa , Serafino Annalucia , Barca Alessandra
Published: July 2002

We have previously shown that saposin C (Sap C), a glucosylceramidase activator protein, interacts with phosphatidylserine (PS) large unilamellar vesicles (LUV), promoting the glucosylceramidase binding to the bilayer [(1993) FEBS Lett. 336, 159–162]. In the present paper the consequences of the Sap C interaction on the lipid organization of the vesicles are reported. It was found that Sap C perturbs the PS bilayer as shown by the release of an encapsulated fluorescent dye. Three different procedures, resonance energy transfer, gel filtration and electron microscopy, indicated that the activator protein is also able to make PS liposomes fuse. The effects of Sap C on PS vesicles were observed at low but not at neutral pH. The lipid composition of the bilayer also affected the Sap C‐induced destabilization; in fact, the presence of PS in mixed LUV was essential for significant leakage to occur. These results demonstrate for the first time that Sap C is a protein capable of destabilizing and fusing acidic phospholipid‐containing membranes in a pH‐dependent fashion.
Effect of aging and acetyl‐l‐carnitine on the activity of cytochrome oxidase and adenine nucleotide translocase in rat heart mitochondria
Authors: Paradies Giuseppe , Ruggiero Francesca Maria , Petrosillo Giuseppe , Gadaleta Maria Nicola , Quagliariello Ernesto
Published: July 2002

The effect of aging and treatment with acetyl‐l‐carnitine on the activity of cytochrome oxidase and adenine nucleotide translocase in rat heart mitochondria was studied. It was found that the activity of both these mitochondrial protein systems was reduced (by around 30%) in aged animals. Treatment of aged rats with acetyl‐l‐carnitine almost completely reversed this effect. Changes in the mitochondrial cardiolipin content appear to be responsible for these effects of acetyl‐l‐carnitine.
Essential role of phosphatidylinositol 3‐kinase in insulin‐induced activation and phosphorylation of the cGMP‐inhibited cAMP phosphodiesterase in rat adipocytes studies using the selective inhibitor wortmannin
Authors: Rahn Tova , Ridderstråle Martin , Tornqvist Hans , Manganiello Vincent , Fredrikson Gudrun , Belfrage Per , Degerman Eva
Published: July 2002

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3‐kinase (PI 3‐kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC50≈ 100 nM), the insulin‐induced activation and phosphorylation of cGMP‐inhibited cAMP phosphodiesterase (cGI‐PDE) as well as the activation of the insulin‐stimulated cGI‐PDE kinase (IC50≈ 10–30 nM). No direct effects of the inhibitor on the insulin‐stimulated cGI‐PDE kinase, the cGI‐PDE and the hormone‐sensitive lipase were observed. These data suggest that activation of PI 3‐kinase upstream of the insulin‐stimulated cGI‐PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin‐induced cGI‐PDE activation/ phosphorylation and anti‐lipolysis.
Troponin T is capable of binding dystrophin via a leucine zipper
Authors: Pearlman Joel A. , Powaser Peter A. , Elledge Stephen J. , Caskey C.Thomas
Published: July 2002

Using genetic and physical assays for protein—protein interactions, we identified a fast isoform of troponin T that binds to dystrophin. Troponin T specifically bound to the first of two highly conserved leucine zipper motifs in the carboxy terminus of dystrophin [1,2]. Single amino acid changes in the zipper predicted to disrupt α‐helix formation or cause steric hindrance abolished this binding. These data support the hypothesis that dystrophin couples the contractile apparatus to the sarcolemma and indicate that leucine zipper mediated protein—protein interactions are functionally important in the cytoskeleton as well as the nucleus.
ADP ribosyl cyclase activity of a novel bone marrow stromal cell surface molecule, BST‐1
Authors: Hirata Yuichi , Kimura Naoki , Sato Koh , Ohsugi Yoshiyuki , Takasawa Shin , Okamoto Hiroshi , Ishikawa Jun , Kaisho Tsuneyasu , Ishihara Katsuhiko , Hirano Toshio
Published: July 2002

Human BST‐1, a bone marrow stromal cell surface molecule, is a GPI‐anchored protein that facilitates the growth of pre‐B cells. The deduced amino acid sequences of human and mouse BST‐1 show around 30% homology with those of CD38 and Aplysia ADP ribosyl cyclase. Therefore, like CD38, BST‐1 might possess ADP ribosyl cyclase activity. Here, we report the establishment of a stable transformant CHO cell line which secretes truncated human soluble BST‐1, and show that purified soluble BST‐1 displays both ADP ribosyl cyclase cADPR hydrolase activities.
ORL1, a novel member of the opioid receptor family
Authors: Mollereau Catherine , Parmentier Marc , Mailleux Pierre , Butour Jean-Luc , Moisand Christiane , Chalon Pascale , Caput Daniel , Vassart Gilbert , Meunier Jean-Claude
Published: July 2002

Selective PCR amplification of human and mouse genomic DNAs with oligonucleotides encoding highly conserved regions of the δ‐opioid and somatostatin receptors generated a human DNA probe (hOP01, 761 bp) and its murine counterpart (mOP86, 447 bp). hOP01 was used to screen a cDNA library from human brainstem. A clone (named hORL1) was isolated, sequenced and found to encode a protein of 370 amino acids whose primary structure displays the seven putative membrane‐spanning domains of a G protein‐coupled membrane receptor. The hORL1 receptor is most closely related to opioid receptors not only on structural (sequence) but also on functional grounds: hORLl is 49–50% identical to the murine μ‐, δ‐ and κ‐opioid receptors and, in CHO‐K1 cells stably transfected with a pRc/CMV:hORLl construct, ORL1 mediates inhibition of adenylyl cyclase by etorphine, a ‘universal’ (nonselective) opiate agonist. Yet, hORLl appears not to be a typical opioid receptor. Neither is it a somatostatin or σ (N‐allylnormetazocine) receptor. mRNAs hybridizing with synthetic oligonucleotides complementary to mOP86 are present in many regions of the mouse brain and spinal cord, particularly in limbic (amygdala, hippocampus, septum, habenula,⋯) and hypothalamic structures. We conclude that the hORL1 receptor is a new member of the opioid receptor family with a potential role in modulating a number of brain functions, including instinctive behaviours and emotions.
A novel prenyltransferase, farnesylgeranyl diphosphate synthase, from the haloalkaliphilic archaeon, Natronobacterium pharaonis
Authors: Tachibana Akira
Published: July 2002

A novel prenyltransferase, farnesylgeranyl diphosphate (FGPP) synthase (EC 2.5.1.X), which synthesizes C25‐prenyl diphosphate, was found in the haloalkaliphilic archaeon Natronobacterium pharaonis. It was separated from geranylgeranyl diphosphate (GGPP) synthase (EC 2.5.1.29), which synthesizes C20‐prenyl diphosphate, a major prenyltransferase in this organism. The highest activity of FGPP synthase was observed when GGPP was used as the allylic substrate. FGPP synthase may synthesize a precursor for the C25 moiety of C20,C25 diether lipids using a longer allylic diphosphate, such as GGPP synthesized by GGPP synthase, rather than dimethylallyl diphosphate, which is the product of isopentenyl diphosphate isomerase.
The actin binding site in the tail domain of Dictyostelium myosin 1C (myoC) resides within the glycine‐ and proline‐rich sequence (tail homology region 2)
Authors: Jung Goeh , Hammer John A.
Published: July 2002

The majority of protozoan myosins I possess tail domains composed of three distinct and conserved regions of sequence, referred to as tail homology regions 1,2 and 3 (TH · 1, TH · 2 and TH · 3). While the N‐tenninal ~ half of the tail (corresponding to TH·1 ) has been implicated in membrane binding, all or some portion of the C‐terminal ~half of the tail (corresponding to TH·2 plus TH·3) has been implicated in binding to F‐actin in a nucleotide‐insensitive fashion. Here we show, using fusion proteins containing portions of the Dictyostelium myosin 1C (myoC) tail domain and F‐actin sedimentation assays, that the ability of the myoC tail to bind to actin resides entirely within the glycine‐ and proline‐rich TH·2 domain. The src‐like TH·3 domain does not bind to actin, nor does it augment the binding properties of the TH·2 domain. In addition to defining more precisely the location of the actin binding site in the tail domain of a protozoan myosin I, these results have implications for the function of the src‐like TH·3 domain in myosins I and other proteins.
Identification of the 23 kDa subunit of tau protein kinase II as a putative activator of cdk5 in bovine brain
Authors: Ishiguro Koichi , Kobayashi Shunsuke , Onion Akira , Takamatsu Masako , Yonekura Sayuri , Anzai Kaijiro , Imahori Kazutomo , Uchida Tsuneko
Published: July 2002

Tau protein kinase II (TPKII) was reported previously to be composed of a neuron‐rich cdc2‐related kinase (PSSALRE/cdk5) and 23 kDa subunit. Here we show that the 23 kDa subunit is a putative activator for the kinase activity. Amino acid sequence analysis revealed that the protein was novel and included a partial similarity of amino acids to a cyclin box important for the interaction with cdc2‐related kinase. These results suggest that the 23 kDa subunit, but not cyclin, activates cdk5 in neuronal cells, which no longer exhibit cell cycling but are terminally differentiated cells.