bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of neuropeptide Y from porcine intestine
Authors: Tatemoto K. , Siimesmaa S. , Jörnvall H. , Allen J.M. , Polak J.M. , Bloom S.R. , Mutt V.
Published: July 2002

The isolation and primary structure of intestinal neuropeptide Y (NPY) is described. The peptide was purified from porcine intestinal extracts using a chemical assay and radioimmunoassay for NPY. The amino acid sequence of this peptide is: Tyr‐Pro‐Ser‐Lys‐Pro‐Asp‐Asn‐Pro‐Gly‐Glu‐Asp‐Ala‐Pro‐Ala‐Glu‐Asp‐LeuAla‐Arg‐Tyr‐Tyr‐Ser‐Ala‐Leu‐Arg‐His‐Tyr‐Ile‐Asn‐Leu‐Ile‐Thr‐Arg‐Gln‐Arg‐Tyr‐NH2. This indicates that the structure of intestinal NPY is identical to the NPY of brain origin.
Hydralazine binds covalently to complement component C4
Authors: Sim Edith , Law Sai-Kit A.
Published: July 2002

Long‐term treatment with hydralazine is sometimes associated with deposition of immune complexes and development of systemic lupus erythematosus (SLE) as an adverse side‐effect. Hydralazine inhibits the covalent binding reaction of the complement protein C4. We show that when hydralazine inhibits C4, it becomes covalently bound to the polypeptide chain containing the active site thiol ester. C4 is encoded at 2 adjacent polymorphic loci, C4A and C4B, within the major histocompatibility complex. We show that hydralazine binds more efficiently to the C4A than to the C4B gene product and suggest that C4 type may predispose patients to hydralazine‐induced SLE.
Subtype ayw variant of hepatitis B virus
Authors: Bichko V. , Pushko P. , Dreilina D. , Pumpen P. , Gren E.
Published: July 2002

The entire genome of human hepatitis B virus (HBV) occurring in Latvia was sequenced. This sequence, which is 3182 nucleotides long, was compared with the other previously published HBV genomes and was shown to share maximum homology with HBV subtype ayw DNA. The coordinates of 4 main open reading frames as well as hairpin structures are very well conserved in the two genomes. The distribution of nucleotide substitutions among different HBV genomes suggests that the open reading frames P and X can fulfil a coding function. On the basis of primary stucture comparison for hepadnaviral DNAs several evolutionary conclusions can be drawn.
Isolation and characterization of syncytiotrophoblast plasma membrane from human placenta
Authors: Khalfoun B. , Degenne D. , Arbeille-Brassart B. , Gutman N. , Bardos P.
Published: July 2002

Human full‐term syncytiotrophoblast plasma membranes isolated by mechanical procedures (sieving and ultrasonic disintegration), purified by phase centrifugation, form a single band of 1.052 ± 0.002 density in percoll gradient. The purity of the preparation was assessed by electron microscopy, enzyme analysis and β2‐microglobulin determination.
Peptides of postulated inhibin activity
Authors: Kohan Sergio , Fröysa Berit , Cederlund Ella , Fairwell Thomas , Lerner Richard , Johansson Jan , Khan Shafiq , Ritzen Martin , Jörnvall Hans , Cekan Sten , Diczfalusy Egon
Published: July 2002

A 94‐residue polypeptide isolated from human seminal plasma and its chemically synthesized C‐terminal 28‐residue segment were studied in an in vitro inhibin bioassay utilizing rat pituitary cell cultures. Both peptides have previously been claimed to have inhibin activities, and the effects on the secretion and cellular content of gonadotrophins (FSH and LH) were now assessed in the in vitro assay. No inhibition was found. After 72 h of culture, both the cellular content and the spontaneous as well as the LHRH‐stimulated release of bioactive or immunoactive FSH and LH remained unaffected. Similarly, no effects were found on the storage and/or release of prolactin, growth hormone, or thyrotropin. We conclude that both the native 94‐residue peptide and the synthetic replicate of its C‐terminal 28‐residue segment, do not influence the pituitary FSH secretion when assessed in this in vitro system.
Activation of polyphosphoinositide phospholipase C by fluoride in WRK1 cell membranes
Authors: Guillon Gilles , Mouillac Bernard , Balestre Marie-Noëlle
Published: July 2002

Partially purified plasma membranes prepared from myo‐[3H]inositol‐prelabeled WRK1 cells exhibit a phosphatidylinositol 4,5‐bisphosphate (PIP2) phospholipase C activity sensitive to NaF. NaF increased the production of IP2 and IP3 in a time‐ and concentration‐dependent manner. The maximal increase in IP2 and IP3 production rates represented 400 ± 18 and 360 ± 40% of the basal production rate, respectively. Half‐maximum stimulation was reached with 2–4 mM NaF. The observed effect was specific for F−. Aluminium potentiated fluoride‐induced IP3 and IP2, accumulation in a concentration‐dependent manner. The effect of fluoride on the PIP2 phospholipase C from WRK1 cell membranes appears to be similar to the well‐documented effect of F− on the well‐characterized Ns. Ni and transducin GTP‐binding proteins. This observation constitutes an additional argument to suggest that a GTP‐binding protein is involved in the process of receptor‐mediated activation of PIP2 phospholipase C.
Two processes for the breakdown of the QB protein of chloroplasts
Authors: Hilditch Paul , Thomas Howard , Rogers Lyndon J.
Published: July 2002

The behaviour of the 32 kDa QB protein of the chloroplast thylakoid membrane was investigated in senescing leaves of Festuca pratensis. Bf 993, a non‐yellowing Festuca mutant, is known to be unable to degrade at least some of its thylakoid proteins during senescence. A high rate of QB protein turnover under illumination was observed in both Bf 993 and a normally yellowing cultivar. In the dark, however, the QB protein of Bf 993 exhibited the unusual stability of other thylakoid proteins that have been investigated in this genotype, whereas the normal cultivar was able to degrade its QB protein at an appreciable rate. These observations suggest that two independent processes exist for the degradation of the QB protein, one of which is impaired in mutant genotype Bf 993.
Structure‐linked latency of muscarinic receptors in axonal transport
Authors: Laduron Pierre M. , Scholtus Thierry
Published: July 2002

Muscarinic receptors that accumulated above a ligature in rat sciatic nerves were labelled in vitro under isotonic conditions withN[3H]methylscopolamine. The addition of 0.005% of digitonin doubled the binding in proximal segments above and close to the ligature but not in the intermediary segments between two ligatures. Osmotic shock and freeze‐thawing treatments also enhanced the binding. Digitonin did not affect the affinity of muscarinic receptors but revealed a greater number of sites by increasing the membrane permeability to the hydrophilic ligand. We conclude that presynaptic muscarinic receptors that undergo fast axonal transport in rat sciatic nerves exist under a latent form because they are associated with vesicles. This is the first demonstration of a structure‐linked latency for receptors.
Location of penicilloyl groups on CNBr fragments of the albumin from penicillin‐treated patients
Authors: Lafaye Pierre , Lapresle Claude
Published: July 2002

Two fixation sites for penicilloyl groups on human albumin were demonstrated. Using CNBr cleavage the first site was located between methionine 123 and methionine 297 and the second one between methionine 297 and the C‐terminal residue. In both cases, penicilloyl groups were unmasked by pronase degradation or disulfide bond reduction.
Dimethylallylpyrophosphate:3,9‐dihydroxypterocarpan 10‐dimethylallyl transferase from Phaseolus vulgaris Identification of the reaction product and properties of the enzyme
Authors: Biggs David R. , Welle Roland , Visser Frans R. , Grisebach Hans
Published: July 2002

A microsomal fraction of elicitor‐challenged bean (Phaseolus vulgaris) cell suspension cultures catalyses the prenylation of 3,9‐dihydroxypterocarpan (DHP) to 3,9‐dihydroxy‐10‐dimethylallylpterocarpan (phaseollidin) with dimethylallylpyrophosphate (DMAPP) as the prenyl donor. K
m values for DMAPP and DHP are in the range 1–3 μM. Strong product inhibition with phaseollidin and phaseollin is observed.
Glycogen synthesis from pyruvate in the periportal and from glucose in the perivenous zone in perfused livers from fasted rats
Authors: Bartels Heidrun , Vogt Beate , Jungermann Kurt
Published: July 2002

The isolated liver of 24 h fasted rats was perfused in a non‐recirculating manner in the orthograde or retrograde direction with media containing glucose and/or gluconeogenic precursors. Glycogen formation was determined biochemically and demonstrated histochemically. With glucose as the only exogenous substrate glycogen was formed exclusively in the perivenous area during both orthograde and retrograde perfusion. With gluconeogenic precursors as the exogenous substrates glycogen was deposited in the periportal zone during orthograde perfusion and in the intermediate zone during retrograde perfusion. Supply of glucose and gluconeogenic substrates initiated glycogen synthesis only in the upstream region, i.e. in the periportal zone during orthograde and in the perivenous zone during retrograde perfusion. This localization of glycogen synthesis was probably due to an unavoidable, insufficient oxygen supply of the respective downstream area. In general, the results confirm the hypothesis that periportal and perivenous glycogen was synthesized from different substrates.
Antibody used to identify penicillin‐binding protein 2' in methicillin‐resistant strains of Staphylococcus aureus (MRSA)
Authors: O'Hara Denise M. , Reynolds Peter E.
Published: July 2002

Polyclonal antibodies were raised against the membrane‐bound penicillin‐binding protein (PBP 2') present in methicillin‐resistant strains of Staphylococcusaureus (MRSA) and were used to detect its presence in membranes from strains grown under varying conditions for the expression of resistance. The antibody preparation reacted with another membrane protein from methicillin‐sensitive S. aureus (MSSA) which migrated to the same position as PBP 2' on SDS gel electrophoresis. Pretreatment of the antisera with membranes of sensitive strains removed antibodies recognising high‐M
r, proteins common to both MRSA and MSSA: the residual antibody then reacted specifically with PBP 2'. Antisera pretreated in this manner can be used as a probe for the definitive identification of MRSA strains.
The neurotoxin histrionicotoxin interacts with the putative ion channel of the nicotinic acetylcholine receptors in the central nervous system
Authors: Rapier Catherine , Wonnacott Susan , Lunt George G. , Albuquerque Edson X.
Published: July 2002

Perhydrohistrionicotoxin at micromolar concentrations blocked the nicotine‐evoked transmitter release from perfused striatal (dopaminergic) and hippocampal (cholinergic) nerve terminals. Perhydrohistrionicotoxin failed to compete with [3H]nicotine for its high‐affinity binding site in rat brain, suggesting that the action of this toxin on central nicotinic receptors is noncompetitive. From the dose‐response curve, 50% inhibition of nicotine‐evoked striatal dopamine release occurred at 5 μM perhydrohistrionicotoxin, a value similar to that obtained in frog sartorius muscle and Electrophorus electroplax. This close agreement may suggest that the ionic channel of the presynaptic nicotinic acetylcholine receptor of brain neurons has similar properties to those of the peripheral receptor.
1,4‐Dihydropyridine receptor associated with Ca2+ channels in human embryonic fibroblasts
Authors: Dudkin Sergei M. , Gnedoj Sergei N. , Chernyuk Natalia N. , Soldatov Nikolai M.
Published: July 2002

By using the radioactively labeled 1,4‐dihydropyridine (DHP) probe, [3H]PMD, we have demonstrated that cultured human embryonic fibroblasts grown at a low density in Eagle's medium supplemented with serum contain a single class of non‐interacting DHP binding sites (B
max, 1.2±0.3 pmol/ 106 cells; K
d, 3.9 nM). After inhibition of the DHP receptor biosynthesis by cycloheximide, the number of [3H]PMD binding sites is reduced with a half‐time of 12 h, which implies a turnover rate of 30 000±7500 receptors/h per cell. With progression to confluency, the B
max value decreased up to 0.28±0.08 pmol/106 cells without significant change in K
d value. When cells were grown at a low density in serum‐free conditions, the number of [3H]PMD binding sites gradually increased 1.9‐fold within 3 days. Addition of serum reversed this effect with the same time course. These results imply that the DHP‐sensitive Ca2+ channels are involved in the control of the proliferation of human embryonic fibroblasts.
Cloning, sequencing and expression of a full‐length rabbit fast skeletal troponin‐C cDNA
Authors: Chen Qian , Taljanidisz Janos , Sarkar Satyapriya , Tao Terence , Gergely John
Published: July 2002

A full‐length cDNA coding for troponin‐C isolated from an adult rabbit fast skeletal muscle library has been sequenced and expressed in an E. coli host. The amino acid sequence derived from the coding region agrees with the published protein sequence except that the first two residues are reversed. The expressed protein was found to be identical to purified rabbit skeletal troponin‐C based on electrophoretic mobilities in polyacrylamide gels containing either SDS or urea, and on immunoblotting. These results establish that our troponin‐C cDNA clone is suitable for site‐directed mutagenesis studies on the structure and function of troponin‐C
In vivo studies on the binding sites for lipoprotein (a) on parenchymal and non‐parenchymal rat liver cells
Authors: Harkes Leen , Jürgens Günther , Holasek Anton , van Berkel Theo J.C.
Published: July 2002

The direct correlation between lipoprotein (a) (Lp(a)) concentrations and atherosclerosis stimulated us to investigate the in vivo interaction of Lp(a) with the liver and the various liver cell types. In untreated rats the serum decay of Lp(a) is comparable to that of LDL. By estrogen treatment the interaction of LDL with parenchymal liver cells is increased 17‐fold whereas only a 2‐fold effect on Lp(a) is found. The decay of Lp(a) in estrogen‐treated rats is slower than for LDL. The data indicate that Lp(a) in vivo shows a less efficient interaction than LDL with the estrogen‐induced apo‐B,E receptor on parenchymal liver cells. It is suggested that the inability of Lp(a) to interact efficiently with the LDL removal system of the liver might be related to its atherogenic action.
The human medulloblastoma cell line TE671 expresses a muscle‐like acetylcholine receptor Cloning of the α‐subunit cDNA
Authors: Schoepfer Ralf , Luther Michael , Lindstrom Jon
Published: July 2002

Nicotinic acetylcholine receptors (AChRs) from muscle bind α‐bungarotoxin (αBgt) and are composed of four kinds of subunits, whereas AChRs from mammalian brains do not bind αBgt and are composed of two kinds of subunits. αBgt‐binding proteins whose function is unknown are also found in brain. All these proteins belong to the same gene family. The human medulloblastoma cell line TE671 expresses a functional AChR which binds αBgt. Surprisingly, the AChR of this neuron‐derived cell line has electrophysiological, immunological and biochemical properties different from neuronal AChRs and very similar to muscle AChRs. The TE671 AChR binds αBgt, but is different from αBgt‐binding proteins in brain. Here we show that TE671 expresses the α‐subunit mRNA coding for the muscle AChR, thereby proving that TE671 expresses a muscle‐type AChR that is not expressed in adult brain. The isolated cDNA clones should prove useful for expression of large amounts of human muscle‐type AChR α‐subunit protein for studies of the autoimmune response to muscle AChRs in human myasthenia gravis.
3′‐Fluoro‐3′‐deoxyribonucleoside 5′‐triphosphates: Synthesis and use as terminators of RNA biosynthesis
Authors: Mikhailopulo Igor A. , Pricota Tamara I. , Poopeiko Nicolai E. , Sivets Grigorii G. , Kvasyuk Eugenii I. , Sviryaeva Tatiana V. , Savochkina Larissa P. , Beabealashvilli Robert S.
Published: July 2002

3′‐Fluoro‐3′‐deoxy‐uridine, ‐cytidine, ‐adenosine and ‐guanosine have been synthesized by glycosylation of the corresponding silylated bases with 1‐O‐acetyl‐2,5‐di‐O‐benzoyl‐3‐fluoro‐3‐deoxy‐D‐ribofuranose in the presence of Friedel‐Crafts catalysts and were converted to the 5′‐ triphosphates, NTP(3′‐F). It was shown that NTP(3′‐F) are terminators of RNA synthesis catalyzed by DNA‐dependent RNA polymerase from E. coli and may thus serve as tools for DNA sequencing.
Conformational behavior of the linear hexapeptide senktide: A receptor specific tachykinin analog
Authors: Sumner Susan C.J. , Ferretti James A.
Published: July 2002

A receptor selective linear hexapeptide tachykinin analog, senktide, is shown to be highly ordered in solution. The conformational restriction is attributed to steric and electrostatic interactions produced by N‐methylation of the third amino acid residue in the sequence and the negatively charged N‐terminus. The structure of senktide is described as a dynamic mixture of similar conformations where the predominant one is a distorted antiparallel hydrogen bonded β‐pleated sheet. The observed senktide‐receptor specificity is suggested to result from a selection of this or a closely related conformation.
Differential sensitivity to guanine nucleotides of basal and insulin‐stimulated glucose transporter activity reconstituted from adipocyte membrane fractions
Authors: Schürmann A. , Rosenthal W. , Hinsch K.D. , Joost H.G.
Published: July 2002

The effects of GTPγS on glucose transport activity reconstituted from adipocyte membrane fractions were studied in order to test the hypothesis that intrinsic activity changes of the insulin‐sensitive glucose transporter may be mediated by guanine nucleotide‐dependent mechanisms. GTPγS and GTP inhibited reconstituted glucose transport activity by 50% in membrane fractions from insulin‐treated cells in a concentration‐dependent manner; no inhibitory effect was observed in membrane fractions obtained from basal cells. GDP, GMP and guanosine were less effective than GTP, whereas the adenine nucleotides ATPγS and AMP failed to reduce the reconstituted transport activity. The data indicate that guanine nucleotides may modulate the activity of the adipocyte glucose transporter. Since the effect is dependent on treatment of cells with insulin, the hormone appears to induce a specific functional alteration of the glucose transporter.
Carboxyl methylation of 21–23 kDa membrane proteins in intact neuroblastoma cells is increased with differentiation
Authors: Haklai R. , Kloog Y.
Published: July 2002

Evidence is presented for specific enzymatic methylation of 21–23 kDa membrane proteins in intact neuroblastoma N1E 115 cells, which is increased in dimethylsulfoxide‐induced differentiated cells. Methylation of these proteins has characteristics typical of enzymatic reactions in which base labile volatile methyl groups are incorporated into proteins, consistent with the formation of protein carboxyl methylesters. However, these methylesters of the 21–23 kDa proteins are relatively stable compared to other protein carboxyl methylesters. The 3‐fold increase in methylated 21–23 kDa proteins in the differentiated cells suggest biological significance in differentiation of the cell membranes.
Effect of transforming growth factor β on fibroblasts in three‐dimensional lattice cultures
Authors: Coustry Françoise , Gillery Philippe , Maquart François-Xavier , Borel Jacques-Paul
Published: July 2002

Human skin fibroblasts were cultivated in three‐dimensional fibrin or collagen lattices, under retracting or non‐retracting conditions, and the influence of transforming growth factor β (TGFβ) was tested. TGFβ stimulated the synthesis of non‐collagen protein and of collagen in all the systems. However, only in non‐retracting fibrin lattices did it restore a level of protein synthesis comparable to that found in monolayers. The effects of TGFβ greatly depended on the type of substratum and on the presence or absence of retraction.
1H NMR assignments of sidechain conformations in proteins using a high‐dimensional potential in the simulated annealing calculations
Authors: Habazetti J. , Cieslar C. , Oschkinat H. , Holak T.A.
Published: July 2002

A high‐dimensional potential representing distance constraints for stereospecifically assignable diastereotopic proton or methyl pairs was incorporated into the dynamical simulated annealing protocol to calculate structures with stereospecifically determined sidechain conformations. The protocol is tested on nuclear magnetic resonance cross‐relaxation data of a trypsin inhibitor from squash seeds, CMTI‐I, and compared with two other methods of stereospecific assignment, the floating chirality and coupling constant methods. There is good agreement between the three methods in predicting the same stereospecific assignments. Because the high‐dimensional potential uses more relaxed absolute distance constraints and also takes into account the relative distance constraint patterns, it avoids possible overinterpretation of the NOE data.
The detection and characterization of G‐proteins in the eyespot of Chlamydomonas reinhardtii
Authors: Korolkov S.N. , Garnovskaya M.N. , Basov A.S. , Chunaev A.S. , Dumler I.L.
Published: July 2002

The presence of G‐proteins in the eyespot fraction of Chlamydomonas reinhardtii is shown. This fraction is capable of binding (GTPγ[35S], possesses the GTPase activity and interacts with antibodies raised against a highly conserved peptide of most G‐proteins' α‐subunit. Cross‐reaction with a 24‐kDa protein is detected on immunoblots. Using an antiserum prepared from vertebrate β‐subunit peptide, two additional proteins with apparent M
r 21 and 29 kDa could be revealed. The light‐dependence of GTPase extraction from eyespot membranes is shown. The results make it possible to suggest the participation of G‐proteins in the photosensory transduction chain of Ch. reinhardtii.
Site‐directed mutagenesis of the putative catalytic residues of Trichoderma reesci cellobiohydrolase I and endoglucanase I
Authors: Mitsuishi Yasushi , Nitisinprasert Sunee , Saloheimo Markku , Biese Isa , Reinikainen Tapani , Claeyssens Marc , Keränen Sirkka , Knowles Jonathan K.C. , Teeri Tuula T.
Published: July 2002

Site directed mutagenesis has been performed to test hypotheses concerning the putative active sites of Trichoderma reesci cellobiohydrolase I and endoglucanase I. It is shown that mutagenesis of the residue 1:126, previously proposed to be the proton donor in CBHI, did not totally inactive the enzyme while mutagenesis of the residue 1:127 in the homologous enzyme EG1 resulted in complete loss of activity. These results are compared with those obtained in similar studies of other glucanases and the effects on enzymatic activity of hyperglycosylation of the yeast produced cellulases are discussed.
Rapid aggregation and tight junction formation in single cell suspensions of tumor cells after very low dose trypsin treatment
Authors: Kemmner W. , Zaar K.
Published: July 2002

The possible participation of tight junction formation in intercellular adhesion of tumor cells was studied in single cell suspensions of HT29 adenocarcinoma cells. Very low dose trypsin treatment (0.15 μg/ml, 30 min, 37°C) induced rapid intercellular adhesion of suspended HT29 colon carcinoma cells. Intercellular adhesion was independent of the presence of divalent cations. Electron microscopy of freeze‐fractured membrane fragments of trypsin‐treated HT29 cells revealed a dramatic increase of typical tight junction structures during aggregation.
Activation mechanism of rabbit skeletal muscle myosin light chain kinase 5′‐p‐Fluorosulfonylbenzoyl adenosine as a probe of the MgATP‐binding site of the calmodulin‐bound and calmodulin‐free enzyme
Authors: Kennelly Peter J. , Colburn Joel C. , Lorenzen James , Edelman Arthur M. , Stull James T. , Krebs Edwin G.
Published: July 2002

5′‐p‐Fluorosulfonylbenzoyl adenosine (FSBA), an ATP‐like affinity labelling reagent, reacted with rabbit skeletal muscle myosin light chain kinase (skMLCK) and its calmodulin complex in a site‐specific manner. Reaction was dependent upon the presence of the adenosine moiety of FSBA, saturated with increasing FSBA, was inhibited by MgATP, and was accompanied by stoichiometric incorporation of [14C]FSBA. The kinetic constants describing the reaction were similar for skMLCK and its calmodulin complex: k
3= −0.040 min−1 and −0.038 mint‐1, and K
i=0.18 mM and 0.40 mM, respectively. It is concluded that the MgATP‐binding site on skMLCK remains accessible at all times and maintains a near constant conformation.
Inhibition of rat liver microsomal fatty acid chain elongation by gemfibrozil in vitro
Authors: Sánchez R.M. , Viñals M. , Alegret M. , Vázquez M. , Adzet T. , Merlos M. , Laguna J.C.
Published: July 2002

Gemfibrozil, a hypolipidemic drug mainly used in the treatment of hypertriglyceridemic states, strongly inhibits the rat hepatic microsomal fatty acid chain elongation system in vitro. The inhibition is independent on the reducing cofactor used in the assay. Furthermore, gemfibrozil seems to act by inhibiting the rate‐limiting step of the elongation process, the condensing reaction, without discriminating among the proposed three different condensing enzymes, devoted to condensation of saturated, mono‐unsaturated and polyunsaturated acyl‐CoA substrates.
cDNA cloning of rat proteasome subunit RC1, a homologue of RING10 located in the human MHC class II region
Authors: Aki Masashi , Tamura Tomohiro , Tokunaga Fuminori , Iwanaga Sadaaki , Kawamura Yoshihiro , Shimbara Naoki , Kagawa Susumu , Tanaka Keiji , Ichihara Akira
Published: July 2002

The nucleotide sequence of a cDNA that encodes a new subunit, named RC1, of rat proteasomes (multicatalytic proteinase complexes) has been determined. The polypeptide predicted from the open reading frame consisted of 208 amino acid residues with a calculated molecular mass of 23,130, which is consistent with the size obtained by electrophoretic analysis of purified RC1. The partial amino acid sequences of several fragments of RC1, obtained by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Surprisingly, the overall structure of RC1 was found to be almost identical to that of recently isolated RING10, whose gene is located in the class II region of the human MHC gene cluster. This finding suggests that RC1 is a homologue of human RING10, supporting the proposal that proteasomes are involved in the antigen processing pathway.
Ca2+‐induced Ca2+ release in insulin‐secreting cells
Authors: Islam Md.Shahidul , Rorsman Patrik , Berggren Per-Olof
Published: July 2002

The sulphydryl reagent thimerosal (50 μM) released Ca2+ from a non‐mitochondrial intracellular Ca2+ pool in a dose‐dependent manner in permeabilized insulin‐secreting RINm5F cells. This release was reversed after addition of the reducing agent dithiothreitol, Ca2+ was released from an Ins(1,4,5)P3‐insensitive pool, since release was observed even after depletion of the Ins(1,4,5)P3‐sensitive pool by a supramaximal dose of Ins(2,4,5)P3 or thapsigargin. The Ins(1,4,5)P3‐sensitive pool remained essentially unaltered by thimerosal. Thimerosal‐induced Ca2+ release was potentiated by caffeine. These findings suggest the existence of Ca2+‐induced Ca2+ release also in insulin‐secreting cells.
Tumorigenicity‐associated expression of protein kinase C isoforms in rhabdomyosarcoma‐derived cells
Authors: Hanania Nicole , Lezenes Jacqueline Robert , Castagna Monique
Published: July 2002

Cell sublines were derived from Ni‐induced rat rhabdomyosarcoma which differed in their degree of tumorigenicity. We compared the expression of protein kinase C isoforms α, β, γ and ε in three sublines developing tumors in syngeneic rats (Sy+) and in two sublines devoid of tumorigenicity in these animals (Sy−), with that of normal skeletal muscle. Northern and Western blotting experiments showed that PKC α was dramatically overexpressed in Sy− cells whereas it was underexpressed in Sy+ cells. Southern blot analysis provided evidence for a 3‐fold increase of PKC α‐related DNA fragments in Sy− cells. Steady‐state levels of the PKC ε‐related transcript were also markedly decreased in Sy+ cells. However, the expression of PKC β‐related RNA was increased in these cells. Our data support the concept that the differential expression of the PKC isoforms may play a critical role in determining the neoplastic phenotype.
Characterisation of a near infra‐red absorption band of the Escherichia coli quinol oxidase, cytochrome o, which is attributable to the high‐spin ferrous haem of the binuclear site
Authors: Ingledew W.John , Bacon Mark , Rich Peter R.
Published: July 2002

The bacterial quinol oxidase, cytochrome o, is an enzyme which is highly analogous to the better known cytochrome c oxidase, cytochrome aa3
but with the important difference that it lacks the near infra‐red absorbing pigment CuA. In this article we report an absorption band in the near IR spectrum of cytochrome o with a maximal absorption at 738 nm, and which is attributable to the ferrous high‐spin haem. The 758 nm band has an extinction coefficient or 0.2–0.3 mM−1·cm−1 at 758–800 nm. This region in cytochrome aa3
, is dominated by the CuA absorption. The 758 nm absorption is lost on addition of CO or cyanide to the reduced enzyme. The carbon monoxide compound of cytochrome o also has absorbance bands in the near infra‐red, and these may be attributable to a low‐spin ferrous haem compound.
Experimental evidence of oligomeric organization of antenna bacteriochlorophyll c in green bacterium Chloroflexus aurantiacus by spectral hole burning
Authors: Fetisova Z.G. , Mauring K.
Published: July 2002

Spectral hole burning has been used to prove experimentally the existence in natural antenna of one of the predicted structural optimizing factors — antenna pigment oligomerization [J. Theor. Biol. 140 (1989) 167]—ensuring high efficiency of excitation energy transfer from antenna to reaction center, This point has been examined for the chlorosomal antenna of green bacterium Chloroflexus auranticus by hole burning in fluorescence excitation and emission spectra of intact cells at 1.8 K. The persistent hole spectra have been found to be consistent with a strongly exciton‐coupled bacteriochloraphyll c (BChl c) chromophore system. The lowest exciton state of BChl c oligomers has been direclly detected and separated as the lowest energy inhomogeneously broadened band (FWHM ∼90 cm−1, position of maximum, at ∼752 nm) from the near‐infrared BChl c band (FWHM ∼350 cm−1, position or maximum, at ∼74 nm) of 1.8 K excitation spectrum.
Mono ADP‐ribosylation of transducin catalyzed by rod outer segment extract
Authors: Ehret-Hilberer Sylviane , Nullans Gërard , Aunis Dominique , Virmaux Noélle
Published: July 2002

Transducin is the retinal rod outer segment (ROS)‐specific G protein coupling the photoexcited rhodopsin to cyclic GMP‐phosphodiesterase. The α subunit of transducin is known to be ADP‐ribosylated by bacterial toxins. We investigated the possibility that transducin is modified in vitro by an endogenous ADP‐ribosyltransferase activity. By using either ROS, cytosolic extract of ROS or purified transducin in the presence of [α??P]nicotinamide adenine dinucleotide (NAD*), the α and β subunits of transducin were found to be radiolabeled, The labeling was decreased by snake venom phosphodiesterase I (PDE 1). The modification was shown to be mono ADP‐ribosylation by analyses on thin layer chromatography of the PDE 1‐hydrolyzed products which revealed only 5′AMP residues. In addition we report that sodium nitroprusside activates the ADP‐ribosylation of transducin.
The complete primary structure of the spermadhesin AWN, a zona pellucida‐binding protein isolated from boar spermatozoa
Authors: Sanz Libia , Calvete Juan José , Mann Karlheinz , Schäfer Wolfram , Schmid Erich R. , Amselgruber Werner , Sinowatz Fred , Ehrhard Michael , Töpfer-Petersen Edda
Published: July 2002

AWN is a boar protein which originates in secretions of the male accessory glands and which becomes sperm surface‐associated upon ejaculation. It is one of the components thought to mediate sperm adhesion to the egg's zona pellucida through a carbohydrate‐recognition mechanism. AWN may, thus, participate in the initial events of fertilization in the pig. In this report we describe its complete primary structure by combination of protein‐chemical and mass spectrometric methods. AWN exists as two isoforms, AWN‐1 and AWN‐2, which differ in that AWN‐2 is N‐terminally acetylated. The amino acid sequence of AWN contains 133 amino acid residues and two disulphide bridges between nearest‐neighbour cysteine residues. Analysis of the amino acid sequence of the AWN proteins showed significant similarity only to AQN‐1 and AQN‐3, two other boar spermadhesins.
Monoclonal antibody engineering in plants
Authors: Hiatt Andrew , Ma Julian K-C.
Published: July 2002

Techniques for plant transformation have been developed to such an extent that a number of foreign genes are currently being introduced into transgenic plants. Tobacco plants that produce monoclonal antibodies are of interest, because in addition to synthesis of two gene products (i.e. the heavy and light chains), the two polypeptides need to be assembled correctly, inorder to result in a functional antibody. The studies on a catalytic antibody suggest that this is the case, and that the antibody functions identically to the native murine‐derived antibody. The only difference observed was in the glycosylation of the heavy chain. Further transgenic plants are being generated to produce monoclonal antibodies that may be used therapeutically (and are therefore required in large quantities), or to provide disease resistance in plants. In addition, the ability of plants to assemble antibody complexes is being investigated further, to study the possibility of generating secretory IgA, which consists of heavy and light chains as wall as two additional polypeptide units.
Antibody to the inositol trisphosphate receptor blocks thimerosalenhanced Ca2+‐induced Ca2+ release and Ca2+ oscillations in hamster eggs
Authors: Miyazaki Shun-ichi , Shirakawa Hideki , Nakada Ken , Honda Yoshiko , Yuzaki Michisuke , Nakade Shinji , Mikoshiba Katsuhiko
Published: July 2002

The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5‐trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP3 receptor blocked both the InsP3‐induced Ca2+ release (IICR) and Ca2+‐induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3‐insensitive pools, and causes repetitive Ca2+ releases from InsP3‐sensitive pools.
The cDNA structure of the porcine pro‐hormone convertase PC2 and the comparative processing by PC1 and PC2 of the N‐terminal glycopeptide segment of porcine POMC
Authors: Seidah N.G. , Fournier Hélèrie , Boileau Guy , Benjannet Suzanne , Rondeau Nomand , Chrétien Michel
Published: July 2002

The complete cDNA structure of the porcine (p) pro‐protein and pro‐hormone convertase PC2 (pPC2) was obtained from a cDNA library of pituitary neurointermediate lobes mRNA. The deduced amino acid sequence revealed that pPC2 exhibits a 99‐97% sequence identity to the human, mouse and rat homologues. The 3′ end of the 2.1 kb cDNA is the least conserved segment. On Northern blots of pars intermedia poly A+ RNA two transcripts of 3 and 5 kb were detected. Molecular analysis of the N‐terminal glycopeptide products of porcine pro‐opiomelanocortin (pPOMC) co‐expressed with vaccinia virus recombinants of PC1 or PC2, revealed that in cells devoid or containing secretary granules both convertases can cleave pPOMC with PC1 releasing the 1–80, 1–107 and 1–148 glycopeptide fragments, and PC2 cleaving pPOMC directly into pPOMC 1–107.
The light‐harvesting core‐complex and the B820‐subunit from Rhodopseudomonas marina. Part II. Electron microscopic characterisation
Authors: Meckenstock Rainer U. , Krusche Katarina , Brunisholz René A. , Zuber Herbert
Published: July 2002

Electron micrographs of photosynthetic membranes of the BChla‐containing bacterium Rp, marina showed a quasi‐crystalline structure. The photoreceptor units are arranged in a hexagonal lattice with a reaction center to reaction center distance of 102± Å. Purified B880‐complex was concentrated up to an OD? of 60 which induced the formation of large protein vesicles. The protein complexes within these vesicles were highly ordered and showed a hexagonal lattice with the same center to center distance of 102±3 Å as was observed in the native membranes. Image processing of the micrographs revealed a ring‐like structure of the B880‐complex at 26 Å resolution and suggests that the B880‐complex consists of 5 or 6 subunits. For the first time it can be shown that an isolated core‐complex is in a stable, ring‐like structure even without the reaction center which is supposed to be located in the middle of the B880‐ring. The data indicate that the isolated B880‐complex exhibits the same structure as in the native membrane.
Neuronal‐type nicotinic receptors in human neuroblastoma and small‐cell lung carcinoma cell lines
Authors: Tarroni Paola , Rubboli Francesca , Chini Bice , Zwart Ruud , Oortgiesen Marga , Sher Emanuele , Clementi Francesco
Published: July 2002

A β subunit of the neuronal nicotinic receptor, sharing 88% homology with the rat β4 subunit, has been cloned from a human neuroblastoma cell line. The gene encoding the human β4 subunit is expressed in association with the α3 gene in neuroblastoma and small‐cell lung carcinoma cell lines. Patch‐clamp experiments and radioligand binding assays confirm that these neuroendocrine tumor cell lines express functional neuronal nicotinic receptors. We suggest that these receptors might play a crucial role in the control of neurotransmitter and hormone secretion from neurosecretory human tumors.
Repression of histone H5 gene expression in chicken mature erythrocytes is correlated with reduced DNA‐binding activities of transcription factors SP1 and GATA‐1
Authors: Sun Jian-Min , Penner C.Gail , Davie James R.
Published: July 2002

During the final stages of erythroid maturation, the expression of the chicken histone H5 gene ceases. The histone H5 promoter has binding sites for Sp1 and UPE‐binding protein. The 3' histone H5 enhancer has binding sites for Sp1, GATA‐1 and NF1. Here, we show that the DNA‐binding activities of transcription factors Sp1 and GATA‐1 is reduced 5‐ to 10‐fold in mature cells, while the activities of UPE‐binding protein and NF1 remain the same in mature and immature erythrocytes. The reduced activities of Sp1 and GATA‐1 may contribute to the inactivation of the histone H5 gene in mature erythrocytes.
Endoproteolysis of non‐CAAX‐containing isoprenylated peptides
Authors: Ma Yu-Ting , Rando Robert R.
Published: July 2002

A microsomal endoprotease specifically cleaves isoprenylated peptides of the CAAX motif, such as N‐acetyl‐S‐all‐trans‐farnesyl‐l‐cysteine (AFC‐VIM), at the isoprenylated cysteine residue. It is shown here that endoproteolysis will also occur with peptides which are not of the CAAX type. Peptide substrates modeled after the Delta virus large antigen carboxyl‐terminus (CRPQ) are endoproteolytically hydrolyzed by liver microsomes. AFC‐RPQ is hydrolyzed with a K
M = 12.4 μM and a V
max= 0.27 nmolmin/mg, and AGGC‐RPQ is hydrolyzed with a K
M= 7.9 μM and a V
max = 0.042 nmolmin/mg. Moreover, a series of potent inhibitors of the endoproteolysis of AFC‐AAX‐containing peptides are ineffective at inhibiting the hydrolysis of AFC‐RPQ and AGGC‐RPQ, suggesting the existence of isoforms of the endoprotease.
Relaxation study of the backbone dynamics of human profilin by two‐dimensional 1H‐15N NMR
Authors: Constantine Keith L. , Friedrichs Mark S. , Bell Aneka J. , Lavoie Thomas B. , Mueller Luciano , Metzler William J.
Published: July 2002

The dynamic properties of 111 backbone HN sites in uncomplexed human profilin, a protein of 139 residues, have been characterized by two‐dimensional inverse‐detected 1H‐15N NMR spectroscopy. Heteronuclear {1H}‐15N nuclear Overhauser effects and 15N longitudinal and transverse relaxation rates have been analyzed in terms of model‐free spectral density functions and exchange contributions to transverse relaxation rates. Relatively high mobilities on the nanosecond timescale are observed for Asp26 and Ser27, which form part of a loop connecting β‐strands A and B, and for Thr92 through Ala95, which are in a loop connecting β‐strands E and F. Significant exchange contributions, indicative of motions on the microsecond to millisecond timescale, have been obtained for 30 residues. These include Leu77, Asp80 and Gly81 of a loop between β‐strands D and E, Ser84 and Met85 of β‐strand E, Gly121 of a loop connecting β‐strand G and the C‐terminal helix, and Gln138, which is next to the C‐terminal residue Tyr139. Some of the regions showing high flexibility in profilin are known to be involved in poly‐L‐proline binding.
Immunogold localisation of ubiquitin‐protein conjugates in Sf9 insect cells
Authors: Löw Péter , Doherty Fergus J. , Sass Miklos , Kovács János , Mayer R.John , László Lajos
Published: July 2002

Immunogold electron microscopy with antibodies which primarily detect ubiquitin‐protein conjugates shows conjugate‐specific gold particles enriched severalfold in acid phosphatase‐positive lysosomes and multivesicular bodies in insect Sf9 cells. The observations demonstrate that ubiquitinated proteins are associated with small acid phosphatase‐containing primary lysosomes (transport vesicles) and indicate a pathway in which primary lysosomes fuse with multivesicular bodies to generate mature lysosome‐related structures.
Stoichiometry of the complex of human interleukin‐4 with its receptor
Authors: Hoffman Ross C. , Schalk-Hihi Céline , Castner Beverly J. , Gibson Marylou G. , Rasmussen Brian D. , Zdanov Alexander , Gustchina Alla , March Carl J. , Wlodawer Alexander
Published: July 2002

A large number of cytokines have been shown to possess a four‐helix bundle structure with a unique up‐up‐down‐down connectivity. The receptors for this family of cytokines have been shown to be homologous as well, each possessing two tandem repeats of a fibronectin type III‐like domain. The crystal structure of human growth hormone bound to the soluble portion of its receptor has served as the only experimentally‐determined example of the interaction between the four‐helix bundle cytokines and their receptors: Two identical receptor subunits bind to different epitopes on the same growth hormone ligand. We have conducted a series of experiments to determine if this structural paradigm is true for interleukin‐4 and interleukin‐4 receptor. Native polyacrylamide gel electrophoresis and gel filtration chromatography reveal that interleukin‐4 forms a tight 1:1 complex with the soluble portion of interleukin‐4 receptor and is thus unlike the growth hormone system.
The functional expression of p47‐phox and p67‐phox may contribute to the generation of superoxide by an NADPH oxidase‐like system in human fibroblasts
Authors: Jones S.A. , Wood J.D. , Coffey M.J. , Jones O.T.G.
Published: July 2002

Recent evidence suggests that a number of non‐phagocytic cell types may contain a superoxide generating NADPH oxidase. Studies to date on cultured human fibroblasts have primarily concerned the identification of cytochrome b
558, whilst expression of other NADPH oxidase components have not been addressed. In this study we have investigated the expression of NADPH oxidase with particular reference to the cytosolic factors p47‐phox and p67‐phox. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) showed that human fibroblasts express mRNA for p47‐phox, p67‐phox and p22‐phox. Expression of the gp91‐phox transcript was not detected, indicating that human fibroblasts may possess an NADPH oxidase isoenzyme. Western blot analysis of human fibroblast cytosol, using an anti‐p47‐phox antibody (JW‐1), identified a 47 kDa protein. Cell‐free reconstitution assays showed that fibroblast cytosol could initiate superoxide generation when mixed with either human fibroblast membranes (0.16 nmol superoxide/min/μg membrane protein), or resting human neutrophil membranes (0.20 nmol superoxide/min/μg membrane protein). These data indicate that the expression of p47‐phox and p67‐phox by human fibroblasts may contribute to the cells' generation of superoxide.
Nitric oxide blocks the cell cycle of mouse macrophage‐like cells in the early G2+M phase
Authors: Takagi Kuniaki , Isobe Yuji , Yasukawa Kiyoshi , Okouchi Etsuko , Suketa Yasunobu
Published: July 2002

The effects of nitric oxide produced by macrophage‐like cells (Mml) on the cell cycle were investigated. Mml cells lost proliferative activity in the presence of interleukin‐6 (IL‐6) and a subpopulation accumulated in the G2+ M phase. This level increased in proportion to the incubation time. The DNA content of the cells was slightly lower than that of Mml cells treated with vinbrastine or demecolcine, drugs which block the cell cycle in the M phase. The peak of the early G2+M phase was reduced by treatment with N
G‐mono‐methyl‐l‐arginine. However, after treatment with exogenous nitric oxide or sodium nitroprusside, the G0/G1 phase increased, but the early‐G2+M and the S phase decreased. The flow cytometry pattern in IL‐6‐treated Mml was the same as that of cytochalasin B‐treated Mml. These data suggest that endogenous nitric oxide affects the microfilament system of IL‐6‐treated Mml cells and blocks the cell cycle in the early G2+M phase.
Mutagenesis of Phe381 and Phe382 in the extracellular domain of the insulin receptor: effects on receptor biosynthesis, processing, and ligand‐dependent internalization
Authors: Accili Domenico , Mosthaf Luitgard , Levy-Toledano Rachel , Ullrich Axel , Taylor Simeon I.
Published: July 2002

Mutations of the extracellular domain of the insulin receptor impair processing and transport of receptors to the plasma membrane. We have previously reported that a mutation substituting Val for Phe382 in the α‐subunit of the insulin receptor impairs intracellular processing and insulin‐induced autophosphorylation of the mutant receptor. In this investigation, we have generated two independent mutations of amino acids Phe381 and Phe382 of the insulin receptor: Val for Phe381 and Leu for Phe382. These substitutions cause a slight impairment of intracellular processing and transport of the mutant receptors. Furthermore, insulin‐dependent internalization of the mutant receptors is unaffected by these mutations. Thus, of the three substitutions studied to date, Val for Phe382 is the only mutation of the Phe381‐Phe382 sequence that causes a major defect in posttranslational processing of the receptor.
Generation of a stable folding intermediate which can be rescued by the chaperonins GroEL and GroES
Authors: Peralta Dadna , Hartman Dallas J. , Hoogenraad Nicholas J. , Høj Peter B.
Published: July 2002

Pig heart mitochondrial malate dehydrogenase was chemically denatured in guanidine HCl. Upon 50‐fold dilution of the denaturant spontaneous refolding could be observed in the temperature range 12–32°C. At 36°C spontaneous refolding was not observed but a stable folding intermediate that is fairly resistant to aggregation was formed. This intermediate is readily refolded by the chaperonins GroEL and GroES and may prove useful in future attempts to describe several aspects of chaperonin action at physiological temperatures.
Restoration of down‐regulated PDGF receptors by TGF‐β in human embryonic fibroblasts
Authors: Psarras Stelios , Kletsas Dimitris , Stathakos Dimitri
Published: July 2002

The study of [125I]PDGF‐BB binding to normal human embryonic lung fibroblasts, quiescent when cultured at sparsity in the presence of minute concentrations of homologous PDS, reveals approximately 2 × 105 binding sites for PDGF per cell; this number significantly increases during prolonged quiescence of the culture. As late as 48 h after down‐regulation of PDGF receptors, the cells restore only partially their capacity to bind PDGF, with aged cells (above CPD 45) responding more rapidly and efficiently than younger ones. TGF‐β significantly enhances restoration of PDGF receptors and, in aged cells in particular, its presence results in total receptor recovery within 24 h, suggesting a concerted action of PDGF and TGF‐β regulating the proliferation of human fibroblasts in tissue regeneration.