bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibodies to human plasma Protein X alias complement S-protein
Authors: Jenne Dieter , Hugo Ferdinand , Bhakdi Sucharit
Published: February 2005

Protein X alias complement S-protein was isolated by dissociation from purified XCSb-9 (fluid-phase terminal C5b-9) complexes with 250 mM deoxycholate and subsequent sucrose density gradient centrifugation and Sephacryl gel chromatography. Polyclonal rabbit and monoclonal mouse antibodies were used to preliminarily characterize the protein in human serum and plasma. In plasma, Protein X yielded a symmetrical immuno-precipitate of α2-mobility in a crossed immunoelectrophoresis assay. However, a second immunoprecipitate of Oh-mobility was observed when serum was analysed; this precipitate represented Protein X in complex with antithrombin-III. The co-precipitation of Protein X with serum antithrombin-III was exploited for establishing a simple screening test for unequivocal identification of monocJonal anti – Protein X antibodies. SDS-PAGE immunoblotting with monoclonal antibodies showed that Protein X exhibits pronounced microheterogeneity, migrating as a diffuse moiety of approx. Mr 80–90 000. Additionally, a small amount of polymeric aggregates appear to be present in plasma. Reduction of disulfide bonds led to liberation of a polypeptide of approx. 15 K as discerned by two-dimensional SDS-PAGE immunoblotting. Protein X is not cleaved to lower molecular weight entities during the process of blood coagulation or during formation of fluid-phase terminal complement complexes. The plasma concentrations in healthy adults were in the range of 500–700 pg/ml. The availability of methods for isolating Protein X and raising monoclonal antibodies will facilitate further studies on the dual role of this protein in the terminal complement and coagulation cascades.
Effect of glucagon on hepatic phenylalanine hydroxylase in vivo
Authors: Beirne Edel , Carty Michael P. , Donlon John
Published: February 2005

Moderate doses of glucagon (20 μg/kg I.V.) are sufficient to stimulate rat hepatic phenylalanine hydroxylase in vivo. In addition, the stimulation of the tetrahydrobiopterin-dependent phenylalanine hydroxylase activity in livers of animals fed on a high-protein diet has been correlated with an elevated phosphate content. The tetrahydrobiopterin-dependent hydroxylase activity in these animals can be further elevated by glucagon-stimulated phosphorylation. These results indicate that physiological changes in glucagon concentration modulate rat liver phenylalanine hydroxylase activity in vivo. The current understanding of the role of phosphorylation in regulating human phenylalanine hydroxylase is also considered.
Hydrolysis of phosphatidylcholine liposomes by lysosomal phospholipase A is maximal at the phase transition temperature of the lipid substrate
Authors: Vandenbranden Michel , De Gand Georges , Brasseur Robert , Defrise-Quertain Fabienne , Ruysschaert Jean-Marie
Published: February 2005

We have measured the rate of hydrolysis of liposomes made of DL-α-dipalmitoylphosphatidylcholine (DPPC) and L-α-dimyristoylphosphatidylcholine by a soluble fraction of highly purified lysosomes isolated from rat liver. Phospholipids are hydrolyzed into lysophospho-lipids and fatty acids at a rate which is maximal near the temperature characteristic of the gel to liquid crystalline phase transition of the lipid bilayer. This strong influence of the physical properties of the substrate on the enzyme activity suggests a structural analogy between the lysosomal phospholipases of the A type (EC 3.1.1.32 and EC 3.1.1.4) and the pancreatic phospholipase A2.
Regulation of acetyl-CoA carboxylase by glucagon in HeLa cells
Authors: Bhullar R. P. , Dakshinamurti K.
Published: February 2005

A new method of purification of rat liver L-threonine deaminase has been developed, and the results obtained are compared with values obtained by other authors. Some properties of this enzyme (pH optimum, temperature optimum, thermal stability, specificity, etc.) have been examined and we found that the enzyme is inhibited by carbonate ions, that L-cysteine (a competitive inhibitor) is also an inactivator of the enzyme and that it is bound to the enzyme in a ratio of 0.25 mole of cysteine per mole of enzyme, supporting the hypothesis that the enzyme consists of 4 subunits.
Double-strand cleavage at a two-base deletion mismatch in a DNA heteroduplex by nuclease S1
Authors: Burdon Michael G. , Lees Jennifer H.
Published: February 2005

A two-base deletion mismatch was generated in a DNA heteroduplex by hybridization of two linear plasmid DNA molecules differing only by the presence of a two-base deletion in one of them. The heteroduplex was shown to be sensitive to double-strand cleavage by nuclease S1, thus demonstrating the potential value of single-stranded probes for the detection of polymorphisms in genomic DNA due to very small deletions.
Monoclonal antibodies against neoantigens of the terminal C5b-9 complex of human complement
Authors: Hugo Ferdinand , Jenne Dieter , Bhakdi Sucharit
Published: February 2005

Assembly of the terminal C5b-C9 complement components into the cytolytic C5b-9 complex is accompanied by exposure of characteristic neoantigens on the macromolecule. We report the production and characterization of mouse monoclonal antibodies to C9-dependent neoantigens of human C5b-9. Binding-inhibition assays with EDTA-human plasma and micro-ELISA assays with purified C9 showed that the antibodies did not react with native complement components and thus confirmed the specificity of the antibodies for the neoantigens. The monoclonal antibodies did, however, cross-react with cytolyticaIly inactive, fluid-phase C5b-9 complexes, Thus, expression of the neoantigenic determinants was not dependent on the formation of high molecular weight C9 polymers with the complex, since these are absent in fluid-phase C5b-9. Radioiodinated antibodies could be utilized in immunoradiometric assays for the detection and quantitation of C5b-9 on cell membranes. Cross-reactivities of the antibodies with C9-dependent neoantigens of several other animal species were examined and antibody clones cross-reacting with rabbit (clones 3BI, 3Dg, and 2F3), sheep (clones 3Dg and 2F3) and guinea-pig (clone 3D8) neoantigens were identified. Three of four tested clones (3D8, 2F3, IA12) precipitated C5b-9 complexes in double-diffusion assays, probably due to their interaction with multiple and repeating C9-epitopes on the terminal complexes. The monoclonal antibodies will be of value for definitive identification and quantitation of C5b-9 on cell membranes and in tissues, and for establishing immunoassays for detection and quantitation of terminal fluid-phase C5b-9 complexes in plasma.
Isolation of the first trypsin inhibitor from the genus Aspergillus
Authors: Ali Rashda , Zaidi Zafar H.
Published: February 2005

The fungus Aspergillus flavipes was grown on a Czapeck sucrose medium; the biomass so obtained was treated with high concentration of sucrose to release intracellular metabolites. Sephadex G-75 chromatography of the latter yielded a pure protein having anti-trypsin activity in vitro.
Rabbit antibodies with specificity for tyrosine phosphate are not reactive with tyrosine sulphate
Authors: Jones Jane A. , Cushley William
Published: February 2005

Tyrosine phosphate is a frequently observed modification in a limited number of protein molecules, principally the products of viral oncogenes and membrane receptors for mitogenic growth factors. Such a group is therefore an attractive candidate for affinity selection of such phosphoproteins by antibodies with specificity for tyrosine phosphate. This report demonstrates that antisera raised against 4-aminobenzylphosphonic acid have specificity for tyrosine phosphate, but are not inhibited by tyrosine sulphate. This observation has important ramifications for the detection and isolation of phosphotyrosine-containing proteins without co-purification or detection of tyrosine sulphate-containing proteins, which are present in cells of all lineages. Furthermore, we demonstrate that antisera raised against the sulphanilate moiety are not reactive with tyrosine sulphate.
The role of serine hydroxymethyltransferase in cell proliferation: DNA synthesis from serine following mitogenic stimulation of lymphocytes
Authors: Eichler Hans-Georg , Hubbard Ron , Snell Keith
Published: February 2005

It is shown that the time-course of incorporation of radioactivity from [3-14C]serine into nucleic acids parallels DNA synthesis following mitogenic stimulation of human peripheral blood lymphocytes by phytohaemagglutinin (PHA). The activity of serine hydroxymethyltransferase was elevated about four-fold in PHA-stimulated lymphocytes compared to that in unstimulated control ceils. It is suggested that lymphocytes, in common with other proliferating cell systems:, may synthesize serine de novo for utilization in pathways of nucleotide biosynthesis following mitogenic stimulation.
The effect of staphylococcal σ-haemolysin on the secretory activity of the pancreatic β-cell
Authors: Morgan Noel G. , Fitzhugh Alexandra J. , Montague William
Published: February 2005

The secretion of insulin from isolated rat islets of Langerhans was found to be stimulated by the surface-active staphylococcal exotoxin, σ-haemolysin. The response was dependent on the concentration of σ-haemolysin, was rapid in onset, and could be maintained for at least an hour in the presence of the agent. The rate of secretion rapidly declined on removal of σ-haemolysin and the islets remained responsive to glucose follow!ng toxin treatment.

Further characterization of the interaction of this agent with the σ-cell plasma membrane may provide valuable information concerning the role played by this membrane in the regulation of insulin secretion.
The photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis
Authors: Deisenhofer Johann , Michel Harmut
Published: February 2005

We first describe the history and methods of membrane protein crystallization, and show how the structure of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis was solved. The structure of this membrane protein complex is correlated with its function as a light-driven electron pump across the photosynthetic membrane. Finally we draw conclusions on the structure of the photosystem II reaction centre from plants and discuss the aspects of membrane protein structure.
Analysis by simulation of the transient rectification in molluscan neurons
Authors: Barbi M. , Chillemi S.
Published: February 2005

Central neurons of Lymnaea stagnalis exhibit, by the dne of a hyperpolarizing current pulse, a breaking-off of the rise of the membrane potential to the resting level. By using an electrical model of the membrane, this effect is accounted for by the activation of the fast outward current.
Use of purified antipeptide sera of selected specificity for the detection of the simian virus 40 large T antigen by western blotting
Authors: Dietrich Jean Bernard
Published: February 2005

Western blotting was used as a powerful alternative to immunoprecipitation for the detection of the simian virus 40 (SV40) large tumor (T) antigen. After resolution by electrophoresis on a SDS-polyacrylamide gel of a [15S]methionine labeled crude extract from SV40 infected monkey kidney cells, the separated proteins were transferred electrophoretically on nitrocellulose paper. T antigen was detected on nitrocellulose strips by using for the first time, specific, purified antipeptide monoclonal antibodies directed against the N- and C-terminal portions of the molecule, and125I-labeled Protein A.
High affinity GDP binding sites on brown adipose tissue mitochondria of genetically obese ‘fa/fa’ rats
Authors: French R. R. , Holt S. J. , York D. A.
Published: February 2005

The number of high affinity [3H]GDP binding sites in brown adipose tissue mitochondria is normal in obese (f a / f a) rats in contrast to the reduced number of low affinity GDP binding sites. Adrenalectomy corrected the loss of low affinity binding sites in fa/fa rats but had no effect on the number of high affinity sites in either lean or obese rats. Equilibrium dialysis was used to show the presence of both high and low affinity binding sites on the purified 32 kdalton protein.
A characterization of heam uptake and intracellular distribution by isolated hepatocytes
Authors: Lodola A.
Published: February 2005

The uptake and intracellular distribution of haem by isolated rat hepatocyte suspensions was studied. An increase in cell haem content occurred after a challenge with 5, 10 or 20 μM haem, supplied as methaemalbumin. The rate of haem uptake was temperature dependent; no non-specific binding occurred. Intracellular haem distribution data are consistent with a rapid association of haem with the endoplasmic reticulum fraction prior to its accumulation in the cytosol and at the mitochondrion.
Interaction of tyrosine residues with the chromophore in bacteriorhodopsin
Authors: Takeda Kazuo , Iwasa Tatsuo , Tokunaga Fumio , Packer Lester
Published: February 2005

We previously reported that the absorption spectrum at low temperatures of iodinated bacteriorhodopsin can be separated into four components with maxima at shorter wavelengths than in native bacteriorhodopsin. In this study, the time course of the formation of each spectral component after iodination was analyzed, revealing that these four components correspond to four different iodinated states of tyrosine residues interacting with the retinal chromophore of bacteriorhodopsin. Therefore at least two tyrosine residues interact with the chromophore of bacteriorhodopsin.
Inhibition and reversal of denervation-induced atrophy by the β-agonist growth promoter, clenbuterol
Authors: Maltin C. A. , Reeds P. J. , Delday M. I. , Hay S. M. , Smith F. G. , Lobley G. E.
Published: February 2005

Dietary administration of the growth promoter, clenbuterol, ameliorated denervation-induced atrophy in rat soleus muscles. In acutely denervated muscles the drug inhibited the appearance of atrophy, and in chronically denervated muscles the atrophy was almost fully reversed. Responses in slow twitch oxidative fibres were particularly marked.
In vitro studies on the subcellular location of glucosidase I and glucosidase II in dog pancreas
Authors: Bause Ernst , Günther Roland , Schweden Jürgen , Tillmann Ulrich
Published: February 2005

When programmed with yeast prepro-α-factor mRNA, the heterologous reticulocyte/dog pancreas translation system synthesizes two pheromone related polypeptides, a cytosolically located primary translation product (pp-α-Fcyt, 21 kDa) and a membrane-specific and multiply glycosylated e-factor precursor (pp-α-F3, 27.5 kDa). Glycosylation of the membrane specific pp-α-F3 species is competitively inhibited by synthetic peptides containing the consensus sequence Asn-Xaa-Thr as indicated by a shift of its molecular mass from 27.5 kDa to about 19.5 kDa (pp-α-F0), whereas the primary translation product pp-α-F cyt is not affected. Likewise, only the glycosylated pp-α-F3 structure is digested by Endo H yielding a polypeptide with a molecular mass between PP-α-F0 and pp-α-F cyt. These observations strongly suggest that the primary translation product is proteolytically processed during/on its translocation into the lumen of the microsomal vesicles. We believe that this proteolytic processing is due to the cleavage of a signal sequence from the pp-α-F cyt species, although this interpretation contradicts previous data from other groups. The distinct effect exerted by various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-dNM, 1-deoxymannojirimycin) on the electrophoretic mobility of the pp-α-F3 polypeptide indicates that its oligosaccharide chains are processed to presumbly Man9-GlcNAc2 structures under the in vitro conditions of translation. This oligosaccharide processing is most likely to involve the action of glucosidase I and glucosidase II as follows from the specificity of the glycosidase inhibitors applied and the differences of the molecular mass observed in their presence. In addition, several arguments suggest that both trimming enzymes are located in the lumen of the microsomal vesicles derived from endoplasmic reticulum membranes.
Semliki Forest virus induced cell-cell fusion at neutral extracellular pH
Authors: Kempf Christoph , Michel Marcel R. , Omar Adames , Jentsch Pia , Morell Andreas
Published: February 2005

Semliki Forest virus-induced cell-cell fusion from within was considered to exclusively occur at mildly acidic pH (<6.2). Data of this study show that such cell fusion can also be triggered by transient acidification of the cytoplasm of infected cells at an extracellular, neutral pH. Results were obtained by utilizing NH4Cl pulses combined with covalent modification of cell surface proteins. The observation implies a revision of the current consensus regarding the mechanism of Semliki Forest virus induced cell-cell fusion. We propose a model in which at least two peptide segments of the viral spike protein E1 may be involved in triggering the fusion event.
The pharmacodynamic action of the cyclic AMP phosphodiesterase inhibitor rolipram on prolactin producing rat pituitary adenoma (GH4C1) cells
Authors: Gordeladze Jan O.
Published: February 2005

Rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) represents a new class of specific low Km cAMP phosphodiesterase (PDE) inhibitors. This compound enhances basal, hormone- and forskolin-elicited cAMP accumulation in prolactin (PRL) producing rat pituitary adenoma (GH4C1) cells in culture (ED50=5·10−8 M). This effect is due to a selective inhibition of the low Km cAMP PDE (type III), since neither basal nor hormone-stimulated adenylate cyclase (AC) nor the Ca2+/calmodulin-dependent PDE were affected by rolipram. The drug enhanced vasoactive intestinal polypeptide (VIP)-stimulated PRL-secretion, while thyroliberin (TRH)- and 12-0-tetradecanoyl phorbol-13-acetate (TPA)-elicited PRL egress were slightly reduced indicating a cAMP-mediated reduction of protein kinase C (PK-C) mediated PRL release. Interestingly, inhibition of PRL secretion by somatostatin (SRIH) was completely suppressed suggesting cAMP-mediated inactivation of some GTP-binding protein(s) of the αi family (Gαi2 or Gk). Rolipram did not affect phosphoinositide metabolism (i.e. IP3 accumulation), neither acutely nor after long term administration. Rolipram, like the cAMP PDE inhibitor Ro 20–1724, did not influence AC and PDE I, but dose-dependently inhibited PDE III activity.

Long term incubation of GH4C1 cells with rolipram in the presence of noradrenaline (NA) exerted a marginal decrease of β-receptor number, AC activation and cAMP accumulation, while Ro 20–1724 brought about a marked down-regulation and desensitization of the AC complex.

In summary, rolipram selectively interacts with PDE III in rat pituitary adenoma cells in culture and does not result in β-adrenoceptor AC downregulation. These features are not shared by the other drugs tested.
Inhibition of protein kinase C by oxidation products of retinol
Authors: Epand Richard M. , McCallum Christine
Published: February 2005

Retinol has little effect on the activity of protein kinase C. However, air oxidation of retinol produces products which are inhibitory to this enzyme. These results are consistent with a suggestion that the activity of protein kinase C is modulated by the bulk biophysical properties of its environment. The facile susceptibility of retinol to oxidation, as demonstrated by HPLC analysis, explains some of the discrepant reports of its effects on the activity of protein kinase C.
Purification of thymocyte growth peptide (TGP) from sheep thymus. Relationship to FTS/thymulin
Authors: Ernström Ulf , Gafvelin Guro , Rudja Jane-Marie
Published: February 2005

Thymocyte growth peptide (TGP) initiates DNA synthesis in immature thymocytes and has previously been characterized as an acidic peptide isolated from calf thymus. We now report the isolation of TGP from sheep thymus and show it to be a nonapeptide with a large N-terminal blocking moiety characterized by high UV absorbance. The amino acid composition is identical to FTS, consisting of 2 Gly, 2 Ser, 2 Glx, 1 Ala, 1 Lys, 1 Asx. In contrast to FTS, TGP is acidic with an apparent isoelectric point of 4.2 and a high UV absorbance at 270–280 nm. Reverse phase chromatography of TGP at an acidic pH results in a change of the molecule and the appearance of two new compounds TGP-A and TGP-B, both with less than 50% of the original TGP activity. Full activity could be restored by the addition of ZnCl2 to TGP-A. Both TGP-A and B have some amino acid composition and high UV absorbance as native TGP. We propose that TGP consists of a non-peptide moiety bound to the N-terminal of the nonapeptide Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn and that the active molecule is stabilized by Zn2+.
Effects of membrane lipid and fluidity modifications on HIV-1 infectibility of primate lymphocytesin vitro
Authors: Zimmer J. Pascal , Lehr Hans A. , Hübner Christoph , Lindner Stephan G. , Ramsperger Ralf , Claussen Matthias , Kohlschütter Alfried , Schmitz Herbert
Published: February 2005

Although most non-human primates, except the chimpanzee and the gibbon in vivo are not infectible by HIV-1, lymphocytes of several of these species can be infected by HIV-1 in vitro.

In order to investigate whether the in vitro infectibility of primate lymphocytes might be attributed to plasma membrane adaptation processes or to serum factors, we compared HIV-1 infectibility of cultivated peripheral blood lymphocytes of macaques and of baboons on day one and on day ten of cultivation. These data were correlated to plasma membrane lipid composition and membrane fluidity.

We found a correlation between increased HIV-1 in vitro infectibility and changes in plasma membrane lipid composition resulting in decreased membrane fluidity of cultured primate lymphocytes.
Induction of membrane proliferation in mouse CNS by gold sodium thiomalate with reference to increased virulence of the avirulent Semliki Forest virus
Authors: Mehta S. , Pathak S. , Webb H. E.
Published: February 2005

Separation of smooth membrane vesicles from whole mouse brain by isopycnic centrifugation in discontinuous sucrose density gradients show an increased membrane proliferation in gold sodium thiomalate (GSTM) treated mice. Induction of membrane proliferation by GSTM seems to be an important factor in converting the avirulent Semliki Forest virus infection into a lethal one.
Comparative action of calpain on erythrocyte Ca2+-pumping ATPase in sickle cell anaemia, essential hypertension and kwashiorkor
Authors: Olorunsogo O. O. , Agbolade F. O. , Owojuyigbe S. O. , Adebisi J. A. , Adebayo A. O. , Okunade W. G.
Published: February 2005

Calpain, a calcium-dependent, neutral cysteine-protease was purified from the erythrocyte cytosol of subjects having essential hypertension (HTN), sickle cell anaemia, (SCA), or kwashiorkor (KWA). Identical electrophoretic mobility on SDS-polyacrylamide gradient gel, sensitivity to micromolar amounts of Ca2+, absolute requirement for a reducing environment and a high susceptibility to inhibition by leupeptin and thiol-group modifying reagents confirm that calpain preparations from these erythrocytes are equivalent to calpain I. Whereas the extent of calpain activation of erythrocyte membrane Ca2+-pumping ATPase of normal subjects was almost equal to that due to calmodulin, calpain activation of the HTN and SCA pump was greater than activation by calmodulin. Like in normal membranes, exogenous calmodulin protected the Ca2+-pumping ATPase of these erythrocytes against calpainization; the degree of protection by calmodulin is least in SCA and HTN. Electrophoretic separation of erythrocyte membranes and the purified Ca2+-pumping ATPase of HTN, SCA and KWA subjects does not indicate the presence of fragments resulting from the proteolytic action of calpain.
Acute effects of thyroid hormone on sodium currents in neonatal myocytes
Authors: Craelius W. , Green W. L. , Harris D. R.
Published: February 2005

Sodium currents and action potentials were recorded from myocytes of neonatal rats during acute exposure to thyroid hormone (5–20 nM). One to 5 minutes after addition of thyroid hormone to the bath, decay from peak Na current was slowed, with the fractional current flowing 20 ms after onset (relative to peak current) increasing from 6±5% to 17±13% (p<0.01, n=12). Action potential durations were increased from 55±14 to 86±36 msec (p<0.05, n=6). The effects of thyroid hormone were partially reversed by lidocaine (60 μM, n=5), a specific blocker of a slow sub-population of Na channels. Thus thyroid hormone interacts directly with myocyte membrane, probably by slowing of inactivation of Na channels.
Polyhydroxybutyrate: An intriguing biopolymer
Authors: Dawes Edwin A.
Published: February 2005

The microbial polymer poly-3-hydroxybutyrate (PHB) and related poly-hydroxyalkanoates, such as poly-3-hydroxyvalerate and poly-3-hydroxyoctanoate, are unique biodegradable thermoplastics of considerable commercial importance. The structure, properties and regulation of synthesis and degradation of PHB are reviewed and the microbial production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate, with properties varying according to copolymer composition, is discussed.
A simple method for identification of gap-bridging subclones in DNA sequencing
Authors: Hong G. F.
Published: February 2005

A simple method for the identification of gap-bridging subclones in DNA sequencing is described.
Development and differentiation in plants
Authors: Johri M. M.
Published: February 2005

An overview of plant development has been presented. In lower plants like mosses, auxin and cytokinin regulate the creation and the stability of the differentiated state of various cell types. The differentiated state is plastic and all cell types dedifferentiate to ground state, the chloronema. Even in higher plants, embryonic cells become only roughly committed during shoot meristem formation. Their terminal destiny becomes specified during the post-germination phase when the rough outline gets refined. The lack of a firm determined state, clonally heritable through mitosis, indicates that the development in plants is unlikely to be specified by a rigid programme.
A novel role of Ca2+ and Zn2+: Protection of cells against membrane damage
Authors: Pasternak C. A.
Published: February 2005

Certain cytotoxic agents damage cells by the induction of pores across their plasma membrane. Ca2+ and Zn2+ protect against such damage by promoting pore closure. Zn2+ may play a beneficial role in this regard in certain disease states.
Seminalplasmin: A protein with many biological properties
Authors: Shivaji Sisinthy
Published: February 2005

Proteins present in the seminal plasma of mammals are known to influence functions associated with ejaculated spermatozoa such as motility, capacitation, acrosome reaction and fertilising ability. The proteins isolated and characterised so far influence only one of the above functions of spermatozoa. Seminalplasmin, a protein isolated from the seminal plasma of bull is exceptional in that it influences many of the above spermatozoal functions. It is also a potent antimicrobial protein and capable of lysing microbial and mammalian cells. The physiological function of seminalplasmin as nature's own antifertility agent is discussed.
Translational control in E. coli: The case of threonyl-tRNA synthetase
Authors: Springer Mathias , Graffe Monique , Dondon Jacques , Grunberg-Manago Marianne , Romby Pascale , Ehresmann Bernard , Ehresmann Chantal , Ebel Jean-Pierre
Published: February 2005

Genetic studies have shown that expression of the E. coli threonyl-tRNA synthetase (thrS) gene is negatively auto-regulated at the translational level. A region called the operator, located 110 nucleotides downstream of the 5′ end of the mRNA and between 10 and 50bp upstream of the translational initiation codon in the thrS gene, is directly involved in that control. The conformation of an in vitro RNA fragment extending over the thrS regulatory region has been investigated with chemical and enzymatic probes. The operator locus displays structural similarities to the anti-codon arm of threonyl tRNA. The conformation of 3 constitutent mutants containing single base changes in the operator region shows that replacement of a base in the anti-codon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in regulation. However mutation in or close to the anti-codon-like stem results in a partial or complete rearrangement of the structure of the operator region. Further experiments indicate that there is a clear correlation between the way the synthetase recognises each operator, causing translational repression, and threonyl-tRNA.
A possible adenine nucleotide storage form in normal and ischaemic rat heart
Authors: Fitt Peter S. , Korecky Borivoj , Sharma Nishi
Published: February 2005

Perfusion of Langendorff rat hearts with [14C]adenosine yields an acid-insoluble, radioactive product whose concentration falls during ischaemia. The properties of the substance show that it is a polyribonucleotide. It is suggested that it may be mitochondrial poly A acting as a storage form of adenine nucleotides.
Oxytocin contracts the human uterus at term by inhibiting the myometrial Ca2+-extrusion pump
Authors: Popescu L. M. , Nutu O. , Panoiu Cristina
Published: February 2005

The sarcolemma of smooth-muscle ceils from human pregnant myometrium possesses a high-affinity Ca2+-ATPase, which has the characteristics of an active Ca2+-extrusion pump. This pump enzyme, either membrane-bound or solubilized, was strongly inhibited by oxytocin (half-maximal inhibition at about 4 μU/ml or 10 pM). However, under similar conditions, oxytocin did not inhibit Ca2+extrusion ATPase of the erythrocyte membrane. The inhibitory concentrations of oxytocin correspond to hormone plasma levels which initiate spontaneous labor.
Stable maintenance in chemostat-grown Escherichia coli of pBR322 and pACYC184 by disruption of the tetracycline resistance gene
Authors: Vernet Thierry , McDonald Ian J. , Cameron Dave R. , Visentin Louis P.
Published: February 2005

Plasmid stability was studied in antibiotic-free chemo-stat cultures. Disruption, either by deletion or insertion, of the tetracycline resistance gene in the EcoRl/EcoRV region of the cloning vector pBR322 or in the HindIII]BamHl region of pACYCI84 yields plasmids markedly more stable than the parent plasmids. Thus, at least for these two instances, cloning of a partitioning (par) locus is not prerequisite for plasmid maintenance.
DNAse I – actin complex: An immunological study
Authors: Boyer M. , Roustan C. , Benyamin Y.
Published: February 2005

DNAse I – actin complex formation is studied in the presence of different anti actin antibody populations. The binding of DNAse I to actin is shown to be affected by antibodies specific to a central region in actin sequence (168–226). The C- and N-extremities of actin are shown to be in spatial proximity at the surface of the actin monomer and far from the binding area of DNAse I.
Control of tissue carnitine contents: Effects of partial hepatectomy and liver regeneration on carnitine concentrations in liver and extrahepatic tissues of the rat
Authors: French Timothy J. , Goode Anthony W. , Schofield Paul S. , Sugden Mary C.
Published: February 2005

The liver is the sole site of carnitine biosynthesis in the rat. However, the first 24 h after the surgical removal of two-thirds of the liver mass are not associated with depletion of carnitine either in the liver remnant or in a number of extrahepatic tissues with relatively short turnover times of carnitine (<24 h; heart, spleen, kidney). Dietary carnitine was not supplied. The results suggest that the capacity of the remnant liver for carnitine biosynthesis is sufficient to maintain tissue carnitine contents. Liver regeneration influenced the relative proportions of hepatic free and acylated carnitines in a manner compatible with changes in fat disposition in the proliferating tissue.
Resonance electroconformational coupling: A proposed mechanism for energy and signal transductions by membrane proteins
Authors: Tsong Tian Yow , Liu Dao-Sheng , Chauvin Francoise , Dean Astumian R.
Published: February 2005

Recent experiments show that membrane ATPases are capable of absorbing free energy from an applied oscillating electric field and converting it to chemical bond energy of ATP or chemical potential energy of concentration gradients. Presumably these enzymes would also respond to endogenous transmembrane electric fields of similar intensity and waveform. A mechanism is proposed in which energy coupling is achieved via Coulombic interaction of an electric field and the conformational equilibria of an ATPase. Analysis indicates that only an oscillating or fluctuating electric field can be used by an enzyme to drive a chemical reaction away from equilibrium. In vivo, the stationary transmembrane potential of a cell must be modulated to become “locally” oscillatory if it is to derive energy and signal transduction processes.
Restriction fragment analysis of the HLA Class II region: Application to tissue transplantation
Authors: Bushell Andrew , Wood Kathryn J.
Published: February 2005

Southern blotting and hybridization with locus specific probes is a reliable method for the determination of HLA Class II specificities and in conjunction with several other techniques is providing valuable information on the nature of Class II polymorphisms in man. In terms of the identification of Class II specificities in transplantation however, the advanced nature of DR serology means that DRβ restriction fragment analysis is probably limited only to those situations where serological methods have been unsuccessful.

Whether DQ and DP incompatibilities can evoke rejection responses in vivo remains to be fully ascertained although our preliminary data suggest that DQ mismatches probably have little effect upon graft outcome. Restriction fragment analysis is particularly attractive in that in enables reliable retrospective studies to be performed in conjunction with prolonged clinical follow up, and the technique will clearly continue to contribute significantly to our understanding of the importance of these molecules in transplantation.
ATP inhibits onset of exocytosis in permeabilised mast cells
Authors: Tatham P. E. R. , Gomperts B. D.
Published: February 2005

ATP is not required for exocytosis from permeabilised mast cells, and therefore there is no direct role for protein phosphorylation in the late stages of the activation pathway. We have measured the timecourse of exocytosis from permeabilised cells triggered to release hexosaminidase following addition of Ca2+ to cells equilibrated for 2 min with GTP-γ-S. If ATP is included at the time of permeabilisation, then exocytosis commences after a delay, the duration of which depends on the square root of the product [Ca2+][GTP-γ-S], and which may extend to beyond 3 min. When ATP is excluded then the maximal rate of exocytosis is established within 3 secs of completing the effector combination. These results suggest that the achievement of a new steady-state, induced by Ca2+ and GTP-γ-S, and required for exocytosis is inhibited by ATP. From this we conclude that dephosphorylation of an unknown regulator protein may comprise a step in the exocytotic pathway.
Lateral diffusion of PDGF β-receptors in human fibroblasts
Authors: Ljungquist-Höddelius Pia , Lirvall Margareta , Wasteson Åke , Magnusson Karl-Eric
Published: February 2005

When platelet-derived growth factor (PDGF) binds to its receptors a number of biochemical reactions are elicited in the cell. Several models have been presented for the effects of ligand-induced receptor conformation and aggregation on signal transduction but little is known about the direct effects on receptor diffusion. This study concerns the lateral mobility of PDGF receptors in fibroblasts. It was assessed with fluorescence recovery after photobleaching (FRAP), using rhodaminated receptor antibodies or Fab-fragments of the antibody as ligands. The aims of the investigation were: (a) to compare the lateral mobility of membrane receptors of human fibroblasts labelled with either antibodies against the PDGF receptor or Fab-fragments of the same antibodies, and (b) to study the effects of serum or PDGF on the mobility of the receptors. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard or under serum-free conditions yielding “normal” and “starved” cells, respectively. Two parameters of the diffusion were evaluated; the diffusion coefficient (D) and the mobile fraction (R) of the receptors. We found that normal fibroblasts had a smaller diffusion coefficient and a lower mobile fraction compared to starved cells using antibodies for receptor labelling. The addition of PDGF, just before the measurement, increased the D and R for normal cells, while starved cells, showing higher initial values, displayed slightly reduced values of D and R. After the addition of serum, D increased and R remained low for normal cells, whereas for starved cells both D and R increased to upper limits of 11.0×10−10 cm2s−1 and >90% respectively. In general, the D and R values, both in normal and starved cells, were higher for cells labelled with Fab-fragments than for antibody-labelled cells. The results are discussed in relation to the natural complexity of the receptor, and how PDGF, serum, antibodies and Fab-fragments might interfere with receptor structure, aggregation state and membrane diffusion characteristics.
Evidence for the regulation of the activity of protein kinase C through changes in membrane properties
Authors: Epand Richard M. , Epand Raquel F. , Leon Bryan T.-C. , Menger Fredric M. , Kuo J. F.
Published: February 2005

We measured the effects of two branched-chain analogs of distearoyl-phosphatidylcholine, containing either a methyl or an n-butyl group at the 8 position, on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. The former compound raised the bilayer to hexagonal phase transition temperature while the latter compound lowered it. The opposite effects of these amphiphiles on protein kinase C activity (inhibition and activation, respectively) correlated with their effects on lipid polymorphism. Because of the similarity of the structures of these two compounds, it seems likely that their opposite effects on the activity of protein kinase C is a result of their alteration of the lipid environment of the membrane rather than to binding to a specific site on the protein.

We also compared the effects of hexachlorophene on lipid polymorphism and protein kinase C activity at high and at low calcium concentrations. We also found that the effect of hexachlorophene forming a complex with Ca2+ is to increase both the hexagonal phase forming propensity of the membrane as well as to increase the activity of protein kinase C, again demonstrating the correlation between lipid phase propensity and effects on protein kinase C activity.
Starvation is associated with changes in the elemental composition of the pancreatic β-cell
Authors: Juntti-Berggren L. , Lindh U. , Berggren P.-O.
Published: February 2005

By using the proton microprobe technique we have investigated the elemental composition of both pancreatic β-cells and exocrine pancreas from fed and 24 h or 48 h starved obese hyperglycemic mice. Among the 15 elements measured in the β-cells both Ca and Fe increased while Mg and S decreased significantly after 24 h of starvation, the effects being more pronounced after 48 h. When animals were starved for 48 h there was a decrease in the contents of Cl, Rb and Cu, whereas that of Al and Mn increased with 152 and 55%, respectively. There was an initial decrease in Na after 24 h of starvation, which was followed by an increase after 48 h. This is in contrast to Cd, which first increased and then decreased to a value lower than that obtained in the fed animal. The content of K showed a small decrease and that of Pb showed an increase only in the 24 h starved group. In the β-cells the contents of Zn and P did not change subsequent to starvation. In the exocrine pancreas Na, Cl and P decreased after 24 h of starvation and except for Na, the decrease was maintained when the starvation period was increased to 48 h. After 24 h there was a significant, though transient, increase in K, Mg and Rb. With regard to the contents of Zn, Cu and S there was a progressive decrease as the starvation continued. In contrast to the endocrine pancreas the content of Al in the exocrine pancreas did not change after 48 h of starvation. There was no change in islet insulin content subsequent to starvation. The extent to which the observed changes in β-cell elemental composition is involved in the impaired insulin release associated with starvation, merits further investigations.
Lipid fluidity and membrane protein dynamics
Authors: Lenaz Giorgio
Published: February 2005

Membrane fluidity plays an important role in cellular functions. Membrane proteins are mobile in the lipid fluid environment; lateral diffusion of membrane proteins is slower than expected by theory, due to both the effect of protein crowding in the membrane and to constraints from the aqueous matrix. A major aspect of diffusion is in macromolecular associations: reduction of dimensionality for membrane diffusion facilitates collisional encounters, as those concerned with receptor-mediated signal transduction and with electron transfer chains. In mitochondrial electron transfer, diffusional control is prevented by the excess of collisional encounters between fast-diffusing ubiquinone and the respiratory complexes. Another aspect of dynamics of membrane proteins is their conformational flexibility. Lipids may induce the optimal conformation for catalytic activity. Breaks in Arrhenius plots of membrane-bound enzymes may be related to lipid fluidity: the break could occur when a limiting viscosity is reached for catalytic activity. Viscosity can affect protein conformational changes by inhibiting thermal fluctuations to the inner core of the protein molecule.
Cytosolic epoxide hydrolase from liver of control and clofibrate-treated mice. Structural comparison by HPLC peptide mapping
Authors: Meijer Johan , DePierre Joseph W. , Jörnvall Hans
Published: February 2005

Cytosolic epoxide hydrolases purified from livers of control and clofibrate-induced male C57B1/6 mice were compared. The proteins were reduced, alkylated and cleaved with trypsin and chymotrypsin. The digests were analyzed by HPLC and no qualitative differences were observed in the peptide mapping profiles of the two types of epoxide hydrolase preparation. The amino acid compositions and N-terminal residues of selected tryptic peptides also gave identical results for the control and clofibrate-induced mice. Both intact proteins have e-amino-blocked N-termini. The two enzyme forms are concluded to have highly similar, if not identical, primary structures.
Synthesis of (+) and (−) RNA molecules of potato spindle tuber viroid (PSTV) in isolated nuclei and its impairment by transcription inhibitors
Authors: Spiesmacher Ellen , Mühlbach Hans-Peter , Tabler Martin , Sänger Heinz L.
Published: February 2005

Transcription studies with highly purified potato cell nuclei in combination with a ‘transcription-hybridization analysis’ unequivocally demonstrate that the nucleus is the subcellular site where the entire process of PSTV replication takes place. Inhibition experiments with actinomycin D and α-amanitin furthermore suggest that the nuclear DNA-dependent RNA polymerases I and II are involved in the synthesis of PSTV (+) and (−) RNA, respectively.
Alanine and inter-organ relationships in branched-chain amino and 2-oxo acid metabolism
Authors: Palmer T. Norman , Caldecourt Margaret A. , Snell Keith , Sugden Mary C.
Published: February 2005

Branched-chain amino acid metabolism in skeletal muscte promotes the production of alanine, an important precursor in hepatic gluconeogenesis. There is controversy concerning the origin of the carbon skeleton of alanine produced in muscle, specifically whether it is derived from carbohydrate via glycolysis (the glucose-alanine cycle) or from amino acid precursors (viz. glutamate, valine, isoleucine, methionine, aspartate, asparagine) via a pathway involving phosphoenolpyruvate (PEP) carboxykinase and pyruvate kinase, or NADP-malate dehydrogenase (malic enzyme). The relevant literature is reviewed and it is concluded that neogenic flux from amino acids is unlikely to be of major quantitative importance for provision of the carbon skeleton of alanine either in vitro or in vivo. Evidence is presented that branched-chain amino acid oxidation in muscle is incomplete and that the branched-chain 2-oxo acids and the products of their partial oxidation (including glutamine) are released. The role of these metabolites is discussed in the context of fuel homeostasis in starvation.
Some properties of 2-5A binding/nucleolytic activities in gel filtered rabbit reticulocyte lysates
Authors: Wu Joseph M. , Wertheimer Stanley J. , Eslami Behruz , Figuereido Joanne C. , Goswami Biswendu B.
Published: February 2005

Rabbit reticulocyte lysates, gel filtered on Sephadex G-25 with or without ATP (or its analogs), were preincubated at 37°C and their subsequent binding to p3A4,3′-[32P]pCp was studied. Lysates filtered without ATP or in the presence of 0.1 mM 8-bromo-ATP, 1,N6-etheno-ATP, or ITP showed a time-dependent decrease in binding activity. This decrease was completely prevented when lysates were filtered with 0.1 mM ATP, 2′-deoxy-ATP, β-γ-methylene-ATP, or ATP-γ-S. The stability of binding provided by ATP or 2′-deoxy-ATP analogs corresponds to a more active 2–5A dependent endonucleolytic (RNAase L) activity based on studies using [3H] viral mRNA. Chromatography on heparin-agarose showed that ATP-supplemented gel-filtered reticulocyte lysates had a different p3A4,3′-[32P]pCp binding activity elution-profile than lysates gel-filtered in the absence of ATP. Covalent cross-linking of periodate-oxidized p3A4,3′-[32P]pC to gelfiltered lysates, preincubated at 0°C or 37°C for 30 min, showed the following results: (1) all lysates gave a major cross-linking of the radioactive ligand to an 80 000 dalton polypeptide, regardless of the temperature of preincubation, (2) Iysates gel-filtered without ATP, with 0.1 mM ITP, or β-γ-methylene-ATP, showed a significant reduction in the cross-linking of the 80 000 dalton protein, after preincubation at 37°C for 30 min. This decrease was accompanied by an increase in the labeling of two smaller polypeptides.
Post-meiotic transcription of phosphoglycerate-kinase 2 in mouse testes
Authors: Erickson Robert P. , Michelson Alan M. , Rosenberg Michael P. , Sánchez Edwin , Orkin Stuart H.
Published: February 2005

We have used a human phosphoglycerate kinase-1 (PGK-1) cDNA clone to study expression of PGK-2 during mouse spermatogenesis. Hybrid selection, in vitro translation with product identification by 2-D gel electrophoresis demon-strated that the PGK-1 cDNA clone hybridized to PGK-2 mRNA in mouse testes. Northern analyses of RNA purified from separated spermatogenic cells demonstrated a large increase in abundance of PGK-2 mRNA in post-meiotic cells. Thus, post-meiotic transcription of PGK-2 mRNA is demonstrable with cloned DNA probes.