bluuebunny/crossref_metadata_2025 huggingface dataset:
Washout phenomena in dialyzed mast cells allow discrimination of different steps in stimulus-secretion coupling
Authors: Penner Reinhold , Pusch Michael , Neher Erwin
Published: February 2005

Transient increases of intracellular calcium and exocytotic activity of rat peritoneal mast cells following stimulation with compound 48/80 were monitored using the Ca-indicator dye fura-2 and the capacitance measurement technique. It is known that mast cells very rapidly lose their secretory response towards antigenic or compound 48/80-induced stimulation in the whole-cell recording configuration of the patch-clamp technique due to “washout” of signal mediators. In contrast, we found that calcium transients remained unaffected by intracellular dialysis for as long as 10 min.

The fast “washout” phenomenon of exocytosis could be overcome by supplementing the pipette filling solution with guanosinetriphosphate (GTP) indicating a major role for GTP-binding proteins in secretion. The restoration of exocytosis was transient and decayed within three minutes, suggesting diffusional escape of one or several other cytoplasmic substances involved in stimulus-secretion coupling. Quantitative aspects of this process and the implications of its differential effects on Ca-transients versus secretion are discussed.
Intracellular Ca2+ in pancreatic acinar cells: Regulation and role in stimulation of enzyme secretion
Authors: Dormer Robert L. , Brown Graham R. , Doughney Claire , McPherson Margaret A.
Published: February 2005

Evidence for a primary role for intracellular Ca2+ in the stimulation of pancreatic enzyme secretion is reviewed. Measurements of cytoplasmic free Ca2+ concentration have allowed direct demonstration of its importance in triggering enzyme secretion and defined the concentration range over which membrane Ca2+ pumps must work to regulate intracellular Ca2+. Current evidence suggests a key role for the Ca2+ Mg-ATPase of rough endoplasmic reticulum in regulating intracellular Ca2+ and accumulating a Ca2+ store which is released by the action of inositol-l,4,5 trisphosphate following stimulation of secretion.
ph-Dependent hydrophobicity profile of hemagglutinin of influenza virus and its possible relevance in virus fusion
Authors: Korte Thomas , Ludwig Kai , Herrmann Andreas
Published: February 2005

The hydropathy profile of hemagglutinin (HA) subunits HA1 and HA2 of influenza virus X31 and A/PR 8/34 is analyzed at different pH. At neutral pH (7.4) pronounced hydrophobic sequences of HA correspond to the N-terminus and the transmembrane spanning sequence of HA2. At pH 5.0 where influenza virus is known to fuse with biological membranes several hydrophobic sequences in the ectodomain exist which are comparable in both the hydrophobicity and length of the N-terminus of HA2. It is suggested that these hydrophobic stretches are important for the fusion complex, in addition to the N-terminal site of HA2.
Virally induced alterations in cellular permeability: A basis of cellular and physiological damage?
Authors: Pasternak C. A. , Micklem K. J.
Published: February 2005

Virally induced permeability changes occur when haemolytic paramyxoviruses are added to cells; similar (though not identical) changes take place during infection of ceils with viruses from several families (including paramyxoviruses). These changes occur in intact) viable cells) and precede subsequent cytopathic effects) to which they are likely to contribute. There is accumulating evidence to suggest that virally induced permeability changes may also underlie the physiological and clinical consequences of viral infection in certain situations.
Metabolic studies of whole animals and humans using phosphorus nuclear magnetic resonance
Authors: Gadian David G.
Published: February 2005

Conclusions

31P NMR now provides a totally non-invasive method of studying the metabolism of selected tissues and organs within whole animals and humans. A wide range of studies therefore becomes feasible. For example, there have been problems associated with metabolic studies of isolated organs such as the brain which are particularly sensitive to hypoxia; these problems can now be circumvented by studying the organ within the whole animal. Theories of metabolic control are based largely on studies performed in vitro, and it should now prove possible to test some of these theories directly in vivo. NMR could provide significant advances in our understanding of organ preservation and function. Finally,31P NMR provides a new method of studying the metabolic basis and effects of disease in humans and in animal models. The technique could also provide an objective way of assessing the effectiveness of procedures that are used in the treatment of human disease.
Intramolecular general acid catalysis in the binding reactions of α2 and complement components C3 and C4
Authors: Davies Stephen G. , Sim Robert B.
Published: February 2005

The complement system proteins C3 and C4 and the plasma protease inhibitor α2-macroglobulin~ when activated by limited proteolysis, can bind covalently to other macromolecules. The three proteins also exhibit an unusual internal peptide-bond cleavage reaction when denatured. The covalent binding reaction is likely to occur by a transacylation mechanism involving an internal thioiester in the three proteins. However, the activated species of these proteins are much more reactive than simple thiolesters. Studies of molecular models of the thiolester region in C3 show that an intramolecular acid catalysis mechanism can both account for the exceptional reactivity of the activated form of these proteins and provide an explanation for the denaturation-induced peptide bond cleavage.
Recognition of base sequences by regulatory proteins in procaryotes and eucaryotes
Authors: Hélène Claude
Published: February 2005

A model is described whereby (i) regulatory proteins recognize one face of the DNA double helix on non-adjacent DNA regions brought close together in space through folding around nucleosomes, (if) regulatory sequences may occur inside gene structures, and (iii) the recognition of regulatory sequences might be modulated by short (4–6 base-pairs) insertions or deletions in introns or upstream from the transcription start site. This model might apply not only to eucaryotes but also to procaryotic organisms whose DNA is organized through interactions with histone-like proteins. Consequences of the model regarding the binding of regulatory proteins in procaryotes are suggested.
6-Dimethyl amino purine and 2-amino purine inhibit the induction of expresion of milk protein genes by prolactin
Authors: Bayat-Sarmadi Mahasti , Maliénou-Ngassa Rachel , Puissant Claudine , Houdebine Louis-Marie
Published: February 2005

Two protein kinase-inhibitors, 6-dimethyl amino purine and 2-amino purine inhibited induction of β-casein synthesis by prolactin when added to the culture medium of rabbit mammary explant and cells. The accumulation of the mRNA for αs1- and β-caseins and for whey acidic protein did not take place in the presence of the inhibitors whereas β-actin mRNA concentration was not altered. In the same experimental conditions, H7, an inhibitor of protein kinase C and, to a lower extent, of protein kinase A did not prevent prolactin from acting. These data suggest for the first time that specific protein kinases are involved in the transduction of the prolactin signal to milk protein genes.
Decreased protein kinase C activity is associated with programmed cell death (apoptosis) in freshly isolated rat hepatocytes
Authors: Sanchez Victor , Lucas Miguel , Sanz Aureo , Goberna Raimundo
Published: February 2005

Apoptosis of freshly isolated rat hepatocytes was induced by either the omission of fetal bovine serum in the culture medium or addition of the protein kinase C inhibitors polymyxin B or staurosporin. The time-course of DNA breakdown into oligonucleosome-sized fragments and the activity of protein kinase C was determined. Hepatocytes were found to be sensitive to bleomycin which induced a high degree of DNA breakdown even within 30 min incubation. Both staurosporin and polymyxin B induced DNA degradation in hepatocytes after three hours incubation, an effect that was partially prevented by phorbol myristate acetate (PMA). After eight hours incubation, PMA failed to counteract this action and itself produced the apoptosis of rat hepatocytes. The results suggest the involvement of protein kinase C in hepatocyte survival.
Seasonal commitment of HMGCoA reductase activity to vitellogenin production
Authors: Manzi Pamela , Bruscalupi Giovannella , Castellano Flavia , Trentalance Anna
Published: February 2005

In female frogs (Rana Esculenta) during gametogenesis the cholesterol synthesized in the liver by 3-hydroxy-3-methylglutaryl coenzyme A reductase is mostly exported into the blood and taken up by the oocytes.

In order to understand the fate of the neosynthesized cholesterol, female and male frogs and estrogenized male controls were injected with the labelled precursor14C mevalonate.

In females and in estrogenized controls, mevalonate-derived radioactivity is found in a plasmatic lipoprotein that has been identified as vitellogenin by immunological detection.

The increased 3-hydroxy-3-methylglutaryl coenzyme A reductase activity present in females in Fall is likely to be committed to provide cholesterol for the lipidation of this cholesterol-rich protein.
Changes in membrane permeability during semliki forest virus induced cell fusion
Authors: Lanzrein Markus , Käsermann Nicole , Kempf Christoph
Published: February 2005

The infection of Aedes albopictus cells by Semliki Forest virus (SFV) is a non lytic event. Exposure of infected cells to mildly acidic pH (<6.2) leads to syncytium formation. This polykaryon formation is accompanied by an influex of protons into the cells (Kempf et al. Biosci. Rep. 7, 761–769, 1987). We have further investigated this permeability change using various fluorescent or radiolabeled compounds. A significant, pH dependent increase of the membrane permeability to low molecular weight compounds (Mr<1000) was observed when infected cells were exposed to a pH<6.2. The pH dependence of the peremability change was very similar to the pH dependence of cell-cell fusion. The permeability change was sensitive to divalent cations, protons and anionic antiviral drugs such as trypan blue. The nature of this virus induced, pH dependent permeability change is discussed.
Transfection of a DNA/protein complex into nuclei of mammalian cells using polyoma capsids and electroporation
Authors: Bertling Wolf
Published: February 2005

We used empty capsids ofpolyoma virus to transfer DNA fragments and DNA/protein complexes into human cells. We encapsulated labeled and unlabeled single stranded DNA fragments by viral capsids. A complex of DNA with a DNA binding protein, recA, will also be taken up by the capsids, whereas the free protein is not incorporated. We further compared this gentle biological method of DNA transfection with a well-established physical method, electroporation. Electroporation also allows the transfer of DNA as well as protein into cells, although there is no proof that a DNA/protein complex can survive the procedure functionally. Whereas the viability of capsid transfected cells is unaffected (100%), electroporation reduces the viability to 90–95%. On the other hand, the amount of DNA found in the nucleus of electroporated cells is higher than for cells treated with loaded viral capsids.
Decreased brown adipose tissue thermogenic activity following a reduction in brain serotonin by intraventricular p-chlorophenylalanine
Authors: Fuller Nigel J. , Stirling Dorothy M. , Dunnett Stephen , Reynolds Gavin P. , Ashwell Margaret
Published: February 2005

The effects of reducing brain serotonin (5-HT) levels by means of intracerebral-ventricular injections of the tryptophan antagonist p-chlorophenylalanine (PCPA) were investigated in male rats. Six days after the operation, PCPA-treated rats, either fed ad libitum or pair-fed to the food intake of control rats, showed decreased thermogenic activity and capacity in their interscapular brown adipose tissue (BAT) and also increased fat storage in their white adipose tissue (WAT). These results indicate that serotonergic synapses might play a regulatory role in the sympathetic control of BAT thermogenesis and in the rate of WAT deposition (by an as yet unidentified mechanism), in addition to their well established role in controlling food intake.
Aldrin epoxidase activity in liver microsomes from normal or streptozotocin-diabetic rats: Comparison with activity in isolated hepatocytes from normal rats incubated with glucagon
Authors: Rouer Evelyne , Rouet Patricia , Leroux Jean Paul
Published: February 2005

Aldrin epoxidase activity in liver microsomes from streptozotocin-diabetic rats is only 40% of that from normal rats. Epoxidation of aldrin has also been assayed in freshly isolated hepatocytes from normal rats. Addition of 10−7 M glucagon to the incubation medium leads to a decreased aldrin epoxidase activity. Owing to the previously reported phosphorylation of a purified cytochrome P-450 isozyme, it is postulated that the cytochrome P-450 dependent aldrin epoxidase may be regulated by a glucagon induced phosphorylation process.
Up- and down-regulation of membrane receptors as possible mechanisms related to the antiulcer actions of milk in rat gastric mucosa
Authors: Gespach C. , Emami S. , Chastre E. , Launay J.-M. , Rosselin G.
Published: February 2005

The effects of a cow's milk diet on receptor activity and histamine metabolism in gastric glands and mucosa isolated from adult rats were examined. The milk diet was associated with (1) a decreased mobilization of H2 receptors by histamine and (2) an increased mobilization of PGE2 (prostaglandin E2) receptors in mucous cells (cytoprotective effect) and parietal cells (antiacid effect). These changes are not observed for the receptors reducing pentagastrin- and histamine-induced gastric acid secretion (pancreatic/enteroglucagons, somatostatin) and stimulating mucus, bicarbonate and pepsin secretions in the rat (secretin). Cimetidine produced a parallel displacement of the histamine dose-response curve, suggesting competitive inhibition between this classical H2 receptor antagonist and histamine in the two experimental groups. Prostaglandins and other components in milk such as EGF (epidermal growth factor) and somatostatin might therefore protect gastric mucosa by a differential control of PGE2 and histamine H2 receptor activity either directly (PGE2 in milk) or indirectly (inhibition of endogeneous histamine synthesis/release and stimulation of PGE-I synthesis/release).
The effect of the anabolic agent, clenbuterol, on overloaded rat skeletal muscle
Authors: Maltin C. A. , Delday M. I. , Hay S. M. , Smith F. G. , Lobley G. E. , Reeds P. J.
Published: February 2005

The dietary administration of clenbuterol to young male rats has been shown to produce a muscle specific hypertrophic growth response. This paper demonstrates that the combined effect of drug treatment and hypertrophic stimulus induced by tenotomy produced an additive effect on muscle growth. This effect was demonstrated in terms of both muscle composition (protein and RNA) and fibre size.
Investigations on myelinogenesis in vitro: I. The occurrence of endogenous protein kinase and its role in the phosphorylation of myelin basic proteins in “Myelin-like membranes” isolated from cerebral cell cultures
Authors: Shanker G. , Pieringer R. A.
Published: February 2005

The presence of a protein kinase capable of phosphorylating endogenous as well as exogenously added myelin basic proteins has been demonstrated in a myelin-like membrane fraction isolated from reaggregating and surface adhering, primary cultures of cells dissociated from embryonic mouse brain. Only the large and small components of myelin basic proteins were found to be phosphorylated when myelin-like membrane fraction was incubated with [γ-32P]ATP. The protein kinase endogenous to the myelin-like membrane fraction was mainly of the cyclic AMP independent type. There was very little cyclic AMP dependent or cyclic GMP dependent protein kinase activities in this myelin-like fraction. Although the myelin basic proteins were the only endogenous proteins phosphorylated, protein kinase of the myelin-like membrane was capable of catalyzing the phosphorylation of exogenous substrates, such as histones.
Binding of benzo[a]pyrene to different chromatin domains following activation at the nuclear membrane
Authors: Obi F. O. , Billett M. A.
Published: February 2005

When isolated liver nuclei from methylchol-anthrene-treated rats are incubated with benzopyrene, covalent adducts are formed between DNA and the ultimate carcinogen, benzopyrene diol epoxide. Brief digestion with DNaseI, or micrococcal nuclease has been used to demonstrate that benzopyrene metabolites bind more readily to DNA in chromatin regions with a more open, active conformation than to inactive chromatin.
Determination of the internal volume of reconstituted Sendai virus envelopes by quenching of calcein fluorescence
Authors: Bartzatt Ronald L. , Volsky David J.
Published: February 2005

Sendal virus envelopes (SVE) were isolated from Sendal virus particles by Triton X-100 solubilization and ultracentrifugation. The envelopes were reconstituted in the presence of the fluorescent dye calcein by gradual removal of the detergent with Bio-beads SM-2. The internal volume of reconstituted Sendal virus envelopes (RSVE) was determined by quenching the fluorescence of calcein with cobalt (II) ions. The internal volume of RSVE was found to be proportional to the initial SVE protein concentration in the recon-stitution mixture, reaching about 18% of the total volume with 5.6 mg of SVE protein per ml. When radiolabelled cloned Epstein-Barr virus DNA fragment was included in the reconstitution mixture, the proportion of DNA associated with the vesicles much exceeded the trapping volume, indicating adsorption of DNA to the internal surface of RSVE. These deter-minations will allow optimization of the use of RSVE as gene-transfer vehicles.
The relationship between secretion and intracellular free calcium in bovine adrenal chromaffin cells
Authors: Burgoyne Robert D.
Published: February 2005

The effect of carbamylcholine and the calcium ionophore A23187 on catecholamine release and intracellular free calcium, [Ca2+]i, in bovine adrenal chromaffin cells was determined. At 10−4M carbamylcholine maximal release occurred with an accompanying increase in [Ca2+]i from a basal level of 168 nM to less than 300 nM. An increase in [Ca2+]i of a similar magnitude was found following challenge with 40 nM A23187. However, in this case, no catecholamine release occurred. These results suggest that stimulation of secretion from chromaffin cells by carbamylcholine may involve additional triggers which stimulate secretion at low [Ca2+]i.
An active-site carboxyl group in liquefying α-amylase: specific chemical modification
Authors: Kochhar Sunil , Dua R. D.
Published: February 2005

Bacillus amyloliquefaciens α-amylase activity is pH-dependent and the plot log (Vmax/Km) versus pH implicated a carboxyl group of aspartic acid/glutamic acid at the active site. Chemical modification of α-amylase with EDC confirmed this view. Further, analysis of inactivation kinetics showed that modification of a single carboxyl group led to complete loss of the enzymic activity.
Initiation of DNA synthesis in the parotid salivary gland of adult mice by a factor isolated from acellular fluid of the Ehrlich ascites carcinoma
Authors: Burns E. Robert , Yeh Hsing-Wu , Yeh Yun-Chi
Published: February 2005

The rate of DNA synthesis in the parotid salivary gland of adult mice is very low. We have purified about 5 000-fold a mitogen from the acellular ascitic fluid of the Ehrlich ascites carcinoma which stimulates DNA synthesis in the parotid salivary gland in vivo. This stimulation of DNA synthesis was produced with a protein concentration of this mitogen of 3 μg per 25 g of body weight. The purification procedure included ammonium sulfate fractionation and DEAE Sephacel column chromatography. This potent, heat-labile mitogen is presumed to be a protein with a mol. wt, of 3.5 × 103 to 1.3 × 104. The data indicate that this new factor is quite different from epidermal growth factor and tumor growth factor. Hypophysectomy did not prevent the stimulatory effect of this mitogen on the parotid salivary gland, indicating that the pituitary gland is not involved directly in mediating the mitogenic effect.
13C nuclear magnetic resonance studies of anaerobic glycolysis in Trypanosoma brucei spp.
Authors: Mackenzie N. E. , Hall James Edwin , Flynn I. W. , Scott A. I.
Published: February 2005

Anaerobic glycolysis in Trypanosoma brucei spp. has been studied by 13C NMR at 50 and 75.5 MHz. The uptake of [U-13C]glucose by cell suspensions of T. b. brucei was monitored by time-course spectroscopy, and while no anomeric specificity was found, the end -products of glycolysis were confirmed as glycerol and pyruvate together with alanine and dihydroxypropionat e. The intermediacy of L-glycerol-3-phosphate was also ascertained. The incorporation of C-I of [1-13C]glucose and of C-6 of [6-13C]glucose into glycerol and pyruvate in T. b. gambiense was quantified by measurement of the longitudinal relaxation times of the species involved. An incorporation to the extent of 66% of each substrate into equimolar amounts of glycerol and pyruvate indicate that Keq for the triosephosphate-isomerase-mediated reaction approaches unity.
Effect of several nucleotides on the phosphorylating activities of the Rous-sarcoma-virus transforming protein pp60v-sic and its cellular homologue, pp60c-sic
Authors: Barnekow Angelika
Published: February 2005

In the presence of ADP and GDP the tyrosine-phosphorylating activities of the viral as well as the cellular pp60src show a similar concentration-dependent inhibition in vitro. Addition of diadenosine 5′,5′″-pIp4 tetraphosphate (Ap4A) to the kinase assay leads to an inhibition of the viral kinase activity, whereas the cellular kinase is not influenced.
Activation of a procollagenase by low-molecular-weight angiogenesis factor
Authors: Weiss Jacqueline B. , Hill C. R. , Davis R. J. , McLaughlin B. , Sedowofia K. A. , Brown R. A.
Published: February 2005

Avascular tumours have the ability to establish a blood supply for themselves by secreting a humoral factor which stimulates their host's endothelial cells to proliferate and to migrate towards the tumour source. The mechanism of action of such a humoral anglo-genesis factor is more than that of an endothelial-cell growth factor since it requires an oriented migration of cells towards the tumour. We report here the activation of pure skin-fibroblast procollagenase by a low-molecular-weight angiogenesis factor capable of stimulating endothelial-cell growth in vitro. The activation was observed when either Type I or III collagen was used as substrate. It is suggested that at least one function of angiogenesis factor is to promote limited degradation of the connective tissue through which it passes causing channeling in the matrix along which stimulated endothelial cells may
Early alterations in rat liver chromatin structure after a single dose of diethylnitrosamine
Authors: Letnansky Karl , Vardapetjan Hratschik R.
Published: February 2005

In the chromatin of 24-h regenerating rat livers, derivative melting profiles are characterized by a high proportion of transitions above 90°C. After the injection of diethylnitrosamine there is a rapid shift to lower melting temperatures. This is due to a rearrangement of the chromatin to higher amounts of nucleosomal components but possibly also a consequence of chemical modifications and conformational alterations of the DNA. In the nonregenerating liver essentially the same observations can be made, although reactions proceed significantly slower. These results are in good agreement with the observation that carcinogens are more active in tissues stimulated to rapid proliferation as compared to resting tissues.
The lysine residues implicated in the gene 5 protein association sites
Authors: Bayne S. , Rasched I.
Published: February 2005

Gene 5 protein, a DNA unwinding protein encoded by the bacteriophage fd, is self-associative in presence of DNA or oligonucleotides. The lysine residues implicated in the protein-protein binding domains have been identified after modification with acetimidate by means of peptide and amino acid analyses. These residues are Lys-7 and Lys-69.
Processing endopeptidase deficiency in neurohypohysial secretory granules of the diabetes insipidus (Brattleboro) rat
Authors: Chauyet J. , Rouille Y. , Spang A. , Cardine A. M. , Acher R.
Published: February 2005

The homozygote Brattleboro rat exhibits a hereditary diabetes insipidus due to a deficiency of vasopressin, the antidiuretic hormone. It has previously been shown that in this animal a single nucleotide deletion in the provasopressin gene leads to a mutant precursor with a C-terminal amino acid sequence different from that of the wild-type. However the N-terminal region including the hormone moiety, the processing signal as well as the first two-thirds of the neurophysin is entirely preserved and absence of maturation has to be explained by an additional cause. We show here that the neurohypophysis of the homozygote Brattleboro rat, in contrast to the adenohypophysis, displays a significant decrease in the Lys-Arg processing endopeptidase activity when compared to the heterozygote or the wild-type Wistar. It is suggested that hypothalamic vasopressinergic neurons of the homozygote Brattleboro rat display a deficiency in the processing enzyme in contrast to the oxytocinergic neurons in which processing of prooxytocin is normal.
The simultaneous exposition of galactose and mannose-specific receptors on rat liver macrophages is developmentally regulated
Authors: Lentini Alessandro , Falasca Laura , Autuori Francesco , Dini Luciana
Published: February 2005

We studied the simultaneous binding of galactose and mannose-exposing ligands in sinusoidal rat liver cells during development and aging. The galactose-specific receptors were visualized using 17 nm diameter colloidal gold particles coupled with Lactosylated bovine serum albumine (LacBSA), while mannose-specific receptors were localized by means of 5 nm diameter particles adsorbed with mannan. We observed the presence of four different classes of Kupffer cells in relation to the ligands bound. The percentage of each group of Kupffer cells varied in relation to the age of the subject from which the sample was taken. There were few double-labelled cells in the livers from newborn rats, with numbers increasing with age to adulthood, and decreasing again in the older animals. Cells without labelling were in the majority after birth, but they decreased in number up to adulthood and increased again during subsequent aging. The numbers of single-labelled cells did not change significantly during liver maturation. We hypothesize that the exposition of galactose and mannose-specific receptorial systems is regulated by developmental conditions.
G-Proteins mediate inhibition and activation of Ca2+-induced exocytosis from SLO-permeabilized peptidergic nerve endings
Authors: Ahnert-Hilger Gudrun , Dayanithi Govindan , Spicher Karsten , Nordmann Jean J.
Published: February 2005

In SLO-permeabilized isolated nerve endings from the rat neurohypophysis, GTP, guanosine 5′[y-thio]triphosphate (GTPyS) and guanosine 5′(ßy-imido]triphosphate (GMPPNP) inhibit the Ca2+-evoked vasopressin release. Pretreatment with pertussis toxin enhances the inhibitory effects of both GTP-analogues. Omission of Mg2+ overcomes the effect of GMPPNP and reverses the inhibitory effect of GTP and GTPyS. In the absence of Mg2+, GTP and GTPyS now potentiate Ca2+-evoked secretion.
Processing and characterization of the low density lipoprotein receptor in the human colonic carcinoma cell subclone HT29-18: A potential pathway for delivering therapeutic drugs and genes
Authors: Mazière J. C. , Mazière C. , Emami S. , Noel B. , Poumay Y. , Ronveaux M. F. , Chastre E. , Porte H. , Barbu V. , Biade S. , Santus R. , Gespach C.
Published: February 2005

Low density lipoprotein (LDL) processing has been investigated in the subcloned human colonic carcinoma cell line HT29-18. LDL binding at 4°C was a saturable process in relation to time and LDL concentration. The Kd for LDL binding was 11 μg/ml. ApoE-free HDL3 or acetylated LDL did not significantly compete with125I-LDL binding, up to 500 μg/ml.125I-LDL binding was decreased by 70% in HT29-18 cells preincubated for 24 hours in culture medium containing 100 μg/ml unlabelled LDL. Ligand blotting studies performed on HT29-18 homogenates using colloidal gold labelled LDL indicated the presence of one autoradiographic band corresponding to an apparent molecular weight of 130 kDa, which is consistent with the previously reported molecular weight of the LDL receptor in human fibroblasts. At 37°C,125I-LDL was actively internalized by HT29-18 cells and lysosomal degradation occurred as demonstrated by the inhibitory effect of chloroquine. LDL uptake and degradation by HT29-18 cells also resulted in a marked decrease in endogenous sterol synthesis. These data demonstrate that the HT29-18 human cancerous intestinal cells are able to specifically bind and internalize LDL, and that LDL processing results in down-regulation of sterol biosynthesis. Thus, intestinal epithelial cells possess specific LDL receptors that can be exploited to accomplish drug delivery and gene transfer via the receptor-mediated endocytosis pathway.
Exo-endocytosis in isolated peptidergic nerve terminals occurs in the sub-second range
Authors: Knoll Gerd , Plattner Helmut , Nordmann Jean J.
Published: February 2005

Exo- and endocytotic processes induced by depolarization of isolated neurosecretory nerve terminals show a close temporal correlation, which suggests a short time of integration of the neurosecretory granule membrane with the plasma membrane. In order to determine minimal time requirements for exocytosis-coupled endocytosis to occur, we have analyzed by electron microscopy uptake of horserdish peroxidase (HRP) as a fluid phase marker at the onset of depolarization. We have applied rapid mixing and sampling (quenched flow) to assess events in subsecond time peroids after stimulation. A significant number of labelled endocytotic vacuoles was observed during the first second of depolarization. This number then further increased by a factor of about 2 (within 5 s) and 4 (within 50s). Thus, as for exocytosis, the rate of endocytosis decreased considerably during prolonged stimulation. These data indicate i) that a substantial proportion of secretory granules undergoes exocytosis very shortly after stimulation, and ii) that, following exocytosis, the minimal time required for consecutive membrane retrieval is in the sub-second range.
Biosynthesis of the C-terminal amide in peptide hormones
Authors: Bradbury A. F. , Smyth D. G.
Published: February 2005

Recent developments in the study of peptide amidation are reviewed. The main areas covered are assay procedures, purification of amidating enzymes, co-fact0rs and regulation; mechanism and specificity of the amidating reaction, and multiple forms of the amidating enzyme and glycosylation. Discussion is presented on aspects that are poorly understood and new areas open to investigation are indicated.
Tissue distribution and age-dependent expression of β-4-N-acetylgalactosaminyltransferase in guinea-pig
Authors: Dall'Olio F. , Malagolini N. , Serafini-Cessi F.
Published: February 2005

Guinea-pig kidney contains β-4-N-acetylgalactosaminyltransferase which may be involved in the biosynthesis of the Sd a determinant expressed on Tamm-Horsfall glycoprotein. In the present study we show that this enzyme is expressed far more in the medulla than in the cortex of the kidney and that, among the other organs tested, is expressed only in colon and caecum. This transferase is ontogenically regulated, in that its activity is low at birth and increases as a function of age. From several aspects, the tissue distribution and the ontogenic expression of β-4-N-acetylgalactosaminyl-transferase and Tamm-Horsfall glycoprotein are similar.
The GC box as a silencer
Authors: Jankowski Jacek M. , Dixon Gordon H.
Published: February 2005

A DNA control sequenceTGGGGCGGAATGGC, or the “GC” box, has been described in the promoter regions upstream of a number of eukaryotic genes transcribed by polymerase II (for review, see Dynan, W. S. and Tjian, R., Nature316:774, 1985). The “GC” box can occur in single or multiple copies and is the binding site for a protein factor, Spl, which activates initiation of transcription. We have observed in the rainbow trout protamine gene 3′ to the TATA box, three “GC” boxes spaced at 80 bp intervals. The first is 5′ to the cap site and possesses the ability to “silence” transcription from the protamine promoter in constructs linking this promoter to the bacterial chloramphenicol acetyl transferase (CAT) coding sequence following transfection to COS-1 cells. A model is proposed to account for the silencing of the protamine gene in all tissues except developing sperm cells.
Expression in Escherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation
Authors: Höög Jan-Olov , Weis Marianne , Zeppezauer Michael , Jörnvall Hans , von Bahr-Lindstrom Hedvig
Published: February 2005

Human alcohol dehydrogenase (ADH, tiff isozyme of class I) was expressed in Escherichia coli, purified to homogeneity, and characterized regarding N-terminal processing. The expression system was obtained by ligation of a cDNA fragment corresponding to the fl-subunit of human liver alcohol dehydrogenase into the vector pKK 223-3 containing the tac promoter. The enzyme, detected by Western-blot analysis and ethanol oxidizing activity, constituted up to 3% of the total amount of protein. Recombinant ADH was separated from E. coli ADH by ion-exchange chromatography and the isolated enzyme was essentially pure as judged by SDS-polyacrylamide gel electrophoresis and sequence analysis. The N-terminal sequence was identical to that of the authentic fl-subunit except that the N-terminus was non-acetylated, indicating a correct removal of the initiator methionine, but lack of further processing.
Microinjection of cultured cells using red-cell-mediated fusion and osmotic lysis of pinosomes: A review of methods and applications
Authors: McElligott Mary Ann , Dice J. Fred
Published: February 2005

Proteins and other macromolecules can be injected into cultured cells by several different methods. Here we review the strengths and limitations of two of these methods, red-cell-mediated microinjection and osmotic: lysis of pinosomes, and indicate how they may be successfully applied to the study of cultured cells.
Pro-opiocortin: The mutiple adrenal hormone precursor
Authors: Lowry P. J.
Published: February 2005

Corticotropin (ACTH) is biosynthesized in the human pituitary gland as a long polypeptide precursor (pro-opiocortin) of some 240 residues. When ACTH is secreted in response to stress, the peptides derived from the rest or this precursor, pro-γ-melanotropin (γ-MSH) and β-1ipotropin (β-LPH), are also secreted (Fig. 1). This article will describe the search for a biological significance for this phenomenon.
Estimation of kinetic parameters of androgen-synthesizing enzyme activities in superfused Leydig cells from rat testes: Difference between endogenous and exogenous substrates
Authors: Kühn-Velten Nikolaus , Wolff Joachim , Staib Wolfgang
Published: February 2005

Kinetic parameters of 3β-hydroxysteroid dehydrogenase/isomerase, steroid-17α-monooxygenase, and steroid-17,20-lyase activities were estimated under steady-state conditions. Purified Leydig cells from rat testes were superfused with pregnenolone, progesterone, or 17α-hydroxyprogesterone. The Km values for both the monooxygenase- and the lyase-catalyzed reactions were by factors of five to ten higher if analyzed with the exogenously added substrate (0.98 and 0.65 μM, respectively) than if calculated from endogenous substrate derived from a precursor (0.10 and 0.13 μM, respectively). This discrepancy may be explained by different substrate partition between the intra- and extraceIJular spaces and by different substrate concentration at the active site of the respective enzyme, depending on whether the actual substrate is of exogenous or endogenous source.
Cyclic-AMP-dependent phosphorylation of glicentin
Authors: Conlon J. M. , Thim L. , Moody A. J. , Söling H. D.
Published: February 2005

Highly purified glicentin, a 69-amino-acid-residue peptide isolated from porcine intestine that contains the full sequence of glucagon and is probably biosynthetically related to glucagon, is a substrate for cyclic-AMP-dependent protein kinase in a cell-free system, Glicentin-related pancreatic peptide (residues 1–30 of glicentin) and glucagon were not phosphorylated under the same reaction conditions. It is postulated that the serine residue at position 34 of glicentin (position 2 of glucagon), that is part of the sequence Lys.Arg. His.Ser., is the probable site of phosphorylation.
Viral immunoblotting: A sensitive method for detecting viral-specific oliogoclonal bands in unconcentrated cerebrospinal fluid
Authors: Moyle S. , Keir G. , Thompson E. J.
Published: February 2005

A new method for detecting viral antibodies in cerebrospinal fluid is described. The technique has many advantages over previously published methods in that it is highly sensitive eliminating the need to concentrate the CSF, takes 5 h to complete, avoids the use of radionucleides, and most importantly circumvents problems associated with prozone effects which occur in immunoprecipitation reaction since the viral antigen is immobilized on nitrocellulose membranes.
Inhibitory effect of clonidine upon adenylate cyclase activity, cyclic AMP production, and insulin release in rat pancreatic islets
Authors: Garcia-Morales P. , Dufrane S. P. , Sener A. , Valverde I. , Malaisse W. J.
Published: February 2005

Conflicting opinions were recently expressed concerning the possible effect of α2-adrenergic agonists upon cyclic AMP production in pancreatic islets. In the present: study, clonidine inhibited glucose-induced insulin release from rat pancreatic islets, this inhibitory effect being abolished by idazoxan. Clonidine did not suppress the capacity of forskolin to augment glucose-induced insulin release. In a particulate subcellular fraction derived from the islets, adenylate cyclase was activated by calmodulin (in the presence of Ca2+), NaF, GTP, L-arginine, and forskolin, and slightly inhibited by clonidine. The inhibitory action of clonidine upon basal adenylate cyclase activity was more pronounced in islet crude homogenates. The inhibitory effect of clonidine was antagonized by forskolin whether in the particulate fraction or crude homogenate. At variance with the modest effects of glucagon, D-glucose, L-arginine, or a tumor-promoting phorbol ester upon cyclic AMP production by intact islets, forskolin caused a six-fold increase in cyclic AMP production. Clonidine inhibited cyclic AMP production by intact islets, whether in the absence or presence of forskolin. It is proposed that the inhibitory action of clonidine upon insulin release is attributable, in part at least, to inhibition of adenylate cyclase.
Acute influences on the two GDP-binding sites in brown-adipose-tissue mitochondria
Authors: Bryant K. R. , Rothwell N. J. , Stock M. J.
Published: February 2005

Scatchard analysis of3H-guanosine diphosphate (GDP) binding to rat brown-adipose-tissue mitochondria demonstrated that binding to the high- and low-affinity sites (Kd = 0.05 and 2.0 μM) was abolished by denaturation at 100°C but non-specific binding remained constant (0.2% of free-GDP). Prior incubation of mitochondria at 37°C reduced binding to the high-affinity site, but this could be reversed by incubating samples at 0°C. Addition of palmitic acid (5–40 nmole/mg of mitochondrial protein) did not affect GDP-binding, but similar concentrations of palmitoyl CoA caused a slight reduction in the number of high-affinity sites and a significant decrease in the number of lower-affinity sites. Acute treatments known to stimulate thermogenesis in vivo (a single meal, cold exposure, or noradrenaline injection 40–80 min before sacrifice) all increased binding to both binding sites, and tended to raise the dissociation constants, whereas injection of 2-deoxy-D-glucose, which depresses metabolic rate in the rat, decreased dissociation constants of both sites and the maximum number of high-affinity sites. These data indicate that both GDP-binding sites respond rapidly to acute thermogenic stimuli, possibly due to conformational changes in the mitochondrial inner membrane, and that palmitoyl CoA may influence mitochondrial proton conductance via an association with purine nucleotide binding sites.
Amino acid sequence restriction in relation to proteolysis
Authors: Jórnvall Hans , Persson Bengt
Published: February 2005

Distributions of amino acid residues in proteins show that proline is overrepresented in sequence positions following two basic residues ({LysArg}−{LysArg}), i.e. at sites similar to those susceptible to proteolytic cleavages of hormonal pro-forms. Conformational correlations further show that {LysArg}−{LysArg}-Pro sequences are often (8/11) not adiacent to elements of secondary structure, whereas the opposite applies to {LysArg}−{LysArg}-nonPro sequences (82/103 adjacent to elements of secondary structure). These distribution patterns from proteins in general also seem applicable in individual protein groups as demonstrated for some dehydrogenases. It appears possible that {LysArg}−{LysArg}-nonPro constitutes a restricted sequence, n proteins, and that proline, in addition to elements of secondary structure, contributes a means of avoiding unacceptable proteolytic processings of proteins in general.
Studies on glutathione metabolism in ventral prostate and chemically induced prostatic carcinoma in rats
Authors: Singhal Radhey L. , Vijayvargiya Rameshwar
Published: February 2005

Glutathione content and the activity of glutathione reductase were examined in ventral prostate and chemically induced 11095 squamous-cell prostatic carcinoma in rats, Castration produced a significant reduction in the levels of reduced (GSH) and oxidized (GSSG) glutathione and glutathione reductase activity in the prostate. Replacement of testosterone (50 mg/kg) daily for 7 days to castrated animals elevated the reduced glutathione level and the activity of glutathione reductase almost to normal limits, Squamous-cell carcinoma was implanted in castrated and intact animals, Tumor growth in normal rats produced a decrease of almost 30% in the weight of the ventral prostate at 21 days post-implantation, although the glutathione levels remained unaffected. Much greater activity of glutathione reductase was detected in the tumor in comparison to the values noted for the normal tissue, The tumor also showed significantly higher values for the GSH/GSSG ratio, No apparent difference could be found in the rate of the growth of tumors whether implanted in normal or castrated animals, The levels of reduced and oxidized glutathione and glutathione reductase activity also seemed identical in tumors obtained from both groups of animals, Administration of testosterone (50 mg/kg) or β-estradiol (2 mg/kg) daily for 11 days to tumor-bearing castrated animals did not alter the levels of glutathione and glutathione reductase activity. A significantly higher level of blood reduced glutathione was found in tumor-bearing rats in comparison to that seen for the normal subjects.

Our results demonstrate that androgen depletion and replacement therapy influence the metabolism of glutathione in rat ventral prostate. Squamous-cell carcinoma of the prostate appears to differ from the normal tissue with respect to the observed androgen effects, There is dissimilarity in the metabolism of glutathione in the two tissues since greater activity of glutathione reductase and lower values of reduced glutathione were seen in the tumor as compared to those of the ventral prostate. Treatment with β-estradiol, an antiprostatic agent, does not seem to influence the growth or glutathione metabolism of squamous-cell carcinoma of the prostate. The observed changes in blood glutathione levels might prove to be useful as an index of rapid growth of the neoplastic tissue.
Thrombin, a stimulator of bone resorption
Authors: Gustafson G. T. , Lerner U.
Published: February 2005

It is shown that thrombin (0.1–7 units/ml) stimulates calcium mobilization and bone matrix degradation, as indicated by release of [3H]proline, from cultured calvarial bones. The second finding in this paper, that indomethacin blocks the stimulating effect of thrombin on bone resorption, is consistent with the concept that prostaglandin synthesis may be involved in this process. It is suggested that thrombin is a potential mediator of bone resorption associated with inflammatory and malignant processes.
Effect of some hormones on the rate of the triacylglycerol/fatty-acid substrate cycle in adipose tissue of the mouse in vivo
Authors: Brooks B. J. , Arch J. R. S. , Newsholme E. A.
Published: February 2005

A method is described for the measurement of the rate of the triacylglycerol/fatty-acid cycle in adipose tissue of the mouse in vivo, which depends upon the incor-poration of tritium from [3H]H2O into the glycerol and fatty-acid moieties of triacyiglycerol. The rate of the cycling is increased two-fold by feeding, an effect that is completely abolished by the β-adrenergic blocker propranolol. The β-adrenergic agonist fenoterol increased the rate of cycling five-fold in white adipose tissue and three-fold in brown adipose tissue. Cold exposure had no effect on the rate of cycling in white adipose tissue but increased the rate almost two-fold in brown adipose tissue. The increased rate of cycling during feeding, which may be due to increased sympathetic nervous activity, is consistent with the view that the role of cycling is to increase sensitivity of metabolic control systems when required.
An analysis of amino acid composition of a few oxidoreductases by different methods
Authors: Naqui Ali , Vincent Jean-Claude
Published: February 2005

1. All methods used to analyze amino acid composition of different enzymes agree well with each other. 2. They lead to a relationship between amino acid composition and function of proteins. 3. Cytochrome oxidase is in a class by itself; copper-containing oxidases are closely related as expected.
Trypsin digestion of core chromatin
Authors: Diaz B. M. , Walker I. O.
Published: February 2005

Chicken erythrocyte core chromatin was digested with trypsin for 18 h. Five major limit petides were produced with mol. wts, from 10 800 to 8000 which arose from the central regions of each core histone. The basic amino- and carboxyl-terminal regions of each core histone were digested to small peptides having an average size of less than six amino acids. The small basic peptides, 25% by weight of the total histone, dissociated from the complex and could be removed by dialysis. The five major limit peptides remained bound to the DNA and contained all the secondary structure originally present in the native histones. Trypsin digestion decreased the supercoiling of the DNA in the complex and perturbed the tertiary structure of the histones. By contrast, there were no changes in the secondary structure of the large degraded histone fragments. However, when these were dissociated from the DNA, the secondary structure, which is predominantly α-helix, decreased by 50%. It is concluded that DNA binds strongly to the central regions of the core histones via α-helical segments on the polypeptide chains.