Bovine Brain Cortex Research Articles

Latrotoxin-like properties of a protein from brain

In bovine brain cortex cytoplasm we have identified a soluble protein (L-protein) of M r ∼90 kDa interacting with polyclonal antibodies to α-latrotoxin. The L-protein forms potential-dependent and cation-selective ion channels in BLM, which are blocked by Cd 2+. The fusogenic activity of the L-protein was demonstrated on liposomes. We have arrived at the conclusion that the action mechanisms of the L-protein and α-latrotoxin are similar.

The 16K Fragment of Prolactin Specifically Inhibits Basal or Fibroblast Growth Factor Stimulated Growth of Capillary Endothelial Cells

Intact 23 kilodalton (kDa), rat PRL is enzymatically cleaved in many target tissues to a 16 kDa (16K PRL) and an 8 kDa fragment. After reduction of an internal disulfide bond the fragments are released. 16K PRL was shown to be a potent mitogen on mammary epithelial cells via PRL receptors. Since estradiol-induced prolactinomas develop a new blood supply we tested the action of intact PRL and 16K PRL on growth of new vessels (angiogenesis). The angiogenic action of intact PRL and 16K PRL was tested in cultured bovine brain and adrenal cortex endothelial cells. Basal (b) or b-fibroblast growth factor (FGF) stimulated growth was estimated by counting cells or measuring the level of incorporation of 3H-thymidine into DNA. Paradoxically, 16K PRL inhibited the basal and FGF-stimulated growth of cultured endothelial cells in a dose-dependent fashion. Intact PRL or the cleaved but not reduced PRL were inactive even at a 100-fold higher concentration. When reformation of disulfide bonds was inhibited by carbamidomethylation of 16K PRL the preparations were more potent. 16K PRL had no effect on the mitogenic action of bFGF on baby hamster kidney cells which are known to have FGF receptors. These data demonstrate that in vitro 16K PRL is a potent and specific angiolytic factor, i.e. it inhibits angiogenesis. Furthermore, the action of 16K PRL does not appear to be via the known PRL or FGF receptors. Since angiogenesis is an essential component of tumor growth 16K PRL has potential as a therapeutic agent for the treatment of cancer.

Purification and characterization of metabolically active capillaries of the blood-brain barrier

Microvessels were isolated from bovine and rat cerebral cortex by simple procedures involving mechanical homogenization, differential and density-gradient centrifugation, and chromatography on a column of glass beads. The preparations were composed of short capillaries with a diameter of 1-10 microns. Both purifications were monitored by assaying the activity of the marker enzyme gamma-glutamyl transpeptidase (gamma-GTase). The final bovine and rat preparations were enriched 20- and 14-fold over the homogenate respectively. gamma-GTase activity was measured in different fractions after bovine and rat membranes were solubilized with 0.5% and 0.3% Triton X-100 respectively. Measurement of 5'-nucleotidase and acetylcholinesterase activities indicated very low levels of contamination of the microvessel preparations by glial cells and neurons. The integrity of the capillary membranes was confirmed by the assay of a cytosolic marker enzyme, lactate dehydrogenase. Viability of the microvessels was demonstrated by the presence of detectable levels of adenylates and by tissue respiration induced by glucose and succinate. Comparison of the proteins of homogenized bovine and rat brain cortex with those of purified capillaries separated by SDS/PAGE revealed enrichment of at least three predominant proteins of 14, 16 and 18 kDa in the capillary preparations. It is concluded that these methods allow rapid isolation of small blood vessels of the blood-brain barrier which are suitable for metabolic and structural studies in vitro.

A proper transmembrane Ca 2+ gradient is essential for the higher enzymatic activity of adenylate cyclase

Adenylate cyclase from bovine brain cortex was reconstituted into liposomes with (1000 fold) or without transmembrane Ca 2+ gradient. The highest enzyme activity (the active center of enzyme exposing outside) was observed in the vesicles with lower Ca 2+ concentration outside (≈10 −6 M, similar to physiological condition). If the transmembrane Ca 2+ gradient was in the inverse direction (i.e. higher Ca 2+ concentration outside, 1 mM), a lowest enzyme activity would appear. Such a difference could be diminished following addition of A23187. Obtained results showed that a proper transmembrane Ca 2+ gradient is essential for the optimal fluidity of phospholipid bilayer, favouring the formation of suitable conformation of adenylate cyclase with higher enzyme activity.

Monoclonal antibodies against various forms of the adenylyl cyclase catalytic subunit and associated proteins

Monoclonal antibodies against partially purified adenylyl cyclase from bovine brain cortex were raised in mice. Three types of antibody were obtained. Type 1 was specific for the calmodulin-sensitive enzyme. Type II also recognized this enzyme, but recognized the calmodulin-insensitive enzymes from a variety of species and tissues as well. Type I antibodies precipitated their antigens in both the native and denatured forms, while type II strongly favored the denatured forms. Type III antibodies precipitated adenylyl cyclase activity, but as shown by Western blot analysis, were directed against 38-kDa and 45-kDa glycoproteins. The 38-kDa protein was identified as synaptophysin.

Brain-type glucose transporter (GLUT-1) is selectively localized to the blood-brain barrier. Studies with quantitative western blotting and in situ hybridization.

The hypothesis that the GLUT-1 glucose transporter isoform is expressed selectively in brain at the capillary endothelium, i.e. the blood-brain barrier (BBB), was tested by using quantitative Western blotting, cytochalasin B binding, and in situ hybridization in bovine brain cortex. Purified human red cell glucose transporter was used as the standard for quantitative Western blots, because the mobility of the human erythrocyte and BBB glucose transporters in electrophoretic gels was identical. The concentration of immunoreactive glucose transporter in bovine BBB plasma membranes was 10.8 +/- 0.9 pmol/mgp (mean +/- S.E., n = 6). This value was not statistically different from the estimate of the maximal binding sites of D-glucose-displaceable [3H]cytochalasin B binding in the BBB membrane preparations, 11.7 +/- 3.5 pmol/mgp. In situ hybridization experiments using 35S-labeled antisense and sense riboprobes corresponding to nucleotides 385-932 of the GLUT-1 cDNA showed prominent hybridization of the antisense probe over brain microvascular endothelium, but no hybridization over neuropil greater than that found with the 35S-labeled sense probe. These studies are consistent with the following conclusion: (a) essentially 100% of the glucose transporter binding sites at the BBB can be accounted for by the GLUT-1 isoform; (b) in situ hybridization studies confirm previous Northern blot analysis and indicate the GLUT-1 gene is expressed selectively in microvascular endothelium in brain with minimal, if any, expression of this gene in neurons or glial cells in vivo.

Involvement of protein kinase C in the mitogenic and chemotaxis effects of basic fibroblast growth factor on bovine cerebral cortex capillary endothelial cells

Basic fibroblast growth factor is increasingly implicated in cellular growth, differentiation, angiogenesis and oncogenesis. In culture, basic fibroblast growth factor greatly improved the growth rate of bovine brain cortex capillary endothelial cells. Down-regulation of protein kinase C by prolonged treatment with phorbol esters prevented the mitogenic effect of basic fibroblast growth factor on capillary endothelial cells. Furthermore, staurosporine, a potent protein kinase inhibitor, showed strong antiproliferative activity against basic fibroblast growth factor-induced endothelial cell growth. Similarly, the chemotaxis effect of basic fibroblast growth factor on capillary endothelial cells was abolished by down-regulation of protein kinase C or by staurosporine treatment. Therefore, it is suggested that protein kinase C could account for part of the angiogenic effect of basic fibroblast growth factor.

Molecular species analysis of phospholipids by negative ion fast atom bombardment mass spectrometry: application of surface precipitation technique.

Eight phospholipidic classes from bovine brain cortex and soybean were prepared and purified by preparative high-performance and liquid chromatography, and their molecular species were identified by negative ion fast atom bombardment mass spectrometry using the 'surface precipitation' method. Its main advantages are: (i) clear-cut and abundant diagnostic ions for structural elucidation of the species; (ii) fragments characteristic of the main fatty acids, the polar head-group and the molecule frame in phospholipids; (iii) less background caused by the liquid matrix.

The presence of ATP + ubiquitin-dependent proteinase and multicatalytic proteinase complex in bovine brain

The presence of two distinct high-molecular-weight proteases with similar pH optima in the weakly alkaline region was shown in cytosol of the bovine brain cortex. They were separated by ammonium sulfate fractionation and each was further purified by DEAE-Sephacel, Sephacryl S-300, DEAE-Cibacron Blue 3GA-agarose, heparin-agarose, and Sepharose 6B chromatography. The larger enzyme (Mr 1,400 kDa), which precipitates at 0-38% ammonium sulfate saturation, seems to be active in ATP + ubiquitin (Ub)-dependent proteolysis; it has low basal caseinolytic activity that is stimulated 3-fold by ATP, and when Ub is present ATP causes a 4.5-fold stimulation. A second proteinase was also found to be present (Mr 700 kDa) that precipitates at 38-80% ammonium sulfate saturation, is composed of multiple subunits ranging in Mr from 18 to 30 kDa, and degrades both protein and peptide substrates, demonstrating trypsin-, chymotrypsin- and cucumisin-like activities. Catalytic, biochemical, and immunological characteristics of this proteinase indicate that it is a multicatalytic proteinase complex (MPC), whose enzyme activity, in contrast to that of MPC from bovine pituitaries (1-3), is stimulated 1.7-fold by addition of ATP in the absence of ubiquitin at the early steps of purification; this property is lost during the course of further purification. Both proteinases are present in the nerve cells, since the primary chicken embryonic telencephalon neuronal cell culture extracts contain both ATP + Ub-dependent proteinase and MPC activities.

Calmodulin-independent bovine brain adenylate cyclase Amino acid sequence and nucleotide sequence of the corresponding cDNA

An individual catalytic component of calmodulin-independent adenylate cyclase has been isolated from bovine brain cortex. Affinity chromatography on an immunosorbent was used. The amino acid sequence of adenylate cyclase as well as the corresponding nucleotide sequence of the cDNA has been determined. cDNA of adenylate cyclase encodes a protein consisting of 834 amino acid residues and the signal peptide (19 amino acid residues). A series of adenylate cyclase isoforms has been found. A homology between adenylate cyclases from bovine brain, E. coli and Bordetella pertussis has been revealed.

Tumor-promoting phorbol esters stimulate bovine cerebral cortex capillary endothelial cell growth in vitro

The active tumor promoters, 12-O-tetradecanoyl phorbol 13-acetate (TPA) and phorbol 12, 13-dibutyrate (PDBu), which activate protein kinase C (PKC), were found to stimulate bovine brain cortex capillary endothelial cell (CEC) proliferation in a dose-dependent manner. 4 alpha-phorbol-12, 13-didecanoate (4 alpha-PDD), known to be inactive for PKC, was without effect in stimulating CEC proliferation. Furthermore, prolonged incubation with TPA led to a decrease in the number of [3H]-PDBu binding sites with a parallel loss of responses of the cells to TPA. Finally, staurosporine, a potent PKC inhibitor, showed a strong antiproliferative effect on CEC (IC50 = 1.3 nM). Therefore, this work suggests that PKC plays a fundamental role in CEC growth.

Multiple species and isoforms of Bordetella pertussis toxin substrates

In purified G proteins from bovine brain cortex the ADP-ribosylated substrates of Bordetella pertussis toxin (PT) can be resolved in three polypeptides by polyacrylamide gel electrophoresis: a 39 kDa major substrate, corresponding to Go alpha and two others (40 and 41 kDa) assigned to alpha subunits of Gi-like proteins. These three polypeptides were also detected in membranes of normal cells or tissues from neuronal and endocrine origins. In contrast, in membranes from other origins, only two PT substrates at 41 and 40 kDa were resolved; the latter being the most abundant ADP-ribosylated substrate in human platelets and C6 glioma cells. In these cells, electrophoretic patterns of PT-radiolabeled proteolytic fragments derived from the 40 kDa peptide were different to those from the 39 and 41 kDa polypeptides of purified G proteins. However, isoelectrofocusing and two dimensional analyses showed that the 40 kDa and 39 kDa (but not the 41 kDa) PT substrate of purified G proteins exhibited similar isoforms.

Substrate specificity of cerebral cathepsin D and high-Mr aspartic endopeptidase.

The specificity of action of bovine brain cortex cathepsin D (EC 3.4.23.5) and high-Mr aspartic endopeptidase (EC 3.4.23.-) was studied with the vasoactive peptides renin substrate tetradecapeptide (RSTP), substance P (SP), and angiotensins I and II, and with model peptides--Lys-Pro-Ala-Glu-Phe-Phe (NO2)-Ala-Leu (I), Gly-Gly-His-Phe (NO2)-Phe-Ala-Leu-NH2 (II), and Abz-Ala-Ala-Phe-Phe-pNA (III). Cerebral aspartic peptidases show identical substrate specificity, cleaving the Leu10-Leu bond in RSTP and Phe-Phe in SP and peptide I-III, and not splitting angiotensins I and II. Because of the higher catalytic efficiency of cathepsin D (Kcat value), the specificity constants (Kcat/Km) for cathepsin D-catalyzed hydrolysis of substrates 1-111 are much higher than those for the high-Mr enzyme. High-Mr aspartic peptidase shares a number of properties with cathepsin D (sensitivity to pepstatin, substrate specificity, pH activity profile) and shows partial immunological identity; however, high-Mr aspartic peptidase has a specific activity 7-10 times lower than that of cathepsin D. The kinetic parameters of proteolysis of model peptides presented indicate that the high-Mr enzyme may be a complex of a single-chain cathepsin D with another polypeptide, although the possibility that it is an independent aspartic peptidase cannot be excluded.

5′-Nucleotidase from bovine brain cortex: effect of solubilization on enzyme kinetics and modulation

The kinetic characteristics and the EDTA inhibition of microsomal 5?-nucleotidase from bovine brain cortex were studied and compared with the properties of the enzyme solubilized with Lubrol WX. The K(m) value after enzyme solubilization was not significantly different from that of the membrane-bound enzyme. Likewise, di- and trinucleotides performed a similar competitive inhibition of the two forms of the enzyme. In contrast, divalent cations inhibited the intact microsomal enzyme activity at the same concentrations in which they increased the soluble-enzyme activity. The solubilization of microsomal 5?-nucleotidase did not change the progressive and irreversible character of the EDTA inhibition, but the mechanism of the irreversible inhibition was different. The addition of divalent metal cations did not affect the irreversibility of either inhibition, even though the effect on the residual activities was different. The Arrhenius plot of the 5?-nucleotidase activity in intact microsomal fraction exhibited a well-defined break at 31 +/- 0.1 degrees C, whereas that of the solubilized enzyme was a straight line. It is concluded then that microsomal 5?-nucleotidase from bovine brain cortex does not require the membrane environment to express its activity, although the influence of this lipidic environment was evident in the differences observed in the enzyme activity modulation by EDTA, cations and temperature.

Solubilization of serotonin1a and serotonin1b binding sites from bovine brain.

Serotonin1 (5-hydroxytryptamine1, 5-HT1) binding sites have been solubilized from bovine brain cortex using a mixture of 0.1% Nonidet P-40 and 0.3% digitonin in a low-salt buffer containing 0.1% ascorbic acid. The affinity of [3H]5-HT for the soluble cortical binding sites (2.1 nM) is identical to its affinity at membrane-bound binding sites (2.1 nM). [3H]8-Hydroxy-2-(di-n-propylamino)tetralin ([3H]DPAT), a selective 5-HT1a radioligand, also binds to soluble cortical binding sites with high affinity (1.8 nM) comparable with its affinity in the crude membranes (1.7 nM). A significant correlation exists in the rank order potency of serotonergic agents for [3H]5-HT binding and for [3H]DPAT binding to crude and soluble membranes. The density of [3H]DPAT binding sites relative to the [3H]5-HT sites in the solubilized cortical membranes (35%) corresponds well with the proportion of 5-HT1a sites in the crude membranes determined by spiperone displacement (33%), suggesting that both the 5-HT1a and 5-HT1b binding sites have been cosolubilized. [3H]5-HT binding in the soluble preparations was inhibited by GTP, suggesting that a receptor complex may have been solubilized. [3H]Spiperone-specific binding was not detectable in this preparation, suggesting that 5-HT2 sites were not cosolubilized.

Neuron localization and neuroblastoma cell expression of brain-derived growth factor

Bovine brain-derived growth factor (BDGF) is a approximately 16-17 kD polypeptide mitogen with a broad spectrum of cell specificity. Using a highly specific mouse polyclonal anti-BDGF antiserum for indirect immunoperoxidase and immunofluorescent stainings, BDGF was found to be specifically localized in the neurons of bovine brain cortex. The indirect immunofluorescent staining was blocked by the presence of excess purified BDGF. Human neuroblastoma cells showed cytoplasmic staining with anti-BDGF antiserum. The cell lysates of neuroblastoma cells elicited a BDGF-like activity which could be completely inhibited by preincubation with anti-BDGF antiserum.

Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex, and corpus luteum capillaries.

Bovine adrenal and brain cortex and corpus luteum-derived capillary endothelial cells have been established in culture, taking advantage of their ability to proliferate at clonal density when maintained on extracellular matrix (ECM) coated dishes in the presence of serum supplemented medium. All three cell types formed at confluency a monolayer of small, tightly packed, contact inhibited cells that express factor VIII related antigen. Their proliferative response to basic and acidic FGF when cells were maintained on plastic and exposed to serum supplemented medium was similar to that previously reported for endothelial cells derived from large vessels, with acidic FGF being 30-fold less potent than basic FGF. Their requirement for high density lipoproteins and transferrin in order to proliferate actively when maintained on ECM-coated dishes and exposed to serum-free conditions was also similar to that previously reported for endothelial cells derived from large vessels. Heparin strongly reduced the proliferative response of capillary endothelial cells to either basic or acidic FGF, as well as their response to serum alone, regardless of whether cells were maintained on plastic or on ECM-coated dishes. The present data indicate that bovine endothelial cells derived from large or small vessels are indistinguishable in so far as their response to growth factors, plasma factors, and substrata are concerned.

Antibodies to blood-brain barrier bind selectively to brain capillary endothelial lateral membranes and to a 46K protein.

To begin elucidating the biochemical basis of the polarized membrane features of the blood-brain barrier (BBB), a series of immunochemical and immunoperoxidase studies were initiated with bovine brain microvessels that make up the BBB in vivo. A rabbit antiserum was prepared against isolated bovine brain BBB plasma membranes. The bovine microvessel plasma membranes were radioiodinated with chloramine-T, and the antiserum selectively immunoprecipitated a 46K protein. The antibodies directed against the 46K protein were quantitatively absorbed with bovine brain capillaries but not with rat kidney or liver powder. Only the capillaries of brain reacted with the rat kidney-absorbed antiserum in immunoperoxidase studies of ethanol-fixed, 8-micron sections of bovine brain cortex, whereas the capillaries in heart, liver, and kidney did not react. This antiserum also strongly illuminated the lateral membranes of isolated bovine brain capillary endothelial cells grown in primary tissue culture. These studies provide evidence for a polarized distribution of a surface antigen in bovine brain capillary endothelial cells that is not present in capillary endothelia of liver, heart, or kidney. The correlation of the immunoperoxidase and immunoprecipitation techniques suggests that a candidate for the asymmetrically distributed surface antigen in the BBB is the 46K protein. The relationship between the 46K protein and the composition of BBB tight junctions remains to be determined.

3H]mepyramine binding to histamine H 1 receptors in bovine retina

The promethazine-sensitive [ 3H]mepyramine binding was used to determine the presence of histamine H 1 receptors in membranes from bovine retina. Specific mepyramine binding to retinal membranes was reversible, saturable and of high affinity. The apparent dissociation constant (K D=2.2±0.4 nM) and the density of binding sites (B max=60.9±5.1 fmol/mg protein), obtained in equilibrium studies, were similar to those found in bovine brain cortex. Binding was stereospecific and the inhibitory potencies of H 1 and H 2 antagonists indicated that [ 3H]mepyramine binding sites in the retina have characteristics of H 1 receptors.

Adenylate cyclase from bovine brain cortex: purification and characterization of the catalytic unit.

The non-stimulated (basal) adenylate cyclase from bovine brain cortical membranes was purified 10 000-fold to apparent homogeneity by Lubrol PX extraction and two cycles of affinity chromatography on forskolin-agarose. The final product appears as one major band (mol. wt. 115 000) on SDS-polyacrylamide gels. Further identification was achieved by affinity cross-linking using Gs (stimulatory GTP-binding protein) that was [32P]ADP-ribosylated by cholera-toxin/[32P]NAD: cross-linking with disuccinimidyl suberate gave products with mol. wts. of 160 000, approximately 270 000 and higher. The distribution of these products was dependent on the concentration of cross-linker, suggesting aggregation of two or more adenylate cyclase complexes. In contrast, photo-affinity cross-linking with 4-azidobenzoyl-[32P]Gs yielded a single product with a mol. wt. of 160 000. Purified adenylate cyclase was completely unresponsive towards stimulators (GTP-analogs, NaF) acting via Gs suggesting that this component was removed during purification. On the other hand, stimulation by forskolin and by added activated Gs was preserved but to a smaller degree as compared with the crude enzyme. In contrast, the stimulation of Ca2+/calmodulin was only marginal. Purified adenylate cyclase reversibly bound to wheat germ agglutinin-Sepharose. This suggests that bovine brain adenylate cyclase is a glycoprotein.