Interferon inhibits the accumulation of glycerol phosphate dehydrogenase mRNA in oligodendrocytes and C6 cells

We investigated the effect of rat interferon-/ (IFN) on the expression of glycerol phosphate dehydrogenase (E.C.1.1.1.8; GPDH), in both C6 cells and pure cultures of oligodendrocytes. IFNs are naturally produced inhibitors of cell growth that can also affect differentiated cell functions. GPDH is a biochemical marker for oligodendrocytes and is known to be developmentally regulated and steroid inducible. GPDH activity is induced by hydrocortisone (HC) 3.5 fold in C6 cells and 5 fold in oligodendrocytes compared to untreated cultures. A pretreatment of these cells with 75 U/ml of rat IFN-/ resulted in an inhibition of the HC induction of GPDH enzymatic activity by 50% and 40% in C6 cells and oligodendrocytes respectively. We also found that IFN impaired the accumulation of GPDH mRNA in both cell types. These results demonstrate that IFNs are capable of modifying the cellular response to hormones in cells of neuroepithelial origin, and suggest the possibility that IFNs may be able to influence the development and function of the brain.Special issue dedicated to Dr. Paola S. Timiras     

Phospholipase C activation in rat pituitary adenoma (GH) cells

The presence of the pertussis toxin (PTX) insensitive GTP-binding proteins (G-proteins) G_q and/or G₁₁ has been demonstrated in three different prolactin (PRL) and growth hormone (GH) producing pituitary adenoma cell lines. Immunoblocking of their coupling to hormone receptors indicates that G_q and/or G₁₁ confer throliberin (TRH) responsive phospholipase C (PL-C) activity in these cells. The contention was substantiated by immunoprecipitation analyses snowing that anti G_q/11-sera coprecipitated PL-C activity. In essence, only G_q/11 (but neither G_i2, G_i3 nor G_o) seems to mediate the TRH-sensitive PL-C activity, while G_o may be coupled to a basal or constitutive PL-C activity. Immunoblocking studies imply that the B-complex also, to some extent, may stimulate GH₃ pituitary cell line PL-C activity. Finally, the steady state levels of G_q/11 mRNA and protein were downregulated upon long term exposure of the GH3 cells to TRH (but not to vasoactive intestinal peptide = VIP).     

The heterogeneous nature of microbial products as shown by solid-state13C CP/MAS NMR spectroscopy

Homoionic Na-, Ca-, and Al-clays were prepared from the <2 m fractions of Georgia kaolinite and Wyoming bentonite and mixed with sand to give artificial soils with 5, and 25% clay. The artificial soils were inoculated with microbes from a natural soil before incubation. Unlabelled and uniformly¹³C-labelled (99.9% atom) glucose were incorporated into the artificial soils to study the effects of clay types, exchangeable cations and clay contents on the mineralization of glucose-carbon and glucose-derived organic materials. Chemical transformation of glucose-carbon upon incorporation into microbial products and metabolites, was followed using solid-state¹³C CP/MAS NMR spectroscopy.There was a significant influence of exchangeable cations on the mineralization of glucose-carbon over a period of 33 days. At 25% clay content, mineralization of glucose-carbon was highest in Ca-soils and lowest in Al-soils. The influence of exchangeable cations on mineralization of glucose-carbon was more pronounced in soils with bentonite clay than those with kaolinite clay. Statistical analysis of data showed no overall effect of clay type on mineralization of glucose-carbon. However, the interactions of clay type with clay content and clay type with clay content and exchangeable cations were highly significant. At 25% clay content, the mineralization of glucose-carbon was significantly lower in Na- and Al-soils with Wyoming bentonite compared with Na- and Al-soils with Georgia kaolinite. For Ca-soils this difference was not significant. Due to the increased osmotic tension induced by the added glucose, mineralization of glucose-carbon was slower in soils with 5% clay than soils with 25% clay.Despite the differences in the chemical and physical characteristics of soils with Ca-, Na- and Al-clays, the chemical composition of organic materials synthesised in these soils were similar in nature. Assuming CP/MAS is quantitative, incorporation of uniformly¹³C-labelled glucose (99.9% atom) in these soils resulted in distribution of carbon in alkyl (24–25%), O-alkyl (56–63%), carbonyl (11–15%) and small amounts of aromatic and olefinic carbon (2–4%). However, as decomposition proceeded, the chemistry of synthesised material showed some changes with time. In the Ca- and Na-soils, the proportions of alkyl and carbonyl carbon decreased and that of O-alkyl carbon increased with time of incubation. However, the opposite trend was found for the Al-soil.Proton-spin relaxation editing (PSRE) subspectra clearly showed heterogeneity within the microbial products. Subspectra of the slowly-relaxing (long T₁(H)) domains were dominated by alkyl carbon in long- and short-chain structures. The signals due to N-alkyl (55 ppm) and carbonyl carbon were also strong in these subspectra. These subspectra were very similar to those obtained for microbial and fungal materials and were probably microbial tissues attached to clay surfaces by polysaccharide extracellular mucilage. Subspectra of fast-relaxing (short T₁(H)) domains comprised mostly O-alkyl and carbonyl carbon and were probably microbial metabolites released as neutral and acidic sugars into the extracellular environment, and strongly sorbed by clay surfaces.     

A cell-based reporter assay for the identification of protein kinase C activators and inhibitors

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes. (Mol Cell Biochem141: 129–134, 1994)     

Depolarization and Neurotransmitters Increase Neuronal Protein Tyrosine Phosphorylation

Abstract: In rat hippocampal slices and in neurons in primary culture, K⁺-induced depolarization increased markedly and rapidly tyrosine phosphorylation of a 110-kDa protein (pp110) and, to a lesser degree, of a 120-kDa protein (pp120), in a calcium-dependent fashion. Qlutamate, 1-aminocyclopentane- trans -1,3-dicarboxylic acid (an agonist of metabotropic glutamate receptors), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (an agonist of ionotropic glutamate receptors) stimulated also tyrosine phosphorylation of pp110 and pp120. These effects were not observed in astrocytes in primary culture. In hippocampal slices tyrosine phosphorylation of pp110 and pp120 was stimulated by Ca²⁺-ionophores and by phorbol esters and antagonized by a chelator of intracellular Ca²⁺and by drugs that inhibit protein kinase C. Stimulation of muscarinic and α₁,-adrenergic receptors increased also tyrosine phosphorylation of pp110 and pp120. These results demonstrate that membrane depolarization and stimulation of neurotransmitter receptors activate a tyrosine phosphorylation pathway in neurons. This pathway involves an increase in intracellular Ca²⁺ concentrations and the activation of protein kinase C. It may provide a biochemical basis for some neurotrophic effects of electrical activity and neurotransmitters and may contribute to the role of tyrosine phosphorylation in long-term potentiation.     

Identification of N-terminal helix capping boxes by means of 13C chemical shifts

Summary We have examined the ¹³C and ¹³C chemical shifts of a number of proteins and found that their values at the N-terminal end of a helix provide a good predictor for the presence of a capping box. A capping box consists of a hydrogen-bonded cycle of four amino acids in which the side chain of the N-cap residue forms a hydrogen bond with the backbone amide of the N3 residue, whose side chain in turn may accept a hydrogen bond from the amide of the N-cap residue. The N-cap residue exhibits characteristic values for its backbone torsion angles, with and clustering around 94±15° and 167±5°, respectively. This is manifested by a 1–2 ppm upfield shift of the ¹³C resonance and a 1–4 ppm downfield shift of the ¹³C resonance, relative to their random coil values, and is mainly associated with the unusually large value of . The residues following the N-cap residue exhibit downfield shifts of 1–3 ppm for the ¹³C resonances and small upfield shifts for the ¹³C ones, typical of an -helix.     

Regional Distribution and Quantitative Measurement of the Phosphoinositidase C-Linked Guanine Nucleotide Binding Proteins G1α and Gqα in Rat Brain

Abstract: Levels of the guanine nucleotide binding proteins G₁₁α and G_qα, which produce receptor regulation of phosphoinositidase C., were measured immunologically in 13 regions of rat central nervous system. This was achieved by immunoblotting membranes from these regions with antisera (CQ series) that identify these two polypeptides equally, following separation of the membranes using sodium dodecyl sulphate-polyacrylamide gel electrophoresis conditions that can resolve G_qα and G₁₁α. In all regions examined, G_qα was more highly expressed than G₁₁α. Ratios of levels of G_qα to G₁₁α varied between the regions from 5:1 to 2:1. Quantitative measurements of the levels of G_qα and G₁₁α in each region were obtained by comparison with known amounts of purified liver G_qα and G₁₁α and with E. coli expressed recombinant G_qα. Areas that expressed G_qα highly included olfactory bulb (930 ng/ mg of membrane protein), frontal cortex (700 ng/mg of membrane protein), parietal occipital cortex (670 ng/mg of membrane protein), caudate putamen (1,003 ng/mg of membrane protein), hippocampus (1,045 ng/mg of membrane protein), hypothalamus (790 ng/mg of membrane protein), and cerebellum (950 ng/mg of membrane protein). More modest levels were observed in thalamus (450 ng/mg of membrane protein), pituitary (480 ng/mg of membrane protein), optic chiasma (330 ng/mg of membrane protein), and spinal cord (350 ng/mg of membrane protein). Gna was more evenly expressed with values ranging from about 170 ng/mg of membrane protein in spinal cord and optic chiasma to close to 300 ng/mg of membrane protein in regions expressing high levels of G_qα. A third polypeptide could be identified by the CQ antisera in all brain regions. The possibility that this polypeptide is the α subunit of G₁₄ is discussed.     

Prolactin-Stimulated Mitogenesis of Cultured Astrocytes Is Mediated by a Protein Kinase C-Dependent Mechanism

Abstract: Prolactin (PRL) has been reported to activate cellular proliferation in nonreproductive tissue, such as liver, spleen, and thymus. Recently, we have extended the possible role of PRL as a mammalian mitogen by demonstrating a mitogenic effect of PRL in cultured astrocytes. Although the cellular mechanisms by which PRL regulates cell growth are not fully understood, protein kinase C (PKC) has been implicated as one of the transmembrane signaling systems involved in the regulation of PRL-induced cell proliferation in Nb2 lymphoma cells and liver. In the present studies, we examined the possible role of PKC in PRL-induced proliferation of cultured astrocytes. Incubation of cultured astrocytes with 1 nM PRL resulted in a rapid translocation of PKC from the cytosol to the membrane, with maximal PKC activity in the membrane occurring 30 min after exposure to PRL. Translocation of PKC activity occurred over a physiological range of PRL, with maximal PKC activation occurring at 1 nM. At concentrations greater than 10 nM PRL, there was a decrease in the amount of PKC activity associated with the membrane fraction compared with that of cells stimulated with 1 nM PRL. Incubation of astrocytes with PRL in the presence of the PKC inhibitors staurosporine, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine, or polymyxin B blocked the PRL-induced increase in cell number with IC₅₀ values of approximately 2 nM, 10 μM, and 6 μM, respectively. PKC is the only known cellular receptor for 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulates the translocation of PKC from the cytosol to the membrane. Incubation of astrocytes with 20 nM TPA resulted in an increase in the expression of proliferating cell nuclear antigen and cell number, whereas 4α-phorbol 12,13-didecanoate, an inactive phorbol ester, was ineffective. To examine further the effect of TPA and PRL on cellular proliferation, cultured astrocytes were incubated with increasing concentrations of TPA in the presence or absence of a minimal effective dose of PRL (100 pM). In the absence of PRL, incubation with TPA resulted in an inverted U-shaped dose-response curve, with 100 nM TPA resulting in a maximal increase in cell number. In the presence of 100 pM PRL, the TPA dose-response curve was shifted to the left, with maximal activity occurring with 10 nM TPA. Chronic stimulation of astrocytes with 500 nM TPA depleted the cells of PKC and blocked the PRL-induced increase in cell number. Finally, TPA treatment decreased cell-surface binding of ¹²⁵I-PRL. These data indicate that the PKC is involved in the mitogenic effect of PRL in cultured astrocytes.     

Improved pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins

Summary NMR pulse sequences for measuring coupling constants in ¹³C, ¹⁵N-labeled proteins are presented. These pulse sequences represent improvements over earlier experiments with respect to resolution and number of radiofrequency pulses. The experiments are useful for measuring J_NH , J_NCO, J_NC , J_H ^N _CO and J_H ^N _H . Applications to chymotrypsin inhibitor 2 (CI-2) are shown.