Differential induction of cytochrome P450 gene expression by 4n-alkyl-methylenedioxybenzenes in primary rat hepatocyte cultures

A well-characterized primary rat hepatocyte culture system was used to examine induction patterns of cytochrome 450 gene expression by a series of 4-n -alkyl-methylenedioxybenzene (MDBs) derivatives. Hepatocytes were treated for 24, 48, or 72 hours with 0–500 μ M of the MDB compounds, and total cellular RNA and protein from each treatment was evaluated by hybridization and immunochemical techniques. Exposure to MDB congeners possessing increasing 4-n -alkyl side-chain length (C₀–C₈) resulted in dose- and structure-dependent activation of CYP2B1, 2B2, 3A1, 1A1, and 1A2 gene expression. At equivalent 100 μ M concentrations, the C₆ and C₈ MDB congeners were more effective than the prototypical inducer phenobarbital (PB) with respect to induction potency of CYP2B1, CYP2B2, and CYP3A1 gene expression. In contrast to PB, longer side-chain–substituted MDBs effectively induced CYP1A1 and CYP1A2 gene expression, in addition to the CYP2B and CYP3A genes. At equivalent molar concentrations, the catechol derivative of C₆-MDB was ineffective in its ability to induce CYP gene expression, indicating the importance of the intact methylenedioxy bridge in the induction mechanism. Levels of MDB-inducible CYP2B1 and CYP2B2 mRNA were highly correlated with CYP2B1/2 apoprotein levels, ascertained by immunoblot analysis of cultured hepatocyte S9 fractions. Compared with results from previous in vivo analysis (12), the current data indicate that pharmacodynamic factors may influence MDB induction profiles and that differences in MDB effects on CYP gene expression result depending on distinct structure-activity relationships. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 253–262, 1998     

Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures

A cDNA of tobacco BY-2 cells corresponding to an mRNA species which was rapidly induced by methyl jasmonate (MeJA) in the presence of cycloheximide (CHX) was found to encode ornithine decarboxylase (ODC). Another cDNA from a MeJA-inducible mRNA encoded S-adenosylmethionine synthase (SAMS). Although these enzymes could be involved in the biosynthesis of polyamines, the level of putrescine, a reaction product of ODC, increased slowly and while the levels of spermidine and spermine did not change following treatment of cells with MeJA. However, N-methylputrescine, which is a precursor of pyrrolidine ring of nicotine, started to increase shortly after MeJA-treatment of cells and the production of nicotine occured thereafter. The levels of mRNA for arginine decarboxylase (ADC), an alternative enzyme for putrescine synthesis, and that for S-adenosylmethionine decarboxylase (SAMDC), required for polyamine synthesis, were not affected by MeJA. In addition to mRNAs for ODC and SAMS, mRNA for putrescine N-methyltransferase (PMT) was also induced by MeJA. Unlike the MeJA-induction of ODC mRNA, MeJA-induction of SAMS and PMT mRNAs were blocked by CHX. The level of ODC mRNA declined after 1 to 4 h following MeJA treatment, while the levels of mRNAs for SAMS and PMT continued to increase. Auxin significantly reduced the MeJA-inducible accumulation of mRNAs for ODC, SAMS and PMT. These results indicate that MeJA sequentially induces expression of a series of genes involved in nicotine biosynthesis by multiple regulatory mechanisms.p>     

Expression of genes coding NGF and BDNF human proteins in embryos of transgenic stocks of Drosophila melanogaster

Transgenic Drosophila stocks were selected. These stocks contained human ngf and bdnf genes under Drosophila heat-shock promoter. The expression of foreign genes during Drosophila development was demonstrated with Northern and whole mount hybridization.     

Voltage dependent calcium channels in cerebellar granule cell primary cultures

Voltage activated calcium channels were studied in rat cerebellar granule cells in primary culture. Macroscopic currents, carried by 20mM Ba2+, were measured in the whole-cell configuration. Slowly inactivating macroscopic currents, with a maximum value at a membrane potential around 5 mV, were recorded between the 1st and the 4th day in culture. These currents were completely blocked by 5mM Co2+, partially blocked by 10 microM nifedipine, and increased by 2 to 5 microM BAY K-8644. Two types of channels, in the presence of 80 mM Ba2+, were identified by single channel recording in cell-attached patches. The first type, which was dihydropyridine agonist sensitive, had a conductance of 18 pS, a half activation potential of more than 10 mV and did not inactivate. This type of channel was the only type found during the first four days in culture, although it was also present up to the 11th day. The second type of channel was dihydropyridine insensitive, had a conductance of 10 pS, a half activation potential less than -15 mV, and displayed voltage dependent inactivation. This second type of channel was found in cells for more than four days in culture.     

Differential induction of enzyme in soybean cell cultures by elicitor or osmotic stress

Soybean cell cultures were challenged either by glucan elicitor from Phytophthora megasperma f.sp. glycinea or by osmotic stress (0.4 M glucose). Osmotic stress induced production of a microsomal NADPH-dependent flavone synthase (flavone synthase II) which catalyses conversion of (2S)-naringenin to apigenin. In one of our cell-lines this enzyme activity was not detected either in unchallenged cells or in cells treated with glucan elicitor. Inducibility of flavone synthase II by 0.4 M glucose was highest at the end of the linear growth phase. Changes in the activities of a number of other enzymes were determined after treatment of the cells with elicitor or 0.4 M glucose. The activities of phenylalanine ammonialyase, cinnamate 4-hydroxylase, chalcone synthase and dihydroxypterocarpan 6a-hydroxylase all increased with elicitor and with osmoticum, albeit to a different degree. The rise in enzyme activity occurred later with osmoticum than with elicitor. The prenyltransferase involved in glyceollin synthesis was induced strongly by elicitor but only very weakly by osmoticum, whereas isoflavone synthase and NADPH: cytochrome-c reductase were only induced by elicitor. The activity of glucose-6-phosphate dehydrogenase did not change with elicitor or with osmoticum. Different product patterns were also obtained: whereas with elicitor, glyceollin I was the major product, intermediates of the glyceollin pathway (7,4-dihydroxyflavanone, trihydroxypterocarpan) accumulated with osmoticum.     

Epidermal growth factor stimulates DNA-synthesis of astrocytes in primary cerebellar cultures

Summary The capability of epidermal growth factor (EGF) to stimulate DNA synthesis in neural cells was investigated in primary cultures of early postnatal mouse cerebellum. At concentrations of 10^-8M, EGF stimulates DNA synthesis in astrocytes, which were identified immunocytologically by the cell type-specific marker, glial fibrillary acidic (GFA) protein. Astrocytes express cell-surface receptors for EGF as can be shown by binding of [¹²⁵I]-labeled EGF to live monolayer cultures. In the presence of 10% horse serum, EGF stimulates DNA-synthesis by a factor of about two-fold. Stimulation by EGF over control values is approximately 4-fold in the presence of 1% serum and 6 to 10-fold in the absence of serum. Absolute numbers of astrocytes are increased after more prolonged action of EGF. DNA-synthesis in neurons or oligodendroglia is not significantly stimulated by EGF. EGF enhances cell survival of serum-deprived cerebellar cultures. Fibroblast growth factor does not increase DNA-synthesis in astrocytes under the conditions used in this study.Abbreviations BME basal medium, Eagle's - BME-BSA basal medium, Eagle's containing 0.1% bovine serum albumin - EDTA ethylene-diamino-N, N-tetraacetic acid - EGF epidermal growth factor - FGF fibroblast growth factor - GFA glial fibrillary acidic - HS horse serum - [³ H] TdR tritium-labeled thymidine - PAP peroxidase-anti-peroxidase - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

Pax genes and eye organogenesis

Pax6 is a highly conserved gene that controls eye development in all species where it has been tested. In spite of this common 'master control regulator', the eyes of different animals are morphologically very different and it is believed that they have evolved independently multiple times through evolution. Recent works looking at eye development in 'primitive' species offer some explanation as to the surprising amount of conservation in genetic and morphogenetic pathways involved in eye development. These studies not only implicate the Pax genes but also the So/Six gene family in playing a crucial ancestral role in visual system development.     

Differential sensitization to deoxyribonuclease I of Xenopus vitellogenin and albumin genes during primary and secondary induction of vitellogenesis by oestradiol.

The sensitivity to DNAase (deoxyribonuclease) I (which preferentially digests transcribed sequences) of vitellogenin and albumin genes in liver and erythrocytes of male Xenopus after primary and secondary induction of vitellogenesis by oestrogen was measured by hybridization to cDNA (complementary DNA) of the residual DNA after enzymic digestion of isolated nuclei. Vitellogenin sequences were rendered selectively more sensitive to limited DNAase-I digestion (15-20% of DNA rendered acid-soluble) during primary hormonal activation (5 days) of vitellogenin genes in liver, but not erythrocyte, nuclei. Hormone withdrawal (25 days after first injection) did not result in reversion to a pre-activation gene configuration, nor did secondary hormonal stimulation (5 days after second and 25 days after first injection) augment the sensitivity of the genes to digestion by the nuclease. Similar hormone treatment did not affect the sensitivity of the constitutively expressed albumin genes in liver nuclei, nor their insensitivity in erythrocyte nuclei. Under the same conditions, globin genes remained indigestible in liver nuclei. It is concluded that primary induction of vitellogenesis in male Xenopus liver is accompanied by relatively long-lasting (3-4 weeks) change in the configuration of vitellogenin genes in hepatic nuclei which is not reversed or further modified during short-term oestrogen withdrawal or upon secondary stimulation.     

Metallothionein-III prevents glutamate and nitric oxide neurotoxicity in primary cultures of cerebellar neurons

Metallothionein (MT)-III, a member of the MT family of metal-binding proteins, is mainly expressed in the CNS and is abundant in glutamatergic neurons. Results in genetically altered mice indicate that MT-III may play neuroprotective roles in the brain, but the mechanisms through which this protein functions have not been elucidated. The aim of this work was to assess whether MT-III is able to prevent glutamate neurotoxicity and to identify the step of the neurotoxic process interfered with by MT-III. Glutamate neurotoxicity in cerebellar neurons in culture is mediated by excessive activation of glutamate receptors, increased intracellular calcium, and increased nitric oxide. It is shown that MT-III prevented glutamate- and nitric oxide-induced neurotoxicity in a dose-dependent manner, with nearly complete protection at 0.3-1 microgram/ml. MT-III did not prevent the glutamate-induced rise of intracellular calcium level but reduced significantly the nitric oxide-induced formation of cyclic GMP. Circular dichroism analysis revealed that nitric oxide triggers the release of the metals coordinated to the cysteine residues of MT-III, indicative of the S(Cys)-nitrosylation of the protein. Therefore, the present results indicate that MT-III can quench pathological levels of nitric oxide, thus preventing glutamate and nitric oxide neurotoxicity.     

The metabotrophic NGF and BDNF: an emerging concept

The field of neurotrophins, particularly, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), has witnessed a number of breakthroughs in recent years. There is evidence now that NGF and BDNF mediate multiple biological phenomena, ranging from the Rita Levi Montalcini's neurotrophic through immunotrophic to epitheliotrophic and nociceptive effects. In 2003 we, for the first time, enriched the "NGFome" with one more expression presented in our concept of NGF metabotrophicity, also that of BDNF. This envisages that these two factors may operate as metabotrophins, that is, involved in the maintenance of cardiometabolic homeostasis (glucose and lipid metabolism as well as energy balance, cardioprotection, and wound healing). Recent results also demonstrated that the circulating and/or tissue levels of NGF and BDNF are altered in cardiometabolic diseases (atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, and type 3 diabetes). Altogether, a hypothesis of metabotrophic deficit due to the reduction of NGF/BDNF availability and/or utilization was raised, and implicated in the pathogenesis of cardiometabolic diseases. This may cultivate a novel pathogenic and therapeutic thinking for these diseases.     

Unipolar brush cells develop a set of characteristic features in primary cerebellar cultures

Brain Cell Biology - The unipolar brush cells (UBCs) are a class of excitatory interneurons recently discovered in the cerebellar granular layer. UBCs differ morphologically and biochemically from...