Adrenergic nerves in the pars intermedia of the pituitary in the toad,Bufo arenarum

Summary By means of a highly sensitive and specific histochemical method for the demonstration of certain biogenic monoamines a plexus of nerves containing a primary catecholamine has been demonstrated in the pars intermedia of the toad, Bufo arenarum. These nerves are restricted in distribution to the part of the gland which contains colloid vesicles (stored MSH ?) in the cells. The view is put forward, based on the results of pharmacological and surgical experiments, that the adrenergic nerves inhibit the release of the MSH from the pars intermedia. The origin of the nerves in the brain is unknown, but experiments with lesions have shown that it is not to be found in the nucleus periventricularis arcuatus.The research reported in this document has been sponsored by the Swedish Medical Research Council and by the Swedish Natural Science Research Council.Research fellow of the Commission of Scientific Research of the Province of Buenos Aires, Argentina.     

Aluminium interactions with K+(86Rb+) and 45Ca2+ fluxes in three cultivars of sugar beet (Beta vulgaris)

Three cultivars of sugar beet (Beta vulgaris L.), which are sensitive to aluminium (Al) in the order Primahill > Monohill > Regina, were grown in water culture for 2 weeks. Nutrients were supplied at 15% increase of amounts daily, corresponding to the nutrient demand for maximal growth. The 2.4-dinitrophenol (DNP)-sensitive (metabolic) and DNP-insensitive (non-metabolic) uptake of aluminium, phosphate. ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in roots were measured as well as transport to shoots of intact plants. All 3 cultivars absorbed more aluminium if DNP was present during the aluminium treatment than in its absence. It is suggested that sugar beets are able to extrude aluminium activity or that they possess an active mechanism to keep Al outside the cell. The presence of Al in the medium during the 1-h experiment affected the metabolic and non-metabolic fluxes of ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in different ways. In the presence of DNP, the influx of both ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) and the efflux of ⁴⁵Ca²⁺ were inhibited by Al in a competitive way. At inhibition of ⁴⁵Ca²⁺ influx, 2 Al ions are probably bound per Ca²⁺ uptake site in cv. Regina (Al-tolerant), but in cvs Primahill and Monohill only one Al ion is bound (more Al sensitive). Aluminium competitively inhibited the active efflux of ⁴⁵Ca²⁺ (absence of DNP) in almost the same way in the 3 cultivars. In contrast, aluminium stimulated the influx of K⁺(⁸⁶Rb⁺) in cvs Primahill, Monohill and Regina in the absence of DNP. Thus, the Al effects on active and passive K⁺(⁸⁶Rb⁺) influx are different. The total influx of K⁺(⁸⁶Rb⁺) increased in the presence of Al and might be connected to an active exclusion of Al. Regina is the least Al-sensitive cultivar, probably because Al interferes less with the Ca²⁺ fluxes and because this cultivar actively excludes phosphate in the presence of Al. Thus Al-phosphate precipitation within the plant could be avoided.     

Site-directed mutagenesis of cytochrome P450s CYP2A1 and CYP2A2: influence of the distal helix on the kinetics of testosterone hydroxylation.

Cytochrome P450s CYP2A1 and CYP2A2 exhibit 88% sequence similarity, yet CYP2A1 metabolizes testosterone almost exclusively (90%) at the 7 alpha-position, whereas CYP2A2 forms several metabolites, with 15 alpha-hydroxytestosterone as a major metabolite. One of the regions with relatively low sequence homology corresponds by sequence alignment to the I and J helices of P450cam. Since this region is known to be part of the active site for P450cam, 26 single point and two double point mutants were prepared where the amino acid for one form was substituted with that of the other. Mutant and wild-type enzymes were expressed in Hep G2 cells using the vaccinia virus vector. Analysis of testosterone regioselectivity revealed that 25 of the mutants show the same regioselectivity as the parent wild-type enzymes and three are inactive, suggesting that no single amino acid in this region is totally responsible for the different selectivities of CYP2A1 and CYP2A2. Kinetic analysis of the CYP2A1 mutants showed that four of the mutants with changes near the conserved oxygen-binding region had Km values with much higher and Vmax values much lower than those of the wild-type enzyme and one mutant had a Vmax value twice as high as that of the wild-type enzyme. Deuterium isotope effects on 7 alpha-hydroxxylation were used to determine changes in the rate of reduction and estimate the relative amount of excess water formation. Changes in reduction rates and the amount of water produced are not sufficient to account for the differences in Vmax values, suggesting that the amount of hydrogen peroxide released is a primary determinant for changes in Vmax.     

Sea snake (Microcephalophis gracilis) hemoglobin: Primary structure and relationships to other forms

The hemoglobin of the sea snakeMicrocephalophis gracilis was purified and the primary structure of the α and β chains determined. This is the first sea snake hemoglobin structure characterized, and apparently also the first complete structure of any snake hemoglobin (an α chain of a viper was known), allowing judgments of reptilian variants. Variations between the sea snake form and other reptilian forms are large (52–65 differences for the α chains), of similar order as those between the sea snake and avian (56–65 differences) or human (58 differences) forms. Functionally, 19 residues at α/β contact areas and 7 at heme contacts are exchanged in relation to the human α and β chains. Four positions of the sea snake hemoglobin contain residues thus far unique to this form. However, all replacements appear compatible with conserved overall functional properties.     

Activated platelet-derived growth factor autocrine pathway drives the transformed phenotype of a human glioblastoma cell line

Human glioblastoma cells (A172) were found to concomitantly express PDGF-BB and PDGF β-receptors. The receptors were constitutively autophosphorylated in the absence of exogenous ligand, suggesting the presence of an autocrine PDGF pathway. Neutralizing PDGF antibodies as well as suramin inhibited the autonomous PDGF receptor tyrosine kinase activity and resulted in up-regulation of receptor protein. The interruption of the autocrine loop by the PDGF antibodies reversed the transformed phenotype of the glioblastoma cell, as determined by (1) diminished DNA synthesis, (2) inhibition of tumor colony growth, and (3) reversion of the transformed morphology of the tumor cells. The PDGF antibodies showed no effect on the DNA synthesis of another glioblastoma cells line (U343MGa 31L) or on Ki-ras-transformed fibroblasts. The present study demonstrates an endogenously activated PDGF pathway in a spontaneous human glioblastoma cell line. Furthermore, we provide evidence that the autocrine PDGF pathway drives the transtormed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists. © 1994 Wiley-Liss, Inc.     

Effect of temperature and glucose supply on the production of polyunsaturated fatty acids by the fungus Mortierella alpina CBS 343.66 in fermentor cultures

The fungus Mortierella alpina CBS 343.66 was grown in a fermentor at different pH, temperatures and supplies of carbon source (glucose) in order to optimize the culture conditions for rapid biomass and lipid production with a high proportion of polyunsaturated fatty acids, especially arachidonic acid (AA). Good growth and lipid production with 31% AA was obtained at pH 6.5 and 25°C. A temperature decrease to 18°C gave a significantly higher degree of polyunsaturated fatty acids. Eicosapentaenoic acid was not detected at 25°C, but was formed at 18°C at about 10%. The AA concentration but was similar for 18°C about 10%. The AA concentration was similar for 18°C and 25°C (around 30%): 18°C allowed relatively good growth and had a beneficial effect on the fungus morphology, i.e. pellets were formed. Best lipid production and a AA content of up to 33% was achieved at an excess of glucose (carbon source) and a deficit of ammonium chloride (nitrogen source). The percentage of AA of the total fatty acid composition was constant as ong as glucose was present. At glucose exhaustion, the proportion of AA increased to 57%. The increase in AA corresponded to a decrease in palmitic acid, stearic acid and oleic acid. Correspondence to: G. Molin     

Z → B transition in poly[d(G-m5C)2] induced by interaction with 4′,6-diamidino-2-phenylindole

The Z form of poly[d(G-m⁵C)₂], in presence of Mg²⁺ ion, is found to be transformed into B form upon interaction with 4′,6-diamidino-2-phenylindole (DAPI). The Z → B transformation is complete at a mixing ratio of about 0.07 DAPI per DNA base pairs, i.e., each DAPI molecule may be related to the conversion of 6–7 base pairs. An interaction between DAPI and poly[d(G-m⁵C)₂] in its Z form at low drug: DNA ratios is suggested from optical dichroism and time-resolved luminescence anisotropy results. The spectroscopic behaviour of DAPI indicates that the Z conformation of DNA does not provide normal binding sites for DAPI, such as groove or intercalation sites, but that the initial association may be of external nature. © 1993 John Wiley & Sons, Inc.