Cloning and expression in Escherichia coli of the obtusifoliol 14α-demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14α-demethylases (CYP51) from fungi and mammals

Obtusifoliol 14β-demethylase from Sorghum bicolor (L.) Moench has been cloned using a gene-specific probe generated using PCR primers designed from an internal 14 amino acid sequence. The sequence identifies sorghum obtusifoliol 14α-demethylase as a cytochrome P450 and it is assigned to the CYP51 family together with the sterol 14α-demethylases from fungi and mammals. The presence of highly conserved regions in the amino acid sequences, analogous substrates and the same metabolic role demonstrate that the sterol 14α-demethylases are orthologous enzymes. The sterol 14α-demethylases catalyse an essential step in sterol biosynthesis as evidenced by the absence of a 14α-methyl group in all known functional sterols. A functional sorghum obtusifoliol 14α-demethylase was expressed at high levels in Escherichia coli and purified using an efficient method based on temperature-induced Triton X-114 phase partitioning. The recombinant purified enzyme produced a type I spectrum with obtusifoliol as substrate. Reconstitution of purified recombinant enzyme with sorghum NADPH—cytochrome P450 reductase in dilaurylphosphatidylcholine micelles confirms that obtusifoliol 14α-demethylase catalyses the 14α-demethylation of obtusifoliol to 4α-methyl-5α-ergosta-8,14,24(28)-trien-3β-ol as evidenced by GC—MS. The isolation of a cDNA clone encoding the plant sterol 14α-demethylase, combined with the previously isolated cDNA clones for fungal and mammalian sterol 14α-demethylases, provides an important tool in the rational design of specific inhibitors towards the individual sterol 14α-demethylases.     

The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes

The Arabidopsis thaliana MALE STERILITY 2 ( MS2 ) gene product is involved in male gametogenesis. The first abnormalities in pollen development of ms2 mutants are seen at the stage in microsporogenesis when microspores are released from tetrads. Expression of the MS2 gene is observed in tapetum of wild-type flowers at, and shortly after, the release of microspores from tetrads. The MS2 promoter controls GUS expression at a comparable stage in the tapetum of transgenic tobacco containing an MS2 promoter–GUS fusion. The occasional pollen grains produced by mutant ms2 plants have very thin pollen walls. They are also sensitive to acetolysis treatment, which is a test for the presence of an exine layer. The MS2 gene product shows sequence similarity to a jojoba protein that converts wax fatty acids to fatty alcohols. A possible function of the MS2 protein as a fatty acyl reductase in the formation of pollen wall substances is discussed.     

Nicotine Enhances the Cyclic AMP-Dependent Protein Kinase-Mediated Phosphorylation of α4 Subunits of Neuronal Nicotinic Receptors

Abstract: Studies determined whether α4β2 or α3β2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 n M for α4β2 and 500 n M for α3β2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing α4β2 receptors were incubated with [γ-³²P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the α4 subunit was present. Phosphorylation of α4 subunits of α4β2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing α3β2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the α3 subunit. Results suggest that the PKA-mediated phosphorylation of α4 and not α3 subunits may explain the differential inactivation by nicotine of these receptors subtypes expressed in oocytes.     

Adventitious regeneration of Juglans nigra L. (eastern black walnut)

Somatic embryos and adventitious shoots were initiated from immature cotyledons 10–14 weeks after anthesis. Maximum embryogenesis occurred 12 weeks after anthesis and maximum shoot organogenesis occurred 14 weeks after anthesis. The best treatment for induction of somatic embryos and adventitious shoots from immature cotyledon explants was on agar-solidified WPM supplemented with 0.1 M 2,4-D and 5.0 M TDZ and incubated in light for the first four weeks. Rooting of adventitious shoots was best if they were quickdipped in 2.5 mM IBA and 1.25 mM NAA in 1% dimethyl formamide and 3.9% ethanol (120 Wood's Rooting Compound: water, by volume). Plantlets from rooted adventitious shoots were acclimatized to the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - DKW Driver and Kuniyuki (1984) walnut medium - IBA indolebutyric acid - LP Long and Preece medium described herein - NAA naphthaleneacetic acid - PPF photosynthetic photon flux - TDZ thidiazuron - WPM Woody Plant Medium of Lloyd and McCown (1980)     

Isolation of a cDNA encoding a growth-arrest associated gene and characterization of its regulation

We are interested in understanding the molecular events associated with the growth-arrest of vascular SMCs. We constructed a subtracted cDNA library enriched in nucleotide sequences associated with quiescent SMCs. This library was screened with similarly subtracted ³²P-labeled cDNAs to identify growth-arrest associated cDNA clones. Characterization of 19 of these cDNA clones revealed that 9 hybridized to mRNAs that exhibited a 2–3 fold increase in growth-arrested SMCs. In addition, two other cDNAs hybridized to a 5 Kb mRNA that was elevated approximately 10-fold in high density growth-arrested SMCs. Genomic Southern blot hybridization and DNA sequencing analysis indicated that these cDNAs encoded the same gene (LG7) and that this gene may be a member of a multigene family or that it may contain a sequence shared by other unrelated genes. Augmented expression of LG7 was associated with both high cell density and serum deprivation induced growth-arrest. LG7 mRNA expression was down-regulated when SMCs were incubated with FBS or with reagents that arrest cells in early S-phase. Additional analysis with cell cycle specific inhibitors indicated that LG7 mRNA levels were also low when cells were blocked at the G₂ phase of the cell cycle but blockage at mitosis resulted in an elevated level of LG7 mRNA. We further demonstrated that the expression of LG7 was dependent on the presence of a relatively labile protein since protein synthesis inhibitors specifically blocked the expression of this mRNA but not the mRNA expression of α₁(III) collagen or ferritin H-chain. Finally, we demonstrated that Bt₂cAMP was able to induce mRNA expression of LG7 within 2 h, suggesting that this gene may be directly regulated via the cyclic-AMP-dependent protein kinase pathway.     

Reflective organelles in the anterior pigment epithelium of the iris of the European starling Sturnus vulgaris

The ultrastructure and chemical composition of reflective organelles in the anterior pigment epithelium of the iris of the European starling Sturnus vulgaris were examined. The reflective organelles produced a diffuse white reflectance at the iris mid-section which was visible only when the stroma was removed. The pigment granules were clear, angular, and birefringent under the light microscope. In electron micrographs the granules were irregular in shape and density, sometimes crystalline in appearance, but more often they were lost during sectioning or staining. Guanine was abundant in the modified pigment epithelium of the starling, but not in the pigment epithelia of other birds that lacked birefringent granules. Pteridines, such as xanthopterin and leucopterin, were present in small amounts. Pteridines were also present in the iris stroma which had no reflective organelles. The reflective organelles in the starling pigment epithelium resemble both the reflecting platelets of lower vertebrate chromatophores and the reflective granules in the tapeta of various vertebrates. Possible derivation of the organelles from these sources is discussed.     

Evidence for a major gene (RP10) for autosomal dominant retinitis pigmentosa on chromosome 7q: linkage mapping in a second,unrelated family

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration, which has X-linked, autosomal recessive and autosomal dominant forms. The disease genes in families with autosomal dominant retinitis pigmentosa (adRP) have been linked to six loci, on 3q, 6p, 7p, 7q, 8q and 19q. In a large American family with late-onset adRP, microsatellite markers were used to test for linkage to the loci on 3q, 6p, 7p, 7q and 8q. Linkage was found to 7q using the marker D7S480. Additional microsatellite markers from 7q were then tested. In total, five markers, D7S480, D7S514, D7S633, D7S650 and D7S677, show statistically significant evidence for link-age in this family, with a maximum two-point lod score of 5.3 at 0% recombination from D7S514. These results confirm an earlier report of linkage to an adRP locus (RP10) in an unrelated family of Spanish origin and indicate that RP10 may be a significant gene for inherited retinal degeneration. In addition, we used recently reported microsatellite markers from 7q to refine the linkage map of the RP10 locus.     

Therapy of bovine ocular squamous-cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up

We have tested the therapeutic potency of peritumorally injected low doses of interleukin-2(IL-2). Seventy tumours of the bovine ocular squamous-cell carcinoma (BOSCC), 1–3 cm in diameter, were treated with 5000, 20 000 or 200 000 U IL-2 from Eurocetus (Chiron) to find the optimal dose for treatment. Injections were given peritumorally on Monday to Friday on 2 consecutive weeks. The size of the tumours was measured before treatment and 1, 3, 4, 9 and 20 months after treatment. After 9 months complete regression was observed in 89% of the tumours treated with 5000 U IL-2, 80% treated with 20 000 U and 67% treated with 200 000 U. After 20 months, there was complete regression of 35%, 31% and 67% of the tumours respectively. The 9-and 20-month results of the 200 000-U treatment are significantly better than those of the 5000-U and 20 000-U treatments taken together. This protocol may be useful to treat advanced inoperable tumours (e.g. of the nasopharynx or skin) of human patients.