Sugarbeet leaf disc culture: an improved procedure for inducing morphogenesis

In preparation for gene transfer experiments we investigated factors that might affect the production of shoots and somatic embryos from the wound callus of cultured sugarbeet leaf discs. A complex interaction was found between the leaf disc plating density, the disc culture medium, the source-shoot culture medium and the frequency of disc transfer to fresh medium. The most productive protocol utilized: source shoots maintained on MS medium containing 0.25 mg 1^-1 BA; multiple leaf discs (ten 4-mm discs/plate) plated onto an enriched modification of MS medium (RV) containing 1.0 mg 1^-1 BA and solidified with 0.3% Gelrite (not permitted to dry during hardening); and transfer of the discs to fresh medium every two weeks during the first month. This standard protocol produced more callus per plate and higher rates of morphogenesis per unit dry weight of callus than did the one-step method of Saunders and Doley. Water availability considerations were found to be critical to obtaining high morphogenic rates. Root induction frequency and quality was superior on shoots transplanted to MS medium containing 1 mg 1^-1 NAA as the sole growth regulator compared to IAA at the same concentration.Abbreviations BA N⁶-benzyladenine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Mouse pulmonary cytochrome P-450 naphthalene hydroxylase: cDNA cloning, sequence, and expression in Saccharomyces cerevisiae.

We have isolated a cDNA clone, Nah-2, encoding the cytochrome P-450Nah (naphthalene hydroxylase) from a mouse lung lambda ZAP cDNA library using anti-cytochrome P-450Nah IgG as a probe. This same antibody selectively blocked [Nagata, K., Martin, B.M., Gillette, J.R., & Sasame, H.A. (1990) Drug Metab. Dispos. 18, 557-564] the cytochrome P-450 in mouse lung microsomes that catalyzed the conversion of naphthalene to (1R,2S)-naphthalene 1,2-oxide, which has been postulated as a causative agent in the naphthalene-induced tissue-specific necrosis of Clara cells in mouse lung. The toxic effect is seen in mouse and not in rat. The cDNA encodes a polypeptide of 491 amino acids with a molecular mass of 50 kDa. Northern blot analysis with an Nah-2-specific probe revealed that the mRNA is expressed in a species- and tissue-specific manner, present only in mouse lung and liver and not in that of rat. The mRNA encoding Nah-2 is constitutively expressed and is not induced by either phenobarbital, pyrazole, pregnenolone 16 alpha-carbonitrile, or 3-methylcholanthrene. Comparative amino acid sequence analyses with other documented members of the P-450 gene superfamily revealed that this encoded protein is in the IIF subfamily. To analyze its substrate specificity, the cDNA was inserted into the vector, pAAH5, and expressed in the Saccharomyces cerevisiae strain, AH22. The presence of cytochrome P-450Nah in the microsomes isolated from transformed cells and analyzed by Western blot was confirmed by immunocomplexing product with anti-cytochrome P450Nah IgG. Furthermore, activity toward naphthalene in the microsomes from the transformed cells established that this clone encodes a naphthalene hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypercholesterolaemia: simvastatin and pravastatin alter cholesterol metabolism by different mechanisms

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor simvastatin, reduced low-density-lipoprotein (LDL) cholesterol in hypercholesterolaemic patients by 40% (P less than 0.001). The reduction in LDL cholesterol was accompanied by a significant decrease in the esterified/free cholesterol ratio of the patients' LDL from 2.51 +/- 0.13 to 2.06 +/- 0.14 (P less than 0.01). This change led to a significant increase (P less than 0.05) in the capacity of the LDL to suppress [14C]acetate incorporation into cholesterol in mononuclear leucocytes. Furthermore, [14C]acetate incorporation into the patients mononuclear leucocytes was significantly lower (P less than 0.02) following drug treatment (117 +/- 22 vs. 162 +/- 29 nmol/mg cell protein). Comparison of simvastatin with another HMG-CoA reductase inhibitor pravastatin, showed similar reduction in LDL cholesterol. Pravastatin treatment however, did not result in a reduction in the LDL esterified/free cholesterol ratio or in the changes in cellular cholesterol synthesis and its regulation by LDL which accompanied simvastatin treatment. The activity of the enzyme acyl-coenzyme A: cholesterol acyltransferase (ACAT) in patients' mononuclear cells remained unchanged after treatment with either drug. Results of the study show that while the drugs are equally effective in lowering LDL cholesterol, simvastatin has additional compositional effects on LDL which increase its capacity to regulate mononuclear leucocyte cholesterologenesis.     

Structure-activity relationship of the neurotransmitter alpha-bag cell peptide on Aplysia LUQ neurons: Implications regarding its inactivation in the extracellular space

Alpha-bag cell peptide [α-BCP (Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu)] is a neurotransmitter that mediates bag cell-induced inhibition of left-upper-quadrant (LUQ) neurons L₂, L₃, L₄, and L₆ in the abdominal ganglion of Aplysia. Our recent biochemical studies have shown that α-BCP[1–9] is cleaved into α-BCP[1–2], [3–9], [1–5], [6–9], and [7–9] by a combination of three distinct peptidase activities located within the extracellular spaces of the CNS: A diaminopeptidase-IV (DAP-IV)-like enzyme cleaves α-BCP[1–9] at the 2–3 peptide bond; a neutral metalloendopeptidase (NEP)-like enzyme cleaves either α-BCP[1–9] or α-BCP[3–9] at the 5–6 bond; an aminopeptidase M-II (APM-II)-like enzyme cleaves α-BCP[6–9] at the 6–7 bond, but cleaves neither α-BCP[1–9], nor the other ganglionic peptidase products. To further understand the manner in which α-BCP is inactivated after release, that is loses its electro-physiological activity, we studied its structure-activity relationship by recording intracellularly from LUQ neurons in isolated abdominal ganglia that were arterially perfused with peptides dissolved in artificial sea water. The effects of α-BCP[1–9] and 15 of its fragments ([1–8], [1–7], [1–6], [1–5], [2–9], [3–9], [3–8], [6–9], [7–9], [8–9], [6–7], [6–8], [1–2], Phe, Tyr) indicated that the sequence Phe⁶-Tyr⁷ was both necessary and sufficient to produce LUQ inhibitory activity. The combined results of our electrophysiological and biochemical studies strongly suggest that α-BCP[1–9] is inactivated by the serial actions of the NEP-like and APM-II-like peptidases; that is, the NEP-like enzyme yields an electro-physiologically active product, α-BCP[6–9], that is cleaved by the APM-II-like enzyme to yield inactive α-BCP[7–9]. Furthermore, because α-BCP[6–9] is more active than α-BCP[1–9], cleavage by the NEP-like enzyme potentiates α-BCP's activity. © 1992 John Wiley & Sons, Inc.     

Fibroblast heterogeneity in glucocorticoid regulation of collagen metabolism: Genetic or epigenetic?

Summary Cultured fibroblasts derived from normal human dermis show a consistent 62% inhibition of collagen synthesis by hydrocortisone, whereas cultures derived from keloids average only 30% inhibition and show a much larger strain to strain variation ranging from 75% inhibition to 49% stimulation. Examination of fibroblast clones indicates that this high variation among keloid strains is not due to differences in the proportion of normal and keloid cells in the mass culture populations. Small but significant differences in the effect of hydrocortisone on collagen deposition are also seen among these clonal populations, but are not related to the type of tissue from which cultures were derived. Two to three-fold differences among clones derived from a single individual were observed, possibly suggesting functional heterogeneity of dermal fibroblasts with regard to collagen metabolism under control conditions and in response to hydrocortisone. However, this variation among clones may simply reflect differences in clonal growth, inasmuch as both collagen synthesis and deposition, and the effect of hydrocortisone on these processes, are strongly affected by population density. This work was supported in part by PHS grants, CA-17229 from the National Cancer Institute and AG-02046 from the National Institute on Aging, DHHS; and by Grant RIM 78-17313 from the National Science Foundation.     

A light and electron microscopic study of stigmas inAneilema andCommelina species (Commelinaceae)

Summary The stigmas of species inAneilema andCommelina are trifid and comprise elongate papillae. Progressive degeneration of papular cells is observed in stigmas from open flowers and at anthesis papillae may be moribund and collapsed. Fluid emanating from the hollow style flows onto the surface through ruptures in the cuticle at the interpapillar junctions into the interstices at maturity. This secretion stains positively for protein. Stigmas are of the wet type.The cuticle overlying the papillar cells is ridged and at the final stages prior to flowering this cuticle becomes detached from the underlying cellulosic wall. The sub-cuticular space so formed is filled with secretion. InAneilema species detachment of cuticle is at the papillar tip and along the lateral walls. InCommelina species the anticlinal walls of adjacent papillae are strongly attached for much of their length and thus detachment of cuticle is restricted to the papillar tip. The cell wall at the tip in both genera may proliferate forming a rudimentary transfer-cell type wall. The secretion is considered to be produced by the papillar cells. It is PAS positive but fails to stain for protein and in both the light and electron microscopes appears heterogenous.Pollen attachment, hydration, germination and early tube growth are very rapid following self-pollination, the pollen tubes entering the neck of the style within ten minutes of attachment.A unique character combination involving pollen and stigmas in these genera indicates a monophyletic origin.     

Characteristics of teratomas regenerated in vitro from octopine-type crown gall

Crown galls induced by infection of tobacco plants with Agrobacterium tumefaciens strain C58-Cl(pTiB6S3) were excised and cultured in vitro. After about one year of culture on medium-lacking phytohormones, two noncloned lines spontaneously formed shoots. Leaf explants from shoots of tumor-line T5 were capable of growing on hormone-free medium, and the resulting mixture of organized and unorganized tissue synthesized octopine. Detached leaves from T5 shoots also synthesized octopine. These results establish that shoots from this octopine-type tumor contain transformed cells and are true crown-gall teratomas.