Biological characterization of purified native 20-kDa human growth hormone

Because of the propensity of the 20-kDa variant of human growth hormone (GH) to aggregate with itself and with 22-kDa human GH, it has been difficult to prepare monomeric 20-kDa GH in highly purified form. This has been a major complicating factor in determining whether 20-kDa GH has a biological activity profile distinct from that of 22-kDa GH. In the present study, native 20-kDa GH was isolated from a human GH dimer concentrate and purified by a procedure that included column electrophoresis in agarose suspension as a final separation step. This procedure yielded highly purified monomeric 20-kDa GH, which was contaminated to an extent of less than 1% with 22-kDa GH, and which exhibited only a small degree of dimerization upon storage. The native 20-kDa GH was quite active in stimulating growth in hypophysectomized rats, when growth was assessed by body weight gain, longitudinal bone growth, the stimulation of sulfation of cartilage, and the elevation of serum IGF-1 level. However, in all of these growth assays, the 20-kDa GH was somewhat less active than the native 22-kDa GH to which it was compared; e.g., in the body weight gain and longitudinal bone growth assays, it had an estimated potency of 0.6 relative to the 22-kDa GH. The 20-kDa GH exhibited substantial diabetogenic activity when tested for the ability to raise fasting blood glucose concentration and to impair glucose tolerance in ob/ob mice. Also, the native 20-kDa GH had significant in vitro insulin-like activity, although its potency was approximately 20% that of the native 22-kDa GH to which it was compared. Thus, the biological activity profile of native 20-kDa GH differs from that of 22-kDa GH primarily in that insulin-like activity is markedly attenuated.     

31P nuclear magnetic resonance study of the effect of azide on xylose fermentation by Candida tropicalis

Maximal ethanol production by Candida tropicalis grown on xylose was obtained at an oxygen transfer rate of 5 to 7 mmol/liter per h. Addition of 0.2 mM azide increased the ethanol yield by a factor of 3 to 4, based on the cell mass produced, and decreased the formation of the by-product xylitol by 80%. In the presence of azide, ethanol was reassimilated before the carbon source was depleted. At all oxygenation levels studied, azide caused 25 to 60% of the carbon to be lost, most probably as carbon dioxide. Identical spectra were obtained with 31P nuclear magnetic resonance spectroscopy performed on extracts of C. tropicalis grown on xylose in the absence and presence of azide. Azide lowered the levels of sugar phosphates. Enzymatic analysis showed extremely low levels of fructose 1,6-diphosphate compared with the levels obtained in the absence of azide, while the level of malate, a citric acid cycle intermediate, was not influenced by azide. 31P nuclear magnetic resonance spectroscopy performed on xylose-grown whole cells of C. tropicalis showed that azide lowered the intracellular pH, inhibited the uptake of external Pi, and decreased the buildup of polyphosphate in relation to results with untreated cells. Similar results were obtained with the uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), except that CCCP treatment led to extremely high levels of internal Pi. The dual effect of azide as a respiratory inhibitor and as an uncoupler is discussed with respect to the metabolism and product formation in xylose-assimilating C. tropicalis.     

Flower formation in excised tobacco stem segments; I. Methodology and effects of plant hormones

The formation of flowers has been studied in stem tissue excised from flowering plants of Nicotiana tabacum variety Wisconsin No. 38, and cultured in vitro on Murashige and Skoog nutrient medium. A procedure for quantitative evaluation of factors influencing floral expression has been developed and effects of the growth substances, indole-3-acetic acid (IAA), kinetin and gibberellic acid (GA₃), on the process are reported.     

Flower Formation in Excised Tobacco Stem Segments; II. Reversible Removal of IAA Inhibition by RNA Base Analogues

The RNA base analogues, 2-thiouracil, 6-azauracil and 8-azaguanine incorporated singly into the medium, increased the number of floral buds in excised stem segments of Nicotiana tabacum variety Wisconsin No. 38 cultured in vitro. Combined treatments with 2 and 3 base analogues were even more effective. The effects were prevented by the corresponding natural counterparts, uracil, uridine, and guanosine respectively. These nucleic acid constituents added to cultures without base analogues did not affect the number of floral buds formed. In stem segments from the lower internodes treatments with the analogues effected a transition from vegetative to floral bud formation, thus in a sense removing the floral gradient as defined by Chouard and Aghion.     

Cytology and immunocytochemistry of bilateral breast metastases from prostatic cancer. Report of a case

The cytologic findings in fine needle aspirates of a previously treated prostatic cancer metastatic to both breasts in a 65-year-old man are described. The prostatic origin of the poorly differentiated adenocarcinoma cells was demonstrated by identification of cytoplasmic prostate-specific antigen. The clinical importance of a conclusive diagnosis differentiating a primary from a metastatic lesion is discussed; this case illustrates the value of immunocytochemical analysis as an aid to cytomorphologic diagnosis in making such determinations.     

Cleavage of synthetic peptides by purified poliovirus 3C proteinase

Synthetic peptides, 14-16 residues in length, were used as substrates for purified recombinant poliovirus proteinase 3C. The sequences of the substrates correspond to the sequences of authentic cleavage sites in the poliovirus polyprotein, all of which contain Gln-Gly at the scissile bond. Specificity of cleavages was demonstrated by analysis of 3C digests of synthetic peptides. Relative rate constants for the cleavages were derived by competition experiments. The rate constants roughly correlated with the estimated half-life of the homologous precursor proteins detected in poliovirus-infected cells. The peptide most resistant to cleavage corresponded to the 3C/3D junction, a site known to be cleaved very slowly by 3C in vivo. Substitution of threonine for alanine in P4 position of this peptide, however, resulted in significant cleavage. This observation supports the hypothesis that the residue in P4 position, in addition to the Gln-Gly in P1 and P1', respectively, contributes to substrate recognition. Ac-Gln-Gly-NH2 was not a substrate for 3C.     

Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.