Amber mutations in Escherichia coli essential genes: isolation of mutants affected in the ribosomes.

Summary A method to obtain amber mutations in ribosomal protein genes is described. It relies on the P1-mediated localized mutagenesis (Hong and Ames, 1971) and on the fact that the recipient strain contains (a) an efficient but genetically unstable suppressor, (b) a particular thermoinducible prophage which kills suppressor hosts at 42° C. Exposure of these bacteria to the high temperature yields frequent suppressor-free derivatives while none will be found if the strain carries an amber mutation in an essential gene. Eleven mutants have been isolated by this method, of which at least six appear to carry amber mutations. All of them map close to, and to the right of spcA, in a region which codes mostly for ribosomal proteins. Three mutants were studied biochemically; all three show defective ribosomal assembly in vivo upon loss of suppression.     

Syntheses of 3-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-α-d-galactopyranose (“lacto-N-biose II”) and 3,4-di-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-d-galactopyranose

3- O-(2-Acetamido-2-deoxy-β-d-glucopyranosyl)-α-d-galactopyranose (10, “Lacto-N-biose II”) was synthesized by treatment of benzyl 6-O-allyl-2,4-di-O-benzyl-β-d-galactopyranoside with 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-glucopyrano)[2,1-d]-2-oxazoline (5), followed by selective O-deallylation, O-deacetylation, and catalytic hydrogenolysis. Condensation of 5 with benzyl 6-O-allyl-2-O-benzyl-α-d-galactopyranoside, followed by removal of the protecting groups, gave 10 and a new, branched trisaccharide, 3,4-di-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-d-galactopyranose (27).     

Effects of methylazoxymethanol given at different stages of postnatal life on development of the rat brain. Comparison with those of thyroid deficiency

Newborn rats were treated at different stages of their development with low doses of methylazoxymethanol acetate. The postnatal increase of the DNA content of the cerebrum did not differ from that of controls. In the cerebellum, the DNA content was transitorily reduced, but later, the external granular layer became thicker and DNA deposition increased in comparison with controls; finally, the cerebellar DNA returned to a normal value. Morphological abnormalities of the cerebellum, abnormal orientation of migrating cells, scattering of Purkinje cell bodies within the internal granule cells and specially striking abnormalities of the morphology and orientation of Purkinje cell dendrites were noted in rats treated with MAM from birth to day 3. The effects on the Purkinje cell morphogenesis persisted but were much less marked when MAM was given from 4 to 7 or from 8 to 11 days. Neonatal thyroid deficiency, as MAM-treatment between days 0 and 3, leads to an abnormal position of Purkinje cell bodies within the cerebellar cortex; it also leads to morphological abnormalities of their dendritic arborization which closely resemble those observed after MAM-treatment during the second postnatal week. It also alters the cell formation in the cerebellum. Thyroid deficiency probably exerts its effect on cell formation earlier than previous biochemical studies have shown. On another hand, the morphological abnormalities of Purkinje cell arborizations in the thyroid-deficient animals may be partly due to the perturbations of cell formation which persist later in the cerebellum.     

Assessment of interleukin 1 and interleukin 2 effects on cycling and noncycling murine thymocytes

When mouse thymocytes are stimulated with PHA, the proliferative response is very low, unless the culture medium is enriched with interleukin 1 (IL-1)- or interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analyses show, however, that PHA stimulation generates a significant number of cells with increased RNA content (transition from the G₀ to G₁ phase of the cell cycle). If IL-2 is added to such cultures, the activated cells complete their process of RNA synthesis and then enter the S phase. The use of IL-2-containing culture medium thus permits one to obtain a high correlation between the number of g₁ cells and [³H]thymidine incorporation (r = 0.97). Enrichment with IL-1-containing supernatants also results in a statistically significant correlation (r = 0.68), but the regression lines are markedly different for the two interleukins (s = 20.3 for IL-2 and s = 9.2 for IL-1), when analyzed after 48 hr of incubation. These observations suggest that the G₁ phase must be divided into two subcompartments, G_1a and G_1b, the G_1a-G_1b transition being an IL-2-dependent event. If the number of G_1b cells is used to establish correlations with [³H]thymidine incorporation, all values fall on the same regression line, regardless of culture conditions and of the addition of interleukins. It is concluded that IL-2 regulates lymphocyte proliferation at the level of RNA synthesis (G_1a-G_1b transition) rather than that of DNA synthesis (G₁-S transition).     

The effect of electrochemical regeneration upon the enzymatic catalysis of a thermodynamically unfavorable reaction

The association between enzymatic and electrochemical reactions, enzymatic electrocatalysis, had proven to be a very powerful tooth in both analytical and synthetic fields. However, most of the combinations studied have involved enzymatic catalysis of irreversible or quasi-irreversible reaction. In the present work, we have investigated the possibility of applying enzymatic electrocatalysis to a case where the electrochemical reaction drives a thermodynamically unfavorable reversible reaction. Such thermodynamically unfavorable reactions include most of the oxidations catalyzed by dehydrogenases. Yeast alcohol dehydrogenase (E.C. 1.1.1.1) was chosen as a model enzyme because the oxidation of ethanol is thermodynamically very unfavorable and because its kinetics are well known. The electrochemical reaction was the oxidation of NADH which is particularly attractive as a method of cofactor regeneration. Both the electrochemical and enzymatic reactions occur in the same batch reactor in such a way that electrical energy is the only external driving force. Two cases were experimentally and theoretically developed with the enzyme either in solution or immobilized onto the electrode's surface. In both cases, the electrochemical reaction could drive the enzymatic reaction by NADH consumption in solution or directly in the enzyme's microenvironment. However even for a high efficiency of NADH consumption, the rate of enzymatic catalysis was limited by product (acetaldedehyde) inhibition. Extending this observation to the subject of organic synthesis catalyzed by dehydrogenases, we concluded that thermodynamically unfavorable reaction and can only be used in a process if efficient NAD regeneration and product elimination are simultaneously carried out within the reactor.     

v-myb Transformation of Xeroderma pigmentosum human fibroblasts: Overexpression of the c-Ha-ras oncogene in the transformed cells

Human Xeroderma pigmentosum “normal” fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental “normal” AS16 cells and a revertant clone (ASKXA Cl 1.1 G). Our results lead to the conclusion that the XP fibroblasts are phenotipically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.     

Anatomy and ultrastructure of the proboscis in Mesorhynchus terminostylis (Platyhelminthes,Rhabdocoela)

The ultrastructural organization of the proboscis in Mesorhynchus terminostylis is distinctly different from that in other members of the Polycystididae in which it is currently classified. The sheath epithelium is formed by three belts, all with intra-epithelial nuclei. The apical belt of the bipartite cone epithelium has a single intrabulbar nucleus, and the basal belt possesses five insunk nucleiferous cell parts behind the bulb. Six types of glands surface through the epithelia; the three types emerging through the cone epithelium can be homologized with those described for Polycistis naegelii. Only uniciliary receptors are found in the epithelium. The musculature in the bulb has a very loose appearance, and the bulbar septum appears to be a bipartite basement membrane. The septum can be considered the basement membrane of the cone epithelium as if the contractile portion of the inner longitudinal muscles have invaded the epithelium and come to lie between the epithelial cells and the basement membrane. Thus the inner musculature of the bulb is entirely intraepithelial as is the case for Psammorhynchus tubulipenis and Cytocystis clitellatus. The systematic position of M. terminostylisremains uncertain but seems to lie between Psammorhynchus and Cytocystis on one hand and Koinocystididae and Polycystididae on the other.     

Steroid glucuronides: Human circulatory levels and formation by LNCaP cells

We studied the relationship between circulating androsterone glucuronide, androstane-3,17β-diol glucuronide and androstane-3β,17β-diol glucuronide concentrations and adrenal as well as testicular C-19 steroids in men. Among the three 5-reduced steroid glucuronides, androsterone glucuronide is the predominant C-19 steroid measured in plasma and its levels are markedly elevated compared to those of the non-conjugated steroid. The marked rise in testosterone during puberty was strongly correlated with the increase in both androsterone glucuronide and androstane-3,17β-diol glucuronide, thus suggesting that testicular C-19 steroids are the main precursors of the steroid glucuronides. We also found that the presence of testicular androgen in plasma contributes to approx. 70% of plasma androsterone glucuronide and androstane-3,17β-diol glucuronide. Our data suggest that the adrenal C-19 steroids remaining in circulation after castration in men are converted into potent androgen which are then glucuronidated by UDP-glucuronyltransferase. We also demonstrated that the human prostate cell line LNCaP is capable of converting to a large extent androstenedione into androsterone glucuronide. Our data further confirm that glucuronidation is a major pathway of steroid metabolism in steroid target tissues.     

DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin.

Summary We have determined the sequence of the lamB gene from Klebsiella pneumoniae. It encodes the precursor to the LamB protein, a 429 amino acid polypeptide with maltoporin function. Comparison with the Escherichia coli LamB protein reveals a high degree of homology, with 325 residues strictly identical. The N-terminal third of the protein is the most conserved part of the molecule (1 change in the signal sequence, and 13 changes up to residue 146 of the mature protein). Differences between the two mature proteins are clustered mainly in six regions comprising residues 145–167, 173–187, 197–226, 237–300, 311–329, and 367–387 (K. pneumoniae LamB sequence). The most important changes were found in regions predicted by the two-dimensional model of LamB folding to form loops on the cell surface. In vivo maltose and maltodextrin transport properties of E. coli K 12 and K. pneumoniae strains were identical. However, none of the E. coli K12 LamB-specific phages was able to plaque onto K. pneumoniae. Native K. pneumoniae LamB protein forms highly stable trimers. The protein could be purified by affinity chromatography on starch-Sepharose as efficiently as the E. coli K12 LamB protein, indicating a conservation of the binding site for dextrins. However, none of the monoclonal antibodies directed against native E. coli K12 LamB protein recognized native purified K. pneumoniae LamB protein. These data indicate that most of the variability occurs within exposed regions of the protein and provide additional support for the proposed model of LamB folding. The fact that the N-terminal third of the protein is highly conserved is in agreement with the idea that it is part of, or constitutes, the pore domain located within the transmembranous channel and that it is not accessible from the cell surface.     

Hormonal effects of zinc on growth in children

Zinc deficiency impairs the metabolism of thyroid hormones, androgens, and above all growth hormones. In view of their important role in growth, it is not surprising to find growth disorders associated with zinc deficiency. Stunted growth linked to zinc deficiency is found during gestation, and also in the newborn and children up to adolescence. Depending on the country, 5–30% of children suffer from moderate zinc deficiency, responsible for small-for-age height. Zinc supplementation has proven effective in many studies, mainly in children where zinc deficiency has first been found. Finally, zinc supplementation makes it possible in certain cases to overcome resistance to growth hormone treatment.