Telomere end-replication problem and cell aging.

Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.     

Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60)

The nuclear sugar-binding components (i.e., lectinlike molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.     

Reverse-phase high-pressure liquid chromatography of uridine diphosphate N-acetylmuramyl peptide precursors of bacterial cell wall peptidoglycan

A series of seven different UDP-N-acetylmuramyl peptide precursors of bacterial cell wall peptidoglycan was examined by reverse-phase high-pressure liquid chromatography. Mixtures of these compounds were successfully and rapidly analyzed by using a Waters μBondapak C₁₈ column as a stationary phase and isocratic elutions with 0.05 m ammonium phosphate or formate buffers of appopriate pH. Furthermore, their accurate quantitation could also be readily achieved by reverse-phase high-pressure liquid chromatography. All these techniques should be extremely useful for the purification of these compounds and for a wide range of biochemical studies concerning the cytoplasmic steps of the biosynthesis of peptidoglycan.     

Regeneration of NADH with immobilized systems of alanine dehydrogenase and hydrogen dehydrogenase

Summary An alternative approach to the regeneration of coenzymes using immobilized hydrogen dehydrogenase (hydrogenase) is described. Hydrogenase isolated from Alcaligenes Eutrophus was immobilized to porous glass particles and used in combination with alanine dehydrogenase for formation of alanine, while the NADH consumed was regenerated by molecular hydrogen. Different physical arrangements of the two enzymes were compared. Alanine was conveniently assayed with a specially designed enzyme thermistor method.     

Organic acid production from CO2/H2 and CO/H2 by mixed-culture anaerobes

The gases CO, CO₂, and H₂ were used as substrates in anaerobic fermentations producing organic acids. Various mixed bacterial sources were used, including sewage sludge digester effluent, rabbit feces, and soil. Nonsterile microorganism selection was carried out using CO₂/H₂ and CO/H₂ as the primary carbon and energy sources. Cultures were grown in specially designed, high-pressure (to 70 psig) flasks. Methanogenic bacteria were eliminated from the cultures. Liquid products of the fermentations were acetic through caproic acids, with the even-numbered acids predominating. Carbon balances showed conclusively that acetic acid was formed from carbon contained in the CO or CO₂ feed gas. Measurements made included rates of acid product formation, cell density, and degree of gas utilization. Limited characterization of the microorganisms was also performed. Production of organic acids by mixed culture inocula from CO₂/H₂ or CO/H₂ had not been reported previously. Application of this work is to the production of organic chemicals from synthesis gas (SNG), produced by the gasification of fossil fuels (peat, lignite, and various ranks of coals), biomass (agricultural and forest residues, and various biomass crops grown expressly for energy recovery), and municipal solid waste.     

L'Ichthyofaune des marnes messiniennes des environs de senigallia (Marche,Italie): Signification paleoecologique et paleogeographique

The revision of the fish fauna from the Messinian marls of Senigallia (Marche, Italy) leads to the reduction of the faunal list of the fossil Fishes found in this locality to only four species. One new genus is created: Cryptolebias new. gen.. This fish fauna characterizes an environment with a variable salt content: it may have been more or less temporarily connected with the sea, although it was also exposed to noticeable continental influences, as demonstrated by the occurrence of Amphibians and Reptiles (Ophidians).     

Biosynthesis of sesquiterpenes in Hymenaea inferred from their quantitative co-occurrence

Leaf pocket resins of 11 species of the tropical arborescent genus Hymenaea are virtually identical qualitatively, but of widely varying quantitative proportions. Within this large range of variability, several strong positive quantitative correlations between resin constituents were found, especially between caryophyllene and β-humulene and between γ-muurolene and δ-cadinene. These data lead to clarification of sesquiterpene biosynthetic routes in Hymenaea. In addition, quantitative relationships found among caryophyllene, α- and β-selinene, γ-muurolene and δ-cadinene are explained only with difficulty by long accepted biosynthetic pathways, and the intermediacy of germacrenes is suggested.