Senescence-induced alteration in cell surface carbohydrates correlated using proton NMR spectroscopy and a lectin-based affinity-binding assay

Changes in the cell surface oligosaccharides in human fetal lung fibroblasts (IMR-90) are studied as the cells progress to senescence using nuclear magnetic resonance spectroscopy (NMR) and a biochemical assay. A lectin-based affinity-binding technique is used which measures the organization of carbohydrates on the cell surface. Proton NMR studies of the water in samples of frozen cell suspensions of young and old cells provide information on the local dynamics of the cell surface by monitoring the motion of bound water. Changes in the lectin binding density and affinity class distribution correlate with a decrease in the water proton linewidth in frozen cells. These observations reflect alterations in the conformation or structure of the cell surface oligosaccharides and local constituent water.     

Generation of nitric oxide by human neutrophils

Human neutrophils were evaluated for their ability to generate nitric oxide. Neutrophils incubated with superoxide dismutase at 37 degrees C produce nitrite anion at a rate of 1.8 nmols/2 x 10(6) cells/30 min, providing indirect evidence of nitric oxide production. Incubation of the neutrophils with concentrations of serum-opsonized zymosan, N-formyl-methionyl-leucyl-phenylalanine, or phorbol myristate acetate sufficient to stimulate the respiratory burst and lysosomal enzyme release caused no additional nitrite anion production. Glass wool-adherent neutrophils exhibited a similar dissociation of nitrite anion production from the respiratory burst and lysosomal enzyme release. Direct evidence for nitric oxide production was also obtained using nitric oxide-specific chemiluminescence. These results demonstrate that human neutrophils are capable of generating nitric oxide.     

The phagovar variability of Listeria strains under the influence of virulent and temperate bacteriophages

Selected strains of Listeria spp. were challenged against a variety of bacteriophages usually employed for phage typing. The resistant mutants derived were characterized by the loss of sensitivity to defined groups of phages. When different phages were used in succession multiple mutants could be obtained. They eventually became insensitive to all phages employed. Our results indicate the possibility of shifting phagovars among Listeria strains grown in mixed culture, due to the potential action of free bacteriophages.     

Long term oscillation in glycolysis

To increase the period of glycolytic oscillations in yeast extracts (Saccharomyces uvarum), the dependence of the period on pH, on concentrations of phosphate and enzymes, and on temperature has been studied. Stable oscillatory trans were obtained at a pH value of about 6.5. Increasing the phosphate and decreasing the enzyme concentrations as well as decreasing temperature lengthened the period. By dilution of the extract with buffer while maintaining the metabolite concentrations at their initial level the period could be successively prolonged from 20 min to about 6 h.     

Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

Evolution of crystallins: expression of lens-specific proteins in the blind mammals mole (Talpa europaea) and mole rat (Spalax ehrenbergi)

The mole (Talpa europaea; Insectivora) and the mole rat (Spalax ehrenbergi; Rodentia) both have degenerated eyes as a convergent adaptation to subterranean life. The rudimentary eye lenses of these blind mammals no longer function in a visual process. The crystallin genes, which display a lens-specific expression pattern, were studied in these blind mammals and in related species with normal eyes by hybridizing their genomic DNAs with probes obtained from cDNA clones for alpha A-, alpha B-, and beta Bp-crystallins from calf and gamma 3- crystallin from the rat. For all crystallin genes examined, the hybridization signals of mole and mole rat genomic DNA were comparable, respectively, with those of shrew and of rat and mouse, normal-vision representatives of the orders Insectivora and Rodentia. The expression of the crystallins at the protein level was tested by using antiserum specific for alpha-crystallin in immunofluorescence reactions on lens sections of mole and mole rat eyes and by using antisera against the beta- and gamma-crystallins on sections of the mole eye. All antisera gave positive fluorescence reactions exclusively with lens tissue of these blind mammals, indicating that the crystallins are still normally expressed despite the fact that these lenses have had no function in a visual process in these mammals for at least many million years. These findings apparently imply that some unknown selective advantage has conserved the crystallin genes and their expression after the loss of normal function of the lenses.      

Induction and activity of NO synthase in bone-marrow-derived macrophages are independent of Ca2+.

The aim of the present study was to analyse whether an increase in the intracellular free Ca2+ concentration ([Ca2+]i) plays a role as a signal mediating synthesis of nitric oxide (NO) in bone-marrow-derived macrophages, either by stimulating induction of NO synthase or by regulating the activity of the enzyme. Therefore we compared the effects of various synthetic analogues of bacterial lipopeptide and of lipopolysaccharide (LPS) on NO production (assessed as nitrite formation during an incubation for 24 h) and on [Ca2+]i [measured with the fluorescent probe indo-1 (1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2- 2-(2'-amino-5'-methylphenoxy)ethane-NNN'N'-tetra-acetic acid)]. Strongly dissociating effects were evoked on nitrite formation and on [Ca2+]i by the stimuli. LPS was preferentially effective on nitrite formation, whereas the Ca2+ ionophore ionomycin and AlF3 induced increases only in [Ca2+]i. The lipopeptides N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-(R)-cysteinylalanylglycine, N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)- (2RS)-propyl]-(R)-cysteinylseryl-lysyl-lysyl-lysine and (S)-(1,2- dicarboxyhexadecyl)ethyl-N-palmitoylcysteinylseryl-lysyl-lys yl-lysine stimulated both parameters, but the maximal effects on nitrite formation and the shape of the dose-response curves did not parallel the effects on [Ca2+]i. Reduction of extracellular Ca2+ with EGTA significantly inhibited increases in [Ca2+]i, but did not change nitrite formation. Furthermore, NO synthesis in the cytosolic fraction of stimulated macrophages was not affected by Ca2+ over the concentration range 10 nM-2 microM. We conclude that increases in [Ca2+]i are not required for NO production in bone-marrow-derived macrophages. Thus the cellular regulation of NO production strikingly differs from that in the vascular endothelium, brain and adrenal gland.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.