High resolution nuclear magnetic resonance study of base pairing in four purified transfer RNA molecules

The high resolution nuclear magnetic resonance spectra of hydrogen bonded protons in four purified tRNA molecules are reported. From the temperature and concentration dependence it is shown that these resonances arise from intramolecular hydrogen bonds.     

31P NMR saturation-transfer measurements in Saccharomyces cerevisiae: characterization of phosphate exchange reactions by iodoacetate and antimycin A inhibition

31P nuclear magnetic resonance (NMR) saturation-transfer (ST) techniques have been used to measure steady-state flows through phosphate-adenosine 5'-triphosphate (ATP) exchange reactions in glucose-grown derepressed yeast. Our results have revealed that the reactions catalyzed by glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK) and by the mitochondrial ATPase contribute to the observed ST. Contributions from these reactions were evaluated by performing ST studies under various metabolic conditions in the presence and absence of either iodoacetate, a specific inhibitor of GAPDH, or the respiratory chain inhibitor antimycin A. Intracellular phosphate (Pi) longitudinal relaxation times were determined by performing inversion recovery experiments during steady-state ATP gamma saturation and were used in combination with ST data to determine Pi consumption rates. 13C NMR and O2 electrode measurements were also conducted to monitor changes in rates of glucose consumption and O2 consumption, respectively, under the various metabolic conditions examined. Our results suggest that GAPDH/PGK-catalyzed Pi-ATP exchange is responsible for antimycin-resistant saturation transfer observed in anaerobic and aerobic glucose-fed yeast. Kinetics through GAPDH/PGK were found to depend on metabolic conditions. The coupled system appears to operate in a unidirectional manner during anaerobic glucose metabolism and bidirectionally when the cells are respiring on exogenously supplied ethanol. Additionally, mitochondrial ATPase activity appears to be responsible for the transfer observed in iodoacetate-treated aerobic cells supplied with either glucose or ethanol, with synthesis of ATP occurring unidirectionally.     

Measurement of intracellular sodium concentration and sodium transport in Escherichia coli by 23Na nuclear magnetic resonance

Escherichia coli is known to actively extrude sodium ions, but little is known concerning the concentration gradient it can develop. We report here simultaneous measurements, by 23Na NMR, of intracellular and extracellular Na+ concentrations of E. coli cells before and after energization. 23Na spectra in the presence of a paramagnetic shift reagent (dysprosium tripolyphosphate) consisted of two resonances, an unshifted one corresponding to intracellular Na+ and a shifted one corresponding to Na+ in the extracellular medium, including the periplasm. Extracellular Na+ was found to be completely visible despite the presence of a broad component in its resonance; intracellular Na+ was only 45% visible. Measurements of Na+ were made under aerobic and glycolytic conditions. Na+ extrusion and maintenance of a stable low intracellular Na+ concentration were found to correlate with the development and maintenance of proton motive force, a result that is consistent with proton-driven Na+/H+ exchange as a means of Na+ transport. In both respiring and glycolyzing cells, at an extracellular Na+ concentration of 100 mM, the intracellular Na+ concentration observed (4 mM) corresponded to an inwardly directed Na+ gradient with a concentration ratio of about 25. The kinetics of Na+ transport suggest that rapid extrusion of Na+ against its electrochemical gradient may be regulated by proton motive force or intracellular pH.     

High Bulk Diet for Diverticular Disease of the Colon

In the past century diverticular disease of the colon has changed from being almost unknown to becoming the most common disease of the colon. Studies in Britain indicated that the pathological basis of the disease is a thickening of the colonic musculature, with diverticulosis and diverticulitis developing because of increased intracolonic pressures generated by the thickened colon wall. This pressure can be sharply reduced by increased colonic bulk. Geographical and anthropological data reveal that diverticular disease results from Western civilization''s food habits, specifically the reduced fiber content in food. There is evidence that increasing the dietary intake of fiber by the addition of bran can prevent formation of diverticula and relieve the symptoms of established disease. Large scale studies are recommended both as treatment and to further test the validity of this concept.     

Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses. Received: 8 January 1996 / Revised: 22 March 1996     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Mass mortality of Diadema antillarum on the Caribbean coast of Panama

The ecologically important sea urchin Diadema antillarum suffered mass mortalities in 1983, first noted in Panama and then reported from the rest of the Caribbean. We documented the effects of this mortality at two localities on the Atlantic coast of Panama, Punta Galeta and the San Blas Archipelago. At Punta Galeta, affected by the mortality in January 1983, the numbers of D. antillarum changed from an estimated 14,000 per ha in June 1982 to 0.5 per ha in May 1983; by February 1984 they had increased to 38 per ha. In the San Blas, where mass mortality started in April 1983, the number of D. antillarum in permanent quadrats on 8 reefs was reduced by an average of 94.2%. The average reduction in population density measured in transects on nine reefs was 98.9%. Data taken in permanent quadrats on four reefs in 1978, 1979 and 1980 indicate that population fluctuations of D. antillarum are normally much smaller, justifying the labeling of the 1983 event as mass mortality. Size structure of the San Blas populations was also affected; mean test diameter of D. antillarum on four reefs was reduced from 48.6 mm to 25.0 mm. Other echinoids (Echinometra viridis, E. lucunter, Lytechinus variegatus, L. williamsi, Eucidaris tribuloides, Tripneustes ventricosus, Clypeaster rosaceus and Echinoneus cyclostomus) suffered no ill effects at either Galeta or the San Blas; their population densities remained stable or increased. Density determinations of Diadema mexicanum at the island of Taboguilla on the Pacific side of Panama indicate that Diadema mass mortality did not extend to the eastern Pacific. Sea surface temperatures, tidal levels, rainfall and salinity showed no abnormal fluctuations during the time of D. antillarum mass mortality at Galeta, suggesting that mortality was not due to physical stress. The wide geographical spread and species-specificity of the mortality suggest a water-borne pathogen as the most likely causative agent. Recovery of D. antillarum populations is likely to be slow because there are few, if any, unaffected populations in the Caribbean to contribute larvae for the recolonization of depleted areas. The absence of D. antillarum will probably be reflected by changes in the algal, coral and echinoid communities, and by altered patterns of bioerosion.