Active and passive cation transport and L antigen hertogeneity in low potassium sheep red cells

Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump.     

Cycling of organic and inorganic sulphur in a chestnut oak forest

Summary Sulfur (S) cycling in a chestnut oak forest on Walker Branch Watershed, Tennessee, was dominated by geochemical processes involving sulfate. Even though available SO ₄ ^2- was present far in excess of forest nutritional requirements, the ecosystem as a whole accumulated 60% of incoming SO₄–S. Most (90%) of this accumulation occurred by SO ₄ ^2- adsorption in sesquioxide-rich subsurface soils, with a relatively minor amount accumulating and cycling as SO ₄ ^2- within vegetative components. Organic sulfates are thought to constitute a large proportion of total S in surface soils, also, and to provide a pool of readily mineralized available S within the ecosystem.Research sponsored by Division of Biomedical and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with Union Carbide Corporation. Soil ester sulfate work sponsored by contract RP-1813-1 with the Electric Power Research Institute. Publication No. 1990, Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830     

Measurement of DNA-excision repair in suspensions of freshly isolated rat hepatocytes after exposure to some carcinogenic compounds: its possible use in carcinogenicity screening.

When suspensions of freshly isolated rat hepatocytes were exposed to a number of carcinogenic compounds, it was possible to measure an increased UDS by a rapid procedure via liquid-scintillation counting. For a number of carcinogenic compounds and some of their non-carcinogenic structural analogues a good correlation between the carcinogenic property and the ability to induce UDS was demonstrable. Out of 12 carcinogenic compounds, belonging to several different chemical classes, 10 gave rise to an increased UDS, whereas only 2 compounds, the polycyclic aromatic hydrocarbons benzo[alpha]pyrene and benz[alpha]anthracene, did not. All 4 noncarcinogenic compounds tested were negative. Possibly this method can be of value as a routine screening test, in combination with other short-term test systems, thus improving the predictive value of screening in vitro with respect to carcinogenicity.     

Valine-sensitive acetohydroxy acid synthases in Escherichia coli K-12: unique regulation modulated by multiple genetic sites.

Summary Spontaneous mutants (146) of Escherichia coli K-12 were selected that were resistant to inhibition of growth by 1.2 mM L-valine (Val^r). The Val^r isolates, containing acetohydroxy acid synthase resistant to feedback inhibition by L-valine (AHAS^r), were classed according to cotransduction of the mutation with leu. Several mutations resulting in an AHAS^r phenotype were found to be cotransducible with glyA. However, no mutations causing a Val^r phenotype were linked to ilv. AHAS activity was more closely examined in representatives of three classes of mutants with Val^r linked to leu, labeled ilv-660, ilv-661, and ilv-662. The ilvE503 allele in E. coli K-12, known to cause a two- to three-fold derepression of AHAS, was found to affect regulation of synthesis of both valine-sensitive AHAS (AHAS^s) and AHAS^r in the mutants containing ilv-660 and ilv-661, whereas it affected repression of AHAS^s, only, in the mutant containing ilv-662. Further, both AHAS^s and AHAS^r in the ilv-661 mutant were repressed by valine, whereas valine did not repress AHAS^r synthesis in the strain carrying ilv-660 and only partially repressed AHAS^r in the strain carrying ilv-662. Unexpectedly, AHAS^r synthesis in strains carrying ilv-660 or ilv-662 was repressible by leucine. The ilv-660 locus appears to be similar in position to ilvH and encodes a product that confers valine-sensitivity upon AHAS activity in the wild-type E. coli K-12. The ilv-660 and ilv-662 loci may normally encode products that influence both the feedback sensitivity of AHAS and control of AHAS biosynthesis.     

Assessment of the technical quality of electrocardiograms.

The technical quality of 600 electrocardiograms (ECG''s) was assessed for missing leads and clipping, and graded from 1 to 5 for each of noise, lead drift and beat-to-beat drift. Three subgroups of 200 ECGs each were studied: group A, those obtained by emergency department staff (non-technicians); group B, records obtained by ECG technicians; and group C, telephone-transmitted records obtained by technicians performing all the laboratory work at a smaller, outlying hospital. Records with missing leads, clipping, grade 4 or 5 noise, grade 5 lead drift or grade 5 beat-to-beat drift were classified as unsatisfactory or rejected. With these stringent criteria the rejection rate was 71.0% for group A records, 58.5% for group B and 44.5% for group C. The proportions of records with peak quality (no missing leads or clipping, and grade 1 noise, lead drift or beat-to-beat drift) were 4.5% for group A, 5.5% for group B and 23.0% for group C. Suggested revisions in the grading of technical quality of ECGs are presented.     

The effects of alterations of transmembrane Na+ and K+ gradients by ionophores (nigericin,monensin) on serotonin transport in human blood platelets

Ionophores (monensin, nigericin) capable of transporting both Na⁺ and K⁺ across cell membranes down their concentration gradients reduce the rate and total magnitude of serotonin uptake by platelets. The effect of the ionophores was time dependent, so that inhibition increased progressively until eventually uptake ceased entirely. Nigericin and monensin produced loss of platelet K⁺ and an equivalent molar uptake of Na⁺ thereby abolishing the normal transmembrane Na⁺ and K⁺ gradients. The time course of these ionophore-induced cation shifts at 37° C corresponded to the rate at which inhibition of serotonin transport developed. The ionophores did not affect total ATP concentration of platelets nor the metabolic pool of ATP formed from [¹⁴C] adenine. Nigericin and monensin released about 80% of platelet ¹⁴C and endogenous serotonin over a 30 min period, without release of platelet adenine nucleotides, calcium or β-glucuronidase. The ionophores did not elicit platelet aggregation nor did they prevent maximal aggregation brought about by ADP, collagen or A23187. Replacement of Na⁺ in the medium by K⁺ abolished serotonin uptake but only 10–20% of endogenous serotonin was released. In KCl medium the Na⁺ gradient was initially reversed, but quickly dissipated as Na⁺ reequilibrated with the extracellular fluid. At 37° C the ionophores did not affect either the rate of Na⁺ reequilibration or the efflux of [¹⁴C] serotonin. Na⁺ reequilibration was slower at 20° C and the ionophores significantly increased platelet Na⁺ loss and strongly inhibited the efflux of [¹⁴C] serotonin. The data support a mechanism of serotonin transport due to a Na⁺-dependent carrier-mediated process which need not be directly dependent on metabolic energy, but which does require metabolic energy to maintain normal $\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$ gradients.     

Guanidine hydrochloride-induced shedding of a Dictyostelium discoideum plasma membrane fraction enriched in the cyclic adenosine 3',5'-monophosphate receptor

The cell surface cyclic AMP receptor of Dictyostelium discoideum is under study in a number of laboratories with respect to both its role in development of the organism and the physiology of excitation-response coupling. We report here that when starved amoebae are exposed to the chaotrope guanidine hydrochloride at 1.8 M, they shed a particulate cyclic AMP binding activity into the medium. This activity is due to membrane vesicles which originate from the cell surface. The vesicles are enriched up to 150-fold in cyclic AMP binding activity and up to 14-fold in phospholipid content when compared to the starting amoebae. The cyclic AMP binding activity of the membrane vesicles is identical to that of the cell surface receptor with respect to the following properties; (i) it is lacking in preparations from unstarved, vegetative amoebae; (ii) it is not inhibited by cyclic GMP and is stimulated by calcium ions; (iii) it has very rapid rates of association and dissociation of bound cyclic AMP; (iv) it has two classes of binding sites with dissociation constants similar to those of the surface receptors of whole amoebae. The binding activity of the isolated membranes is stable for several days at 4 degrees C and the lower affinity binding sites are stable up to several months when stored at -80 degrees C. Due to enrichment and stability of the receptor in this preparation, it should be highly suitable for many types of studies. The usefulness is enhanced by the fact that the preparation does not contain detectable cyclic AMP phosphodiesterase activity.     

Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences

Five distinct patterns of DNA replication have been identified during S-phase in asynchronous and synchronous cultures of mammalian cells by conventional fluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. During early S-phase, replicating DNA (as identified by 5-bromodeoxyuridine incorporation) appears to be distributed at sites throughout the nucleoplasm, excluding the nucleolus. In CHO cells, this pattern of replication peaks at 30 min into S-phase and is consistent with the localization of euchromatin. As S-phase continues, replication of euchromatin decreases and the peripheral regions of heterochromatin begin to replicate. This pattern of replication peaks at 2 h into S-phase. At 5 h, perinucleolar chromatin as well as peripheral areas of heterochromatin peak in replication. 7 h into S-phase interconnecting patches of electron-dense chromatin replicate. At the end of S-phase (9 h), replication occurs at a few large regions of electron-dense chromatin. Similar or identical patterns have been identified in a variety of mammalian cell types. The replication of specific chromosomal regions within the context of the BrdU-labeling patterns has been examined on an hourly basis in synchronized HeLa cells. Double labeling of DNA replication sites and chromosome-specific alpha-satellite DNA sequences indicates that the alpha-satellite DNA replicates during mid S-phase (characterized by the third pattern of replication) in a variety of human cell types. Our data demonstrates that specific DNA sequences replicate at spatially and temporally defined points during the cell cycle and supports a spatially dynamic model of DNA replication.