Inactivation kinetics and pharmacology distinguish two calcium currents in mouse pancreatic B-cells

Summary Voltage-dependent calcium currents were studied in cultured adult mouse pancreatic B-cells using the whole-cell voltage-clamp technique. When calcium currents were elicited with 10-sec depolarizing command pulses, the time course of inactivation was well fit by the sum of two exponentials. The more rapidlyinactivating component had a time constant of 75±5 msec at 0 mV and displayed both calcium influx- and voltage-dependent inactivation, while the more slowly-inanctivating component had a time constant of 2750±280 msec at 0 mV and inactivated primarily via voltage. The fast component was subject to greater steady-state inactivation at holding potentials between –100 and –40 mV and activated at a lower voltage threshold. This component was also significantly reduced by nimodipine (0.5 m) when a holding potential of –100 mV was used, whereas the slow component was unaffected. In contrast, the slow component was greatly increased by replacing external calcium with barium, while the fast component was unchanged. Cadmium (1–10 m) displayed a voltage-dependent block of calcium currents consistent with a greater effect on the high-threshold, more-slowly inactivating component. Taken together, the data suggest that cultured mouse B-cells, as with other insulin-secreting cells we have studied, possess at least two distinct calcium currents. The physiological significance of two calcium currents having distinct kinetic and steady-state inactivation characteristics for B-cell burst firing and insulin secretion is discussed.     

Spermatogenesis in XY, XYSxra and XOSxra mice: a quantitative analysis of spermatogenesis throughout puberty

Adult XYSxra mice exhibit varying degrees of spermatogenic deficiency but are usually fertile, while XOSxra mice have severe spermatogenic failure and are always sterile. The present quantitative spermatogenic analysis documents when these anomalies first appear during puberty. The results demonstrate that in XYSxra mice there was increased degeneration of pachytene spermatocytes and, to a lesser extent, meiotic metaphase stages. On average, there were only one-half the number of spermatids compared with the XY controls. The defect in XOSxra mice appeared a little later, with an almost complete arrest and degeneration during the meiotic metaphases, so that the number of spermatids produced was only 3% of the control value. These results are discussed in relation to an hypothesis that links sex chromosome univalence during meiotic prophase with spermatogenic failure.     

Slow voltage inactivation of Ca2+ currents and bursting mechanisms for the mouse pancreatic beta-cell

Summary Recent whole-cell electrophysiological data concerning the properties of the Ca²⁺ currents in mouse -cells are fitted by a two-current model of Ca²⁺ channel kinetics. When the -cell K⁺ currents are added to this model, only large modifications of the measured Ca²⁺ currents will reproduce the bursting pattern normally observed in mouse islets. However, when the measured Ca²⁺ currents are modified only slightly and used in conjunction with a K⁺ conductance that can be modulated dynamically by ATP concentration, reasonable bursting is obtained. Under these conditions it is the K-ATP conductance, rather than the slow voltage inactivation of the Ca²⁺ current, that determines the interburst interval. We find that this latter model can be reconciled with experiments that limit the possible periodic variation of the K-ATP conductance and with recent observations of intracellular Ca²⁺ bursting in isletsThis work was supported in part by NSF grant DIR-90-06104 and the Agricultural Experiment Station of the University of California. P.S. gratefully acknowledges financial support from an NRC Fellowship. We have benefited from numerous conversations with Drs. John Rinzel, Arthur Sherman, Daniel Cook, and Leslie Satin     

Promoter methylation and progressive transgene inactivation inArabidopsis

Agrobacterium-transformedArabidopsis plants were generated and the stability of their T-DNA-encoded resistance to kanamycin was examined. Of seven families, each homozygous for a single insertion event, two showed progressive inactivation of resistance over four generations of inbreeding. Loss of resistance was associated with methylation of anSst II site in thenos promoter of the kanamycin resistance gene. Treatment of plant roots from inactive lines with the demethylating agent 5-azacytidine restored the ability of such lines to form callus on kanamycin-containing media. These observations are consistent with the view that methylation is a factor in the progressive inactivation of transgenes inArabidopsis.     

Ploidy reduction and genome segregation in cultured carrot cell lines. I. Prophase chromosome reduction

Cytological analysis of different carrot cell lines in culture has shown various cytogenetic anomalies generating new levels of ploidy and novel chromosome numbers. Polyploidy may be considered a reservoir of variability that can be released in the form of distinct new segregants of different ploidy. Mechanisms alternative to mitosis (reductional grouping, prophase chromosome reduction) operate from a polyploid state (possibly reached by means of endopolyploidy, endomitosis, nuclear fusion, or restitution nuclei) to generate new levels of ploidy and novel chromosome numbers necessary for selection to operate in vitro. The segregational phenomena require chromosome recognition in haploid set complements and abnormal behaviour of mitoses; the resulting chromosome variability suggests that chromosomes are arranged, in the resting nuclei, in an orderly and predictable manner.The knowledge of the molecular events governing these mechanisms, and how to control them, would be of great help for future applications of plant cell culture.     

Sorbitol dehydrogenase genetics in the mouse: A ';null' mutant in a ';European' C57BL strain

A ';null' activity variant phenotype for sorbitol dehydrogenase (SDH) was observed in C57BL/LiA mice and used to examine the genetics of this enzyme. Linkage studies of the locus ( Sdh-1 ) with non-agouti (a) and a biochemical Iocus encoding liver L-α-hydroxyacid oxidase ( Hao-1 ) demonstrated that it is coincident with or closely linked to the structural locus, previously localized on chromosome 2. Alcohol dehydrogenase (ADH) isozymes were also examined, since the liver A₂ isozyme exhibited some activity as a sorbitol dehydrogenase on cellulose acetate zymograms. It is apparent that SDH activity is not ';essential' in this mouse strain.     

The late-replicating X chromosome in digynous mouse triploid embryos

Using BrdU-labeling and acridine orange staining, the behavior of X-chromosome replication was studied in 28 XXX and 19 XXY digynous mouse triploids. In some of these the paternal and maternal X chromosome could by cytologically distinguished. Such embryos were obtained by mating chromosomally normal females with males carrying Cattanach's X chromosome which contains an autosomal insertion that substantially increases the length of this chromosome. In the XXX triploids there were two distinct cell lines, one with two late-replicating X chromosomes, and the other with only one late-replicating X. The XXY triploids were also composed of two cell populations, one with a single late-replicating X and the other with no late replicating X chromosome. Assuming that the late-replicating X is genetically inactive, in both XXX and XXY triploids, cells from the embryonic region tended to have only one active X chromosome, whereas those from the extra-embryonic membranes tended to have two active X chromosomes. The single active X chromosome was either paternal or maternal in origin, but two active X chromosomes were overwhelmingly maternal in origin, suggesting paternal X-inactivation in extra-embryonic tissues.     

Thermal Denaturation of Native Striatal Tyrosine Hydroxylase: Increased Thermolability of the Phosphorylated Form of the Enzyme

Tyrosine hydroxylase was purified from bovine corpus striatum. The native enzyme had a half-life of 15 +/- 3 min at 50 degrees C. Phosphorylation of tyrosine hydroxylase with protein kinase purified from both corpus striatum and heart activated the enzyme, but activity was rapidly lost with additional preincubation of the enzyme at 30 degrees C. Thermal denaturation studies indicated that phosphorylated tyrosine hydroxylase had a half-life of 5 +/- 2 min at 50 degrees C     

Investigations on radiosensitive and radioresistant populations of Drosophila melanogaster: X. The resistance factor rar 3: genetics

In earlier work, immature oocytes of the irradiated population RÖI₄ of Drosophila melanpgaster were found to be radioresistant relative to those of the basic population RÖI and to those of the control population Berlin wild (+K). The resistance of RÖI₄ relative to RÖI was previously attributed to a hypothetical “factor” rar-3. In the present paper, evidence is presented to show that rar-3 is a single, recessive genetic factor, located on chromosome 3 at a map position of about 49.8. The action of rar-3 is apparently independent of that of rar-1 and rar-2, the factors already present in RÖI.