A DBL-homologous region of the yeast CLS4/CDC24 gene product is important for Ca(2+)-modulated bud assembly.

The CLS4/CDC24 is essential for the budding process of the yeast Saccharomyces cerevisiae. Disruption of the CLS4/CDC24 gene is lethal, and expression of the CLS4 product under the control of the GAL1 promoter is sufficient for cellular growth. The CLS4 product is detected in yeast cell lysate with an apparent molecular mass of 93 kD (854 amino acid residues) and shows homology with the human DBL oncogene product. Temperature-sensitive cdc24-1 mutation is located in the N-terminal portion of the protein whereas Ca(2+)-sensitive cls4-1 mutation is present after the DBL-homologous region (amino acid residues 281-518) near the putative Ca(2+)-binding site. Mutations within the DBL-homologous region are responsible for the Ca(2+)-sensitive phenotype. Thus the CLS4 gene product seems to have several functional domains within the molecule essential for bud assembly.     

Spontaneous and agonist-induced calcium oscillations in pituitary gonadotrophs

Basal and receptor-regulated changes in cytoplasmic calcium concentration ([Ca2+]i) were monitored by fluorescence analysis in individual rat pituitary gonadotrophs loaded with the calcium-sensitive dye indo-1. Most gonadotrophs exhibited low amplitude spontaneous oscillations in basal [Ca2+]i that were interspersed by quiescent periods and abolished by removal of extracellular Ca2+ or addition of calcium channel blockers. Such random fluctuations in [Ca2+]i, which reflect the operation of a plasma membrane oscillator, were not coupled to basal gonadotropin secretion. The physiological agonist GnRH induced high amplitude [Ca2+]i oscillations; when a threshold [Ca2+]i level was reached, a cytoplasmic oscillator began to generate extremely regular Ca2+ transients. The time required to reach the threshold [Ca2+]i level was inversely correlated with agonist dose; the frequency, but not the amplitude, of agonist-induced Ca2+ spiking increased with agonist concentration. The duration of the latent period decreased and the frequency of Ca2+ spiking increased with the increase in ambient temperature. At high GnRH concentrations, the calcium transients merged into biphasic responses similar to those observed in cell suspensions at all GnRH concentrations. The presence of spontaneous fluctuations in basal [Ca2+]i did not significantly change the patterns of agonist-induced [Ca2+]i responses. Also, removal of extracellular Ca2+ did not interfere with the frequency or amplitude of Ca2+ spikes, but caused the loss of the plateau phase. Blockade of intracellular Ca(2+)-ATPase pumps by thapsigargin was usually accompanied by a subthreshold increase in [Ca2+]i. In such cells the agonist-induced oscillatory pattern was transformed into the biphasic response. In about 10% of the cells, however, high thapsigargin concentrations induced coarse [Ca2+]i oscillations; subsequent stimulation of such cells with GnRH was ineffective. The cytoplasmic oscillatory and biphasic responses may represent a mechanism for differential activation of Ca(2+)-dependent enzymes and their dependent cellular processes, including hormone secretion. The membrane oscillator is probably responsible for refilling of agonist-sensitive pools during and after agonist stimulation.     

Isolation and characterization of major urinary amino acid O-glycosides and a dipeptide O-glycoside from a new lysosomal storage disorder (Kanzaki disease). Excessive excretion of serine- and threonine-linked glycan in the patient urine

Four major sialo compounds, termed GP-M1, GP-D1, GP-D2, and GP-D3 have been isolated from the urine of a novel glycoprotein storage disorder patient with angiokeratoma corporis diffusum which was discovered by Kanzaki et al. (Kanzaki, T., Yokota, M., Mizuno, N., Matsumoto, Y., and Hirabayashi, Y. (1989) Lancet April 22, 875-877). Based on the results of fast atom bombardment mass spectrometry, methylation analysis, and proton nuclear magnetic resonance spectroscopy, their chemical structures were concluded to be: (formula; see text) The yields of GP-M1, GP-D1, GP-D2, and GP-D3 were approximately 15, 6, 50, and 5 mg/liter of urine, respectively. The most major compound GP-D2, was further purified into single molecular species, threonine and serine type, by reversed phase high performance liquid chromatography. NMR analysis of the two purified compounds with single molecular species showed that the chemical shifts of anomeric protons of GalNAc were significantly different between threonine- and serine-linked GalNAc. Neither mannose-containing glycopeptides nor glycosphingolipids were excreted in the patient urine. From these results, this disease is thought to be caused by the deficiency of a lysosomal enzyme(s) acting on O-linked glycan chains.     

Human serum deoxyribonuclease I (DNase I) polymorphism: pattern similarities among isozymes from serum, urine, kidney, liver, and pancreas.

We have devised a zymogram method with high sensitivity and resolution for investigating molecular heterogeneity and genetic polymorphism of deoxyribonuclease I. A combination technique of polyacrylamide-gel isoelectric-focusing electrophoresis and the newly developed zymogram method have led to the discovery of genetic polymorphism of human serum DNase I. Family studies showed that the three common phenotypes--DNASE1 1, DNASE1 1-2, and DNASE1 2--and the other five relatively rare phenotypes--DNASE1 1-3, DNASE1 2-3, DNASE1 2-4, and DNASE1 3-4--represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4. The frequencies of DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4 calculated in a Japanese population were .5517, .4358, .0104, and .0021, respectively. Moreover, it was found that urine and extracts of kidney, liver, and pancreas, as well as serum, can be used for DNase I phenotyping.     

Is the IS1-flanked r-determinant of the R plasmid NR1 a transposon?

The 23 kilobase multiple drug resistance r-determinant (r-det) of the R plasmid NR1 is an IS1-mediated transposon, Tn2671. Drug-resistant Escherichia coli transductants isolated after infection with bacteriophage P1::Tn2671 derivatives carry the intact r-det in their chromosomes. Independently isolated transductants carry the r-det at different locations on the chromosome. From the E. coli chromosome, Tn2671 can transpose to various locations on the phage P7 genome. Throughout these processes, r-det is maintained as a stable unit. Various possible molecular mechanisms, which all might contribute with characteristic frequencies to the transposition of Tn2671, are discussed. The results presented are relevant to the understanding of mechanisms for a wide spreading of drug resistance genes.     

Seven sesquiterpene lactones from Inula britannica var. chinensis

Four known sesquiterpene lactones, tomentosin, ivalin, 4-epi-isoinuviscolide and gaillardin, together with three new lactones, inuchinenolides A, B and C, were identified in the whole plant of Inula britannica var. chinensis.     

Interrelation of Two Forms of Ferredoxin-NADP+ Reductase with Different Molecular Weights

The heavier form of ferredoxin-NADP⁺ reductase was extractedfrom spinach leaves or chloroplasts and isolated as the majorfraction at high ionic strengths with ammonium sulfate or sodiumchloride. At low ionic strengths, the form with the higher molecularweight was relatively unstable and was converted gradually intothe lower form. We concluded that the enzyme exists in vivoas the form with the higher molecular weight. ¹Present address: Market Development Department, Shionogi &Co. LTD., 5-Sagisu, Fukushima-ku, Osaka 553, Japan. (Received December 16, 1980; Accepted January 19, 1981)