Silver staining for carboxymethylated metallothioneins in polyacrylamide gels

A sensitive method for detecting metallothioneins (MTs) by use of silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of carboxymethylated MTs was developed. Carboxymethylation of metallothioneins is indispensable because it prevents their aggregation, thereby allowing each of them to be detected as a single band by SDS-PAGE. However, when the gel was subjected to the silver-staining method of C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert [(1981) Science 211, 1437-1438], the image of carboxymethylated purified MTs was totally negative. Pretreatment of the gel with 1% sodium thiosulfate just prior to the silver-staining procedure successfully reversed the negative image of carboxymethylated MTs. Further, they could be detected with a limit of nanogram levels per lane. This method can be applied to MTs in cell extracts from cultured cell lines treated with cadmium or to those from liver of cadmium-intoxicated mice.     

Nerve growth factor, a differentiating agent, and epidermal growth factor, a mitogen, increase the activities of different S6 kinases in PC12 cells

Treatment of PC12 cells with either nerve growth factor, a differentiating agent, or epidermal growth factor, a mitogen, leads to an increase in the phosphorylation of the ribosomal protein S6. The soluble fraction of PC12 cells contains two S6 kinases, separable on heparin-Sepharose. Treatment of the cells with nerve growth factor results in an increase in the activity of one of these kinases; treatment of the cells with epidermal growth factor results in an increase in the activity of the other one. The data suggest that the patterns of phosphorylation and, in turn, the functional properties of S6 are different in cells instructed to differentiate from those in cells instructed to divide.     

Assignment of the gene locus for the sixth component (C6) of complement in mice to chromosome 15

A structural locus (C-6) for the sixth component of complement in mice is assigned to chromosome 15. Three-point linkage analysis indicated that the order of loci is C-6, Gpt-1, Gdc-1, and that the map distances are 25.9±4.9 between C-6 and Gpt-1, and 36.4±5.5 between C-6 and Gdc-1. Since Gdc-1 is more distal than Gpt-1, and C-6 is 26 cM away from Gpt-1, it is estimated that the C-6 is proximal to the centromere. In addition, a new C6 form found in AKR mice is described. We propose the designation C6B for it and C-6 ^b for the allele encoding C6B.Abbreviations used in this paper IEF isoelectric focusing - GPT glutamic-pyruvic transaminase - GDC L-glycerol 3-phosphate dehydrogenase - cM centimorgan     

The mechanism of end-organ resistance to 1 alpha,25-dihydroxycholecalciferol in the common marmoset.

The common marmoset, a New World monkey, requires a large amount of cholecalciferol (110 i.u./day per 100g body wt.) to maintain its normal growth. In a previous report, we demonstrated that the circulating levels of 1 alpha, 25-dihydroxycholecalciferol [1 alpha,25(OH)2D3] in the marmosets are much higher than those in rhesus monkeys and humans, but the marmosets are not hypercalcaemic [Shinki, Shiina, Takahashi, Tanioka, Koizumi & Suda (1983) Biochem. Biophys. Res. Commun. 14, 452-457]. To compare the effect of the daily intake of cholecalciferol, two rhesus monkeys were given a large amount of cholecalciferol (900 i.u./day per 100g body wt). Their serum levels of calcium, 25-hydroxycholecalciferol and 24R,25-dihydroxycholecalciferol were markedly elevated, but the serum 1 alpha,25(OH)2D3 levels remained within a range similar to those in the rhesus monkeys fed the normal diet (intake of cholecalciferol 5 i.u./day per 100g body wt). Intestinal cytosols prepared from both monkeys contained similar 3.5 S macromolecules to which 1 alpha,25(OH)2D3 was bound specifically. However, the cytosols from the marmosets contained only one-sixth as many 1 alpha,25(OH)2D3 receptors as those from the rhesus monkeys. Furthermore, the activity of the 1 alpha,25(OH)2D3-receptor complex in binding to DNA-cellulose was very low in the marmosets. These results suggest that the marmoset possesses an end-organ resistance to 1 alpha,25(OH)2D3 and is a useful animal model for studying the mechanism of vitamin D-dependent rickets, type II.     

Isolation and Characterization of an Escherichia coli ruv Mutant Which Forms Nonseptate Filaments After Low Doses of Ultraviolet Light Irradiation

Two ultraviolet light (UV)-sensitive mutants have been isolated from Escherichia coli K-12. These mutants, designated RuvA(-) and RuvB(-), were controlled by a gene located close to the his gene on the chromosome map. They were sensitive to UV (10- to 20-fold increase) and slightly sensitive to gamma rays (3-fold increase). Host cell reactivation, UV reactivation and genetic recombination were normal in these mutants. Irradiation of the mutants with UV resulted in the production of single-strand breaks in deoxyribonucleic acid, which was repaired upon incubation in a growth medium. After UV irradiation, these mutants resumed deoxyribonucleic acid synthesis at a normal rate, as did the parent wild-type bacteria, and formed nonseptate, multinucleate filaments. From these results we concluded that the mutants have some defect in cell division after low doses of UV irradiation, similar to the lon(-) or fil(+) mutant of E. coli. The ruv locus was divided further into ruvA and ruvB with respect to nalidixic acid sensitivity and the effect of minimal agar or pantoyl lactone on survival of the UV-irradiated cell. The ruvB(-)mutant was more sensitive to nalidixic acid than were ruvA(-) and the parent strain. There was a great increase in the surviving fraction of the UV-irradiated ruvB(-) mutant when it was plated on minimal agar or L agar containing pantoyl lactone. No such increase in survival was observed in the ruvA(-) mutant.     

The androgen-dependent rat prostate protein,probasin, is a heparin-binding protein that co-purifies with heparin-binding growth factor-1

Summary Rat prostate extracts contain an abundant 20–22 kilodalton heparin-binding protein with near identical chromatographic properties, but only 0.2–1% of the mitogenic activity, of bovine brain heparin-binding growth factor-1 (acidic fibroblast growth factor). Amino terminal amino acid sequence (met-met-thr-asp-lys-asn-leu-lys-lys-lys-ile-glu-gly-asn-trp-arg-thr-val-tyr-leu-ala-ala-ser-?-val-glu-lys-ile-asn-glu-gly-ser-pro) and immunochemical analysis revealed that the protein is identical to the androgen-dependent protein “probasin”. This work was supported in part by NCI grant CA37589 (W. L. M., J. W. C.) and the Medical Research Council of Canada (R. J. M.).     

Studies on the life history of the population of an evergreen herb,Pyrola japonica,on the forest floor in a warm temperate region

Photosynthetic and respiratory activities and gross production in relation to temperature conditions were investigated in the population of an evergreen herb,Pyrola japonica, growing on the floor of a deciduous forest in the warm temperate region of central Japan. Analysis of the temperature-photosynthesis relationship ofP. japonica leaves during the growing season indicated distinct seasonal changes in the temperature optimum for photosynthesis. This population was found to be acclimatable to ambient air temperatures exceeding 15C, but this acclimation became less pronounced under thermal conditions below 15 C. This plant possessed narrow photosynthetic optima in the warm season but wide optima in the cold season. The shape of the temperature-respiration curve did not vary significantly with the months except for April. The Q₁₀ for respiration between 10 C and 20 C was calculated to be 1.93–2.65. Annual dry matter loss associated with respiration was estimated to amount to 159.1 g d.w.m⁻² based on the measurements of the seasonal changes in the respiratory activity of each organ. Gross production of this population was estimated to be 219.3 g d.w.m⁻² year⁻¹ as the sum total of the net production (60.2 g d.w.m⁻²year⁻¹) and the respiration. Monthly gross production was high in the early growing season, and low and stable in winter.