A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum: Evidence for a calcium channel

The sarcoplasmic reticulum (S.R.) of rabbit skeletal muscle has been found to contain a single, high affinity binding site for the Ca antagonist drug [³H] -nitrendipine. Two subfractions of the reticulum were studied, the heavy (HSR) and light (LSR) preparations, which exhibited similar nitrendipine equilibrium dissociation constants (K_D) of 1nM. Crude cardiac and brain membranes assayed under the same conditions exhibited K_D values of 0.2–0.3nM. The concentration of binding sites per mg. protein (B_max) in HSR was found to be very high, namely 6.7 picomoles/mg, some four times greater than that of LSR. [³H] -nitrendipine binding to HSR was reversible and inhibited by the Ca antagonists flunarizine and verapamil, and by the intracellular Ca release antagonist TMB-8 (8-diethylamino-octyl 3,4,5- trimethylbenzoate hydrochloride). However, unlabelled nitrendipine at 2 × 10^?5M had no effect on contraction of isolated electrically stimulated rabbit lumbrical or rat diaphragm muscles, nor did it affect the neuromuscular junction as studied in rat phrenic nerve-diaphragm preparations. Also, little effect of 2 × 10^?5M nitrendipine was seen on net ⁴⁵Ca uptake by HSR. These results suggest that [³H] -nitrendipine binding to skeletal muscle S.R. resembles that of brain membranes, which also contain a high affinity binding site for [³H] -nitrendipine and which similarly are pharmacologically insensitive to this dihydropyridine type of Ca channel blocking agent. Since HSR is also enriched in calsequestrin and terminal cysternae from which Ca is released in vivo, it seems likely that the [³H]- nitrendipine binding sites in S.R. are associated with Ca channels in the S.R.     

Effects of ultraviolet-B radiation on the growth and yield of crop plants

This paper reviews growth chamber, greenhouse, and field studies on the effects of ultraviolet-B (UV-B. between 280 and 320 nm) radiation on agricultural crop plants. Our understanding of the physiological effects of UV-B radiation comes primarily from growth chamber studies, where UV-B is artificially supplied via filtered lamps. Both photosystems I and II, as well as carboxylating enzymes, are sensitive to UV-B radiation. Ultraviolet-B radiation also affects stomatal resistance, chlorophyll concentration, soluble leaf proteins, lipids, and carbohydrate pools. In general, the effects of UV-B radiation are accentuated by the low levels of visible radiation typically found inside growth chambers. Ultraviolet-B radiation has also been shown to affect anatomical and morphological plant characteristics. Commonly observed UV-B induced changes include plant stunting, reductions in leaf area and total biomass, and alterations in the pattern of biomass partitioning into various plant organs. In sensitive plants, evidence of cell and tissue damage often appears on the upper leaf epidermis as bronzing, glazing, and chlorosis. Epidermal transmission in the UV region decreases in irradiated leaves. This decrease is primarily associated with a stimulation in flavonoid biosynthesis and is thought to be a protective, screening response to the deleterious effects of UV-B. A considerable degree of variability exists in sensitivity to UV-B radiation between different species. Approximately 30% of the species tested were resistant, another 20% were extremely sensitive, and the remainder were of intermediate sensitivity, in terms of reductions in total dry weight. In addition to this sizable interspecific variability, there appears to be a similarly wide intraspecific variability in UV-B response. The effects of UV-B radiation on crop yield have only been examined in a limited number of field studies, with ambient levels of UV-B radiation being supplemented with fluorescent sun lamps. Due to various deficiencies, all these field experiments to date have only limited utility for assessing the potential impact of enhanced levels of UV-B on crop productivity.     

Metabolism of [3H]gibberellin A4 in somatic suspension cell cultures of carrot

The native gibberellin A₄ (GA₄), in radioactive form ([1,2-³H]GA₄, 1.06 Ci/mmol), was fed to carrot somatic cell cultures (suspension and immobilized cell systems) and its metabolism over a 48 hr period was investigated. It was found that the [³H]GA₄ was metabolized to at least two GAs, [³H]GA₁ and [³H]GA₈, six GA glucosyl conjugates, [³H]GA₁-0(3)-glucoside, [³H]GA₁-0(13)-glucoside, [³H]GA₁-glucosyl ester, [³H]GA₄-glucoside, [³H]GA₄-glucosyl ester, a [³H]GA₈ glucosyl conjugate(s) and a previously unknown [³H]GA₁ glucosyl conjugate ([³H]GA₁-0(3,13)-diglucoside-like compound). The GA₁-diglucoside-like compound was found only in extracts of cells and was present in significant amounts (33 % of total extractable radioactivity). All other metabolites were present in both cells and medium. For extracts of the medium, no differences between the suspension and immobilized cultures existed in types of [³H]GA₄ metabolites although quantitative differences were apparent.     

Tumor-host zinc metabolism the central role of metallothionein

Features of tumor and host zinc metabolism are described. Emphasis is placed on tumor-host interactions. Using the model of the Ehrlich ascites tumor in mice, one clear site of modulation of cellular zinc by the amount of nutrient zinc available in the host is a zinc-binding protein with the properties of metallothionein. The selective depletion of zinc from this protein is correlated with the loss of cell proliferation by tumors injected into zinc-deficient animals. The properties of isolated metallothionein are consistent with a role for it as a reactive pool of intracellular zinc which can be donated to apozinc proteins and other structures. The presence of the Ehrlich tumor in mice also perturbs their distribution of zinc: zinc leaves the plasma and is accumulated by liver in the form of newly synthesized zinc metallothionein. During host zinc deficiency, this redistribution is not observed. This may be caused not only by a lack of mobile plasma zinc, but also by an inhibition of the initiation of this host response at the site of the tumor in the peritoneum.     

Sexual differentiation of rat liver carbonic anhydrase III

Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.     

On the structure of the iron-sulfur complex in the two-iron ferredoxins

Recent spectroscopic and magnetic susceptibility studies of the iron center in the two-iron ferredoxins provide criteria which any model for the iron-sulfur complex in these proteins must satisfy. These criteria are most stringent for parsley and spinach ferredoxin: the reduced proteins contain a high-spin ferric atom antiferromagnetically exchange-coupled (presumably via sulfide bridging ligands) to a high-spin ferrous atom. In the oxidized proteins the iron atoms are antiferromagnetically spin-coupled, high-spin ferric atoms. Arguments are given to substantiate the claim that the ferrous atom in the reduced protein is ligated by four sulfur atoms in a distorted tetrahedral configuration: two are the bridging sulfides, two are cysteinyl sulfurs. A treatment of proton contact shifts based upon the above model is pertinent to proton magnetic resonance data already available and provides a means to identify directly the ligands at both iron atoms via further PMR experiments.     

Characterization of Mutants of Escherichia coli Temperature-Sensitive for Ribonucleic Acid Regulation: an Unusual Phenotype Associated with a Phenylalanyl Transfer Ribonucleic Acid Synthetase Mutant

A mutant strain AA-522, temperature-sensitive for protein synthesis, was isolated from a stringent strain (CP-78) of Escherichia coli K-12. The mutant strain has a relaxed phenotype at the nonpermissive growth temperature. Protein synthesis stops completely at 42 C, whereas the rate of ribonucleic acid (RNA) synthesis is maintained at 20% of the 30 C rate. Sucrose-gradient centrifugation analysis of RNA-containing particles formed at 42 C indicated the presence of “relaxed particles.” These particles possess 16S and 23S RNA and are precursors to normal 50S and 30S ribosomal subunits. A search for the temperature-sensitive protein responsible for the halt in protein synthesis implicated phenylalanyl transfer RNA (tRNA) synthetase. Essentially no enzyme activity is detected in vitro at 30 or 40 C. Analysis of phenylalanyl tRNA synthetase activity in revertants of strain AA-522 indicated the presence of intragenic suppressor mutations. Revertants of strain AA-522 analyzed for the relaxed response at 42 C were all stringent; strain AA-522 was stringent at 30 C. These data indicate that a single mutation in phenylalanyl tRNA synthetase is responsible for both a block in protein synthesis and the relaxed phenotype at 42 C.     

Photoinactivation of Ammonia Oxidation in Nitrosomonas

Photoinactivation of ammonia oxidation in cells of Nitrosomonas was shown to follow first-order kinetics with a rate constant proportional to incident light intensity. The action spectrum for photoinactivation consisted of a broad peak in the ultraviolet range, where both hydroxylamine and ammonia oxidation were affected, and a shoulder at approximately 410 nm where only ammonia oxidation was affected. In photoinactivated cells, hydroxylamine but not ammonia was oxidized to nitrite and hydroxylamine but not ammonia caused reduction of cytochromes in vivo. The amount per cell of the following constituents was not measurably altered by photoinactivation: cytochromes b, c, a, and P460; ubiquinone; phospholipid; free amino acids; hydroxylamine-dependent nitrite synthetase; nitrite reductase; p-phenylenediamine oxidase; and cytochrome c oxidase. Malonaldehyde or lipid peroxides were not detected in photoinactivated cells. Photoinactivation was prevented (i) under anaerobic conditions, (ii) in the presence of methanol, allylthiourea, thiosemicarbazide, hydroxylamine, ethylxanthate, or CO at concentrations wich caused 100% inhibition of ammonia oxidation, and (iii) at concentrations of ammonia or hydroxylamine which gave a rapid rate of nitrite production. Recovery of ammonia oxidation activity in 90% inactivated cells took place in 6 h, required an energy and/or nitrogen source, and was inhibited by 400 mug of chloramphenicol per ml.     

Conjugal Transfer of Plasmid-Borne Multiple Antibiotic Resistance in Streptococcus faecalis var. zymogenes

A strain of Streptococcus faecalis var. zymogenes, designated JH1, had high-level resistance to the antibiotics streptomycin, kanamycin, neomycin, erythromycin, and tetracycline. These resistances were lost en bloc from approximately 0.1% of cells grown in nutrient broth at 45 C. The frequency of resistance loss was not increased by growth in the presence of the "curing" agents acriflavine or acridine orange, but after prolonged storage in nutrient agar 17% of cells became antibiotic sensitive. Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated from the parental strain and from antibiotic-sensitive segregants by using cesium chloride-ethidium bromide gradients. DNA molecular species were identified by using neutral sucrose gradients. Strain JH1 contained two covalently closed circular DNA species of molecular weights 50 x 10(6) and 38 x 10(6). An antibiotic-sensitive segregant, strain JH1-9, had lost the larger molecular species. A second sensitive segregant, strain JH1-5, had also lost the larger molecular species but a new molecular species of approximate molecular weight 6 x 10(6) was present. The antibiotic resistances that were curable from the parental strain were transferred to antibiotic-sensitive strains of S. faecalis and to strain JH1-9, during mixed incubation in nutrient broth at 37 C. Data to be described are interpreted to suggest that the transfer is by a conjugal mechanism. Analysis of the plasmid species in recipient clones showed that all had received the plasmid of molecular weight 50 x 10(6). Strain JH1-5 was not a good recipient. Analysis of one successful recipient clone of JH1-5 revealed that it had gained the 50 x 10(6) molecular weight plasmid but lost the 6 x 10(6) molecular weight species. These data are interpreted to mean that the multiple antibiotic resistance is borne by a transferable plasmid of 50 x 10(6) molecular weight, and that in clone JH1-5 this plasmid suffered a large deletion leaving only a 6 x 10(6) remnant which was incompatible with the complete replicon.     

Reactions of polycyclic hydrocarbon epoxides with RNA and polyribonucleotides

RNA, poly(G) and poly(A) were reacted with benz[a]anthracene 5,6-oxide or with 7,12-dimethylbenz[a]anthracene 5,6-oxide and hydrolysates of the alkylated polymers examined using a combination of Sephadex LH20 column chromatography and thin-layer chromatography on silica gel. The results show that two RNA products are formed in reactions with benz[a]anthracene 5,6-oxide, one resulting from reaction with guanine and the other from reaction with adenine. With 7,12-dimethylbenz[a]anthracene 5,6-oxide, six RNA products appeared to be formed, two resulting from reactions with guanine and three from alkylation of adenine; the other product has not been identified.