Protective mechanism of sodium molybdate against the acute toxicity of cadmium in rats. II. Prevention of cytoplasmic acidification

In order to clarify the protective mechanism of sodium molybdate against the acute toxicity of cadmium chloride in rat, the effect of in vivo sodium molybdate pretreatment on the cytotoxic action of cadmium in isolated hepatocytes was studied. The cytosolic pH of hepatocytes isolated from untreated rats immediately decreased with incubation in either neutral Hank's balanced salt solution (HBS), pH 7.4, containing 5 µM cadmium chloride minimum or acidic HBS (pH 7.1, 6.8, 6.5, and 6.2). The presence of 5 µM cadmium in HBS adjusted to pH 7.1 aggravated cytosalic acidification induced by the acidic medium alone. Cell viability of hepatocytes incubated in HBS at pH 6.2 was significantly reduced as compared to that of control cells in HBS at pH 7.4, but the presence of cadmium in the acidic HBS had no aggravating action against such a toxic action of the acidic medium although cellular uptake of the metal in the medium increased, as compared to that in HBS at pH 7.4. Molybdenum pretreatment alleviated cytoplasmic acidification induced by the treatment with HBS at pH 7.4 or 7.1 containing cadmium or by extracellular acid load wothout cadmium. This pretreatment also prevented the loss of cell viability induced by the treatment with HBS at pH 6.2 but could not attenuate that when cadmium was present in the medium.These facts suggest that molybdenum pretreatment alleviated the acute toxicity of cadmium in rat by preventing cytoplasmic acidification caused by the harmful metal.     

Molecular cloning and nucleotide sequence of cDNA encoding the rat kidney S-adenosylmethionine synthetase.

We previously reported the isolation of a cDNA encoding the liver-specific isozyme of rat S-adenosylmethionine synthetase from a lambda gt11 rat liver cDNA library. Using this cDNA as a probe, we have isolated and sequenced cDNA clones for the rat kidney S-adenosylmethionine synthetase (extrahepatic isoenzyme) from a lambda gt11 rat kidney cDNA library. The complete coding sequence of this enzyme mRNA was obtained from two overlapping cDNA clones. The amino acid sequence deduced from the cDNAs indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,715 Da. The predicted amino acid sequence of this protein shares 85% similarity with that of rat liver S-adenosylmethionine synthetase. This result suggests that kidney and liver isoenzymes may have originated from a common ancestral gene. In addition, comparison of known S-adenosylmethionine synthetase sequences among different species also shows that these proteins have a high degree of similarity. The distribution of kidney- and liver-type S-adenosylmethionine synthetase mRNAs in kidney, liver, brain, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.4-kilobase (kb) mRNA species hybridizable with a probe for kidney S-adenosylmethionine synthetase was found in all tissues examined except for liver, while a 3.4-kb mRNA species hybridizable with a probe for liver S-adenosylmethionine synthetase was only present in the liver. The 3.4-kb kidney-type isozyme mRNA showed the same molecular size as the liver-type isozyme mRNA. Thus, kidney- and liver-type S-adenosylmethionine synthetase isozyme mRNAs were expressed in various tissues with different tissue specificities.     

Inhibited insulin secretion by recombinant human interleukin-1 beta in adrenalectomized rats: involvement of prostaglandin

An involvement of prostaglandin synthesis in reduced insulin secretion by interleukin-1 was investigated in adrenalectomized (ADX) rats. The recombinant human interleukin-1 beta (IL-1) significantly reduced insulin secretion in ADX rats 2 and 4 hr after the injection, although IL-1 stimulated insulin secretion in intact rats. In ADX rat, IL-1 showed dose-dependent inhibition of pancreatic insulin secretion. In addition, insulin response to intravenous glucose loading was also attenuated in ADX rats with pretreatment by IL-1. At 4 hours after injection, ibuprofen (IBP; 0.5-50.0 mg/kg, ip), selective cyclooxygenase blocker, attenuated insulin inhibition by IL-1 in a dose-dependent manner. These data suggest that IL-1 may suppress in vivo insulin release at least in part through the mediation of prostaglandin synthesis in the absence of adrenal glands.     

Induction and stimulation of 92-kDa gelatinase/type IV collagenase production in osteosarcoma and fibrosarcoma cell lines by tumor necrosis factor alpha

Production of a 92-kDa gelatinase/type IV collagenase and tissue inhibitor of metalloproteinases (TIMP) was investigated with human sarcoma cell lines. Among the cytokines and growth factors examined, only human recombinant tumor necrosis factor alpha (TNF alpha) induced and stimulated the proteinase with concomitant increase in TIMP expression, but matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) expression was unchanged. These data suggest that gene expression of the two metalloproteinases is regulated in a different fashion and TNF alpha may be important to allow cancer cells to be more invasive and metastatic.     

Application of a ribosomal DNA integration vector in the construction of a brewer's yeast having alpha-acetolactate decarboxylase activity.

An integration plasmid, pIARL28, containing the ribosomal DNA gene as a homologous recombination sequence was constructed for introduction of the alpha-acetolactate decarboxylase gene into brewer's yeast. The transformation efficiency of pIARL28 was 20- to 50-fold higher than those of the other YIp vectors, as yeast cells had approximately 140 copies of the ribosomal DNA gene. All transformants showed very high alpha-acetolactate decarboxylase activity due to the multiple integrated copies of the plasmid. The transformants were grown in nonselective conditions, and segregants which had maintained the alpha-acetolactate decarboxylase expression cassette but no other vector sequences were isolated. Southern analysis showed that these marker-excised segregants contained more than 20 copies of the alpha-acetolactate decarboxylase gene and were stably maintained under nonselective conditions. Fermentation tests confirmed that the diacetyl concentration was considerably reduced in wort fermented by these marker-excised segregants. The degree of reduction was related to the copy number of the alpha-acetolactate decarboxylase gene.     

Role of prostaglandin endoperoxides in the serum thiobarbituric acid reaction

In the presence of heme and reduced glutathione, prostaglandin (PG) endoperoxides underwent rapid conversion to malondialdehyde and 12l-hydroxy-5,8,10-heptadecatrienoic acid. In addition, PG endoperoxides as well as lipid peroxides produced malondialdehyde to yield a red pigment during the thiobarbituric acid reaction with different efficiencies. The relative rates of the reaction were: 1,1,3,3-tetraethoxypropane, 100; PGG₂, 55; PGH₂, 32; and 15-hydroperoxyarachidonic acid, 6. The thiobarbituric acid reactive materials in rabbit serum decreased by 25–60%, after intravenous administration of aspirin (a cyclo-oxygenase inhibitor) and with a concomitant decline of serum PG levels. These results, taken together, suggested that serum thiobarbituric acid values, considered to be an indicator of lipid peroxide levels, were to a significant extent due to PG endoperoxides and their derivatives.     

Angeloylcumambrin-B,an antimicrobial sesquiterpene lactone from Chrysanthemum ornatum var. Spontaneum

Angeloylcumambrin-B, a new antimicrobial guaianolide sesquiterpene lactone, was isolated from Chrysanthemum ornatum and the structure was determined by a combination of chemical and physical methods.     

Mineral nutrition, of cultured chlorophyllous cells of tobacco VII. Effects of the NH4/NO3 ratio and of Cl in the media on the growth and ionic balance of cells

NG, a strain of cultured tobacco cells of Nicotiana glutinosahad high growth rates and carboxylate contents (C—A) of100 to 130 meq/100 g of dry cells on media containing 42 meqNO₃/liter as the sole N source. (C—A) is the amount ofinorganic cations minus inorganic anions in meq per 100 g ofdry cells. NG, cultured on media containing NH₄ 10+NO₃ 42 in meq/liter,had lower growth rates and lower (C—A) values as comparedwith NG on media containing NO₃ as the sole N source. NG, cultured on media containing NH₄ 30+NO₃ 42 in meq/liter,had high growth rates and (A—C) values of 22 to 53 meq/100gof dry cells. In this case, the (A—C) content may correspondto organic cations, basic organic N compounds such as free asprotein-bound basic amino acids. The easily absorbed Cl mayhave been required maintain good growth conditions such as ionicbalance and a favorable pH in the cells. Thus cultured cells of Nicotiana glutinosa may have physiologicaladaptability against variations in a relatively wide range of|C—A| contents [|C—A| being the absolute valuesof (C—A)]. (Received May 15, 1980; )     

A spin-label study of the effects of drugs on calcium release from isolated sarcoplasmic reticulum vesicles

The effects of caffeine, thymol, and procaine on calcium release from fragmented sarcoplasmic reticulum (FSR) from rabbit skeletal white muscle were investigated by the spin label method at the organellar level. Two thiol-directed spin labels, 4-maleimide-2,2,6,6-tetramethylpiperidinooxyl and 4-(2-iodoacetamide)-2,2,6,6-tetramethylpiperidinooxyl, were used for the labeling of SR proteins. The ratio (W/S) of the weakly (W) and strongly (S) immobilized ESR signals was measured for the maleimide and iodoacetamide labeled FSR. The two labels gave different W/S values, which means that the two labels report conformational changes at different loci of SR proteins. The dependences of the W/S ratios on the concentration of the drugs showed that conformational changes of SR proteins induced by these drugs are not the same. From measurements of the distribution of 5-doxyldecanoic acid methylester between the lipid and water phases, it was found that the conformational changes of the SR proteins caused by thymol or procaine induced a disorder in local regions of the phospholipid bilayers of FSR, while such disordering was not induced by caffeine. On the other hand, caffeine and thymol showed definite effects on calcium release from FSR, while procaine did not. These results indicate that the effects of the drugs on the protein conformations can be well characterized at the organellar level by means of the spin label technique and that some specific changes in the conformations of SR proteins are necessary for calcium release from FSR.