Formation of functionally active chloroplasts is determined at a limited stage of leaf development in virescent mutants of rice

The ability to form functionally active chloroplasts is determined at a certain early stage of leaf development in three non-allelic temperature-sensitive virescent mutants of rice. Temperature-shift analysis, together with anatomical observations, indicates that the intrinsic developmental signals of the virescent genes are expressed at the stage immediately following the formation of basic leaf structure, but just before the onset of leaf elongation. These signals control the expression of chloroplast-encoded genes but do not affect the subsequent morphological development of the leaf or the photo-regulation of the expression of nuclear genes encoding chloroplast proteins.     

Two Forms of GO Type G Proteins: Identification and Distribution in Various Rat Tissues and Cloned Cells

A G(o) type G protein distinct from the major species of G(o) was recently isolated from bovine brain and designated G(o)*. The cDNAs encoding two forms of mammalian G(o) alpha were also isolated and designated GoA alpha and GoB alpha. To recognize two forms of G(o) type G proteins, we raised antibodies in rabbits against two peptides with sequences found only in the respective proteins of murine GoA alpha (SNTYEDAAAYIQTQF) and GoB alpha (TEAVAHIQGQYWSK). Purified anti-GoA alpha antibodies reacted with the major species of G(o) alpha purified from bovine and rat brain, whereas anti-GoB alpha antibodies reacted only with rat G(o)*alpha, but not with the major species of G(o) alpha or bovine G(o)*alpha. These results indicate that the major species of G(o) alpha is encoded by GoA alpha cDNA and G(o)*alpha is encoded by GoB alpha cDNA. Using these antibodies, the distribution of GoA and GoB was studied in various rat tissues and cloned cells. Both GoA and GoB were present in many tissues, but their distribution in peripheral tissues was distinct. GoA alpha seemed to associate mainly with neural tissues. On the other hand, relatively high concentrations of GoB alpha were present in the brain, pituitary gland, adipose tissue, lung, and testis. The concentrations of both GoA and GoB in the brain increased during ontogenic development, but the increase in GoB was observed at a later age. Both GoA and GoB were found in such cloned cells as PC12, NG108-15, C6, GA-1, G8, and 3T3-L1 cells. Treatment of PC12 cells with nerve growth factor caused the extension of neuron-like processes and the increase in the level of GoA, but not in the level of GoB.     

Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.

Immunoreactive alpha B crystallin and a 28-kDa protein in an extract of human pectoral muscle were precipitated by (NH4)2SO4 at 40% saturation, and coeluted during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. The two proteins were separated on a column of S-Sepharose HP in the presence of 7 M urea. Further chromatography of each of the two resultant fractions on a column of Superdex 75 pg and on a TSK-SP 5PW column in the presence of urea yielded preparations of alpha B crystallin and the 28-kDa protein each of which gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation of 28-kDa protein contained at least two subtypes, which were separable on the TSK-SP column. However, fragmentation patterns of the two major 28-kDa proteins after digestion with endoproteinase Asp-N were identical. Amino acid sequences of peptides formed by cleavage of the purified 28-kDa protein and alpha B crystallin were identical to those of particular regions of the deduced amino acid sequences of human small heat shock protein (HSP28) and lens alpha B crystallin, respectively. Using an immunoassay method, with antibodies raised in rabbits, we found that HSP28 was present in all human tissues tested and at high levels (greater than 1 micrograms/mg protein) in the heart and other tissues composed of striated and smooth muscles. HSP28, found with alpha B crystallin, in extracts of several human and bovine tissues was trapped on and coeluted with alpha B crystallin from an affinity column prepared with antibodies against alpha B crystallin. This result suggests that the two proteins are associated in cells.     

Lipase modification by various synthetic polymers for use in chloroform

Summary Lipase was modified with several hydrophilic and hydrophobic synthetic polymers. The modified lipase was solubilized into chloroform by. The catalytic esterification activity of modified lipase increased linearly with the increase of its solubility in chloroform.     

Excretion of peroxidase from horseradish hairy root in combination with ion supplementation

Summary The effect of ion-supplemented medium on peroxidase excretion from horseradish (Armoracia rusticana) hairy roots was studied. Supplementation of mannitol instead of ions revealed that the excretion was stimulated not by osmotic pressure in the medium but by ionic properties. Extracellular peroxidase activity per dry cell was proportionally correlated with the ionic strength of the cations. CaCl₂ or MgCl₂ was found to be the most effective agent for excretion among other combinations. CaCl₂ supplementation at the beginning of the culture caused higher peroxidase production in the medium without a significant loss of final cell mass compared with CaCl₂ addition during the culture. Repeated batch culture with 50 mM CaCl₂ supplementation allowed a continuous retention of cell viability over 149 days and produced a great amount of extracellular peroxidase, 12-fold higher than that achieved in a 40-day-old batch culture with 50 mM CaCl₂ supplementation. Correspondence to: T. Kobayashi     

Semisynthesis and properties of some insulin analogs

The trypsin-catalyzed coupling of bovine (Boc)₂-desoctapeptide (B23-B30)-insulin with synthetic octapeptides, H-Gly-X₂-X₃-X₄-Thr-Pro-Lys(Boc)-Thr-OH (X₂ = Phe or Ala, X₃ = Phe or Ala, X₄ = Tyr or Ala), followed by deprotection and purification produced the [Ala^B24, Thr^B30]-, [Ala^B25, Thr^B30]-, and [Ala^B26, Thr^B30]-analogs of bovine insulin in yields of 32, 35, and 32%, respectively. The biological activity of these analogs decreased in the order, normal insulin ([Thr^B30]-bovine insulin) = Ala^B26-insulin > Ala^B25-insulin > Ala^B24-insulin, as assayed for receptor binding and some other biological effects, in contrast with the corresponding Leu-analogs of human insulin, in which the activity decreased in the order, normal insulin > Leu^B24-insulin > Leu^B25-insulin. The affinity to insulin antibodies greatly diminished in both Ala^B24-insulin and Leu^B24-insulin but not in the B25-substituted analogs. The CD spectra of the Leu- and the Ala-analogs were compared with those of normal insulins to show that no apparent correlation seems to exist between the decrease in biological activity and the conformational changes observed in solution. The effects of organic solvents on the peptide-bond equilibrium and on the stability of trypsin are also discussed.     

A high-potential nonheme iron protein (HiPIP)-linked,thiosulfate-oxidizing enzyme derived fromChromatium vinosum

A thiosulfate-oxidizing enzyme was partially purified fromChromatium vinosum, and some of its properties were studied. The enzyme rapidly reducede HiPIP (high-potential nonheme iron protein) in the presence of thiosulfate. Cytochromesc of yeast and tuna and ferricyanide also acted well as electron acceptors for the enzyme; horse cytochromec was a poor electron acceptor. Cytochromec-552, cytochromec′, and cytochromec-553 did not act as electron acceptors. The enzyme was inhibited by cyanide and sulfite. On the basis of the stoichiometry in reduction of ferricyanide catalyzed by the enzyme in the presence of thiosulfate, the oxidized product of thiosulfate was inferred to be tetrathionate.     

Acyl migrations in diacyl derivatives of 2-methylmercaptobenzimidazole : A model of biotin

Diacyl derivatives of 2-methylmercaptobenzimidazole undergo the tautomerization 2 1 2′. Thermodynamic predominancy of one isomer over the others depends on the substituents on carbonyl groups. It has been found that electron-withdrawing substituents tend to favor 2-type compounds, whereas electron-releasing substituents make 1-type compounds more stable. The migration has been extended to include the carboethoxy group, and the results are discussed in relation to the mechanism of biotin-dependent enzymic carboxylation.     

Effect of phenformin on the response of plasma intestinal glucagon-like immunoreactivity (GLI) to oral glucose in gastrectomized subjects.

The effect of phenformin (DBI) on the plasma intestinal glucagon-like immunoreactivity (GLI) and pancreatic glucagon (IRG) responses to oral and intravenous glucose loads were studied in 26 gastrectomized subjects, using a cross-reacting and an IRG-specific anti-serum. The drug produced no significant changes in fasting GLI and IRG levels. Thirty minutes after oral glucose alone, the total GLI level rose to a peak of 1.55 +/- 0.17 ng/ml in the untreated subjects and to a maximum level of 1.67 +/- 0.18 ng/ml in the DBI-pretreated subjects. However, the mean GLI levels obtained 120 and 180 min after oral glucose were significantly higher after treatment with DBI. The blood sugar and IRI responses to oral glucose were lowered significantly by DBI pretreatment. DBI did not alter the glucose, IRI, IRG and GLI response to intravenous glucose. These results suggest that the release of intestinal GLI is not related to the intestinal absorption of glucose.