Effects of purine nucleotides on photosynthetic electron transport in isolated chloroplasts

The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H⁺ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H⁺ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 µM for the first and 110 and 160 µM for the second step, respectively (at pH 8.3, 15°C). Above pH 9, the ratio (P/e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi + phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexylcarbodiimide was taken to be the basal activity, the P/e ratio remained almost constant ( 1) from pH 7.0 up to 10.     

MECHANISM OF ACTION OF NOTEXIN AND NOTECHIS 11-5 ON SYNAPTOSOMES

The neurochemical activity of notexin and notechis II-5 was investigated using a synaptosomal preparation of rat cerebral cortices. In preparations preincubated with [³H]choline in order to label acetylcholine, the toxins caused a rapid release of the transmitter which was calcium-dependent. The toxins were also potent inhibitors of high affinity choline uptake. Both agents produced a marked depolarization of the synaptosomal preparation as measured by a fluorescent dye and at high concentrations lysed the preparation. At a concentration of 0.1 μM, notexin and notechis II-5 caused a 50% increase in the efflux of lactate dehydrogenase activity. These results, together with electron microscopic observations, indicated that the toxins disrupt the synaptosomal membranes presumably by their inherent phospholipase activity. The release of acetylcholine and inhibition of choline uptake, together with the depolarization of synaptosomal membranes noted in this study, could explain the observed electrophysiological effects of these toxins.     

Rhythm in potassium uptake by a duckweed, Lemna gibba G3

The potassium uptake activity of the "flow-medium culture" ofa long-day duckweed, Lemna gibba G3, followed a circadian rhythmwhich persisted for more than 5 days under continuous light.The period of the rhythm was about 25 hr under 3000 lux at 26?Cand was slightly over-compensated against temperature, Q₁₀ beinga little less than 1.0. The amplitude of the rhythm was dependenton light intensity, and there was no potassium uptake in thedark. Magnesium uptake was affected by the potassium movementand showed circadian rhythmicity with a small amplitude underconditions where the potassium uptake was already saturated.Calcium uptake did not show any obvious rhythm. In Contrastto L. gibba, a short-day duckweed L. perpusilla 6746 displayedcircadian rhythm of potassium uptake only in the dark and notin the light. This rhythm did not persist beyond the secondcycle. (Received June 13, 1978; )     

The crystal structure of di-D-fructose anhydride III,produced by inulin D-fructotransferase

Reaction of methyl α-d-glucopyranoside and methyl α-d-mannopyranoside with alkaline hydrogen peroxide and a ferrous salt, at room temperature and below, afforded the corresponding d-glycosiduronic acids. On dehydration, the acids gave the corresponding gamma lactones, with a shift of the pyranoid ring to a furanoid ring. Surprisingly, the glycosidic methyl group was retained during the oxidation reactions and pyranose-furanose interconversions. This retention is rationalized by a mechanism involving formation of a pseudo-acyclic intermediate. Another unexpected reaction was the conversion of slightly moist methyl d-glucopyranosiduronolactone syrup, on standing for 5–6 days at room temperature, into crystalline d-glucofuranurono-6,3-lactone, and of methyl α-d-mannopyranosidurono-6,3-lactone into crystalline d-mannofuranurono-6,3-lactone.     

Modulation of somatostatin release by endogenous glucagon and insulin: physiological relationship between A, B and D cells in rat pancreatic islets

In order to understand the physiological role of endogenous insulin or glucagon in somatostatin release, isolated rat pancreatic islets were treated with antiinsulin or antiglucagon antiserum in the presence of physiological amounts of glucose. The release of somatostatin was unchanged by treatment with antiinsulin antiserum which neutralized insulin released by 3.3, 8.3 and 16.7 mM of glucose. However, somatostatin release after treatment with antiglucagon antiserum was much reduced at all concentrations of glucose when compared with the release from control serum. Exogenous rat insulin (0.11, 1.11 micrograms/ml) had no effect, but exogenous glucagon (1, 5 micrograms/ml) resulted in a significant increase. Somatostatin release was stimulated by glucose, but the effect was insignificant. These results clearly indicate the physiological role of endogenous glucagon in the modulation of somatostatin release from the islets of Langerhans. Furthermore, the physiological relationship between A, B and D cells may be mediated through the paracrine mechanism.     

Evidence for insertion sequence-mediated spread of the thermostable direct hemolysin gene among Vibrio species.

The tdh gene of Vibrio parahaemolyticus which encodes the thermostable direct hemolysin has been found in some strains of other Vibrio species. Analysis of seven tdh genes cloned from V. parahaemolyticus, Vibrio mimicus, and non-O1 Vibrio cholerae revealed that all tdh genes were flanked by insertion sequence-like elements (collectively named ISVs) or related sequences derived from genetic rearrangement of ISVs. The ISVs possessed 18-bp terminal inverted repeats highly homologous to those of IS903 (2- to 4-bp mismatch) and were 881 to 1,058 bp long with less than 33.6% sequence divergence. These features and nucleotide sequence similarities among ISVs and IS903 (overall homologies between ISVs and IS903, ca. 50%) strongly suggest that they were derived from a common ancestral sequence. A family of ISVs were widely distributed in Vibrio species, often regardless of the possession of the tdh genes, and one to several copies of the ISVs per organism were detected. A strain of V. mimicus possessed two copies of the ISVs flanking the tdh gene and three copies unrelated to the tdh gene. However, the transposition activity of the ISVs could not be demonstrated, probably because they had suffered from base changes and insertions and deletions within the transposase gene. The possible mode of ISV-mediated spread of the tdh gene is discussed from an evolutionary standpoint.     

A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.     

Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.     

The micronucleus test with peripheral reticulocytes from phenacetin-treated mice.

The in vivo micronucleus test is conventionally performed using mouse bone marrow cells (BM assay). Using phenacetin as a test chemical, an alternative method using reticulocytes (RET assay) was examined to determine if this could be substituted for the BM assay. Single doses of 400, 600, and 800 mg/kg gave negative results 24 h after i.p. administration, but positive results were obtained with 600 and 800 mg/kg after 48 h. Responses were weak at 72 h. Double treatment enhanced the responses; 400 mg/kg gave a positive result. Maximum responses were generally reached 24 h after the second treatment, 48 h if doses were highly toxic. When the BM and RET assays were compared, the BM assay seemed to be slightly more sensitive than the RET assay; double treatment was superior to a single treatment in both BM and RET assays. Both assays can be used routinely but in the RET assay, sequential samples can be obtained from the same individuals without killing them, providing a firm basis to substitute it for the BM assay. Taking advantage of this characteristic of the RET assay, a regimen of double treatments and double sampling at 24 and 48 h is recommended for a wide range of doses. These data were obtained with CD-1 mice; MS/Ae mice gave a higher incidence of micronuclei than did the CD-1 strain.