Photosystem I electron transport and phosphorylation supported by electron donation to the plastoquinone region

In chloroplasts, tetramethyl-p-hydroquinone supports high rates of phosphorylation-coupled, noncyclic electron flow through Photosystem I to methylviologen. The reaction is totally sensitive to dibromothymoquinone, indicating an electron donation to the plastoquinone region of the photosynthetic chain. The uncoupled electron flow rate exceeds 1000 μequivalents per hour per mg chlorophyll. The phosphorylation efficiency ($\text{P}\text{e}{}_2$) at the optimal pH of 8 is 0.6–0.65. Presumably this ratio represents the efficiency of energy coupling in the electron transfer step plastoquinone → cytochrome f.     

Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds

Using a rice maturing seed pUC9 expression library, we isolated a cDNA clone corresponding to 10 kDa sulfurrich prolamin by immunoscreening. A longer cDNA clone was obtained from a gtll library by plaque hybridization using this ³²P-labeled cDNA as a probe. A polypeptide sequence composed of 134 amino acids was deduced from the nucleotide sequence. A 24 amino acid signal peptide was assigned by computer calculation for the membrane spanning region and Edman sequencing of the purified mature polypeptide. Remarkably, 20% of methionine and 10% of cysteine were found in the mature polypeptide as well as high contents of glutamine, and hydrophobic amino acids. Part of the amino acid sequence was homologous with a conserved cysteine-rich region found in other plant prolamins. Two repeats of amino acid sequence were found in the polypeptide.     

The Site of Synthesis and Accumulation of Rice Storage Proteins

Electron microscopy showed that the two types of protein bodies(PB) in starchy endosperms of rice were formed differently duringthe period of storage protein accumulation. Two routes for thetransport of storage protein from the site of synthesis at therough endoplasmic reticulum (RER) to the site of accumulationwere also proposed. PB-I, bound by a single membrane to whichribosomes were attached, was thought to develop inside the cisternaeof RER, while the PB-II membrane was thought to originate fromthe vacuole. In the wheat germ cell-free translation system, storage protein-relatedpolypeptides of developing rice endosperms, including a precursorof glutelin and putative precursors of prolamin, were directedby membrane-bound polysomes but not by free-polysomes. Immunoassayof the total translation products directed by a PB fractionshowed that 46% were storage protein-related polypeptides. Rice storage proteins (prolamin) that accumulate in PB-I appearto be synthesized by membrane-bound polysomes attached to PB-Ior RER and to pass through the membrane into the lumen wherethey aggregate and are deposited. The proteins (glutelin andglobulin) that accumulate in PB-II, however, seem to be synthesizedby membrane-bound polysomes as a large precursor and to becomesequestered into the cisternal space of RER, from where theyare transferred to the vacuolar precursor of PB-II. (Received August 6, 1985; Accepted November 6, 1985)     

Characterization of extracellular substance of Vibrio anguillarum toxic for rainbow trout and mice

An extracellular toxic substance was separated from the cell-free culture filtrate of Vibrio anguillarum (strain NCMB571). Two fractions (GI and GII + III) obtained by Sephadex G-200 chromatography following DEAE-cellulose chromatography were lethal to rainbow trout and mice. Material separated from the GI fraction by Sepharose 4B affinity chromatography (GI-A fraction) was lethal to these animals. By sodium dodecylsulfate polyacrylamide gel electrophoresis, the GI and GI-A fractions were found to be composed of components with molecular weights of 44K and 34K, and 44K, respectively. The 44K protein band was associated with carbohydrate. Peripheral vascular disorder was observed in fish and mice that died after inoculation with GI or GI-A fraction. The toxic substance was sensitive to potassium periodate but was resistant to trypsin and acetone. Heat inactivation of the toxic substance was almost complete at 100 C for 20 min and complete at 121 C for 20 min. The toxic activity was not associated with hemolytic or proteolytic activity. Homologous antitoxin completely neutralized the toxic activity.     

Quantitative measurement of endotoxin in rainbow trout (Salmo gairdneri) serum by the chromogenic substrate method

The amount of endotoxin in serum collected from normal rainbow trout ( Salmo gairdneri) and trout inoculated with viable Vibrio anguillarum or lipopolysaccharide (LPS) extracted from bacteria was determined by the chromogenic substrate method. The mean values of endotoxin in four different groups of normal rainbow trout sera ranged from 31.9 to 65.3 pg/ml. When fish were inoculated with viable bacteria (1 × 10⁸), they became septicaemic and a large amount of endotoxin (> 14 ng/ml) was detected in the sera. In fish inoculated with a smaller number of bacteria the amount of endotoxin was several times higher than that of normal fish in spite of failure of bacterial isolation. Although the endotoxin level in serum increased rapidly (> 100 ng/ml) after intraperitoneal inoculation with purified V. anguillarum LPS (540 μg), no fish died during the experiment. The high level of endotoxin in normal rainbow trout and the resistance of trout to endotoxin are in striking contrast to those of mammalian and avian species.     

Receptor recycling: liberation of glucocorticoid receptors from liver nuclei in vitro

Dynamics of the steroid receptors seems to be the consequence of receptor recycling. In the present study, as a clue to elucidate the mechanism of receptor recycling, factors which affect the rate of liberation of nuclear bound 3H-glucocorticoids were examined in vitro. Among the factors examined, NAD, NADPH, cAMP and p-nitrophenyl phosphate accelerated the liberation of radioactivity from nuclei in a temperature-dependent manner when added to the incubation mixture. The presence of a large amount of unlabeled dexamethasone (Dex) did not modify the rate of liberation. From these results, it was concluded that the metabolism of ligand bound to the receptor is not a necessary step in the liberation of receptor from nuclei. These agents did not influence the binding process of 3H-Dex-receptor complex to DNA-cellulose. Therefore the stimulation of receptor release does not seem to be mediated by reducing the binding affinity between nuclei and receptor complexes. The liberated radioactivity was eluted on a Sephadex G-100 column in the void volume and in macromolecule-unbound fractions. In both fractions, the majority of the radioactivity comigrated with authentic glucocorticoids on thin-layer chromatography.     

Proposal of leukotoxin, 9,10-epoxy-12-octadecenoate, as a burn toxin

It is postulated that toxic substances (burn toxin) synthesized in burned skin are transferred into general circulation and cause multiple organ failure. We found a highly cytotoxic substance, leukotoxin, a linoleate epoxide, exists in burned skin. Leukotoxin, as the name indicates, was synthesized by leukocytes from linoleate as a substrate. The aim of this study is to evaluate the possibility of leukotoxin as a burn toxin. We studied plasma leukotoxin level of four patients with extensive burns (over 50% of body surface area) and examined coagulation studies in these patients. We detected considerable amounts of leukotoxin (11.4 nmol/ml-37.0 nmol/ml) in all patients. Leukotoxin was not detected in the control subjects. Pulmonary edema, cardiac failure, and coagulation abnormalities were found in these patients. Exogeneously administered leukotoxin induced similar pathological conditions in experimental animals to those observed in patients with extensive burns. Hence, it is concluded that leukotoxin is a responsible substance as a burn toxin.