Structural Studies on “Isosclerotan”, a New Glucan Isolated from Sclerotinia Fungus,by Physical,Chemical and Enzymatic Methods

“Isosclerotan”, a polysaccharide constituent extracted with a sodium hydroxide solution from sclerotia of Sclerotinia libertiana, could be purified by the successive precipitation with the followings; a mixture of copper sulfate and sodium hydroxide, ammonium sulfate, and ethyl alcohol. The preparation proved homogeneous by ultracentrifugal analysis. From sedimentation and viscosity measurements, the molecular weight of isosclerotan was calculated as 6.13 × 10⁶, andas 1.60 × 10⁵ after treatment with a dilute oxalic acid solution. Isosclerotan was scarecely soluble in cold water but soluble in hot water, yielding a highly viscous solution. It exhibited a low positive optical rotation, + 23.0° (in water), and infrared spectrum had a sharp absorption at 890~898 cm^?1, which indicated the prevalence of the β-glycosidic linkage in isosclerotan. Through degradation by acids and enzymes of isosclerotan, there were obtained various oligosaccharides containing β-1.3, β-1.4, and β-1.6 linkages. From results obtained by periodate oxidation and methylation, it is assumed that the polysaccharide involves the 1.3, 1.4, and 1.6 linkages in 47.7%, 16.6% and 35.7%, respectively, and a branching structure about 12.5%.     

D1 agonist SKF 38393 antagonizes pentobarbital-induced narcosis and depression of hippocampal and cortical cholinergic activity in rats

SKF 38393 (5 mg/kg), but not quinpirole, shortened the duration of loss of righting reflex produced in pentobarbital-narcotized rats. This effect was blocked by atropine (2 mg/kg), but not by atropine methylbromide, suggesting involvement of central cholinergic mechanisms. The analeptic effect was also blocked by SCH 23390 (0.2 mg/kg) or raclopride (2 mg/kg). SKF 38393 also increased sodium dependent high affinity choline uptake (HACU) in cortical and hippocampal synaptosomes that had been depressed by pentobarbital. SCH 23390 or raclopride prevented the SKF 38393 reversal of the depressed HACU, indicating that both D1 and D2 mechanisms were involved mediating the analeptic effect. These results provide neurochemical evidence that cortical and hippocampal D1-mediated cholinergic activation results in a behavioral arousal (analeptic) response. They also suggest that DA mechanisms may be involved in regulation of cortical and hippocampal cholinergic neurons.     

Crystal structure of Clostridium perfringens enterotoxin displays features of beta-pore-forming toxins

Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å. The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of β-pore-forming toxins such as aerolysin, C. perfringens ϵ-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of β-strands, two of which span its entire length. Domain II and domain III have three short β-strands each, by which they are distinguished. In addition, domain II has an α-helix lying on the β-strands. The sequence of amino acids composing the α-helix and preceding β-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming β-barrel-made pores. These structural features imply that CPE is a β-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long β-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins.     

Molecular identification of Ehrlichia species and 'Candidatus Neoehrlichia mikurensis' from ticks and wild rodents in Shizuoka and Nagano Prefectures, Japan

A total of 293 ticks and 111 wild rodents that were collected in Shizuoka and Nagano Prefectures, Japan, were examined for infection of Ehrlichia species and 'Candidatus Neoehrlichia mikurensis.' The 16S rDNA or the omp-1 gene of these bacterial DNAs were detected from the spleens of tick-inoculated mice (5 positive/total 29 mice) or from the spleens of wild rodents (25 positive/total 111 rodents) by PCR amplifi-cation. Sequencing of the 16S rDNA revealed Ehrlichia spp. from the 5 tick-inoculated mice and 8 wild rodents, and 'Candidatus N. mikurensis' from 17 wild rodents. The data suggest the presence of additional genetic variants, and potential vectors and/or reservoirs for these bacteria in central Japan.     

MAIGO2 is involved in exit of seed storage proteins from the endoplasmic reticulum in Arabidopsis thaliana

Seed storage proteins are synthesized on the endoplasmic reticulum (ER) as precursors and then transported to protein storage vacuoles, where they are processed into mature forms. Here, we isolated an Arabidopsis thaliana mutant, maigo2 (mag2), that accumulated the precursors of two major storage proteins, 2S albumin and 12S globulin, in dry seeds. mag2 seed cells contained many novel structures, with an electron-dense core that was composed of the precursor forms of 2S albumin. 12S globulins were segregated from 2S albumin and were localized in the matrix region of the structures together with the ER chaperones lumenal binding protein and protein disulfide isomerase, which were more abundant in mag2 seeds. The MAG2 gene was identified as At3g47700, and the MAG2 protein had a RINT-1/TIP20 domain in the C-terminal region. We found that some MAG2 molecules were peripherally associated with the ER membrane. MAG2 had an ability to bind to two ER-localized t-SNAREs (for target-soluble NSF [N-ethylmaleimide-sensitive fusion protein] attachment protein receptor; At Sec20 and At Ufe1). Our findings suggest that MAG2 functions in the transport of storage protein precursors between the ER and Golgi complex in plants.     

Chemical modification and 1H-NMR studies on the receptor-binding region of human interleukin 6

Oxidation of the Met residues of human interleukin 6 (IL-6) molecule has been performed. Reactivity of Met for the oxidation reaction was found to decrease in the order of Met50, Met118, Met185, Met162, and Met68. Chemical modifications involving oxidation and carboxypeptidase A digestion of IL-6 have led to the assignments of the methyl proton resonances of Met162 and Met185, respectively. The hydroxynitrobenzyl chromophore attached to Trp158 in the IL-6 molecule showed a different absorption spectrum when the labeled IL-6 was bound to the soluble IL-6 receptor. This result indicates that Trp158 is near the receptor-binding region in IL-6. On the basis of the 1H-NMR and chemical modification data, it has been concluded that Trp158 is in spatial proximity to Met162, His165 and Met185. The receptor-binding activity decreased with an increase in the number of oxidized Met residues. Of these five Met residues, Met162 was the residue in which the receptor-binding activity decreased in the most parallel degree with that of the oxidation reaction.     

Discrimination and characterization of photosystem I-and photosystem II-induced 515-nm absorbance changes in chloroplasts II. Separate local fields in thylakoids indicated by differential responses of the 515-nm absorbance change

Flash-induced 515-nm and 475-nm absorbance changes in spinachchloroplasts were investigated in the presence of 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU). DCMU reduced the magnitude of the 515-nmabsorbance change by half and almost completely diminished theabsorbance change at 475-nm. The reduction of the 475-nm absorbancechange paralleled the inhibition of the photosystem II (PS II)light reaction. When chloroplasts were illuminated with red or far-red light,the ratio of A₅₁₅/A₄₇₅ changed depending on the photosystemactivated. Wide variations in the A₅₁₅/A₄₇₅ ratio observed insubchloroplast particle preparations were probably due to theenrichment and activation of one of the photosystems. We suggest that the photosynthetic pigments in the thylakoidmembrane are heterogeneously distributed, and chlorophyll bmolecules that may be responsible for the 475- nm absorbancechange are affected by the local field formed by the PS II lightreaction. On the other hand, an electric field due to the PSI reaction probably induced the absorbance change at 515-nm (Received February 24, 1978; )     

Light-induced chemiluminescence of luminol in spinach chloroplast fragments: Reaction of O2- with electron transfer components

Chemiluminescence of luminol (CLL) was induced by illuminatedspinach chloroplast fragments. CLL was diminished by superoxidedismutase or under anaerobic conditions and increased by anautoxidizable electron acceptor, methyl viologen. The optimumpH for CLL was 10.0-10.5. Ferredoxin and cytochrome c reducing substance (CRS) did notaffect the intensity of CLL, but accelerated the dark decayin the absence of methyl viologen. In the presence of methylviologen, ferredoxin and CRS lowered the intensity and acceleratedthe dark decay. 3-(4-Chlorophenyl)-1,1-dimethylurea diminishedCLL. Carbonylcyanide m-chlorophenylhydrazone accelerated theinitial rate of CLL increase at low concentration and inhibitedit at high concentration. Half-decay time of CLL after the cessationof light was shortened by inhibiting electron transfer on theoxidizing side of photosystem II. We conclude that most of the CLL observed in illuminated chloroplastsis dependent on O₂^–. The results also suggest that O₂^–is reduced by reduced ferredoxin or CRS and oxidized on theoxidizing side of photosystem II. The half life of O₂^–in illuminated chloroplasts was estimated from the half-decaytime of CLL to be a few sec. ¹ Present address: Kyushu Dental College, Department of Biology,Kitakyushu 803, Japan. (Received May 30, 1977; )