Water-soluble chlorophyll protein of Brassica oleracea var. Botrys (cauliflower)

A water-soluble chlorophyll protein was prepared from Brassica oleracea var. Botrys (cauliflower) and purified by (NH₄)₂SO₄ fractionation and by chromatography on a DEAE-cellulose column. The chlorophyll protein contained chlorophylls a and b in the ratio 6:1, and no carotenoids. The molecular weight, determined by means of gel filtration on Sephadex G-100, was 78000. The chlorophyll protein showed absorption peaks at 273, 340, 384, 420, 438, 465, 628, 674 and 700 nm. Since the three bands at 384, 420 and 438 nm all have approximately the same height, the spectrum is different from that of chlorophyll a in organic solvents. The fluorescence of the chlorophyll protein showed a peak at 683 nm, with shoulders at 706 and 745 nm at room temperature, and peaks at 685, 706 and 744 nm at the temperature of liquid N₂. An apo-protein was prepared by removing the chlorophylls with 2-butanone and purified by precipitation with (NH₄)₂SO₄. The apo-protein thus prepared had an absorption band at 273 nm but none at longer wavelengths. The apo-protein could be combined with chlorophylls, forming a chlorophyll protein which had spectral characteristics similar to those of the original.     

Mislocalization of fukutin protein by disease-causing missense mutations can be rescued with treatments directed at folding amelioration

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common childhood muscular dystrophy in Japan, is caused by alterations in the fukutin gene. Mutations in fukutin cause abnormal glycosylation of α-dystroglycan, a cell surface laminin receptor; however, the exact function and pathophysiological role of fukutin are unclear. Although the most prevalent mutation in Japan is a founder retrotransposal insertion, point mutations leading to abnormal glycosylation of α-dystroglycan have been reported, both in Japan and elsewhere. To understand better the molecular pathogenesis of fukutin-deficient muscular dystrophies, we constructed 13 disease-causing missense fukutin mutations and examined their pathological impact on cellular localization and α-dystroglycan glycosylation. When expressed in C2C12 myoblast cells, wild-type fukutin localizes to the Golgi apparatus, whereas the missense mutants A170E, H172R, H186R, and Y371C instead accumulated in the endoplasmic reticulum. Protein O-mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) also mislocalizes when co-expressed with these missense mutants. The results of nocodazole and brefeldin A experiments suggested that these mutant proteins were not transported to the Golgi via the anterograde pathway. Furthermore, we found that low temperature culture or curcumin treatment corrected the subcellular location of these missense mutants. Expression studies using fukutin-null mouse embryonic stem cells showed that the activity responsible for generating the laminin-binding glycan of α-dystroglycan was retained in these mutants. Together, our results suggest that some disease-causing missense mutations cause abnormal folding and localization of fukutin protein, and therefore we propose that folding amelioration directed at correcting the cellular localization may provide a therapeutic benefit to glycosylation-deficient muscular dystrophies.     

Discovery of a novel EP2/EP4 dual agonist with high subtype-selectivity

A series of γ-lactam prostaglandin E(1) analogs bearing a 16-phenyl moiety in the ω-chain and aryl moiety in the α-chain were synthesized and biologically evaluated. Among the tested compounds, γ-lactam PGE analog 3 designed as a structural hybrid of 1 and 2 was discovered as the most optimized EP2/EP4 dual agonist with excellent subtype-selectivity (K(i) values: mEP2=9.3 nM, mEP4=0.41 nM). A structure-activity relationship study is presented.     

Revision of Asian species of the Drosophila melanica species group (Diptera: Drosophilidae), with a description of a new species from Vietnam

A new species of the Drosophila (Drosophila) melanica species group (Diptera: Drosophilidae), Drosophila denruoi Suwito & Watabe, sp. nov., is described from northern Vietnam. It is proved by crossing experiments that reproductive isolation is complete between this species and other morphologically similar species. Larval ganglion cells of D. denruoi possess 2n = 12 chromosomes, comprised of four pairs of acrocentric, one pair of metacentric and one pair of micro‐chromosomes. This is the largest number of acrocentric chromosomes in the group and implies that the species has the most primitive chromosomal configuration in the melanica group. Based on the results of crossing experiments and close morphological examination, Drosophila pengi Okada & Kurokawa, 1957 from central Japan, once regarded as a synonym of Drosophila tsigana Burla & Gloor, 1952, is shown to be distinct from D. tsigana. Drosophila pengi is thus resurrected as a member of the melanica group. We synonymize Drosophila bisetata Toda, 1988, described from central Myanmar, with D. pengi. In addition, a population from Guizhou, southwestern China, once regarded as D. tsigana, is also identified as D. pengi. These populations from central Myanmar and southwestern China share the diagnostic characters of D. pengi. At the same time, it is reconfirmed that the Hokkaido population is conspecific with European D. tsigana. Furthermore, another species, Drosophila longiserrata Toda, 1988 described from central Myanmar, is synonymized with Drosophila afer Tan, Hsu & Sheng, 1949 from China, based on their having identical karyotypes and external morphology.     

Morphological changes in diabetic kidney are associated with increased <Emphasis Type="Italic">O</Emphasis>-GlcNAcylation of cytoskeletal proteins including α-actinin 4

Purpose  

The objective of the present study is to identify proteins that change in the extent of the modification with O-linked N-acetylglucosamine (O-GlcNAcylation) in the kidney from diabetic model Goto-Kakizaki (GK) rats, and to discuss the relation between O-GlcNAcylation and the pathological condition in diabetes.     

Natural Abundance 13C Nuclear Magnetic Resonance Spectra of Intact Fungal Mycelia

We examined the change of the composition of the cell wall polysaccharides prepared from cells of the salt-tolerant yeast Zygosaccharomyces rouxii grown in two media containing 20% NaCl and 0% NaCl. Comparative analysis of their walls showed that the wall obtained from salt-free medium had greater quantities of alkali-insoluble fraction and smaller quantities of mannan than the walls obtained from 20% NaCl medium. The alkali-insoluble fractions from the cell walls obtained from salt-free medium contained a large amount of glucosamine and a smaller amount of linear β-1,3-glucosidic linkage than the fractions from the cell walls obtained from 20% NaCl medium. Structural analyses showed that the mannans from each cell wall contained an α-1,6-mannbsidic linked backbone to which single mannose and mannobiose units were connected as side chains by α-1,2-mannosidic linkages. However, when cells were grown in the presence of 20% NaCl, the side chains of the mannans consisting of a mannobiose unit were largely reduced.

These results indicated that the structure of alkali-insoluble glucan and mannan were greatly affected by the presence of NaCl in the final medium.     

Structure and expression of elongation factor 2 gene during development of Dictyostelium discoideum

A cDNA library constructed from poly(A)+ RNA isolated from Dictyostelium discoideum cells at 12 h of development was screened with the hamster elongation factor 2 (EF-2) cDNA. Several different cDNA clones which hybridized were isolated after a second screening. A cDNA clone representing the 5'-end of the mRNA was obtained by primer extension. By comparing the amino acid sequence deduced from the nucleotide sequences of these clones with that of hamster EF-2, we found enough homology between them to conclude that the isolated clones were complementary to the mRNA of D. discoideum EF-2. The N terminus which is the GTP-binding domain and the C-terminal half where it interacts with a ribosome showed a high degree of homology. The amino acid sequence of the carboxyl half includes that it contain a site of ADP-ribosylation by diphtheria toxin. From the Northern blotting analysis, the size of the mRNA was estimated to be 2.6 kilobases. The expression of the mRNA was high in vegetative cells, became maximal at the aggregation stage, and decreased thereafter through development. Upon differentiation of prespore and prestalk cells, the mRNA was highly enriched in the former over the latter. ADP-ribosylation assay of EF-2 protein by diphtheria toxin showed nearly the same developmental changes for the protein as the mRNA. However, prestalk cells were found to contain the same amount of the protein as prespore cells. The Southern blot analyses indicated that the gene encoding EF-2 is unique.     

Expression of low density lipoprotein receptor-related protein 4 (Lrp4) gene in the mouse germ cells

The low density lipoprotein receptor-related protein 4 gene (Lrp4) was identified by subtractive screening of cDNAs of the migratory primordial germ cells (PGCs) of E8.5-9.5 embryo and E3.5 blastocysts. Lrp4 is expressed in PGCs in the hindgut and the dorsal mesentery of E9.5 embryos, and in germ cells in the genital ridges of male and female E10.5-13.5 embryos. Lrp4 is also expressed in spermatogonia of the neonatal and adult testes and in the immature oocytes and follicular cells of the adult ovary. The absence of Lrp4 expression in the blastocyst, embryonic stem cells and embryonic germ cells suggests the Lrp4 is a molecular marker that distinguishes the germ cells from embryo-derived pluripotent stem cells.     

Suppression of Glial Tumor Growth by Expression of Glial Fibrillary Acidic Protein

We examined the effect of expression of glial fibrillary acidic protein (GFAP) on the tumor growth of astrocytoma in vivo. When rat astrocytoma C6 cells were injected subcutaneously in athymic mice, the cells produced tumors that grew rapidly. The tumor growth of C6 cells transfected with GFAP cDNA was significantly reduced compared to that of control NeoC6 cells transfected only with the neomycin resistant gene. After implantation of C6 cells transfected with mutated GFAP cDNA at the phosphorylation sites, the tumor growth was suppressed similar to that of the wild GFAP transfectants. To determine whether the cell growth suppression by GFAP is specific for astroglial cells, we assessed the effect of GFAP on the cell growth of an L cell of fibroblast origin in vitro. By GFAP cDNA transfection, L cells showed morphological changes, but the cell growth was not reduced. These results suggest that GFAP is a critical regulator of the tumor growth of astrocytoma.     

Down-Regulation of KCC2 Expression and Phosphorylation in Motoneurons,and Increases the Number of in Primary Afferent Projections to Motoneurons in Mice with Post-Stroke Spasticity

Spasticity obstructs motor function recovery post-stroke, and has been reported to occur in spinal cord injury and electrophysiological studies. The purpose of the present study was to assess spinal cord circuit spasticity in post-stroke mice. At 3, 7, 21, and 42 d after photothrombotic ischemic cortical injury in C57BL/6J mice, we observed decreased rate-dependent depression (RDD) of the Hoffmann reflex (H reflex) in the affected forelimb of mice compared with the limbs of sham mice and the non-affected forelimb. This finding suggests a hyper-excitable stretch reflex in the affected forelimb. We then performed immunohistochemical and western blot analyses to examine the expression of the potassium-chloride cotransporter 2 (KCC2) and phosphorylation of the KCC2 serine residue, 940 (S940), since this is the main chloride extruder that affects neuronal excitability. We also performed immunohistochemical analyses on the number of vesicular glutamate transporter 1 (vGluT1)-positive boutons to count the number of Ia afferent fibers that connect to motoneurons. Western bolts revealed that, compared with sham mice, experimental mice had significantly reduced KCC2 expression at 7 d post-stroke, and dephosphorylated S940 at 3 and 7 d post-stroke in motoneuron plasma membranes. We also observed a lower density of KCC2-positive areas in the plasma membrane of motoneurons at 3 and 7 d post-stroke. However, western blot and immunohistochemical analyses revealed that there were no differences between groups 21 and 42 d post-stroke, respectively. In addition, at 7 and 42 d post-stroke, experimental mice exhibited a significant increase in vGluT1 boutons compared with sham mice. Our findings suggest that both the down-regulation of KCC2 and increases in Ia afferent fibers are involved in post-stroke spasticity.