Synthesis of 1-(2-Deoxy-β-D-ribofuranosyl)benzimidazole via Cyclonucleosides

Abstract

Starting from 2-chlorobenzimidazole and 1-O-acetyl-2,3,5-tri-O-benzoylribofuranose a β-D-ribonucleoside of 2-chlorobenzimidazole was obtained using Vorbrüggen's procedure. This compound was derivatized to a 2,2′-S-cyclonucleoside via 2′-O-tosylation and thiourea treatment. The cyclonucleoside was converted to 1-(2-deoxy-β-D-ribo-furanosyl) benzimidazole by Raney nickel desulfurization.     

Brassinosteroids Act as Regulators of Tracheary-Element Differentiation in Isolated Zinnia Mesophyll Cells

Uniconazole [S-3307; (E)-l-(4-chlorophenyl)-4,4-dimethyl-2-(l,2,4-triazol-l-yl)-l-penten-3-ol],a synthetic plant-growth retardant, inhibited the differentiationof isolated mesophyll cells of Zinnia elegans L. into trachearyelements (TEs) but had no effect on cell division when it wasadded to the culture medium at a concentration of 3.4 µM.In the presence of uniconazole, none of the cytological eventscharacteristic of the processes of TE differentiation, suchas aggregation of actin filaments, bundling of microtubulesor localized thickening and lignification of secondary walls,was observed. Uniconazole was effective when it was added tothe medium within 36 h after the start of culture. Brassinosteroids(0.2 nM brassinolide or 2 µM homobrassinolide), but notgibberellin A₃, counteracted the inhibitory effect of uniconazoleon TE differentiation. Brassinosteroids were most effectivewhen they were added to cultures between 24 and 30 h after thestart of culture. Exogenously applied brassinosteroids promotedTE differentiation. It is suggested that the synthesis of brassinosteroidsis essential for the differentiation of the cells into TEs andthat uniconazole inhibits this differentiation through its inhibitoryeffect on the biosynthesis of brassinosteroids. (Received May 9, 1991; )     

Involvement of a Calcium-Dependent Protein Kinase in Hypoosmotic Turgor Regulation in a Brackish Water Characeae Lamprothamnium succinctum

Calcium ion is a key messenger in turgor regulation of internodalcells of Lamprothamnium succinctum in response to hypoosmotictreatment. An increase in the concentration of cytosolic freecalcium ion ([Ca²⁺]_c) is prerequisite for the turgor regulation[Okazaki and Tazawa (1990) J. Membr. Biol. 114: 189], We examinedwhether or not a calcium-dependent protein kinase (CDPK) isinvolved in the Ca²⁺-mediated turgor regulation of Lamprothamniumcells. A 53-kDa CDPK which phosphorylated preferentially histoneH1 but poorly myelin basic protein or casein, was detected inthe cell extract of Lamprothamnium by an in-gel protein kinaseassay. This protein kinase was detected by Western blottingand was immunoprecipitated using an anti-Dunaliella tertiolectaCDPK antibody which can neutralize the Dunaliella CDPK activity[Yuasa et al. (1995) Plant Cell Physiol. 36: 699]. The 53-kDaCDPK was partially purified from Lamprothamnium and its activitywas shown to be inhibited by the antibody and K-252a, a proteinkinase inhibitor. Microinjection of the antibody into the cytosblof Lamprothamnium cells inhibited the decrease in turgor pressurein response to hypoosmotic treatment. However, a transient increasein [Ca²⁺]_c, which was suggested by a transient reduction ofthe velocity of cytoplasmic streaming, was induced in antibody-injectedcells after hypoosmotic treatment. Turgor regulation upon hypoosmotictreatment was inhibited when the cells were treated with K-252a.These results imply that CDPK of Lamprothamnium functions ata down-stream position of Ca²⁺-mobilization in processing turgorregulation in response to hypoosmotic treatment. ² These authors contributed equally to the work.     

Chaihu-Shugan-San Administration Ameliorates Perimenopausal Anxiety and Depression in Rats

Chaihu-Shugan-San (CSS) is a traditional Chinese herbal formula that is widely used for treating perimenopausal symptoms in China; however, its mechanisms remain unknown. The present study was designed to investigate potential CSS mechanisms in rats with unpredicted chronic mild stress (UCMS) and normally aging rats (52 weeks of age). We performed the sucrose consumption test along with the forced swimming test to confirm depression-like behavior and the open field test (OFT) to confirm anxiety-like behavior in the animals. In addition, we used an enzyme-linked immunosorbent assay to measure serum and hippocampal estradiol (E₂) levels and a quantitative real-time polymerase chain reaction to assess hippocampal mRNA levels of estrogen receptors (ERs) α and β as well as G protein-coupled receptor 30 (GPR30). We found that CSS administration resulted in a significant increase in the ratio of hippocampal ERα and ERβ mRNA (ERα/ERβ ratio) in UCMS rats (p<0.001). However, no significant changes were observed in E₂ levels, ERα mRNA expression, and GPR30 mRNA expression. In contrast, changes in ERα/ERβ mRNA ratio were sensitively associated with changes in mood states in the animal models. These findings suggest that enhancement of ERα/ERβ ratio may play a role in the pharmacological mechanisms of CSS. Furthermore, this ratio can be employed as a potential index for evaluating mood states in animal models and can be considered as a therapeutic target for perimenopausal anxiety and depression in the future.     

Resolution of DL-Glutamic Acid with Optically Active Neutral Amino Acid Amides

DL-Glutamic acid has been resolved into optically active forms as the diastereoisomeric salt of optically active neutral amino acid amides, and the salt is easily converted to the sodium salt of the active forms. By resolution with L-tyrosinamide and L-leucinamide, sodium L-glutamate was obtained in 65 and 80 per cent yield respectively. Attempts to extend this method to resolution of DL-glutamic acid using L-phenylalaninamide as resolving agent resulted in poor yield of less pure D-glutamic acid.

By the infrared spectroscopy and X-ray diffraction analysis it has been confirmed that the diastereoisomeric salts of L-leucinamide or L-tyrosinamide with L- or D-glutamic acid compose a combined salt structure in solid state, whereas L-phenylalaninamide with L- or D-glutamic acid does not compose a characteristic diastereoisomeric salt but rather the mechanical mixture of L-phenylalaninamide and L- or D-glutamate anion in solid state.

In the previous study¹⁾, it was reported that L-leucinamide forms the characteristic diastereoisomeric salts with racemic N-acyl mono amino acids, most of which are fairly resolved into their antipodes.     

Selenium Compound Protects Corneal Epithelium against Oxidative Stress

The ocular surface is strongly affected by oxidative stress, and anti-oxidative systems are maintained in corneal epithelial cells and tear fluid. Dry eye is recognized as an oxidative stress-induced disease. Selenium compound eye drops are expected to be a candidate for the treatment of dry eye. We estimated the efficacy of several selenium compounds in the treatment of dry eye using a dry eye rat model. All of the studied selenium compounds were uptaken into corneal epithelial cells in vitro. However, when the selenium compounds were administered as eye drops in the dry eye rat model, most of the selenium compounds did not show effectiveness except for Se-lactoferrin. Se-lactoferrin is a lactoferrin that we prepared that binds selenium instead of iron. Se-lactoferrin eye drops suppressed the up-regulated expression of heme oxygenase-1, cyclooxygenase-2, matrix metallopeptidase-9, and interleukin-6 and also suppressed 8-OHdG production in the cornea induced by surgical removal of the lacrimal glands. Compared with Se-lactoferrin, apolactoferrin eye drops weakly improved dry eye in high dose. The effect of Se-lactoferrin eye drops on dry eye is possibly due to the effect of selenium and also the effect of apolactoferrin. Se-lactoferrin is a candidate for the treatment of dry eye via regulation of oxidative stress in the corneal epithelium.     

Restoration of Mismatch Repair Functions in Human Cell Line Nalm-6, Which Has High Efficiency for Gene Targeting

Gene targeting is a powerful approach in reverse genetics. The approach has been hampered in most of human cell lines, however, by the poor targeting efficiency. Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, exhibits exceptionally high gene targeting efficiency and is used in DNA repair and the related research fields. Nonetheless, usage of the cell line is still limited partly because it lacks expression of MSH2, a component of mismatch repair complex, which leads to increased genome instability. Here, we report successful restoration of MSH2 expression in Nalm-6 cells and demonstrate that the recovery does not affect the high targeting efficiency. We recovered the expression by introduction of cDNA sequences corresponding to exons 9 to 16 at downstream of exon 8 of the MSH2 gene. Endogenous exons 9 to 16 were deleted in the cell line. The MSH2 expression substantially reduced spontaneous HPRT mutation frequency. Moreover, gene targeting efficiency in the MSH2-expressing cells was similar to that in the MSH2-lacking cells. In fact, we generated heterozygously REV3L knockout and the catalytically dead mutants in the MSH2-proficient Nalm-6 cells with efficiency of 20–30%. The established cell line, Nalm-6-MSH+, is useful for reverse genetics in human cells.     

Studies on Streptomycetes

Investigation was made on the mycological and antibacterial properties of a strain No. 46408 isolated from a soil sample collected in Shimoueda, Wakayama Prefecture. The strain No. 46408 was compared with similar strains, St. hygroscopicus and St. halstedii, and it was judged to belong to a new species and therefore named Streptomyces atratus nov. sp. Also a strain A-165-Z1, which produces ilamycin, was referred to on its mycological properties and assumed to resemble St. atratus. St. atratus was found to produce new antibiotics, rufomycin A and B, specially active against acid-fast bacteria.