Production of bioactive triterpenes by Eriobotrya japonica calli

Callus tissue cultures induced from an axenic leaf of Eriobotrya japonica (Rosaceae) produced triterpenes in large amounts (ca. 50 mg/g dry wt). Nine triterpenes were characterized as ursolic acid, oleanolic acid, 2alpha-hydoxyursolic acid, maslinic acid, tormentic acid, 2alpha, 19alpha-dihydroxy-3-oxo-urs-12-en-28-oic acid, hyptadienic acid and a mixture of 3-O-cis-p-coumaroyltormentic acid and 3-O-trans-p-coumaroyltormentic acid. The triterpene composition in the callus tissues was noticeably different from that in intact leaves. The contents of tormentic acid with antidiabetic action, and 2alpha, 19alpha-dihydroxy-3-oxo-urs-12-en-28-oic acid with anti-HIV activity, were much larger than those in the intact leaves. All of the triterpenes isolated from the callus tissues showed an inhibitory effect comparable to (-)-epigallocatechin gallate (EGCG) of green tea on the activation of Epstein-Barr virus early antigen (EBV-EA) induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). 2alpha, 19alpha-Dihydroxy-3-oxo-urs-12-en-28-oic acid was the most potent inhibitor among them and caused a significant delay of two-stage carcinogenesis on mouse skin.     

Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes.

Recent studies using mice with genetically engineered gap junction protein connexin (Cx) genes have provided evidence that reduced gap-junctional coupling in ventricular cardiomyocytes predisposes to ventricular arrhythmia. However, the pathological processes of arrhythmogenesis due to abnormalities in gap junctions are poorly understood. We have postulated a hypothesis that dysfunction of gap junctions at the single-cell level may affect synchronization of calcium transients among cardiomyocytes. To examine this hypothesis, we developed a novel system in which gap-junctional intercellular communication in primary neonatal rat cardiomyocytes was inhibited by a mutated (Delta130-137) Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP), and calcium transients were imaged in real time while the mutated Cx43-EGFP-expressing cardiomyocytes were identified. The mutated Cx43-EGFP inhibited dye coupling not only in the liver epithelial cell line IAR 20 but also in primary neonatal rat cardiomyocytes in a dominant-negative manner, whereas wild-type Cx43-EGFP made functional gap junctions in otherwise communication-deficient HeLa cells. The mutated Cx43-EGFP induced desynchronization of calcium transients among cardiomyocytes with significantly higher frequency than wild-type Cx43-EGFP. These results suggest that dysfunction of gap-junctional intercellular communication at the single-cell level could hamper synchronous beating among cardiomyocytes as a result of desynchronization of calcium transients.     

Effects of soy protein diet on the expression of adipose genes and plasma adiponectin.

Many studies have reported the cholesterol-lowering, anti-lipogenic, anti-obesity and anti-hypertensive effects of soy protein. Adipose tissue-specific plasma protein, adiponectin, has anti-atherogenic and anti-insulin-resistance properties. Here, we investigated the effects of soy protein diet on body fat composition, plasma glucose, lipid and adiponectin levels and expression of genes involved in glucose and fatty acid metabolism in obese KK-A y mice. Body weights and adipose tissue weights of mesenteric, epididymal, and brown fat were lower in mice on calorie-restricted diet containing soy protein isolate. Plasma cholesterol, triglyceride, free fatty acid, and glucose levels were also decreased by this diet. Body fat content and plasma glucose levels in mice on a soy protein isolate diet were still lower than those treated with an isocaloric casein-protein-diet. Among the genes related to glucose and fatty acid metabolism, adiponectin mRNA levels in adipose tissue and adiponectin plasma concentrations were elevated in mice on a calorie-restricted diet, although there were no significant differences between soy protein and casein protein groups. Our results indicate that that soy protein diet decreased body fat content and plasma glucose levels more effectively than isocaloric casein-protein diet in obese mice.     

Removal of Heparan Sulfate Chains Halted Epithelial Branching Morphogenesis of the Developing Mouse Submandibular Gland in vitro

The physiological function of heparan sulfate chains in the mouse embryonic submandibular gland was studied by the use of heparitinases purified from Flavobacteriu heparinum . Heparitinase I, which catalyzes the cleavage of specific glycosaminidic linkages adjacent to non-or monosulfated disaccharides of heparan sulfate chains, in the culture medium of the mid and late 12-day gland inhibited the branch-initiation and changed their round epithelial shape to elongated one, together with a concommitant reduction in lobular growth. [³H]Thymidine incorporation experiments indicated that heparitinase I treatment blocked 24% of the DNA synthesis compared with controls. Analysis of ³⁵S-inorganic sulfate labeled glycosaminoglycans extracted from cultured rudiments revealed that the glands with heparitinase I contained no heparan sulfate, while in the glands without the enzyme more than 20% of total glycosaminoglycans was heparan sulfate.
The heparitinase effect on morphogenesis was mimicked by the addition of heparan sulfate (1 mg ml⁻¹) or heparin (75 μg ml⁻¹), but not by chondroitin sulfate (1 mg ml⁻¹) in the culture medium. Transmission electron microscopic study indicated that at the epithelial-mesenchymal interface close contacts between the fibroblast and epithelial cells were much fewer in heparitinase-treated glands than in controls. Immunohistochemical analysis demonstrated that the core protein of basement membrane heparan sulfate proteoglycan and type IV collagen accumulated abnormally inside the epithelial lobules of glands cultured with heparitinase I. These results strongly suggested that glycosaminoglycan chains of heparan sulfate or heparin is involved in the epithelial morphogenesis of the mouse embryonic submandibular gland.     

Phase I/II trial of didanosine (2',3'-dideoxyinosine) in hemophiliac patients with AIDS or AIDS-related complex

Forty-three hemophiliacs with AIDS or ARC received a daily dose of 334 or 500 mg didanosine (2',3'-dideoxyinosine or ddI) orally in 2 divided doses in phase I/II, open-label clinical trial conducted in Japan. Twenty-eight patients completed 6 months of therapy. There was an increase in circulating CD4(+) cells in 19 valuable patients from 91 +/- 25 (mean +/- SE) at entry to 131 +/- 38 at 24 weeks of therapy P = 0.01; Wilcoxon signed rank). Fourteen of 37 patients met the criteria for CD4 rise >/= 50/mm3 rise or >/= 50% increase from entry values) for more than 4 consecutive weeks. Twenty patients were p24 positive at entry. Nine out of the 10 evaluable patients (90%) showed a decline in p24 antigen at weeks 20-24 (P = 0.02). Thirty-five patients had symptoms related to HIV-1 infection at entry. Twenty-seven patients reported improvements in constitutional symptoms during therapy. Nine patients presented with possible drug-related adverse effects, and didanosine was discontinued in 6 patients (one each with edema; abdominal pain with anorexia; hematuria with edema and rash; sense of abdominal distension with anorexia; diarrhea and abdominal pain; and irritability). One patient had a transient increase in serum amylase level to twice the upper limit of normal, but he continued to receive the drug. These data suggest that didanosine was generally well tolerated in hemophiliacs with AIDS or ARC, and its administration correlated with improvement in constitutional symptoms and laboratory findings. The adverse effects of didanosine seen in this population were moderate to mild, and no complications related to hemorrhagic diathesis were observed, although the relative risk of acute pancreatitis in this population (while not seen in the present study to date) requires more study.     

Improvement of production of l-aspartic acid using immobilized microbial cells

Summary In our laboratory, EAPc-7 a strain having higher aspartase activity was derived from Escherichia coli ATCC 11303. For the improvement of l-aspartic acid productivity using EAPc-7 cells immobilized in -carrageenan, it was necessary to eliminate the fumarase activity which converts fumaric acid to l-malic acid. Several treatments for specifically eliminating fumarase activity from EAPc-7 cells were tested and it was found that when EAPc-7 cells were treated in a culture broth (pH 4.9) containing 50 mM l-aspartic acid at 45° C for 1 h, fumarase activity was almost completely eliminated without inactivation of the aspartase.The treated cells, immobilized in -carrageenan, were used for continuous production of l-aspartic acid from ammonium fumarate. The formation of l-malic acid was negligible and the half-life of the immobilized preparation was 126 days.Productivity of immobilized preparation of treated EAPc-7 cells in l-aspartic acid production was six times of that of the parent cell preparation.     

The 5'-terminal sequence of TMV RNA. Question on the polymorphism found in vulgare strain

The complete nucleotide sequence of TMV RNA (common strain) reported in [Proc. Natl. Acad. Sci. USA (1982) 79, 5818] its 5'-end to be represented by two variants which differed in length. We have tested that result and sequenced the 5'-terminal regions of two strains of TMV RNA (common strain OM and tomato strain L) using cloned cDNA copies. The results showed that the 5'-terminal region of the TMV genome is not polymorphic and that one of the two variants cited above represents a tomato strain but not the common strain.     

Preferential inhibitory action of sodium cholate on an Escherichia coli strain carrying a plasmid in an integrated state.

Sodium cholate was shown to be preferentially more active on Escherichia coli strains carrying an integrated plasmid, i.e., on Hfr strains, than on their parental strains with or without a plasmid in an autonomous state.     

Cytofluorometry on cells isolated from paraffin sections after blocking of the background fluorescence by azocarmin G

Summary A technique for isolation of cells from paraffin embedded tissue is indispensable for the performance of Feulgen-DNA cytofluorometry in parallel with the definition of histological characteristics. Background fluorescence due to nonspecific dye-binding by a pseudo-plasmal reaction is usually found to be so intense on cells isolated from formalin-fixed tissues, that we are often forced to abandon quantitative DNA determinations. In the present work, we report that fixation of tissues with Carnoy's fixative for 12 h at 5° C not only reduces nonspecific dye-binding but also facilitates the process of cell isolation. Furthermore, we find that pre-treatment of cells isolated from Carnoy-fixed tissues with acidic azocarmin G solution completely blocks nonspecific dye-binding in subsequent acriflavine Feulgen nuclear staining. This combination of techniques for specimen preparation enables us to carry out Feulgen-DNA cytofluorometry on cells isolated from histological sections with satisfactorily low coefficients of variation (less than 8%). The techniques should be widely applicable for parallel DNA determinations and histology.     

Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase

The occurrence of adenosine 5-triphosphate-3-diphosphate-synthesizing activity was detected in five strains of actinomycetes; Streptomyces morookaensis, Streptomyces aspergilloides, Streptomyces hachijoensis, Actinomyces violascens and Streptoverticillium septatum, out of 825 strains of actinomycetes, bacteria, fungi and imperfecti. Purine nucleotide pyrophosphotransferase were extracellularly excreted associating with the cell growth, and were purified partially or to apparent homogeniety from the culture filtrate. The enzymes are a monomeric protein with a molecular weight of 18000–26000 and synthesize adenosine, guanosine and inosine 5-phosphate (mono, di or tri)-3-diphosphate such as pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp and pppIpp by transferring a pyrophosphoryl group from the 5-position of ATP, dATP and pppApp to the 3-position of purine nucleotides in the presence of a divalent cation and in alkaline state.Abbreviations pppApp adenosine 5-triphosphate 3-diphosphate - ppApp adenosine 5-diphosphate 3-diphosphate - pApp adenosine 5-monophosphate 3-diphosphate - pppGpp guanosine 5-triphosphate 3-diphosphate