Phospholipase D production using immobilized cells of Streptoverticillium cinnamoneum

Summary Production of phospholipase D (PLD) by Streptoverticillium cinnamoneum immobilized within porous particles was investigated in repeated batch fermentation. The enzyme productivity in repeated batch fermentation was 2.2-fold that obtained in batch fermentation without immobilization, since many of the immobilized cells could be utilized as seed cells for each subsequent batch cycle.     

Repression by a differentiation-specific factor of the human cytomegalovirus enhancer.

We detected a novel nuclear protein, MRF, that binds to multiple sites on the modulator which is located upstream of the human cytomegalovirus major immediate early gene enhancer. The expression of MRF is differentiation specific; the DNA binding activity is present in nuclear extracts from undifferentiated Tera-2 and THP-1 cells, but significantly reduced after these cells are induced to differentiate. In undifferentiated cells the enhancer activity is repressed by the modulator and upon differentiation the enhancer becomes active. Competitive binding assays demonstrate that MRF requires the presence of multiple A+T stretches for binding to DNA, rather than binding to a specific DNA sequence. Mutations of these stretches in the modulator reduce the binding activity of MRF, as well as the repressing activity on the enhancer. These results suggest that MRF may act as a repressor of enhancer function. We propose that MRF binds over the entire modulator and exerts repressor activity.     

Immunocytochemical detection of Ca2+-dependent subspecies of protein kinase C in mouse embryos before and during compaction

Summary To confirm the possibility that protein kinase C is involved in compaction of mouse embryos, the presence and distribution pattern of Ca²⁺-dependent subspecies of this enzyme in mouse embryos, before and during compaction, were examined immunocytochemically with three different monoclonal antibodies. These were MC-1a, MC-2a and MC-3a, which selectively interact with the subspecies of the enzyme known as types I, II and III, respectively. Only when embryos were incubated with MC-3a, was immunofluorescence clearly detected in all cells of embryos before and during compaction. This result demonstrates the presence of type III protein kinase C in embryos before and during compaction and suggests the possibility that the type III enzyme may be involved in compaction. No marked differences were found in the distribution pattern of the type III enzyme between embryos examined before and during compaction.     

Tumor Necrosis Factor-α (TNF-α), Interferon-γ, and Interleukin-6 but Not TNF-β Induce Differentiation of Neuroblastoma Cells: The Role of Nitric Oxide

Abstract: Tumor necrosis factor-a (TNF-α), interferon-γ (IFN-7), and interleukin-6 (IL-6), but not TNF-β, can induce the in vitro differentiation of the neuroblastoma cell line N103 in a dose-dependent manner. Differentiation of N103 was accompanied by the arrest of cell growth and neurite formation. The induction of neuroblastoma cell differentiation by TNF-α and IFN-γ can be specifically inhibited by a nitric oxide (NO) synthase inhibitor, l -N^G-monomethylarginine. In contrast, the differentiation of N103 cells by IL-6 was not affected by l -N^G-monomethylarginine. These results indicate that TNF-α and IFN-γ, but not IL-6, induce the differentiation of neuroblastoma cells via NO. This is confirmed by the finding that the culture super- natants of N103 cells induced by TNF-α and IFN-γ, but not that by IL-6, contained high levels of NO₂⁻, the production of which was inhibited by l - N ^G-monomethylarginine. Furthermore, the differentiation of N103 cells can be induced directly in a dose-dependent manner by the addition of nitroprusside, a generator of NO, into the culture medium. These data therefore indicate that NO may be an important mediator in the induction of neuronal cell differentiation by certain cytokines such as TNF-α and IFN-γ and that neuronal cells, in addition to the macrophagelike brain cells, can be induced by immunological stimuli to produce large quantities of NO.     

A new yeast gene, HTR1, required for growth at high temperature, is needed for recovery from mating pheromone-induced G1 arrest

A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original is mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).     

A Novel Assay System for Anti-Human Immunodeficiency Virus Type 1 (HIV-1) Activity Using a Subclone of a Monocytic Cell Line,U937

A subclonal cl.1–14 cell was established from a monocytic cell line U937 by a limiting dilution method. The anti-HIV-1 activity of some antiviral compounds was evaluated in HIV-1-infected cl.1–14 cells. The results demonstrated that although AZT was a potent inhibitor of HIV-1 replication in cl.1–14 cells, its 50% effective concentration (EC₅₀) values was 80 times higher than that in HIV-1 infected MT-4 cells; the EC₅₀ of AZT was 0.16 μM and 0.002 μM in cl.1–14 and MT-4 cells, respectively. In contrast, the anti-HIV-1 activity of ddA, ddI and ddC in cl.1–14 cells was comparable to that in MT-4 cells. The antiviral activity of nevirapine, dextran sulfate, curdlan sulfate and T22 did not differ significantly between the cl. 1–14 and MT-4 cells. The antiviral activity of several compounds in the HIV-1-infected cl.1–14 cells was similar to that in the HIV-1jr -fl -infected human peripheral macrophages. Our results suggest that cl.1–14 cell cultures are very useful for estimating antiviral activity and more advantageous than the use of peripheral blood macrophages.     

Restriction Fragment Length Polymorphism (RFLP) of Genomic DNA of Moraxella (Branhamella) catarrhalis Isolates in a Hospital

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1,000 kb to 30 kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.     

A comparative study of floral development inTrillium apetalon andT. kamtschaticum (Liliaceae)

Trillium apetalon Makino is unique amongTrillium in having apetalous flowers. Using scanning electron microscope, the early floral development was observed in comparison with that ofT. kamtschaticum Pallas ex Pursh having petalous flowers. Morphologically petal primordia closely resemble stamen primordia in their more or less narrow and radially symmetric shape and are clearly distinct from sepal primordia with broad bases. Early in floral development sepal primordia are first initiated and subsequently two whorls of three primordia each are formed in rapid sequence, the first three at the corners and the second three at the sides of the triangular floral apex. Based on comparison in position and early developmental processes of their primordia, petals and outer stamens ofTrillium kamtschaticum are equivalent to outer stamens and inner stamens ofT. apetalon. The replacement of petals by outer stamens apparently leads to the loss of petals inTrillium apetalon flowers. Such a replacement can be interpreted in terms of homeosis. The replacement of the petal whorl leads to the serial replacement of the subsequent whorls: outer stamens by inner stamens, and inner stamens by gynoecium inTrillium apetalon. The term ‘serial homeosis’ is introduced for this serial replacement.     

Synthesis of sulfated derivatives of curdlan and their anti-HIV activity

Sulfopropyl curdlan was synthesized, its structure was determined, and the anti-HIV activity was compared with that of standard curdlan sulfates obtained with piperidine N-sulfonic acid in dimethyl sulfoxide. It was shown that sulfopropyl curdlan exhibits weaker anti-HIV activity than curdlan sulfate. Curdlan sulfates were synthesized with a SO₃-pyridine complex in a heterogeneous phase. It was shown from ¹³C-NMR spectra of acetylated curdlan sulfates that they had a different substituent distribution from standard curdlan sulfate. The cytotoxicity of the curdlan sulfates was attributed to their heterogeneous structure.