Re-examination ofPyrenomonas andRhodomonas (class cryptophyceae) through ultrastructural survey of red pigmented cryptomonads

Several taxa of cryptomonads, including species of marineChroomonas, Cryptomonas and freshwaterRhodomonas were examined using transmission electron microscopy. They have cellular structures fundamentally in common: a single bilobed chlorplast, a single pyrenoid between the chloroplast lobes, and a nuclemorph embedded within a cleft of the pyrenoidal matrix. These features are in accordance with the taxonomic characteristics of the recently established genusPyrenomonas. The algae also have similar pigmentation to that ofRhodomonas andPyrenomonas which is red or reddish-brown. On the basis of these observations, the genusRhodomonas Karsten (1898) is redescribed in this paper and the genusPyrenomonas Santore is considered to be synonymous withRhodomonas.     

Effects of combinations of transforming growth factor-beta 1 and tumor necrosis factor on induction of differentiation of human myelogenous leukemic cell lines

Effects of transforming growth factor-beta 1 (TGF-beta 1), either alone or in combination with TNF, on the induction of differentiation of human myelogenous leukemic cell lines were examined. TGF-beta 1 alone induced differentiation of a human monocytic leukemia U-937 line into the cells with macrophage characteristics. When combined with TNF, TGF-beta 1 synergistically or additively induced differentiation associated properties. A human myeloblastic leukemia cell line, ML-1, differently responded to TGF-beta 1 in induction of differentiation. FcR activity and phagocytic activity induced by TNF were suppressed by TGF-beta 1. However, nitroblue tetrazolium reducing activity was synergistically induced by combinations of TGF-beta 1 and TNF. Scatchard analysis of TNF receptors indicated that the number of binding sites and dissociation constant of TNF for its receptors on U-937 or ML-1 cells were not changed by treatment with TGF-beta 1. Although IFN-gamma, IL-6, granulocyte CSF, and granulocyte-macrophage CSF-induced nitroblue tetrazolium reducing activity of U-937 cells, only IFN-gamma, and TNF induced it synergistically in combination with TGF-beta 1. Synergism between TGF-beta 1 and TNF was also observed in inhibition of growth of U-937 and ML-1 cells. Although TGF-beta 1 induction of differentiation of other monocytoid leukemic THP-1 cells was similar to that of U-937 cells, TGF-beta 1 only slightly induced differentiation of promyelocytic leukemic HL-60 cells, either alone or in combination with TNF. Our observations indicate that TGF-beta 1 strongly modulates differentiation and proliferation of human myelogenous leukemia cells, macrophage precursors.     

Metabolism of exogenous galactosylceramide in the twitcher mouse brain

The in vivo metabolism of galactosylceramide (gal-cer) in normal mice and in twitcher mice, a model of human GLD, was examined following intracerebral administration of gal-cer containing [1-¹⁴C]stearic acid. In normal mice, gal-cer was hydrolyzed to ceramide within 6 hours and ceramide was hydrolyzed to sphingosine and fatty acid. Most of the released fatty acid was immediately incorporated into other lipids. About 75% of injected gal-cer was hydrolyzed 80 hours after the injection, while in the twitcher mouse, only 17% of gal-cer was hydrolyzed. These results show that degradation of gal-cer is impaired in the twitcher mouse brain, but contradict to the fact that there was no evidence of any accumulation of gal-cer in the brain. This discrepancy may be due to the different sorting routes of biosynthesized and exogenously-administered gal-cer in the mouse brain. Most of the biosynthesized gal-cer is incorporated into myelin, while the injected gal-cer is incorporated into lysosomes.     

Studies on chemical synthesis of human cystatin A gene and its expression in Escherichia coli

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immunoblotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH2-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.     

Multiple tachykinins are produced and secreted upon post-translational processing of the three substance P precursor proteins, alpha-, beta-, and gamma-preprotachykinin. Expression of the preprotachykinins in AtT-20 cells infected with vaccinia virus recombinants

The rat preprotachykinin I gene mRNA is alternatively spliced to yield three different mRNA species differing in their protein coding regions. We have produced recombinant vaccinia viruses expressing alpha-, beta-, and gamma-preprotachykinin to examine the tachykinin-related peptides produced upon post-translational processing of each individual precursor. Infection of BSC-40 or AtT-20 cell lines with a beta-preprotachykinin-encoding vaccinia virus recombinant results in the expression of the precursor protein. The pro-form (signal peptide removed) can be immunoprecipitated from extracts of infected cells. Infected cells of both types secrete into the culture medium a product(s) which reacts in radioimmunoassay with an antiserum shown to recognize precursor as well as mature substance P. Infected AtT-20, but not BSC-40, cells secrete into the culture medium a processed form(s) of beta-preprotachykinin which reacts in radioimmunoassay with an anti-serum which recognizes the amidated carboxyl terminus of substance P. The molecular nature of the tachykinin products produced in and secreted from AtT-20 cells infected with alpha-, beta-, and gamma-preprotachykinin-encoding recombinants was analyzed by combined high performance liquid chromatography and radioimmunoassay. Peptides were identified based on comigration with synthetic standards and antisera cross-reactivity. We determined that alpha-preprotachykinin is processed to the mature undecapeptide, substance P. beta-Preprotachykinin was processed into multiple products, including substance P, neurokinin A, neurokinin A(3-10), and neuropeptide K. gamma-Preprotachykinin was processed into substance P, neurokinin A, neurokinin A(3-10), and neuropeptide gamma. These five tachykinin peptide products were all routed through the regulated secretory pathway and were secreted into the medium in a cAMP-stimulatable fashion. Since all of these peptides have been shown to be biologically active, it is important to consider the biological consequences of their co-secretion in vivo.     

Acceleration of chromosome aberrations in senescence-accelerated strains of mice

Age-related changes in the frequency of chromosome aberrations were examined using bone marrow cells of senescence-accelerated strains of mice (SAM). An accelerated senescence-prone strain, SAM-P/1, showed a striking increase in the frequency of chromosome aberrations, from age 3 to 8 months, whereas an accelerated senescence-resistant strain, SAM-R/1, at the same ages showed only a slight increase. Both these strains were derived from the same ancestral strain (AKR/J). The rate of increase of chromosome aberration frequency paralleled the advancement of senescence in both strains. These observations suggest that there are genetic factors which closely relate to chromosomal instability and acceleration of the senescence processes.     

Collection and Chemical Composition of Pure Phloem Sap from Zea mays L.

Pure phloem sap was collected from leaf sheaths of Zea maysL. by the insect laser technique, and its chemical compositionwas analyzed. Sucrose was the only sugar detected. The predominantinorganic ions were K⁺ and Cl^–. The adenylate energy chargeof phloem sap was between 0.72 and 0.86. (Received October 18, 1989; Accepted May 11, 1990)     

Rubber-degrading enzyme from a bacterial culture

Rubber-degrading activity was found in the extracellular culture medium of Xanthomonas sp. strain 35Y which was grown on natural rubber latex. Natural rubber in the latex state was degraded by the crude enzyme, and two fractions were separately observed by gel permeation chromatography of the reaction products. One fraction was of higher molecular weight (HMW) with a very wide MW distribution from 10 to 10, and the other fraction was of lower molecular weight (LMW) with a MW of a few hundred. H-nuclear magnetic resonance spectra of the partially purified fractions were those expected of cis-1,4-polyisoprene mixtures with the structure OHC-CH(2)-(-CH(2)-C(-CH(3)) = CH-CH(2)-)(n)-CH(2)-C(=O)-CH(3), with average values of n of about 113 and 2 for HMW and LMW fractions, respectively. The LMW fraction consisted mostly of one component in gas-liquid chromatography as well as in gel permeation chromatography, and the main component was identified as 12-oxo-4,8-dimethyl trideca-4,8-diene-1-al (acetonyl diprenyl acetoaldehyde, A(l)P(2)A(t)) by C-nuclear magnetic resonance and gas chromatography-mass spectra. Not only the latices of natural and synthetic isoprene rubber, but also some kinds of low-MW polyisoprene compounds of cis-1,4 type, were degraded by the crude enzyme. The rubber-degrading reaction was found to be at least partly oxygenase catalyzed from the incorporation of O into A(l)P(2)A(t) under an O(2) atmosphere.