CYTOPLASMIC MASSES PRESERVED IN EARLY HOLOCENE DIATOMS: A POSSIBLE TAPHONOMIC PROCESS AND ITS PALEO‐ECOLOGICAL IMPLICATIONS1

In Lake Suigetsu, central Japan, greenish/light‐brown granules identified as cytoplasmic masses had been preserved in siliceous cell walls of freshwater diatoms in annual layers of lacustrine muds since the early Holocene. The lacustrine muds consisted of alternating dark‐colored (rich in diatom valves, clay, and organic matter) and light‐colored (mainly diatom valves) laminae. The greenish/light‐brown granules were predominately preserved in frustules of the genus Aulacoseira preserved in the dark‐colored laminae. The dark‐colored laminae were inferred to have formed annually under stratified water caused by surface water warming in summer that caused the formation of an organic‐rich anoxic layer on the lake bottom that favored granule preservation. The good preservation of cytoplasmic masses in dark‐colored laminae suggested a cause for diatom assemblage periodicity, a phenomenon that was commonly noted in temperate lakes: the cells containing these masses could be potential seed stocks for subsequent spring blooms. Frustules of the most abundant granule‐containing species, Aulacoseira nipponica (Skvortzow) Tuji, in the dark‐colored laminae of the Early Holocene muds were abundant in the overlying light‐colored laminae, suggesting that these species reproduced abundantly in springtime yielding a massive diatom bloom.     

Increased susceptibility to Staphylococcus aureus colonization of the skin of the NOA mouse: a potentially useful animal model for evaluating antiseptic effects.

Isolation of bacteria from wet skin lesions was attempted using Naruto Research Institute Otsuka Atrichia (NOA) mice, which develop such lesions spontaneously at a high rate. As a result, Staphylococcus aureus was demonstrated to have colonized the wet skin lesions at high density. In addition, the isolated S. aureus was found to be similar to the strain of S. aureus thought to colonize the eczematous lesions seen in humans with atopic dermatitis. Furthermore, a survey of the S. aureus colonization status of NOA mice with no wet skin lesions confirmed colonization at higher density than in HR-1 mice as control, indicating that the skin of the NOA mouse has the novel characteristic of increased susceptibility to S. aureus colonization. Thus, by using changes in S. aureus counts as an index, the NOA mouse can be expected to serve as a useful animal model for evaluating the effects of topical antiseptics. The antiseptic effects of an ointment and a lotion containing chlorhexidine gluconate were confirmed using this animal model.     

Differentiation ofMelampsora rust species on willows in Japan using PCR-RFLP analysis of ITS regions of ribosomal DNA

To develop a reliable method for identifyingMelampsora species parasitic on willows in Japan, we differentiated 10Melampsora species by PCR-RFLP analysis. Internal transcribed, spacer (ITS) regions, including 5.8S ribosomal DNA, of 63 collections of 10Melampsora species and 4 collections of unidentified species were amplified by PCR. The fragments from the 67 collections varied in size (approximately 880 bp, 860 bp and 840 bp). The restriction sites in the amplified DNA fragments were mapped after the RFLP analysis using four restriction enzymes,Dra I,EcoRI,SspI andTaqI. All the collections were divided into 11 RFLP types. In the 6 species,M. caprearum, M. epiphylla, M. kamikotica, M. larici-urbaniana, M. microsora andM. yezoensis, the RFLP type was species-specific. The RFLP type ofM. chelidonii-pierotii andM. coleosporioides was identical. The collections ofM. epitea were separated into three RFLP types. One of these three types was identical with the type ofM. humilis. It is suggested that the PCR-RFLP analysis of ITS regions is a useful and reliable method for species identification ofMelampsora. Contribution No. 131, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

The third egg envelope subunit in fish: cDNA cloning and analysis, and gene expression

The inner layer of the egg envelope of a teleost fish, the medaka, Oryzias latipes, consists of two major subunit groups, Zl-1,2 and Zl-3. On SDS-PAGE, the Zl-1,2 group presents three glycoprotein bands that were considered to be composed of a common polypeptide moiety derived from their precursor, choriogenin H (Chg H). Zl-3 is a single glycoprotein derived from the precursor, choriogenin L (Chg L). In the present study, a fraction of a novel subunit protein was found in the V8 protease digest of Zl-1,2 that was partially purified from oocyte envelopes. This protein fraction was not present in the purified precursor, Chg H. By RT-PCR employing the primers based on the amino acid sequence of this fraction, a cDNA for the novel subunit was amplified, and a full-length clone of the cDNA was obtained by screening a cDNA library constructed from the spawning female liver. The clone consisted of 2025 b.p. and contained an open reading frame encoding the novel protein of 634 amino acids. This protein included Pro-X-Y repeat sequences in two-fifths of the whole length from its N-terminus. Northern blot analysis revealed that the gene expression for this protein occurred in the liver but not in the ovary of spawning female fish. This protein is considered as the third major subunit of the inner layer of the egg envelope of medaka.     

Formation of Keto and Hydroxy Compounds of Linoleic Acid in Submitochondrial Particles of Bovine Heart

To observe lipid peroxidation of additive-free submitochondrial particles, we incubated submitochondrial particles in the absence of exogenous irons and t-butyl hydroperoxide. After the incubation, the phospholipids were hydrolyzed by phopholipase A₂, and the fatty acid constituents were analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. Contrary to a commonly accepted theory, lipid peroxidation in the submitochondrial particles did not need the addition of NADH. In the phospholipid constituent fatty acids of the oxidized submitochondrial particles, derivatives of hydroperoxides of linoleic acid such as keto, hydroxy, trihydroxy, and hydroxyepoxy compounds were generated. Lipid peroxidation in the submitochondrial particles was not inhibited by the addition of catalase, superoxide dismutase, hydroxyl radical scavengers, or ethylenediaminetetraacetic acid, but was inhibited by the addition of KCN, antimycin-A, NADH, ubiquinol, deferoxamine mesylate, ascorbic acid, and -tocopherol. The cardiolipin–cytochrome c lipid peroxidation system could mimic the lipid peroxidation of the submitochondrial particles, in terms of linoleic acid products and the inhibitory patterns of radical scavengers and electron transfer chain inhibitors. Thus, lipid peroxidation in the submitochondrial particles seems to be due to phospholipid–hemoprotein lipid peroxidation systems such as the cardiolipin–cytochrome c system.     

Effect of DNA preparation from laver (Porphyra yezoensis) thalli on reproducibility of RAPD (random amplified polymorphic DNA) patterns

Laver (Porphyra yezoensis) DNAs were extracted from thalli with five different procedures and used for RAPD (random amplified polymorphic DNA) analysis as templates. Restriction enzyme-digestive DNAs were obtained with all procedures examined. However, RAPD patterns generated with these DNAs appeared highly irreproducible and were considerably different from each other. When DNAs purified with CsCl gradient centrifugation were used for RAPD analysis as templates, highly reproducible RAPD patterns were obtained, suggesting that unpurified DNAs extracted from thalli with all five extraction procedures contained an excess of RNA, polysaccharides and/or other materials which affected the RAPD reproducibility. Thus, results indicated that purification of DNA is essential to produce reproducible RAPD patterns of Porphyra DNA. This revised version was published online in June 2006 with corrections to the Cover Date.     

Generation mutant mice with large chromosomal deletion by use of irradiated ES cells—analysis of large deletion around hprt locus of ES cell

A method of generating mice from embryonic stem (ES) cells with a large chromosomal deletion produced by X-ray irradiation has been developed. Fifty-two mutant ES clones were made that carried a nested set of chromosomal deletions up to approximately 10 cM in length around the hprt locus on the X Chromosome (Chr). Germline chimeras were generated from three ES clones with deletions ranging from 200 to 700 kb. In germline male mice from two independent clones, deletions around the hprt locus yielded a runty phenotype or caused death at birth. The runty mice had approximately ⅓ the body weight and size of wild littermates and did not survive more than 3 weeks after birth. The most plausible cause of these phenotypes is defects in regions flanking the hprt locus. This method of creating mutant mice with a large chromosomal deletion is very useful for the identification and understanding of gene functions. Received: 31 October 1996 / Accepted: 16 December 1997     

Indole and benzimidazole derivatives as steroid 5α-reductase inhibitors in the rat prostate

A novel series of indole and benzimidazole derivatives were synthesized and evaluated for their inhibitory activity of rat prostatic 5α-reductase. Among these compounds, 4-{2-[1-(4,4′-dipropylbenzhydryl)indole-5-carboxamido]phenoxy}butyric acid (15) and its benzimidazole analogue 25 showed potent inhibitory activities for rat prostatic 5α-reductase (IC₅₀ values of 9.6 ± 1.0 and 13 ± 1.5nM, respectively), with the potency very close to that of finasteride. Compound 30, in which the moiety between the benzene ring and amide bond was replaced by quinolin-4-one ring, showed almost equipotent activity (IC₅₀ = 19 ± 6.2nM) with the correspondent amide derivative 13. This result was consistent with the previous observation that the coplanarity of this moiety might contribute to the potent inhibitory activity.     

Different Modulation of Protein Kinase C Isozymes in Dibutyryl Cyclic AMP-Differentiated PC12h Cells

Abstract: PC12h cells can be differentiated into sympathetic neuron-like cells by various agents, including nerve growth factor, basic fibroblast growth factor, cyclic AMP analogues, and protein kinase C (PKC) activators. To study the involvement of PKC in the process of PC12h cell differentiation by cyclic AMP treatment, PKC isozymes (α, βI, βII, and γ) were analyzed using column chromatography and immunoblotting. Two PKC isozymes, PKC(α) and PKC(βII), were predominantly detected in PC12h cells. When stimulated by dibutyryl cyclic AMP, PKC(α) levels declined in the cytosolic fraction of the cells, whereas PKC(βII) levels increased. Increased PKC(βII) levels were also detected in the particulate fraction, whereas particulate PKC(α) levels did not change. The total PKC activity decreased in the cytosolic fraction following cyclic AMP stimulation of PC12h cells, whereas it stayed constant in the particulate fraction. Fractionation on a hydroxyapatite column showed a decreased level of PKC(α) activity and a transient increase followed by a decreased level of PKC(βII) activity. This discrepancy between increased PKC(βII) immunoreactivity and reduced PKC(βII) activity suggested the presence of nonactivatable PKC(βII) in cyclic AMP-treated PC12h extract. These findings indicate that PKC(α) and PKC(βII) are differentially regulated during the differentiation of PC12h cells. In addition, the differentiation of PC12h cells triggered by cyclic AMP seems to involve characteristic alterations of PKC isozymes.