Subnuclear localization and antitransforming activity of N-myc:beta-galactosidase fusion proteins.

N-myc expression is under stage- and tissue-specific regulation in mammalian development, but its function is totally unknown. We sought agents to block N-myc activity in order to infer from the effect the possible function of N-myc in the apparently complex processes. As candidates for such agents, we tested fusion genes encoding N-myc:beta-galactosidase fusion proteins for their effects on the formation of transformed foci of rat embryo primary fibroblasts as the result of transfection with N-myc and activated H-ras. One of the gene constructs very efficiently antagonized N-myc activity, as assessed by its effect on focus formation, but did not appreciably affect cell viability. The product of this gene was not only targeted to the nucleus but also accumulated in subnuclear loci which may represent the sites where normal N-myc proteins reside. The occurrence of antagonistic effect at a low stoichiometric ratio suggested that the fusion protein gene competed with the N-myc gene in a fashion analogous to a dominant negative mutation.     

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.     

Purification of a fibrolast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4

Abstract A factor showing inhibitory activity against human gingival fibrolasts was extracted from the cytosol fraction of Actinobacillus actinomycetemcomitans Y4. The activity markedly inhibited the proliferation of human gingival fibrolasts, but had no effect on cell viability or gross morphology. No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101. The extract from A. actinomycetemcomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa. The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.     

Feedbacks between nutrient cycling and vegetation predict plant species coexistence and invasion

To investigate the role of species‐specific litter decomposability in determining plant community structure, we constructed a theoretical model of the codevelopmental dynamics of soil and vegetation. This model incorporates feedback between vegetation and soil. Vegetation changes the nutrient conditions of soil by affecting mineralization processes; soil, in turn, has an impact on plant community structure. The model shows that species‐level traits (decomposability, reproductive and competitive abilities) determine whether litter feedback effects are positive or negative. The feedback determines community‐level properties, such as species composition and community stability against invasion. The model predicts that positive feedback may generate multiple alternative steady states of the plant community, which differ in species richness or community composition. In such cases, the realized state is determined by initial abundance of co‐occurring species. Further, the model shows that the importance of species‐level traits depends on environmental conditions such as system fertility.     

Requirement of caspase and p38MAPK activation in zinc-induced apoptosis in human leukemia HL-60 cells.

Zinc (Zn), an endogenous regulator of apoptosis, and has abilities both to induce apoptosis and inhibit the induction of apoptosis via the modulation of caspase activity. Due to the multifunctions of Zn, the intracellular Zn level is strictly regulated by a complex system in physiological and pathological conditions. The commitment of Zn to the regulation of apoptosis is not fully understood. In the present study, we investigated the role of intracellular Zn level in the induction of apoptosis in human leukemia cells (HL-60 cells) using a Zn ionophore [pyrithione (Py)]. Treatment of HL-60 cells with Zn for 6 h in the presence of Py (1 micro m) exhibited cytotoxicity in a Zn dose-dependent manner (25-200 micro m). Necrotic cells, assayed by trypan blue permeability, increased in number in a Zn dose-dependent fashion (50-100 micro m), but the appearance of apoptotic cells, assayed by formation of a DNA ladder and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method, peaked at 25 micro m, suggesting the dependence of intracellular Zn level on the execution of apoptosis. In fact, treatment with Py resulted in increases in intracellular Zn levels, and N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine, a cell-permeable Zn chelator, inhibited DNA ladder formation induced by Py/Zn treatment (1 micro m Py and 25 micro m Zn). Py/Zn treatment activated the caspases, as assessed by the proteolysis of poly(ADP-ribose) polymerase (PARP), which is a substrate of caspase, and activated p38 mitogen-activated protein kinase (p38MAPK), which is a transducer of apoptotic stimuli to the apparatus of the apoptosis execution. Z-Asp-CH2-DCB, a broad-spectrum inhibitor of caspase, attenuated proteolysis of PARP and DNA ladder formation by Py/Zn, indicating that apoptosis induced by Py/Zn is mediated by caspase activation. The p38MAPK-specific inhibitor SB203580 also inhibited induction of apoptosis by Py/Zn. Although SB203580 suppressed the proteolysis of PARP, Z-Asp-CH2-DCB did not inhibit the phosphorylation of p38MAPK, raising the possibility that apoptosis triggered by Py/Zn might be mediated by the p38MAPK/caspase pathway.     

Effect of Exogenous Gangliosides on Amino Acid Uptake and Na+,K+-ATPase Activity in Superior Cervical and Nodose Ganglia of Rats

The effects of some gangliosides on active uptake of nonmetabolizable alpha-aminoisobutyric acid (AIB) and Na+, K+-ATPase and Ca2+, Mg2+-ATPase activities in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined during aerobic incubation at 37 degrees C for 2 h. In NG, amino acid uptake was greatly accelerated with the addition of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylgluc osyl ceramide (GM1) (85%) and also with N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide (GM2) or [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetyl- neuraminyl]-galactosylglucosyl ceramide (GD1a) (43% each) compared with a nonaddition control at a 5 nM concentration. Under identical conditions, Na+, K+-ATPase activity was strongly stimulated with GM1 (180%) and GD1a (93%), whereas Ca2+, Mg2+-ATPase activity showed no change. In SCG, on the other hand, AIB uptake was apparently inhibited (-27%) by addition of GM1, with a slight decrease in Na+, K+-ATPase but no change in Ca2+, Mg2+-ATPase activity in the tissue. Both asialo-GM1, in which N-acetylneuraminic acid is deficient, and Forssman glycolipid, which is not present in nervous tissue, failed to produce any significant increase in both SCG and NG not only in amino acid uptake, but also in Na+, K+-ATPase activity. A kinetic study of active AIB uptake showed that GM1 ganglioside produced an increase in Km with no change in Vmax in SCG, whereas it caused a decrease in Km with a slight increase in Vmax in NG. Treatment of NG and SCG with neuraminidase from Vibrio cholerae, an enzyme that split off sialic acid from polysialoganglioside, leaving GM1 intact, caused little inhibition of the amino acid uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteinase production and pathogenicity of Candida albicans. I. Invasion into chorioallantoic membrane by C. albicans strains of different proteinase activity

In order to investigate a role of proteinase in the pathogenesis of Candida infections, invasion of C. albicans strains of different proteinase activity into the chorioallantoic membrane (CAM) of developing chicks was studied. Eight strains were used after examining the inducible proteinase activity in the culture containing bovine serum albumin as the sole source of nitrogen. Six were proteinase-producing strains (type I) and two were proteinase-deficient ones (type II). Type I strains were subdivided into type Ia strains in which the proteinase activity persisted for a week in the in vitro culture and type Ib ones in which the enzyme activity was lost by the 7th day after inoculation. By inoculation onto CAM, the type I strains could invade the tissue in which secreted proteinase was detected on the periphery of the invading Candida cells by immunohistochemical method. At an early stage of the infection, proteinase secretion was detected on the surface of the yeast cells before their entry into the tissue. The type II strains remained on the surface of the CAM and did not invade the tissue where the secretion of the enzyme was not detected. The mortality rate of the chick embryo was not correlated with the degree of proteinase production of these strains. Two type Ib strains invaded the CAM tissue and elicited some tissue reactions by the host, yielding a low mortality rate of the chick embryos. These results suggested that the secretion of proteinase was an important factor for the invasion of CAM but other factors were also involved for the pathogenicity of C. albicans.     

Two distinct types of endothelin receptors are present on chick cardiac membranes

Competitive displacement experiments of 125I-endothelin (ET)-1, -2, or -3 binding to chick cardiac membranes were performed with unlabeled ET-1, -2, -3, and sarafotoxin S6b (STX) as competitors. 125I-ET-1 and -2 binding was competitively inhibited by increasing concentrations of these unlabeled peptides in the same order; i.e. ET-2 greater than or equal to ET-1 greater than ET-3 greater than STX. In contrast, the order of potency in displacing 125I-ET-3 binding was ET-3 greater than ET-2 greater than or equal to ET-1 greater than STX. Affinity labeling of the membranes by cross-linking with 125I-ET-1 and -2 via disuccinimidyl tartarate yielded one major specific band with an apparent Mr = 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. On the other hand, affinity labeling with 125I-ET-3 showed that two major and one minor bands of Mr = 34,000, 46,000, and 53,000, respectively, were specifically labeled. These results indicate the presence of two distinct types of ET receptors, one of which has higher affinity for ET-1 and -2 than ET-3 and the other is conversely ET-3-preferring.