大鼠肝癌中α_2-巨球蛋白等几种分泌性蛋白基因表达的变化

用Northern blot方法对二乙基亚硝胺所诱发的大鼠肝癌中内源性蛋白酶抑制因子α_2-巨球蛋白(α_2-M)、非特异性免疫抑制剂α_1-酸性糖蛋白(α_1-AGP)及雄性激素正调控的α-2u球蛋白(α-2u)三种分泌性蛋白基因表达情况进行了分析。结果表明在大部分(14/16)肝癌样品中α_2-M RNA水平显著降低;而α_1-AGP RNA水平显著高于正常对照水平;α-2u RNA水平明显下降,但在某些雄性大鼠肝癌样品中该基因却有一定程度的表达。这些结果说明,一些肿瘤宿主血浆中α_2-M水平的显著下降及α_1-AGP水平的明显升高分别是由于基因表达活性的下降及升高所致。α-2u基因表达的异常提示,在癌变过程中机体的内分泌功能发生了某些变化。     

In vitro study of the proteolytic activity of rumen anaerobic fungi

Abstract To better define the antigenic structure of the outer cell membranes of Legionellae, a panel of 6 monoclonal antibodies was raised against partially purified outer membranes of Legionella pneumophila serogroup 1, Corby strain. This study describes the purification and characterization of one of these monoclonal antibodies reacting with a 135-kDa protein, which was shown to be common to all 14 serogroups of Legionella pneumophila . It shows no cross-reactivity with 20 other Legionella species, or 9 other Gram-negative species tested by SDS-PAGE and Western blotting procedures. The epitope would appear to be predominantly surface exposed and, from preliminary detergent extraction studies, not peptidoglycan-associated.     

Inhibition of Bordetella pertussis filamentous hemagglutinin-mediated cell adherence with monoclonal antibodies

Abstract Filamentous hemagglutinin (FHA), a 220-kDa protein located on the surface of Bordetella pertussis , is one of the major cell adhesins of this bacterium. We have produced three hybridoma cell lines that express monoclonal antibodies (mAbs) against FHA: X3C, X3E and X4B. The anti-FHA mAbs X3C and X3E reacted with 220-kDa FHA protein bands on Western blots. The mAb X4B, which reacted with FHA in ELISA, did not bind to FHA in a Western blot assay. All three mAbs seemed to be directed to the same epitope or to epitopes in close proximity as suggested by competition ELISAs. All three mAbs were able to inhibit the adherence of Chinese hamster ovary cells to purified FHA, and they could also inhibit the FHA-mediated agglutination of goose red blood cells. The attachment of B. pertussis to epithelial cell monolayers was inhibited by the mAb X3C. These antibodies are very useful probes to identify the presence of FHA in bordetellae species and in clinical reagents such as pertussis vaccines, and to characterize the functional domains of this important bacterial adhesin.     

Association between barium exposed,CYP19A1 and central obesity: A cross-sectional study in rural China

Backgroundobesity is a major risk factor for many metabolic diseases such as diabetes and cardiometabolic diseases. This study aimed to evaluate the association of plasma and urinary barium concentrations, CYP19A1 gene polymorphisms, and their interaction with central obesity in a rural Chinese population.Methodsrestricted cubic spline model was used to explore the dose-response relationship between barium and the risk of developing central obesity and waist circumference; logistic regression model was used to assess the association between barium, CYP19A1 gene polymorphisms and their interaction with central obesity.Resultsthe results of the restricted cubic spline model showed that plasma barium concentration was linearly associated with the risk of developing central obesity and non-linearly associated with waist circumference. Logistic regression analysis showed that participants with Q4 plasma barium concentration exhibited a higher risk of central obesity compared to participants with Q1 barium concentration; participants carrying the rs10046-AA gene exhibited a lower risk of central obesity than those carrying the rs10046-G(GG+GA) gene; participants carrying the rs10046-GA genotype showed 1.754 times higher risk of central obesity than those carrying rs10046-GG+AA genotype. There was a significant interaction between plasma barium and CYP19A1 gene polymorphism on central obesity.Conclusionthe development of central obesity was associated with plasma barium and CYP19A1.     

HPLC separation of the enantiomers of nicotine and nicotine-like compounds

A sensitive and reproducible HPLC method utilizing a commercially available chiral α₁-acid glycoprotein (AGP) phase has been developed to separate and quantify the enantiomers of nicotine. The method is suitable for routine use as indicated by column life. The quantification of (R/S:0.05/99.95)-nicotine or (R/S:99/1)-nicotine was possible. In addition, the separation or at least partial separation of the enantiomers of nornicotine and nornicotine-derived compounds was achieved. © 1993 Wiley-Liss, Inc.     

Dual Appearances of Pigment Cells from In Vitro Cultured Embryonic Cells of Japanese Flounder: An Implication for a Differentiation–Associated Clock

The cells dissociated from developing embryos of Japanese flounder (Paralichthys olivaceus) are cultured in vitro to examine the developmental fate of their pigment cells in relation to establishment of bilaterally asymmetric integumental coloration in vivo. When neurula embryos are dissociated using trypsin–EDTA in Dulbecco's modified Ca²⁺–, Mg²⁺–free phosphate buffered saline and then cultured in vitro using L–15–based fetal calf serum–supplemented growth medium at 20°C, numerous pigment cells appear twice in the same culture with an interval of approximately 1 month even under similar culture conditions. The first group of pigment cells, which is relatively larger in cell size (about 70 μm wide) and lower in cell density, emerges within 12 hr after plating, whereas the second, which is far smaller in cell size (about 30 μm) and overwhelmingly higher in cell density than the first, does so about 1 month after plating. The timing of their appearances in vitro is in good accordance, respectively, with that observed for the larvae under normal development in vivo; the first group appears at the period corresponding to hatching, whereas the second at the period corresponding to the completion of metamorphosis. Light microscopic examinations disclose that each group of pigment cells is composed of black melanophores and reflecting leucophores, and that the population density of melanophores and leucophores in the first group at the climax of appearance is approximated as 1:4. Typical xanthophores that are distributed in the skin of the larvae of this species are scarcely observed in culture in vitro. Because of their dual synchronous appearances with about 1 month interval under the similar culture conditions, and because of their low proliferative activity during the periods from the first appearance to the second, it is presumed that both groups of pigment cells are installed with a clock set differently for their differentiation. Light and electron microscopic immunocytochemistry on cultured cells using the HNK–I antibody, which marks avian migratory neural crest cells, both disclose that the antibody cross–reacts with all these pigment cells, and that a certain number of immunoreactive unpigmented cells exist even at the time of the second appearance of pigment cells. These findings would imply that the second group of pigment cells served in a form of undifferentiated propigment cells up to metamorphosis, at which they start to differentiate under control of a clock presumably set during neurulation.     

EFFECTS OF HELPER PROTEINS ENCODED BY p19 AND orf1-orf2 GENES ON Cyt1Aa PROTEIN EXPRESSION IN ACRYSTALLIFEROUS STRAIN OF BACILLUS THURINGIENSIS

A series of plasmids were constructed to examine the effects of p19 and orf1‐orf2 genes from Bacillus thuringiensis on Cyt1Aa synthesis and inclusion formation. The plasmids expressed the cyt1Aa gene along with either p19 or orf1‐orf2, or each of them coordinatively with p20 in the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. No effect on the expression of Cyt1Aa protein was found when P19 or Orf1‐Orf2 co‐expressed with Cyt1Aa. However, when including p20 gene, the constructs with p19 or orf1‐orf2 gene produced lower yield of Cyt1Aa proteins than without p19 or orf1‐orf2 gene. Electron microscopy observation and bioassay showed that P19 and Orf1‐Orf2 have no influence on the crystal size and toxicity of Cyt1Aa protein. It is presumed that P19 and Orf1‐Orf2 might have negative effects on Cyt1Aa synthesis in B. thuringiensis.     

The CD95 (Fas/APO-1) receptor is phosphorylatedin vitro andin vivo and constitutively associates with several cellular proteins

Different CD95 (Fas/APO-1) isoforms and phosphory lated CD95 species were identified in human T and B cell lines. We had shown previously that the CD95 intracellular domain (IC), expressed as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts, was phosphorylatedin vivo. GST-CD95IC was phosphorylatedin vitro by a kinase present in extracts from the human lymphocytic cell lines Jurkat and MP-1 and from murine L929 cells. Phosphoamino acid analysis indicated that phosphorylation occurred at multiple threonine residues and also at tyrosine (Tyr232 and Tyr291) and serine. Amino acids 191 to 275 of CD95 were sufficient for phosphorylation at threonine, tyrosine and serine and also mediated interaction with a 35 kDa cellular protein. Immuno-precipitation of CD95 and chemical cross-linking revealed CD95-associated proteins of approximately 35, 45 and 75 kDa. GST-CD95IC affinity chromatography detected binding of the 35 and 75 kDa protein species. The 75 kDa species may correspond to the CD95-associated proteins RIP or FAF1 and the 35 kDa protein may represent a TRADD analogue. These data indicate that several cellular proteins interact with CD95, possibly in a multi-protein complex, and that a kinase activity is associated with CD95 not onlyin vitro but alsoin vivo. Therefore, receptor phosphorylation may play a role in CD95 signal transduction. This work was in part supported by a grant from the Health Research Council of New Zealand (to JW).     

Multiple xenobiotic-inducible P450s are involved in alkoxycoumarin and alkoxyresorufin metabolism in higher plants

O-Dealkylation of two series of fluorescent 7-alkoxy-coumarins and 7-alkoxyphenoxazones by plant cytochrome P450s was investigated in Helianthus tuberosus tuber tissues treated with prototype P450 inducers, environmental pollutants or agrochemicals. Methoxy-, ethoxy-, propoxy-and butoxycoumarins and methoxy- and ethoxyresorufins were metabolized by fplant microsomes. Dealkylation of pentoxy- and benzyloxyresorufins was not detected. All dealkylating activities were enhanced by aging plant tissues in the presence of xenobiotics, in some cases up to 20-fold relative to the activities detected in control tissues. Increases in total P450 in the same tissues never exceeded 3-fold. The isozymes induced by prototype P450 inducers clearly differed from those in mammalian liver. That multiple P450s with overlapping substrate specificities were involved in the metabolism of both alkoxycoumarins and alkoxyresorufins was demonstrated by (1) the differential induction of the activities in response to exposure to xenobiotics, (2) the differential inhibition of the activities by clotrimazole, paclobutrazole and tetcyclacis in aminopyrine and benzo(a)pyrene-treated tissues, and(3) the selective inhibition observed with antibodies raised against purified ethoxycoumarin deethylase fractions. Our results suggest that the measurement of the dealkylation of such fluorescent substrates in plants might be useful to monitor environmental pollution.