Purification and characterization of RNA polymerase I from a higher plant.

RNA polymerase I was purified from chromatin isolated from auxin-treated soybean hypocotyl. Purification was achieved by using Agarose A-1.5m gel filtration, DEAE-cellulose, CM-sephadex, and phosphocellulose chromatography, and sucrose density gradient centrifugation. With denatured calf thymus DNA as template, the enzyme has a high specific activity (200-300 nmol/mg/30 min at 28 degrees C) which is comparable to other RNA polymerase I enzymes purified from animals and yeast. While the gel profiles indicate that purification to homogeneity (greater than 90%) may not have been achieved, the enzyme appears to be composed of possibly 7 subunits, several of which are similar to the subunits of yeast RNA polymerase I. The putative subunits and molar ratios are 183 000 (1), 136 000 (1), 50 000 (0.5), 46 000 (0.5), 40 000 (0.5), 33 000 (0.2), and 28 000 (2). The purified enzyme strongly prefers a completely denatured template such as poly(dC).     

Reaction mechanism of surfactant‐sensitized chemiluminescence of bis(2,4,6‐trichlorophyenyl) oxalate and hydrogen peroxide induced by gold nanoparticles

The chemiluminescence (CL) of bis(2,4,6‐trichlorophyenyl) oxalate with hydrogen peroxide in the present of cationic surfactant and gold nanoparticles was studied. The CL emission was obviously enhanced in the presence of surfactant at a suitable concentration, with a synergetic catalysis effect exhibited. Different sizes of gold nanoparticles (15 and 50 nm) showed different effects on CL intensity. Mechanisms of the CL reaction and sensitization effect are discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

BJ-HCC-20, a potential novel cancer-testis antigen.

In an effort to identify novel Cancer-Testis genes, we analyzed the sequence in the q26-28 region of human X chromosome by several on-line tools. The candidate sequences were then confirmed by experiments. We have obtained a novel Cancer-Testis gene, BJ-HCC-20. In vivo, it was found to have two isoforms. In samples of liver, colon, gastric and lung cancer tested, the expression frequency of BJ-HCC-20 is 25%, 17%, 21% and 15%, respectively. Full-length cDNAs of both BJ-HCC-20 isoforms were isolated and their gene structures and promoter regions were characterized. BJ-HCC-20 might have implications in theoretical and practical tumor biology.     

In vitro propagation of Alstroemeria ‘Alsaan’

Plantlets were regenerated from Alstroemeria Alsaan rhizome tips cultured in vitro on solid and liquid media based on Murashige and Skoog salt formulation. The quality of the cultures was superior when intact rather than longitudinally sliced rhizome tips were used as explants and when a temperature of 8°C rather than 22°C was used at the initiation stage. More roots were produced on rhizome tips containing a rhizome apical meristem than on rhizome sections lacking such a meristem. Most (90%) of the rooted plantlets were successfully acclimatized and developed into true-to-type flowering plants.     

An electron microscopic study of retinal degeneration in Sprague-Dawley rats

Retinal degeneration in untreated, female Sprague-Dawley rats was studied by electron microscopy and horseradish peroxidase tracer technique. The degeneration appeared to have started at a very young age. The severity of the defect varied from a decrease of photoreceptor nuclei to total loss of receptor cells and the pigment epithelium. In mild degeneration some regions of the retinal pigment epithelium became bilayered and the basal plasma membrane became flattened or formed elaborate infoldings. Breaks in Bruch's membrane occurred in severe degeneration. Degeneration of the pigment epithelium allowed permeation of tracer material from the choroid into the retina.     

Immunocytochemical studies on prolactin cells in the adenohypophysis of the golden hamster

Mammotrophs or prolactin (PRL) cells were identified in the adenohypophysis of adult golden hamsters by immunocytochemical techniques with a polyclonal anti-PRL, that was proved to be specific to PRL by the dot immunoblotting test. Postembedding immunostaining was performed on Araldite thin sections by immunoperoxidase and immunogold methods. PRL cells were classified into three types according to the different size of the secretory granules. The Type A cells were usually small and angular or oval in shape, and had secretory granules ranging in diameter from 100-230 nm, and showed poorly developed organelles. The Type B and C cells were larger and round or ovoid in shape, contained larger granules, 230-280 nm and 280-570 nm, respectively, and displayed well developed organelles. Immunoreactive PRL cells in the male pituitaries were far less numerous than in the nonpregnant female glands, and were mostly of the Type A and B, whereas in the female the Type C and B cells predominated. In pregnant females, Type C cells became activated and increased in number, while the other two types decreased in proportion. In lactating females, Type A and B cells significantly increased in number at the expense of the Type C cells; meanwhile, the exocytosis of secretory granules was frequently found in all types of PRL cells. The present findings suggest that Type C and B PRL cells, especially the former, are potent in producing and releasing PRL and highly responsive to various physiological stimuli, while Type A cells are probably relatively inert in synthetic activity.     

Characterization of the Erwinia carotovora pelB gene and its product pectate lyase.

The pelB gene encodes pectate lyase B, one of three pectate lyases identified in Erwinia carotovora EC. Pectate lyase B was purified from Escherichia coli containing the pelB gene on a recombinant plasmid. The activity of the protein was optimal at a pH of 8.3. The amino acid composition, N-terminal amino acid sequence, and C-terminal peptide sequence were determined and compared with the polypeptide sequence deduced from the DNA sequence of pelB. Purified pectate lyase B started at amino acid 23 of the predicted sequence, suggesting that a 22-amino-acid leader peptide had been removed. Pectate lyase B of E. carotovora EC and pectate lyase B of E. chrysanthemi EC16 contain 352 and 353 amino acids, respectively (N. T. Keen, S. Tanaki, W. Belser, D. Dahlbeck, and B. Staskawicz, J. Bacteriol. 168:595-606, 1986). The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical.     

Identification of cellular mechanisms operational in vivo during the regression of established pulmonary metastases by the systemic administration of high-dose recombinant interleukin 2

The systemic administration of high-dose recombinant IL 2 mediated significant reductions of established 3-day pulmonary micrometastases from both weakly immunogenic and nonimmunogenic sarcomas. However, when treatment with IL 2 was delayed for 10 days after the injection of tumor cells in an attempt to treat grossly visible pulmonary macrometastases, only those established from weakly immunogenic sarcomas remained susceptible. Established 10-day pulmonary nodules from the nonimmunogenic sarcomas became refractory to IL 2 therapy. We utilized selective depletion of lymphocyte subsets in vivo by the systemic administration of specific monoclonal antibodies to cells bearing either the L3T4 or Lyt-2 marker or a heteroantiserum to cells bearing the ASGM-1 glycosphingolipid to identify lymphocytes involved in IL 2-induced tumor regression. Cells with potent lymphokine-activated killer (LAK) activity against fresh tumor targets in vitro were identified in the lungs of IL 2-treated mice. By flow cytometry analysis, the majority of these effector cells were Thy-1+, L3T4-, Lyt-2-, ASGM-1+. Depletion in vivo of ASGM-1+ cells before the onset of IL 2 administration eliminated the successful therapy of 3-day pulmonary metastases from nonimmunogenic sarcomas, with concurrent elimination of LAK cell activity in the lungs. In mice with 3-day pulmonary metastases from weakly immunogenic sarcomas, both Lyt-2+ cells and ASGM-1+ cells were involved in IL 2-mediated tumor regression, but Lyt-2+ cells appeared to be the more potent mediator in the response. Lyt-2+ cells were also involved in the elimination of grossly visible 10-day macrometastases from these weakly immunogenic tumors. Depletion of L3T4+ cells had no effect on tumor regression. Thus, although LAK effectors derived from ASGM-1+ precursors can eliminate pulmonary micrometastases regardless of tumor immunogenicity, Lyt-2+ cells are predominant effectors in the elimination of both pulmonary micro- and macrometastases from weakly immunogenic sarcomas.     

Regulation of β-1,4-Endoglucanase Synthesis in Thermomonospora fusca

In Thermomonospora fusca YX, endocellulase synthesis varies over a 100-fold range depending on the carbon source used. This study shows that the variation is caused by two regulatory mechanisms: an induction mechanism that increases the rate of endocellulase synthesis about 20-fold and a growth rate-dependent repression mechanism that changes the rate of synthesis over a 6-fold range in both induced and noninduced cells. In T. fusca, endocellulase synthesis can be induced by cellulose, cellobiose, or cellodextrin. Cellulase is involved in inducer generation from cellulose. Growth rate-dependent repression can be reversed by limiting cultures for carbon, nitrogen, or, to a lesser extent, phosphorus. Further evidence for two separate regulatory mechanisms is provided by the isolation of mutants (CC-1 and CC-2) whose endocellulases are synthesized constitutively but are still sensitive to growth rate-dependent repression. These conclusions about total endocellulase synthesis were extended to the individual endocellulases by showing that three T. fusca endocellulases are coordinately regulated.