白百利烟草愈伤组织细胞细胞分裂素结合蛋白的研究

利用BA-Sepharose 4B亲和层析技术丛白百利烟草(Nicotiana tabacum Baibaili)愈伤组织细胞分离提纯了分子量为4400±100道尔顿的细胞分裂素结合蛋白(CB-蛋白)。在细胞表面、核糖体、线粒体、叶绿体和细胞核上以及在细胞液中都有CB-蛋白存在,而核糖体上的CB-蛋白含量量高。探讨了CB-蛋白的功能。     

东北白眉蝮蛇毒细胞毒组分的分离制备及活性鉴定

用离子交换层析和凝胶过滤法从东北白眉蝮蛇毒中分离出一种具有较强的细胞毒活性的蛋白质。这种蛋白质对肿瘤细胞的细胞毒作用元选择性,但各种肿瘤细胞的敏感性有一定差别。对正常人体细胞也有一定的细胞毒性作用,与肿瘤细胞相比,敏感性较低。这种蛋白质加热到56℃,30分钟,细胞毒活性稳定。4℃及-20℃保存4个月,细胞毒活性稳定。PH值低于4及高于9时,可使其完全失活。     

Design,synthesis and in vitro biological evaluation of quinazolinone derivatives as EGFR inhibitors for antitumor treatment

Abstract

In this paper, a series of novel 3-methyl-quinazolinone derivatives was designed, synthesised and evaluated for antitumor activity in vitro on wild type epidermal growth factor receptor tyrosine kinase (EGFR^wt-TK) and three human cancer cell lines including A549, PC-3, and SMMC-7721. The results displayed that some of the compounds had good activities, especially 2-{4-[(3-Fluoro-phenylimino)-methyl]-phenoxymethyl}-3-methyl-3H-quinazolin-4-one (5?g), 2-{4-[(3,4-Difluoro-phenylimino)-methyl]-phenoxymethyl}-3-methyl-3H-quinazolin-4-one (5k) and 2-{4-[(3,5-Difluoro-phenylimino)-methyl]-phenoxymethyl}-3-methyl-3H-quinazolin-4-one (5?l) showed high antitumor activities against three cancer cell lines. Moreover, compound 5k could induce late apoptosis of A549 cells at high concentrations and arrest cell cycle of A549 cells in the G2/M phase at tested concentrations. Also, compound 5k could inhibit the EGFR^wt-TK with IC₅₀ value of 10?nM. Molecular docking data indicates that the compound 5k may exert inhibitory activity by forming stable hydrogen bonds with the R817, T830 amino acid residues and cation-Π interaction with the K72 residue of EGFR^wt-TK.     

Regulation of the cyclin D3 promoter by E2F1

We have previously demonstrated that ectopic expression of E2F1 is sufficient to drive quiescent cells into S phase and that E2F1 expression can contribute to oncogenic transformation. Key target genes in this process include master regulators of the cell cycle, such as cyclin E, which regulates G(1) progression, and cyclin A, which is required for the initiation of DNA synthesis. In the present work, we present novel evidence that a second G(1) cyclin, cyclin D3, is also potently activated by E2F1. First, an estrogen receptor-E2F1 fusion protein (ER-E2F1) potently activates the endogenous cyclin D3 mRNA upon treatment with 4-hydroxytamoxifen, which induces nuclear accumulation of the otherwise cytosolic fusion protein. Furthermore, trans-activation of cyclin D3 by ER-E2F1 occurs even in the presence of the protein synthesis inhibitor cycloheximide and thus appears direct. Second, all of the growth-stimulatory members of the E2F family (E2F1, -2, and -3A) potently activate a cyclin D3 promoter reporter, whereas growth-restraining members of the family (E2F4, -5, and -6) have little effect. Third, recombinant E2F1 binds with high affinity to the cyclin D3 promoter in vitro. Fourth, chromatin immunoprecipitation assays demonstrate that endogenous E2F1 is associated with the cyclin D3 promoter in vivo. Finally, mapping experiments localize the essential E2F regulatory element of the cyclin D3 promoter to a noncanonical E2F site in the promoter between nucleotides -143 and -135 relative to the initiating methionine codon. We conclude that in addition to cyclins E and A, E2F family members can also activate one member of the D-type cyclins, further contributing to the ability of the stimulatory E2F family members to drive cellular proliferation.     

Overlapping but distinct gene regulation profiles by glucocorticoids and progestins in human breast cancer cells

Glucocorticoids and progestins bind to receptors that share many structural and functional similarities, including virtually identical DNA recognition specificity. Nonetheless, the two hormones mediate very distinct biological functions. For example, progestins are associated with the incidence and progression of breast cancer, whereas glucocorticoids are growth suppressive in mammary cancer cells. To understand the mechanisms that engender biological specificity, it is necessary to identify genes that are differentially regulated by the two receptors. Here we employ Affymetrix oligonucleotide arrays to compare glucocorticoid- and progestin-regulated gene expression in a human breast cancer cell line. This global analysis reveals that the two hormones regulate overlapping but distinct sets of genes, including 31 genes that are differentially regulated. Surprisingly, the set of differentially regulated genes was almost as large as the set of genes regulated by both hormones. Examination of the set of differentially regulated genes suggests mechanisms behind the distinct growth effects of the two hormones in breast cancer. The differential regulation of four genes representing different regulatory patterns was confirmed by RT-PCR and Northern blot analyses. Treatment with cycloheximide or RU486 indicates that the regulation is a primary, receptor-mediated event. Detailed analyses of genes identified in these studies will furnish a mechanistic understanding of differential regulation by glucocorticoids and progestins.     

Study of Toxin Production by Isolates of Stachybotrys chartarum and Memnoniella echinata Isolated during a Study of Pulmonary Hemosiderosis in Infants

A cluster of cases of pulmonary hemosiderosis among infants was reported in Cleveland, Ohio, during 1993 and 1994. These unusual cases appeared only in infants ranging in age from 1 to 8 months and were characterized by pulmonary hemorrhage, which caused the babies to cough up blood. A case-control study identified major home water damage (from plumbing leaks, roof leaks, or flooding) as a risk factor for development of pulmonary hemorrhage in these infants. Because of an interest in the possibility that trichothecene mycotoxins might be involved in this illness, a number of isolates of Stachybotrys chartarum were grown in the laboratory on rice, and extracts were prepared and analyzed both for cytotoxicity and for specific toxins. Two isolates of Memnoniella echinata, a fungus closely related to S. chartarum, were also included in these studies. S. chartarum isolates collected from the homes were shown to produce a number of highly toxic compounds, and the profiles of toxic compounds from M. echinata were similar; the most notable difference was the fact that the principal metabolites produced by M. echinata were griseofulvins.     

Stoichiometric Structure-Function Analysis of the Prolactin Receptor Signaling Domain by Receptor Chimeras

The intracellular domain of the prolactin (PRL) receptor (PRLr) is required for PRL-induced signaling and proliferation. To identify and test the functional stoichiometry of those PRLr motifs required for transduction and growth, chimeras consisting of the extracellular domain of either the α or β subunit of human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-CSFr) and the intracellular domain of the rat PRLr were synthesized. Because the high-affinity binding of GM-CSF results from the specific pairing of one α- and one β-GM-CSFr, use of GM-CSFr/PRLr chimera enabled targeted dimerization of the PRLr intracellular domain. To that end, the extracellular domains of the α- and β-GM-CSFr were conjugated to one of the following mutations: (i) PRLr C-terminal truncations, termed α278, α294, α300, α322, or β322; (ii) PRLr tyrosine replacements, termed Y309F, Y382F, or Y309+382F; or, (iii) PRLr wild-type short, intermediate, or long isoforms. These chimeras were cotransfected into the cytokine-responsive Ba/F3 line, and their expression was confirmed by ligand binding and Northern and Western blot analyses. Data from these studies revealed that heterodimeric complexes of the wild type with C-terminal truncation mutants of the PRLr intracellular domain were incapable of ligand-induced signaling or proliferation. Replacement of any single tyrosine residue (Y309F or Y382F) in the dimerized PRLr complex resulted in a moderate reduction of receptor-associated Jak2 activation and proliferation. In contrast, trans replacement of these residues (i.e., αY309F and βY382F) markedly reduced ligand-driven Jak2 activation and proliferation, while cis replacement of both tyrosine residues in a single intracellular domain (i.e., αY309+382F) produced an inactive signaling complex. Analysis of these GM-CSFr–PRLr complexes revealed equivalent levels of Jak2 in association with the mutant receptor chains, suggesting that the tyrosine residues at 309 and 382 do not contribute to Jak association, but instead to its activation. Heterodimeric pairings of the intracellular domains from the known PRLr receptor isoforms (short-intermediate, short-long, and intermediate-long) also yielded inactive receptor complexes. These data demonstrate that the tyrosine residues at 309 and 382, as well as additional residues within the C terminus of the dimerized PRLr complex, contribute to PRL-driven signaling and proliferation. Furthermore, these findings indicate a functional requirement for the pairing of Y309 and Y382 in trans within the dimerized receptor complex.     

Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing

This study characterizes the colonization and composition of bacterial flora in dwarf Asian honeybee (Apis florea) larvae and compares bacterial diversity and distribution among different sampling locations. A. florea larvae were collected from 3 locations in Chiang Mai province, Thailand. Bacterial DNA was extracted from each larva using the phenol–chloroform method. Denaturing gradient gel electrophoresis was performed, and the dominant bands were excised from the gels, cloned, and sequenced for bacterial species identification. The result revealed similarities of bacterial community profiles in each individual colony, but differences between colonies from the same and different locations. A. florea larvae harbor bacteria belonging to 2 phyla (Firmicutes and Proteobacteria), 5 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli, and Clostridia), 6 genera (Clostridium, Gilliamella, Melissococcus, Lactobacillus, Saccharibacter, and Snodgrassella), and an unknown genus from uncultured bacterial species. The classes with the highest abundance of bacteria were Alphaproteobacteria (34%), Bacilli (25%), Betaproteobacteria (11%), Gammaproteobacteria (10%), and Clostridia (8%), respectively. Similarly, uncultured bacterial species were identified (12%). Environmental bacterial species, such as Saccharibacter floricola, were also found. This is the first study in which sequences closely related to Melissococcus plutonius, the causal pathogen responsible for European foulbrood, have been identified in Thai A. florea larvae.     

Detecting Static and Dynamic Differences between Eyes-Closed and Eyes-Open Resting States Using ASL and BOLD fMRI

Resting-state fMRI studies have increasingly focused on multi-contrast techniques, such as BOLD and ASL imaging. However, these techniques may reveal different aspects of brain activity (e.g., static vs. dynamic), and little is known about the similarity or disparity of these techniques in detecting resting-state brain activity. It is therefore important to assess the static and dynamic characteristics of these fMRI techniques to guide future applications. Here we acquired fMRI data while subjects were in eyes-closed (EC) and eyes-open (EO) states, using both ASL and BOLD techniques, at two research centers (NIDA and HNU). Static brain activity was calculated as voxel-wise mean cerebral blood flow (CBF) using ASL, i.e., CBF-mean, while dynamic activity was measured by the amplitude of low frequency fluctuations (ALFF) of BOLD, i.e., BOLD-ALFF, at both NIDA and HNU, and CBF, i.e., CBF-ALFF, at NIDA. We showed that mean CBF was lower under EC than EO in the primary visual cortex, while BOLD-ALFF was higher under EC in the primary somatosensory cortices extending to the primary auditory cortices and lower in the lateral occipital area. Interestingly, mean CBF and BOLD-ALFF results overlapped at the visual cortex to a very small degree. Importantly, these findings were largely replicated by the HNU dataset. State differences found by CBF-ALFF were located in the primary auditory cortices, which were generally a subset of BOLD-ALFF and showed no spatial overlap with CBF-mean. In conclusion, static brain activity measured by mean CBF and dynamic brain activity measured by BOLD- and CBF-ALFF may reflect different aspects of resting-state brain activity and a combination of ASL and BOLD may provide complementary information on the biophysical and physiological processes of the brain.