广义估计方程在CT显示方法研究数据分析中的应用

目的:探讨广义估计方程在CT显示方法研究中的应用.方法:采用SAS软件的GENMOD过程,应用广义估计方程方法分析CT显示方法研究实例.结果:给出了广义估计方程SAS程序,并对参数估计和两两比较结果进行解释.结论:广义估计方程能有效的分析CT显示方法研究中反应变量为两分类或多分类的非独立数据.     

Genotoxicity of fractionated organic material in airborne particles from São Paulo, Brazil

Particulate matter less than 10 microns aerodynamic diameter (PM10) is associated with adverse health effects including increased respiratory problems and mortality. PM10 is also associated with increases in cancer in some urban areas. Identification of toxic compounds in PM10 is a step toward estimating exposure to these compounds and evaluating their public health risk. However, the toxic compounds on PM10 are part of a highly complex mixture of compounds that makes chemical characterization difficult. Before this study, there has been little investigation of genotoxic compounds in particulate matter from Latin American cities. Here, both bioassay (mutagenicity) and chemical analyses were conducted with organic solvent extracts of PM10 collected from S?o Paulo, a major Brazilian city. Sequential extraction in dichloromethane (DCM) followed by acetone (ACE) yielded 20.3% and 10.2% of the total mass, respectively. Non-polar and moderately polar organic material solubilized in DCM. ACE extracted more polar organic species and some inorganic ions. Both extracts were fractionated separately using cyanopropyl-bonded silica chromatography with organic solvents of increasing polarity. The mass distribution among the fractions was measured. The mutagenic activity of the fractions was assayed using the microsuspension procedure with the Salmonella typhimurium tester strain TA98, with and without addition of metabolic enzymes (S9). The DCM extract had about four times higher mutagenic activity than the ACE extract. In general, addition of S9 resulted in an increase in mutagenicity of DCM fractions, but a decrease for the ACE extract. Most of the activity was concentrated in fractions in the mid-range of polarity within both the DCM and ACE extracts. The fractions were analyzed by gas chromatography with mass selective detection (GC/MS) without derivatization. The most mutagenic fractions in the DCM extract contained ketones, aldehydes, and quinolines. The most mutagenic ACE fraction had ketones, carboxylic acids, and aldehydes.     

Increased calbindin-D28K immunoreactivity in rat cerebellar Purkinje cell with excitatory amino acids agonists is not dependent on protein synthesis

The calcium binding protein Calbindin-D28K (CaBP) is abundantly expressed in cerebellar Purkinje cells and show increased immunoreactivity (CaBP-IR) when challenged with glutamate or an analog agonist for the ionotropic glutamate receptor (iGluR). Here we report that t-ACPD, a metabotropic glutamate receptor (mGluR) agonist, produced small increases in CaBP-IR which was potentiated by a mGluR antagonist The increase in CaBPIR was not due to de novo protein synthesis because the translational inhibitors (cycloheximide and emetine) or transciptional inhibitors (actinomycine-D and a-amanitine), did not prevent the EAA enhanced CaBP-IR. The CaBP-IR in the PC appears to be coupled to the ionotropic rather than the metabotropic glutamate receptors, but the latter become effective in the presence of their blocker, L-AP3. The results suggest that CaBP may increase its IR through a conformational change of the protein itself.     

First determination of the inhibitor complex structure of human hematopoietic prostaglandin D synthase

Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is responsible for the production of PGD(2) as an allergy or inflammation mediator in mast and Th2 cells. We determined the X-ray structure of human H-PGDS complexed with an inhibitor, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT) at 1.9 A resolution in the presence of Mg(2+). The styryl group of the inhibitor penetrated to the bottom of the active site cleft, and the tetrazole ring was stabilized by the stacking interaction with Trp104, inducing large movement around the alpha5-helix, which caused the space group of the complex crystal to change from P2(1) to P1 upon binding of BSPT. The phthalhydrazidyl group of BSPT exhibited steric hindrance due to the cofactor, glutathione (GSH), increasing the IC(50) value of BSPT for human H-PGDS from 36.2 micro M to 98.1 micro M upon binding of Mg(2+), because the K(m) value of GSH for human H-PGDS was decreased from 0.60 micro M in the presence of EDTA to 0.14 micro M in the presence of Mg(2+). We have to avoid steric hindrance of the GSH molecule that was stabilized by intracellular Mg(2+) in the mM range in the cytosol for further development of structure-based anti-allergic drugs.     

An Isoflavonoid-Inducible Efflux Pump in Agrobacterium tumefaciens Is Involved in Competitive Colonization of Roots

Agrobacterium tumefaciens 1D1609, which was originally isolated from alfalfa (Medicago sativa L.), contains genes that increase competitive root colonization on that plant by reducing the accumulation of alfalfa isoflavonoids in the bacterial cells. Mutant strain I-1 was isolated by its isoflavonoid-inducible neomycin resistance following mutagenesis with the transposable promoter probe Tn5-B30. Nucleotide sequence analysis showed the transposon had inserted in the first open reading frame, ifeA, of a three-gene locus (ifeA, ifeB, and ifeR), which shows high homology to bacterial efflux pump operons. Assays on alfalfa showed that mutant strain I-1 colonized roots normally in single-strain tests but was impaired significantly (P ≤ 0.01) in competition against wild-type strain 1D1609. Site-directed mutagenesis experiments, which produced strains I-4 (ifeA::gusA) and I-6 (ifeA::Ω-Tc), confirmed the importance of ifeA for competitive root colonization. Exposure to the isoflavonoid coumestrol increased β-glucuronidase activity in strain I-4 21-fold during the period when coumestrol accumulation in wild-type cells declined. In the same test, coumestrol accumulation in mutant strain I-6 did not decline. Expression of the ifeA-gusA reporter was also induced by the alfalfa root isoflavonoids formononetin and medicarpin but not by two triterpenoids present in alfalfa. These results show that an efflux pump can confer measurable ecological benefits on A. tumefaciens in an environment where the inducing molecules are known to be present.     

Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens

Abstract The virB operon of the Agrobacterium tumefaciens Ti plasmid encodes 11 proteins. Specific antisera to VirB2, VirB3 and VirB9 were used to locate these virulence proteins in the A. tumefaciens cell. Immunoblot analysis located VirB2 protein to the inner and outer membranes; VirB3 and VirB9 were likewise associated with both membranes, but mainly in the outer membrane. VirB2 is processed from a 12.3-kDa protein into a 7.2-kDa polypeptide. Such sized protein results from cleavage at residue Ala⁴⁷, upstream of which two additional alanine residues Ala⁴⁵-Ala⁴⁶ are contained and bearing resemblance to a signal peptide peptidase-I cleavage sequence. VirB2 and VirB3 sequences are strikingly similar to the pilin biosynthetic proteins TraA and TraL encoded by the tra operon of F and R1-19 plasmids. Since traA encodes a propilin that is cleaved into a 7.2-kDa conjugative pilin product and since this cleavage site is present in both TraA and VirB2, we propose that virB2 encodes a pilin-like protein which together with VirB3 and VirB9 as well as other VirB proteins may be used for interkingdom T-DNA transfer between bacteria and plants.     

The virD4 gene is required for virulence while virD3 and orf5 are not required for virulence of Agrobacterium tumefaciens

The virD operon of the resident Ti plasmid of Agrobacterium tumefaciens contains loci involved in T-DNA processing and undefined virulence functions. Nucleotide sequence of the entire virD operon of pTiC58 revealed similarities to the virD operon of the root-inducing plasmid pRiA4b and to that of the octopine-type plasmid pTiA6NC. However, comparative sequence data show that virD of pTiC58 is more akin to that of the pRiA4b than to that of the pTiA6NC. T7f10::virD gene fusions were used to generate polypeptides that confirm the presence of four open reading frames virD1, virD2, virD3, and virD4 within virD which have a coding capacity for proteins of 16.1, 49.5, 72.6, and 73.5 kDa, respectively. virD3 therefore encodes a polypeptide 3.4 times larger (72.6 versus 21.3 kDa) than that encoded by virD3 of octopine Ti plasmids. Non-polar virD4 mutants could not be complemented by a distant homologue, TraG protein of plasmid RP4. An independently regulated fifth ORF (orf5) is located immediately downstream of 3′ end of virD4 and encodes a polypeptide of 97.4 kDa. The expression of orf5 is dependent on its own promoter and is independent of acetosyringone induction in A. tumefaciens. Recently, it has been shown that virD3 of octopine Ri or Ti plasmids is not required for virulence. In this report, we confirm and extend these findings on a nopaline Ti plasmid by using several virD non-polar mutants that were tested for virulence. virD3 and orf5 non-polar mutants showed no effect on tumorigenicity on 14 different plant species, while virD4 mutants lost their tumorigenicity completely on all these test plants. These data suggest that virD3 and orfS are not essential for virulence whereas virD4 is absolutely required on a wide range of host plants.     

Escherichia coli plasmid pBR313 insertion into plant protoplasts and into their nuclei

Summary Cowpea mesophyll protoplasts were shown to bind irreversibly up to 3% input radioactive pBR313 plasmid DNA after 15 min of contact. Maximum uptake occurred in the presence of 5mM ZnSO₄ and 5 g/ml poly-L-ornithine. Under these conditions about one half of the TCA precipitable radioactivity was associated with the nuclear fraction and behaved as linear plasmid molecules. These could not be chased from the protoplasts upon further incubation with unlabeled plasmid DNA. The presence of donor DNA within the nuclear fraction is most probably not due to an artifactual redistribution of adsorbed plasmid DNA. Prolonged incubation periods resulted in extensive degradation of plasmid in the incubation medium but little degradation occurred in the protoplasts. The donor DNA was not covalently associated with the protoplast nuclear DNA.     

The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.