实验室条件下达乌尔黄鼠的越冬繁殖

达乌尔黄鼠（Spermophilusdauricus）是国内研究冬眠生理学的主要模式动物,且日益为医学研究所关注。实验室条件下繁殖种群具有明确生活史特征,是相关研究的理想对象,但以往研究中,达乌尔黄鼠实验室条件下繁殖成功率及后代成活率均很低。本研究通过采取丰富饲料种类、雄性出眠后的暖温暂养、“双笼配对”等措施,对实验室条件下越冬达乌尔黄鼠进行配对,从21对动物中成功繁殖出18窝子代,繁殖成功率85.7 %。78只子代饲养至3月龄,仍有67只存活,存活率为85.9 %。通过本实验,得出改进达乌尔黄鼠实验室条件下繁殖的主要措施如下：（1）繁殖期母鼠及发育期幼鼠,除饲喂标准大鼠饲料外,适量添加幼犬粮、胡萝卜,以补充营养;（2）出眠的雄性达乌尔黄鼠放入18℃房间暂养2周,有助于其性腺发育,促进繁殖成功;（3）“双笼配对”能有效地降低外界干扰,减少动物的杀婴行为。       

TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells

Leprosy enables investigation of mechanisms by which the innate immune system contributes to host defense against infection, because in one form, the disease progresses, and in the other, the infection is limited. We report that Toll-like receptor (TLR) activation of human monocytes induces rapid differentiation into two distinct subsets: DC-SIGN+ CD16+ macrophages and CD1b+ DC-SIGN- dendritic cells. DC-SIGN+ phagocytic macrophages were expanded by TLR-mediated upregulation of interleukin (IL)-15 and IL-15 receptor. CD1b+ dendritic cells were expanded by TLR-mediated upregulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor, promoted T cell activation and secreted proinflammatory cytokines. Whereas DC-SIGN+ macrophages were detected in lesions and after TLR activation in all leprosy patients, CD1b+ dendritic cells were not detected in lesions or after TLR activation of peripheral monocytes in individuals with the progressive lepromatous form, except during reversal reactions in which bacilli were cleared by T helper type 1 (TH1) responses. In tuberculoid lepromatous lesions, DC-SIGN+ cells were positive for macrophage markers, but negative for dendritic cell markers. Thus, TLR-induced differentiation of monocytes into either macrophages or dendritic cells seems to crucially influence effective host defenses in human infectious disease.     

Structural characterization and kinetics of nitric-oxide synthase inhibition by novel N5-(iminoalkyl)- and N5-(iminoalkenyl)-ornithines

Isoform-specific nitric-oxide synthase (NOS) inhibitors may prove clinically useful in reducing the pathophysiological effects associated with increased neuronal NOS (nNOS) or inducible NOS (iNOS) activity in a variety of neurological and inflammatory disorders. Analogs of the NOS substrate L-arginine are pharmacologically attractive inhibitors because of their stability, reliable cell uptake, and good selectivity for NOS over other heme proteins. Some inhibitory arginine analogs show significant isoform selectivity although the structural or mechanistic basis of such selectivity is generally poorly understood. In the present studies, we determined by x-ray crystallography the binding interactions between rat nNOS and N5-(1-imino-3-butenyl)-L-ornithine (L-VNIO), a previously identified mechanism-based, irreversible inactivator with moderate nNOS selectivity. We have also synthesized and mechanistically characterized several L-VNIO analogs and find, surprisingly, that even relatively minor structural changes produce inhibitors that are either iNOS-selective or non-selective. Furthermore, derivatives having a methyl group added to the butenyl moiety of L-VNIO and L-VNIO derivatives that are analogs of homoarginine rather than arginine display slow-on, slow-off kinetics rather than irreversible inactivation. These results elucidate some of the structural requirements for isoform-selective inhibition by L-VNIO and its related alkyl- and alkenyl-imino ornithine and lysine derivatives and may provide information useful in the ongoing rational design of isoform-selective inhibitors.     

Crystal structures of the ferric,ferrous, and ferrous-NO forms of the Asp140Ala mutant of human heme oxygenase-1: catalytic implications

Site-directed mutagenesis studies have shown that Asp140 in both human and rat heme oxygenase-1 is critical for enzyme activity. Here, we report the D140A mutant crystal structure in the Fe(III) and Fe(II) redox states as well as the Fe(II)-NO complex as a model for the Fe(II)-oxy complex. These structures are compared to the corresponding wild-type structures. The mutant and wild-type structures are very similar, except for the distal heme pocket solvent structure. In the Fe(III) D140A mutant one water molecule takes the place of the missing Asp140 carboxylate side-chain and a second water molecule, novel to the mutant, binds in the distal pocket. Upon reduction to the Fe(II) state, the distal helix running along one face of the heme moves closer to the heme in both the wild-type and mutant structures thus tightening the active site. NO binds to both the wild-type and mutant in a bent conformation that orients the NO O atom toward the alpha-meso heme carbon atom. A network of water molecules provides a H-bonded network to the NO ligand, suggesting a possible proton shuttle pathway required to activate dioxygen for catalysis. In the wild-type structure, Asp140 exhibits two conformations, suggesting a dynamic role for Asp140 in shuttling protons from bulk solvent via the water network to the iron-linked oxy complex. On the basis of these structures, we consider why the D140A mutant is inactive as a heme oxygenase but active as a peroxidase.     

Genome-wide linkage analysis using cross-sectional and longitudinal traits for body mass index in a subsample of the Framingham Heart Study

To evaluate linkage evidence for body mass index (BMI) using both cross-sectional and longitudinal data, we performed genome-wide multipoint linkage analyses on subjects who had complete data at four selected time points (initial, 8th, 12th, and 16th year following the initial visit) from the Framingham Heart Study. The cross-sectional measures included BMI at each of the four selected time points and the longitudinal measure was the within-subject mean of BMI at the above four time points. Using the variance components method, we consistently observed the maximum LOD score out of the genome scan using BMI at each time point and the mean of BMI between 049xd2 and GATA71H05 on chromosome 16. The highest LOD score (3.0) was at time point 1, while the lowest (1.9) was at time point 4. We also observed other suggestive linkages on chromosome 6, 10, and 18 at time point 1 only. The longitudinal measure we studied (mean of BMI) did not provide greater power to identify a positive linkage than some of the cross-sectional measures (e.g., time point 1). The changing of linkage evidence over time provided some insights on the variation of genetic effect on BMI with aging. There may be a QTL on chromosome 16 that contributes to BMI and this locus, and maybe others, is more likely to affect BMI during early adulthood.     

Bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo

Primary cultures of rat type II alveolar epithelial cells (AECs) or human AEC-derived A549 cells, when exposed to bleomycin (Bleo), exhibited concentration-dependent apoptosis detected by altered nuclear morphology, fragmentation of DNA, activation of caspase-3, and net cell loss over time. In both cell culture models, exposure to Bleo caused time-dependent increases in angiotensinogen (ANGEN) mRNA. Antisense oligonucleotides against ANGEN mRNA inhibited Bleo-induced apoptosis of rat AEC or A549 cells by 83 and 84%, respectively (P < 0.01 and P < 0.05), and prevented Bleo-induced net cell loss. Apoptosis of rat AECs or A549 cells in response to Bleo was inhibited 91% by the ANG-converting enzyme inhibitor captopril or 82%, respectively, by neutralizing antibodies specific for ANG II (both P < 0.01). Antagonists of ANG receptor AT(1) (losartan, L-158809, or saralasin), but not an AT(2)-selective blocker (PD-123319), inhibited Bleo-induced apoptosis of either rat AECs (79%, P < 0.01) or A549 cells (83%, P < 0.01) and also reduced the activity of caspase-3 by 52% (P < 0.05). These data indicate that Bleo, like Fas(L) or TNF-alpha, induces transactivation of ANG synthesis de novo that is required for AEC apoptosis. They also support the theory that ANG system antagonists have potential for the blockade of AEC apoptosis in situ.     

A novel NOD2/CARD15 haplotype conferring risk for Crohn disease in Ashkenazi Jews

Crohn disease (CD) exhibits a 2-4-fold increased frequency in Jews as compared with other ethnic/racial groups. Three coding variants of the NOD2/CARD15 have been reported as independent disease-predisposing mutations (DPMs), but these were found in only 30%-40% of patients with CD and could not account for all the linkage between CD and the IBD1 locus. The aim of the present study was to explore whether additional DPMs at the IBD1 locus exist in the high-risk Jewish group. Sixty-four Ashkenazi Jewish and 147 non-Jewish white families were studied. Six microsatellite markers spanning IBD1 were genotyped for linkage analysis in subgroups stratified on NOD2/CARD15 DPM status. SNPs in NOD2/CARD15 (R702W, G908R, 1007fs, and S268P) were then genotyped in family and independent case-control samples. On the basis of initial results, sequencing was done on NOD2/CARD15-translated regions in 12 Jewish individuals. Subsequently, a new NOD2/CARD15 variant was genotyped and analyzed. After excluding the influence of the three DPMs, significant linkage of IBD1 to CD in Jews remained with two peaks at D16S403 (mean allele sharing [MAS] = 0.70] and D16S411 (MAS = 0.59). Further, we observed an increased frequency of a haplotype carrying only the 268S variant in Jewish patients (OR = 3.13, P=.0023) but not in non-Jews, suggesting the existence of a Jewish-specific additional disease-predisposing factor on this haplotype. Sequencing of this haplotype revealed a new variant (IVS8+158; JW1). The 268S-JW1 combination exhibited a further increased risk (OR = 5.75, P=.0005) and the highest population-attributable risk (15.1%) for CD among reported DPMs in Jews. In Ashkenazi Jews, unrecognized population-specific predisposing factor(s) exist on the 268S-JW1 haplotype at the IBD1 locus. This factor may contribute to the higher risk for CD in Ashkenazi Jews as compared with non-Jews.     

Crystal structure of P450cin in a complex with its substrate, 1,8-cineole, a close structural homologue to D-camphor, the substrate for P450cam

Cytochrome P450cin catalyzes the monooxygenation of 1,8-cineole, which is structurally very similar to d-camphor, the substrate for the most thoroughly investigated cytochrome P450, cytochrome P450cam. Both 1,8-cineole and d-camphor are C(10) monoterpenes containing a single oxygen atom with very similar molecular volumes. The cytochrome P450cin-substrate complex crystal structure has been solved to 1.7 A resolution and compared with that of cytochrome P450cam. Despite the similarity in substrates, the active site of cytochrome P450cin is substantially different from that of cytochrome P450cam in that the B' helix, essential for substrate binding in many cytochrome P450s including cytochrome P450cam, is replaced by an ordered loop that results in substantial changes in active site topography. In addition, cytochrome P450cin does not have the conserved threonine, Thr252 in cytochrome P450cam, which is generally considered as an integral part of the proton shuttle machinery required for oxygen activation. Instead, the analogous residue in cytochrome P450cin is Asn242, which provides the only direct protein H-bonding interaction with the substrate. Cytochrome P450cin uses a flavodoxin-like redox partner to reduce the heme iron rather than the more traditional ferredoxin-like Fe(2)S(2) redox partner used by cytochrome P450cam and many other bacterial P450s. It thus might be expected that the redox partner docking site of cytochrome P450cin would resemble that of cytochrome P450BM3, which also uses a flavodoxin-like redox partner. Nevertheless, the putative docking site topography more closely resembles cytochrome P450cam than cytochrome P450BM3.     

Structural basis for novel delta-regioselective heme oxygenation in the opportunistic pathogen Pseudomonas aeruginosa

The Gram-negative bacterium Pseudomonas aeruginosa contains a heme oxygenase (pa-HO) that primarily oxygenates the delta-meso heme carbon [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, 14879-14892]. This differs from other previously characterized heme oxygenases, which display regioselectivity for the alpha-meso heme carbon. Here we report the crystal structure of pa-HO at 1.60 A resolution and compare it to the 1.50 A structure of nm-HO from Neisseria meningitidis [Schuller, D. J., Zhu, W., Stojiljkovic, I., Wilks, A., and Poulos, T. L. (2001) Biochemistry 40, 11552-11558]. The crystal structure of pa-HO maintains the same overall fold as other bacterial and mammalian heme oxygenases, including a conserved network of hydrogen-bonded solvent molecules important for dioxygen activation. The novel delta-regioselectivity of heme oxygenation observed by pa-HO is due to the heme being rotated by approximately 100 degrees, which places the delta-meso heme carbon in the same position as the alpha-meso heme carbon in other heme oxygenases. The main interaction in pa-HO that stabilizes the unique heme orientation is a salt bridge between Lys132 and the heme 7-propionate, as well as hydrophobic contacts involving Leu29, Val33, and Phe189 with the heme methyl and vinyl groups.