Enzyme inhibition potency enhancement by active site metal chelating and hydrogen bonding induced conformation-restricted cyclopropanecarbonyl derivatives

Two cyclopropanecarbonyl derivatives were independently found to be 15 and 14 times more potent than the corresponding isopropylcarbonyl analogues as inhibitors of 4-hydroxyphenylpyruvate dioxygenase and dihydroorotate dehydrogenase, respectively. A thorough examination of the co-crystal structures of available enzyme inhibitor complexes and the conformation of X-ray crystal structures of several synthesized cyclopropanecarbonyl derivatives revealed that this enhancement by one order of magnitude of inhibition potency exhibited by cyclopropanecarbonyl derivatives in both enzymes is probably caused by respective metal chelating and hydrogen bonding interactions at the ligand-receptor binding site. These specific interactions subsequently cause the cyclopropyl group of the molecules to adopt a fixed bisected conformation, which is unavailable for isopropylcarbonyl derivatives.     

Structural basis of mercury- and zinc-conjugated complexes as SARS-CoV 3C-like protease inhibitors

Five active metal-conjugated inhibitors (PMA, TDT, EPDTC, JMF1586 and JMF1600) bound with the 3C-like protease of severe acute respiratory syndrome (SARS)-associated coronavirus were analyzed crystallographically. The complex structures reveal two major inhibition modes: Hg(2+)-PMA is coordinated to C(44), M(49) and Y(54) with a square planar geometry at the S3 pocket, whereas each Zn(2+) of the four zinc-inhibitors is tetrahedrally coordinated to the H(41)-C(145) catalytic dyad. For anti-SARS drug design, this Zn(2+)-centered coordination pattern would serve as a starting platform for inhibitor optimization.     

Nuclear transfer in cattle: Effect of nuclear donor cells,cytoplast age,co-culture,and embryo transfer

There are many factors affecting the efficiency of nuclear transfer technology. Some are evaluated here using our novel approach by enucleating oocytes at 20–22 hr after in vitro maturation (IVM), culturing the enucleated oocytes (cytoplasts) for 8–10 hr or 18–20 hr to gain activation competence and then conducting nuclear transfer. In the first experiment, we demonstrated that cumulus cell (CC) monolayer can support some cloned embryos to develop into morulae or blastocysts. Co-culture with CC and bovine oviduct epithelial cell (BOEC) monolayers resulted in no differences (P 0.05) in supporting the development of cloned embryos (Experiment 2). When in vitro matured oocytes were enucleated at 22 hr after IVM followed by nuclear transfer 18–20 hr later, cleavage and morula or blastocyst development of the cloned embryos were similar to those resulting from the enucleated oocytes which had been matured in vivo (Experiment 3). Frozen embryos as nuclear donor cells worked equally well as fresh embryos for cloning in embryo development which was superior to IVF embryos (Experiment 4). However, fresh embryos resulted in a higher proportion (P < 0.05) of blastomere recovery than did frozen or IVF ambryos. Finally, embryo transfer of cloned embryos from our procedure produced a viable calf, demonstrating the commercial value of this novel approach of the technology. © 1993 Wiley-Liss, Inc.     

大鼠睾丸中S-100蛋白生后发育变化的免疫细胞化学研究

本文应用免疫细胞方法,研究大鼠生后４天、７天、１４天、３０天、２个月和３个月睾丸中Ｓ－１００蛋白的分布和变化规律。结果表明：Ｓ－１００蛋白染色反应位于睾丸间质细胞,直到生后３０天才出现阳性细胞,数量少,着色浅,而２月龄和３月龄大鼠睾丸Ｓ－１００蛋白阳性细胞数量多,着色深。提示Ｓ－１００蛋白可能参与间质细胞的合成和分泌睾酮过程。     

Modulation of microglial immune responses by a novel thiourea derivative

Increasing evidence indicates that microglial activation plays an important role in the pathogenesis of Alzheimer's disease (AD). In AD, activated microglia may facilitate the clearance of β-amyloid (Aβ), a neurotoxic component in AD pathogenesis. However, microglial activation comes at the cost of triggering neuro-inflammation, which contributes to cerebral dysfunction. Thus, pharmacological approaches that can achieve a favorable combination of a reduced microglia-mediated neuro-inflammation, and an enhanced Aβ clearance may be beneficial for preventing the progression of the disease. Here, we show that some newly synthesized compounds may exert such a combination of functions. Using mouse primary microglia and RAW264.7 cells, we found that some thiourea derivatives significantly enhanced microglial Aβ phagocytosis and suppressed microglial immune responses, as evidenced by the reduced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Of note, some commercially available inhibitors for iNOS and/or COX-2, such as ibuprofen, dextromethorphan, and N^G-methyl-l-arginine (l-NMA), show negligible effects on microglial Aβ phagocytosis. Among the thiourea derivatives, our data show that a lead compound, designated as compound #326, (1-Naphthalen-1-yl-3-[5-(3-thioureido-phenoxy)-pentyl]-thiourea) appears to be the most potent in promoting Aβ phagocytosis and in inhibiting the LPS-induced expression of iNOS and COX-2 (when used at concentrations in the low μM range). The potency of compound #326 may have beneficial effects on modulating microglial activation in AD. The structure–activity relationship indicates that the thiourea group, alkyl linker, and the hydrophobic aryl group largely influence the dual functions of the compounds. These findings may indicate a structural basis for the improved design of future drug therapies for AD.     

Inhibition of phosphorylated Ser473‐Akt from translocating into the nucleus contributes to 2‐cell arrest and defective zygotic genome activation in mouse preimplantation embryogenesis

Phosphorylated Ser473‐Akt (p‐Ser473‐Akt) is extensively studied as a correlate for the activity of Akt, which plays an important role in mouse oogenesis and preimplantation embryogenesis. However, little progress has been made about its effect on the mouse zygotic genome activation (ZGA) of 2‐cell stage in mouse preimplantation embryos. In this study, we confirmed its localization in the pronuclei of 1‐cell embryos and found that p‐Ser473‐Akt acquired prominent nucleus localization in 2‐cell embryos physiologically. Akt specific inhibitors API‐2 and MK2206 could inhibit the development of mouse preimplantation embryos in vitro, and induce 2‐cell arrest at certain concentrations. 2‐cell embryos exposed to 2.0 μmol/L API‐2 or 30 μmol/L MK2206 displayed attenuated immunofluorescence intensity of p‐Ser473‐Akt in the nucleus. Simultaneously, qRT‐PCR results revealed that 2.0 μmol/L API‐2 treatment significantly downregulated the mRNA pattern of MuERV‐L and eIF‐1A, two marker genes of ZGA, suggesting a defect in ZGA compared with that of control group. Collectively, our work demonstrated the nuclear localization of p‐Ser473‐Akt during major ZGA, and Akt specific inhibitors API‐2 and MK2206 which led to 2‐cell arrest inhibited p‐Ser473‐Akt from translocating into the nucleus of 2‐cell embryos with defective ZGA as well, implying p‐Ser473‐Akt may be a potential player in the major ZGA of 2‐cell mouse embryos.     

Products and bioenergy from the pyrolysis of rice straw via radio frequency plasma and its kinetics

The radio frequency plasma pyrolysis technology, which can overcome the disadvantages of common pyrolysis methods such as less gas products while significant tar formation, was used for pyrolyzing the biomass waste of rice straw. The experiments were performed at various plateau temperatures of 740, 813, 843 and 880 K with corresponding loading powers of 357, 482, 574 and 664 W, respectively. The corresponding yields of gas products (excluding nitrogen) from rice straw are 30.7, 56.6, 62.5 and 66.5 wt.% with respect to the original dried sample and the corresponding specific heating values gained from gas products are about 4548, 4284, 4469 and 4438 kcal kg^?1, respectively, for the said cases. The corresponding combustible portions remained in the solid residues are about 64.7, 35, 28.2 and 23.5 wt.% with specific heating values of 4106, 4438, 4328 and 4251 kcal kg^?1 with respective to solid residues, while that in the original dried sample is 87.2 wt.% with specific heating value of 4042 kcal kg^?1. The results indicated that the amount of combustibles converted into gas products increases with increasing plateau temperature. The kinetic model employed to describe the pyrolytic conversion of rice straw at constant temperatures agrees well with the experimental data. The best curve fittings render the frequency factor of 5759.5 s^?1, activation energy of 74.29 kJ mol^?1 and reaction order of 0.5. Data and information obtained are useful for the future design and operation of pyrolysis of rice straw via radio frequency plasma.     

Cysteine-rich domain of scavenger receptor AI modulates the efficacy of surface targeting and mediates oligomeric Aβ internalization

Background

Insufficient clearance of soluble oligomeric amyloid-β peptide (oAβ) in the central nervous system leads to the synaptic and memory deficits in Alzheimer''s disease (AD). Previously we have identified scavenger receptor class A (SR-A) of microglia mediates oligomeric amyloid-β peptide (oAβ) internalization by siRNA approach. SR-A is a member of cysteine-rich domain (SRCR) superfamily which contains proteins actively modulating the innate immunity and host defense, however the functions of the SRCR domain remain unclear. Whether the SRCR domain of SR-AI modulates the receptor surface targeting and ligand internalization was investigated by expressing truncated SR-A variants in COS-7 cells. Surface targeting of SR-A variants was examined by live immunostaining and surface biotinylation assays. Transfected COS-7 cells were incubated with fluorescent oAβ and acetylated LDL (AcLDL) to assess their ligand-internalization capabilities.

Result

Genetic ablation of SR-A attenuated the internalization of oAβ and AcLDL by microglia. Half of oAβ-containing endocytic vesicles was SR-A positive in both microglia and macrophages. Clathrin and dynamin in SR-AI-mediated oAβ internalization were involved. The SRCR domain of SR-AI is encoded by exons 10 and 11. SR-A variants with truncated exon 11 were intracellularly retained, whereas SR-A variants with further truncations into exon 10 were surface-targeted. The fusion of exon 11 to the surface-targeted SR-A variant lacking the SRCR domain resulted in the intracellular retention and the co-immunoprecipitation of Bip chaperon of the endoplasmic reticulum. Surface-targeted variants were N-glycosylated, whereas intracellularly-retained variants retained in high-mannose states. In addition to the collagenous domain, the SRCR domain is a functional binding domain for oAβ and AcLDL. Our data suggest that inefficient folding of SR-AI variants with truncated SRCR domain was recognized by the endoplasmic reticulum associated degradation which leads to the immature N- glycosylation and intracellular retention.

Conclusion

The novel functions of the SRCR domain on regulating the efficacy of receptor trafficking and ligand binding may lead to possible approaches on modulating the innate immunity in Alzheimer’s disease and atherosclerosis.     

Prevention of mitochondrial dysfunction in post-traumatic mouse brain by superoxide dismutase

Reactive oxygen species (ROS) are known to be involved in the pathogenesis of traumatic brain injury (TBI). Previous studies have shown that the susceptibility of mice to TBI-induced formation of cortical lesion is determined by the expression levels of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD, respectively). However, the underlying biochemical mechanisms are not understood. In this study, we measured the efficiency of mitochondrial respiration in mouse brains with altered expression of these two enzymes. While controlled cortical impact injury (CCII) with a deformation depth of 2 mm caused a drastic decrease in NAD-linked bioenergetic capacity in brain mitochondria of wild-type mice, the functional decrease was not observed in brains of littermate transgenic mice overexpressing CuZnSOD or MnSOD. In addition, a 1 mm CCII greatly compromised brain mitochondrial function in mice deficient in CuZnSOD or MnSOD, but not wild-type mice. Inclusion of the calcium-chelating agent, EGTA, in the assay solution could completely prevent dysfunction of oxidative phosphorylation in all mitochondrial samples, suggesting that the observed impairment of mitochondrial function was a result of calcium overloading. In conclusion, our results imply that mitochondrial dysfunction induced by superoxide anion radical contributes to lesion formation in mouse brain following physical trauma.