Crystal structures of substrate complexes of malic enzyme and insights into the catalytic mechanism

Malic enzymes catalyze the oxidative decarboxylation of L-malate to pyruvate and CO(2) with the reduction of the NAD(P)(+) cofactor in the presence of divalent cations. We report the crystal structures at up to 2.1 A resolution of human mitochondrial NAD(P)(+)-dependent malic enzyme in different pentary complexes with the natural substrate malate or pyruvate, the dinucleotide cofactor NAD(+) or NADH, the divalent cation Mn(2+), and the allosteric activator fumarate. Malate is bound deep in the active site, providing two ligands for the cation, and its C4 carboxylate group is out of plane with the C1-C2-C3 atoms, facilitating decarboxylation. The divalent cation is positioned optimally to catalyze the entire reaction. Lys183 is the general base for the oxidation step, extracting the proton from the C2 hydroxyl of malate. Tyr112-Lys183 functions as the general acid-base pair to catalyze the tautomerization of the enolpyruvate product from decarboxylation to pyruvate.     

长期游泳运动对大鼠铁状态的影响

目的：观察不同的运动时间对铁状态的影响。方法：大鼠随机分为3、6、12个月的三个游泳运动组和相应安静组;运动期满后观察血液学铁状态指标和器官非血红素铁(NHI)含量和NHI总含量(TNHI)的变化。结果：与安静组相比,三种不同时间长度的运动均诱导一种具有血浆铁浓度降低、血浆转铁蛋白铁饱和度降低而血红蛋白浓度和红细胞比容得到雏持的血液学低铁状态;这种低铁状态伴有肝、脾、心、肾NHI浓度显著降低,但与运动时间无关;肝、脾和肾TNHI变化与其浓度变化方向一致,但心脏没有显著变化;上述器官TNHI随时间增加而增多。结论：尽管运动诱导的低铁状态类似于铁缺乏中期表现,但由于器官NHI重分布和铁贮存并没有进行性降低,因此,长期运动引起的低铁状态可能是机体内铁代谢对运动的适应,不存在所谓“运动性铁缺乏”现象。     

Function and regulation of Aurora／Ipllp kinase family in cell division

During mitosis,the parent cell distributes its genetic materials equally into two daughter cells through chromosome segregation,a complex movements orchestrated by mitotic kinases and its effector proteins.Faithful chromosome segregation and cytokinesis ensure that each daughter cell receives a full copy of genetic materials of parent cell.Defects in these processes can lead to aneuploidy or polyploidy.Aurora/Ipllp family, a class of conserved serine/threonine kinases,plays key roles in chromosome segregation and cytokinesis.This article highlights the function and regulation of Aurora/Ipllp family in mitosis and provides potential links between aberrant regulation of Aurora/Ipllp kinases and pathogenesis of human cancer.     

Random analysis of amino acid pairs sensitive to variants in human coagulation factor IX precursor

In this study, we used a random approach to determine which amino acid pairs in human coagulation factor IX precursor are more sensitive to its 99 variants. The results show that the randomly unpredictable amino acid pairs are more sensitive to variants.     

Induction of CD8+ T cells to an HIV-1 antigen through a prime boost regimen with heterologous E1-deleted adenoviral vaccine carriers

E1-deleted adenoviral recombinants most commonly based on the human serotype 5 (AdHu5) have been shown thus far to induce unsurpassed transgene product-specific CD8(+) T cell responses. A large percentage of the adult human population carries neutralizing Abs due to natural exposures to AdHu5 virus. To circumvent reduction of the efficacy of adenovirus (Ad) vector-based vaccines by neutralizing Abs to the vaccine carrier, we developed E1-deleted adenoviral vaccine carriers based on simian serotypes. One of these carriers, termed AdC68, expressing a codon-optimized truncated form of gag of HIV-1 was shown previously to induce a potent transgene product-specific CD8(+) T cell response in mice. We constructed a second chimpanzee adenovirus vaccine vector, termed AdC6, also expressing the truncated gag of HIV-1. This vector, which belongs to a different serotype than the AdC68 virus, induces high frequencies of gag-specific CD8(+) T cells in mice including those pre-exposed to AdHu5 virus. Generation of an additional E1-deleted adenoviral vector of chimpanzee origin allows for sequential booster immunizations with heterologous vaccine carriers. In this study, we show that such heterologous prime boost regimens based on E1-deleted adenoviral vectors of different serotypes expressing the same transgene product are highly efficient in increasing the transgene product-specific CD8(+) T cell response. They are equivalent to sequential vaccinations with an E1-deleted Ad vector followed by booster immunization with a poxvirus vector and they surpass regimens based on DNA vaccine prime followed by a recombinant adenoviral vector boost.     

The role of OX40 ligand interactions in the development of the Th2 response to the gastrointestinal nematode parasite Heligmosomoides polygyrus

In these studies, we examined the effects of OX40 ligand (OX40L) deficiency on the development of Th2 cells during the Th2 immune response to the intestinal nematode parasite Heligmosomoides polygyrus. Elevations in IL-4 production and total and Ag-specific serum IgE levels were partially inhibited during both the primary and memory immune responses to H. polygyrus in OX40L(-/-) mice. The host-protective memory response was compromised in OX40L(-/-) mice, as decreased worm expulsion and increased egg production were observed compared with H. polygyrus-inoculated OX40L(+/+) mice. To further examine the nature of the IL-4 defect during priming, adoptively transferred DO11.10 T cells were analyzed in the context of the H. polygyrus response. Although Ag-specific T cell IL-4 production was reduced in the OX40L(-/-) mice following immunization with OVA peptide plus H. polygyrus, Ag-specific T cell expansion, cell cycle progression, CXCR5 expression, and migration were comparable between OX40L(+/+) and OX40L(-/-) mice inoculated with OVA and H. polygyrus. These studies suggest an important role for OX40/OX40L interactions in specifically promoting IL-4 production, as well as associated IgE elevations, in Th2 responses to H. polygyrus. However, OX40L interactions were not required for serum IgG1 elevations, increases in germinal center formation, and Ag-specific Th2 cell expansion and migration to the B cell zone.     

Altered distribution of mitochondria impairs calcium homeostasis in rat hippocampal neurons in culture

The specificity of Ca2+ signals is conferred in part by limiting changes in cytosolic Ca2+ to subcellular domains. Mitochondria play a major role in regulating Ca2+ in neurons and may participate in its spatial localization. We examined the effects of changes in the distribution of mitochondria on NMDA-induced Ca2+ increases. Hippocampal cultures were treated with the microtubule-destabilizing agent vinblastine, which caused the mitochondria to aggregate and migrate towards one side of the neuron. This treatment did not appear to decrease the energy status of mitochondria, as indicated by a normal membrane potential and pH gradient across the inner membrane. Moreover, electron microscopy showed that vinblastine treatment altered the distribution but not the ultrastructure of mitochondria. NMDA (200 micro m, 1 min) evoked a greater increase in cytosolic Ca2+ in vinblastine-treated cells than in untreated cells. This increase did not result from impaired Ca2+ efflux, enhanced Ca2+ influx, opening of the mitochondrial permeability transition pore or altered function of endoplasmic reticulum Ca2+ stores. Ca2+ uptake into mitochondria was reduced by 53% in vinblastine-treated cells, as reported by mitochondrially targeted aequorin. Thus, the distribution of mitochondria maintained by microtubules is critical for buffering Ca2+ influx. A subset of mitochondria close to a Ca2+ source may preferentially regulate Ca2+ microdomains, set the threshold for Ca2+-induced toxicity and participate in local ATP production.     

Herpes simplex virus type 1 DNA is immunostimulatory in vitro and in vivo

Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically immunostimulatory both in vitro and in vivo, tending to promote Th1-like responses. In contrast, CpG dinucleotides in mammalian DNAs are extensively methylated on cytosines and hence immunologically inert. Since the herpes simplex virus (HSV) genome is unmethylated and G+C rich, we predicted that CpG motifs would be highly prevalent in the HSV genome; hence, we examined the immunostimulatory potential of purified HSV DNA in vitro and in vivo. Mouse splenocyte cultures treated with HSV DNA or HSV-derived oligodeoxyribonucleotides (ODNs) showed strong proliferative responses and production of inflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor [TNF], and interleukin-6 [IL-6]) in vitro, whereas splenocytes treated with mammalian CV-1 DNA or non-CpG ODN did not. After immunization with ovalbumin (OVA), only splenocytes from mice immunized with HSV DNA or HSV-ODN as the adjuvants proliferated strongly and produced typical Th1 responses, including CD8⁺ cytotoxic T-lymphocyte responses, upon restimulation with OVA. Furthermore, HSV-ODN synergized with IFN-γ to induce nitric oxide (NO), IL-6, and TNF production from macrophages. These results demonstrate that HSV DNA and HSV-ODN are immunostimulatory, driving potent Th1 responses both in vitro and in vivo. Considering that HSV DNA has been found to persist in nonneuronal cells, these results fuel speculation that HSV DNA might play a role in pathogenesis, in particular, in diseases like herpes stromal keratitis (HSK) that involve chronic inflammatory responses in the absence of virus or viral antigens.     

In vitro and in vivo characterization of a murine cytomegalovirus with a mutation at open reading frame m166

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the mutants, Rvm166, which contained the transposon sequence at open reading frame m166, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. The viral mutant replicated as well as the wild-type Smith strain in vitro in NIH 3T3 cells, whereas the transposon insertion precluded the expression of >65% of the m166 open reading frame. Compared to the wild-type strain and a rescued virus that restored the m166 region, the viral mutant was significantly attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. At 21 days postinfection, the titers of the viral mutant in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice were lower than the titers of the Smith strain and the rescued virus by about 30000-, 10000-, 1000-, 300-, and 800-fold, respectively. Moreover, the virulence of the mutant virus appears to be severely attenuated because no death was found in SCID mice infected with the viral mutant up to 90 days postinfection, whereas all of the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results suggest that m166 probably encodes a virulence factor and is required for MCMV virulence in killing SCID mice and for optimal viral growth in vivo.