稳定表达人TMPRSS2蛋白质悬浮生长MDCK细胞系的构建

为了构建能稳定表达人Ⅱ型跨膜型丝氨酸蛋白酶(TMPRSS2)的重组MDCK细胞,并考察该重组细胞株对禽流感H9亚型病毒的增殖能力,采用基因工程的方法,将TMPRSS2基因克隆入真核表达载体中获得重组表达载体p IRES-TMPRSS2。采用25 000线性PEI进行悬浮生长MDCK细胞的转染操作,经两轮G418抗性筛选获得能够稳定表达该蛋白质的重组MDCK-Sus-TMPRSS2细胞。RT-PCR、Western blot以及间接免疫荧光检测(IFA)的结果显示,TMPRSS2已在该重组细胞株中正常表达。该重组细胞的悬浮比生长速率达到0.438 d-1;在3 L反应器中,最大细胞密度可达到1 ml6.83×106个。在对6株禽流感H9亚型病毒的连续盲传试验中,经MDCK-Sus-TMPRSS2细胞盲传的子代病毒可以获得较高的血凝效价和半数组织感染滴度。     

The centromeric protein Sgo1 is required to sense lack of tension on mitotic chromosomes

Chromosome alignment on the mitotic spindle is monitored by the spindle checkpoint. We identify Sgo1, a protein involved in meiotic chromosome cohesion, as a spindle checkpoint component. Budding yeast cells with mutations in SGO1 respond normally to microtubule depolymerization but not to lack of tension at the kinetochore, and they have difficulty attaching sister chromatids to opposite poles of the spindle. Sgo1 is thus required for sensing tension between sister chromatids during mitosis, and its degradation when they separate may prevent cell cycle arrest and chromosome loss in anaphase, a time when sister chromatids are no longer under tension.     

A heme export protein is required for red blood cell differentiation and iron homeostasis

Hemoproteins are critical for the function and integrity of aerobic cells. However, free heme is toxic. Therefore, cells must balance heme synthesis with its use. We previously demonstrated that the feline leukemia virus, subgroup C, receptor (FLVCR) exports cytoplasmic heme. Here, we show that FLVCR-null mice lack definitive erythropoiesis, have craniofacial and limb deformities resembling those of patients with Diamond-Blackfan anemia, and die in midgestation. Mice with FLVCR that is deleted neonatally develop a severe macrocytic anemia with proerythroblast maturation arrest, which suggests that erythroid precursors export excess heme to ensure survival. We further demonstrate that FLVCR mediates heme export from macrophages that ingest senescent red cells and regulates hepatic iron. Thus, the trafficking of heme, and not just elemental iron, facilitates erythropoiesis and systemic iron balance.     

The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide

The production of nitric oxide and other reactive nitrogen intermediates (RNI) by macrophages helps to control infection by Mycobacterium tuberculosis (Mtb). However, the protection is imperfect and infection persists. To identify genes that Mtb requires to resist RNI, we screened 10,100 Mtb transposon mutants for hypersusceptibility to acidified nitrite. We found 12 mutants with insertions in seven genes representing six pathways, including the repair of DNA (uvrB) and the synthesis of a flavin cofactor (fbiC). Five mutants had insertions in proteasome-associated genes. An Mtb mutant deficient in a presumptive proteasomal adenosine triphosphatase was attenuated in mice, and exposure to proteasomal protease inhibitors markedly sensitized wild-type Mtb to RNI. Thus, the mycobacterial proteasome serves as a defense against oxidative or nitrosative stress.     

The three-dimensional structure of Asn102 mutant of trypsin: role of Asp102 in serine protease catalysis

The structure of the Asn102 mutant of trypsin was determined in order to distinguish whether the reduced activity of the mutant at neutral pH results from an altered active site conformation or from an inability to stabilize a positive charge on the active site histidine. The active site structure of the Asn102 mutant of trypsin is identical to the native enzyme with respect to the specificity pocket, the oxyanion hole, and the orientation of the nucleophilic serine. The observed decrease in rate results from the loss of nucleophilicity of the active site serine. This decreased nucleophilicity may result from stabilization of a His57 tautomer that is unable to accept the serine hydroxyl proton.     

The protein kinase domain of the ANP receptor is required for signaling

A plasma membrane form of guanylate cyclase is a cell surface receptor for atrial natriuretic peptide (ANP). In response to ANP binding, the receptor-enzyme produces increased amounts of the second messenger, guanosine 3',5'-monophosphate. Maximal activation of the cyclase requires the presence of adenosine 5'-triphosphate (ATP) or nonhydrolyzable ATP analogs. The intracellular region of the receptor contains at least two domains with homology to other proteins, one possessing sequence similarity to protein kinase catalytic domains, the other to regions of unknown function in a cytoplasmic form of guanylate cyclase and in adenylate cyclase. It is now shown that the protein kinase-like domain functions as a regulatory element and that the second domain possesses catalytic activity. When the kinase-like domain was removed by deletion mutagenesis, the resulting ANP receptor retained guanylate cyclase activity, but this activity was independent of ANP and its stimulation by ATP was markedly reduced. A model for signal transduction is suggested in which binding of ANP to the extracellular domain of its receptor initiates a conformational change in the protein kinase-like domain, resulting in derepression of guanylate cyclase activity.     

Juvenile hormone is required to couple imaginal disc formation with nutrition in insects

In starved larvae of the tobacco hornworm moth Manduca sexta, larval and imaginal tissues stop growing, the former because they lack nutrient-dependent signals but the latter because of suppression by juvenile hormone. Without juvenile hormone, imaginal discs form and grow despite severe starvation. This hormone inhibits the intrinsic signaling needed for disc morphogenesis and does so independently of ecdysteroid action. Starvation and juvenile hormone treatments allowed the separation of intrinsic and nutrient-dependent aspects of disc growth and showed that both aspects must occur during the early phases of disc morphogenesis to ensure normal growth leading to typical-sized adults.     

Trim28 is required for epigenetic stability during mouse oocyte to embryo transition

Phenotypic variability in genetic disease is usually attributed to genetic background variation or environmental influence. Here, we show that deletion of a single gene, Trim28 (Kap1 or Tif1β), from the maternal germ line alone, on an otherwise identical genetic background, results in severe phenotypic and epigenetic variability that leads to embryonic lethality. We identify early and minute epigenetic variations in blastomeres of the preimplantation embryo of these animals, suggesting that the embryonic lethality may result from the misregulation of genomic imprinting in mice lacking maternal Trim28. Our results reveal the long-range effects of a maternal gene deletion on epigenetic memory and illustrate the delicate equilibrium of maternal and zygotic factors during nuclear reprogramming.     

The sor gene of HIV-1 is required for efficient virus transmission in vitro

The genome of the human immunodeficiency virus HIV-1 contains at least eight genes, of which three (sor, R, and 3' orf) have no known function. In this study, the role of the sor gene was examined by constructing a series of proviral genomes of HIV-1 that either lacked the coding sequences for sor or contained point mutations in sor. Analysis of four such mutants revealed that although each clone could generate morphologically normal virus particles upon transfection, the mutant viruses were limited in their capacity to establish stable infection. Virus derived from transfection of Cos-1 cells (OKT4-) with sor mutant proviral DNA's was resistant to transmission to OKT4+ "susceptible" cells under cell-free conditions, and was transmitted poorly by coculture. In contrast, virus derived from clones with an intact sor frame was readily propagated by either approach. Normal amounts of gag-, env-, and pol-derived proteins were produced by all four mutants and assays in both lymphoid and nonlymphoid cells indicated that their trans-activating capacity was intact and comparable with wild type. Thus the sor gene, although not absolutely required in HIV virion formation, influences virus transmission in vitro and is crucial in the efficient generation of infectious virus. The data also suggest that sor influences virus replication at a novel, post-translational stage and that its action is independent of the regulatory genes tat and trs.     

The social sense: susceptibility to others' beliefs in human infants and adults

Human social interactions crucially depend on the ability to represent other agents' beliefs even when these contradict our own beliefs, leading to the potentially complex problem of simultaneously holding two conflicting representations in mind. Here, we show that adults and 7-month-olds automatically encode others' beliefs, and that, surprisingly, others' beliefs have similar effects as the participants' own beliefs. In a visual object detection task, participants' beliefs and the beliefs of an agent (whose beliefs were irrelevant to performing the task) both modulated adults' reaction times and infants' looking times. Moreover, the agent's beliefs influenced participants' behavior even after the agent had left the scene, suggesting that participants computed the agent's beliefs online and sustained them, possibly for future predictions about the agent's behavior. Hence, the mere presence of an agent automatically triggers powerful processes of belief computation that may be part of a "social sense" crucial to human societies.     

GRK2-dependent S1PR1 desensitization is required for lymphocytes to overcome their attraction to blood

Lymphocytes egress from lymphoid organs in response to sphingosine-1-phosphate (S1P); minutes later they migrate from blood into tissue against the S1P gradient. The mechanisms facilitating cell movement against the gradient have not been defined. Here, we show that heterotrimeric guanine nucleotide-binding protein-coupled receptor kinase-2 (GRK2) functions in down-regulation of S1P receptor-1 (S1PR1) on blood-exposed lymphocytes. T and B cell movement from blood into lymph nodes is reduced in the absence of GRK2 but is restored in S1P-deficient mice. In the spleen, B cell movement between the blood-rich marginal zone and follicles is disrupted by GRK2 deficiency and by mutation of an S1PR1 desensitization motif. Moreover, delivery of systemic antigen into follicles is impaired. Thus, GRK2-dependent S1PR1 desensitization allows lymphocytes to escape circulatory fluids and migrate into lymphoid tissues.     

The Southern Ocean biological response to aeolian iron deposition

Biogeochemical rate processes in the Southern Ocean have an important impact on the global environment. Here, we summarize an extensive set of published and new data that establishes the pattern of gross primary production and net community production over large areas of the Southern Ocean. We compare these rates with model estimates of dissolved iron that is added to surface waters by aerosols. This comparison shows that net community production, which is comparable to export production, is proportional to modeled input of soluble iron in aerosols. Our results strengthen the evidence that the addition of aerosol iron fertilizes export production in the Southern Ocean. The data also show that aerosol iron input particularly enhances gross primary production over the large area of the Southern Ocean downwind of dry continental areas.     

Human hair growth deficiency is linked to a genetic defect in the phospholipase gene LIPH

The molecular mechanisms controlling human hair growth and scalp hair loss are poorly understood. By screening about 350,000 individuals in two populations from the Volga-Ural region of Russia, we identified a gene mutation in families who show an inherited form of hair loss and a hair growth defect. Affected individuals were homozygous for a deletion in the LIPH gene on chromosome 3q27, caused by short interspersed nuclear element-retrotransposon-mediated recombination. The LIPH gene is expressed in hair follicles and encodes a phospholipase called lipase H (alternatively known as membrane-associated phosphatidic acid-selective phospholipase A1alpha), an enzyme that regulates the production of bioactive lipids. These results suggest that lipase H participates in hair growth and development.     

Cytoplasmic partitioning of P granule components is not required to specify the germline in C. elegans

Asymmetric segregation of P granules during the first four divisions of the Caenorhabditis elegans embryo is a classic example of cytoplasmic partitioning of germline determinants. It is thought that asymmetric partitioning of P granule components during mitosis is essential to distinguish germline from soma. We have identified a mutant (pptr-1) in which P granules become unstable during mitosis and P granule proteins and RNAs are distributed equally to somatic and germline blastomeres. Despite symmetric partitioning of P granule components, pptr-1 mutants segregate a germline that uniquely expresses P granules during postembryonic development. pptr-1 mutants are fertile, except at high temperatures. Hence, asymmetric partitioning of maternal P granules is not essential to specify germ cell fate. Instead, it may serve to protect the nascent germline from stress.     

The new effector AbSCP1 of foliar nematode (Aphelenchoides besseyi) is required for parasitism rice

Plant parasitic nematodes secrete effector proteins to parasitize hosts successfully. Of these proteins, serine carboxypeptidases have critical roles in pathogenicity. This study investigated the role of new effector AbSCP1 in Aphelenchoides besseyi pathogenicity. In situ hybridization and qRT-PCR analyses indicated that AbSCP1 was exclusively expressed in the esophageal glands and upregulated in juveniles. Subcellular localization assays indicated that the protein was expressed in the nucleus. The ability to hydrolyze C-terminal amino acid residues was proven for AbSCP1. Moreover, RNAi significantly reduced the expression of AbSCP1 and RNAi-treated nematodes’ reproductive potential. Pathogenicity assays on rice showed that RNAi-treated nematodes were less pathogenic than the untreated control groups. These results suggest the important role of AbSCP1 in the A. besseyi infection process.     

全自动换血治疗G-6-PD缺陷症并发急性溶血的疗效观察

目的 :观察全自动换血治疗危重G 6 PD缺陷症并发急性溶血的疗效。方法 :对 1 0例危重G 6 PD缺陷症并发急性溶血采用全自动换血治疗。结果 :总胆红素由换血前 (5 33.1 4± 1 1 5 .6 5 ) μmol/L下降至换血后 (2 95 .5 0± 88.5 1 )μmol/L ,换出率是 4 4 .5 7% ,血红蛋白增加 34.1 2 % ,红细胞增加 4 3.30 % ,换血过程无明显不良反应。结论 :全自动换血是抢救危重G 6 PD缺陷症并发急性溶血的简单、实用、安全、有效的方法