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101.
Leibing A 《Culture, medicine and psychiatry》2008,32(2):177-193
When, in the 1980s, Alzheimer's disease became a disease of major public concern, 'personhood' also became an important, related topic of discussion. Those in caring professions (psychology, social work, etc.) and caregiver groups advocated for the 'person within' who was getting lost in a forgetful body and in a reductionist biomedical system. This essay aims to critically approach the dualism of this kind of argument by focusing on the moral positioning of claims such as personhood or biomedical reductionism. 相似文献
102.
Store-operated Ca(2+) entry is controlled by the interaction of stromal interaction molecules (STIMs) acting as endoplasmic reticulum ER Ca(2+) sensors with calcium release-activated calcium (CRAC) channels (CRACM1/2/3 or Orai1/2/3) in the plasma membrane. Here, we report structural requirements of STIM1-mediated activation of CRACM1 and CRACM3 using truncations, point mutations, and CRACM1/CRACM3 chimeras. In accordance with previous studies, truncating the N-terminal region of CRACM1 or CRACM3 revealed a 20-amino acid stretch close to the plasma membrane important for channel gating. Exchanging the N-terminal region of CRACM3 with that of CRACM1 (CRACM3-N(M1)) results in accelerated kinetics and enhanced current amplitudes. Conversely, transplanting the N-terminal region of CRACM3 into CRACM1 (CRACM1-N(M3)) leads to severely reduced store-operated currents. Highly conserved amino acids (K85 in CRACM1 and K60 in CRACM3) in the N-terminal region close to the first transmembrane domain are crucial for STIM1-dependent gating of CRAC channels. Single-point mutations of this residue (K85E and K60E) eliminate store-operated currents induced by inositol 1,4,5-trisphosphate and reduce store-independent gating by 2-aminoethoxydiphenyl borate. However, short fragments of these mutant channels are still able to communicate with the CRAC-activating domain of STIM1. Collectively, these findings identify a single amino acid in the N terminus of CRAC channels as a critical element for store-operated gating of CRAC channels. 相似文献
103.
Modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses 总被引:5,自引:0,他引:5 下载免费PDF全文
Worgall S Busch A Rivara M Bonnyay D Leopold PL Merritt R Hackett NR Rovelink PW Bruder JT Wickham TJ Kovesdi I Crystal RG 《Journal of virology》2004,78(5):2572-2580
Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing beta-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the beta-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing beta-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to beta-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-beta-galactosidase antibody levels following vector administration. However, cellular responses to beta-galactosidase were significantly enhanced, with the frequency of CD4(+) as well as the CD8(+) beta-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P < 0.01). Importantly, this enhanced cellular immune response of the AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing beta-galactosidase: BALB/c mice implanted with the CT26 syngeneic beta-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These data demonstrate that addition of an RGD motif to the Ad fiber knob increases the infectibility of DC and leads to enhanced cellular immune responses to the Ad-transferred transgene, suggesting that the RGD capsid modification may be useful in developing Ad-based vaccines. 相似文献
104.
Arcus VL Bäckbro K Roos A Daniel EL Baker EN 《The Journal of biological chemistry》2004,279(16):16471-16478
Genome sequencing projects have focused attention on the problem of discovering the functions of protein domains that are widely distributed throughout living species but which are, as yet, largely uncharacterized. One such example is the PIN domain, found in eukaryotes, bacteria, and Archaea, and with suggested roles in signaling, RNase editing, and/or nucleotide binding. The first reported crystal structure of a PIN domain (open reading frame PAE2754, derived from the crenarchaeon, Pyrobaculum aerophilum) has been determined to 2.5 A resolution and is presented here. Mapping conserved residues from a multiple sequence alignment onto the structure identifies a putative active site. The discovery of distant structural homology with several exonucleases, including T4 phage RNase H and flap endonuclease (FEN1), further suggests a likely function for PIN domains as Mg2+-dependent exonucleases, a hypothesis that we have confirmed in vitro. The tetrameric structure of PAE2754, with the active sites inside a tunnel, suggests a mechanism for selective cleavage of single-stranded overhangs or flap structures. These results indicate likely DNA or RNA editing roles for prokaryotic PIN domains, which are strikingly numerous in thermophiles, and in organisms such as Mycobacterium tuberculosis. They also support previous hypotheses that eukaryotic PIN domains participate in RNAi and nonsense-mediated RNA degradation. 相似文献
105.
Trudie Weatherford Deborah Chavez Kathleen M. Brasky Stanley M. Lemon Annette Martin Robert E. Lanford 《Journal of virology》2009,83(16):8062-8075
Approximately 3% of the world population is chronically infected with hepatitis C virus (HCV). GB virus B (GBV-B), a surrogate model for HCV, causes hepatitis in tamarins and is the virus phylogenetically most closely related to HCV. Previously we described a chimeric GBV-B containing an HCV insert from the 5′ noncoding region (NCR) that was adapted for efficient replication in tamarins (Saguinus species). We have also demonstrated that wild-type (WT) GBV-B rapidly adapts for efficient replication in a closely related species, the common marmoset (Callithrix jacchus). Here, we demonstrate that the chimeric virus failed to adapt during serial passage in marmosets. The chimeric virus was passaged four times through 24 marmosets. During passage, two marmoset phenotypes were observed: susceptible and partially resistant. Although appearing to adapt in a resistant animal during a prolonged and gradual increase in viremia, the chimeric GBV-B failed to replicate efficiently upon passage to a naïve marmoset. The resistance was specific to the chimeric virus, as the chimeric virus-resistant animals were susceptible to marmoset-adapted WT virus during rechallenge studies. Three isolates of the chimeric virus were sequenced, and 20 nucleotide changes were observed, including eight amino acid changes. Three unique changes were observed in the 5′ NCR chimeric insert, an area that is highly conserved in HCV. We speculate that the failure of the chimeric virus to adapt in marmosets might be due to a bottleneck that occurs at the time of infection of resistant animals, which may lead to a loss of fitness upon serial passage.Worldwide, approximately 170 million people are chronically infected with hepatitis C virus (HCV). The current approved therapy involves the combination of pegylated alpha interferon and ribavirin and has response rates for sustained viral clearance of 42% and 82% for genotypes 1 and 2/3, respectively (15, 29). However, a significant proportion of the population still develops serious disease as a consequence of HCV infection. HCV infection is the leading cause for liver transplantation in the United States (1, 50), and liver cancer due to HCV infection is one of the most rapidly increasing types of cancer in the United States (20).GB virus B (GBV-B) is a hepatotropic virus that causes hepatitis in tamarins and is the virus phylogenetically most closely related to HCV (33, 36, 44), and as such, GBV-B represents an important small-primate surrogate model for HCV infections. The history of the GB agent is complex and originates with the inoculation of tamarins with serum obtained from a surgeon with hepatitis (for a review, see reference 3); however there is little doubt that GBV-B is a tamarin virus, despite the fact that it has never been isolated from tamarins a second time. GBV-B has a very narrow host range for tamarins, marmosets, and other closely related New World monkeys (6, 23, 54). The GBV-B model overcomes a number of limitations encountered when working with HCV (3, 22). Due to the limited availability of tamarins, our lab and others (5, 16, 21, 23) initiated GBV-B studies in common marmosets (Callithrix jacchus), a small New World primate closely related to the tamarin (Saguinus sp.). The marmoset and tamarin represent less expensive, more readily available, and smaller animal models than the chimpanzee. While replication in marmosets is typically higher than what is observed in HCV-infected chimpanzees, reproducible infection profiles require some adaptation to this host (5, 54). Although robust replication of HCV in vitro is now possible using specific adapted strains of HCV (derivatives of the JFH1 and H77-S) and the Huh-7.5 cell line (4, 27, 52, 55, 56), the GBV-B primary hepatocyte culture system (2) may be more suitable for some studies, especially those involving specific aspects of the innate immune response and other viral host interactions.The organization of the GBV-B genome is very similar to that of HCV and the GBV-B polyprotein gene encodes 10 proteins analogous to the HCV proteins. The polyprotein of GBV-B has approximately 25 to 30% homology to that of HCV at the amino acid level (33), while the 5′ and 3′ noncoding regions (NCRs) are more divergent (7, 33, 40). The HCV and GBV-B 5′ NCRs are essential for both replication and translation. The structures are similar; however, GBV-B domain I is predicted to fold into two stem-loops (SL), compared to one SL in HCV, and the GBV-B 5′ NCR is longer due mainly to additional SL IIB and IIC that are not present in HCV (Fig. (Fig.1A)1A) (40). The 5′ NCR contains the internal ribosomal entry site (IRES), which can directly bind the 40S ribosomal subunit in order to initiate translation of the viral RNA (19, 38). cis RNA elements involved in RNA replication are also located in the HCV 5′ NCR (for a review, see reference 49). In GBV-B, 5′ NCR segments essential for genome replication have recently been identified (53).Open in a separate windowFIG. 1.(A) Predicted structures of the GBV-B (left) and HCV (right) 5′ NCRs. The scissors on GBV-B represent the sequence that was excised and replaced by the HCV insert, which is represented by scissors on the HCV 5′ NCR. The locations of mutations detected during sequencing are boxed: the first box identifies the deletion (ΔC) in a run of cytosines, and the second box identifies the polymorphisms present at two adjacent uracils (C, C/U). (Adapted from reference 40.) (B) Schematic of the GBV-B genome. During passage of GB/IIIHC in tamarins, nine mutations were identified in virus from the T2 serum used to inoculate M1, and these nine mutations remained fixed in all marmoset isolates sequenced. Amino acid changes are indicated by dark arrows, silent mutations are indicated by asterisks, and NCR changes are indicated by dotted arrows.The utility of the GBV-B model was increased by the development of infectious cDNA clones that induced hepatitis upon intrahepatic inoculation of tamarins with in vitro-transcribed RNA (7, 31, 45). In order to further increase the use of GBV-B as a model for HCV, chimeras between GBV-B and HCV were constructed (16, 43, 48). In one chimera, a portion of the GBV-B 5′ NCR containing domain III, which is within the IRES functional domain, was replaced by an analogous region of HCV (40-43). The chimeric GB/IIIHC retained IRES translational function and supported replication in tamarins (43). In this study, we examined the host range of this chimeric virus during serial passage in marmosets. We found that chimeric GBV-B failed to adapt during passage in marmosets. Marmosets infected with GB/IIIHC displayed variable phenotypes ranging from susceptible to resistant, which appear to be due to a polymorphism in the marmoset population that also affects wild-type (WT) GBV-B (54). The failure of chimeric virus to adapt to replication in marmosets with the resistant phenotype was specific to the chimeric virus, and not the WT, and may involve several factors, including reduced replication capacity and the requirement to acquire multiple adaptive mutations. These barriers cumulatively may result in GB/IIIHC experiencing a bottleneck in the resistant marmoset host. 相似文献
106.
Loosli F Del Bene F Quiring R Rembold M Martinez-Morales JR Carl M Grabher C Iquel C Krone A Wittbrodt B Winkler S Sasado T Morinaga C Suwa H Niwa K Henrich T Deguchi T Hirose Y Iwanami N Kunimatsu S Osakada M Watanabe T Yasuoka A Yoda H Winkler C Elmasri H Kondoh H Furutani-Seiki M Wittbrodt J 《Mechanisms of development》2004,121(7-8):703-714
In a large scale mutagenesis screen of Medaka we identified 60 recessive zygotic mutations that affect retina development. Based on the onset and type of phenotypic abnormalities, the mutants were grouped into five categories: the first includes 11 mutants that are affected in neural plate and optic vesicle formation. The second group comprises 15 mutants that are impaired in optic vesicle growth. The third group includes 18 mutants that are affected in optic cup development. The fourth group contains 13 mutants with defects in retinal differentiation. 12 of these have smaller eyes, whereas one mutation results in enlarged eyes. The fifth group consists of three mutants with defects in retinal pigmentation. The collection of mutants will be used to address the molecular genetic mechanisms underlying vertebrate eye formation. 相似文献
107.
Entry and transcription as key determinants of differences in CD4 T-cell permissiveness to human immunodeficiency virus type 1 infection 下载免费PDF全文
Ciuffi A Bleiber G Muñoz M Martinez R Loeuillet C Rehr M Fischer M Günthard HF Oxenius A Meylan P Bonhoeffer S Trono D Telenti A 《Journal of virology》2004,78(19):10747-10754
108.
Juana Obreo Luisa Díez-Marques Santiago Lamas Annette Düwell Nélida Eleno Carmelo Bernabéu Atanasio Pandiella José M López-Novoa Alicia Rodríguez-Barbero 《Cellular physiology and biochemistry》2004,14(4-6):301-310
BACKGROUND/AIMS: TGF-beta1 plays a major role in extracellular matrix (ECM) accumulation in tissue fibrosis. Connective tissue growth factor appears to play a critical role in this effect. Endoglin is a component of the transforming growth factor b (TGF-beta) receptor complex. Endoglin is upregulated by TGF-beta1, but its functional role in ECM regulation is unknown. Using rat myoblasts as a model system, we have assessed the role of endoglin on regulating CTGF expression and ECM synthesis and accumulation in the presence or absence of TGF-beta1. METHODS: L6E9 myoblast cell line was transfected with human endoglin, and collagen, fibronectin and CTGF production was assessed by Western blot and by proline incorporation to collagen proteins. RESULTS: Northern blot analysis revealed that parental rat myoblasts L6E9 do not express endogenous endoglin. Upon endoglin transfection, endoglin-expressing cells displayed a decreased CTGF expression and decreased collagen and fibronectin accumulation respect to mock transfectants. Northern blot analysis also revealed a decreased alpha2 (I) procollagen mRNA expression in endoglin transfectants. TGF-beta1 treatment induced an increase in CTGF expression and collagen synthesis and accumulation in L6E9 myoblasts. This effect was significantly lower in endoglin-transfected than in mock-transfected cells. CONCLUSION: These results demonstrate that endoglin expression negatively regulates basal and TGF-beta1-induced CTGF and collagen expression and synthesis. 相似文献
109.
110.
Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart 总被引:21,自引:0,他引:21
Martin CM Meeson AP Robertson SM Hawke TJ Richardson JA Bates S Goetsch SC Gallardo TD Garry DJ 《Developmental biology》2004,265(1):262-275
Stem cells are important in the maintenance and repair of adult tissues. A population of cells, termed side population (SP) cells, has stem cell characteristics as they have been shown to contribute to diverse lineages. In this study, we confirm that Abcg2 is a determinant of the SP cell phenotype. Therefore, we examined Abcg2 expression during murine embryogenesis and observed robust expression in the blood islands of the E8.5 yolk sac and in developing tissues including the heart. During the latter stages of embryogenesis, Abcg2 identifies a rare cell population in the developing organs. We further establish that the adult heart contains an Abcg2 expressing SP cell population and these progenitor cells are capable of proliferation and differentiation. We define the molecular signature of cardiac SP cells and compare it to embryonic stem cells and adult cardiomyocytes using emerging technologies. We propose that the cardiac SP cell population functions as a progenitor cell population for the development, maintenance, and repair of the heart. 相似文献