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191.
Martin MD Wirth TC Lauer P Harty JT Badovinac VP 《Journal of immunology (Baltimore, Md. : 1950)》2011,187(6):2923-2931
One goal of immunization is to generate memory CD8 T cells of sufficient quality and quantity to confer protection against infection. It has been shown that memory CD8 T cell differentiation in vivo is controlled, at least in part, by the amount and duration of infection, Ag, and inflammatory cytokines present early after the initiation of the response. In this study, we used models of anti-vectorial immunity to investigate the impact of pre-existing immunity on the development and differentiation of vector-induced primary CD8 T cell responses. We showed that existing CD8 T cell memory influences the magnitude of naive CD8 T cell responses. However, the differentiation of newly recruited (either TCR-transgenic or endogenous) primary CD8 T cells into populations with the phenotype (CD62L(hi), CD27(hi), KLRG-1(low)) and function (tissue distribution, Ag-driven proliferation, cytokine production) of long-term memory was facilitated when they were primed in the presence of vector-specific memory CD8 T cells of the same or unrelated specificity. Therefore, these data suggested that the presence of anti-vectorial immunity impacts the rate of differentiation of vector-induced naive CD8 T cells, a notion with important implications for the design of future vaccination strategies. 相似文献
192.
Janelle L. Lauer Manishabrata Bhowmick Dorota Tokmina-Roszyk Yan Lin Steven R. Van Doren Gregg B. Fields 《The Journal of biological chemistry》2014,289(4):1981-1992
Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-l-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, kcat/Km values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P23–P23′ subsites of collagenous substrates. 相似文献
193.
Elizabeth Greene Daniela Cazacu Ninkka Tamot Steven Castellano Akshita Datar Anthony Kronkaitis Douglas Gebhard Jon Reed Paul Mawson Lore Florin Nicholas Rossi Anthony Lauer Laura Juckem Andrew Nixon Till Wenger Saurabh Sen 《Biotechnology journal》2021,16(4):2000251
Transient gene expression (TGE) using mammalian cells is an extensively used technology for the production of antibodies and recombinant proteins and has been widely adopted by both academic and industrial labs. Chinese Hamster Ovary (CHO) cells have become one of the major workhorses for TGE of recombinant antibodies due to their attractive features: post-translational modifications, adaptation to high cell densities, and use of serum-free media. In this study, we describe the optimization of parameters for TGE for antibodies from CHO cells. Through a matrix evaluation of multiple factors including inoculum, transfection conditions, amount and type of DNA used, and post-transfection culture conditions, we arrived at an uniquely optimized process with higher titer and reduced costs and time, thus increasing the overall efficiency of early antibody material supply. We further investigated the amount of coding DNA used in TGE and the influence of kinetics and size of the transfection complex on the in vitro efficiency of the transfection. We present here the first report of an optimized TGE platform using Filler DNA in an early drug discovery setting for the screening and production of therapeutic mAbs. 相似文献
194.
Morphometric variation in bluegill Lepomis macrochirus and green sunfish Lepomis cyanellus in lentic and lotic systems 下载免费PDF全文
Bluegill Lepomis macrochirus and green sunfish Lepomis cyanellus were examined using geometric morphometrics to evaluate the variation in morphology between fishes that reside in lentic (e.g. lakes) and lotic (e.g. streams) ecosystems. Live fishes were collected from reservoirs and rivers in central Indiana, while additional fishes were sampled from museum collections at Ball State University and the Illinois Natural History Survey. Male and female L. macrochirus and female L. cyanellus from lentic systems display a deeper body than those from lotic systems, while no differences were found in male L. cyanellus morphometry. A deeper body promotes greater manoeuverability, typically desirable in lentic systems. In contrast, the more streamlined body of the fishes found in lotic systems reduces drag as it contends with flowing water, ultimately maximizing energy efficiency. The absence of morphological differences, such as those found in male L. cyanellus, may be caused by fish occupying both lentic and lotic systems, from the population not having been present in the body of water long enough to display any adaptations, or from a lack of statistical power caused by the small sample size. 相似文献
195.
Characterization of initiation factor 3 from wheat germ. 1. Effects of proteolysis on activity and subunit composition 总被引:1,自引:0,他引:1
Wheat germ initiation factor 3 (eIF-3) is a large (15 S) particle containing 10 subunits with molecular weights ranging from 28 000 to 116 000. Two forms of wheat germ eIF-3 which differ in ability to support polypeptide synthesis in vitro have been obtained by chromatography on carboxymethyl-Sephadex (CM-Sephadex). The less active form is not retained on CM-Sephadex in 50 mM KCl and contains lower amounts of two subunits, the 116 000-dalton polypeptide (pp116) and the 36 000-dalton polypeptide (pp36). The more active form is retained on CM-Sephadex in 50 mM KCl and is eluted by 150 mM KCl. Treatment of the more active form with small amounts of trypsin results in a rapid degradation of four of the subunits (pp116, pp107, pp87, and pp36) and in a rapid loss in the ability to support polypeptide synthesis. Trypsin treatment also diminishes the ability of eIF-3 to support the binding of mRNA to 40S ribosomal subunits. These findings indicate that pp116, pp107, pp87, and pp36 are in exposed positions in the eIF-3 particle and that pp116 and/or pp36 are essential for activity. 相似文献
196.
T Lauer T Rassaf C Planitz R Preuss R Krause T Henle A Deussen 《Hormones et métabolisme》2008,40(4):233-238
Direct interaction between Maillard reaction products (MRPs) and nitric oxide (NO) has been suggested as a pathophysiological mechanism involved in enhanced diabetic arteriosclerosis. Only MRPs without structural characterization have been studied to date. Using chemically synthesized and analytically well defined individual MRPs, we investigated whether the native nitric oxide concentration is directly affected by the Amadori compound N-epsilon-fructosyllysine or the advanced glycation end product N-epsilon-carboxymethyllysine. MRPs were incubated with nitric oxide solution or NO donors (SNAP, spermine-NONOate). Changes in the nitrite (oxidative metabolite of NO) concentration served as indicator of NO availability. MRPs, either as free amino acids or covalently bound to bovine serum albumin (BSA), had no influence on nitrite concentration when using NO solution. In contrast, incubation of the respective NO donors with several covalently protein-bound MRPs as well as native BSA significantly reduced nitrite concentration. If SNAP was co-incubated with EDTA or with Fe (2+) ions, nitrite concentration was decreased or increased, respectively, suggesting a metal ion-dependent alteration of the NO liberation rate. Native NO concentration was not affected by the MRPs tested. Substitution of native NO by NO-releasing substances may be inadequate as a model of NO-MRP interaction, as metal ions or chelators present in compound preparations may affect the NO-liberating mechanism of the donor. 相似文献
197.
Roth J Minond D Darout E Liu Q Lauer J Hodder P Fields GB Roush WR 《Bioorganic & medicinal chemistry letters》2011,21(23):7180-7184
Matrix metalloproteinase-13 (MMP-13) has been implicated as the protease responsible for collagen degradation in cartilage during osteoarthritis (OA). Compounds that inhibit the metalloproteinase at the Zn binding site typically lack specificity among MMP family members. Analogs of the low-micromolar lead MMP-13 inhibitor 4, discovered through high-throughput screening, were synthesized to investigate structure-activity relationships in this inhibitor series. Systematic modifications of 4 led to the discovery of MMP-13 inhibitors 20 and 24 which are more selective than 4 against other MMPs. Compound 20 is also approximately fivefold more potent as an MMP-13 inhibitor than the original HTS-derived lead compound 4. 相似文献
198.
Carla Winkler Elena N Atochina-Vasserman Olaf Holz Michael F Beers Veit J Erpenbeck Norbert Krug Stefan Roepcke Gereon Lauer Martin Elmlinger Jens M Hohlfeld 《Respiratory research》2011,12(1):29
Background
Pulmonary surfactant protein D (SP-D) is considered as a candidate biomarker for the functional integrity of the lung and for disease progression, which can be detected in serum. The origin of SP-D in serum and how serum concentrations are related to pulmonary concentrations under inflammatory conditions is still unclear.Methods
In a cross-sectional study comprising non-smokers (n = 10), young - (n = 10), elderly smokers (n = 20), and smokers with COPD (n = 20) we simultaneously analysed pulmonary and serum SP-D levels with regard to pulmonary function, exercise, repeatability and its quaternary structure by native gel electrophoresis. Statistical comparisons were conducted by ANOVA and post-hoc testing for multiple comparisons; repeatability was assessed by Bland-Altman analysis.Results
In COPD, median (IQR) pulmonary SP-D levels were lower (129(68) ng/ml) compared to smokers (young: 299(190), elderly: 296(158) ng/ml; p < 0.01) and non-smokers (967(708) ng/ml; p < 0.001). The opposite was observed in serum, with higher concentrations in COPD (140(89) ng/ml) as compared to non-smokers (76(47) ng/ml; p < 0.01). SP-D levels were reproducible and correlated with the degree of airway obstruction in all smokers. In addition, smoking lead to disruption of the quaternary structure.Conclusions
Pulmonary and serum SP-D levels are stable markers influenced by smoking and related to airflow obstruction and disease state. Smaller subunits of pulmonary SP-D and the rapid increase of serum SP-D levels in COPD due to exercise support the translocation hypothesis and its use as a COPD biomarker.Trial registration
no interventional trial 相似文献199.
Listeria monocytogenes is a facultative intracellular bacterial pathogen that can infect the placenta, a chimeric organ made of maternal and fetal cells. Extravillous trophoblasts (EVT) are specialized fetal cells that invade the uterine implantation site, where they come into direct contact with maternal cells. We have shown previously that EVT are the preferred site of initial placental infection. In this report, we infected primary human EVT with L. monocytogenes. EVT eliminated ~80% of intracellular bacteria over 24-hours. Bacteria were unable to escape into the cytoplasm and remained confined to vacuolar compartments that became acidified and co-localized with LAMP1, consistent with bacterial degradation in lysosomes. In human placental organ cultures bacterial vacuolar escape rates differed between specific trophoblast subpopulations. The most invasive EVT-those that would be in direct contact with maternal cells in vivo-had lower escape rates than trophoblasts that were surrounded by fetal cells and tissues. Our results suggest that EVT present a bottleneck in the spread of L. monocytogenes from mother to fetus by inhibiting vacuolar escape, and thus intracellular bacterial growth. However, if L. monocytogenes is able to spread beyond EVT it can find a more hospitable environment. Our results elucidate a novel aspect of the maternal-fetal barrier. 相似文献
200.
Gentschev I Müller M Adelfinger M Weibel S Grummt F Zimmermann M Bitzer M Heisig M Zhang Q Yu YA Chen NG Stritzker J Lauer UM Szalay AA 《PloS one》2011,6(7):e22069
Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man. 相似文献