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61.
Hilpert K Wessner H Schneider-Mergener J Welfle K Misselwitz R Welfle H Hocke AC Hippenstiel S Höhne W 《The Journal of biological chemistry》2003,278(27):24986-24993
Studying protease/peptide inhibitor interactions is a useful tool for understanding molecular recognition in general and is particularly relevant for the rational design of inhibitors with therapeutic potential. An inhibitory peptide (PMTLEYR) derived from the third domain of turkey ovomucoid inhibitor and optimized for specific porcine pancreatic elastase inhibition was introduced into an inhibitor scaffold to increase the proteolytic stability of the peptide. The trypsin-specific squash inhibitor EETI II from Ecballium elaterium was chosen as the scaffold. The resulting hybrid inhibitor HEI-TOE I (hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor) shows a specificity and affinity to porcine pancreatic elastase similar to the free inhibitory peptide but with significantly higher proteolytic stability. Isothermal titration calorimetry revealed that elastase binding of HEI-TOE I occurs with a small unfavorable positive enthalpy contribution, a large favorable positive entropy change, and a large negative heat capacity change. In addition, the inhibitory peptide and the hybrid inhibitor HEI-TOE I protected endothelial cells against degradation following treatment with porcine pancreatic elastase. 相似文献
62.
Single nucleotide polymorphisms (SNPs) are becoming widely recognized as the new currency for gene mapping as increasing numbers are discovered. Here we outline a method for their rapid analysis based on an allele-specific polymerase chain reaction (PCR) which employs a competitive approach, whereby both allele-specific primers are present in the same reaction and carry different fluorescent labels. This procedure is simple and amenable to high-throughput genotyping using conventional automated sequencing equipment, and no post-PCR modifications are required. Verification of the procedure was carried out by comparison of results derived by this method with those from restriction enzyme digestion of the ALDH2 exon 12 functional polymorphism (Glu-487-Lys) in 109 individuals. Additionally, we have examined all combinations of nucleotide substitutions and shown them to be differentiated by this method. As proof of concept, several assays were combined and loaded on a single gel lane/capillary to substantially improve throughput. This was made possible by designing the PCR products to be of different lengths and no interference was observed between products differing in size by only six nucleotides. We outline a number of test assays for well-characterized SNPs in human candidate genes for behavioral disorders. 相似文献
63.
Jurevics H Largent C Hostettler J Sammond DW Matsushima GK Kleindienst A Toews AD Morell P 《Journal of neurochemistry》2002,82(1):126-136
Exposure of mice to the copper chelator, cuprizone, results in CNS demyelination. There is remyelination after removal of the metabolic insult. We present brain regional studies identifying corpus callosum as particularly severely affected; 65% of cerebroside is lost after 6 weeks of exposure. We examined recovery of cerebroside and ability to synthesize cerebroside and cholesterol following removal of the toxicant. The temporal pattern for concentration of myelin basic protein resembled that of cerebroside. We applied Affymetrix GeneChip technology to corpus callosum to identify temporal changes in levels of mRNAs during demyelination and remyelination. Genes coding for myelin structural components were greatly down-regulated during demyelination and up-regulated during remyelination. Genes related to microglia/macrophages appeared in a time-course (peaking at 6 weeks) correlating with phagocytosis of myelin and repair of lesions. mRNAs coding for many cytokines had peak expression at 4 weeks, compatible with intercellular signaling roles. Of interest were other genes with temporal patterns correlating with one of the three above patterns, but of function not obviously related to demyelination/remyelination. The ability to correlate gene expression with known pathophysiological events should help in elucidating further function of such genes as related to demyelination/remyelination. 相似文献
64.
We investigated in an aviary experiment the behavioral and hormonal responses of European starlings (Sturnus vulgaris) that were moved from a same sex group to an aviary containing either a nest box alone, a nest box and another male, or a nest box and a female. Luteinizing hormone (LH) and testosterone (T) levels increased significantly and independently of the situation, suggesting that nest boxes were the most important stimulus affecting the levels of these hormones. Some birds occupied more than two boxes (winners), and others a single or no box (losers). Levels of T increased less in males that did not acquire a nest box. However, the increase in LH was similar in all males after the test. Singing was positively correlated with T levels. Winners started singing earlier and sang more during a contest than losers. In the presence of females LH increased more in winners than in losers, while the increase in T was similar in both groups. In females, there was no increase in T but LH increased in the presence of males. Levels were higher in females paired with winners than in females paired with losers. Finally, winners advertised their nest boxes more frequently than losers. These results indicate that within a relatively short time frame levels of LH and T increase following the transfer from a flock to a territorial situation and can react independently from each other depending on reproductive circumstances. For males, the possession of a nest box and, for females, the qualities of the male seemed to be the most important factors stimulating reproduction. 相似文献
65.
Rosbottom A Scudamore CL von der Mark H Thornton EM Wright SH Miller HR 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(10):5689-5695
Mucosal mast cells (MMC) or their precursors migrate through the intestinal lamina propria to reside intraepithelially, where expression of mouse mast cell protease-1 indicates the mature phenotype. Alterations in expression of integrins that govern cell adhesion to the extracellular matrix may regulate this process. As the key cytokine mediating differentiation of mouse mast cell protease-1-expressing MMC homologues in vitro, TGF-beta1 was considered a likely candidate for regulation of the integrins that facilitate intraepithelial migration of MMC. Therefore, we examined adhesion of bone marrow-derived mast cells cultured with and without TGF-beta1 to laminin-1, fibronectin, and vitronectin along with expression of integrins likely to regulate this adhesion. Adhesion of PMA-stimulated cultured mast cells to laminin-1 increased from 5.3 +/- 3.6% (mean +/- SEM) in the absence of TGF-beta1 to 58.7 +/- 4.0% (p < 0.05) when cultured mast cells had differentiated into MMC homologues in the presence of TGF-beta1. Increased adhesion of MMC homologues to laminin-1 was also stimulated by FcepsilonRI cross-linking and the calcium ionophore A23187. Expression of the laminin-binding integrin alpha(7) by MMC homologues grown in the presence of TGF-beta1 was demonstrated by RT-PCR and flow cytometry, and preincubation of MMC homologues with the alpha(7)-neutralizing Ab 6A11 inhibited adhesion to laminin-1 by 98% (p < 0.05), demonstrating a novel role for this molecule in adhesion of a hemopoietic cell to laminin-1. 相似文献
66.
The motility patterns of the reticulorumen evoke mainly mixing of the ingesta. So far unknown, intrinsic neural circuits of the enteric nervous system are involved in the control of these motility patterns. The aim of the study was to characterize neurochemically sheep ruminal myenteric neurones, in particular the neural pathways innervating the ruminal muscle layers. Cell bodies within the myenteric plexus projecting to the longitudinal or circular muscle layer were retrogradely labelled by direct application of the fluorescent tracer 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) onto the circular or longitudinal muscle. The neurochemical code of myenteric neurones was identified by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). According to their neurochemical code, ruminal myenteric neurones were divided into three populations: ChAT/SP (68% of all myenteric neurones), NOS/VIP (26% of all myenteric neurones) and ChAT/- (5% of all myenteric neurones). Application of DiI onto the circular or longitudinal muscle revealed on average 64 or 44 labelled cell bodies in the myenteric plexus, respectively. DiI-labelled neurones expressed the code ChAT/SP or NOS/VIP. In the pathways to circular or longitudinal muscle, ChAT/SP-positive neurones outnumbered NOS/VIP-immunoreactive neurones by 5:1 and 2:1. Pathways to the circular or longitudinal muscle did not exhibit any pronounced polarized innervation patterns. This study demonstrated specific projections of myenteric neurones to the ruminal muscle. Neurones expressing the code ChAT/SP might function as excitatory muscle motor neurones, whereas NOS/VIP neurones are likely to act as inhibitory muscle motor neurones. 相似文献
67.
Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants 总被引:3,自引:1,他引:2 下载免费PDF全文
Martin Dominik Vollmer Helga Hoier Hans-Jürgen Hecht Ursula Schell Janosch Grning Adrian Goldman Michael Schlmann 《Applied microbiology》1998,64(9):3290-3299
Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other. 相似文献
68.
PCR Detection of Coxiella burnetii from Different Clinical Specimens, Especially Bovine Milk, on the Basis of DNA Preparation with a Silica Matrix 下载免费PDF全文
For PCR detection of Coxiella burnetii in various clinical specimens we developed a sample preparation method in which silica binding of DNA was used. This method was found to be fast, easily performed with large numbers of samples, and equally sensitive for all of the specimens tested (livers, spleens, placentas, heart valves, milk, blood). The DNA preparation method described here can also be used as an initial step in any PCR-based examination of specimens. The procedure was tested with more than 600 milk samples, which were taken from 21 cows that were seropositive for C. burnetii and reportedly had fertility problems (and therefore were suspected of shedding the agent through milk intermittently or continuously). Of the 21 cows tested, 6 were shedding C. burnetii through milk. Altogether, C. burnetii DNA was detected in 6% of the samples. There was no correlation between the shedding pattern and the serological results. 相似文献
69.
Catherin Niemann Volker Brinkmann Eva Spitzer Guido Hartmann Martin Sachs Helga Naundorf Walter Birchmeier 《The Journal of cell biology》1998,143(2):533-545
We have established a cell culture system that reproduces morphogenic processes in the developing mammary gland. EpH4 mouse mammary epithelial cells cultured in matrigel form branched tubules in the presence of hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the c-met tyrosine kinase receptor. In contrast, alveolar structures are formed in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. These distinct morphogenic responses can also be observed with selected human mammary carcinoma tissue in explant culture. HGF/SF-induced branching was abrogated by the PI3 kinase inhibitors wortmannin and . In contrast, neuregulin- induced alveolar morphogenesis was inhibited by the MAPK kinase inhibitor PD98059. The c-met–mediated response could also be evoked by transfection of a c-met specific substrate, Gab1, which can activate the PI3 kinase pathway. An activated hybrid receptor that contained the intracellular domain of c-erbB2 receptor suffices to induce alveolar morphogenesis, and was observed in the presence of tyrosine residues Y1028, Y1144, Y1201, and Y1226/27 in the substrate-binding domain of c-erbB2. Our data demonstrate that c-met and c-erbB2 signaling elicit distinct morphogenic programs in mammary epithelial cells: formation of branched tubules relies on a pathway involving PI3 kinase, whereas alveolar morphogenesis requires MAPK kinase. LY294002相似文献
70.
Karl Henrik Larsson Helga Grosse-Brauckmann Jean Keller 《Nordic Journal of Botany》1998,18(2):239-242
The new species Hyphoderma tibia (Aphyllophorales, Basidiomycotina) is described and illustrated using drawings and scanning electron microscope. 相似文献