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101.
Santa H Kammonen J Lehtonen O Karimäki J Pastinen O Leisola M Turunen O 《Biochimica et biophysica acta》2005,1749(1):65-73
We used molecular dynamics simulations to study how a non-natural substrate, L-ribose, interacts with the active site of Actinoplanes missouriensis xylose isomerase. The simulations showed that L-ribose does not stay liganded in the active site in the same way as D-xylose, in which the oxygens O2 and O4 are liganded to the metal M1. The oxygen O4 of L-ribose moved away from the metal M1 to an upside down position. Furthermore, the distances of the carbons C1 and C2 of L-ribose to the catalytic metal M2 were higher than in the case of D-xylose. These findings explain the extremely low reaction rate of xylose isomerase with L-ribose. The mutation V135N close to the C5-OH of the substrate increased the reaction efficiency 2- to 4-fold with L-ribose. V135N did not affect the reaction with D-xylose and L-arabinose, whereas the reaction with D-glucose was impaired, probably due to a hydrogen bond between Asn-135 and the substrate. When L-ribose was the substrate, Asn-135 formed a hydrogen bond to Glu-181. As a consequence, O4 of L-ribose stayed liganded to the metal M1 in the V135N mutant in molecular dynamics simulations. This explains the decreased K(m) of the V135N mutant with L-ribose. 相似文献
102.
Vitikainen M Lappalainen I Seppala R Antelmann H Boer H Taira S Savilahti H Hecker M Vihinen M Sarvas M Kontinen VP 《The Journal of biological chemistry》2004,279(18):19302-19314
The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains. 相似文献
103.
In the present study, we describe a novel mouse model for inducible germ cell ablation. The mice express herpes simplex virus thymidine kinase (HSV-TK) under the inhibin-alpha subunit promoter (Inhalpha). When adult transgenic (TG) mice were treated with famciclovir (FCV) for 4 wk, their spermatogenesis was totally abolished, with only Sertoli cells and few spermatids remaining in the seminiferous tubules. However, testicular steroidogenesis was not affected. Shorter treatment periods allowed us to follow up the progression of germ cell death: After 3 days, spermatogonia and preleptotene spermatocytes were no longer present. After a 1-wk treatment, spermatogonia, preleptotene, and zygotene spermatocytes were missing and the amount of pachytene spermatocytes was decreased. After a 2-wk treatment, round and elongating spermatids were present. During the third week, round spermatids were lost and, finally, after a 4-wk treatment, only Sertoli cells and few spermatids were present. Interestingly, the transgene is detected in Leydig and Sertoli cells but not in spermatogonia. This suggests that FCV is phosphorylated in Sertoli cells, and thereafter, leaks to neighboring spermatogonia, apparently through cell-cell junctions present, enabling trafficking of phosphorylated FCV. Because of the many mitotic divisions they pass through, the spermatogonia are very sensitive to toxins interfering with DNA replication, while nondividing Sertoli cells are protected. Using transillumination-assisted microdissection of the seminiferous tubules, the gene-expression patterns analyzed corresponded closely to the histologically observed progression of cell death. Thus, the model offers a new tool for studies on germ cell-Sertoli cell interactions by accurate alteration of the germ cell composition in seminiferous tubules. 相似文献
104.
Mutka AL Lusa S Linder MD Jokitalo E Kopra O Jauhiainen M Ikonen E 《The Journal of biological chemistry》2004,279(47):48654-48662
Astrocytes secrete cholesterol in lipoprotein particles. Here we show that primary murine embryonic astrocytes secrete endogenously synthesized cholesterol but also the cholesterol precursors desmosterol and lathosterol. In astrocyte membranes, desmosterol and cholesterol were the predominant sterols. Astrocytes derived from Niemann-Pick type C lipidosis (NPC1-/-) mice displayed late endosomal cholesterol deposits, but the secretion of biosynthetic sterols from the cells was not inhibited. Both wild-type and NPC1-/- astrocytes secreted the NPC2 protein. Size-exclusion chromatography combined with electron microscopy showed that the majority of sterols were secreted separately from NPC2 in heterogeneous spherical particles with an average diameter of 20 nm. These data suggest that NPC2 and the majority of sterols secreted from astrocytes are not released together and that the secretion of neither sterols nor NPC2 requires NPC1 function. In addition, the findings reveal a complexity of sterol species in astrocytes and bring up the possibility that some of the effects assigned to astrocyte cholesterol may be attributed to its penultimate precursors. 相似文献
105.
106.
Teija Ojala Veera Kuparinen J. Patrik Koskinen Edward Alatalo Liisa Holm Petri Auvinen Sanna Edelman Benita Westerlund-Wikstr?m Timo K. Korhonen Lars Paulin Matti Kankainen 《Journal of bacteriology》2010,192(13):3547-3548
Lactobacillus crispatus is a common member of the beneficial microbiota present in the vertebrate gastrointestinal and human genitourinary tracts. Here, we report the genome sequence of L. crispatus ST1, a chicken isolate displaying strong adherence to vaginal epithelial cells.Lactobacillus crispatus can persist in the vertebrate gastrointestinal tract and is among the most prevalent species of the Lactobacillus-dominated human vaginal microbiota (2, 9, 13, 14). It belongs to the so-called acidophilus group (3), which has attracted interest because some of its species are important factors in the production of fermented foods (12) and some can, at least transiently, colonize the human host (2, 9, 13, 14). Moreover, some specific strains, mainly L. acidophilus NCFM and L. johnsonii NCC 533, have received prominence as intestinal-health-promoting microbes (4). Although the genomes of seven members of the acidophilus complex have been sequenced to date (12), the genome sequences of L. crispatus and other predominant lactobacillar species in the urogenital flora have mostly remained obscure. Vaginal lactobacilli can have an important role in controlling the health of the host (2, 14). They can, for example, positively influence and stabilize the host''s vaginal microbiota via the production of compounds that are acidic or exert a direct inhibiting action toward pathogenic bacteria (2, 14). In addition to the antimicrobial compounds, the competitive exclusion of pathogens is another mechanism by which the host''s microbiota can be balanced (2). L. crispatus ST1 was originally isolated from the crop of a chicken, and PCR profiling of L. crispatus isolates has verified it to be an abundant colonizer of the chicken crop (6, 8). It also displays a strong protein-dependent adhesion to the epithelial cells of the human vagina and has been shown to inhibit the adhesion of avian pathogenic Escherichia coli (6, 7).The genome was sequenced (18× coverage) using a 454 pyrosequencer with GS FLX chemistry (Roche). The contig order was confirmed and gaps were filled by sequencing PCR fragments from the genomic DNA template using ABI 3730 and Big Dye chemistry (Applied Biosystems). Genomic data were processed using the Staden Package (11) and gsAssembler (Roche). Coding sequences (CDSs) were predicted using Glimmer3 (5) followed by manual curation of the start sites. The remaining intergenic regions were reanalyzed for missed CDSs by using BlastX (1). Annotation transfer was performed based on a BlastP search, followed by Blannotator analysis using default settings (http://ekhidna.biocenter.helsinki.fi/poxo/blannotator) and manual verification. Orthologous groups between the different lactobacillar proteomes were identified using OrthoMCL (10).The genome of L. crispatus ST1 consists of a single circular chromosome 2.04 Mbp in size, with an overall G+C content of 37%, without any plasmids. There are 64 tRNA genes, 4 rRNA operons, and 2 CRISPR loci. Out of the 2,024 predicted CDSs, a putative function was assigned to 77%, whereas 10% of the CDSs were annotated as conserved and 13% as novel. Based on the orthologous grouping, 302 (15%) of the CDSs encoded by ST1 have no detectable homologs in any of the Lactobacillus proteomes published to date. 相似文献
107.
Lee-Rueckert M Vikstedt R Metso J Ehnholm C Kovanen PT Jauhiainen M 《Journal of lipid research》2006,47(8):1725-1732
In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study. 相似文献
108.
Wu GH Auvinen A Yen AM Hakama M Walter SD Chen HH 《Biometrical journal. Biometrische Zeitschrift》2012,54(1):20-44
To compare the survival between screen-detected and clinically detected cancers, we applied a series of non-homogeneous stochastic processes to deal with leadtime, length bias, and over-detection by using full information on detection modes obtained from the Finnish randomized controlled trial for prostate cancer screening. The results show after 9-year follow-up the hazard ratio of prostate cancer death for screen-detected cases against clinically detected cases increased from 0.24 (95% CI: 0.16-0.35) without correction for these biases, to 0.76 after correction for leadtime and length biases, and finally to 1.03 (95% CI: 0.79-1.33) for a further adjustment for over-detection. Adjustment for leadtime and length bias but no over-detection led to a 24% reduction in prostate cancer death as a result of prostate-specific antigen test. The further calibration of over-detection indicates no gain in survival of screen-detected prostate cancers (excluding over-detected case as stayer considered in the mover-stayer model) as compared with the control group in the absence of screening that is considered as the mover. However, whether the model assumption on over-detection is robust should be validated with other data sets and longer follow-up. 相似文献
109.
Nguyen SD Oörni K Lee-Rueckert M Pihlajamaa T Metso J Jauhiainen M Kovanen PT 《Journal of lipid research》2012,53(10):2115-2125
HDL particles may enter atherosclerotic lesions having an acidic intimal fluid. Therefore, we investigated whether acidic pH would affect their structural and functional properties. For this purpose, HDL(2) and HDL(3) subfractions were incubated for various periods of time at different pH values ranging from 5.5 to 7.5, after which their protein and lipid compositions, size, structure, and cholesterol efflux capacity were analyzed. Incubation of either subfraction at acidic pH induced unfolding of apolipoproteins, which was followed by release of lipid-poor apoA-I and ensuing fusion of the HDL particles. The acidic pH-modified HDL particles exhibited an enhanced ability to promote cholesterol efflux from cholesterol-laden primary human macrophages. Importantly, treatment of the acidic pH-modified HDL with the mast cell-derived protease chymase completely depleted the newly generated lipid-poor apoA-I, and prevented the acidic pH-dependent increase in cholesterol efflux. The above-found pH-dependent structural and functional changes were stronger in HDL(3) than in HDL(2). Spontaneous acidic pH-induced remodeling of mature spherical HDL particles increases HDL-induced cholesterol efflux from macrophage foam cells, and therefore may have atheroprotective effects. 相似文献
110.