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951.
The anaerobic veratrol O-demethylase mediates the transfer of the methyl group of the phenyl methyl ether veratrol to tetrahydrofolate. The primary
methyl group acceptor is the cobalt of a corrinoid protein, which has to be in the +1 oxidation state to bind the methyl group.
Due to the negative redox potential of the cob(II)/cob(I)alamin couple, autoxidation of the cobalt may accidentally occur.
In this study, the reduction of the corrinoid to the superreduced [CoI] state was investigated. The ATP-dependent reduction of the corrinoid protein of the veratrol O-demethylase was shown to be dependent on titanium(III) citrate as electron donor and on an activating enzyme. In the presence
of ATP, activating enzyme, and Ti(III), the redox potential versus the standard hydrogen electrode (E
SHE) of the cob(II)alamin/cob(I)alamin couple in the corrinoid protein was determined to be −290 mV (pH 7.5), whereas E
SHE at pH 7.5 was lower than −450 mV in the absence of either activating enzyme or ATP. ADP, AMP, or GTP could not replace ATP
in the activation reaction. The ATP analogue adenosine-5′-(β,γ-imido)triphosphate (AMP-PNP, 2–4 mM) completely inhibited the
corrinoid reduction in the presence of ATP (2 mM). 相似文献
952.
We investigated the effects of the interaction between flavanols and related procyanidins (dimer to hexamer) with both cell and synthetic membranes, on bilayer fluidity and susceptibility to oxidation. Cocoa derived dimers (0.05 to 1 microg/ml) protected Jurkat T cells from AMVN-mediated oxidation and increased plasma membrane fluidity. These effects occurred in a concentration- and chain length-dependent manner. In liposomes, procyanidins prevented the Fe2+ -induced permeabilization of the membrane. Together, these results support the hypothesis that procyanidins could interact with the polar headgroup of lipids, increasing membrane fluidity and also, preventing the access of molecules that could affect membrane integrity. 相似文献
953.
Dominic Eberle Tiago Santos-Ferreira Sandra Grahl Marius Ader 《Journal of visualized experiments : JoVE》2014,(84)
Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions. 相似文献
954.
Bearzatto M Naserke H Piquer S Koczwara K Lampasona V Williams A Christie MR Bingley PJ Ziegler AG Bonifacio E 《Journal of immunology (Baltimore, Md. : 1950)》2002,168(8):4202-4208
The related tyrosine phosphatase-like proteins islet Ag (IA)-2 and IA-2beta are autoantigens of type 1 diabetes in humans. Autoantibodies are predominantly against IA-2, and IA-2-specific epitopes are major autoantibody targets. We used the close homology of IA-2 and IA-2beta to design chimeras and mutants to identify humoral IA-2-specific epitopes. Two major IA-2 epitopes that are absent from the related autoantigens IA-2beta and IA-2Delta 13 splice variant ICA512.bdc were found contiguous to each other within IA-2 juxtamembrane amino acids 611-620 (epitope JM1) and 621-630 (epitope JM2). JM1 and JM2 are recognized by sera from 67% of patients with IA-2 Abs, and relatives of patients with type 1 diabetes having Abs to either JM epitope had a >50% risk for developing type 1 diabetes within 6 years, even in the absence of diabetes-associated HLA genotypes. Remarkably, the presence of Abs to one of these two epitopes was mutually exclusive of the other; JM2 Abs and not JM1 Abs were found in relatives with HLA DR3/4, DR4/13, or DR1/4 genotypes; and the binding of autoantibodies to the JM2 epitope, but not the JM1 epitope, markedly affected proteolysis of IA-2. This is a unique demonstration of HLA-associated B cell responses to epitopes within a single autoantigen in humans and is consistent with modification of Ag processing by specific Ab-influencing peptide presentation by HLA molecules. 相似文献
955.
The A167Y mutation converts the herpes simplex virus type 1 thymidine kinase into a guanosine analogue kinase 总被引:1,自引:0,他引:1
The thymidine (dThd) kinase (TK) encoded by herpes simplex virus type 1 (HSV-1) is not only endowed with dThd kinase, but also with thymidylate (dTMP) kinase and 2'-deoxycytidine (dCyd) kinase (dCK) activity. HSV-1 TK also recognizes a variety of antiherpetic guanine nucleoside analogues such as acyclovir (ACV), ganciclovir (GCV), lobucavir (LBV), penciclovir (PCV), and others (i.e., A5021). Site-directed mutagenesis of the highly conserved Ala-167 to Tyr in HSV-1 TK completely abolished TK, dTMP-K, and dCK activity, but maintained ACV-, GCV-, LBV-, PCV-, and A5021-phosphorylating capacity. A variety of 5-substituted pyrimidine nucleoside substrates, but also a number of selective HSV-1 TK inhibitors structurally related to thymine lost significant binding affinity for the mutant enzyme and did not markedly compete with GCV phosphorylation by the mutant enzyme. These findings could be explained by computer-assisted modeling data that revealed steric hindrance of the pyrimidine ring in the HSV-1 TK active site by the large 4-hydroxybenzyl ring of 167-Tyr, while the positioning of the purine ring of guanine-based HIV-1 TK substrates in the active site was kept virtually unaltered. Surprisingly, the efficiency of conversion the antiherpetic 2'-deoxyguanosine analogues ACV, GCV, LBV, PCV, and A5021 to their phosphorylated forms by the A167Y mutant HSV-1 TK was far more pronounced than for the wild-type enzyme. Therefore, the single A167Y mutation converts the wild-type HSV-1 TK from a predominantly pyrimidine nucleos(t)ide kinase into a virtually exclusive purine (guanine) nucleoside analogue kinase. 相似文献
956.
Robert Blackburn Sandra Galoforo Christine M. Berns Mark Ireland Joong M. Cho Peter M. Corry Yong J. Lee 《Molecular and cellular biochemistry》1996,155(1):51-60
We investigated the role of B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10–1 isosurvival was 1.7. Expression of B-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected B-crystallin can contribute to increased thermoresistance. 相似文献
957.
Thyroid peroxidase (TPO) autoantibody epitopes are largely restricted to an immunodominant region (IDR) on the extracellular region of the native molecule. Localization of the IDR has been a longstanding and difficult goal. The TPO extracellular region comprises a large myeloperoxidase-like domain, linked to the plasma membrane by two smaller domains with homology to complement control protein (CCP) and epidermal growth factor (EGF), respectively. Recent studies have focused on the CCP- and EGF-like domains as the putative location of the TPO autoantibody IDR. To address this issue, we attempted to express on the surface of transfected cells native TPO in which the CCP- and EGF-like domains were deleted, either together or individually. We used a quartet of human monoclonal autoantibodies that define the TPO IDR, as well as polyclonal TPO autoantibodies in patients' sera, to detect these mutated TPO molecules by flow cytometry. The combined CCP/EGF-like domain deletion did not produce a signal with TPO autoantibodies but did not traffic to the cell surface. In contrast, both monoclonal and polyclonal autoantibodies recognized TPO with the juxtamembrane EGF-like domain deleted equally as well as the wild-type TPO on the cell surface. TPO with the CCP-like domain deleted expressed normally on the cell surface, as determined using the polyclonal mouse antiserum. Nevertheless, this modified TPO molecule was recognized very poorly by both the human monoclonal autoantibodies and the polyclonal autoantibodies in patients' sera. In conclusion, we have clearly excluded the juxtamembrane EGF-like domain as being part of the IDR. In contrast, a component of the CCP-like domain does contribute to the IDR. These data, together with findings from other studies, localize the TPO autoantibody IDR to the junction of the CCP-like domain and the much larger myeloperoxidase-like domain on TPO. 相似文献
958.
Robert Y. Kanterman Christian C. Felder Douglas E. Brenneman Alice L. Ma Sandra Fitzgerald Julius Axelrod 《Journal of neurochemistry》1990,54(4):1225-1232
The alpha 1-adrenergic receptor has been shown to mediate the release of arachidonic acid in FRTL5 thyroid cells and MDCK kidney cells. In primary cultures of spinal cord cells, norepinephrine stimulated release of arachidonic acid (from neurons only) and turnover of inositol phospholipids (from neurons and glia) via alpha 1-adrenergic receptors. These two responses were dissociated by treatment with phorbol ester and pertussis toxin, which inhibited production of inositol phosphates with no appreciable effect on release of arachidonic acid. Extracellular calcium was required for release of arachidonic acid, but not for production of inositol phosphates. The calcium channel blockers nifedipine and verapamil inhibited release of arachidonic acid only. However, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a compound that blocks intracellular calcium release, diminished production of inositol phosphates, but had little effect on release of arachidonic acid. These results suggest that alpha 1-adrenergic receptors couple to release of arachidonic acid in primary cultures of spinal cord cells by a mechanism independent of activation of phospholipase C, possibly via the activation of phospholipase A2. 相似文献
959.
A Splice-Isoform of Vesicle-associated Membrane Protein-1
(VAMP-1) Contains a Mitochondrial Targeting Signal 总被引:6,自引:1,他引:6 下载免费PDF全文
Sandra Isenmann Yeesim Khew-Goodall Jennifer Gamble Mathew Vadas Binks W. Wattenberg 《Molecular biology of the cell》1998,9(7):1649-1660
Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell. 相似文献
960.
Shmulewitz D Auerbach SB Lehner T Blundell ML Winick JD Youngman LD Skilling V Heath SC Ott J Stoffel M Breslow JL Friedman JM 《Human heredity》2001,51(1-2):8-19
OBJECTIVES: Obesity, type II diabetes, hypertension, and dyslipidemia are major causes of morbidity and mortality throughout the world. Though these disorders often cluster in individuals and families and are collectively known as syndrome X, the basis for this aggregation is not well understood. To further understand the pathogenesis of syndrome X, a comprehensive epidemiological study was undertaken on the Pacific Island of Kosrae, Federated States of Micronesia (FSM). METHODS: The entire adult (>20 years of age) population of Kosrae underwent a clinical evaluation that included a questionnaire that noted the participants' sex, family data including listing of biological parents, siblings, and children, smoking status, village of residence, age and health status. The medical evaluation included: anthropometric measures (weight, height, waist, hip), serum chemistries (leptin, fasting blood sugar (FBS), insulin, total cholesterol (TC), triglycerides (TG), and apolipoproteins B and A-I (apo B and apo A-I) and blood pressure (BP) measurements. RESULTS: Obesity (BMI >/=35) was found in 24%, diabetes (FBS >/=126 or 2-hour oral glucose tolerance test >/=200) in 12%, hypertension (SBP >/=140 or DBP >/=90) in 17%, and dyslipidemia (TC >/=240 or TG >/=200 or apo B >/=120 or apo A-I =88) in 20% of the population. Significant covariate effects after multivariate analysis were as follows: sex affected the frequency of all four disorders, parity affected the frequency of dyslipidemia, smoking affected the frequency of obesity and diabetes, village of residence affected the frequency of obesity, hypertension, and dyslipidemia, and age affected the frequency of all four disorders. Factor analysis identified four independent factors that explained 73% of the total variance of the entire data set: factor 1 (weight, waist, leptin, insulin, and TG), factor 2 (TC, TG, apo B, apo A-I, and insulin), factor 3 (systolic and diastolic BP, FBS, waist and weight), and factor 4 (apo A-I, TG, leptin, and weight). CONCLUSIONS: This population-based study on the Island of Kosrae suggests that syndrome X is a composite of 4 independent factors: obesity with diabetes and hypertriglyceridemia, combined hyperlipidemia with diabetes, hypertension with obesity and diabetes, and increased HDL-low TG with thinness and high leptin. Further studies to identify the genetic components of these factors as well as the individual traits are under way. 相似文献