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1.
A. A. Chukalovsky T. V. Rakhimova K. S. Klopovsky N. A. Popov Yu. A. Mankelevich O. V. Proshina 《Plasma Physics Reports》2014,40(1):34-51
Available data on the kinetic processes in H2-O2-O2(a 1Δ g ) mixtures are analyzed theoretically, and the ranges in which the rate constants of these processes can vary are determined. The processes of energy transformation in an O2(a 1Δ g )-H2-H-HO2 system in the approximations of the fast and slow (in comparison with the vibrational relaxation time of the HO2 radical) quenching of the electronically excited state are considered. The experiments on the quenching of singlet delta oxygen (SDO) molecules O2(a 1Δ g ) excited in a microwave discharge at a temperature of 300 K and pressure of 6 Torr in a lean hydrogen-oxygen mixture are simulated (by a lean fuel mixture is meant a mixture in which the ratio of the fuel to the oxidizer mass fraction is less than the stoichiometric ratio, which is 2: 1 for a hydrogen-oxygen mixture). It is shown that, over the experimental observation times, the SDO quenching frequency depends on the concentration of molecular hydrogen, the residual odd oxygen fraction in the gas flow, and the ratio between the rate constants of kinetic processes involving HO2 and HO2* radicals. Simulations of the microwave discharge and the transport of excited oxygen along the drift tube make it possible to determine the residual odd oxygen concentration in the gas flow. Recommendations on the choice of the rate constants for the O2(a 1Δ g ) + HO2)A″, v3″ = 0) ? O2 + HO2*(A′, v3′ = 1), HO2*(A′v3′ ≤ 1) + O2(a 1Δ g ) → HO2*(A′,v3′ ≥ 6) + O2, and HO2*(A′,v3′ ≤ 1) + O2(a 1Δ g ) → H + O2 + O2 processes are given. It is shown that, in the case of slow quenching in a H2-O2-O2(a 1Δ g ) mixture at a low temperature, the intensity of hydrogen oxidation is enhanced due to the reaction + HO2*(A′,v3′ ≤ 1) + O2(1Δ) → H + O2 + O2. 相似文献
2.
Summary The type II singlet molecular oxygen [O2(1
g
)]-mediated photo-oxidation of the tripeptide gly-tyr-ala was studied. It has two non-oxidizable amino-acids (gly and ala) bonded to the oxidizable one, tyr. Overall (k
t) and reactive (k
r) rate constants for the interaction were determined by time-resolved methods (IR emission of O2(1
g
)) and stationary photolysis, in water at pH 11.5 as well as in alkaline non-aqueous etOH-MeCN (80:20, v/v, 10 mM in KOH) solutions. An important solvent polarity effect onk
t was detected; the rate constant increasing one order of magnitude in going from the organic mixture to water (k
t H2O = 2 × 109 M–1 s–1). Nevertheless,k
r does not parallel this trend; gly-tyr-ala being less photooxidizable in a more polar environment. The effective quantum yield (
r
) forTPE photooxidation is much higher in etOH-MeCN (
r
= 0.056) than in water (
r
= 0.023). Results are discussed on the basis of the formation of an exciplex with polar character between the TPE and O2(1
g
).Two remarkable points should be taken into account: a) the rate costants for the interaction of O2(1
g
) with gly-tyr-ala are practically the same as for free tyr. b) New -NH2 groups are generated upon sensitized irradiation. Both findings indicate that the peptide bonds in the TPE break as a result of the photooxidation. A thorough analysis with data for tyrosine and related dipeptides is undertaken.Abbreviations
O
2(1g)
singlet molecular oxygen
-
AA
amino-acid
-
TPE
tripeptide
-
gly-tyr-ala
glycyl-L-tyrosine-alanine
-
tyr-gly
L-tyrosyl-glycine
-
FFA
furfuryl alcohol
-
tyr
L-tyrosine
-
gly-tyr
glycyl-L-tyrosine
-
DPE
dipeptide
-
NaN
3
sodium azide
-
RB
rose bengal
-
ZnTPP
Zinc tetraphenyl porphyrine
-
MeCN
acetonitrile
-
DMA
9,10-dimethyl anthracene
-
etOH
ethanol
-
TRPD
time-resolved phosphorescence detection
-
NH
2 loss
loss of primary amine reactivity 相似文献
3.
We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F2alpha and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca2+]i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca2+]i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF2alpha and ET-1. Response amplitudes and number of responding cells were affected significantly by agonist concentration, luteal development, and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF2alpha increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF2alpha and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF2alpha steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF2alpha action. The lower efficacy of PGF2alpha in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF2alpha to up-regulate ET-1. 相似文献
4.
A novel 1,2-cis stereoselective synthesis of protected α-D-Gal-(1→2)-D-Glc fragments was developed. Methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranoside (13), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-glucopyranoside (15), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside (17), and methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-β-D-glucopyranoside (19) were favorably obtained by coupling a new donor, isopropyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (2), with acceptors, methyl 3-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranoside (4), methyl 3,4,6-tri-O-benzoyl-α-D-glucopyranoside (5), methyl 3-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside (8), and methyl 3,4,6-tri-O-benzoyl-β-D-glucopyranoside (12), respectively. By virtue of the concerted 1,2-cis α-directing action induced by the 3-O-allyl and 4,6-O-benzylidene groups in donor 2 with a C-2 acetyl group capable of neighboring-group participation, the couplings were achieved with a high degree of α selectivity. In particular, higher α/β stereoselective galactosylation (5.0:1.0) was noted in the case of the coupling of donor 2 with acceptor 12 having a β-CH(3) at C-1 and benzoyl groups at C-4 and C-6. 相似文献
5.
6.
Shawn J. Rice Shao-Chiang Lai Lauren W. Wood Kaitlin R. Helsley E. Aaron Runkle Monte M. Winslow David Mu 《The Journal of biological chemistry》2013,288(23):16348-16360
In lung cancers, TTF-1 displays seemingly paradoxical activities. Although TTF-1 is amplified in primary human lung cancers, it inhibits primary lung tumors from metastasizing in a mouse model system. It was reported that the oncogenic proepithelial mesenchymal transition (EMT) high mobility group AT-hook 2 gene (HMGA2) mediates the antimetastatic function of TTF-1. To gain mechanistic insight into the metastasis-critical signaling axis of TTF-1 to HMGA2, we used both reverse and forward strategies and discovered that microRNA-33a (miR-33a) is under direct positive regulation of TTF-1. By chromatin immunoprecipitation, we determined that TTF-1 binds to the promoter of SREBF2, the host gene of miR-33a. The 3′-untranslated region (UTR) of HMGA2 contains three predicted binding sites of miR-33a. We showed that the first two highly conserved sites are conducive to HMGA2 repression by miR-33a, establishing HMGA2 as a genuine target of miR-33a. Functional studies revealed that enforced expression of miR-33a inhibits the motility of lung cancer cells, and this inhibition can be rescued by overexpression of the form of HMGA2 without the 3′-UTR, suggesting that TTF-1 keeps the prometastasis gene HMGA2 in check via up-regulating miR-33a. This study reports the first miRNAs directly regulated by TTF-1 and clarifies how TTF-1 controls HMGA2 expression. Moreover, the documented importance of SREBF2 and miR-33a in regulating cholesterol metabolism suggests that TTF-1 may be a modulator of cholesterol homeostasis in the lung. Future studies will be dedicated to understanding how miRNAs influence the oncogenic activity of TTF-1 and the role of TTF-1 in cholesterol metabolism. 相似文献
7.
8.
Belinda Schegg Andreas J. Hülsmeier Christoph Rutschmann Charlotte Maag Thierry Hennet 《Molecular and cellular biology》2009,29(4):943-952
Collagen is a trimer of three left-handed alpha chains representing repeats of the motif Gly-X-Y, where (hydroxy)proline and (hydroxy)lysine residues are often found at positions X and Y. Selected hydroxylysines are further modified by the addition of galactose and glucose-galactose units. Collagen glycosylation takes place in the endoplasmic reticulum before triple-helix formation and is mediated by β(1-O)galactosyl- and α(1-2)glucosyltransferase enzymes. We have identified two collagen galactosyltransferases using affinity chromatography and tandem mass spectrometry protein sequencing. The two collagen β(1-O)galactosyltransferases corresponded to the GLT25D1 and GLT25D2 proteins. Recombinant GLT25D1 and GLT25D2 enzymes showed a strong galactosyltransferase activity toward various types of collagen and toward the serum mannose-binding lectin MBL, which contains a collagen domain. Amino acid analysis of the products of GLT25D1 and GLT25D2 reactions confirmed the transfer of galactose to hydroxylysine residues. The GLT25D1 gene is constitutively expressed in human tissues, whereas the GLT25D2 gene is expressed only at low levels in the nervous system. The GLT25D1 and GLT25D2 enzymes are similar to CEECAM1, to which we could not attribute any collagen galactosyltransferase activity. The GLT25D1 and GLT25D2 genes now allow addressing of the biological significance of collagen glycosylation and the importance of this posttranslational modification in the etiology of connective tissue disorders.Collagens are the most abundant proteins in the human body. To date, 29 types of collagen have been described, which are encoded by at least 44 genes (21, 37, 45). Collagens are characterized by domains representing repeats of the triplet Gly-X-Y, where proline and lysine are often found at positions X and Y. The Gly-X-Y repeats are not confined to collagens but are also found in several other proteins, such as the hormone adiponectin (29), the mannose-binding lectin (MBL) (11), the C1q complement protein (35), the COLQ subunit of the acetylcholine esterase complex (4), and the surfactant proteins SP-A and SP-D (11).After synthesis in the endoplasmic reticulum (ER), three procollagen subunits associate to build a right-handed triple helix. However, before the formation of the triple-helix structure, the nascent procollagen polypeptides undergo several posttranslational modifications. These modifications comprise the hydroxylation of selected proline (20) and lysine (33) residues, which are catalyzed by three prolyl-4-hydroxylases (17), one prolyl-3-hydroxylase (46), and three lysyl hydroxylases (43). Hydroxylysine can be further modified by the addition of the monosaccharide Gal(β1-O) or the disaccharide Glc(α1-2)Gal(β1-O) (39).Whereas the glycosylation of collagen was first described by Grassmann and Schleich in 1935 (9) and the structure of the glycan determined by Spiro in 1967 as being Glc(α1-2)Gal(β1-O)Hyl (40), the molecular nature of the collagen glycosyltransferase enzymes has remained elusive up to now. Collagen galactosyltransferase (ColGalT) and glucosyltransferase activities have been characterized using partially purified proteins (24, 31, 32), which appeared to be unstable. Recently the lysyl hydroxylase 3 (LH3) enzyme has been shown to catalyze a modest galactosyl and glucosyltransferase activity, suggesting that this enzyme is a combined hydroxylase and glycosyltransferase (12).Prolyl and lysyl hydroxylation contribute to the stability of the collagen triple helix, where hydroxylysine is essential for the cross-linking of collagen molecules, thus ensuring the strength of collagen fibrils (28). In contrast, the biological significance of collagen glycosylation is still unclear. The collagen domain of adiponectin and mannose-binding lectin also carry glycosylated hydroxylysine residues, which appear to be important for the oligomerization and proper secretion of these proteins (6, 29).The importance of collagen posttranslational modifications is reflected by the diseases caused by defective collagen modifying enzymes. Mutations of the LH1 lysyl hydroxylase 1 gene lead to the connective tissue disorder Ehlers-Danlos syndrome type VI (14), and mutations in the LH2 lysyl hydroxylase 2 gene lead to the Bruck syndrome (44). A deficiency in the prolyl 3-hydroxylase 1 gene causes a severe form of osteogenesis imperfecta (5). The availability of the collagen glycosyltransferase genes will enable comprehensive investigation of this posttranslational modification in cellular and animal models and possibly in human diseases. 相似文献
9.
Birk Poller Jürgen Drewe Stephan Krähenbühl Jörg Huwyler Heike Gutmann 《Cellular and molecular neurobiology》2010,30(1):63-70
Brain capillary endothelial cells form the blood–brain barrier (BBB), a highly selective permeability membrane between the
blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain
tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances
penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp).
During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the
systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6
(IL-6) and tumor necrosis factor-α (TNF-α) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line.
BCRP mRNA levels were significantly reduced by IL-1β, IL-6 and TNF-α. The strongest BCRP suppression at the protein level
was observed after IL-1β treatment. IL-1β, IL-6 and TNF-α also significantly reduced the BCRP activity as assessed by mitoxantrone
uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-α treatment. TNF-α
also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the
cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by
acute inflammation, possibly affecting the penetration of their substrates into the brain. 相似文献
10.
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyra-nosyl chloride was condensed with the N-(benzyloxycarbonyl) derivatives of, respectively, L-seryl-glycine ethyl, L-seryl-L-alanine methyl, L-seryl-L-phenylalanine methyl, and L-seryl-L-aspartic dibenzyl esters to give (3-O-GlcpNAc-CbzN-L-Ser)-GlyOEt (8), (3-O-GlcpNAc-CbzN-L-Ser)-L-AlaOMe (9), (3-O-GlcpNAc-CbzN-L-Ser)-L-PheOMe (10), and (3-O-GlcpNAc-CbzN-L-Ser)-L-Asp(diOBzl) (11), respectively; O-(2-acetamido-3,4,6-tri-O-acetyl-β-D-glucopyranosy-l)-N-(benzyloxycarbonyl)-L-serine methyl ester was deblocked by treatment with hydrobromic acid in glacial acetic acid, followed by triethylamine, to give a glycoamino acid that was condensed with the N-(benzyloxycarbonyl) derivatives of the p-nitrophenyl ester of glycine, L-alanine, and L-proline, respectively, to give CbzNGly-(3-O)-Glcp NAc-L-SerOMe) (17), CbzN-L-Ala-(3-O-GlcpNAc-L-SerOMe), and CbzN-L-Pro-(3-O-GlcpNAc-L-SerOMe), respectively. Similarly, the glycopeptide resulting from 8 was condensed with the activated esters of glycine, L-alanine, L-phenylalanine, L-proline, and L-serine, respectively, to give CbzNGly-(3-OGlcpNAc-L-Ser)-GlyOEt, CbzN-L-Ala-(3-O-GlcpNAc-L-Ser)-GlyOEt, CbzN-L-Phe-(3-O-GlcpNAc-L-Ser)-GlyOEt, and CbzN-L-Ser-(3-O-GlcpNAc-L-Ser)-GlyOEt, respectively; that from 9, with the p-nitrophenyl esters of glycine1,L-alanine, L-phenylalanine, L-proline, and L-leucine, respectively, to give CbzNGly-(3-O-GlcpNAc-L-Ser)-L-AlaOMe, CbzN-L-Ala(3-O-GlcpNAc-L-Ser)-L-AlaOMe, CbzN-L-Phe-(3-O-GlcpNAc-L-Ser)-L]-AlaOMe, CbzN-L-Pro-(3-O-GlcpNAc-L-Ser)-L-AlaOMe, and CbzN-L-Leu-(3-O-GlcpNAc- L-Ser)-L-AlaOMe, respectively; that from 10, with the derivatives of glycine, L-alanine, L-phenylalanine, and L-leucine, respectively, to give CbzNGly-(3-O-GlcpNAc-L-Ser)-L-PheOMe, CbzN-L-Phe-(3-O-GlcpNAc-L-Ser)-L-PheOMe, CbzN-L-Phe-(3-O-GlcpNAc-L-Ser)-L-PheOMe, and CbzN-L-Leu-(3-O-GlcpNAc-L-Ser)-L-PheOMe, respectively; and that from 11, with the derivatives of glycine, L-alanine, L-phenylalanine, L-proline, and L-leucine, respectively, to give CbzNGly-(3-O-GlcpNAc-L-Ser)-L-Asp(diOBzl), CbzN-L-Ala-(3-O-GlcpNAc-L-Ser)-L-Asp(diOBzl), CbzN-L-Phe-(3-O-GlcpNAc-L-Ser)-L-Asp(diOBzl), CbzN-L-Pro-(3-O-GlcpNAc-L-Ser)-L-Asp(diOBzl), and CbzN-L-Leu-(3-O-GlcpNAc-L-Ser)-L-Asp-(diOBzl), respectively. O-(2-Acetamido-3,4,5-tri-O-acetyl-2-deoxy-β-D-gluco-pyranosyl)-N-(benzyloxycarbonyl)- L-asparaginylglycyl-L-serine methyl ester (20) was synthesized by treating the free amine of 17 with the p-nitrophenyl ester of N-(benzyloxycarbonyl)-L-asparagine. 2-Acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbo-nyl)-L-aspart-1-oyl-(glycyl-L-serine methyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine (41) was synthesized by the condensation of 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbo-nyl)-L-aspart-4-oyl]-2-deoxy-β-D-glucopyranosylamine with glycyl-L-serine methyl ester. Attempts to transfer the 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranosyl group from the hydroxyl group of L-serine in 20 to the amido group of L-asparagine, to give 41, were unsuccessful. The β-elimination of some of the glycodi- and glycotri-peptides was studied. 相似文献
11.
Summary
86Rb uptake into LLC-PK1 cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na+ and Cl– for activity, and a leak component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of86Rb,22Na and36Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na+K+Cl– cotransport system. The kinetic parameters for Na+ and K+ interaction have been analyzed under initial linear zerotrans conditions. The following values were obtained:K
mNa+=0.42±0.05 mmol/liter,V
max=303±24 pmol/mg/6 sec;K
mK+=11.9±1.0 mmol/liter,V
maxK+=307±27 pmol/mg/6 sec. For Cl– interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na+1K+2Cl– can be calculated. It is concluded that the apical membrane of LLC-PK1 cells contains a Na+K+2Cl– cotransport system with properties similar to those described for the thick ascending limb of the loop of Henle. 相似文献
12.
Kátia Daniela Silveira Lívia Corrêa Barroso Angélica Thomáz Vieira Daniel Cisalpino Cristiano Xavier Lima Michael Bader Rosa Maria Esteves Arantes Robson Augusto Souza dos Santos Ana Cristina Sim?es-e-Silva Mauro Martins Teixeira 《PloS one》2013,8(6)
Angiotensin-(1–7) [Ang-(1–7)] is a biologically active heptapeptide that may counterbalance the physiological actions of angiotensin II (Ang II) within the renin-angiotensin system (RAS). Here, we evaluated whether activation of the Mas receptor with the oral agonist, AVE 0991, would have renoprotective effects in a model of adriamycin (ADR)-induced nephropathy. We also evaluated whether the Mas receptor contributed for the protective effects of treatment with AT1 receptor blockers. ADR (10 mg/kg) induced significant renal injury and dysfunction that was maximal at day 14 after injection. Treatment with the Mas receptor agonist AVE 0991 improved renal function parameters, reduced urinary protein loss and attenuated histological changes. Renoprotection was associated with reduction in urinary levels of TGF-β. Similar renoprotection was observed after treatment with the AT1 receptor antagonist, Losartan. AT1 and Mas receptor mRNA levels dropped after ADR administration and treatment with losartan reestablished the expression of Mas receptor and increased the expression of ACE2. ADR-induced nephropathy was similar in wild type (Mas+/+) and Mas knockout (Mas
−/−) mice, suggesting there was no endogenous role for Mas receptor activation. However, treatment with Losartan was able to reduce renal injury only in Mas+/+, but not in Mas
−/− mice. Therefore, these findings suggest that exogenous activation of the Mas receptor protects from ADR-induced nephropathy and contributes to the beneficial effects of AT1 receptor blockade. Medications which target specifically the ACE2/Ang-(1–7)/Mas axis may offer new therapeutic opportunities to treat human nephropathies. 相似文献
13.
The N-terminal α-amino groups of β1-bungarotoxin (β1-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the α-amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between β1-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that β1-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca2+ and ANS, indicating that the N-terminal region is not involved in Ca2+ and substrate binding. A fluorescence study revealed that the α-amino group of the A chain was in the vicinity of substrate binding site and that the TNP α-amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for β1-Bgt. 相似文献
14.
Calderón-Rivera A Andrade A Hernández-Hernández O González-Ramírez R Sandoval A Rivera M Gomora JC Felix R 《Cell calcium》2012,51(1):22-30
Voltage-gated calcium (Ca(V)) channels are transmembrane proteins that form Ca(2+)-selective pores gated by depolarization and are essential regulators of the intracellular Ca(2+) concentration. By providing a pathway for rapid Ca(2+) influx, Ca(V) channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. Ca(V) channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α(1) subunit, which typically contains associated β and α(2)δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α(2)δ-1 is post-translationally cleaved into an extracellular α(2) polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α(2)δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α(2)) and Cys1047 (in the extracellular domain of δ) prevented the association of the α(2) and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α(2)δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (Ca(V)2.2α(1)/β(3)) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α(2)δ-1 function. 相似文献
15.
Kari Seppälä Jyrki Heinämäki Juha Hatara Lassi Seppälä Jouko Yliruusi 《AAPS PharmSciTech》2010,11(1):402-408
In powder technology, it is often important to directly measure real powder flow rate from a small amount of powder. For example, in pharmaceutical industry, a frequent problem is to determine powder flow properties of new active pharmaceutical ingredient (API) in an early stage of the development when the amount of API is limited. The purpose of this paper is to introduce a new direct method to measure powder flow when the material is poorly flowing (cohesive) and the amount of material is about 1 to 2 g. The measuring system was simple, consisting of a flow chamber and electronic balance and an automated optical detection system, and for each measurement, only 1 to 2 g of sample was required. Based on the results obtained with this testing method, three selected sugar excipients, three grades of microcrystalline cellulose, and APIs (caffeine, carbamazepine, and paracetamol) can be classified as freely flowing, intermediate flowing, and poorly flowing powders, respectively. The average relative standard deviation for the flow time determinations was not more than 2–10%. The present novel flowability testing method provides a new tool for a rapid determination of flowing characteristics of powders (e.g., inhalation powders) and granules at a small scale. 相似文献
16.
Carlo Bavassano Letizia Marvaldi Michiel Langeslag Bettina Sarg Herbert Lindner Lars Klimaschewski Michaela Kress Antonio Ferrer-Montiel Hans-Günther Knaus 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2013,1833(12):3166-3175
The Transient Receptor Potential Vanilloid 1 (TRPV1, vanilloid receptor 1) ion channel plays a key role in the perception of thermal and inflammatory pain, however, its molecular environment in dorsal root ganglia (DRG) is largely unexplored. Utilizing a panel of sequence-directed antibodies against TRPV1 protein and mouse DRG membranes, the channel complex from mouse DRG was detergent-solubilized, isolated by immunoprecipitation and subsequently analyzed by mass spectrometry. A number of potential TRPV1 interaction partners were identified, among them cytoskeletal proteins, signal transduction molecules, and established ion channel subunits. Based on stringent specificity criteria, the voltage-gated K+ channel beta 2 subunit (Kvβ2), an accessory subunit of voltage-gated K+ channels, was identified of being associated with native TRPV1 channels. Reverse co-immunoprecipitation and antibody co-staining experiments confirmed TRPV1/Kvβ2 association. Biotinylation assays in the presence of Kvβ2 demonstrated increased cell surface expression levels of TRPV1, while patch-clamp experiments resulted in a significant increase of TRPV1 sensitivity to capsaicin. Our work shows, for the first time, the association of a Kvβ subunit with TRPV1 channels, and suggests that such interaction may play a role in TRPV1 channel trafficking to the plasma membrane. 相似文献
17.
18.
The tetrasaccharide 2-(p-trifluoroacetamidophenyl)ethylO-α-l-fucopyranosyl-(1–3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1–3)-O-β-d-galactopyranosyl-(1–4)-β-d-glucopyranoside was synthesized from thioglycoside intermediates. The key step was a methyl triflate promoted glycosidation of a lactose-derived 3′,4′-diol with a disaccharide thioglycoside to give a β(1–3)-linked tetrasaccharide derivative in 67% yield. 相似文献
19.
The extracellular matrix (ECM) provides the microenvironment that is pivotal for cell growth, motility, attachment, and differentiation. Advances in cell culture techniques have led to the development of cell-derived ECM model systems that are more reflective of the in vivo architecture of the ECM in tissue. In this study, a fibroblast-derived ECM (fd-ECM) was used to study the feedback regulation of type I collagen synthesis in fibroblasts. Fibroblasts plated on a preformed fd-ECM showed a significant decrease in the production of type I collagen and pro-α2(1) collagen mRNA compared to cells grown in the absence of a matrix. Function-blocking antibodies showed that this downregulation of type I collagen gene expression is mediated via α2β1 integrin. The use of several kinase inhibitors and a dominant negative ras construct (N17Ras) showed that the matrix-mediated downregulation of COL1A2 occurs via Ras-dependent activation of the MEK/ERK signaling pathway. Deletion analysis of the COL1A2 promoter implicated the region between -375 and -107 as containing a potential matrix responsive element. The use of Sp1 siRNA demonstrated that Sp1 is an important mediator of this feedback inhibition. This study provides some new insights into the feedback regulation of COL1A2 gene expression. 相似文献