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961.
962.
963.
Cerebral blood vessels are frequently damaged in traumatic brain injury. Mechanical properties of fresh human cerebral vessels obtained through surgeries have been reported. Because surgical sources of human specimens are rare and produce a limited amount of material, we sought to compare the properties of more readily available cerebral arteries and veins obtained from cadavers to fresh vessel data. Additionally, because the previous study was limited to small vessels available in surgery, it was unknown how generally applicable the results were to larger cerebral arteries and veins. In the current study, large and small cerebral vessels from autopsy were stretched axially. Data from these and similar tests on fresh vessels were combined to determine the significance of source and size on mechanical properties. Structural comparisons of histological samples were additionally utilized to characterize differences. Results indicate that specimens from autopsy and surgery behave similarly except that vessels from autopsy tend to be less extensible. While tests on large vessels were limited, small arteries obtained from autopsy tended to be slightly stiffer than large arteries. In contrast, bridging veins from cadavers were typically stiffer and stretched less before structural failure than cortical veins from the same source. These effects are, however, secondary to differences identified between arteries and veins in the previous study. 相似文献
964.
CaMKII tethers to L-type Ca2+ channels, establishing a local and dedicated integrator of Ca2+ signals for facilitation
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Hudmon A Schulman H Kim J Maltez JM Tsien RW Pitt GS 《The Journal of cell biology》2005,171(3):537-547
Ca2+-dependent facilitation (CDF) of voltage-gated calcium current is a powerful mechanism for up-regulation of Ca2+ influx during repeated membrane depolarization. CDF of L-type Ca2+ channels (Ca(v)1.2) contributes to the positive force-frequency effect in the heart and is believed to involve the activation of Ca2+/calmodulin-dependent kinase II (CaMKII). How CaMKII is activated and what its substrates are have not yet been determined. We show that the pore-forming subunit alpha(1C) (Ca(v)alpha1.2) is a CaMKII substrate and that CaMKII interaction with the COOH terminus of alpha1C is essential for CDF of L-type channels. Ca2+ influx triggers distinct features of CaMKII targeting and activity. After Ca2+-induced targeting to alpha1C, CaMKII becomes tightly tethered to the channel, even after calcium returns to normal levels. In contrast, activity of the tethered CaMKII remains fully Ca2+/CaM dependent, explaining its ability to operate as a calcium spike frequency detector. These findings clarify the molecular basis of CDF and demonstrate a novel enzymatic mechanism by which ion channel gating can be modulated by activity. 相似文献
965.
966.
Rusling DA Le Strat L Powers VE Broughton-Head VJ Booth J Lack O Brown T Fox KR 《FEBS letters》2005,579(29):6616-6620
We have used DNase I footprinting to examine DNA triple helix formation at a 12 base pair oligopurine.oligopyrimidine sequence, using oligonucleotides that contain combinations of 2'-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine (bis-amino-U, BAU) and 3-methyl-2-aminopyridine (MeP) in place of T and C, respectively. This combination acts cooperatively to enable high affinity triple helix formation at physiological pH. The affinity depends on the number of substitutions and their arrangement; oligonucleotides in which these analogues are evenly distributed throughout the third strand bind much better than those in which they are clustered together. 相似文献
967.
Metal ion binding to human hemopexin 总被引:1,自引:0,他引:1
Binding of divalent metal ions to human hemopexin (Hx) purified by a new protocol has been characterized by metal ion affinity chromatography and potentiometric titration in the presence and absence of bound protoheme IX. ApoHx was retained by variously charged metal affinity chelate resins in the following order: Ni(2+) > Cu(2+) > Co(2+) > Zn(2+) > Mn(2+). The Hx-heme complex exhibited similar behavior except the order of retention of the complex on Zn(2+)- and Co(2+)-charged columns was reversed. One-dimensional (1)H NMR of apoHx in the presence of Ni(2+) implicates at least two His residues and possibly an Asp, Glu, or Met residue in Ni(2+) binding. Potentiometric titrations establish that apoHx possesses more than two metal ion binding sites and that the capacity and/or affinity for metal ion binding is diminished when heme binds. For most metal ions that have been studied, potentiometric data did not fit to binding isotherms that assume one or two independent binding sites. For Mn(2+), however, these data were consistent with a high-affinity site [K(A) = (15 +/- 3) x 10(6) M(-)(1)] and a low-affinity site (K(A) 相似文献
968.
The requirement for d-alanine in the peptidoglycan layer of bacterial cell walls is fulfilled in part by alanine racemase (EC 5.1.1.1), a pyridoxal 5'-phosphate (PLP)-assisted enzyme. The enzyme utilizes two antiparallel bases focused at the C(alpha) position and oriented perpendicular to the PLP ring to facilitate the equilibration of alanine enantiomers. Understanding how this two-base system is utilized and controlled to yield reaction specificity is therefore a potential means for designing antibiotics. Cycloserine is a known alanine racemase suicide substrate, although its mechanism of inactivation is based on transaminase chemistry. Here we characterize the effects of a Y265F mutant (Tyr265 acts as the catalytic base in the l-isomer case) of Bacillus stearothermophilus alanine racemase on cycloserine inactivation. The Y265F mutant reduces racemization activity 1600-fold [Watanabe, A., Yoshimura, T., Mikami, B., and Esaki, N. (1999) J. Biochem. 126, 781-786] and only leads to formation of the isoxazole end product (the result of the transaminase pathway) in the case of d-cycloserine, in contrast to results obtained using the wild-type enzyme. l-Cycloserine, on the other hand, utilizes a number of alternative pathways in the absence of Y265, emphasizing the importance of Y265 in both the inactivation and racemization pathway. In combination with the kinetics of inactivation, these results suggest roles for each of the two catalytic bases in racemization and inactivation, as well as the importance of Y265 in "steering" the chemistry to favor one pathway over another. 相似文献
969.
Harrington DJ Soper R Edwards C Savidge GF Hodges SJ Shearer MJ 《Journal of lipid research》2005,46(5):1053-1060
We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status. 相似文献
970.
Maximizing interferon-gamma production by Chinese hamster ovary cells through temperature shift optimization: experimental and modeling 总被引:3,自引:0,他引:3
The Chinese hamster ovary (CHO) cell line producing interferon-gamma (IFN-gamma) exhibits a 2-fold increase in specific productivity when grown at 32 degrees C compared to 37 degrees C. Low temperature also causes growth arrest, meaning that the cell density is significantly lower at 32 degrees C, nutrients are consumed at a slower rate and the batch culture can be run for a longer period of time prior to the onset of cell death. At the end of the batch, product concentration is doubled at the low temperature. However, the batch time is nearly doubled as well, and this causes volumetric productivity to only marginally improve by using low temperature. One approach to alleviate the problem of slow growth at low temperature is to utilize a biphasic process, wherein cells are cultured at 37 degrees C for a period of time in order to obtain reasonably high cell density and then the temperature is shifted to 32 degrees C to achieve high specific productivity. Using this approach, it is hypothesized that IFN-gamma volumetric productivity would be maximized. We developed and validated a model for predicting the optimal point in time at which to shift the culture temperature from 37 degrees C to 32 degrees C. It was found that by shifting the temperature after 3 days of growth, the IFN-gamma volumetric productivity is increased by 40% compared to growth and production at 32 degrees C and by 90% compared to 37 degrees C, without any decrease in total production relative to culturing at 32 degrees C alone. The modeling framework presented here is applicable for optimizing controlled proliferation processes in general. 相似文献