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51.
The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 > or = 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A "tail" domain alone, or the synergistic effect of lamin "head" and the "tail" domains together, interferes with assembly into 10-nm filaments. 相似文献
52.
Calmyrin is a myristoylated calcium binding protein that contains four putative EF-hands. Calmyrin interacts with a number of proteins, including presenilin-2 (PS2). However, the biophysical properties of calmyrin, and the molecular mechanisms that regulate its binding to different partners, are not well understood. By site-directed mutagenesis and Ca2+ binding studies, we found that calmyrin binds two Ca2+ ions with a dissociation constant of approximately 53 microM, and that the two C-terminal EF-hands 3 and 4 bind calcium. Using ultraviolet spectroscopy, circular dichroism (CD), and NMR, we found that Ca(2+)-free and -bound calmyrin have substantially different protein conformations. By yeast two-hybrid assays, we found that both EF-hands 3 and 4 of calmyrin must be intact for calmyrin to interact with PS2-loop sequences. Pulse-chase studies of HeLa cells transfected with calmyrin expression constructs indicated that wild-type (Wt) calmyrin has a half-life of approximately 75 min, whereas a mutant defective in myristoylation turns over more rapidly (half-life of 35 min). By contrast, the half-lives of calmyrin mutants with a disrupted EF-hand 3 or EF-hand 4 were 52 and 170 min, respectively. Using immunofluorescence staining of HeLa cells transfected with Wt and mutant calmyrin cDNAs, we found that both calcium binding and myristoylation are important for dynamic intracellular targeting of calmyrin. Double immunofluorescence microscopy indicated that Wt and myristoylation-defective calmyrin proteins colocalize efficiently and to the same extent with PS2, whereas calmyrin mutants defective in calcium binding display less colocalization with PS2. Our results suggest that calmyrin functions as a calcium sensor and that calcium binding sequences in calmyrin are important for interaction with the PS2 loop. 相似文献
53.
Bittsánszky A Gyulai G Humphreys M Gullner G Csintalan Z Kiss J Szabó Z Lágler R Tóth Z Rennenberg H Heszky L Komíves T 《Zeitschrift für Naturforschung. C, Journal of biosciences》2006,61(9-10):699-703
Stress response capacity (Fv/Fm at 690 nm and F690/F735 at Fmax) of untransformed hybrid poplar, Populus x canescens (P tremula x P alba), and two transgenic lines overexpressing gamma-ECS (gamma-glutamylcysteine synthetase) either in the cytosol (cyt-ECS) or in the chloroplast (chl-ECS) was studied in response to the herbicide paraquat (4.0 x 10(-9) to 4.0 x 10(-6) M) for 21 days. Significant differences at sublethal (4.0 x 10(-7) M) and bleaching (4.0 x 10(-6) M) concentrations of paraquat were observed with about a two-fold and eight-fold decrease in the photosynthetic activity (Fv/Fm at 690 nm and F690/F735 at Fmax), respectively. None of the gshI transgenic lines (cyt-ECS, chl-ECS) with elevated GSH content exhibited significant tolerance to paraquat. Semiquantitative RT-PCR of the cyt-ECS clone was used for gene expression analysis of the nuclear encoded rbcS gene and the stress responsive gst gene. Expression of the constitutively expressed 26SrRNA ribosomal gene was probed as a control for all RT-PCR reactions. The relative intensities of gene expressions normalized to the level of 26SrRNA intensity showed a 50% decrease in the nuclear encoded rbcS expression and a 120% increase in the stress responsive gst gene expression of the paraquat treated (4.0 x 10(-7) M) samples of the transgenic poplar line (cyt-ECS). 相似文献
54.
Ubiquilin proteins have been shown to interact with a wide variety of other cellular proteins, often regulating the stability and degradation of the interacting protein. Ubiquilin contains a UBL (ubiquitin-like) domain at the N-terminus and a UBA (ubiquitin-associated) domain at the C-terminus, separated by a central region containing Sti1-like repeats. Little is known about regulation of the interaction of ubiquilin with other proteins. In the present study, we show that ubiquilin is capable of forming dimers, and that dimerization requires the central region of ubiquilin, but not its UBL or the UBA domains. Furthermore, we provide evidence suggesting that monomeric ubiquilin is likely to be the active form that is involved in binding presenilin proteins. Our results provide new insight into the regulatory mechanism underlying the interaction of ubiquilin with presenilins. 相似文献
55.
Mullen MP Elia G Hilliard M Parr MH Diskin MG Evans AC Crowe MA 《Journal of proteome research》2012,11(5):3004-3018
Uterine secretions, or histotroph, are a critical component for early embryo survival, functioning as the sole supply of vitamins, minerals, enzymes, and other myriad of nutrients required by the developing conceptus before implantation. Histotroph is therefore a promising source for biomarkers of uterine function and for enhancing our understanding of the environment supporting early embryo development and survival. Utilizing label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics, we characterized the uterine proteome at two key preimplantation stages of the estrous cycle in high fertility cattle. We identified 300 proteins on Day 7 and 510 proteins on Day 13 including 281 proteins shared between days. Five proteins were more abundant (P < 0.05) on Day 7 compared with Day 13 and included novel histotroph proteins cytokeratin 10 and stathmin. Twenty-nine proteins were more abundant (P < 0.05) including 13 unique on Day 13 compared with Day 7 and included previously identified legumain, metalloprotease inhibitor-2, and novel histotroph proteins chromogranin A and pyridoxal kinase. Functional analysis of the 34 differentially expressed proteins (including 14 novel to histotroph) revealed distinct biological roles putatively involved in early pregnancy, including remodelling of the uterine environment in preparation for implantation; nutrient metabolism; embryo growth, development and protection; maintenance of uterine health; and maternal immune modulation. This study is the first reported LC-MS/MS based global proteomic characterization of the uterine environment in any domesticated species before implantation and provides novel information on the temporal alterations in histotroph composition during critical stages for early embryo development and uterine function during the early establishment of pregnancy. 相似文献
56.
Yang T Zhuang L Rei Fidalgo AM Petrides E Terrando N Wu X Sanders RD Robertson NJ Johnson MR Maze M Ma D 《PloS one》2012,7(5):e37020
It is not possible to identify all pregnancies at risk of neonatal hypoxic-ischemic encephalopathy (HIE). Many women use some form of analgesia during childbirth and some anesthetic agents have been shown to be neuroprotective when used as analgesics at subanesthetic concentrations. In this study we sought to understand the effects of two anesthetic agents with presumptive analgesic activity and known preconditioning-neuroprotective properties (sevoflurane or xenon), in reducing hypoxia-induced brain damage in a model of intrauterine perinatal asphyxia. The analgesic and neuroprotective effects at subanesthetic levels of sevoflurane (0.35%) or xenon (35%) were tested in a rat model of intrauterine perinatal asphyxia. Analgesic effects were measured by assessing maternal behavior and spinal cord dorsal horn neuronal activation using c-Fos. In separate experiments, intrauterine fetal asphyxia was induced four hours after gas exposure; on post-insult day 3 apoptotic cell death was measured by caspase-3 immunostaining in hippocampal neurons and correlated with the number of viable neurons on postnatal day (PND) 7. A separate cohort of pups was nurtured by a surrogate mother for 50 days when cognitive testing with Morris water maze was performed. Both anesthetic agents provided analgesia as reflected by a reduction in the number of stretching movements and decreased c-Fos expression in the dorsal horn of the spinal cord. Both agents also reduced the number of caspase-3 positive (apoptotic) neurons and increased cell viability in the hippocampus at PND7. These acute histological changes were mirrored by improved cognitive function measured remotely after birth on PND 50 compared to control group. Subanesthetic doses of sevoflurane or xenon provided both analgesia and neuroprotection in this model of intrauterine perinatal asphyxia. These data suggest that anesthetic agents with neuroprotective properties may be effective in preventing HIE and should be tested in clinical trials in the future. 相似文献
57.
Moira McKendry Helen McGloin Debbie Saberi Libby Caudwell Anthony R Brady Mervyn Singer 《BMJ (Clinical research ed.)》2004,329(7460):258
Objective To assess whether a nurse led, flow monitored protocol for optimising circulatory status in patients after cardiac surgery reduces complications and shortens stay in intensive care and hospital.Design Randomised controlled trial.Setting Intensive care unit and cardiothoracic unit of a university teaching hospital.Participants 174 patients who underwent cardiac surgery between April 2000 and January 2003.Interventions Patients were allocated to conventional haemodynamic management or to an algorithm guided by oesophageal Doppler flowmetry to maintain a stroke index above 35 ml/m2.Results 26 control patients had postoperative complications (two deaths) compared with 17 (four deaths) protocol patients (P = 0.08). Duration of hospital stay in the protocol group was significantly reduced from a median of nine (interquartile range 7-12) days to seven (7-10) days (P = 0.02). The mean duration of hospital stay was reduced from 13.9 to 11.4 days, a saving in hospital bed days of 18% (95% confidence interval -12% to 47%). Usage of intensive care beds was reduced by 23% (-8% to 59%).Conclusion A nurse delivered protocol for optimising circulatory status in the early postoperative period after cardiac surgery may significantly shorten hospital stay. 相似文献
58.
59.
David Lloyd Katie L Thomas George Cowie Jonathan D Tammam Alan G Williams 《Journal of microbiological methods》2002,48(2-3):289-302
Direct measurement of dissolved gases and low molecular weight volatiles through permeable membranes (e.g. 50-microm-thick silicone rubber), provides an invaluable tool for the investigation of the activities of microorganisms in the laboratory and in their natural environments. Multiple molecular species are monitored at a single point. Fast response times (t(90%)<1 min) and long-term stability, (<1% week(-1)); high specificity and high sensitivity (e.g. 0.2 microM for O(2), <0.5 mM for ethanol), provides a technique that can provide information on the kinetics of processes over many decades (10(0)-10(6)) of minutes. Spatial resolution of <1 mm enables 3D mapping of gases in complex ecosystems (sediments, peat, soils, biofilms, foodstuffs). Results with membrane inlet mass spectrometry (MIMS) when used in conjunction with confocal scanning laser microscopy, provides a powerful approach to the analysis of kinetic and spatial aspects of natural environments. Examples discussed are peat cores and cheese. 相似文献
60.
Robert H.Cowie J. S. Jones 《Biological journal of the Linnean Society. Linnean Society of London》1998,65(3):233-255
In 1985 we resurveyed the sites on the Marlborough Downs in southern England at which Cain and Currey in 1960/61 sampled Cepaea snails and thence introduced the term 'area effects' to describe large areas of uniform morph frequency. Some sites no longer harboured Cepaea and at others the species composition had changed, with a general spread of Cepaea hortensis at the expense of Cepaea nemoralis. The majority, however, permitted comparison of morph frequencies between the two surveys. In C. nemoralis, we detected a significant overall decrease in the frequency of the brown morph and estimate selection as 5–9% per generation. There was no apparent change in frequencies of banded morphs. In C. hortensis we detected a significant overall increase in the frequency of unbanded shells (1–3% selection per generation) and an almost significant decrease in the frequency of fusions within the banded class. There was insufficient colour polymorphism in C. hortensis to allow analysis of colour morph frequencies. These changes—all in the direction of reduced absorption of solar energy—resemble others detected in both species at other localities in southern England. Possible explanations include large-scale climatic effects and changes in vegetation. 相似文献