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71.
In this study, a detailed characterization of Monocarboxylic Acid Transporter-1 (Mct1) in cytoplasmic vesicles of cultured rat brain microvascular endothelial cells shows them to be a diverse population of endosomes intrinsic to the regulation of the transporter by a brief 25 to 30 minute exposure to the membrane permeant cAMP analog, 8Br-cAMP. The vesicles are heterogeneous in size, mobility, internal pH, and co-localize with discreet markers of particular types of endosomes including early endosomes, clathrin coated vesicles, caveolar vesicles, trans-golgi, and lysosomes. The vesicular localization of Mct1 was not dependent on its N or C termini, however, the size and pH of Mct1 vesicles was increased by deletion of either terminus demonstrating a role for the termini in vesicular trafficking of Mct1. Using a novel BCECF-AM based assay developed in this study, 8Br-cAMP was shown to decrease the pH of Mct1 vesicles after 25 minutes. This result and method were confirmed in experiments with a ratiometric pH-sensitive EGFP-mCherry dual tagged Mct1 construct. Overall, the results indicate that cAMP signaling reduces the functionality of Mct1 in cerebrovascular endothelial cells by facilitating its entry into a highly dynamic vesicular trafficking pathway that appears to lead to the transporter''s trafficking to autophagosomes and lysosomes. 相似文献
72.
Protein glycosylation may contribute to the evolution of mammalian brain complexity by adapting excitatory neurotransmission in response to environmental and social cues. Balanced excitatory synaptic transmission is primarily mediated by glutamatergic neurotransmission. Previous studies have found that subunits of the AMPA subtype of glutamate receptor are N-glycosylated, which may play a critical role in AMPA receptor trafficking and function at the cell membrane. Studies have predominantly used rodent models to address altered glycosylation in human pathological conditions. Given the rate of mammalian brain evolution and the predicted rate of change in the brain-specific glycoproteome, we asked if there are species-specific changes in glycoprotein expression, focusing on the AMPA receptor. N-glycosylation of AMPA receptor subunits was investigated in rat (Rattus norvegicus), tree shrew (Tupaia glis belangeri), macaque (Macaca nemestrina), and human frontal cortex tissue using a combination of enzymatic deglycosylation and Western blot analysis, as well as lectin binding assays. We found that two AMPA receptor subunits, GluA2 and GluA4, are sensitive to deglycosylation with Endo H and PNGase F. When we enriched for glycosylated proteins using lectin binding assays, we found that all four AMPA receptor subunits are glycosylated, and were predominantly recognized by lectins that bind to glucose or mannose, N-acetylglucosamine (GlcNAc), or 1-6αfucose. We found differences in glycosylation between different subunits, as well as modest differences in glycosylation of homologous subunits between different species. 相似文献
73.
74.
Lo CM Buxton DB Chua GC Dembo M Adelstein RS Wang YL 《Molecular biology of the cell》2004,15(3):982-989
Although myosin II is known to play an important role in cell migration, little is known about its specific functions. We have addressed the function of one of the isoforms of myosin II, myosin IIB, by analyzing the movement and mechanical characteristics of fibroblasts where this protein has been ablated by gene disruption. Myosin IIB null cells displayed multiple unstable and disorganized protrusions, although they were still able to generate a large fraction of traction forces when cultured on flexible polyacrylamide substrates. However, the traction forces were highly disorganized relative to the direction of cell migration. Analysis of cell migration patterns indicated an increase in speed and decrease in persistence, which were likely responsible for the defects in directional movements as demonstrated with Boyden chambers. In addition, unlike control cells, mutant cells failed to respond to mechanical signals such as compressing forces and changes in substrate rigidity. Immunofluorescence staining indicated that myosin IIB was localized preferentially along stress fibers in the interior region of the cell. Our results suggest that myosin IIB is involved not in propelling but in directing the cell movement, by coordinating protrusive activities and stabilizing the cell polarity. 相似文献
75.
High mobility group B (HMGB) proteins contain two HMG box domains known to bind without sequence specificity into the DNA minor groove, slightly intercalating between basepairs and producing a strong bend in the DNA backbone. We use optical tweezers to measure the forces required to stretch single DNA molecules. Parameters describing DNA flexibility, including contour length and persistence length, are revealed. In the presence of nanomolar concentrations of isolated HMG box A from HMGB2, DNA shows a decrease in its persistence length, where the protein induces an average DNA bend angle of 114 +/- 21 degrees for 50 mM Na+, and 87 +/- 9 degrees for 100 mM Na+. The DNA contour length increases from 0.341 +/- 0.003 to 0.397 +/- 0.012 nm per basepair, independent of salt concentration. In 50 mM Na+, the protein does not unbind even at high DNA extension, whereas in 100 mM Na+, the protein appears to unbind only below concentrations of 2 nM. These observations support a flexible hinge model for noncooperative HMG binding at low protein concentrations. However, at higher protein concentrations, a cooperative filament mode is observed instead of the hinge binding. This mode may be uniquely characterized by this high-force optical tweezers experiment. 相似文献
76.
Jarrahy R Huang W Rudkin GH Lee JM Ishida K Berry MD Sukkarieh M Wu BM Yamaguchi DT Miller TA 《American journal of physiology. Cell physiology》2005,289(2):C408-C414
Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(L-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions. gene expression; osteogenesis; angiogenesis 相似文献
77.
Drummond MJ Vehrs PR Schaalje GB Parcell AC 《Journal of strength and conditioning research / National Strength & Conditioning Association》2005,19(2):332-337
Excess postexercise oxygen consumption (EPOC) may describe the impact of previous exercise on energy metabolism. Ten males completed Resistance Only, Run Only, Resistance-Run, and Run-Resistance experimental conditions. Resistance exercise consisted of 7 lifts. Running consisted of 25 minutes of treadmill exercise. Vo(2) was determined during treadmill exercise and after each exercise treatment. Our findings indicated that treadmill exercise Vo(2) was significantly higher for Resistance-Run compared with Run-Resistance and Resistance Only at all time intervals. At 10 minutes postexercise, Vo(2) was greater for Resistance Only and Run-Resistance than for Resistance-Run. At 20 and 30 minutes, Vo(2) following Resistance Only was significantly greater than following Run Only. In conclusion, EPOC is greatest following Run-Resistance; however, treadmill exercise is more physiologically difficult following resistance exercise. Furthermore, the sequence of resistance and treadmill exercise influences EPOC, primarily because of the effects of resistance exercise rather than the exercise combination. We recommend performing aerobic exercise before resistance exercise when combining them into 1 exercise session. 相似文献
78.
West SA Drummond MJ Vanness JM Ciccolella ME 《Journal of strength and conditioning research / National Strength & Conditioning Association》2005,19(4):772-776
Controlled frequency breathing (CFB) is a training technique used by swimmers in an effort to simulate high-intensity workloads by limiting oxygen availability to the body and stimulating anaerobic metabolism. During CFB, a swimmer voluntarily restricts breathing, which, theoretically, limits oxygen availability and stimulates anaerobic metabolism. The purpose of this study was to determine the influence of CFB on blood lactate and metabolic responses during graded increases in swimming intensity. A free swimming (FS) protocol was used to determine blood lactate and heart rate (HR) responses to CFB, while a tethered swimming (TS) protocol was used to determine blood lactate, HR, and ventilatory responses to CFB. The subjects swam four 3-minute trials at workloads of 55, 65, 75, and 85% of peak intensity during both protocols. A total of 46 competitive collegiate swimmers participated in the study. Thirty-four subjects (14 men and 20 women) completed the FS protocol, and 12 subjects (7 men and 5 women) completed the TS protocol. CFB reduced ventilation and Vo(2) (p < 0.05) during the TS protocol and reduced HR (p < 0.05) during the FS protocol when compared to normal breathing. However, CFB did not alter blood lactate concentrations for either protocol (p > 0.05). Our findings demonstrate that although CFB does not alter the blood lactate response to graded increases in swimming intensity, it appears to reduce the ventilatory and HR responses to exercise. Thus, swim coaches can use CFB at moderate intensities to simulate high-intensity training but should consider adjusting HR training zones to reflect the reduction in HR associated with reduced ventilation. 相似文献
79.
Meriem Bahira Micah J. McCauley Ali A. Almaqwashi Per Lincoln Fredrik Westerlund Ioulia Rouzina Mark C. Williams 《Nucleic acids research》2015,43(18):8856-8867
Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [μ-C4(cpdppz)2(phen)4Ru2]4+, which consists of two Ru(phen)2dppz2+ moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (Kd ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins. 相似文献
80.
The Drosophila stomatogastric nervous system (SNS) is a compact collection of neurons that arises from the migration of neural precursors. Here we describe genetic tools allowing functional analysis of the SNS during the migratory phase of development. We constructed GAL4 lines driven by fragments of the Ret promoter, which yielded expression in a subset of migrating neural SNS precursors and also included a distinct set of midgut associated cells. Screening of additional GAL4 lines driven by fragments of the Gfrl/Munin, forkhead, twist and goosecoid (Gsc) promoters identified a Gsc fragment with expression from initial selection of SNS precursors until the end of embryogenesis. Inhibition of EGFR signaling using three identified lines disrupted the correct patterning of the frontal and recurrent nerves. To manipulate the environment traveled by SNS precursors, a FasII-GAL4 line with strong expression throughout the entire intestinal tract was identified. The transgenic lines described offer the ability to specifically manipulate the migration of SNS precursors and will allow the modeling and in-depth analysis of neuronal migration in ENS disorders such as Hirschsprung’s disease. 相似文献