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831.
832.
Dotta BT Buckner CA Cameron D Lafrenie RF Persinger MA 《General physiology and biophysics》2011,30(3):301-309
Photon emissions were measured at ambient temperature (21°C) in complete darkness once per min from cultures of 10(6) cells during the 12 h following removal from 37°C. The energy of emission was about 10(-20) J/s/cell. Of 8 different cell lines, B16-BL6 (mouse melanoma cells) demonstrated the most conspicuous emission profile. Acridine orange and ethidium bromide indicated the membranes were intact with no indication of (trypan blue) cell necrosis. Treatments with EGF and ionomycin produced rapid early (first 3 h) increases in energy emission while glutamine-free, sodium azide and wortmanin-treated cells showed a general diminishment 3 to 9 h later. The results suggested the most probable origin of the photon emission was the plasma cell membrane. Measures from cells synchronized at the M- and S-phase supported this inference. 相似文献
833.
Ma J Guo L Fiene SJ Anson BD Thomson JA Kamp TJ Kolaja KL Swanson BJ January CT 《American journal of physiology. Heart and circulatory physiology》2011,301(5):H2006-H2017
Human-induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes; however, the electrophysiological properties of hiPSC-derived cardiomyocytes have yet to be fully characterized. We performed detailed electrophysiological characterization of highly pure hiPSC-derived cardiomyocytes. Action potentials (APs) were recorded from spontaneously beating cardiomyocytes using a perforated patch method and had atrial-, nodal-, and ventricular-like properties. Ventricular-like APs were more common and had maximum diastolic potentials close to those of human cardiac myocytes, AP durations were within the range of the normal human electrocardiographic QT interval, and APs showed expected sensitivity to multiple drugs (tetrodotoxin, nifedipine, and E4031). Early afterdepolarizations (EADs) were induced with E4031 and were bradycardia dependent, and EAD peak voltage varied inversely with the EAD take-off potential. Gating properties of seven ionic currents were studied including sodium (I(Na)), L-type calcium (I(Ca)), hyperpolarization-activated pacemaker (I(f)), transient outward potassium (I(to)), inward rectifier potassium (I(K1)), and the rapidly and slowly activating components of delayed rectifier potassium (I(Kr) and I(Ks), respectively) current. The high purity and large cell numbers also enabled automated patch-clamp analysis. We conclude that these hiPSC-derived cardiomyocytes have ionic currents and channel gating properties underlying their APs and EADs that are quantitatively similar to those reported for human cardiac myocytes. These hiPSC-derived cardiomyocytes have the added advantage that they can be used in high-throughput assays, and they have the potential to impact multiple areas of cardiovascular research and therapeutic applications. 相似文献
834.
Brandes G Khayami M Peck CT Baumgärtner W Bugday H Wewetzer K 《Histochemistry and cell biology》2011,135(4):397-408
Olfactory ensheathing cells (OECs) are Schwann cell-like glial cells of the olfactory system that promote neural regeneration
after transplantation into the injured central nervous system. Compared to the closely related Schwann cells, however, the
biological characterization of OECs has remained fragmentary. This is due to the fact that the expression of OEC-specific
markers is subject to complex regulation and that intricate ultrastructural analysis is essential to determine their localization.
The p75 neurotrophin receptor (p75NTR) as the prototype OEC marker, for example, is only expressed by a minor population of neonatal rat OECs in situ. The major
population carries O4-positive axonal fragments on their surface after dissociation and up-regulates p75NTR during culturing (Wewetzer et al. in Glia 49:577–587, 2005). In the present study, we investigated whether the cell surface determinant 27C7, defined by a monoclonal antibody to Schwann
cells, is also expressed by neonatal rat OECs in situ and in vitro. Primary cell suspensions of the olfactory bulb displayed
27C7 expression of both p75NTR-negative and p75NTR-positive OECs, while immature oligodendrocytes and astrocytes were devoid of any 27C7 labeling. This together with the finding
that the intrafascicular OECs of the olfactory nerves in the mucosa expressed 27C7 but not p75NTR, suggests that 27C7 was expressed by the entire OEC population in situ. Maintenance of OECs in the absence of olfactory neurons
in organotypic slice culture up-regulated p75NTR but did not alter 27C7 expression. It is concluded that 27C7 unlike p75NTR is constitutively expressed by OECs and may, therefore, be a useful marker for characterization of neonatal OECs in situ
and in vitro. 相似文献
835.
Xu R Banka A Blake JF Mitchell IS Wallace EM Bencsik JR Kallan NC Spencer KL Gloor SL Martinson M Risom T Gross SD Morales TH Wu WI Vigers GP Brandhuber BJ Skelton NJ 《Bioorganic & medicinal chemistry letters》2011,21(8):2335-2340
We describe the design and synthesis of novel bicyclic spiro sulfonamides as potent Akt inhibitors. Through structure-based rational design, we have successfully improved PKA selectivity of previously disclosed spirochromanes. Representative compounds showed favorable Akt potency while exhibiting up to 1000-fold selectivity against PKA. 相似文献
836.
S.A. Morley V. Lemmon J.I. Spicer L.S. Peck 《Journal of experimental marine biology and ecology》2011,399(1):39-42
The limpet, Nacella concinna, collected from the Antarctic Peninsula (67°S), was incubated at − 0.3 °C and 2.9 °C for 9 months to test if the previously reported absence of acclimation capacity in Antarctic marine ectotherms could be due to the extended time it takes for them to adjust their physiology to a new stable state. Acclimation was tested through acute measurements of upper lethal limit and a modified measure of tenacity, that tested muscle capacity by measuring the length of time that N. concinna were able to remain attached to the substratum at different temperatures. Both measures acclimated in response to incubation to the higher temperature. Lethal limits were elevated in N. concinna incubated at 2.9 °C (8.1 ± 0.3 °C) compared to those incubated at − 0.3 °C (6.9 ± 0.4 °C). 2.9 °C incubated N. concinna also had a maximum tenacity at 2.1 °C, a higher temperature than the maximum tenacity of those incubated at − 0.3 °C, which occurred at − 1.0 °C. This study is the first to show that the Antarctic limpet can acclimate its physiology, but that it requires a greater period of time for acclimation to occur than previous studies have allowed for. 相似文献
837.
Nguyen CQ Gao JH Kim H Saban DR Cornelius JG Peck AB 《Journal of immunology (Baltimore, Md. : 1950)》2007,179(1):382-390
NOD.B10-H2(b) and NOD/LtJ mice manifest, respectively, many features of primary and secondary Sj?gren's syndrome (SjS), an autoimmune disease affecting primarily the salivary and lacrimal glands leading to xerostomia (dry mouth) and xerophthalmia (dry eyes). B lymphocytes play a central role in the onset of SjS with clinical manifestations dependent on the appearance of autoantibodies reactive to multiple components of acinar cells. Previous studies with NOD.IL4(-/-) and NOD.B10-H2(b).IL4(-/-) mice suggest that the Th2 cytokine, IL-4, plays a vital role in the development and onset of SjS-like disease in the NOD mouse model. To investigate the molecular mechanisms by which IL-4 controls SjS development, a Stat6 gene knockout mouse, NOD.B10-H2(b).C-Stat6(-/-), was constructed and its disease profile was defined and compared with that of NOD.B10-H2(b).C-Stat6(+/+) mice. As the NOD.B10-H2(b).C-Stat6(-/-) mice aged from 4 to 24 wk, they exhibited leukocyte infiltration of the exocrine glands, produced anti-nuclear autoantibodies, and showed loss and gain of saliva-associated proteolytic enzymes, similar to NOD.B10-H2(b).C-Stat6(+/+) mice. In contrast, NOD.B10-H2(b).C-Stat6(-/-) mice failed to develop glandular dysfunction, maintaining normal saliva flow rates. NOD.B10-H2(b).C-Stat6(-/-) mice were found to lack IgG1 isotype-specific anti-muscarinic acetylcholine type-3 receptor autoantibodies. Furthermore, the IgG fractions from NOD.B10-H2(b).C-Stat6(-/-) sera were unable to induce glandular dysfunction when injected into naive recipient C57BL/6 mice. NOD.B10-H2(b).C-Stat6(-/-) mice, like NOD.B10-H2(b).IL4(-/-) mice, are unable to synthesize IgG1 Abs, an observation that correlates with an inability to develop end-stage clinical SjS-like disease. These data imply a requirement for the IL-4/STAT6-pathway for onset of the clinical phase of SjS-like disease in the NOD mouse model. 相似文献
838.
Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV8]) and Epstein-Barr virus (EBV/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and retarding its own synthesis to reduce defective ribosomal product processing. We show here that the LANA1 QED-rich central repeat (CR) region, particularly the CR2CR3 subdomain, also retards LANA1 synthesis and markedly enhances LANA1 stability in vitro and in vivo. LANA1 isoforms have half-lives greater than 24 h, and fusion of the LANA1 CR2CR3 domain to a destabilized heterologous protein markedly decreases protein turnover. Unlike EBNA1, the LANA1 CR2CR3 subdomain retards translation regardless of whether it is fused to the 5' or 3' end of a heterologous gene construct. Manipulation of sequence order, orientation, and composition of the CR2 and CR3 subdomains suggests that specific peptide sequences rather than RNA structures are responsible for synthesis retardation. Although mechanistic differences exist between LANA1 and EBNA1, the primary structures of both proteins have evolved to minimize provoking CTL immune responses. Simple strategies to eliminate these viral inhibitory regions may markedly improve vaccine effectiveness by maximizing CTL responses. 相似文献
839.
Aladin F Lautscham G Humphries E Coulson J Blake N 《Cancer immunology, immunotherapy : CII》2007,56(8):1143-1152
It is becoming increasingly apparent that the majority of tumours display defects in the MHC class I antigen processing pathway, particularly low levels of the transporters-associated with antigen processing (TAP) and tapasin. Thus, immunotherapy approaches targeting such tumours with CD8+ cytotoxic T lymphocytes (CTL) requires strategies to overcome these defects. Previously we had identified an antigen processing pathway by which cytosolically derived hydrophobic peptides could be presented in the absence of TAP. Here we show in the tapasin-negative cell line 721.220 that a number of these hydrophobic TAP-independent peptides can also be presented in a tapasin-independent manner. Yet when these experiments were extended to tumour cell lines derived from small cell lung cancer (SCLC), which we show to be tapasin deficient in addition to TAP-negative, the TAP-, tapasin-independent peptides were not presented. This lack of presentation could be rectified by pre-treatment of SCLC cells with IFNgamma. Alternatively, by directing the TAP-, tapasin-independent peptides into the endoplasmic reticulum (ER) via an ER signal sequence, these peptides were presented efficiently by SCLC cells. We infer from this data that the TAP-independent pathway for presentation of hydrophobic peptides generates a low concentration of peptide in the ER and, for tumour cells which also lack tapasin, this concentration of antigenic peptide is insufficient to load onto MHC class I molecules. Thus, for immunotherapeutic approaches to target SCLC and other tumours with defects in the MHC class I antigen processing pathway it will be important to consider strategies that address tapasin-defects. 相似文献
840.
Bowgen AD Fraser KP Peck LS Clarke A 《American journal of physiology. Regulatory, integrative and comparative physiology》2007,292(6):R2266-R2274
The energetic cost of protein synthesis is thought to account for a significant proportion of total metabolism. However, attempts to estimate the energetic cost of synthesizing proteins have resulted in surprisingly variable results, particularly for the small number of polar organisms studied, where cost estimates vary by two orders of magnitude. Much of this variability is probably the result of differing methodologies and experimental designs. Here we have used two different, carefully validated methods to measure the costs of protein synthesis in Antarctic limpets. One method, which utilized a specific protein synthesis inhibitor, was used to measure the cost of protein synthesis at two temperatures to test the hypothesis that the cost of protein synthesis varies with temperature. The cost of protein synthesis measured using the inhibitor cycloheximide was 13.95 +/- 0.77 micromol O2/mg protein, while correlation of absolute protein synthesis with oxygen consumption suggested the cost of protein synthesis was 19.58 micromol O2/mg protein. Water temperature did not alter the cost of protein synthesis in Nacella concinna (Student's t-test, P = 0.849, t = 0.19, df = 12). In a meta-analysis of literature values for the cost of protein synthesis there was no significant effect of temperature, but there was a significant relationship between the concentration of cycloheximide used to inhibit protein synthesis and the measured cost. 相似文献