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101.
酪氨酸羟化酶基因载体-pCMVTH 质粒的构建及在大鼠骨骼肌内的表达 总被引:4,自引:0,他引:4
为用转基因方法治疗巴金森氏病大鼠模型,本研究采用分子克隆技术,将合成多巴胺的关键酶-酪氨酸羟化酶(TH)的基因,克隆进入以巨细胞病毒CMV为启动子的载体质粒内,经限制性内切酶定位分析证实该重组的DNA质粒的可靠性。携带TH基因的PCMVTH质粒以LIPO-FECTIN介导,在培养的原代骨骼肌细胞中高效表达。本研究为进一步用转基因的细胞植入脑内以治疗巴金森氏病打下一定基础。 相似文献
102.
The roles of Ca2+ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca2+]i) that declined to a steady-state level. The peak increase in [Ca2+]i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca2+]i did not. Removal of extracellular Ca2+ had little effect on peak [Ca2+]i but greatly reduced steady-state increases in [Ca2+]i and tension. Verapamil inhibited steady-state [Ca2+]i only at [ACh]<1 µM. After depletion of internal Ca2+ stores by 10 min exposure to ACh in Ca2+-free solution and then washout of ACh for 5 min in Ca2+-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca2+ increased [Ca2+]i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca2+]i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca2+]i is necessary for tension maintenance and is dependent on Ca2+ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists. 相似文献
103.
Iciar Martinez Bent Dreyer Aasta Agersborg Annick Leroux Gilles Boeuf 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1995,112(4)
Rearing of 1-year-old Arctic charr (Salvelinus alpinus) at 12°C, as well as the administration of 50 or 75 mgT3/kg feed, accelerated the neonatal to adult fast myosin heavy chain transition, but the effect of temperature was more dramatic than the effect of T3 administration. The endogenous plasma levels of T3 in charrs reared at 12°C were higher than those of analogous groups reared at natural temperature, which in the period under study was between 0.5 and 12°C. As in other species, T3 seemed to play a role in the regulation of the neonatal to adult fast myosin isoform transition by down-regulating the levels of the neonatal and increasing the levels of an adult fast myosin heavy chain. Temperature seemed to accelerate this transition at least, but not only, by inducing an increase in the endogenous levels of T3 in the Arctic charr. 相似文献
104.
Calsequestrin is the major Ca2+-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its -helical content and the intrinsic fluorescence upon binding of Ca2+. Calsequestrin binds Ca2+ with high-capacity and with moderate affinity and it functions as a Ca2+ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca2+ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsquestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart. 相似文献
105.
To study the physiological role of the creatine kinase/phosphocreatine (CK/PCr) system in cells and tissues with a high and fluctuating energy demand we have concentrated on the site-directed inactivation of the B- and M-CK genes encoding the cytosolic CK protein subunits. In our approach we used homologous recombination in mouse embryonic stem (ES) cells from strain 129/Sv. Using targeting constructs based on strain 129/Sv isogenic DNA we managed to ablate the essential exons of the B-CK and M-CK genes at reasonably high frequencies. ES clones with fully disrupted B-CK and two types of M-CK gene mutations, a null (M-CK–) and leaky (M-CK1) mutation, were used to generate chimaeric mutant mice via injection in strain C57BL/6 derived blastocysts. Chimaeras with the B-CK null mutation have no overt abnormalities but failed to transmit the mutation to their offspring. For the M-CK– and M-CK1 mutations successful transmission was achieved and heterozygous and homozygous mutant mice were bred. Animals deficient in MM-CK are phenotypically normal but lack muscular burst activity. Fluxes through the CK reaction in skeletal muscle are highly impaired and fast fibres show adaptation in cellular architecture and storage of glycogen. Mice homozygous for the leaky M-CK allele, which have 3-fold reduced MM-CK activity, show normal fast fibres but CK fluxes and burst activity are still not restored to wildtype levels. 相似文献
106.
E. R. Chin H. J. Green F. Grange J. D. Mercer P. J. O'Brien 《Molecular and cellular biochemistry》1994,139(1):41-52
A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca++-ATPase activity and Ca++-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle.In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96±0.1 and 0.99±0.1 mg/g in WG and RG, respectively. The percent Ca++-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca++-activated Ca++-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P<0.05) in frozen HOM (5.12±0.18–3.98±0.20 mole/g tissue/min in WG and from 5.39±0.20–4.48±0.24 mole/g tissue/min in RG). Ca++-uptake was measured at a range of physiological free [Ca++] using the Ca++ fluorescent dye Indo-1. Maximum Ca++-uptake rates when corrected for initial [Ca++]f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca++ was reduced (P<0.05) in quick frozen HOM (1.30±0.1–0.66±0.1 mole/g tissue/min in WG and 1.04±0.2–0.60±0.1 mole/g tissue/min in RG). Linear correlations between Ca++-uptake and Ca++-ATPase activity measured in the presence of the Ca++ ionophore A23187 were r=+0.25, (P<0.05) and r=+0.74 (P<0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca++-uptake (r=+0.44, P<0.05) and between HOM and CM Ca++-ATPase activity (r=+0.34, P<0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca++-uptake function and maximal Ca++-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage. (Mol Cell Biochem139, 41–52, 1994) 相似文献
107.
Sajal Chakraborti Sandip K. Batabyal John R. Michael Tapati Sanyal 《Molecular and cellular biochemistry》1994,130(2):121-127
Exposure of rabbit pulmonary arterial smooth muscle cells to 10 M of the calcium ionophore A23187 dramatically stimulates cell membrane-associated phospholipase A2 activity and arachidonic acid release. In addition, A23187 also enhances cell membrane-associated serine esterase activity. Serine esterase inhibitors phenylmethylsulfonylfuoride and diisopropyl fluorophosphate prevent the increase in serine esterase and phospholipase A2 activities and arachidonic acid release caused by A23187. A23187 still stimulated serine esterase and phospholipase A2 activities and arachidonic acid release in cells pretreated with nominal Ca2+ free buffer. Treatment of the cell membrane with A23187 does not cause any appreciable change in serine esterase and phospholipase A2 activities. Pretreatment of the cells with actinomycin D or cycloheximide did not prevent the increase in the cell membrane associated serine esterase and phospholipase A2 activities, and arachidonic acid release caused by A23187. These results suggest that (i) a membrane-associated serine esterase plays an important role in stimulating the smooth muscle cell membrane associated phospholipase A2 activity (ii) in addition to the presence of extracellular Ca2+, release of Ca2+ from intracellular storage site(s) by A23187 also appears to play a role in stimulating the cell membrane-associated serine esterase and phospholipase A2 activities, and (iii) the increase in the cell membrane-associated serine esterase and phospholipase A2 activities does not appear to require new RNA or protein synthesis.Abbreviations A23187
calcium ionophore
- AA
arachidonic acid
- PMSF
phenylmethyl sulfonylfuoride
- DFP
diisopropyl-fluorophosphate
- DMEM
Dulbecco's modified Eagles medium
- FCS
fetal calf serum
- PBS
phosphate buffered saline
- HBPS
Hank's buffered physiological saline
- PLA2
phospholipase A2 相似文献
108.
109.
110.
In a controlled animal experiment the effects of dietary subacute Zn deficiency on growth, Zn concentration, and tissue 42-K
distribution were studied. Growth retardation caused lower body weight because both skeletal and heart muscle showed a reduction
in cell mass. Zn concentrations were reduced in most tissues, however, they remained unaltered in heart muscle. 42-K activity
increased in skeletal muscle and pancreas. We hypothesize the latter reflects the organs rate of metabolism, inducing the
exocrine pancreas to increase Zn absorption; in skeletal muscle it may induce also alterations in cell potentiation, causing
restless behavior. As suggested by the calculated specific K activity (Bq/mol), the K uptake was highest in liver and bone,
high in pancreas and skeletal muscle and low in heart muscle. The latter suggests K retention in heart muscle. Specific activity
in plasma and jejunum remained unaltered: K status and absorption seem unaffected. Zn deficiency causes different 42-K activities
in the various tissues, that respond by alterations in K metabolism without the induction of K deficiency. 相似文献