Folding mechanism of porcine ribonuclease

The kinetics of unfolding and refolding of porcine ribonuclease were investigated. The unfolded state of this protein was found to consist of a fast-refolding species (UF) and two slow-refolding species (UIS and UIIS). After the rapid collapse of the structure during the N (native)----UF unfolding reaction, UIS and UIIS are produced from UF by two independent slow isomerizations of the unfolded polypeptide chain, leading ultimately to a mixture of about 10% UF, 20% UIIS and 70% UIS molecules at equilibrium. This is at variance with all other ribonucleases investigated to date, which show a distribution of 20% UF, 60 to 70% UIIS and only 10 to 20% UIS. The two isomerizations of the unfolded porcine protein differ strongly in rate. The first process with tau = 250 seconds (10 degrees C) leads to an increase in the fluorescence of Tyr92 and was identified as cis in equilibrium trans isomerization of Pro93. At equilibrium, most unfolded molecules contain an incorrect trans Pro93. The second isomerization is much slower (tau = 1300 s at 10 degrees C) and leads to a predominance of the incorrect isomer as well. Like isomerization of Pro93, it is governed by an activation enthalpy of 22 kcal/mol (92 kJ/mol) and it was tentatively assigned to the Pro114-Pro115 sequence of porcine ribonuclease. Because of the wide separation in rate between the two reactions, molecules with an incorrect isomer only at Pro93 accumulate transiently after unfolding. These are the UIIS molecules. Most of them are finally converted to UIS by the 1300 second process. All molecules that have undergone this isomerization refold very slowly, i.e. the UIS molecules. The major fraction contains two incorrect isomers. A 1300 second isomerization after unfolding and a predominant very slow refolding reaction were observed only for the porcine protein. We suggest that these changes in the folding mechanism may be correlated with the presence of the Pro114-Pro115 sequence, which occurs only in porcine ribonuclease. The refolding pathway of porcine UIIS involves the rapid formation of a native-like intermediate with an incorrect trans Pro93 as was found previously for the bovine ribonuclease, where the UIIS species predominates in the unfolded state.     

牙鲆一株弹状病毒病原的分离与鉴定

从患病牙鲆中分离鉴定了一株弹状病毒（Paralichthys olivaceus rhabdovirus,PoRV）。用过滤除菌后的患病牙鲆组织匀浆液,接种不同的鱼类细胞,其中有7种鱼类细胞出现明显的病变在对病毒进行挑斑分离后,测定了PoRV的滴度,显示PoRV在敏感鱼类细胞（Grass Carp Ovary,GCO）中的滴度达到106.5TCID50/mL;绘制了PoRV生长曲线;经蔗糖密度梯度离心提纯PoRV,负染及宿主细胞超薄切片的电镜观察,显示PoRV大小约为60nm×200nm。测定了PoRV的理化性质,显示该病毒对有机溶剂和温度敏感,但对DNA抑制剂阿糖胞苷（Ara-c）不敏感。经SDS-PAGE电泳,对PoRV的蛋白图谱进行了分析,表明该病毒有5种主要蛋白带,其分子量大小分别约为：250kD、67kD、44kD、30kD、23kD。     

Hypertrophy of skeletal muscle in diabetic rats in response to chronic resistance exercise.

This study had the following objectives: 1) to determine whether diabetic rats could increase muscle mass due to a physiological manipulation (chronic resistance exercise), 2) to determine whether exercise training status modifies the effect of the last bout of exercise on elevations in rates of protein synthesis, and 3) to determine whether chronic resistance exercise alters basal glycemia. Groups consisted of diabetic or nondiabetic rats that performed progressive resistance exercise for 8 wk, performed acute resistance exercise, or remained sedentary. Arterial plasma insulin in diabetic groups was reduced by about one-half (P < 0.05) compared with nondiabetic groups. Soleus and gastrocnemius-plantaris complex muscle wet weights were lower because of diabetes, but in response to chronic exercise these muscles hypertrophied in diabetic (0.028 +/- 0.003 vs. 0.032 +/- 0.0015 g/cm for sedentary vs. exercised soleus and 0.42 +/- 0.068 vs. 0.53 +/- 0.041 g/cm for sedentary vs. exercised gastrocnemius-plantaris, both P < 0.05) but not in nondiabetic (0.041 +/- 0.0026 vs. 0.042 +/- 0.003 g/cm for sedentary vs. exercised soleus and 0.72 +/- 0.015 vs. 0.69 +/- 0.013 g/cm for sedentary vs. exercised gastrocnemius-plantaris) rats when muscle weight was expressed relative to tibial length or body weight (data not shown). Another group of diabetic rats that lifted heavier weights showed muscle hypertrophy. Rates of protein synthesis were higher in red gastrocnemius in chronically exercised than in sedentary rats: 155 +/- 11 and 170 +/- 7 nmol phenylalanine incorporated x g muscle(-1) x h(-1) in exercised diabetic and nondiabetic rats vs. 110 +/- 14 and 143 +/- 7 nmol phenylalanine incorporated x g muscle(-1) x h(-1) in sedentary diabetic and nondiabetic rats. These elevations, however, were lower than in acutely exercised (but untrained) rats: 176 +/- 15 and 193 +/- 8 nmol phenylalanine incorporated x g muscle(-1) x h(-1) in diabetic and nondiabetic rats. Finally, chronic exercise training in diabetic rats was associated with reductions in basal glycemia, and such reductions did not occur in sedentary diabetic groups. These data demonstrate that, despite lower circulating insulin concentrations, diabetic rats can increase muscle mass in response to a physiological stimulus.     

Bacillus megaterium mutant deficient in membrane-bound adenosine triphosphatase activity.

An adenosine triphosphatase (ATPase) mutant of Bacillus megaterium was isolated and characterized. This mutant (designated A37) was unable to grow on nonfermentable carbon sources and possessed less than 5% of the wild-type ATPase activity. Oxygen uptake by the mutant was comparable to that in the wild type. Sporulation in the wild type occurred in both glucose- and nitrogen-limiting media; however, A37 sporulated only in the nitrogen-limiting medium. The inability of A37 to sporulate in glucose-limiting medium seemed to be due to insufficient adenosine 5'-triphosphate (ATP) levels during the sporulation stages. Fructose, which can generate ATP via substrate-level phosphorylation, is equally efficient in stimulating ATP synthesis in the wild type and A37. Malate-stimulated ATP synthesis in the wild type was shown to have many characteristics associated with oxidative phosphorylation and was absent in the mutant. These data suggest that the ATPase deficiency results in the loss of oxidative phosphorylation.     

Stimulation of the creatine transporter SLC6A8 by the protein kinase mTOR

Cellular accumulation of creatine is accomplished by the Na(+), Cl(-), and creatine transporter CreaT (SLC6A8). The mammalian target of rapamycin (mTOR) is a kinase stimulating cellular nutrient uptake. The present experiments explored whether SLC6A8 is regulated by mTOR. In Xenopus oocytes expressing SLC6A8 but not in water injected oocytes, creatine-induced a current which was significantly enhanced by coexpression of mTOR. Kinetic analysis revealed that mTOR enhanced maximal current without significantly altering affinity. Preincubation of the oocytes for 32 h with rapamycin (50 nM) decreased the creatine-induced current and abrogated its stimulation by mTOR. The effect of mTOR on CreaT was blunted by additional coexpression of the inactive mutant of the serum and glucocorticoid-inducible kinase (K119N)SGK1 and mimicked by coexpression of wild type SGK1. In conclusion, mTOR stimulates the creatine transporter SLC6A8 through mechanisms at least partially shared by the serum and glucocorticoid-inducible kinase SGK1.