Analytical assays of human HSP27 and thermal-stress survival of Escherichia coli cells that overexpress it

HSP27 is a small heat-shock protein (sHSP). Such proteins are produced in all organisms. These small HSPs exhibit chaperone-like activity that can bind to unfolded polypeptides and prevent uncontrolled protein aggregation in vitro. Cellular anti-apoptosis function and enhanced cell survival are correlated with increased expression of HSPs. This study presents a thermal-stress survival model for cells using the Escherichia coli expression system for which human HSP27, a recombinant protein, is inducible. Results show that E. coli cells overexpressing human HSP27 have enhanced tolerance to 50 degrees C thermal stress.     

真鲷仔鱼用微粒子饲料中肽酶与大豆卵磷脂添加效果的研究

本研究以酪蛋白分解物为蛋白源配制三种微粒子饲料MD-S、MD-T和MD-U对真鲷开口仔鱼进行饲养试验。以MD-S的配方为基准,MD-T采用粉状大豆卵磷脂和麸质代替液状大豆卵磷脂;MD-U则另外添加0.1%的肽酶。结果表明,微粒子饲料在水中浸泡15min后,MD-T的溶出率(35.5%)低于MD-S(46.8%)和MD-U(45.8%);实验结束时(20日龄),仔鱼的成活率以生物饵料(轮虫)组为最高(86.3%),其次是MD-T组为20.7%,显著高于(P<0.05)MD-S组(13.3%)和MD-U组(13.6%);生物饵料组的仔鱼全长(6.14±0.49mm)显著大于微粒子饲料组(4.23±0.30mm~4.46±0.30mm),各微粒子饲料组之间仔鱼的全长并不存在显著差异(P>0.05)。在孵化后第12d,微粒子饲料组的仔鱼肠上皮细胞发育良好,但至孵化后第18d,仔鱼肠上皮细胞大部分萎缩、并发生脱落。鱼体的蛋白质、DNA与RNA日间增长率微粒子饲料MD-T组高于MD-S和MD-U组,但都低于生物饵料组。由此可见,微粒子饲料中添加肽酶并无助真鲷仔鱼对其消化吸收;可是,使用粉状大豆卵磷脂与麸质代替液状卵磷脂能增强微粒子饲料的黏合性,可减少其营养成分的溶出率,从而提高微粒子饲料的饲育效果。     

Entamoeba dispar: Cultivation with Sterilized Crithidia fasciculata1

Four isolates of Entamoeba dispar identified by their hexokinase and phosphoglucomutase isoenzyme profile and by their failure to react with Entamoeba histolytica-specific monoclonal antibody (4G6) could be grown in either Diamond's BI-S-33 medium, newly developed BCSI-S (Biosate cysteine starch iron-serum) medium, or casein-free YI-S medium in the presence of Crithidia fasciculata (ReF-1:PRR) sterilized by heating 56° C for 30 min and subsequent incubation with 1% hydrogen peroxide for 24 hours at 4° C. After the cultures were maintained for over 50 passages, the amebae were identified as E. dispar by isoenzyme analysis, polymerase chain reaction with E. histolytica- and E. dispar-specific primers, i.e. p11 plus p12 and p13 plus p14, respectively, and by negative reactivity with monoclonal antibody 4G6. The flagellates added to the culture were judged to be metabolically inactive based on the results of nuclear magnetic resonance spectroscopy, electron microscopy, and polarographic analysis. All of these findings suggest that E. dispar can grow in vitro with metabolically inactive C. fasciculata as a culture associate.     

The production of antibodies that distinguish rat choline acetyltransferase from its splice variant product of a peripheral type

To produce antibodies that permit the immunohistochemical discrimination of choline acetyltransferase of the common type (cChAT) from its splice variant of a peripheral type (pChAT), we immunized rabbits with a cChAT specific recombinant protein encoded by ChAT exons 7 and 8 of the rat cChAT gene. Successful antibody production was proved by Western blotting on rat brain and on HEK293 cells expressing green fluorescent protein (GFP), cChAT-GFP and pChAT-GFP. By immunohistochemistry our antiserum clearly labeled known cholinergic structures in rat brain, but gave no positive staining in the trigeminal ganglion which contained many neurons positive with pChAT antiserum.     

E6AP ubiquitin ligase mediates ubiquitylation and degradation of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.     

Flagellin glycosylation island in Pseudomonas syringae pv. glycinea and its role in host specificity

The deduced amino acid sequences of the flagellins of Pseudomonas syringae pv. tabaci and P. syringae pv. glycinea are identical; however, their abilities to induce a hypersensitive reaction are clearly different. The reason for the difference seems to depend on the posttranslational modification of the flagellins. To investigate the role of this posttranslational modification in the interactions between plants and bacterial pathogens, we isolated genes that are potentially involved in the posttranslational modification of flagellin in P. syringae pv. glycinea (glycosylation island); then defective mutants with mutations in these genes were generated. There are three open reading frames in the glycosylation island, designated orf1, orf2, and orf3. orf1 and orf2 encode putative glycosyltransferases, and mutants with defects in these open reading frames, deltaorf1 and deltaorf2, secreted nonglycosylated and slightly glycosylated flagellins, respectively. Inoculation tests performed with these mutants and original nonhost tobacco leaves revealed that deltaorf1 and deltaorf2 could grow on tobacco leaves and caused symptom-like changes. In contrast, these mutants failed to cause symptoms on original host soybean leaves. These data indicate that putative glycosyltransferases encoded in the flagellin glycosylation island are strongly involved in recognition by plants and could be the specific determinants of compatibility between phytopathogenic bacteria and plant species.     

Decreases of action potential amplitudes, in sodium-free and calcium-free conditions, of identifiable giant neurons of an African giant snail (Achatina fulica Férussac)--I. The right parietal ganglion

Decreases of the action potential amplitude in sodium- and calcium-free states were observed with respect to the four giant neurons, PON (periodically oscillating neuron), Tan (tonically autoactive neuron), RAPN (right anterior pallial neuron) and d-RPLN (dorsal-right parietal large neuron), identified in the right parietal ganglion of the suboesophageal ganglia of an African giant snail (Achatina fulica Férussac). The decrease of the PON action potential amplitude, caused in the sodium-free state, was observed to be 25.4 +/- 2.1% (23.0 +/- 2.0 mV), expressed by M +/- SE, while that of the calcium-free state was 35.0 +/- 2.1% (30.9 +/- 1.7 mV). Then, the two ionic dependencies of the PON action potential were estimated to be about 40-50% on sodium and 50-60% on calcium. The decrease of the TAN action potential in the sodium-free state, was observed to be 20.7 +/- 1.2% (18.8 +/- 1.3 mV), whereas that of the calcium-free state was 42.2 +/- 2.7% (39.0 +/- 2.2 mV), indicating that the two ionic dependencies were 30-40% on sodium and 60-70% on calcium. The decrease of the RAPN action potential in the sodium-free state, was 45.8 +/- 3.7% (40.3 +/- 3.1 mV), whereas that of the calcium-free state was 21.7 +/- 2.5% (17.8 +/- 2.0 mV), indicating that the two ionic dependencies were about 70% on sodium and about 30% on calcium. The decrease of the d-RPLN action potential in the sodium-free state was found to be 17.6 +/- 2.4% (15.2 +/- 1.8 mV), whereas that of the calcium-free state was 23.1 +/- 1.4% (20.8 +/- 1.4 mV), indicating that the two ionic dependencies were 40-50% on sodium and 50-60% on calcium. The action potential amplitudes of all the neurons tested were decreased in both sodium-free and calcium-free states. However, their ionic dependencies were estimated to vary from 70% on sodium (30% on calcium) to 30% on the sodium (70% on the calcium), according to the neurons tested.     

Precursor of the nuclear-encoded extrinsic 30 kDa protein in photosystem II of Euglena gracilis Z is not a polyprotein

Polyprotein-type precursors have been reported for the nuclear-encoded proteins such as the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the apoproteins of light-harvesting chlorophyll-protein (LHC) in Euglena. We report here that the precursor of the extrinsic 30 kDa protein of photosystem II (PS II) encoded by nuclear DNA is not a polyprotein. The precursor was identified as a 45 kDa protein by immunoprecipitation of in vitro translation products of mRNA and by a pulse-chase experiment. It is probable that the structure of the precursor of the nuclear-encoded protein in Euglena chloroplast is closely related to the feature of assembly, as well as of transport, of the protein in chloroplast.