Alpha-crystallin can function as a molecular chaperone.

The alpha-crystallins (alpha A and alpha B) are major lens structural proteins of the vertebrate eye that are related to the small heat shock protein family. In addition, crystallins (especially alpha B) are found in many cells and organs outside the lens, and alpha B is overexpressed in several neurological disorders and in cell lines under stress conditions. Here I show that alpha-crystallin can function as a molecular chaperone. Stoichiometric amounts of alpha A and alpha B suppress thermally induced aggregation of various enzymes. In particular, alpha-crystallin is very efficient in suppressing the thermally induced aggregation of beta- and gamma-crystallins, the two other major mammalian structural lens proteins. alpha-Crystallin was also effective in preventing aggregation and in refolding guanidine hydrochloride-denatured gamma-crystallin, as judged by circular dichroism spectroscopy. My results thus indicate that alpha-crystallin refracts light and protects proteins from aggregation in the transparent eye lens and that in nonlens cells alpha-crystallin may have other functions in addition to its capacity to suppress aggregation of proteins.     

Paradoxical interplay of viral and cellular functions

Some cellular editing functions can restrict the replication of some viruses but contribute to completion of the life cycle of others. A recent study has identified an isoform of the adenosine deaminase acting on RNA type 1 (ADAR 1) as required for embryogenesis, and as a restriction factor for a number of important RNA virus pathogens [1]. The dual implication of key cellular functions in the innate immunity against viruses, or, paradoxically, as mediators of virus replication is interpreted in the light of the concept of virus-host coevolution and tinkering proposed for general evolution by François Jacob decades ago.     

Structure of Vps75 and implications for histone chaperone function

The vacuolar protein sorting 75 (Vps75) histone chaperone participates in chromatin assembly and disassembly at both active and inactive genes through the preferential binding to histone H3–H4. Vps75 is also one of two histone chaperones, along with antisilencing factor 1, that promotes histone H3-Lys-56 acetylation by the regulation of Ty1 transposition protein 109 (Rtt109) histone acetyltransferase. Here, we report the x-ray crystal structure of Vps75 and carry out biochemical studies to characterize its interaction with Rtt109. We find that the Vps75 structure forms a homodimeric “headphone” architecture that includes an extended helical dimerization domain and earmuff domains at opposite ends and sides of the dimerization domain. Despite the similar overall architecture with the yeast nucleosome assembly protein 1 and human SET/TAF-1β/INHAT histone chaperones, Vps75 shows several unique features including the relative disposition of the earmuff domains to the dimerization domain, characteristics of the earmuff domains, and a pronounced cleft at the center of the Vps75 dimer. These differences appear to correlate with the unique function of Vps75 to interact with Rtt109 for histone acetylation. Our biochemical studies reveal that two surfaces on the earmuff domain of Vps75 participate in Rtt109 interaction with a stoichiometry of 2:1, thus leaving the pronounced central cleft of the Vps75 dimer largely accessible for histone binding. Taken together, our data provide a structural framework for understanding how Vps75 mediates both nucleosome assembly and histone acetylation by Rtt109.     

Thyroid function tests in viral chronic hepatitis

BACKGROUND: One hundred and twenty five patients with virus B or C chronic active hepatitis and postnecrotic cirrhosis and different degrees of liver dysfunction were studied. AIM: 1) To determine a thyroid hormonal profile; 2) to evaluate the prognostic value of these tests in relation to the progression of the disease and mortality; 3) compare these findings with Child-Pugh classification. PATIENTS AND METHODS: The patients were divided in four groups: a) 31 with chronic active hepatitis; b) 41 with postnecrotic cirrhosis Child A; c) 35 with postnecrotic cirrhosis Child B and d) 18 with postnecrotic cirrhosis Child C. The protocol comprised serum measurements of albumin and bilirrubin, estimates of prothrombin time and clinical evaluation of ascites and encephalopathy, measurement of total serum triiodothyronine, thyroxine, thyroid-stimulating hormone, free thyroxine, reverse triiosothyronine, calculated rT3/T3 index (IrT3) and thyrotropin-releasing hormone test. RESULTS: Total serum triiodothyromnine showed the most significant difference among the groups, gradually lower as the disease became more advanced (CAH: 149.2 +/- 42.3 ng/dL; PNC-A: 137.4 +/- 37.2 ng/dL; PNC-B: 88.0 +/- 28.4 ng/dL and PNC-C: 41.8 +/- 21.9 ng/dL). Low levels of T4 (4.5 +/- 2.0 micrograms/dL) and FT4 (0.7 +/- 0.4 ng/dL) and elevated levels of thyroid-stimulating hormone (7.2 +/- 11.5 microIU/mL), reverse triiosothyronine (60.8 +/- 52.1 ng/dL) and calculated rT3/T3 index (2.2 +/- 2.6) were more frequent in patients with postnecrotic cirrhosis Child C. Thyrotropin-releasing hormone test was normal in the majority of the patients. CONCLUSION: The present study shows a positive relationship between the low serum levels of T3 and elevated serum levels of rT3 and IrT3/T3 with the degree of hepatic dysfunction according to the Child-Pugh classification.     

Ageing-related changes in the in vivo function of rat liver macroautophagy and proteolysis

Autophagy is a universal, highly regulated mechanism responsible for the degradation of long-lived proteins, cytomembranes and organelles during fasting and may be the cell repair mechanism that mediates the anti-ageing effects of calorie restriction (Bergamini and Gori, 1995). The function of autophagy was studied in vivo on male Sprague Dawley rats fed ad libitum or 40% food restricted. Autophagy was induced in overnight fasted rats by the injection of an anti-lipolytic agent and was investigated by electron microscopy. Changes in regulatory plasma nutrients and hormones were assessed and rate of proteolysis was calculated from the release of 14C(6)-valine from pre-labelled resident proteins. Results in rats fed ad libitum showed that autophagic-proteolytic response to antilypolitic agents was paramount in one month-old rats; was high but delayed in 2 month-old rats, decreased remarkably in 6 month-old rats and almost negligible at older age. Parallel ageing-related changes were observed in the effects of treatment lowering glucose and insulin plasma levels. Calorie restriction prevented all changes. In view of the known suppressive effects of insulin, it may be concluded that the age-changes of autophagy are secondary to the ageing-related alteration in glucose metabolism and hormone levels, whose appearance is delayed by calorie restriction. Data may support the hypothesis that ad libitum feeding accelerates the rate of ageing by raising insulin plasma levels and suppressing autophagy and membrane maintenance, and that calorie restriction may break this vicious circle.     

Abnormal gastric motor function in viral gastroenteritis

Nausea and vomiting occur commonly with gastroenteritis caused by parvovirus-like agents. Infection results in histologic injury to the small bowel mucosa, but the gastric mucosa remains unaffected. We have studied gastric emptying of liquids serially in 10 volunteers before and after ingestion of the parvovirus-like agents, Norwalk and Hawaii viruses. The five subjects who developed illness all showed marked delays in gastric emptying, while the five well subjects had no alteration of emptying. Five addition volunteers who developed Norwalk virus gastroenteritis underwent serial studies of gastric secretion of hydrochloric acid, pepsin, and intrinsic factor. No change was detected in either basal or betazole-stimulated secretion of these three substances during the course of illness. The nausea and vomiting accompanying this type of viral gastroenteritis may result from abnormal gastric motor function.     

Chaperone function and chaperone overload in the aged. A preliminary analysis

Chaperones have an important role in the repair of proteotoxic damage, which is greatly increased in aged subjects. Chaperone levels and expression were subject of numerous studies in aged organisms. However, there were only very few attempts to measure chaperone activity in aged animals. Here, we report our initial studies showing a decreased chaperone capacity of liver cytosol from aged rats compared to those of young counterparts. The amount of Hsc70/Hsp70 was not significantly different in livers of young and aged rats. On the contrary, old animals showed a significant decrease in their hepatic Hsp90 content, which may explain their decreased chaperone activity. The observed decrease in chaperone capacity may also reflect a direct proteotoxic damage of chaperones, or an increase in chaperone occupancy, i.e. a 'chaperone overload' due to the increased amount of damaged hepatic proteins in aged rats. Experiments are in progress to elucidate the mechanism of the observed age-induced changes in chaperone function.     

The antioxidant protein alkylhydroperoxide reductase of Helicobacter pylori switches from a peroxide reductase to a molecular chaperone function

Helicobacter pylori, an oxygen-sensitive microaerophilic bacterium, contains many antioxidant proteins, among which alkylhydroperoxide reductase (AhpC) is the most abundant. The function of AhpC is to protect H. pylori from a hyperoxidative environment by reduction of toxic organic hydroperoxides. We have found that the sequence of AhpC from H. pylori is more homologous to mammalian peroxiredoxins than to eubacterial AhpC. We have also found that the protein structure of AhpC could shift from low-molecular-weight oligomers with peroxide-reductase activity to high-molecular-weight complexes with molecular-chaperone function under oxidative stresses. Time-course study by following the quaternary structural change of AhpC in vivo revealed that this enzyme changes from low-molecular-weight oligomers under normal microaerobic conditions or short-term oxidative shock to high-molecular-weight complexes after severe long-term oxidative stress. This study revealed that AhpC of H. pylori acts as a peroxide reductase in reducing organic hydroperoxides and as a molecular chaperone for prevention of protein misfolding under oxidative stress.     

LSm14A is a processing body-associated sensor of viral nucleic acids that initiates cellular antiviral response in the early phase of viral infection

Recognition of viral nucleic acids by pattern recognition receptors initiates type I IFN induction and innate antiviral immune response. Here we show that LSm14A, a member of the LSm family involved in RNA processing in the processing bodies, binds to synthetic or viral RNA and DNA and mediates IRF3 activation and IFN-β induction. Knockdown of LSm14A inhibits cytosolic RNA- and DNA-trigger type I IFN production and cellular antiviral response. Moreover, LSm14A is essential for early-phase induction of IFN-β after either RNA or DNA virus infection. We further found that LSm14A-mediated IFN-β induction requires RIG-I-VISA or MITA after RNA or DNA virus infection, respectively, and viral infection causes translocation of LSm14A to peroxisomes, where RIG-I, VISA, and MITA are located. These findings suggest that LSm14A is a sensor for both viral RNA and DNA and plays an important role in initiating IFN-β induction in the early phase of viral infection.     

Molecular kinesis in cellular function and plasticity

Intracellular transport and localization of cellular components are essential for the functional organization and plasticity of eukaryotic cells. Although the elucidation of protein transport mechanisms has made impressive progress in recent years, intracellular transport of RNA remains less well understood. The National Academy of Sciences Colloquium on Molecular Kinesis in Cellular Function and Plasticity therefore was devised as an interdisciplinary platform for participants to discuss intracellular molecular transport from a variety of different perspectives. Topics covered at the meeting included RNA metabolism and transport, mechanisms of protein synthesis and localization, the formation of complex interactive protein ensembles, and the relevance of such mechanisms for activity-dependent regulation and synaptic plasticity in neurons. It was the overall objective of the colloquium to generate momentum and cohesion for the emerging research field of molecular kinesis.     

Effect of treatment on ventricular function and troponin I proteolysis in reperfused myocardium

Effects of ischemia time and treatment interventions upon troponin I (TnI) proteolysis and function of reperfused myocardium were examined in isolated, perfused rabbit hearts. Hearts were randomized to 90 min aerobic perfusion, 15 min low-flow (1 ml/min) ischemia (I) and 60 min reperfusion (R) or 60 min low-flow I and 60 min R. Hearts subject to 60 min I and 60 min R received either no treatment, l -arginine treatment, or treatment with oxygen free radical (OFR) scavengers (mercapto-proponyl-glycine, catalase and superoxide dismutase). Hearts from cholesterol-fed rabbits were also studied after 60 min I and R. Isovolumic LV pressure and heart rate were recorded throughout and Western analysis of ventricular myocardium, using 3 specific antibodies, detected intact TnI (29 kDa) and TnI fragment (25 kDa). Hearts subject to 15 min I had minimal irreversible injury (TTC negative region=0.6+/-0.4% LV) but hearts subject to 60 min I had more extensive injury (TTC negative=40.7+/-5.8% LV). Recovery of rate-pressure product after 15 min I and 60 min R (56+/-9% of baseline) was better than after 60 min I and 60 min R (23+/-9%, P<0.01). Both l -arginine and OFR scavengers were associated with better recovery of function after 60 min I, (66+/-7% and 72+/-3% of baseline respectively, P<0.01 v no treatment) but cholesterol hearts had poor recovery after 60 min I (37+/-8%). The 25 kDa TnI (% total TnI immunoreactivity) was 8.7+/-0.9% in controls, 10.0+/-1.6% after 15 min I and 60 min R, and 17.4+/-2.4% after 60 min I and 60 min R (P<0.01 v controls and 15 min I). The proportion of 25 kDa TnI was increased in all hearts after 60 min I and did not change with treatment (l -arginine 16.8+/-1.8%, OFR scavengers 16.0+/-3.2%, cholesterol 14.0+/-1.9%). There was no relation between proportion of 25 kDa TnI and recovery of function. Samples from freshly excised rabbit hearts and human right atria also had 25 kDa TnI (relative intensities 8.5+/-2.3% and 5.1+/-2.6% respectively). Although TnI fragmentation increases after prolonged ischemia and reperfusion, the functional recovery of stunned myocardium is independent of degree of TnI fragmentation.     

SIV-associated myocarditis: viral and cellular correlates of inflammation severity

Myocarditis is a common finding in HIV-infected people. Cardiac inflammatory lesions and functional abnormalities similar to those documented in HIV infection are frequently seen in SIV infection of rhesus monkeys, suggesting a shared disease mechanism. A retrospective analysis of cardiac tissue collected at necropsy was performed to assess correlates of myocardial inflammation in SIV-infected rhesus monkeys. Intramyocardial SIV-infected cells were identified in 7 of 21 hearts from SIV-infected animals, with viral protein consistently colocalizing with the macrophage marker HAM 56. Productively infected cells occurred in low numbers, and did not correlate with the presence or quantity of inflammation or necrosis. Intramyocardial CMV was identified in 6 of 21 hearts from SIV+ animals, but also did not correlate with the presence or quantity of inflammation or necrosis. In contrast, T cell infiltration correlated inversely with DC-SIGN+ cell numbers, which occurred in significantly higher numbers in SIV+ animals with histologically normal myocardium than in SIV+ animals with active or borderline myocarditis or in uninfected controls (p < 0.001), suggesting an important immunoregulatory role for this population within the myocardium.     

乙型肝炎病毒蛋白反式激活基因的研究

0引言乙型肝炎病毒(HBV)的感染,不仅引起急?慢性病毒性肝炎,而且与肝纤维化?肝细胞癌的发生?发展过程密切相关[1-5].病毒进入到肝细胞之后,肝炎病毒蛋白在肝细胞内不是孤立存在的,病毒基因组在肝细胞内具有两种调控方式:其一是顺式调节如启动子及增强子序列,以直接方式影响另外一些基因组的功能,是基因组内部调节方式;其二是反式调节,即基因组中一部分基因片段通过其转录或翻译产物产生对另一部分基因片段表达的调节方式,如病毒基因组表达的反式激活蛋白,以其表达产物的间接方式参与另外一些基因的功能调节,可以对基因组内部的基因片段,甚…     

Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules.

During biosynthesis, class II major histocompatibility complex molecules are intimately associated with invariant chain (Ii). The Ii-class II association has been shown to block peptide-class II binding and to affect the ultimate conformation of class II expressed on the cell surface. To assess the biochemical basis for the effects of Ii on class II, we have analyzed the biosynthesis of class II in EL4 cells transfected with I-Ad with and without Ii. In these studies, we found that Ii had a profound effect on the biosynthesis of I-Ad. In the absence of Ii, class II could form dimers efficiently, but these dimers appeared to be misfolded and this altered conformation resulted in the loss of some monoclonal antibody epitopes and inefficient transport from the endoplasmic reticulum to the Golgi. In addition, class II that was transported through the Golgi accumulated an abnormally increased molecular mass associated with N-linked glycosylation. Subsequent transfection of Ii into these cells resulted in recovery of normal class II conformation, causing a restoration of monoclonal antibody epitopes, efficient intracellular transport, and normal glycosylation. Together, these data indicate that Ii can have a profound effect on the folding, transport, and modification of class II molecules and suggest that one function of Ii may be to act as a class II-specific chaperone.     

Prognostic value of quantitative liver function tests in viral cirrhosis: a prospective study

BACKGROUND AND AIMS: Widespread application of quantitative liver function tests as a prognostic tool is controversial. In this study we assessed the predictivity of serial evaluations of galactose elimination capacity (GEC) and the monoethylglycinexylidide (MEGX) test on survival in viral cirrhosis, and secondarily we compared these tests with Child-Turcotte-Pugh (CTP) and Model for End Stage Liver Disease (MELD) scores. METHODS: In a cohort of 35 patients with viral cirrhosis, GEC and MEGX were evaluated every 6 months for 24 months and compared with CTP and MELD scores at the same time intervals. The end points were patient death or liver transplantation. RESULTS: Statistically significant differences between dead/transplanted patients and survivors were found for basal values of GEC, MEGX, CTP and MELD. Receiver-operating characteristics curves of CTP and MELD scores showed a higher prognostic accuracy than GEC and MEGX. On multivariate analysis, neither GEC nor MEGX were independent predictors of survival. Repeated-measures analysis of GEC and MEGX did not increase the prognostic accuracy of these tests and did not add useful prognostic information on patient outcome during the following 6 months. CONCLUSIONS: Our data suggest that neither single nor repeated determinations of GEC and MEGX are superior to CTP and MELD scores in predicting prognosis of patients with viral cirrhosis.