Group IIA secreted phospholipase A2 (PLA2G2A) augments adipose tissue thermogenesis.

Group IIA secreted phospholipase A2 (PLA2G2A) hydrolyzes glycerophospholipids at the sn-2 position resulting in the release of fatty acids and lysophospholipids. C57BL/6 mice do not express Pla2g2a due to a frameshift mutation (wild-type [WT] mice). We previously reported that transgenic expression of human PLA2G2A in C57BL/6 mice (IIA+ mice) protects against weight gain and insulin resistance, in part by increasing total energy expenditure. Additionally, we found that brown and white adipocytes from IIA+ mice have increased expression of mitochondrial uncoupling markers, such as uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor-gamma coactivator, and PR domain containing 16, suggesting that the energy expenditure phenotype might be due to an increased thermogenic capacity in adipose tissue. Here, we further characterize the impact of PLA2G2A on thermogenic mechanisms in adipose tissue. Metabolic analysis of WT and IIA+ mice revealed that even when housed within their thermoneutral zone, IIA+ mice have elevated energy expenditure compared to WT littermates. Increased energy expenditure in IIA+ mice is associated with increased citrate synthase activity in brown adipose tissue (BAT) and increased mitochondrial respiration in both brown and white adipocytes. We also observed that direct addition of recombinant PLA2G2A enzyme to in vitro cultured adipocytes results in the marked induction of UCP1 protein expression. Finally, we report that PLA2G2A induces the expression of numerous transcripts related to energy substrate transport and metabolism in BAT, suggestive of an increase in substrate flux to fuel BAT activity. These data demonstrate that PLA2G2A enhances adipose tissue thermogenesis, in part, through elevated substrate delivery and increased mitochondrial content in BAT.

Insulin-induced de novo lipid synthesis occurs mainly via mTOR-dependent regulation of proteostasis of SREBP-1c.

Insulin stimulates de novo lipid synthesis in the liver and in cultured hepatocytes via its ability to activate sterol regulatory element-binding protein 1c (SREBP-1c). Although PI3K-AKT-mTORC1-p70S6K-signaling kinases are known to drive feed-forward expression of SREBP-1c, the identity of the phosphorylated amino acid residue(s) putatively involved in insulin-stimulated de novo lipogenesis remains elusive. We obtained in silico and mass spectrometry evidence, that was combined with siRNA strategies, to discover that insulin-induced phosphorylation of serine 418, serine 419, and serine 422 in rat SREBP-1c was most likely mediated by p70S6 kinase. Here, for the first time, we show that insulin-induced phosphorylation of these 3 serine residues mainly impinged on the mechanisms of proteostasis of both full-length and mature SREBP-1c in the McArdle-RH7777 hepatoma cells. Consistent with this conclusion, nascent SREBP-1c, substituted with phosphomimetic aspartic acid residues at these 3 sites, was resistant to proteasomal degradation. As a consequence, endoplasmic reticulum to Golgi migration and proteolytic maturation of pSREBP-1c was significantly enhanced which led to increased accumulation of mature nSREBP-1c, even in the absence of insulin. Remarkably, aspartic acid substitutions at S418, S419 and S422 also protected the nascent SREBP-1c from ubiquitin-mediated proteasome degradation thus increasing its steady-state levels and transactivation potential in the nucleus. These complementary effects of p70S6K-mediated phosphorylation on proteostasis of pSREBP-1c were necessary and sufficient to account for insulin's ability to enhance transcription of genes controlling de novo lipogenesis in hepatocytes.

Docosahexaenoic acid inhibits proteolytic processing of sterol regulatory element-binding protein-1c (SREBP-1c) via activation of AMP-activated kinase.

In hyperinsulinemic states including obesity and T2DM, overproduction of fatty acid and triglyceride contributes to steatosis of the liver, hyperlipidemia and hepatic insulin resistance. This effect is mediated in part by the transcriptional regulator sterol responsive element binding protein-1c (SREBP-1c), which stimulates the expression of genes involved in hepatic fatty acid and triglyceride synthesis. SREBP-1c is up regulated by insulin both via increased transcription of nascent full-length SREBP-1c and by enhanced proteolytic processing of the endoplasmic reticulum (ER)-bound precursor to yield the transcriptionally active n-terminal form, nSREBP-1c. Polyunsaturated fatty acids of marine origin (n-3 PUFA) prevent induction of SREBP-1c by insulin thereby reducing plasma and hepatic triglycerides. Despite widespread use of n-3 PUFA supplements to reduce triglycerides in clinical practice, the exact mechanisms underlying their hypotriglyceridemic effect remain elusive. Here we demonstrate that the n-3 PUFA docosahexaenoic acid (DHA; 22:5 n-3) reduces nSREBP-1c by inhibiting regulated intramembrane proteolysis (RIP) of the nascent SREBP-1c. We further show that this effect of DHA is mediated both via activation of AMP-activated protein kinase (AMPK) and by inhibition of mechanistic target of rapamycin complex 1 (mTORC1). The inhibitory effect of AMPK on SREBP-1c processing is linked to phosphorylation of serine 365 of SREBP-1c in the rat. We have defined a novel regulatory mechanism by which n-3 PUFA inhibit induction of SREBP-1c by insulin. These findings identify AMPK as an important negative regulator of hepatic lipid synthesis and as a potential therapeutic target for hyperlipidemia in obesity and T2DM.

Phosphorylation of sterol regulatory element binding protein-1a by protein kinase A (PKA) regulates transcriptional activity.

The counter-regulatory hormone glucagon inhibits lipogenesis via downregulation of sterol regulatory element binding protein 1 (SREBP-1). The effect of glucagon is mediated via protein kinase A (PKA). To determine if SREBP-1 is a direct phosphorylation target of PKA, we conducted mass spectrometry analysis of recombinant n-terminal SREBP-1a following PKA treatment in vitro. This analysis identified serines 331/332 as bona-fide phosphorylation targets of PKA. To determine the functional consequences of phosphorylation at these sites, we constructed mammalian expression vector for both nSREBP-1a and 1c isoforms in which the candidate PKA phosphorylation sites were mutated to active phosphomimetic or non-phosphorylatable amino acids. The transcriptional activity of SREBP was reduced by the phosphomimetic mutation of S332 of nSREBP-1a and the corresponding serine (S308) of nSREBP-1c. This site is a strong candidate for mediating the negative regulatory effect of glucagon on SREBP-1 and lipogenesis.

FoxO1 inhibits sterol regulatory element-binding protein-1c (SREBP-1c) gene expression via transcription factors Sp1 and SREBP-1c.

Induction of lipogenesis in response to insulin is critically dependent on the transcription factor, sterol regulatory element-binding protein-1c (SREBP-1c). FoxO1, a forkhead box class-O transcription factor, is an important mediator of insulin action, but its role in the regulation of lipid metabolism has not been clearly defined. We examined the effects of FoxO1 on srebp1 gene expression in vivo and in vitro. In vivo studies showed that constitutively active (CA) FoxO1 (CA-FoxO1) reduced basal expression of SREBP-1c mRNA in liver by ∼60% and blunted induction of SREBP-1c in response to feeding. In liver-specific FoxO knock-out mice, SREBP-1c expression was increased ∼2-fold. Similarly, in primary hepatocytes, CA-FoxO1 suppressed SREBP1-c expression and inhibited basal and insulin-induced SREBP-1c promoter activity. SREBP-1c gene expression is induced by the liver X receptor (LXR), but CA-FoxO1 did not block the activation of SREBP-1c by the LXR agonist TO9. Insulin stimulates SREBP-1c transcription through Sp1 and via "feed forward" regulation by newly synthesized SREBP-1c. CA-FoxO1 inhibited SREBP-1c by reducing the transactivational capacity of both Sp1 and SREBP-1c. In addition, chromatin immunoprecipitation assays indicate that FoxO1 can associate with the proximal promoter region of the srebp1 gene and disrupt the assembly of key components of the transcriptional complex of the SREBP-1c promoter. We conclude that FoxO1 inhibits SREBP-1c transcription via combined actions on multiple transcription factors and that this effect is exerted at least in part through reduced transcriptional activity of Sp1 and SREBP-1c and disrupted assembly of the transcriptional initiation complex on the SREBP-1c promoter.

Effect of statin treatment on metabolites, lipids and prostanoids in patients with Statin Associated Muscle Symptoms (SAMS).

BACKGROUND: Between 5-10% of patients discontinue statin therapy due to statin-associated adverse reactions, primarily statin associated muscle symptoms (SAMS). The absence of a clear clinical phenotype or of biomarkers poses a challenge for diagnosis and management of SAMS. Similarly, our incomplete understanding of the pathogenesis of SAMS hinders the identification of treatments for SAMS. Metabolomics, the profiling of metabolites in biofluids, cells and tissues is an important tool for biomarker discovery and provides important insight into the origins of symptomatology. In order to better understand the pathophysiology of this common disorder and to identify biomarkers, we undertook comprehensive metabolomic and lipidomic profiling of plasma samples from patients with SAMS who were undergoing statin rechallenge as part of their clinical care. METHODS AND FINDINGS: We report our findings in 67 patients, 28 with SAMS (cases) and 39 statin-tolerant controls. SAMS patients were studied during statin rechallenge and statin tolerant controls were studied while on statin. Plasma samples were analyzed using untargeted LC-MS metabolomics and lipidomics to detect differences between cases and controls. Differences in lipid species in plasma were observed between cases and controls. These included higher levels of linoleic acid containing phospholipids and lower ether lipids and sphingolipids. Reduced levels of acylcarnitines and altered amino acid profile (tryptophan, tyrosine, proline, arginine, and taurine) were observed in cases relative to controls. Pathway analysis identified significant increase of urea cycle metabolites and arginine and proline metabolites among cases along with downregulation of pathways mediating oxidation of branched chain fatty acids, carnitine synthesis, and transfer of acetyl groups into mitochondria. CONCLUSIONS: The plasma metabolome of patients with SAMS exhibited reduced content of long chain fatty acids and increased levels of linoleic acid (18:2) in phospholipids, altered energy production pathways (ß-oxidation, citric acid cycle and urea cycles) as well as reduced levels of carnitine, an essential mediator of mitochondrial energy production. Our findings support the hypothesis that alterations in pro-inflammatory lipids (arachidonic acid pathway) and impaired mitochondrial energy metabolism underlie the muscle symptoms of patients with statin associated muscle symptoms (SAMS).

Viral induction of the zinc finger antiviral protein is IRF3-dependent but NF-kappaB-independent.

The zinc finger antiviral protein (ZAP) is an interferon-stimulated gene that restricts the replication of retroviruses, alphaviruses, and filoviruses. Relatively little is known, however, regarding the detailed mechanism of ZAP induction during viral infections. We show that, although being inducible by either interferon or virus, expression of ZAP is more efficiently activated by virus than are several other classical interferon-stimulated genes and that viral induction of ZAP occurs under the direct control of interferon regulatory factor 3 (IRF3) independent of interferon paracrine/autocrine signaling. ZAP was up-regulated in cells unresponsive to type I and III interferons upon engagement of TLR3, retinoic inducible gene I/melanoma differentiation-associated gene 5 pathways, or ectopic expression of a constitutively active IRF3 mutant. Conversely, induction of ZAP by virus or dsRNA was severely impaired in cells expressing a dominant-negative mutant IRF3 and completely abrogated in cells lacking IRF3. In contrast to IRF3, ZAP induction was independent of NF-kappaB activity. Mutational analysis of the human ZAP promoter revealed that multiple interferon-stimulated response elements far distal to the transcription start site serve redundantly to control IRF3-dependent induction of ZAP transcription. Chromatin immunoprecipitation assays demonstrated that IRF3 selectively binds the distal interferon-stimulated response elements in human ZAP promoter following viral infection. Collectively, these data suggest that ZAP is a direct target gene of IRF3 action in cellular antiviral responses.

Breakup of a leaky dielectric drop in a uniform electric field.

Electrohydrodynamic (EHD) breakup phenomena for a leaky dielectric drop suspended in another immiscible viscous dielectric and subjected to a uniform electric field are examined using the leaky dielectric theory and the explicit forcing lattice Boltzmann method, by taking into account full nonlinear inertia effects. The breakup modes are first computed for varied conductivity of the drop fluid, as the viscosity ratio λ (=µ_{in}/µ_{out}) is momentarily set to unity, that is, for the slightly conducting (R=σ_{in}/σ_{out}<10), moderately conducting (10≤R≤20), and highly conducting (R>20) cases. For slightly conducting drops (R=5) only one breakup mode via two symmetrical necks persists for permittivity ratios 0.05Ca_{E,critical} (ratio of electric and surface tension forces), despite significant length-scale variation of mother and daughter drops. At higher Q (for increased drop permittivity) two necks move closer to the bulbous midpart of the extended droplet, which helps enlarge two daughter drops. However, in the case of moderately conducting drops (10≤R≤20) the number of necks increased to four for increased Ca_{E}. Accordingly two pairs of symmetrical daughter drops are created because of recurrent fluctuations of the electrical shear stress and centerline momentum flux. For highly conducting cases of R>20, depending on Ca_{E}, three distinctly elongated droplet states are formed prior to breakup, which results in the onset of three different breakup modes, namely, via formations of lobed ends (Ca_{E}≤0.264), pointed ends (Ca_{E}≤0.68), and nonpointed ends (Ca_{E}>0.83). While being consistent with past measurements, here we precisely characterize the associated breakup mechanisms and physics in terms of the interactive electric pressure, electric shear stress, and hydrodynamic pressure plus velocity gradients. Since the EHD drop breakup is a dynamic process, on an elongated slender drop the activated locally distinct driving forces, i.e., electric pressure at the end regions and tangential electric stress in the midsection, effectively lead to neck formations by virtue of the created high centerline velocity gradient. Accordingly, resulting variations of local extension rate and net mass flux toward drop ends or into intermediate bulbous regions facilitate the multiple-mode drop breakup via the inertia effect, whereas the developed negative curvature around a neck encourages capillary breakup. We also explicitly reveal the effect of the viscosity contrast λ, which particularly influences the breakup characteristics over a broader range of conductivity ratios.

Phosphorylation dependent proteostasis of sterol regulatory element binding proteins.

Lipid homeostasis is critically dependent on the liver. Hepatic genes involved in lipid biosynthesis are controlled by combinatorial actions of multiple transcription factors that include three sterol regulatory element binding proteins (SREBPs), carbohydrate responsive element binding protein, liver X receptors, and others. SREBP-1c, a seminal regulator of de novo lipogenesis, resides in the endoplasmic reticulum as a transcriptionally inert precursor and must undergo a regulated intramembrane proteolysis (RIP) prior to its nuclear translocation as a bone fide transcription factor. The regulation of biosynthesis, turnover and actions of SREBP-1c and lipogenesis are mechanistically linked to signaling kinases, canonically induced by macronutrients and insulin. Here, we briefly review the evidence showing that phosphorylation of SREBP-1c and its interacting partners, catalyzed by phosphatidyl inositol-3-kinase, protein kinase B, mechanistic target of rapamycin complex 1 and 2, mitogen activated protein kinases, glycogen synthase kinase-3ß, protein kinase A and 5' adenosine monophosphate-activated protein kinase regulates the mechanisms of RIP and stability of SREBP-1c and de novo lipogenesis.

Inhibition of HBV gene expression and replication by stably expressed interferon-alpha1 via adeno-associated viral vectors.

BACKGROUND: Interferon-alpha2 (IFNalpha2) is routinely used for anti-hepatitis B virus (HBV) treatment. However, the therapeutic efficiency is unsatisfactory, particularly in East Asia. Such inefficiency might be a result of the short half-life, relatively low local concentration and strong side-effects of interferons. Frequent and repeated injection is also a big burden for patients. In the present study, a single dose of vector-delivered IFNalpha1 was tested for its anti-HBV effects. METHODS: Adeno-associated viral vector (AAV-IFNalpha1) was generated to deliver the IFNalpha1 gene into hepatocytes. IFNalpha1, hepatitis B surface (HBsAg) and e (HBeAg) antigens were measured by enzyme-linked immunosorbent assay and/or western blotting. The level of viral DNA was measured by quantitative real-time polymerase chain reaction. RESULTS: AAV-IFNalpha1 effectively transduced HBV-producing cells (HepAD38) and mouse hepatocytes, where IFNalpha1 was expressed in a stable manner. Both intracellular and extracellular HBsAg and HBeAg were significantly reduced in vitro. In the HBV-producing mice, the concentration of IFNalpha1 in the liver was eight-fold higher than that in plasma. Compared with control groups, HBeAg/HBsAg antigen levels were reduced by more than ten-fold from day 1-5, and dropped to an undetectable level on day 9 in the AAV-IFNalpha1 group. Concurrently, the level of viral DNA decreased over 30-fold for several weeks. CONCLUSIONS: A single dose administration of AAV-IFNalpha1 viral vector displayed prolonged transgene expression and superior antiviral effects both in vitro and in vivo. Therefore, the use of AAV-IFNalpha1 might be a potential alternative strategy for anti-HBV therapy.

A1762T/G1764A mutations of hepatitis B virus, associated with the increased risk of hepatocellular carcinoma, reduce basal core promoter activities.

Hepatitis B virus (HBV) infection is a major global health problem that causes over one million deaths annually. A1762T and G1764A mutations in the basal core promoter are often present in HBV patients but seldom in asymptomatic carriers, and are highly correlated with the increased risk of HBV-associated hepatocellular carcinoma (HCC). In this study, for the first time, we show that the basal core promoter activity of HBV strains isolated from asymptomatic carriers is decreased when 1762A is mutated to 1762T or 1764G is mutated to 1764A by site directed mutagenesis. By contrast, the promoter activity of HBV strains isolated from HCC patients is increased when 1762T and 1764A are reversely mutated into 1762A and 1764G, respectively. 1764G contributes more promoter activity than 1762T. We also show that T1762A and G1764A double mutations synergize the reduction of the promoter activity. A mechanism of HBV evasion from host immunoresponse that may facilitate disease development is also discussed.

Sex-specific differences in hepatic steatosis in obese spontaneously hypertensive (SHROB) rats.

BACKGROUND: Patients with metabolic syndrome, who are characterized by co-existence of insulin resistance, hypertension, hyperlipidemia, and obesity, are also prone to develop non-alcoholic fatty liver disease (NAFLD). Although the prevalence and severity of NAFLD is significantly greater in men than women, the mechanisms by which gender modulates the pathogenesis of hepatic steatosis are poorly defined. The obese spontaneously hypertensive (SHROB) rats represent an attractive model of metabolic syndrome without overt type 2 diabetes. Although pathological manifestation caused by the absence of a functional leptin receptor has been extensively studied in SHROB rats, it is unknown whether these animals elicited sex-specific differences in the development of hepatic steatosis. METHODS: We compared hepatic pathology in male and female SHROB rats. Additionally, we examined key biochemical and molecular parameters of signaling pathways linked with hyperinsulinemia and hyperlipidemia. Finally, using methods of quantitative polymerase chain reaction (qPCR) and western blot analysis, we quantified expression of 45 genes related to lipid biosynthesis and metabolism in the livers of male and female SHROB rats. RESULTS: We show that all SHROB rats developed hepatic steatosis that was accompanied by enhanced expression of SREBP1, SREBP2, ACC1, and FASN proteins. The livers of male rats also elicited higher induction of Pparg, Ppara, Slc2a4, Atox1, Skp1, Angptl3, and Pnpla3 mRNAs. In contrast, the livers of female SHROB rats elicited constitutively higher levels of phosphorylated JNK and AMPK and enhanced expression of Cd36. CONCLUSION: Based on these data, we conclude that the severity of hepatic steatosis in male and female SHROB rats was mainly driven by increased de novo lipogenesis. Moreover, male and female SHROB rats also elicited differential severity of hepatic steatosis that was coupled with sex-specific differences in fatty acid transport and esterification.

Using patient-collected clinical samples and sera to detect and quantify the severe acute respiratory syndrome coronavirus (SARS-CoV).

BACKGROUND: Severe acute respiratory syndrome (SARS) caused a large outbreak of pneumonia in Beijing, China, in 2003. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect and quantify SARS-CoV in 934 sera and self-collected throat washes and fecal samples from 271 patients with laboratory-confirmed SARS managed at a single institution. RESULTS: SARS-CoV detection rates in sera were highest in the first 9 days of illness, whereas detection was highest in throat washes 5-14 days after onset of symptoms. The highest SARS-CoV RT-PCR rates (70.4-86.3%) and viral loads (log10 4.5-6.1) were seen in fecal samples collected 2-4 weeks after the onset of clinical illness. Fecal samples were frequently SARS-CoV RT-PCR positive beyond 40 days, and occasional sera still had SARS-CoV detected after 3 weeks of illness. CONCLUSION: In the context of an extensive outbreak with major pressure on hospital resources, patient self-collected samples are an alternative to nasopharyngeal aspirates for laboratory confirmation of SARS-CoV infection.

Genetic variability of CYP2B6 polymorphisms in four southern Chinese populations.

AIM: To investigate the genotype and allelic frequencies of Cytochrome P450 2B6 polymorphisms in four southern Chinese populations. METHODS: DNA was obtained from blood samples from Han Chinese from Hong Kong and three minority groups, the Wa, Bulang and Lahu from Yunnan in southern China. Genotyping was performed using real-time PCR and confirmed by direct sequencing. RESULTS: A total of 507 subjects from southern China were studied. Results showed there is a high prevalence of 516G > T (34.5%) in ethnic Chinese compared to literature reports on other Asian populations and Caucasians. The frequency of the 516TT genotype is higher in the Han majority (23.1%) than in three other ethnic minority groups (i.e., 7.4%, 9.1% and 15.8%) in southern China. CONCLUSION: This was the first study to document the spectrum of CYP2B6 allelic variants and genotypes in a southern Chinese population. The 516G > T allele is associated with a defective metabolism of efavirenz (EFV), which therefore may predispose to drug toxicity. Treatment regimens for human immunodeficiency virus (HIV) and heroin addiction may need to be optimized in different populations because of the marked variability of the key metabolizing enzyme.

Effects of severe acute respiratory syndrome (SARS) coronavirus infection on peripheral blood lymphocytes and their subsets.

INTRODUCTION: Severe acute respiratory syndrome (SARS) caused large outbreaks of atypical pneumonia in 2003, with the largest localized outbreak occurring in Beijing, China. Lymphopenia was prominent amongst the laboratory abnormalities reported in acute SARS. METHODS: The effect of SARS on peripheral blood lymphocytes and their subsets was examined in 271 SARS coronavirus-infected individuals. RESULTS: There was a significant decrease in the CD45+, CD3+, CD4+, CD8+, CD19+ and CD16+/56+ cell counts over the five weeks of the SARS illness although CD4+/CD8+ ratios did not change significantly. The lymphopenia was prolonged, reaching a nadir during days 7-9 in the second week of illness before returning towards normal after five weeks, with the lowest mean CD4+ cell count of 317 cellsx10(6)/L at day 7, and CD8+ cell count of 239 cellsx10(6)/L at day 8. Patients with more severe clinical illness, or patients who died, had significantly more profound CD4+ and CD8+ lymphopenia. DISCUSSION: Lymphopenia is a prominent part of SARS-CoV infection and lymphocyte counts may be useful in predicting the severity and clinical outcomes. Possible reasons for the SARS-associated lymphopenia may be direct infection of lymphocytes by SARS-CoV, lymphocyte sequestration in the lung or cytokine-mediated lymphocyte trafficking. There may also be immune-mediated lymphocyte destruction, bone marrow or thymus suppression, or apoptosis.

[Inhibition of HBsAg and HBeAg secretion by RNA interference of the polymerase gene sequence of hepatitis B virus: an experimental study].

OBJECTIVE: To investigate the effects of small interfering RNA (siRNA) targeting the polymerase (P) gene sequence of hepatitis B virus (HBV) on the replication and antigen secretion of HBV. METHODS: From the 29 base sequences of the HBV in the HepG2.2.15 cells that accord with the demands of siRNA designing five sequences targeting the P gene of HBV were selected and cloned into the siRNA expressing vector pGE-1. Then the plasmid pGE-HBVP was transfected into the cultured HepG2.2.15 cells. Chemiluminescent immunoassay was used to determine the levels of HBsAg and HBeAg in the supernatant of culture medium 24, 48, 72, and 96 hours after the transfection and the expression of HBsAg in the 2.2.15 cells 24 hours after the transfection so as to observe the inhibitory effects. Untransfected cells and cells transfected with blank pGE-1 vector were used as controls. RESULTS: Five vectors expressing the siRNAs targeting the HBV P region, pGE-HBVP1-pGE-HBV5 were successfully constructed. The efficiency of transfection of the vectors into the 2.2.15 cells were 30% to 40%. 24, 48, 72, and 96 hours after the transfection of pGE-HBVP2, the strongest inhibitor among the five, the inhibitory rates of HBsAg secretion in the supernatant were 28.88%, 32.28%, 29.10%, and 18.42% respectively; and the inhibitory rates of HBeAg secretion were 38.33%, 27.50%, 33.41%, and 12.60% respectively. In view of the transfection efficiency of 30%-40%, the actual inhibitory rate of HBV antigen secretion might reach 80% and over. 24 hours after the transfection the expression rate of HBsAg in the 2.2.15 cells transfected with pGE-HBVP2 was 50%, significantly lower than that in the cells transfected with the blank vector pGE-1 (82%). CONCLUSION: siRNAs targeting the HBV P gene effectively prevent the HBV gene expression and replication and may play an important role in the clinical anti-viral treatment.

[Relationship between B virus hepatitis genotypes and therapeutic efficacy in early treatment for chronic hepatitis B by using lamivudine].

OBJECTIVE: To investigate the relationship between hepatitis B virus (HBV) genotype and therapeutic efficacy during the early phase of lamivudine treatment. METHODS: Totally 595 patients with chronic hepatitis B were treated with lamivudine 100 mg/day for 12 months. HBV genotypes, contents of HBV DNA, HBeAg/anti-HBe and YMDD mutation after lamivudine treatment for 12 months were determined. The data were analyzed with SPSS software. RESULTS: In 595 patients, 8 (1.4%) were genotype A; 53 (8.9%) genotype B; 360 (60.5%) genotype C; 112 (18.8%) were coinfection of genotype B and C; 14 (2.4%) of A and C; 15 (2.5%) A and B; 6 (1.0%) of A, B, and C, and remaining 27 (4.5%) were unspecified. Patients were treated with lamivudine 100 mg/day for 12 months. Genotype B with HBV DNA levels turned to be negative (HBV DNA < 0.1 ng/L) was 87.2%, genotype C was 89.51%, coinfection of genotype B and C was 93.04% (P > 0.05). HBeAg seroconversion of genotype B was 11.65%, of genotype C was 20.64%, and of coinfection of genotype B and C was 18.57% (P > 0.05). All 69 strains of YMDD mutation were detected after lamivudine treatment for 12 months, in which genotype B was in 16.98%, genotype C in 15.38%, and coinfection of genotype B and C was in 13.86% (P > 0.05). CONCLUSION: There was no difference in HBV genotypes and the rate of development of YMDD mutations, HBeAg seroconversion, descending of HBV DNA level in Chinese patients with chronic hepatitis B.

[Distribution and clinical significance of hepatitis B virus (HBV) genotypes and subtypes in HBV-infected patients].

OBJECTIVE: To study hepatitis B virus (HBV) genotype and subtype distribution and its clinical significance in HBV-infected patients. METHODS: We used type/subtype-specific primers and PCR to detect HBV genotype and subtype of 445 HBV-infected patients from Beijing, Changchun, Hanchuan Shenzhen, Qingyuan and Nanjing, including 7 acute hepatitis (AH), 36 asymptomatic HBV carriers (ASC), 352 chronic hepatitis (CH), 28 liver cirrhosis (LC), and 22 hepatocellular carcinoma (HCC) cases. Genotyping results were confirmed by PCR product sequencing. RESULTS: Among 445 HBV-infected patients, the proportions of genotype B, C, and B/C were 32.6% (145/445), 53.7% (239/445), and 13.7% (61/445), respectively. In genotype C, 13 (5.4%) were subtype C1, 135 (56.5%) were subtype C2, and the remaining 91 (38.1%) were neither C1 nor C2. In genotype B, 100 (69.0%) were subtype Ba, 25 (17.2%) subtype Bj, and the other 20 (13.8%) were neither Ba nor Bj. In genotype B/C, 15 (24.6%) were Ba/C2, 8 (13.1%) Bj/C2, 6 (9.8%) Ba/C1, 3 (4.9%) Bj/C1, 11 (18.0%) Ba/neither C1 nor C2, 7 (11.5%) Bj/neither C1 nor C2, and 6 (9.8%) neither Ba nor Bj/neither C1 nor C2, 2 (3.3%) neither Ba nor Bj/C1, 3 (4.9%) neither Ba nor Bj/C2. The HBV genotype and subtype distribution we found exhibited significant differences in the various clinical types of HBV infection tested, and showed that genotype C was predominant among patients with liver cirrhosis (78.6%) and hepatocellular carcinoma (86.4%) while genotype B was predominant in asymptomatic carriers (72.2%). In addition, genotype and subtype distribution showed no significant differences between male and female patients, but genotype and subtype distribution showed significant differences in patients positive or negative with HBeAg. CONCLUSION: Subtypes Ba and C2 are predominant in patients with hepatitis B from these 6 cities, and genotype C may be associated with the development of liver cirrhosis and hepatocellular carcinoma.

Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation.

Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c-SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCF(Fbw7) ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3ß at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver.

[Detection for severe acute respiratory syndrome (SARS) coronavirus RNA in stool of SARS patients].

OBJECTIVE: To investigate excretion of severe acute respiratory syndrome coronavirus RNA (SARS-CoV) in stool of SARS patients. METHODS: SARS-CoV RNA was detected in stool specimens with fluorescent quantitative polymerase chain reactions (FQ-PCR) in 101 SARS patients on the 10 to 55 days after onset, 27 non-SARS patients and 400 individuals with health check-up. RESULTS: SARS-CoV RNA was positive in stool specimens by FQ-PCR in 58 of 101 SARS patients (57.4%), and all negative in 27 non-SARS patients and 400 healthy individuals. Positive rate of SARS-CoV RNA was 100% (8/8), 67.7% (21/31), 47.4% (27/57) and 40.0% (2/5) on the 10 - 19, 20 - 29, 30 - 39 and 40 - 55 days after onset of fever, respectively, with values of logarithm of SARS-CoV RNA load of 6.06 +/- 2.05, 4.51 +/- 1.23, 3.82 +/- 1.44 and 3.57 +/- 1.25, respectively. CONCLUSION: Positive rate and load of SARS-CoV RNA in stool of SARS patients was the highest at their acute phase, and decreased with the extension of its course.