Gestational and Age-Specific Cystatin C Reference Intervals for Newborns.

OBJECTIVE: Cystatin C (CysC) references are useful for the monitoring of renal function in neonates. However, the standard CysC references in newborn infants have not been determined. The aim of this study was to establish a useful reference range for CysC in newborns. STUDY DESIGN: Serum CysC levels were measured in 1,919 blood samples from 1,044 newborns during their first 28 days of life. CysC levels were analyzed for associations between subgroups dichotomized by postnatal age (PA) and gestational age (GA). The serum CysC reference intervals were determined according to the PA and GA. The associations between the serum CysC level and other biochemical parameters as well as perinatal factors were also analyzed. RESULTS: In this study, the mean GA was 35.8 ± 2.9 weeks and the birth weight (BW) was 2,614 ± 697 g. Reference ranges of serum CysC were determined, and a general decreasing trend of CysC levels was observed as the GA increased. CysC levels differed significantly among the PA and GA categories (p < 0.001). Serum CysC levels were relatively stable throughout the GA range but were impacted by the white blood cell count within the first postnatal 24 hours. Moreover, CysC levels always correlated positively with serum creatinine concentrations (p < 0.001). Serum Cr levels were influenced by multiple factors, including BW, GA, total bilirubin, direct bilirubin, white blood cell count, C-reactive protein, and blood urea nitrogen. CONCLUSION: Reference levels of serum CysC should be determined according to the PA and GA. In contrast to Cr, serum CysC is a reliable index for assessing renal function in neonates as it is influenced by very few factors. The CysC reference levels will allow neonatologists to accurately evaluate renal function in the neonatal population. KEY POINTS: · Cystatin C is a useful marker of the glomerular filtration rate in neonates.. · A reference range for cystatin C using 1,919 blood samples of 1,044 newborns was determined.. · In contrast to creatinine, only a few nonrenal factors influence serum cystatin C..

PPARγ activation improves the microenvironment of perivascular adipose tissue and attenuates aortic stiffening in obesity.

BACKGROUND: Obesity-related cardiovascular risk, end points, and mortality are strongly related to arterial stiffening. Current therapeutic approaches for arterial stiffening are not focused on direct targeting within the vessel. Perivascular adipose tissue (PVAT) surrounding the artery has been shown to modulate vascular function and inflammation. Peroxisome proliferator-activated receptor γ (PPARγ) activation significantly decreases arterial stiffness and inflammation in diabetic patients with coronary artery disease. Thus, we hypothesized that PPARγ activation alters the PVAT microenvironment, thereby creating a favorable environment for the attenuation of arterial stiffening in obesity. METHODS: Obese ob/ob mice were used to investigate the effect of PPARγ activation on the attenuation of arterial stiffening. Various cell types, including macrophages, fibroblasts, adipocytes, and vascular smooth muscle cells, were used to test the inhibitory effect of pioglitazone, a PPARγ agonist, on the expression of elastolytic enzymes. RESULTS: PPARγ activation by pioglitazone effectively attenuated arterial stiffening in ob/ob mice. This beneficial effect was not associated with the repartitioning of fat from or changes in the browning of the PVAT depot but was strongly related to improvement of the PVAT microenvironment, as evidenced by reduction in the expression of pro-inflammatory and pro-oxidative factors. Pioglitazone treatment attenuated obesity-induced elastin fiber fragmentation and elastolytic activity and ameliorated the obesity-induced upregulation of cathepsin S and metalloproteinase 12, predominantly in the PVAT. In vitro, pioglitazone downregulated Ctss and Mmp12 in macrophages, fibroblasts, and adipocytes-cell types residing within the adventitia and PVAT. Ultimately, several PPARγ binding sites were found in Ctss and Mmp12 in Raw 264.7 and 3T3-L1 cells, suggesting a direct regulatory mechanism by which PPARγ activation repressed the expression of Ctss and Mmp-12 in macrophages and fibroblasts. CONCLUSIONS: PPARγ activation attenuated obesity-induced arterial stiffening and reduced the inflammatory and oxidative status of PVAT. The improvement of the PVAT microenvironment further contributed to the amelioration of elastin fiber fragmentation, elastolytic activity, and upregulated expression of Ctss and Mmp12. Our data highlight the PVAT microenvironment as an important target against arterial stiffening in obesity and provide a novel strategy for the potential clinical use of PPARγ agonists as a therapeutic against arterial stiffness through modulation of PVAT function.

Valproate is protective against 6-OHDA-induced dopaminergic neurodegeneration in rodent midbrain: A potential role of BDNF up-regulation.

BACKGROUND/PURPOSE: The main purpose of this study was to extend previously reported showing potent neuroprotective effect of valproic acid (VPA) in primary midbrain neuro-glial cultures to investigate whether VPA could protect dopamine (DA) neurons in vivo against 6-hydroxydopamine (6-OHDA)-induced neurodegeneration and to determine the underlying mechanism. METHODS: Male adult rats received a daily intraperitoneal injection of VPA or saline for two weeks before and after injection of 5, 10, or 15 µg of 6-OHDA into the brain. All rats were evaluated for motor function by rotarod performance. Brain samples were prepared for immunohistochemical staining and for determination of levels of dopamine, dopamine metabolites, and neurotrophic factors. RESULTS: 6-OHDA injection showed significant and dose-dependent damage of dopaminergic neurons and decrease of striatal dopamine content. Rats in the VPA-treated group were markedly protected from the loss of dopaminergic neurons and showed improvements in motor performance, compared to the control group at the moderate 6-OHDA dose (10 µg). VPA-treated rats also showed significantly increased brain-derived neurotrophic factor (BDNF) levels in the striatum and substantia nigra compared to the levels in control animals. CONCLUSION: Our studies demonstrate that VPA exerts neuroprotective effects in a rat model of 6-OHDA-induced Parkinson's disease (PD), likely in part by up-regulation BDNF.

Intracellular Chloride and Scaffold Protein Mo25 Cooperatively Regulate Transepithelial Ion Transport through WNK Signaling in the Malpighian Tubule.

Background With No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Drosophila Malpighian tubule.Methods Using in vitro assays and transgenic Drosophila lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux.ResultsIn vitro, autophosphorylation of purified Drosophila WNK decreased as chloride concentration increased. In conditions in which tubule intracellular chloride concentration decreased from 30 to 15 mM as measured using a transgenic sensor, Drosophila WNK activity acutely increased. Drosophila WNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of Drosophila WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In in vitro kinase assays, Drosophila Mo25 enhanced the activity of the Drosophila WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of Drosophila Mo25 in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type Drosophila WNK, with or without Drosophila Mo25, did not affect transepithelial ion transport, Drosophila Mo25 overexpressed with chloride-insensitive Drosophila WNK increased ion flux.Conclusions Cooperative interactions between chloride and Mo25 regulate WNK signaling in a transporting renal epithelium.

Recruitment and activation of the ATM kinase in the absence of DNA-damage sensors.

Two kinases, ATM and DNA-PKcs, control rapid responses to DNA double-strand breaks (DSBs). The paradigm for ATM control is recruitment and activation by the Mre11-Rad50-NBS1 (MRN) sensor complex, whereas DNA-PKcs requires the sensor Ku (Ku70-Ku80). Using mouse cells containing targeted mutant alleles of Mre11 (Mre11a) and/or Ku70 (Xrcc6), together with pharmacologic kinase inhibition, we demonstrate that ATM can be activated by DSBs in the absence of MRN. When MRN is deficient, DNA-PKcs efficiently substitutes for ATM in facilitating local chromatin responses. In the absence of both MRN and Ku, ATM is recruited to chromatin, where it phosphorylates H2AX and triggers the G2-M cell-cycle checkpoint, but the DNA-repair functions of MRN are not restored. These results suggest that, in contrast to straightforward recruitment and activation by MRN, a complex interplay between sensors has a substantial role in ATM control.

Attenuation of hyperoxia-induced lung injury in neonatal rats by 1α,25-Dihydroxyvitamin D3.

BACKGROUND: Mounting evidence suggests that Toll-like receptor (TLRs) plays an important role in oxidative stress and is implicated in the pathogenesis of hyperoxic lung injury. 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D, not only plays an essential role in mineral balance, but also possesses immunomodulatory and antioxidant properties. Besides, Vitamin D3 is involved in the regulation of TLRs signaling. The present study was designed to investigate whether 1,25(OH)2D3 attenuates hyperoxia-induced lung injury by regulating TLRs signaling in neonatal rats. METHODS: Pups were divided into four groups: normoxia control group (NC), normoxia plus 1,25(OH)2D3 treatment group (ND), hyperoxia control group (HC), and hyperoxia plus 1,25(OH)2D3 treatment group (HD). Lung tissues were collected for histological examination and detection of mRNA and protein expressions. RESULTS: Treatment of hyperoxia-exposed animals with 1,25(OH)2D3 resulted in significantly increased body weight and reduced hyperoxia-induced lung injury. Moreover, 1,25(OH)2D3 significantly downregulated the expression of TLR4, NF-κB, and the inflammatory cytokines TNF-α, IL-1ß, and IL-6. CONCLUSIONS: 1,25(OH)2D3 could attenuate hyperoxia-induced lung injury in neonatal rats, possibly by regulating TLR4/NF-κB signaling.

Two inwardly rectifying potassium channels, Irk1 and Irk2, play redundant roles in Drosophila renal tubule function.

Inwardly rectifying potassium channels play essential roles in renal physiology across phyla. Barium-sensitive K(+) conductances are found on the basolateral membrane of a variety of insect Malpighian (renal) tubules, including Drosophila melanogaster. We found that barium decreases the lumen-positive transepithelial potential difference in isolated perfused Drosophila tubules and decreases fluid secretion and transepithelial K(+) flux. In those insect species in which it has been studied, transcripts from multiple genes encoding inwardly rectifying K(+) channels are expressed in the renal (Malpighian) tubule. In Drosophila melanogaster, this includes transcripts of the Irk1, Irk2, and Irk3 genes. The role of each of these gene products in renal tubule function is unknown. We found that simultaneous knockdown of Irk1 and Irk2 in the principal cell of the fly tubule decreases transepithelial K(+) flux, with no additive effect of Irk3 knockdown, and decreases barium sensitivity of transepithelial K(+) flux by ∼50%. Knockdown of any of the three inwardly rectifying K(+) channels individually has no effect, nor does knocking down Irk3 simultaneously with Irk1 or Irk2. Irk1/Irk2 principal cell double-knockdown tubules remain sensitive to the kaliuretic effect of cAMP. Inhibition of the Na(+)/K(+)-ATPase with ouabain and Irk1/Irk2 double knockdown have additive effects on K(+) flux, and 75% of transepithelial K(+) transport is due to Irk1/Irk2 or ouabain-sensitive pathways. In conclusion, Irk1 and Irk2 play redundant roles in transepithelial ion transport in the Drosophila melanogaster renal tubule and are additive to Na(+)/K(+)-ATPase-dependent pathways.

Hypotonicity stimulates potassium flux through the WNK-SPAK/OSR1 kinase cascade and the Ncc69 sodium-potassium-2-chloride cotransporter in the Drosophila renal tubule.

The ability to osmoregulate is fundamental to life. Adult Drosophila melanogaster maintain hemolymph osmolarity within a narrow range. Osmolarity modulates transepithelial ion and water flux in the Malpighian (renal) tubules of the fly, which are in direct contact with hemolymph in vivo, but the mechanisms causing increased transepithelial flux in response to hypotonicity are unknown. Fly renal tubules secrete a KCl-rich fluid. We have previously demonstrated a requirement for Ncc69, the fly sodium-potassium-2-chloride cotransporter (NKCC), in tubule K(+) secretion. Mammalian NKCCs are regulated by a kinase cascade consisting of the with-no-lysine (WNK) and Ste20-related proline/alanine-rich (SPAK)/oxidative stress response (OSR1) kinases. Here, we show that decreasing Drosophila WNK activity causes a reduction in K(+) flux. Similarly, knocking down the SPAK/OSR1 homolog fray also decreases K(+) flux. We demonstrate that a hierarchical WNK-Fray signaling cascade regulates K(+) flux through Ncc69, because (i) a constitutively active Fray mutant rescues the wnk knockdown phenotype, (ii) Fray directly phosphorylates Ncc69 in vitro, and (iii) the effect of wnk and fray knockdown is abolished in Ncc69 mutants. The stimulatory effect of hypotonicity on K(+) flux is absent in wnk, fray, or Ncc69 mutant tubules, suggesting that the Drosophila WNK-SPAK/OSR1-NKCC cascade is an essential molecular pathway for osmoregulation, through its effect on transepithelial ion flux and fluid generation by the renal tubule.

Analysis of induced pluripotent stem cells from a BRCA1 mutant family.

Understanding BRCA1 mutant cancers is hampered by difficulties in obtaining primary cells from patients. We therefore generated and characterized 24 induced pluripotent stem cell (iPSC) lines from fibroblasts of eight individuals from a BRCA1 5382insC mutant family. All BRCA1 5382insC heterozygous fibroblasts, iPSCs, and teratomas maintained equivalent expression of both wild-type and mutant BRCA1 transcripts. Although no difference in differentiation capacity was observed between BRCA1 wild-type and mutant iPSCs, there was elevated protein kinase C-theta (PKC-theta) in BRCA1 mutant iPSCs. Cancer cell lines with BRCA1 mutations and hormone-receptor-negative breast cancers also displayed elevated PKC-theta. Genome sequencing of the 24 iPSC lines showed a similar frequency of reprogramming-associated de novo mutations in BRCA1 mutant and wild-type iPSCs. These data indicate that iPSC lines can be derived from BRCA1 mutant fibroblasts to study the effects of the mutation on gene expression and genome stability.

Expression of BCR/ABL p210 from a knockin allele enhances bone marrow engraftment without inducing neoplasia.

Chronic myeloid leukemia (CML) and some acute lymphoblastic leukemias are characterized by the t(9;22) chromosome, which encodes the BCR/ABL oncogene. Multiple mouse models of CML express BCR/ABL at high levels from non-Bcr promoters, resulting in the development of leukemias. In contrast, a significant fraction of healthy humans have been found to have BCR/ABL-positive hematopoietic cells. To bridge the gap between the information derived from current mouse models and nonleukemic humans with the BCR/ABL oncogene, we generated a knockin model with BCR/ABL p210 expressed from the Bcr locus. Unlike previous models, expression of BCR/ABL from the knockin allele did not induce leukemia. BCR/ABL mutant cells did exhibit favorable bone marrow engraftment compared to control cells. These data suggest that BCR/ABL expression alone is insufficient to induce disease. This model allows for inducible spatial and temporal control of BCR/ABL expression for analysis of early steps in the pathogenesis of BCR/ABL-expressing leukemias.

A polyglutamine expansion disease protein sequesters PTIP to attenuate DNA repair and increase genomic instability.

Glutamine (Q) expansion diseases are a family of degenerative disorders caused by the lengthening of CAG triplet repeats present in the coding sequences of seemingly unrelated genes whose mutant proteins drive pathogenesis. Despite all the molecular evidence for the genetic basis of these diseases, how mutant poly-Q proteins promote cell death and drive pathogenesis remains controversial. In this report, we show a specific interaction between the mutant androgen receptor (AR), a protein associated with spinal and bulbar muscular atrophy (SBMA), and the nuclear protein PTIP (Pax Transactivation-domain Interacting Protein), a protein with an unusually long Q-rich domain that functions in DNA repair. Upon exposure to ionizing radiation, PTIP localizes to nuclear foci that are sites of DNA damage and repair. However, the expression of poly-Q AR sequesters PTIP away from radiation-induced nuclear foci. This results in sensitivity to DNA-damaging agents and chromosomal instabilities. In a mouse model of SBMA, evidence for DNA damage is detected in muscle cell nuclei and muscular atrophy is accelerated when one copy of the gene encoding PTIP is removed. These data provide a new paradigm for understanding the mechanisms of cellular degeneration observed in poly-Q expansion diseases.

Chfr and RNF8 synergistically regulate ATM activation.

Protein ubiquitination is a crucial component of the DNA damage response. To study the mechanism of the DNA damage-induced ubiquitination pathway, we analyzed the impact of the loss of two E3 ubiquitin ligases, RNF8 and Chfr. Notably, DNA damage-induced activation of ATM kinase is suppressed in cells deficient in both RNF8 and Chfr (double-knockout, or DKO), and DKO mice develop thymic lymphomas that are nearly diploid but harbor clonal chromosome translocations. Moreover, DKO mice and cells are hypersensitive to ionizing radiation. We present evidence that RNF8 and Chfr synergistically regulate histone ubiquitination to control histone H4 Lys16 acetylation through MRG15-dependent acetyltransferase complexes. Through these complexes, RNF8 and Chfr affect chromatin relaxation and modulate ATM activation and DNA damage response pathways. Collectively, our findings demonstrate that two chromatin-remodeling factors, RNF8 and Chfr, function together to activate ATM and maintain genomic stability in vivo.

Identification of cancer stem-like CD44+ cells in human nasopharyngeal carcinoma cell line.

BACKGROUND AND AIMS: Recent studies suggest that cancer stem cells (CSC) may be responsible for tumorigenesis and contribute to some individuals' resistance to cancer therapy. Although research is rapidly advancing in this field, to our knowledge there are few published reports about the CSC in human nasopharyngeal carcinoma (NPC). We undertook this study to separate, expand, and explore the biological features of CD44+ stem-like cancer cells from the human NPC SUNE-1 5-8F cell line. METHODS: Immunocytochemistry and flow cytometry were used to detect the expression of CD44 in SUNE-1 5-8F. Fluorescence-activated cell sorting was applied to purify CD44+ cells. MTT assay or clone formation assay was used to detect the differences of CD44+ and CD44- cells in proliferation, differentiation, radiosensitivity and chemosensitivity in vitro. The expression of stem cell markers Oct-4 and Bmi-1 was examined by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: CD44 was positively expressed in ∼52.5% of NPC SUNE-1 5-8F cell line. Regardless of serum-free medium and serum medium culture conditions, freshly sorted CD44+ cells showed stronger proliferative capacity than CD44- and unsorted cells. The expression levels of Bmi-1 and Oct-4 mRNA in CD44+ cells were significantly higher than CD44- cells. After 2 Gy radiation, the average clone formation efficiency for CD44+ and CD44- cells was 22.17 ± 6.65% and 11.50 ± 5.00%, respectively (p <0.05). After cisplatin and docetaxel treatment with the same drug concentration, CD44+ cells showed a higher survival rate compared with CD44- cells. CONCLUSIONS: CD44+ cells have the biological characteristics of tumor stem cell and may be assumed as one of the markers of NPC tumor stem cells.

Mammalian Rif1 contributes to replication stress survival and homology-directed repair.

Rif1, originally recognized for its role at telomeres in budding yeast, has been implicated in a wide variety of cellular processes in mammals, including pluripotency of stem cells, response to double-strand breaks, and breast cancer development. As the molecular function of Rif1 is not known, we examined the consequences of Rif1 deficiency in mouse cells. Rif1 deficiency leads to failure in embryonic development, and conditional deletion of Rif1 from mouse embryo fibroblasts affects S-phase progression, rendering cells hypersensitive to replication poisons. Rif1 deficiency does not alter the activation of the DNA replication checkpoint but rather affects the execution of repair. RNA interference to human Rif1 decreases the efficiency of homology-directed repair (HDR), and Rif1 deficiency results in aberrant aggregates of the HDR factor Rad51. Consistent with a role in S-phase progression, Rif1 accumulates at stalled replication forks, preferentially around pericentromeric heterochromatin. Collectively, these findings reveal a function for Rif1 in the repair of stalled forks by facilitating HDR.

Multiple functions of MRN in end-joining pathways during isotype class switching.

The Mre11-Rad50-NBS1 (MRN) complex has many roles in response to DNA double-strand breaks, but its functions in repair by nonhomologous end joining (NHEJ) pathways are poorly understood. We have investigated requirements for MRN in class switch recombination (CSR), a programmed DNA rearrangement in B lymphocytes that requires NHEJ. To this end, we have engineered mice that lack the entire MRN complex in B lymphocytes or that possess an intact complex that harbors mutant Mre11 lacking DNA nuclease activities. MRN deficiency confers a strong defect in CSR, affecting both the classic and the alternative NHEJ pathways. In contrast, absence of Mre11 nuclease activities causes a milder phenotype, revealing a separation of function within the complex. We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the classical and alternative NHEJ pathways.

Genetic p53 deficiency partially rescues the adrenocortical dysplasia phenotype at the expense of increased tumorigenesis.

Telomere dysfunction and shortening induce chromosomal instability and tumorigenesis. In this study, we analyze the adrenocortical dysplasia (acd) mouse, harboring a mutation in Tpp1/Acd. Additional loss of p53 dramatically rescues the acd phenotype in an organ-specific manner, including skin hyperpigmentation and adrenal morphology, but not germ cell atrophy. Survival to weaning age is significantly increased in Acd(acd/acd) p53(-/-) mice. On the contrary, p53(-/-) and p53(+/-) mice with the Acd(acd/acd) genotype show a decreased tumor-free survival, compared with Acd(+/+) mice. Tumors from Acd(acd/acd) p53(+/-) mice show a striking switch from the classic spectrum of p53(-/-) mice toward carcinomas. The acd mouse model provides further support for an in vivo role of telomere deprotection in tumorigenesis.

Mre11 nuclease activity has essential roles in DNA repair and genomic stability distinct from ATM activation.

The Mre11/Rad50/NBS1 (MRN) complex maintains genomic stability by bridging DNA ends and initiating DNA damage signaling through activation of the ATM kinase. Mre11 possesses DNA nuclease activities that are highly conserved in evolution but play unknown roles in mammals. To define the functions of Mre11, we engineered targeted mouse alleles that either abrogate nuclease activities or inactivate the entire MRN complex. Mre11 nuclease deficiency causes a striking array of phenotypes indistinguishable from the absence of MRN, including early embryonic lethality and dramatic genomic instability. We identify a crucial role for the nuclease activities in homology-directed double-strand-break repair and a contributing role in activating the ATR kinase. However, the nuclease activities are not required to activate ATM after DNA damage or telomere deprotection. Therefore, nucleolytic processing by Mre11 is an essential function of fundamental importance in DNA repair, distinct from MRN control of ATM signaling.

Tpp1/Acd maintains genomic stability through a complex role in telomere protection.

Telomeres serve to protect the ends of chromosomes, and failure to maintain telomeres can lead to dramatic genomic instability. Human TPP1 was identified as a protein which interacts with components of a telomere cap complex, but does not directly bind to telomeric DNA. While biochemical interactions indicate a function in telomere biology, much remains to be learned regarding the roles of TPP1 in vivo. We previously reported the positional cloning of the gene responsible for the adrenocortical dysplasia (acd) mouse phenotype, which revealed a mutation in the mouse homologue encoding TPP1. We find that cells from homozygous acd mice harbor chromosomes fused at telomere sequences, demonstrating a role in telomere protection in vivo. Surprisingly, our studies also reveal fusions and radial structures lacking internal telomere sequences, which are not anticipated from a simple deficiency in telomere protection. Employing spectral karyotyping and telomere FISH in a combined approach, we have uncovered a striking pattern; fusions with telomeric sequences involve nonhomologous chromosomes while those lacking telomeric sequences involve homologues. Together, these studies show that Tpp1/Acd plays a vital role in telomere protection, but likely has additional functions yet to be defined.

[The effect of envelope protein mutation on hepatitis B virus assemble].

OBJECTIVE: To investigate the effect of envelope protein mutation on HBV assembly. METHODS: The envelope protein mutated vectors were constructed by the molecular clone in vitro, and then transfected transiently in the cell HepG2. The expression and secretion of S protein was assay by ELISA. HBV DNA was quantitatively evaluated by PCR. After co-transfection with pHBV-mS1S and adwR9 the DNA was quantitatively evaluated by PCR. RESULTS: There was no significant difference in expression and secretion of S protein assayed by ELISA in the cytoplasm and supernatant among pHBV-mS1, pHBV-mS, pHBV-mS1S and the wild HBV adwR9 plasmid. The DNA detected by real-time fluorescence quantitative PCR from those the cytoplasm of mutants was higher than that from the wild HBV adwR9 cytoplasm, especially from the cytoplasm of pHBV-mS1S plasmid. However, the DNA detected by real-time fluorescence quantitative PCR from the supernatant of those mutants was lower than that from the wild HBV adwR9 supernatant, especially from pHBV-mS1S. The DNA detected by real-time fluorescence quantitative PCR from the supernatant co-transfected with pHBV-mS1S and adwR9 was lower than that from the supernatant co-transfected with pcDNA3 and adwR9. CONCLUSION: There was no effect of envelope protein mutation on the expression and secretion of S protein. Envelope protein mutation could interfere the assembly of HBV particle and cause reduction of secretion of HBV.

[Replication and encapsidation of HBV mutants with the truncated C gene].

OBJECTIVE: To evaluate the replication and encapsidation of HBV mutants with the truncated C gene. METHODS: The HBV mutants with the truncated C gene were constructed by molecular cloning and PCR-based deletion in vitro. The replication and encapsidation of HBV mutants were investigated by Southern blotting, PCR and real-time fluorescence PCR respectively after transfecting the HBV mutants plasmid into HepG2 cells by using liposome. RESULTS: The C-truncated HBV mutant vectors were constructed successfully and confirmed exactly by clone sequencing and enzymes digestion. The C-truncated HBV mutants were replication defective, however, all types of HBV DNA could be detected positive in the cytoplasm and supernatant after co-transfecting the C-truncated HBV mutants plasmid and the helper constructs into HepG2 cells. The C-truncated HBV mutants were proved to produce 3-40 folds more progeny DNA than that of the wild-type HBV by DNA quantitative assay. CONCLUSION: The C-truncated HBV mutants are replication-deficient and could not replicate and encapsulate in the hepatocytes when transfected solely, however, the progeny HBV-variant viruses are encapsidated more effectively to secrete into supernatant when co-transfected with the helper construct which lacks part of 5 prime-proximal HBV RNA packaging signal Epsilon.