To investigate the potential role of hepatitis B virus core protein (HBcAg) in the pathogenesis of hepatitis B and seek for an effective therapeatic approach to the treatment of hepatitis B, the improved yeast two hybrid technique was employed to construct HBcAg bait plasmid, and the plasmids were transformed into yeast AH109. Then the yeast AH109 was mated with yeast Y187 containing liver cDNA library plasmid in 2?YPDA medium, diploid yeast was plated on both synthetic dropout nutrient medium (SD/ Trp Leu His Ade) and synthetic dropout nutrient medium (SD/ Trp Leu His Ade) containing X ? gal to be selected and screened two times. Plasmids were extracted from sixteen positive colonies, and they were sequenced. Among them, two colonies contained metallothionein. The interaction between HBcAg and metallothionein was confirmed by in vitro reticulocyte lysate translocation and immunoprecipitation. The results suggested that the pathogenesis of hepatitis B metallothionein might play an important role in the episode process.

To investigate the biological functions of hepatitis B virus e antigen (HBeAg) on pathogenesis of HBV, and to seek for effective methods to prevent and cure hepatitis B, we performed the yeast two hybrid system 3 technique to construct HBeAg bait plasmid. The bait plasmid was transformed into yeast AH109 and expressed in it. After the expression of HBeAg was identified by SDS page and Western blot, the AH109 yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 YPDA medium to form diploid yeast and plated on synthetic dropout nutrient medium (SD/ Trp Leu His Ade) and synthetic dropout nutrient medium (SD/ Trp Leu His Ade) containing x ? gal for screening. Plasmids of blue colonies were extracted and transformed into Escherichia coli, then analyzed by DNA sequencing and bioinformatics. Thirty nine positive colonies were sequenced, among them three colonies were metallothionein. To further prove the interaction between HBeAg and metallothionein, translation was performed by using reticulocyte lysate, and immunoprecipitation was showed in vitro . The results indicated that hepatitis B virus e antigen could interact with metallothionein in vivo and in vitro.

Objective To identify the arteriosclerosis (AS)changes in peripheral artery and abdominal aorta of patients with familial hypercholesterolemia(FH) during follow-up.Methods Seventeen patients of 6 FH [5 male and 12 female with average age of (16.12?6.65) years old],along with 17 subjects of matching sexes and ages with normal blood cholesterol as healthy control group,underwent examination by color doppler ultrasound,and changes of intima-media thickness (IMT) in peripheral artery and abdominal aorta,severity of stenosis,morphology,and function were observed.Results For 17 patients of FH,the total cholesterol(TC),low density lipoprotein cholesterol(LDL-C) in serum were higher significantly and high density lipoprotein cholesterol (HDL-C) was lower significantly than those in healthy control group(Pa0.05).The IMT of carotid aorta,subclavicular aorta,common abdominal artery,and common femoral artery in 13 patients were showing various degrees of increase,yielding an average of 2.9 mm.As patients aged,this phenomenon was reported to be more profound in common abdominal aorta and femoral artery.Conclusions Patients of FH show AS lesion in early stage and it worsened as they aged,from carotid arteries to common abdominal aorta and femoral artery.Color doppler ultrasound can be a non-invasive examination for monitoring the progress of AS in blood vessels in patients of diagnosed FH.

Objective To identify mutations site and clinical characteristics of a familial hypercholesterolemia(FH) proband diagnosed clinically through DNA sequencing and family analysis in the proband and his family members of 3 generations.Methods Blood samples and clinical data of the kindred of total 29 from 3 generations members were collected.Proband had a physical examination electrocar-diogrom and vascular ultrasound.The proband and his family members took routine clinical exams,and genomic DNA was isolated.The promoter region and the 18 exons of low density liporotein receptor(LDLR) gene were screened by Touch down polymerase chain reaction -single strand conformation polymorphism(PCR-SSCP) and DNA sequencing.The result of sequencing were matched gene sequence published in the BLAST database.Results 1.Increased intima-media thickness and plaque were detected in the common carotid artery,right subclavian artery of the proband.Aortic valve regurgitation was found by echocardiography.2.No mutation R3500Q of ApoB100 was observed.3.Two heterozygous mutations in exon 10 and 13 of LDLR gene (W462X and A606T) were identified.The proband and 5 members of paternal relatives showed W462X heterozygosis mutation in exon 10 of LDLR gene which introduced the change from tryptophone to a new stop codon.The proband's mother and grandmother harboured A606T heterozygous mutation in exon 13 of LDLR gene due to a single base pair substitution of G for A in the codon for residue 1 879.Conclusions Disease causing mutations of proband are W462X and A606T compound heterozygosis mutation in exon 10 and 13 of LDLR gene inherited from mother and father.Proband shows homozyous phenotype though the genotype analysis indicates heterozygous mutations.

Animals ; Disease Models, Animal ; Hypoglycemia ; chemically induced ; metabolism ; Hypothalamus ; drug effects ; metabolism ; Insulin ; pharmacology ; Intracellular Signaling Peptides and Proteins ; metabolism ; Male ; Neuropeptides ; metabolism ; Orexins ; Rats ; Rats, Sprague-Dawley

Objective To explore the molecular basis of familial hypercholesteraemia(FH)by analyzing the phenotype and genotype relationship through identify the low density liporotein receptor(LDL-r)gene mutation in a FH kindred.Methods A male patient of 15 years old was selected to examine the electrocardiogram,lipid.Color Doppler was used to examine heart and great vessels.The promoter region and the 18 exons of the LDL-r gene were screened by touch-down polymerase chain reaction(PCR)and DNA sequencing.Results The caro-tid intima-media thickness(IMT)was increased to 0.23 cm,while coronary flow velocity reserve(CFVR)was decreased to 1.57,and mode-rate mitral regurgitation was found in the proband.The genetic alteration G→A change at 1 448 of exon 10 causing premature stop codon(W462X).The same heterozygous nonsense mutation was also found in his father.The mutation had been reported in other Chinese patients.In vitro experiments showed that W462X mutation leads to low LDL binding and internalization ability.Conclusions The homozygous mutation(W462X)in exon 10 of the LDL-r gene were identified in the clinically heterozygous FH proband.The W462X mutation is the underl-ying cause of hypercholesterolaemia and clinical AS manifestations.W462X is recurrent mutation among Chinese FH patients.It might be a hot spot mutation in LDL-r in Chinese FH.J Appl Clin Pediatr,2009,24(1):18-20

Objective: To establish a mouse hepatocellular carcinoma cell line that can produce mMIP-1α and to evaluate the possibility of cancer gene therapy by mMIP-1α. Methods: mMIP-1α cDNA was cloned into retrovirus vector pBabe puro and pBabe puro-mMIP-1α was constructed, then pBabe puro-mMIP-1α was used to transfect packaging cells, anti-puromycin cells was proliferated, the supernatant was used to infect hepa1-6, the anti-puromycin clone (hepa1-6 mMIP-1α) and hepa1-6 were analysed for the expression of mMIP-1α mRNA and protein by RT-PCR and immunohistochemistry respectively. The growth curve of hepa1-6 and hepa1-6 mMIP-1α was drawn. The chemotaxis of mMIP-1α produced by hepa1-6 mMIP-1α to mouse spleen cells was observed on agarose gel. C57B/L mouse was inoculated with the tumor cell and the tumorigenicity was studied. Results: Recombinant retrovirus vector pBabe puro-mMIP-1α with mMIP-1α cDNA was constructed. Hepa1-6 did not produce mMIP-1α mRNA and protein, while hepa1-6 mMIP-1α could produce mMIP-1α mRNA and protein. The growth curve of hepa1-6 and hepa1-6 mMIP-1α showed no difference. The chemotaxis of mMIP-1α produced by hepa1-6 mMIP-1α to mouse spleen cells was observed. The tumorigenicity was reduced. Conclusion: A mouse hepatocellular carcinoma Hepa1-6 mMIP-1α is established and mMIP-1α can affect the tumorigenecity of hepa1-6.

OBJECTIVEIn recent years, the incidence of fungal infection in neonates has been risen year by year, but there are no widely accepted criteria to identify the clinical significance of the culture results of secretion from lower respiratory tract at present. The aim of the present study was to understand the clinical value and risk factors of fungal infections as suggested by sputum culture in neonates, which may help clinicians to diagnose and treat the neonates with fungal infection of respiratory tract.

METHODSOne hundred and forty nine neonates with positive sputum culture results and suffered from pneumonia hospitalized from October, 2002 to May, 2005 in Children's Hospital of Chongqing Medical University were analyzed. The cases were divided into three groups according to the results of sputum culture. Forty cases who were only fungus positive were enrolled into the fungal group, 30 cases who were positive for both fungus and bacteria were classified into the mixed group, and the remaining 79 cases who were positive for bacteria only were recruited into bacterial group. Several clinical characteristics of neonates from the three groups were compared retrospectively. t test, chi-square test and Logistic regression analysis were used for statistical analyses.

RESULTSSeventy of the 149 patients (47.0%) had fungal infections. Candida was the main genus cultured from sputum. Rate of resistance to fluconazole was 25.7% (9/35). Candida albicans was the most common strain, which was found in 56.9% of cases whose sputum culture was positive for fungi. In fungal group (n = 40), mixed group (n = 30) and bacterial group (n = 79), white blood cell count were (10.3 +/- 3.5) x 10(9)/L (11.7 +/- 5.2) x 10(9)/L and (14.4 +/- 10.5) x 10(9)/L, respectively, F = 3.78, P = 0.03, with neutrophil count (5.1 +/- 3.3) x 10(9)/L, (7.4 +/- 4.7) x 10(9)/L and (9.0 +/- 7.4) x 10(9)/L, respectively, F = 5.50, P = 0.01. Ratios of risk factors were as follows:for preterm infant, 32.5% (13/40), 20% (6/30) and 12.7% (10/79), chi(2) = 6.68, P = 0.04; for antepartum glucocorticoid used, 10.0% (4/40), 6.7% (2/30) and 0% (0/79), P = 0.01; for trilogy of antibiotics used, 10.0% (4/40), 16.7% (5/30) and 2.5% (2/79), P = 0.02; for therapy with carbapenems (Carbenin or Tienam), 32.5% (13/40), 63.3% (19/30) and 17.7% (14/79), chi(2) = 21.26, P = 0.00. There was significant difference among the three groups in the above factors respectively. Using the sputum culture results with or without fungus as the dependent variable, multivariate logistic regression showed that preterm infant (X(1)) and the therapy with carbapenems (X(2)) suited the best regression equation: Logistic (SCF) = beta(0) (0.12) + 1.63X(1) + 1.20X(2) (chi(2) = 43.04, P < 0.05).

CONCLUSIONFungal growth in sputum culture in the neonates with pneumonia was common, Candida was the main genus, and Candida albicans was the most common one. Prematurity and the therapy with carbapenems were the most important independent risk factors associated with fungal growth in culture of sputum specimen from neonates.

Anti-Bacterial Agents ; Candida ; Candidiasis ; drug therapy ; microbiology ; Carbapenems ; therapeutic use ; Child ; Clinical Laboratory Techniques ; Fungi ; Humans ; Incidence ; Infant ; Infant, Newborn ; Mycoses ; drug therapy ; microbiology ; Pneumonia ; drug therapy ; microbiology ; Risk Factors ; Sputum ; microbiology

Animals ; Behavior, Animal ; drug effects ; Depressive Disorder ; drug therapy ; psychology ; Eleutherococcus ; Male ; Plant Extracts ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Stress, Psychological

Objective To observe the effect of hypoxia reoxygenation on activity of superoxide dismutase(SOD)and MDA content as well as[Ca~(2+)],concentration and mitochondria membrane poten- tial of intestinal epithelial cell-6(IEC-6)in IEC culture medium and explore the protective effect and mechanism of serum containing Ziqi-liquid(a preparation of Chinese herbal medicine)on hypoxia reoxy- genation damaged IEC-6.Methods Hypoxia reoxygenation damage model of IEC-6 was made.SOD activity and MPA content in IEC-6 culture medium were determined by ultraviolet spectrometry after hy- poxia reoxygenation and treatment with Ziqi-liquid.Meanwhile,MMP changes and[Ca~(2+)]concentration were detected by laser scanning confocal microscopy.Results After hypoxia reoxygenation,SOD and MMP were significantly decreased,but MDA content and[ Ca~(2+)] concentration significantly increased (P＜0.01),and significantly facilitated by serum containing Ziqi-liquid.Conclusion Hypoxia reoxy- genation can damage IEC-6,but the serum containing Ziqi-liquid has significant protective effect on it.