Molecular epidemiology of hepatitis E virus in Hong Kong.

Hepatitis E virus (HEV) is one of the major causes of acute and self-limiting hepatitis in human. In Hong Kong, the number of notifications increased from 26 to 62 from year 2001 to 2007. This study describes the molecular epidemiology of HEV in Hong Kong in order to determine the movement and distribution of HEV. HEV in 171 serum samples from HEV IgM positive cases from year 2001 to 2007 were amplified using RT-PCR and subjected to nucleotide sequencing. Phylogenetic analysis showed 162 of 171 HEV detected cases (94.7%) belonged to genotype IV and 8 (4.7%) to genotype I. Interestingly, a cluster of 10 cases in year 2007 that had the same sequence of HEV was identified. Epidemiological data however did not detect any relationship between these cases. Since zoonotic transmission is a well known route of HEV infection, close monitoring of the circulating HEV strains in human and food source animals may help to provide additional information on the transmission of HEV and possible source of infection in Hong Kong.

E-cadherin expression is silenced by DNA methylation in cervical cancer cell lines and tumours.

A previous study showed E-cadherin expression was lost in some cervical cancer cell lines and tumours. This study was designed to clarify the significance of DNA methylation in silencing E-cadherin expression. We examined promoter methylation of E-cadherin in five cervical cancer cell lines and 20 cervical cancer tissues using methylation-specific PCR (MSP) and bisulphite DNA sequencing. The correlation of E-cadherin methylation and expression together with methyltransferase (DNMT1) were further studied. We found that hypermethylation of E-cadherin was involved in five cervical cancer cell lines and 40% (8/20) of cervical cancer tissues. E-cadherin protein was lost in 6/8 (75%) samples and 3/5 (60%) cell lines with promoter methylation. E-cadherin methylation was significantly correlated with increased DNMT1. Using an antisense DNMT1 oligo to transfect into SiHa HeLa C33A cell line, E-cadherin protein was re-expressed. We concluded that loss of E-cadherin expression was in part correlated with DNA methylation and DNMT1 expression in cervical cancer.

Detection of human papillomavirus DNA in malignant lesions from Chinese women with carcinomas of the upper genital tract.

OBJECTIVE: The aim of this study was to determine the prevalence of high-risk oncogenic human papillomaviruses (HPVs) in malignant lesions from Hong Kong Chinese women with carcinomas of the upper genital tract. METHODS: The presence of high-risk HPVs in 55 cases of endometrial adenocarcinomas and 60 cases of primary epithelial ovarian cancers was detected by polymerase chain reaction (PCR) using consensus primers complementary to late 1 (L1) gene of the genital HPVs. Amplified PCR products were verified and typed by Southern blot analysis using (32)P-labeled DNA probes prepared from cloned HPV-16 and -18 plasmids. To confirm the presence of high-risk HPV types in the tumor tissues, PCR amplification using HPV type 16- and 18-specific primers for part of the E6 gene were also carried out. RESULTS: While HPV-18 was not detected, HPV-16 DNA sequences were identified in 5 (9.1%) of the 55 studied endometrial carcinoma samples. Of the 5 HPV-16-positive cases, there were 4 stage I, and 1 stage II endometrial cancer. In addition, 6 (10%) of the 60 epithelial ovarian carcinomas were positive for high-risk HPVs, which included 5 cases with HPV-16 and 1 case with HPV-18. Clinical staging revealed that 5 of the 6 HPV-positive cases were stage I and the remaining case was stage III ovarian cancer. Histology of the 6 HPV-positive cases showed that there were 1 case of clear-cell adenocarcinoma, 1 case of mucinous cystadenocarcinoma, and 4 cases of mucinous tumor of borderline malignancy. No other HPV types were detected. CONCLUSION: High-risk HPV was detected in approximately 10% of the tumor samples from women with upper genital tract carcinomas. As compared to the high positive rate of HPV infections in cervical cancer, it appears that HPV infection plays a relatively minor role in the pathogenesis of endometrial and ovarian carcinomas.

pRb-expressing adenovirus Ad5-Rb attenuates the p53-induced apoptosis in cervical cancer cell lines.

The retinoblastoma protein (pRb), the gene product of the first reported tumour suppressor gene, is functionally inactivated by the E7 protein of high-risk human papillomavirus (HPV) found in most human cervical cancers. We have, in this study, constructed an adenoviral vector expressing wild-type pRb (Ad5-Rb) and used the constructed Ad5-Rb to transfect the osteosarcoma cell line Saos-2, and three cervical cancer cell lines HeLa, SiHa and C-33A. Our results showed that pRb caused G1 arrest in Saos-2 cells after transfection with Ad5-Rb. The number of colonies formed by the Ad5-Rb-transfected Saos-2 cells in soft agar was also found to be significantly lower (P<0.05) than those transfected with the adenoviral control expressing Escherichia coli beta-galactosidase (Ad5-LacZ). The transfection of Ad5-Rb caused an increase in the population of SiHa and C-33A cells in the G1 phase from 53.0 and 52.9% to 72.4 and 64.3%, respectively, but not in the HeLa cells. However, Ad5-Rb did not show any inhibitory effect on the growth of SiHa, HeLa and C-33A cells, and inhibition of colony formation in soft agar was not observed either. In contrast, flow cytometric analysis showed that Ad5-p53, a p53-expressing adenovirus, induced apoptosis, i.e. the appearance of sub-G1 peak, in all three tested cervical cancer cell lines. Nevertheless, the Ad5-p53-induced apoptosis was partially inhibited when Ad5-Rb was added simultaneously. These findings suggested that pRb may not be a good candidate for cervical cancer gene therapy. Our data also showed that the use of full-length pRb in combination with TP53 might not be a suitable strategy for cancer gene therapy.

Mitomycin C and cisplatin enhanced the antitumor activity of p53-expressing adenovirus in cervical cancer cells.

This study investigated the enhanced antitumor activity of Ad5-p53 in combination with mitomycin C (MMC) or cisplatin (DDP) in cervical cancer cell lines SiHa and C-33A. MMC and DDP inhibited the growth of SiHa and C-33A cells in a dose-dependent manner, and the combination of MMC or DDP with Ad5-p53 showed a stronger growth inhibition than those treated with either Ad5-p53, MMC, or DDP alone. As evidenced by the formation of the approximately 200 bp DNA ladder and the appearance of sub-G1 peak, both MMC and DDP induced apoptosis in cervical cancer cells. Western blot analysis of p53 showed that MMC/DDP did not induce the increase of p53 protein in SiHa cells nor the increase of the cellular and nuclear p53 protein in Ad5-p53 transfected Saos-2 cells. Taken together, these results suggested that the combination of Ad5-p53 and MMC/DDP may serve as an effective therapeutic regime for human cervical cancer treatment.

A comparative study of gene expression in murine embryos developed in vivo, cultured in vitro, and cocultured with human oviductal cells using messenger ribonucleic acid differential display.

The objectives of this study were to compare the mRNA expression patterns in early mouse embryos in different culture conditions by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Embryos developed in vivo, cultured in vitro, and cocultured with human oviductal epithelial cells were studied at the 2-cell, 4-cell, 8-cell/morula, and blastocyst stages. Messenger RNA profiles were displayed by DDRT-PCR using downstream T11VV (V = A, C, or G) and upstream decamer primers. Total cDNA banding patterns were highly conserved in the three groups studied. Some fragments are unique in different culture conditions. Thirteen out of the 40 selected differentially expressed clones were characterized. The DNA sequence analyses of these clones displayed high sequence homology with cDNA sequences in the mouse expressed sequence tag database. Using semiquantitative RT-PCR, we confirmed differential expression of these DD amplicons in the three groups of embryos. The temporal expression of some of the selected DD amplicons during preimplantation development were studied in the three groups of embryos. In conclusion, DDRT-PCR is an effective tool for contrasting gene expression patterns and characterizing mRNA transcripts in mouse embryo.

P73 gene expression in ovarian cancer tissues and cell lines.

Thep73 gene, a homology of p53, is a new candidate of imprinting and tumor suppressor gene. To investigate the role of p73 in ovarian cancer, we studied the allelic expression in 56 cases of ovarian cancer using StyI polymorphism analysis. We also examined p73 expression by semi-quantitative reverse transcription-PCR as well as by Western blot analysis and DNA methylation study of the CpG island in exon 1 in ovarian cancer tissues and cell lines. Loss of heterozygosity was found in 8.3% (2 of 24) of the cases. Biallelic expression was demonstrated in 91.7% (22 of 24) of the tumor samples, in 70.8% (17 of 24) of the normal samples, and in 1 ovarian cancer cell line. Imbalanced expression and monoallelic expression were found in three and two pairs of matched samples, respectively. Overexpression of p73 was found in advanced ovarian cancer rather than in early-stage disease or in borderline ovarian tumor. No significant difference was found in the p53 expression. Three cell lines with absent p73 protein expression and one tumor sample with monoallelic expression were methylated in the CpG island. Demethylation in SKOV3 cell line using 5-azacytidine can reactivate the expression of this gene in both the mRNA and the protein level. Our results indicated that p73 was not imprinted in most of the ovarian cancer and normal tissues, but it could be involved in the advanced ovarian cancer through overexpression. DNA methylation may contribute to the lack of p73 expression.

Changes in p21WAF1, pRb, Mdm-2, Bax and Bcl-2 expression in cervical cancer cell lines transfected with a p53 expressing adenovirus.

The aim of this study was to provide some insights into the molecular mechanisms involved in p53-dependent apoptosis and growth arrest. Changes in the levels of p53 protein and proteins regulated by p53 were studied in relation to events of the cell cycle and apoptosis in cervical cancer cell lines upon transfection with a p53 expressing adenovirus (Ad5-p53). The post-transfection level of p53 protein in SiHa cells was found to be unchanged during the 24-48 h period. In contrast, the level of p21WAF1 protein was shown to increase to its highest level at 24 h, and decreased gradually up to 48 h after the Ad5-p53 transfection. We further noted that the increase of p21WAF1 was accompanied by G1 arrest at 24 h and the decrease of p21WAF1 was associated with apoptosis at 36-48 h after transfection. An anti-p21WAF1 antibody cross-reactive protein band of approximately 14 kDa was observed in HeLa and C-33A cells when these cells were committed to apoptosis upon Ad5-p53 transfection. In SiHa cells, phosphorylation of pRb was inhibited during the early stage of Ad5-p53 transfection. This was followed by the cleavage of pRb. However, Ad5-p53 transfection did not change the levels of Bax and Bcl-2 proteins. Our results suggested that, Bax and Bcl-2 may not be important for the apoptosis of these cells, whereas cleavage of Rb, and the decrease of p21WAF1 could play important roles in p53-dependent apoptosis.

Loss of imprinting of the IGF-II and H19 genes in epithelial ovarian cancer.

To establish a possible role of genomic imprinting in the carcinogenesis of epithelial ovarian cancer, we determined the imprinting status of both IGF-II and H19 genes in 43 ovarian cancers, 7 low malignant potential ovarian tumors, and their matched normal tissues. In ovarian cancer, loss of heterozygosity (LOH) of IGF-II, H19 RsaI, and H19 AluI was found in 4 of 24 (16.7%), 3 of 20 (15%), and 1 of 16 (6.3%) samples, respectively. All patients with tumor specimens exhibiting LOH are of advanced clinical stages. Loss of imprinting (LOI) was found in 5 of 20 (25%) for IGF-II and in 4 of 17 (23.5%) and 1 of 15 (6.7%) for the RsaI and AluI sites of H19 gene with no LOH. However, no LOH was found in low malignant potential tumors, and only one of them showed LOI in H19 AluI site. Overexpression of IGF-II was demonstrated in all five LOI samples with normal expression of H19. Three of the five tumor specimens exhibiting LOI were transcribed from P1 promoter, whereas the remaining two were from the P3 promoter. These results suggested that LOH of both IGF-II and H19 genes was associated with advanced ovarian cancer. LOI of IGF-II and H19 genes may be involved in the development of ovarian cancer. Transcription of IGF-II from the P1 promoter may account for the biallelic expression of the IGF-II gene.

Glycoproteins present in human follicular fluid that inhibit the zona-binding capacity of spermatozoa.

Previous studies have suggested that human follicular fluid contains factors that reduce the zona-binding capacity of spermatozoa. The present study provides further evidence of the existence of such factors. Using the hemizona binding assay (HZA), we have shown that the inhibitory effect of human follicular fluid on the zona-binding capacity of spermatozoa is concentration-dependent, an inhibitory effect being detected when the concentration of human follicular fluid was > or = 10%. A 1% concentration of human follicular fluid did not possess this inhibitory activity. Heating human follicular fluid at 56 degrees C for 30 min did not affect its inhibitory properties; treatment with proteinase-K abolished such inhibition. Human follicular fluid was fractionated sequentially by concanavalin-A affinity chromatography, Mono Q ion-exchange chromatography and Superose-12 gel filtration. The zona binding inhibitory activity resided in the fraction which bound to the lectin and Mono Q column and contained molecules with native molecular weights of 32 and 192 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis suggested that the 192 kDa glycoprotein was a tetramer, while the 32 kDa glycoprotein remained as a single molecular species under denaturing conditions. We conclude that two glycoproteins were responsible for the zona binding inhibitory activity of human follicular fluid. The physiological role of these factors remains unclear.

Laparoscopic-assisted vs. open surgery for colorectal cancer: comparative study of immune effects.

PURPOSE: Our aim was to test the hypothesis that laparoscopic-assisted resection for colorectal cancer has an immunologic advantage over traditional open surgery. METHODS: Sixteen patients with colorectal cancer were randomized to undergo laparoscopic-assisted resection or open surgery. Basic patient data were recorded, and serum interleukin-6 levels, relative proportions of lymphocytes, and human leukocyte antigen-DR expression on monocytes were determined at specific time intervals. RESULTS: Operating time was longer for laparoscopic-assisted resection (P=0.02), but analgesic requirements were less (P=0.04). All patients exhibited the following: interleukin-6 levels increased to a maximum at 4 hours and returned to preoperative levels within 48 hours. This response appeared greater for open resection (mean peak level, 313 vs. 173 pg/ml; P=0.25). Relative granulocytosis (P < 0.001) was seen within 48 hours, which was offset by a decrease in percentage of lymphocytes (P < 0.001). Changes in lymphocyte subfractions were most significant seven days postsurgery: natural killer cells decreased (P=0.003); T cells increased (P=0.008), with elevation in the CD4/CD8 ratio (P=0.003). B cells were largely unchanged at all time periods. Human leukocyte antigen-DR expression on monocytes was significantly less at 48 hours postsurgery (P < 0.001). All changes were reversed within three weeks of surgery. There were no differences when comparing laparoscopic-assisted resection with open surgery. CONCLUSIONS: Both laparoscopic-assisted resection and open surgery affect the immune response. It would appear that laparoscopic-assisted resection does not have an immunologic advantage over open surgery in patients with colorectal cancer.

Helicobacter pylori and the null genotype of glutathione-S-transferase-mu in patients with gastric adenocarcinoma.

BACKGROUND: Chronic Helicobacter pylori infection now is recognized as an important causative agent for gastric carcinoma. However, only a small minority of infected individuals develop the malignancy, even in areas with a high prevalence of gastric carcinoma. It has been postulated that the absence of glutathione-S-transferase-mu (GST-mu), which impairs detoxification of exogenous carcinogens, might predispose some infected individuals to the development of gastric carcinoma. METHODS: Patients with histologically confirmed adenocarcinoma of the stomach were tested for H. pylori infection and the GST-mu genotype. Prevalence of GST-mu gene deletion was compared with the H. pylori status of the patients. A group of gender- and age-matched control subjects with known H. pylori-related nonulcer dyspepsia also were tested for the GST-mu genotype and compared with patients with H. pylori positive carcinoma. RESULTS: Fifty-one patients with gastric adenocarcinoma were enrolled into the study. Thirty-five were found to have H. pylori in the resected specimens. The null genotype of GST-mu was significantly more common among those patients with H. pylori positive carcinoma compared with the H. pylori negative group (65.7% vs. 31.3%; P < 0.05). Homozygous deletion of GST-mu was significantly higher in the H. pylori positive carcinoma patients than in the H. pylori-infected, nonmalignant control group (65.7% vs. 37.1%; P < 0.05). CONCLUSIONS: The null genotype for GST-mu is found more commonly in gastric carcinoma associated with H. pylori infection. The absence of the GST-mu enzyme may increase the risk of the development of gastric carcinoma in these patients.

The time course and regional variations of lipid peroxidation after diffuse brain injury in rats.

Free radicals are generated after head injury. These radicals rapidly react with polyunsaturated fatty acids in the cell membrane and cause membrane destruction. This process is called lipid peroxidation. Malondialdehyde (MDA) is one of the end products of lipid peroxidation, and it is a frequently used indicator of lipid peroxidation in biological tissues. Using a diffuse head injury animal model, we studied the time course of lipid peroxidation in different regions of injured rat brains. In the present study, the MDA levels were 36.7%, 41.8%, and 35.1% greater than sham at one hour after injury at the frontal, parietal, and brain stem, respectively (p < 0.0001). The MDA levels in these regions continued to increase and peaked a 4 hours after the injury. The levels slowly decreased, and by 24 hours, they were still significantly higher than the sham control's. The elevation of MDA levels was less in the striatum and the temporal regions at one hour. They were 16.9% and 13.3%, respectively (p < 0.002). The MDA levels in these two regions continued to increase even after 4 hours of injury, but the degree of elevation never exceeded 35%. The results demonstrate that there is an immediate, posttraumatic burst of MDA production, suggesting the formation of free radicals after diffuse head injury. Even though all the regions sampled show the same effect, certain regions are less affected by this diffuse head injury animal model.

Effect of carbon dioxide pneumoperitoneum on bacteraemia and endotoxaemia in an animal model of peritonitis.

Laparoscopy is increasingly used in conditions complicated by peritonitis. A theoretical concern is that carbon dioxide pneumoperitoneum may increase bacteraemia. This study examines the effect of carbon dioxide pneumoperitoneum on bacteraemia, endotoxaemia and physiological correlates of sepsis in an animal model of peritonitis. New Zealand white rabbits were assigned to three groups of six animals. Group 1 received an intraperitoneal inoculation of 10(9) colony-forming units of Escherichia coli followed by a 10-cm midline laparotomy. Group 2 received an identical bacterial inoculum followed by a 12-mmHg carbon dioxide pneumoperitoneum for 1 h. Group 3 received no bacteria but had a 12-mmHg carbon dioxide pneumoperitoneum for 1 h. Groups 1 and 2 had significantly higher levels of bacteraemia (P < 0.01) and endotoxaemia (P < 0.01) accompanied by significantly lower mean arterial pressures (P < 0.05) and higher heart rates (P < 0.05) compared with group 3. After 6 h groups 1 and 2 were significantly hypocarbic (P < 0.01), leucopenic (P < 0.01) and thrombocytopenic (P < 0.01). There was no difference between group 1 and group 2. A carbon dioxide pneumoperitoneum of 12 mmHg does not increase bacteraemia or endotoxaemia, nor does it adversely affect physiological or laboratory correlates of sepsis compared with laparotomy in this animal model of peritonitis.

The growth and behaviour of pig retinal pigment epithelial cells in culture.

The retinal pigment epithelium contains melanin and lipofuscin. It is believed that in the in vivo system, the incomplete degradation of phagocytosed outer segment discs leads to the formation of lipofuscin. Our results showed that pig RPE cells can be successfully cultured using standard culture techniques without addition of specific growth factors. In this system, the autofluorescent material is formed mainly from the degradation of pigment granules. This culture system may provide an excellent model for studying of diseases related to the retina.

Systemic absorption of epinephrine after endoscopic submucosal injection in patients with bleeding peptic ulcers.

Epinephrine injection is an effective, simple, and economical method of endoscopic hemostasis for bleeding peptic ulcers. We measured catecholamine levels in 18 patients with actively bleeding ulcers (8 gastric ulcers and 10 duodenal ulcers) treated by endoscopic injection. Injection of epinephrine (1:10,000 IU) was given until bleeding from the ulcers stopped. Catecholamine levels were assayed by high-pressure liquid chromatography. Immediately after the injection the plasma level of epinephrine rose by four to five times above the basal level and returned to the baseline in 20 minutes. Norepinephrine levels were not significantly raised in these patients. No cardiovascular complications were seen. Although adverse cardiac events have not been recorded, it seems prudent to monitor these patients closely during and immediately after epinephrine injection.

Generalization of EMG biofeedback training.

Five young adults received audio biofeedback training to reduce trapezius EMG levels while they engaged in reading in an office, seated at a table. A multiple-baseline-across subjects design was employed in two separate studies. After training, all subjects demonstrated reduced EMG levels while reading in a home or library setting. The first study suggested that subjects reduced EMG levels by minimizing movements and altering their postures; the second study systematically demonstrated changes in such behavior, which was correlated with EMG levels. The data provide evidence that EMG biofeedback resulted in response generalization across several motoric classes, and in stimulus generalization from the training setting to the natural environment. The importance of assessing generalization is discussed.

Purification and characterization of thioredoxin from the N2-fixing cyanobacterium Anabaena cylindrica.

Thioredoxin has been purified to homogeneity from the cyanobacterium Anabaena cylindrica. The protein consists of a single polypeptide chain with a relative molecular mass of about 11 680 which has two cysteine residues (residues 31 and 34) in the sequence-Cys-Gly-Pro-Cys- and an isoelectric point at pH 4.55. The N-terminal amino acid sequence of 39 residues shows distinct homologies with the sequences of Escherichia coli and Corynebacterium nephridii thioredoxins. Anti-(A. cylindrica thioredoxin) antiserum was used to quantify the thioredoxin which constituted about 0.22% of the soluble protein in cell-free extracts of N2-fixing, NO3- -grown or NH4+-grown A. cylindrica. Activation of fructose-1,6-bisphosphatase of A. cylindrica, activation of glutamine synthetase and NADP+-dependent malate dehydrogenase of the green alga Scenedesmus obliquus but not of A. cylindrica, and deactivation of glucose-6-P dehydrogenase of the cyanobacterium Anabaena variabilis were all achieved using the same thioredoxin species. No other thioredoxin species were detected in extracts of A. cylindrica when examined for the activation of these enzymes.

The role of specific cations in regulation of cyanobacterial glutamine synthetase.

Purified glutamine synthetase from the cyanobacterium Anabaena cylindrica required a divalent cation for activity. Maximum biosynthetic activity required Mg2+ (25 mM when supplied alone). Co2+ and Mn2+ each supported up to 20% of this activity; 12 other cations tested were ineffective. At 2.5 - 10 mM Mg2+, 0.1 mM Co2+ or ethylene glycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) stimulated GS activity to maximum rates; other divalent cations (particularly Mn2+) inhibited Mg2+-dependent activity. At 5 mM Mg2+ the Kappm for NH+4 (0.05 mM) was 20-fold lower than at 25 mM Mg2+; added Co2+ did not markedly alter this low Km for NH+4; this could be physiologically important.

Hemagglutinin typing as an aid in identification of biochemically atypical Escherichia coli strains.

Tests for the presence of mannose-sensitive and mannose-resistant, eluting hemagglutinins and fimbriae were helpful in indicating whether biochemically atypical strains of the tribe Escherichieae might be escherichiae or shigellae.