Protein hydration in living cells probed by Fourier transform infrared (FT-IR) spectroscopic imaging.

An application of disrelation mapping to Fourier transform infrared (FT-IR) spectroscopic imaging datasets is provided to reveal different H-bonded water species within a mammalian cell. 2D correlation analysis revealed a disrelation peak at (3420 cm-1, 3220 cm-1), showing the existence of a specific water band at around 3220 cm-1 whose variation of absorbance did not follow the trend of water molecules with a well-coordinated H-bonding network. Disrelation maps constructed with disrelation intensities between (3420 cm-1, 3220 cm-1) and (3420 cm-1, 1540 cm-1) reveal that the disruption of the water network occurs around the interfacial regions between the cell and media, indicating the disintegration of the H-bonding network of bulk water due to the entrapment of water by the protein inside the cell. This hydration effect also becomes apparent around the area adjacent to the cellular nucleus, reflecting the fact that protein synthesis mainly occurs in this region. These results clearly show the presence of different molecular states of water inside living cells, which are not readily identified by conventional analysis methods.

Acute cholestatic hepatitis caused by a probable allergic reaction to paracetamol in an adolescent.

We reported on an adolescent who suffered from cholestatic hepatitis after taking a low dose of paracetamol. It was suspected that the condition was brought about by an allergic reaction to paracetamol. Paracetamol is one of the representative intrinsic hepatotoxic drugs. There have been only a few reports on liver damage due to an allergic reaction to paracetamol. There is a need to call attention to this particular reaction.

Growth inhibition and apoptosis of gastric cancer cell lines by Anemarrhena asphodeloides Bunge.

In this study, we aimed to determine the growth inhibition and the induction of apoptotic cell death brought about by the herb Anemarrhena asphodeloides Bunge in gastric cancer cell lines, and to clarify the mechanism of this apoptosis. Water-soluble ingredients of A. asphodeloides, and the gastric cancer cell lines, MKN45 and KATO-III, were used in vitro. Growth inhibition, induction of cell death, morphological features, the presence of DNA ladders, increases in caspase-3-like activity, the effects of a caspase-3 inhibitor on apoptotic cell death, and the release of cytochrome c by A. asphodeloides were analyzed. A. asphodeloides inhibited the growth and decreased the viability of the gastric cancer cell lines. The viability of normal skin fibroblasts in the presence of low concentrations of A. asphodeloides was higher than that of gastric cancer cells. Apoptotic bodies and DNA ladders were observed to be induced in MKN45 and KATO-III by A. asphodeloides. The caspase 3 inhibitor, Ac-DEVD-CHO, inhibited the apoptotic cell death of gastric cancer cells induced by A. asphodeloides. The caspase 3-like activity in MKN45 and KATO-III cells increased after the addition of A. asphodeloides. Cytochrome c was released from mitochondria into the cytosol 8 h after the addition of A. asphodeloides, and reached a peak at 16 h. The peak of cytochrome c release was earlier than that of caspase 3-like activity. We concluded that A. asphodeloides inhibited the growth of the gastric cancer cell lines MKN45 and KATO-III and induced apoptosis. The apoptosis of MKN45 and KATO-III cells induced by A. asphodeloides was associated with the release of cytochrome c from the mitochondria, followed by an increase in caspase 3-like activity.

Diversity of hepatitis G virus within a single infected individual.

The extent of population diversity among GB virus C (GBV-C)/hepatitis G virus (HGV) within a persistently infected individual (Iw) was investigated by sequence analysis of multiple clones generated from polymerase chain reaction (PCR)-amplified products of cDNA analogous to fragments of 5' non-coding region (5'NC), envelope region 1/2 (E1/E2) and non-structural region 3 (NS3) of viral genome. Although nucleotide substitutions were more common in coding regions than in the 5'NC region, there was no region corresponding to the hypervariable region of hepatitis C virus in the E1/E2 region. Transition substitution exceeded transversion by 7 to 12-fold, and 79.4% of substitutions were synonymous. This bias against substitutions producing amino acid replacements and the use of Pfu DNA polymerase with an error rate 10 times lower than the observed frequency of substitution, suggests that most substitutions were not artefactual. This data suggests that individual genomes of HGV within an infected individual may differ from each other at 0.23-0.84% nucleotide position and at 0.42-0.61% amino acid position.

Spontaneous regression of hepatocellular carcinoma and review of literature.

A 68-year-old man presented with multiple hepatocellular carcinoma, which was considered to be unresectable at the first admission in January 1994. Pathological diagnosis was made by biopsy of the one lesion among them. From January 1994 to December 1997, 10 transarterial chemoembolizations and six percutaneous ethanol injection therapies were performed on the tumours in the cirrhotic liver. In February 1998 the tumour situated in the right lobe began to increase in size. The maximum tumour diameter was 6.3 cm measured by computed tomography (CT). In the beginning of May 1998 moderate ascites was present and mild hepatic encephalopathy was noticed. The patient was in the terminal stage of hepatocellular carcinoma and no further treatment was possible at that time. However, serum alpha-fetoprotein and protein induced by vitamin K absence or antagonist II dramatically decreased in June 1998. The CT scan also showed that the tumour had completely regressed without specific treatment. In February 1999 a new biopsy-proven hepatocellular carcinoma, 2 cm in diameter, developed in the lateral segment of the liver. It was well treated by percutaneous ethanol injection therapy. The patient was alive in good condition without any symptoms or tumour recurrence in June 1999. It was concluded that a rare case of spontaneous regression of hepatocellular carcinoma had occurred.

Lamivudine as an alternative therapy for interferon-resistant chronic hepatitis B and the characteristics of hepatitis B virus: a case report.

A 27-year-old man who had been diagnosed as having chronic hepatitis B suffered disease exacerbation with marked reactivation of hepatitis B virus (HBV). Treatment with interferon (IFN) did not improve his condition, and his serum HBV DNA level increased to over 10 000 pg/ml during IFN administration. Following replacement with lamivudine, there was a substantial reduction in HBV DNA to an undetectable level, and liver function parameters subsequently improved to within the normal range. Quantitative analysis of the precore mutant HBV DNA, which is a variant that cannot express hepatitis B e antigen due to a G-to-A point mutation in the precore region of the viral genome, revealed that the amount present was greater than for the precore wild-type HBV DNA in the serum taken before IFN treatment. This case suggests that lamivudine would be an appropriate alternative to IFN, particularly in patients infected with HBV containing an excess of precore mutants resistant to IFN therapy.

Empty virus-like particle-based enzyme-linked immunosorbent assay for antibodies to hepatitis E virus.

Hepatitis E, an enterically transmitted non-A, non-B hepatitis, is a serious viral infection that occasionally causes large epidemics in developing countries. In developed countries, the disease only appears sporadically due to the transmission routes, and it is considered to be less important. The hepatitis E virus (HEV) cannot grow in cultured cells and no reliable assay system has ever been developed. In addition, the present diagnostic are not perfect, and actual rates of HEV infection may be underestimated. Highly purified empty virus-like particles (VLPs) of HEV have been produced by the use of a recombinant baculovirus vector in insect cells. Using these VLPs as an antigen, an enzyme-linked immunosorbent assay (ELISA) for antibodies to HEV was developed. A panel of 164 sera that were randomized and coded, and sera collected periodically from three patients with hepatitis E were used for the evaluation. The sensitivity of the assay was shown to be equal to or better than that obtained in previous research that used the same serum panel. The ELISA demonstrated that the serum IgM level of the patients was highest at the onset of the clinical illness and then rapidly decreased. In contrast, a high level of circulating IgG antibody titers lasted for more than 4 years. In Japan, a non-endemic country, the prevalence of the IgG class antibody to HEV in healthy individuals was found to range from 1.9% to 14.1%, depending on the geographical area. Only one out of 900 (0.1%) serum samples was IgM-positive. The IgM class antibody to HEV was detected in 10.8% of non-A, non-B, and non-C acute hepatitis patients in northeast China, whereas none of the patients in Korea had the IgM antibody. The ELISA utilizing the VLPs is sensitive and specific in its detection of the IgM and IgG antibodies to HEV. The ELISA is therefore useful for diagnosing HEV infection and for seroepidemiological study of hepatitis E.

High content of lipid hydroperoxides in livers from patients with chronic hepatitis C.

Hepatic lipid hydroperoxides, which are produced when reactive oxygen intermediates react with polyunsaturated lipids, were measured in patients with chronic hepatitis C. Eight patients with chronic persistent hepatitis (chronic persistent hepatitis group), 16 patients with chronic active hepatitis (chronic active hepatitis group), and 11 patients with cirrhosis (cirrhotic group) were included in the study. Measurements from ten patients without liver disease were used as controls. The lipid hydroperoxide levels in the chronic persistent hepatitis, chronic active hepatitis, and cirrhotic groups were significantly higher than that in the controls (p < 0.05, p < 0.005, and p < 0.005, respectively). Among the patients with chronic hepatitis C, the lipid hydroperoxide levels in the chronic active hepatitis and cirrhotic groups were significantly higher than that in the chronic persistent hepatitis group (p < 0.05 in both groups). The activity and progression of liver disease, represented by the Histology Activity Index, were significantly correlated with the hepatic lipid hydroperoxide level (r = 0.654, p < 0.01). Our findings suggest that an increased hepatic lipid hydroperoxide level is closely related to the activity and progression of chronic hepatitis C.

Clinical implications of TT virus superinfection in patients with chronic hepatitis C.

OBJECTIVE: TT virus (TTV) has been identified as a candidate agent of non-A-E hepatitis virus. We investigated superinfection of TTV in patients with chronic hepatitis C and studied the susceptibility to interferon (IFN) treatment and its association with liver disease caused by hepatitis C virus (HCV). METHODS: TTV DNA was examined using the seminested polymerase chain reaction (PCR), and its virus level was measured by the real-time fluorometric PCR. RESULTS: TTV DNA was detected in 20 of 102 (19.6%) patients examined. There was no significant difference in the alanine aminotransferase (ALT) level between patients with or without TTV DNA. Quantitative analysis of HCV RNA and TTV DNA revealed no correlation between virus levels in HCV/TTV-coinfected patients. Both TTV and HCV were sensitive to IFN therapy. Complete response to IFN with a sustained loss of viremia for 24 wk after completion of IFN treatment was found in 11 of 20 (55%) patients with respect to TTV DNA and in five of 20 (25%) patients with respect to HCV RNA. The mean pretreatment HCV RNA level was significantly lower in the complete-response cases than in the no-response cases, but there was no significant difference in the pretreatment TTV DNA levels between them. ALT normalization resulting from IFN therapy was not attributable to the eradication of TTV DNA but was attributable to that of HCV RNA. Superinfection by TTV did not influence the effect of IFN against HCV. No specific TTV genotype correlating with IFN sensitivity was found. CONCLUSIONS: These results suggest that TTV infection stands independent of HCV infection, with no influence on liver injury as a result of HCV infection.

Successful emergency treatment with a transjugular intrahepatic portosystemic shunt for life-threatening Budd-Chiari syndrome with portal thrombotic obstruction.

We report successful treatment of acute severe Budd-Chiari syndrome with portal venous thrombosis. The prognosis of patients with this condition is poor, because the therapeutic options are limited. A 38-year-old woman with polycythemia vera was admitted in a critical condition, and Budd-Chiari syndrome complicated by portal venous thrombosis was diagnosed. Tissue plasminogen activator and urokinase were infused systemically and were partially effective. Transjugular intrahepatic portosystemic shunting to reduce the high portal venous pressure was performed successfully and, eventually, her general condition improved. Our experience indicates that emergency transjugular intrahepatic portosystemic shunting is an effective therapeutic modality for controlling portal hypertension in patients with severe Budd-Chiari syndrome with portal venous thrombosis.

Value of laparoscopic microwave coagulation therapy for hepatocellular carcinoma in relation to tumor size and location.

BACKGROUND AND STUDY AIMS: The indications for laparoscopic microwave coagulation therapy (LMCT) for hepatocellular carcinoma (HCC) have not yet been adequately evaluated. This study investigated the value of LMCT in the treatment of HCC. PATIENTS AND METHODS: Forty-three patients with liver cirrhosis (including five patients in Child Pugh grade C), with 56 HCC lesions, were enrolled in the study. When dynamic computed tomography (CT) showed a loss in HCC enhancement characteristics and a low concentration area after LMCT, a lesion was judged to have undergone complete necrosis. RESULTS: The rate of complete necrosis for lesions measuring 40 mm or less was significantly higher (P<0.01) than for those measuring 41 mm or more. The rate of complete necrosis for lesions located on the liver surface, excluding those located close to the gallbladder or in contact with the diaphragm, was also significantly higher (P<0.01) than for those situated deep within the liver. The outcome for lesions of 40 mm or less was favorable. Intra-abdominal hemorrhage occurred in two patients, pneumothorax in three, and hepatic infarction in one, all associated with LMCT. However, these patients did not suffer any sequelae of clinical significance. CONCLUSIONS: This study suggests that there is a strong indication for LMCT for HCCs measuring 40 mm or less in diameter and those located on the liver surface even if they are as large as 50 mm, but not for those located close to the gallbladder or in contact with the diaphragm. LMCT appears to be applicable in patients with impaired liver function.

Endoscopic observation of squamous metaplasia of the stomach: a report of two cases.

Squamous metaplasia of the stomach is a rare clinical entity that occurs during healing of gastric ulcers or chronic inflammation. We have treated two patients with this condition, which has only occasionally been observed endoscopically. The first was a 60-year-old woman who initially presented with multiple gastric ulcers. Two months after treatment, a white patch about 4 cm in diameter was found in the lesser curvature of the cardiac region of stomach. The second patient was a 65-year-old woman, who also developed a white patch in the same region. Two months later, a small ulcer and inflamed mucosa were seen near the lesion. The white areas in both patients were stained with Lugol's iodine solution, and biopsy specimens confirmed squamous epithelium. The squamous metaplasia was observed as a white mucosal area in the stomach, and the metaplastic area stained positively with Lugol's iodine solution.

Analysis of hepatic oxidative stress status by electron spin resonance spectroscopy and imaging.

Real-time detection of free radicals generated within the body may contribute to clarify the pathophysiological role of free radicals in disease processes. Of the techniques available for studying the generation of free radicals in biological systems, electron spin resonance (ESR) has emerged as a powerful tool for detection and identification. This article begins with a review of spin trapping detection of oxygen-centered radicals using X-band ESR spectroscopy and then describes the detection of superoxide and hydroxyl radicals by the spin trap 5,5-dimethyl-1-pyrroline-N-oxide and ESR spectroscopy in the perfusate from isolated perfused rat livers subjected to ischemia/reperfusion. This article also reviews the current status of ESR for the in vivo detection of free radicals and in vivo imaging of exogenously administered free radicals. Moreover, we show that in vivo ESR-computed tomography with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl may be useful for noninvasive anatomical imaging and also for imaging of hepatic oxidative stress in vivo.

Simultaneous measurements of serum alpha-fetoprotein and protein induced by vitamin K absence for detecting hepatocellular carcinoma. South Tohoku District Study Group.

OBJECTIVE: We evaluated the measurements of serum alpha-fetoprotein (AFP) and the protein induced by vitamin K absence (PIVKA-II) in 734 patients with chronic hepatitis (CH) and liver cirrhosis (LC) who had been followed-up for the development of hepatocellular carcinoma (HCC). METHODS: Serum AFP and PIVKA-II were measured every month and abdominal ultrasonography was performed every 3 months. Youden's index (sensitivity + specificity -1) was calculated. RESULTS: On an average follow-up period of 374.5 days, HCC was detected in three HBsAg-positive LC patients (10.0%/yr), four anti-HCV-positive CH patients (1.35%/yr), 21 anti-HCV-positive LC patients (7.8%/yr), and one patient with both HBsAg- and anti-HCV-positive LC (22.7%/yr). At the time of HCC detection, the size of HCC was 4.7+/-0.6 (mean +/- SD) cm in HBsAg-positive patients and 2.4+/-1.3 cm in anti-HCV-positive patents. Cut-off values of 20 ng/ml for AFP (Youden's index = 0.422) and 60 mAU/ml for PIVKA-II (Youden's index = 0.316) gave the highest index for each marker. When these two markers were combined, cut-off values of 40 ng/ml for AFP and 80 mAU/ml for PIVKA-II gave the highest index (Youden's index = 0.500, sensitivity = 65.5%, specificity = 85.5%, positive predictable value = 14.8%, negative predictable value = 98.3%). The levels of AFP or PIVKA-II increased within three months before the detection of HCC. CONCLUSIONS: Simultaneous measurements of serum AFP and PIVKA-II levels that are performed every 3 months are useful for detecting a developing HCC. The optimal cut-off values for AFP and PIVKA-II may be 40 ng/ml and 80 mAU/ml, respectively.

Analysis of biochemical and virological efficacy of human lymphoblastoid interferon (IFN) in patients with compensated type C liver cirrhosis: comparative study between increase in individual IFN dose and prolonging of treatment period, using a multicenter randomized controlled trial.

A controlled trial was conducted to compare the efficacy of interferon (IFN) between two groups of patients with type C liver. Thirty-five patients were randomly assigned to group A (17 patients) or group B (18 patients). The former received 3 megaunits (MU) of human lymphoblastoid IFN six days per week for two weeks, followed by three days per week for 50 weeks; the latter group received 6 MU six days per week for two weeks followed by three days per week for 24 weeks. The percentages of biological sustained responders (B-SR) and virological sustained responders (V-SR) were 29.4 and 23.5%, respectively, in group B, and 17.6% for both in group A. The therapeutic effects were not different between two groups. HCV genotype 2 accounted for significantly higher percentage of B-SR and V-SR (both 57.1%, respectively). These findings indicate that IFN is effective in type C cirrhosis with genotype 2.

Ex vivo measurement of tissue distribution of a nitroxide radical after intravenous injection and its in vivo imaging using a rapid scan ESR-CT system.

To establish the usefulness of ESR-CT imaging with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) in living animals, we investigated the tissue distribution of carbamoyl-PROXYL after i. v. injection. Ten minutes after injection of carbamoyl-PROXYL, its concentrations in the liver, spleen, kidney, and plasma were higher than those in the small intestine and stomach. However, the inter-organ differences in concentrations were not striking. We selected the liver as a representative organ and attempted to measure the concentration of carbamoyl-PROXYL in it after washing out all of the blood by in situ perfusion with saline. The ESR spectrum of the liver homogenate after complete blood washout revealed that the concentration of carbamoyl-PROXYL was significantly reduced. Thus, at this time, carbamoyl-PROXYL was distributed predominantly in the plasma and/or loosely attached to the surfaces of cells. We obtained high-quality ESR-CT images of the murine abdomen at a measurement time of 40 s and found that a high-intensity area of carbamoyl-PROXYL appeared in the liver and kidneys, indicating an abundant blood circulation. Although the organ specificity of carbamoyl-PROXYL was weak, we consider that ESR-CT imaging with carbamoyl-PROXYL will be a powerful new tool for non-invasive anatomic analysis of the liver and the kidneys.