Apical membrane rupture and backward bile flooding in acetaminophen-induced hepatocyte necrosis.

Morphological changes of hepatocyte death have so far only been described on cells in culture or in tissue sections. Using a high-resolution and high-magnification multiphoton microscopic system, we recorded in living mice serial changes of acetaminophen (APAP)-induced hepatocyte necrosis in relevance to metabolism of a fluorogenic bile solute. Initial changes of hepatocyte injury included basal membrane disruption and loss of mitochondrial membrane potential. An overwhelming event of rupture at adjacent apical membrane resulting in flooding of bile into these hepatocytes might ensue. Belbs formed on basal membrane and then dislodged into the sinusoid circulation. Transmission electron microscopy disclosed a necrotic hepatocyte depicting well the changes after apical membrane rupture and bile flooding. Administration of the antidote N-acetylcysteine dramatically reduced the occurrence of apical membrane rupture. The present results demonstrated a hidden but critical step of apical membrane rupture leading to irreversible APAP-induced hepatocyte injury.

Vertebral bone marrow perfusion evaluated with dynamic contrast-enhanced MR imaging: significance of aging and sex.

PURPOSE: To investigate blood perfusion of nonfractured, normal-appearing vertebral bodies with regard to age and sex. MATERIALS AND METHODS: Dynamic magnetic resonance imaging (160 images obtained in 80 seconds) was performed from T10 to L5 in 66 patients. Patients were assigned to three groups: group 1, those 50 years or younger without compression fracture; group 2, those older than 50 years without compression fracture; or group 3, those older than 50 years with compression fracture. Peak enhancement percentage and enhancement slope were determined from the time-intensity curve of normal (nonfractured) vertebral body. Comparisons were made between groups, and the effect of age and sex interaction was analyzed. RESULTS: Higher peak enhancement percentage was demonstrated for group 1 compared with group 2 (58.21 +/- 44.65 [SD] vs 21.88 +/- 14.77, P <.005). Group 1 women revealed a higher enhancement percentage compared with group 1 men (87.17 +/- 54.13 vs 38.16 +/- 21.69, P <.05), which significantly decreased in those older than 50 years (from 87.17 +/- 54.13 to 17.98 +/- 13.80, P <.005). For men, this decrease in those older than 50 years was not as pronounced (from 38.16 +/- 21.69 to 25.38 +/- 15.43, P >.05). Presence of compression fracture at other levels of the spine (group 3) was not associated with a different enhancement percentage for normal vertebrae. CONCLUSION: Rate of vertebral bone marrow perfusion revealed a significant decrease in subjects older than 50 years. Women demonstrated a higher marrow perfusion rate than men younger than 50 years and a more marked decrease than men older than 50 years.

Paget's disease of the bone: a case report.

Paget's disease of bone is rare in Asia. We report a case of Paget's disease in a 58-year-old Taiwanese man who was admitted with a 3-month history of bilateral numbness in the buttock region. Laboratory data disclosed an elevated serum alkaline phosphatase level (510 U/L). Plain radiographs of the lumbar spine showed generalized increased density at the third lumbar vertebra, associated with cortical thickening, loss of cortico-cancellous definition, and increased anteroposterior diameter. The T1-weighted magnetic resonance image of the lumbar spine showed diffuse, heterogeneous low signal intensity at the third lumbar vertebral body, pedicle, laminae, and spinal process; these areas showed mixed high and low signal intensity on the T2-weighted image. Technetium-99m bone scan revealed abnormal uptake in the involved vertebra. Histologic examination of the third lumbar spinal process confirmed the diagnosis of Paget's disease of bone. The patient remained well during a follow-up period of 6 months.

Morphometric analysis of argyrophilic nucleolar organizer regions (AgNORs) in nasopharyngeal carcinoma.

BACKGROUND: Nucleolar organizer regions (NORs), which are loops of DNA containing ribosomal RNA genes, have been shown to correlate with cell proliferation and malignant transformation. Conventional eyeball measurement of silver staining NORs (AgNORs) is time-consuming and subject to error. The diagnostic value of AgNOR area in nasopharyngeal carcinoma (NPC) by computer-assisted morphometric analysis is evaluated. METHODS: Silver-staining of NORs was applied to 23 paraffin sections of NPC containing both normal squamous epithelial and malignant cells. Various parameters of the AgNORs of these two cell types were analyzed by a computer-assisted image analysis system and then compared. RESULTS: The mean AgNOR area, AgNOR/nuclear area ratio, and AgNOR area/count ratio of malignant tumors were statistically significantly higher than those for the normal epithelium. There was no significant difference in the AgNOR counts between the two cell types. CONCLUSIONS: Computer-assisted morphometric analysis of AgNOR is an objective and reliable assessment method applicable to paraffin sections of NPC. The AgNOR area and its derivatives may aid in the diagnosis of NPC.

Water clusters in life.

The role of water in chemical, biochemical and cellular events has only been recognized as a universal solvent. The conventional wisdom holds that water is a passive agent in biological interaction. However, more and more researchers regard water as an active component in biochemical reactions and hence occupy a crucial role in life. We propose that the active component of water is due to the existence of stable water clusters in aqueous solutions. Our research demonstrated that stable water clusters could be produced in very dilute inorganic and organic water solutions, and also isolated from biological fluids such as bovine serum. Stable water clusters may play an important role in physiological and pathological processes of life.

Amino acid substitutions in codons 9-11 of hepatitis C virus core protein lead to the synthesis of a short core protein product.

BACKGROUND: Previous in vitro experiments have indicated that if the ninth codon of the hepatitis C virus (HCV) core gene is mutated from arginine to lysine, a short 16-kDa (P16) instead of a 21-kDa (P21) core protein will be produced. In this study, we aimed to investigate whether similar mutations existed in patients with chronic HCV infection and whether such mutations led to the expression of P16. METHODS: The core gene was isolated from patients' sera by reverse transcription-polymerase chain reaction and sequenced. RESULTS: Three of 10 patients with hepatocellular carcinoma were found to have mutant viruses with missense mutations at codons 9-11: arginine-to-glycine mutation at codon 9 (case 1); lysine-to-glutamine mutation at codon 10 (case 5); and lysine-to-asparagine/threonine-to-alanine double mutations at codons 10 and 11 (case 8). Site-directed mutagenesis and in vitro translation experiments revealed that P16 was expressed by all three mutants. Using gel-purified P21 and P16 proteins obtained from transformed Escherichia coli, the serum titres of anti-P21 and anti-P16 were assayed. Unequal titres of anti-P16 and anti-P21 were found in only cases 1, 5 and 8. A rabbit antibody directed against P16 but not P21 was thus generated for immunohistochemical analysis. P16 was detected in the nuclei of hepatocytes in the peri-hepatoma tissue of a single case (case 1). CONCLUSIONS: These data indicate that missense mutations at codons 9-11 can occur during chronic HCV infection, which results in the expression of P16 core protein.

Activation of p53 tumor suppressor by hepatitis C virus core protein.

In addition to being a structural protein that packages the viral genomic RNA, hepatitis C virus (HCV) core protein possesses regulatory functions. In this report, we demonstrate that the HCV core protein could enhance the gene transactivation activity of the tumor suppressor p53, regardless of whether p53 was derived from an exogenous or an endogenous gene. The activation of p53 by the HCV core protein was supported by the observation that the HCV core protein could enhance the expression of p21(waf1/Cip1), a downstream effector gene of p53, in a p53-dependent manner. Further studies indicated that the HCV core protein could also suppress hepatocellular growth via p53. The HCV core protein and p53 could bind to each other in vitro, which was evidenced by the coimmunoprecipitation, the GST pull-down, and the Far-Western blot assays. The deletion-mapping analysis indicated that the carboxy-terminal sequence of p53 located between amino acids 366 and 380 was required for the core protein binding. These results raised the possibility that the HCV core protein might activate p53 through direct physical interaction. The persistent perturbation of p53 activity by the HCV core protein during chronic infection may have important consequences in HCV pathogenesis.

Prevalence of TT virus DNA in eastern Taiwan aborigines.

We studied the prevalence of TT virus (TTV) DNA in the general population of the eastern Taiwan aborigine villages, about 11% (34 of 317). There is no association between the presence of HBsAg and TTV DNA or between the presence of HCV RNA and TTV DNA. Therefore, the infection of HBV or HCV and the presence of TTV DNA appear to be independent from each other. The association between the presence of TTV DNA and the elevated alanine aminotransferase (and/or aspartate aminotransferase) activity was also investigated. The presence of TTV DNA was not found to be correlated with abnormal liver function (P = 0.574) when age, gender, and the presence of HBsAg, HCV RNA, and HGV RNA were all considered in the assay. The sequence homology of TTV DNA fragments between different isolates from Taiwan and N22 (the clone obtained from the original prototype strain) from Japan ranged from 84 to 97%. The recombinant protein encoded by the TTV DNA fragment corresponding to the open reading frame of N22 was expressed in E. coli successfully. However, no serum response against the recombinant protein was detected.

Expression and Dimerization of Hepatitis C Virus Core Protein in E. coli.

Hepatitis C virus (HCV) is a positive-stranded RNA virus with a genome size of about 9-10 kb. The genome of this virus encodes a polyprotein with a length of over 3000 amino acids. This polyprotein is cleaved by cellular and viral proteases to generate at least 10 viral gene products. Recent reports have indicated that there are extensive interactions between various HCV proteins: the core (capsid) protein can interact with itself (1) and with the El envelope protein (2); El protein can interact with the E2 envelope protein (3,4) which in turn can be covalently linked to its following p7 protein and interact with another integral membrane protein named NS2 (5); NS2 can also interact with NS5A and NS5B nonstructural proteins (6); and NS3 proteinase/helicase has also been shown to complex with the NS4A protein (6,7). Thus, most of the known HCV proteins interact with at least another HCV protein. These interactions are presumably very important for morphogenesis and replication of HCV.

A randomized comparison of acute phase response and endotoxemia in patients with perforated peptic ulcers receiving laparoscopic or open patch repair.

BACKGROUND: In patients with peritonitis from perforated peptic ulcers, we compared acute stress responses, endotoxemia, and bacteremia following laparoscopic or open surgery. PATIENTS AND METHODS: Consecutive patients with peritonitis from perforated peptic ulcers were randomized to receive laparoscopic sutured or open omental repair. Undiluted peritoneal fluid was obtained at surgery for quantitative bacterial and endotoxin (Limulus Amoebocyte Lysate) assay. Serial blood samples were taken at 0, 30, 60, 90, 120, and 180 minutes, and at 12, 24, 48, 72, and 120 hours for determinations of quantitative bacterial and endotoxin assays, interleukin-6 (IL-6), C-reactive protein (CRP), and cortisol. RESULTS: Twenty-two patients were randomized: laparoscopy group (n = 12), open repair group (n = 10). Conversions were required in 3 patients assigned to laparoscopy, leaving 9 patients for analysis. The two groups were comparable in their demographic data, median duration of perforation (13.5 hours versus 10 hours), severity of peritoneal contamination as indicated by viable bacterial count (5.9 x 102 versus 1.5 x 10(2) colony forming unit/mL) and endotoxin concentration in peritoneal fluid (27.2 versus 24.6 EU/mL). No significant endotoxemia or bacteremia was detected in these patients. Median interleukin-6 was highest at 0 hour (1520 versus 962 pg/mL) and fell rapidly following surgery. C-reactive protein peaked at 24 hours and plateaued thereafter. Cortisol was highest intraoperatively and fell thereafter. No difference was noted between the two treatment groups with respect to these inflammatory markers (IL-6 P = 0.19, CRP P = 0.14, cortisol P = 0.56, multivariate analysis of variance). CONCLUSION: Endotoxemia and bacteremia are insignificant in most patients with perforated peptic ulcers. In patients with perforated peptic ulcers, laparoscopic patch repair does not reduce acute stress responses when compared with open surgery.

Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-beta receptor.

Hepatitis C virus (HCV) core protein is a multifunctional protein. We examined whether it can interact with cellular proteins, thus contributing to viral pathogenesis. Using the HCV core protein as a bait to screen a human liver cDNA library in a yeast two-hybrid screening system, we have isolated several positive clones encoding cellular proteins that interact with the HCV core protein. Interestingly, more than half of these clones encode the cytoplasmic domain of lymphotoxin-beta receptor (LT betaR), which is a member of the tumor necrosis factor receptor family. Their binding was confirmed by in vitro glutathione S-transferase fusion protein binding assay and protein-protein blotting assay to be direct and specific. The binding sites were mapped within a 58-amino-acid region of the cytoplasmic tail of LT betaR. The binding site in the HCV core protein was localized within amino acid residues 36 to 91 from the N terminus, corresponding to the hydrophilic region of the protein. In mammalian cells, the core protein was found to be associated with the membrane-bound LT betaR. Since the LT betaR is involved in germinal center formation and developmental regulation of peripheral lymphoid organs, lymph node development, and apoptotic signaling, the binding of HCV core protein to LT betaR suggests the possibility that this viral protein has an immunomodulating function and may explain the mechanism of viral persistence and pathogenesis of HCV.

Interaction between hepatitis C virus core protein and E1 envelope protein.

Hepatitis C virus has three structural genes named C, E1, and E2. The C gene encodes the core (capsid) protein and the E1 and E2 genes encode the envelope proteins. In an immunoprecipitation experiment, the E1 protein was found to be precipitated by an anti-core antibody in the presence but not in the absence of the core protein, indicating that the E1 protein can interact with the core protein. This interaction is independent of whether the E1 and the C genes are linked in cis or separated in different DNA constructs for expression. The interaction between the core and the E1 proteins is confirmed by the observation that a hybrid protein derived from the core protein and the tissue plasminogen activator is localized in the nucleus in the absence of the E1 protein and in the perinuclear region in the presence of the E1 protein. Deletion-mapping studies indicate that the carboxy-terminal sequences of both the core and the E1 proteins are important for their interaction. Since little E1 sequence is exposed on the cytosolic side of the membrane of the endoplasmic reticulum, the interaction between the core and the E1 proteins most likely takes place in the endoplasmic reticulum membrane. The E2 protein could not be coprecipitated with the core protein by the anti-core antibody in a similar assay and likely does not interact with the core protein. The implications of these findings on the morphogenesis of the hepatitis C virus virion are discussed.

Differential subcellular localization of hepatitis C virus core gene products.

The expression of the core gene of two different hepatitis C virus (HCV) isolates was analyzed. In the presence of its downstream E1 envelope protein sequence, two major core protein products with molecular masses of 21 kDa (P21) and 19 kDa (P19) and a minor protein product with molecular mass of 16 kDa (P16) were detected. In the absence of its downstream E1 envelope protein sequence, P21 and P19 remained the major protein products expressed from the core gene of the HCV-RH isolate, whereas P16 became the major protein product of the core gene of the HCV-1 isolate. Analysis of the amino-terminal sequences of P21 and P16 expressed in Escherichia coli revealed that P21 and P16 were co-amino terminal. Deletion-mapping analysis indicated that P16 lacked the carboxy-terminal sequence of P21. Immunofluorescence analysis of the subcellular localization of different HCV core proteins indicated that P21 and P19 displayed a reticular and punctate staining pattern typical of endoplasmic reticulum-associated proteins, while P16 was localized to the nucleus. The distinct subcellular localization of P16 raises the possibility that P16 may have a biological function very different from those of P21 and P19.

Detection of Cellular Proteins and Viral Core Protein Interacting with the 5' Untranslated Region of Hepatitis C Virus RNA.

Hepatitis C virus (HCV) is a pesti- and flavi-like virus, which contains a highly conserved 5'-untranslated region (UTR). This region is implicated in the regulation of both translation and RNA replication. To examine the possible cellular factors involved in HCV replication, we performed UV cross-linking experiments to detect cellular protein binding to 5'-UTR of HCV RNA. No cytoplasmic proteins were found to cross-link to 5'-UTR. Surprisingly, when nuclear extracts were used for UV cross-linking, a major protein of 110 kD and several other minor proteins were detected. Competition assays confirmed that the binding of the 110-kD protein was specific to the 5'-UTR. The protein-binding site was mapped within the 78-nt region between nucleotides 199 and 277 from the 5' end of the viral RNA. This protein was present in several different cell lines tested. No cellular proteins specifically bound to the complementary strands of the 5'-UTR. We have also shown by an RNA-protein blotting assay that 5'-UTR bound to the HCV core protein, which can be translocated to the nuclei. These findings suggest that HCV RNA may enter nuclei by complexing with the viral core protein and interact with nuclear proteins that are involved in the regulation of RNA replication or translation. It is thus possible that HCV employs a replication strategy distinct from its related pestiviruses or flaviviruses. Copyright 1995 S. Karger AG, Basel

Early detection of anti-HCc antibody in acute hepatitis C virus (HCV) by western blot (immunoblot) using a recombinant HCV core protein fragment.

Crude extract from Escherichia coli which expressed a recombinant protein containing amino acids 2 to 127 of the hepatitis C virus (HCV) core protein was used to detect the antibody against HCV core protein (anti-HCc). After electrophoretic separation of proteins from the extract, Western blot (immunoblot) analysis was performed with the serum samples. This method was compared with a commercially available second-generation enzyme immunoassay (EIA) which employed synthetic peptides corresponding to highly antigenic segments of both structural and nonstructural portions of HCV. Also, reverse transcription PCR for HCV RNA was used for comparison. Seventy-two serum samples from three groups of patients were tested. Groups I and II represented healthy subjects and subjects with acute hepatitis A or B, respectively. Group III included patients with newly acquired acute hepatitis C. By Western blot analysis, 31 of 31 (100%) samples from group I were negative for anti-HCc antibody, whereas 4 of 22 (18%) samples from group II were positive for anti-HCc. One of these four samples was also positive for anti-HCV antibody by the second-generation EIA (1 of 22 [4.5%]). Among 19 patients diagnosed with newly acquired acute hepatitis C, 4 (21%) were positive for anti-HCV by the second-generation EIA, whereas 12 of 19 (63%) were positive for anti-HCc by Western blot analysis. Of EIA-positive subjects, 4 of 4 (100%) were also positive for anti-HCc by Western blot analysis, whereas among EIA-negative subjects, 8 of 15 (53%) were positive. For HCV RNA detected by reverse transcription PCR, 15 of 19 (80%) of this group of samples were positive. Strikingly, the peak bilirubin level for patients with EIA-negative and Western blot-positive results is significantly higher than that for patients with consistent EIA and Western blot results (22.7 versus 7.2 mg/dl). A series of serum samples from a patient with concurrent hepatitis B and C viral infection was also studied by both tests. Although anti-HCc persisted throughout the course of infection, anti-HCV by EIA converted from negative to positive 20 days after admission and then converted back to negative 30 days later.

Comparative studies of the core gene products of two different hepatitis C virus isolates: two alternative forms determined by a single amino acid substitution.

We have compared the expression of the core gene of two different hepatitis C virus (HCV) isolates, HCV-1 and HCV-RH, using the in vitro translation assay. In the absence of the downstream E1 envelope protein sequence, a 16-kDa protein (P16) was the dominant protein product synthesized from the HCV-1 core gene sequence. On the other hand, a 21-kDa protein (P21) was the dominant protein product synthesized from the HCV-RH sequence. Domain-swapping and site-directed mutagenesis experiments indicated that codon 9 of the core protein coding sequence played a crucial role on the synthesis of the core protein: a lysine codon at this position led to the synthesis of P16 and an arginine codon at this position led to the synthesis of P21. For HCV-1 and HCV-RH, this codon encodes lysine and arginine, respectively. Further analyses indicated that P16 was likely co-amino-terminal with P21. In the presence of the downstream E1 envelope protein sequence and microsomal membranes, P16 as well as P21 were synthesized from the HCV-1 core gene sequence whereas P21 remained the only detectable protein product synthesized from the HCV-RH core gene sequence. These results indicate that the pathway leading to the synthesis of the HCV core protein may be more complicated than originally envisioned.

Paraffin section p53 protein immunohistochemistry in neuroectodermal tumors.

In an attempt to characterize the role of p53 alterations in the pathogenesis of intracranial neuroectodermal tumors, 196 tumors were immunostained with a monoclonal antibody against the p53 protein on archival materials of formalin-fixed, paraffin-embedded materials. Only 11% of well differentiated astrocytomas stained positive, whereas up to 40% of high-grade astrocytomas (anaplastic astrocytomas and glioblastoma multiforme) were immunolabelled. The extent of immunolabelling of tumor cells also increased from the low-grade to high-grade astrocytomas. Among the high-grade astrocytomas, the small anaplastic cells were the predominant cell type which exhibited aberrant p53 protein accumulation. Rare cases of oligodendrogliomas and medulloblastomas also stained positive, whereas ependymomas and choroid plexus tumors were uniformly negative. p53 alterations therefore appear to play a role in the progression from low-grade to high-grade astrocytomas and the cell type which appeared to be critically involved appeared to be the small anaplastic cells.

Atmospheric turbidity and the circumsolar radiation.

We investigate the possibility of determining the turbidity from the intensity of the circumsolar radiation and find that such a connection can be made only when the size distribution of the aerosol particles is known. However, measurements of both the turbidity and the aureole intensity can give useful information about the size distribution.