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A highly biased linear current method (HBLCM) for separately extracting source and drain resistance ( ${R}_{S}$ and ${R}_{D}$ ) in MOSFETs is proposed. The technique can be applied to a single device by using simple modeling. Compared to other methods, it provides accurate values of ${R}_{S}$ and ${R}_{D}$ because it considers carrier mobility degradation. The method basically uses linear current versus gate voltage ( ${I}_{\text {DS}} - {V}_{\text {GS}}$ and ${I}_{\text {SD}} - {V}_{\text {GD}}$ ) characteristics before and after the source/drain interchange ( ${I}_{\text {DS}}$ and ${I}_{\text {SD}}$ ). Afterward, by using the traditional Y-function and subsequent resistance modeling in a highly biased linear condition, ${R}_{S}$ and ${R}_{D}$ can be separately extracted. In order to evaluate and verify the accuracy of HBLCM, an external resistor was intentionally connected to a source electrode of a device, and the resulting change in source resistance was detected using the proposed method. Moreover, to demonstrate an application of the proposed method, internal resistance deliberately created by hot-carrier injection (HCI) was linked to a drain electrode, thereby changing drain resistance. The changed drain resistance was also sensed by the HBLCM. Afterward, the HCI-stressed device was cured by electrothermal annealing driven by Joule heating, and the recovery was again clearly observed using the proposed method.

The hepatitis B virus-X protein (HBx) regulates fundamental aspects of mitochondrial physiology. We show that HBx down-regulates mitochondrial enzymes involved in electron transport in oxidative phosphorylation (complexes I, III, IV, and V) and sensitizes the mitochondrial membrane potential in a hepatoma cell line. HBx also increases the level of mitochondrial reactive oxygen species and lipid peroxide production. HBx does not activate apoptotic signaling, although it sensitizes hepatoma cells to apoptotic signaling, which is dependent on reactive oxygen species. Increased intrahepatic lipid peroxidation in HBx transgenic mice demonstrated that oxidative injury occurs as a direct result of HBx expression. Therefore, we conclude that mitochondrial dysfunction is a crucial pathophysiological factor in HBx-expressing hepatoma cells and provides an experimental rationale in the investigation of mitochondrial function in rapidly renewed tissues, as in hepatocellular carcinomas.

The role of integrin-linked kinase (ILK) in transforming growth factor beta (TGFbeta)-mediated epithelial to mesenchymal transition was investigated. A stable transfection of dominant-negative ILK results in the prevention of TGFbeta-mediated E-cadherin delocalization. TGFbeta-mediated phosphorylation of Akt at Ser-473 was inhibited by dominant-negative ILK and PI3K inhibitors, LY294002 and wortmannin. Treatment with TGFbeta stimulated induction of Akt and ILK kinase activity in HaCat control cells. This increased ILK activity by TGFbeta was lowered by PI3K inhibitor, LY294002. In addition, PI3K inhibitor, dominant-negative Akt, and dominant-negative ILK could not block TGFbeta-mediated C-terminal phosphorylation of Smad2. Taken together, these data suggest that PI3K-ILK-Akt pathway that is independent of the TGFbeta-induced Smad pathway is required for TGFbeta-mediated epithelial to mesenchymal transition.

The PTEN gene (phosphatase and tensin homologous on chromosome 10) is frequently mutated or deleted in a number of malignancies including human hepatocellular carcinoma (HCC). We reported previously that the hepatitis B virus X (HBx) protein, known to be a causative agent in the formation of HCC, activates insulin-like growth factor II (IGF-II) expression through Sp1 phosphorylation by protein kinase C (PKC) or mitogen-activated protein kinase (MAPK) signaling. In this report we demonstrate that the PTEN effect on HBx induced IGF-II activation in a hepatoma cell line. Expression of PTEN and IGF-II was inversely related in different hepatoma cell lines. PTEN expression induced decreased Sp1 DNA binding by dephosphorylating Sp1 and interfered with transcriptional transactivation of IGF-II by HBx in hepatoma cells. The protein phosphatase activity was involved in PTEN downregulation of IGF-II transcription through downregulation of MAPK, MAPK kinase phosphorylation and PKC translocation. Our data suggest that PTEN blocks Sp1 phosphorylation in response to HBx, by inactivating PKC, MAPK and MAPK kinase which eventually downregulate IGF-II expression, during the formation of HCC.

The aim of the present study was to investigate the intracellular mediators of the third base mutant of codon 249 in p53 gene (p53mt249) mutation that potentiate IGF-II dependent IGF-I receptor (IGF-IR) signaling. p53mt249 enhanced IGF-II dependent IGF-IR signaling in p53 negative Hep3B hepatoma cells which were specifically prevented by IGF-IR antibody, αIR3 and lovastin. p53mt249 increased the number of IGF-II binding sites with no change in the affinity of IGF-IR. Enhanced levels of IGF-IR expression and transcription were identified in p53mt249 transfected Hep3B cells. Pre-transfection of cultured hepatoma cells with p53mt249 resulted in a three to fourfold increase in IGF-IR phosphorylation and downstream mediator IRS-I phosphorylation but, enhanced more than 15-fold after IGF-II treatment, which coincides well with the cell growth and thymidine uptake results. Our results showed that p53mt249 modulate IGF-II dependent IGF-IR signaling by upregulating IGF-IR and potentiating IGF-IRs where IGF-IRs became more sensitive on treatment with IGF-II. We concluded that p53mt249 stimulates IGF-II dependent IGF-IR signaling by upregulating the expression of both ligand (IGF-II) (Oncogene 19 (2000) 3717) and receptor (IGF-IR) through an autocrine and/or paracrine loop and we outline the physiological significance of potentiation of IGF-IR by p53 mutation in the development of hepatocellular carcinoma (HCC).

Journal of Applied Botany and Food Quality; 87; 24-29, Variation in content of organic acids and soluble sugars, and in physical characteristics was evaluated in apricot (P. armeniaca L. cv. Harcot), plumcot (plum-apricot hybrid, P. salicina ? P. armeniaca L. cv. Harmony), plum (P. salicina Lindl. cv. Formosa), and peach (P. persica L. Batsch cv. Jinmi). The content of organic acids and sugars, as well as parameters of fruit quality (weight, dimensions, firmness, total soluble solids, and total acidity) in Prunus fruits during fruit development were determined. Organic acids, including oxalic acid, quinic acid, malic acid, shikimic acid, citric acid, and quinic acid, sugars, including sucrose, fructose, glucose, and sugar alcohol (sorbitol), were identified and quantified using HPLC. Organic acid mostly increased during the early stages of fruit growth (30 - 60 days after full bloom) and decreased until fruits were fully ripened. In general, plum was the highest in most organic acids compared with the other fruits, while apricot contained the lowest acid content except for citric acid. Sucrose, fructose, and glucose content increased with fruit development, unlike content of sorbitol. Plumcot contained the highest fructose, and peach showed the maximum content of sucrose at full maturation stages. Total soluble solids averaged 17.5, 14.8, 11.9, and 10.6 Brix in apricot, plumcot, plum, and peach, respectively, whereas total acidity was 0.9, 1.4, 0.5, and 0.3% in four Prunus cultivars at ripened stages. Shikimic acid was significantly correlated with oxalic acid in apricot, plumcot, and plum, but not in peach. Fructose and glucose were highly correlated in plumcot, plum, and peach.

Aflatoxin B1 (AFB1) induced mutation of the p53 gene at codon 249 (p53mt249) is critical during the formation of hepatocellular carcinoma (HCC) following hepatitis B virus (HBV) infection. p53mt249 markedly increases insulin-like growth factor II (IGF-II) transcription largely from promoter 4, accumulating the fetal form of IGF-II. Modulation of the transcription factor binding to IGF-II P4 by wild-type p53 and p53mt249 was identified. Wild-type p53 inhibited binding of transcription factors Sp1 and TBP on the P4 promoter, while p53mt249 enhanced the formation of transcriptional complexes through enhanced DNA-protein (Sp1 or TBP) and protein-protein (Sp1 and TBP) interactions. p53mt249 stimulates transcription factor Sp1 phosphorylation which might be a cause of increased transcription factor binding on the P4 promoter while wild-type p53 does not. Transfection of hepatocytes with p53mt249 impaired induction of apoptosis by the HBV-X protein and TNF-alpha. Therefore, the blocking of apoptosis through enhanced production of IGF-II should provide a favorable opportunity for the selection of transformed hepatocytes. These results explain the molecular basis for the genesis of HCC by p53mt249 which was found to be induced by a potent mutagen, AFB1.

Progression through the cell cycle is controlled by the induction of cyclins and activation of cognate cyclin-dependent kinases. The human hepatitis B virus-X (HBV-X) protein functions in gene expression alterations, in the sensitization of cells to apoptotic killing and deregulates cell growth arrest in certain cancer cell types. We have pursued the mechanism of growth arrest in Hep3B cells, a p53-mutant human hepatocellular carcinoma (HCC) cell line. In stable or transient HBV-X transformed Hep3B cells, HBV-X increased protein and mRNA levels of the cyclin-dependent kinase inhibitor (CDKI) p21(waf1/cip1) increased binding of p21(waf1/cip1) with cyclin-dependent kinase 2 (CDK2), markedly inhibited cyclin E and CDK2 associated phosphorylation of histone H1 and induced the activation of a p21 promoter reporter construct. By using p21 promoter deletion constructs, the HBV-X responsive element was mapped to a region between -1185 and -1482, relative to the transcription start site. Promoter mutation analysis indicated that the HBV-X responsive site coincides with the ets factor binding sites. These data indicate that in human hepatocellular carcinoma cells, HBV-X can circumvent the loss of p53 functions and induces critical downstream regulatory events leading to transcriptional activation of p21(waf1/cip1). As a consequence, there is an increased chance of acquisition of mutations which can enhance the genesis of hepatomas. Our results also emphasize the chemotherapeutic potential of p21(waf1/cip1) inhibitors, particularly in the HBV-X infected hepatoma which lacks functional p53.

We prepared human hepatoma cell lines, which expressed the human hepatitis B virus-X gene product. The plasmid pMAMneo-X, containing an HBV-X gene promoter, an enhancer and a structural gene was constructed. Transfected HBV-X gene integration and expression were detected by Southern and Northern blotting, as well as by chloramphenicol acetylase transferase (CAT) assay using various kinds of promoter-CAT reporter systems. HBV-X protein expression in stable transfectants was confirmed by immunofluorescence microscopy. Transfected cell lines showed permanent expression of HBV-X proteins. The HBV-X transfectant activated its target promoters in promoter-CAT constructs as reporters. The HBV-X transfectant enhanced AP-1 transcription factor binding to its target DNA. Therefore, X-transfectants are not only stable, but also have specific biological functions. Cell cycle analysis by flow cytometry showed that the majority of the transfectant cells are arrested in the G1 or G2 phase of the cell cycle. These cell lines may be useful in analyzing the biological functions of HBV-X and its functional role in the formation of hepatocellular carcinomas.

The sequence in the precore and core region of the hepatitis B virus (HBV) genome in the serum of five chronic active hepatitis patients at four different stages in each individual were studied by polymerase chain reaction and DNA sequencing to determine the prevalence and type of pre-core and core mutants in each chronic active hepatitis (CAH) patient. Gradual changes of the virus genome in each CAH patient in precore and core regions were identified. Except for the virus from one patient, the mutant viruses showed gradual changes of genome sequences, which resulted in the generation of stop codons at the precore and core region, causing the association of active hepatitis in each patient even in the presence of anti-HBe. Mutational hot spots in the core region, which includes a clustering of changes in a small region of 14 amino acids (codons 84-97 from the start of the core gene) were found in all patients. This region of mutational hot spots in the core might be a major target of cytotoxic T lymphocytes (CTL), which has evolved under the pressure of immune selections, and these mutants might play a important role in the pathogenesis of viral hepatitis.

A series of C3-substituted cephalosporins with different levels of oxidation was synthesized and the activities of eight compounds, including affinity for penicillin-binding proteins (PBPs), in Staphylococcus aureus and Escherichia coli were studied. The more hydrophobic cephalosporins, with a thiophene moiety at the C7 position, were more active than C7-methoxyiminoaminothiazole derivatives against S. aureus H (beta-lactam-susceptible), and compounds with a thioether function at C3 were more active than those with a sulphonyl function at this position. In contrast, the most active compounds against E. coli were hydrophilic, zwitterionic C7-methoxyimino-aminothiazole C3-thioether cephalosporins. The physicochemical properties of the most active compounds were in accord with those predicted for rapid outer membrane penetration. However, among the thiophene cephalosporins the dianionic sulphonyl compounds were more active than the monoanionic thioether derivatives. There was a reasonably good correlation between MICs and PBP affinities for S. aureus H. The compounds had most affinity for PBP 1 and least for PBP 4. The two most potent compounds had high affinities for PBPs 1, 2 and 3. The compounds had low affinities for the beta-lactam-insensitive PBP 2a of methicillin-resistant S. aureus strain DU4916-K7. The novel cephalosporins had the highest affinities for PBPs 1a/1b and 3 of E. coli DCO. The major morphological response of E. coli to the compounds was cell elongation.

The purpose of this research is to enable anybody to semiautomatically segment the anatomical structures in the MRIs, CTs, and other medical images on the personal computer. The segmented images are used for making 3D images, which are helpful in medical education and research. To achieve this purpose, the following trials were performed. The entire body of a volunteer was scanned to make 557 MRIs. On Adobe Photoshop, contours of 19 anatomical structures in the MRIs were semiautomatically drawn using MAGNETIC LASSO TOOL and manually corrected using either LASSO TOOL or DIRECT SELECTION TOOL to make 557 segmented images. In a likewise manner, 13 anatomical structures in the 8,590 anatomical images were segmented. Also 12 anatomical structures in the 790 brain anatomical images and 10 anatomical structures in the 640 heart anatomical images were segmented. Proper segmentation was verified by making 3D images from the segmented images. The semiautomatic segmentation using Adobe Photoshop is expected to be widely used for segmentation of the anatomical structures in various medical images.

The hepatitis B virus-X (HBx) protein is known as a multifunctional protein that not only coactivates transcription of viral and cellular genes but coordinates the balance between proliferation and programmed cell death, by inducing or blocking apoptosis. In this study the role of the HBx protein in activation of phosphatidylinositol 3-kinase (PI3K) was investigated as a possible cause of anti-apoptosis in liver cells. HBx relieved serum deprivation-induced and pro-apoptic stimuli-induced apoptosis in Chang liver (CHL) cells. Treatment with 1-d-3-deoxy-3-fluoro-myo-inositol, an antagonist to PI3K, which blocks the formation of 3'-phosphorylated phosphatidyl inositol in CHL cells transformed by HBx (CHL-X) but not normal Chang liver (CHL) cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI3K, stimulated apoptosis in HBx-transformed CHL cells but not in normal cells, confirming that HBx blocks apoptosis through the PI3K pathway. The serine 47 threonine kinase, Akt, one of the downstream effectors of PI3K-dependent survival signaling was 2-fold higher in HBx-transformed CHL (CHL-X) cells than CHL cells. Phosphorylation of Akt at serine 473 and Bad at serine 136 were induced by HBx, which were specifically blocked by wortmannin and dominant negative mutants of Akt and Bad, respectively. We also demonstrated that HBx inhibits caspase 3 activity and HBx down-regulation of caspase 3 activity was blocked by the PI3K inhibitor. Regions required for PI3K phosphorylation on the HBx protein overlap with the known transactivation domains. HBx blocks apoptosis induced by serum withdrawal in CHL cells in a p53-independent manner. The results indicate that, unlike other DNA tumor viruses that block apoptosis by inactivating p53, the hepatitis B virus achieves protection from apoptotic death through a HBx-PI3K-Akt-Bad pathway and by inactivating caspase 3 activity that is at least partially p53-independent in liver cells. Moreover, these data suggest that modulation of the PI3K activity may represent a potential therapeutic strategy to counteract the occurrence of apoptosis in human hepatocellular carcinoma.

Human hepatitis B virus (HBV) is one of the causative agents of hepatocellular carcinoma (HCC). The virus encodes a 17 kDa protein, X, which is known to be a causative agent in the formation of HCC. An insulin-like growth factor-II (IGF-II) is expressed during the formation of HCC. Among the four promoters of the IGF-II gene, promoters 2, 3 and 4 become activated during the formation of HCC. The high frequency of detection of hepatitis B virus X (HBV-X) antigen in liver cells from patients with chronic hepatitis, cirrhosis, and liver cancer suggested that the expressions of HBV-X and IGF-II are associated. Studies were carried out to test the relationship between the HBV-X gene product and the activation of IGF-II promoter 4. We demonstrated that the HBV-X protein increases the endogenous IGF-II expression from promoter 3 and 4 of IGF-II gene. Analysis of the fourth promoter of IGF-II gene showed that the HBV-X gene product positively regulates transcription. Two copies of a motif are responsible for conferring HBV-X regulation on the fourth promoter of IGF-II. These motifs have been identified as Sp1 binding sites. Sp1 binding to IGF-II P4 promoter was identified by gel mobility shift assay using purified Sp1. By using a GAL4-Sp1 fusion protein it was demonstrated that HBV-X positively regulates the Sp1 mediated transcriptional activity of IGF-II in vivo. A protein-affinity chromatography experiment showed that HBV-X protein does not bind directly to Sp1, but HBV-X does augment the DNA binding activity of the phosphorylated form of Sp1 in HepG2 cells. Sp1 was phosphorylated by HBV-X and its DNA-binding activity was up-regulated upon HBV-X transfections. Various HBV-X mutant expression vectors were used for the demonstration of specific interactions between Sp1 and HBV-X. These results indicate that HBV-X functions as a positive regulator of transcription, and that Sp1 is a direct target for the transcriptional regulation of IGF-II. Increasing the DNA binding ability of the phosphorylated form of Sp1 by HBV-X might be an important mechanism for regulating the IGF-II gene expression and possibly promoting cell division during hepatic carcinogenesis. Our experimental results suggest that expression of HBV-X might induce the expression of IGF-II and the IGF-II might play a role in hepatitis B virus pathogenesis during the formation of HCC.

The sectioned images (SIs) of the pelvis from a female cadaver are the best source of realistic threedimensional (3D) models of the female urogenital system. The purpose of this research is to present SIs and outlined images of the female pelvis with improved quality, which may be used to produce 3D models to simulate virtual dissection or surgery of the female urogenital and adjacent structures. A pelvis of Korean female cadaver which preserved buttock curve was scanned with 3T MR and CT machines. The pelvis was embedded and milled at 0.1 ㎜ intervals. All sectioned surfaces were photographed to create horizontal SIs. On the Photoshop, 73 structures were outlined in the SIs to create outlined images. Once the structures were outlined, volume and surface models of the structures could be produced. A total of 222 MRIs and 222 CTs of a female pelvis were obtained. 2,220 SIs of the pelvis were obtained (0.1 ×0.1×0.1㎣-sized voxels; 48 bits color). 222 outlined images of 73 structures were prepared at 1㎜ intervals. Once the structures were outlined, 3D volume and surface models of the structures were produced without the help of the computer programmers using MRIcro and Maya software. We have produced high quality SIs of the female pelvis accompanied by corresponding MRI and CT images. 3D volume and surface models of the female pelvic structures have been constructed. These computerized models may serve as the basis for future realistic medical simulation programs that may enhance clinical understanding of pelvic anatomy.