Patterning of catalysts for the selective growth of carbon nanotubes using laser irradiation of nickel nitrate.

We developed a simple method to produce patterned catalysts for the growth of carbon nanotubes (CNTs) on Si substrate using laser irradiation of Ni nitrate. We found that Ni nitrate can easily be decomposed into Ni oxide by KrF laser irradiation and that unexposed Ni nitrate can be removed using deionized (DI) water. Once we obtained patterned Ni oxide, we were able to synthesize multi-walled CNTs using a conventional thermal CVD. This new method does not require any photoresist or vacuum processes. Not only is the method compatible with low-temperature and large-area fabrication, it also significantly reduces the total processing steps required for conventional lithographic patterning technology. A detailed investigation of the decomposition process of this patterned catalyst and the microstructure of the patterned multi-walled CNTs was carried out using IR, SEM and TEM.

Loading behavior of Pt nanoparticles on the surface of multiwalled carbon nanotubes having defects formed via microwave treatment.

We developed a simple and efficient method to load Pt nanoparticles (NPs) uniformly on defects generated in multiwalled CNTs (MWCNTs) without using reduction agents or organic reagents. Defects on the surfaces of MWCNTs were artificially generated by microwave treatment at various exposure times. Nucleation of Pt NPs occurs on the defect sites spontaneously due to an innate electropotential difference. Because of the correlation between defects and Pt NPs, we were able to control the size of Pt NPs by changing defect size, quantity and distribution, which was confirmed by Raman spectroscopy and TEM. After microwave treatment for 3 min, more uniform and smaller Pt NPs were observed. Also, the defects via microwave treatment make adhesion of Pt NPs stronger, which can be helpful to improve the reliability for applications. Finally, the methanol oxidation behavior of MWCNTs with Pt NPs was examined by cyclic voltammetry (CV).

PBK Expression Is Associated With Prognosis of Patients With Oral Squamous Cell Carcinoma Treated With Radiotherapy: A Retrospective Study.

BACKGROUND/AIM: To investigate the impact of PDZ-binding kinase (PBK) on the clinical outcome of patients with oral squamous cell carcinoma (OSCC) who received radiotherapy. PATIENTS AND METHODS: PBK immunoreactivity of cancer specimens obtained from 179 patients with primary OSCC was analyzed by immunohistochemistry. RESULTS: High PBK expression in tumor cells tended to be associated with advanced N-stage. The 5-year survival rate was greater for patients with high total PBK expression than in those with low PBK expression. After adjustment, high PBK remained associated with a favorable outcome. In subgroups according to tumor stage, the prognostic role was significant in patients with stage III/IV rather than those with stage I/II disease. CONCLUSION: We suggest that PBK expression should be used as an independent prognostic marker for patients with OSCC treated with radiotherapy, especially for those with advanced-stage disease.

Regulation of transforming growth factor-beta 1 expression by the hepatitis B virus (HBV) X transactivator. Role in HBV pathogenesis.

TGF-beta 1 has been implicated in the pathogenesis of liver disease. The high frequency of detection of the hepatitis B virus X (HBx) antigen in liver cells from patients with chronic hepatitis, cirrhosis, and liver cancer suggested that expression of HBx and TGF-beta 1 may be associated. To test this possibility, we examined the expression of TGF-beta 1 in the liver of transgenic mice expressing the HBx gene. We show that the patterns of expression of TGF-beta 1 and Hbx protein are similar in these mice and that HBx activates transcription of the TGF-beta 1 gene in transfected hepatoma cells. The cis-acting element within the TGF-beta 1 gene that is responsive to regulation by Hbx is the binding site for the Egr family of transcription factors. We further show that the Egr-1 protein associates with the HBx protein, allowing HBx to participate in the transcriptional regulation of immediate-early genes. Our results suggest that expression of Hbx might induce expression of TGF-beta 1 in the early stages of infection and raise the possibility that TGF-beta 1 may play a role in hepatitis B virus pathogenesis.

Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells.

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.

The retinoblastoma gene product regulates Sp1-mediated transcription.

We have demonstrated that the retinoblastoma gene product (Rb) can positively regulate transcription from the fourth promoter of the insulinlike growth factor II gene. Two copies of a motif (the retinoblastoma control element) similar to that found in the human c-fos, transforming growth factor beta 1, and c-myc promoters are responsible for conferring Rb regulation to the fourth promoter of the insulinlike growth factor II gene. We have shown that the transcription factor Sp1 can bind to and stimulate transcription from the retinoblastoma control element motif. Moreover, by using a GAL4-Sp1 fusion protein, we have directly demonstrated that Rb positively regulates Sp1 transcriptional activity in vivo. These results indicate that Rb can function as a positive regulator of transcription and that Sp1 is one potential target, either directly or indirectly, for transcriptional regulation by Rb.

Increased expression of the insulin-like growth factor I (IGF-I) receptor gene in hepatocellular carcinoma cell lines: implications of IGF-I receptor gene activation by hepatitis B virus X gene product.

Hepatitis B virus infection is associated with acute and chronic liver disease and the development of hepatocellular carcinoma (hcc). Several lines of evidence have suggested that hepatitis B virus X protein (HBx), which is a transcriptional trans-activator, plays a role in the process of liver carcinogenesis. We have investigated the expression of insulin-like growth factor I (IGF-I) receptor in human hepatocellular carcinoma cell lines using SNU368 cells containing HBx and SNU387 cells, which lack HBx gene transcript (J-G. Park et al., Int. J. Cancer, 62: 276-282, 1995), in an attempt to understand its possible relationship to the HBx-induced hcc. The binding of 125I-labeled IGF-I to the SNU368 cells was 5-fold higher than that of SNU387 cells. The Scatchard analysis of the binding data revealed a single class binding site for IGF-I with Kd of 7.6 and 8.8 nM and maximum binding capacities of 169 and 33 fmol/10(5) cells, respectively. Therefore, the difference observed in 125I-labeled IGF-I binding between SNU368 and SNU387 cells was due to an increase in the number of IGF-I binding sites with no change in affinity for the IGF-I receptor. An enhanced level of IGF-I receptors in SNU368 cells was also observed by fluorescence-activated cell sorting analysis using a monoclonal antibody against human IGF-I receptor, alpha IR3. The level of IGF-I receptor RNA and the basal IGF-I receptor gene promoter activity in SNU368 cells were 5 and 10 times higher than those observed in SNU387 cells, respectively. To substantiate further that HBx could transactivate the expression of the endogenous IGF-I receptor gene, Hep G2 cells were transiently transfected with a HBx expression vector. The transfection of Hep G2 cells with an HBx expression vector resulted in increased levels of IGF-I receptor RNA, promoter activity, and 125I-labeled IGF-I binding by 2.6-, 2.8-, and 2-fold, respectively. As a result of higher levels of IGF-I receptor, the mitogenic effect of IGFs (IGF-I and IGF-II) on SNU368 cells was 6 times higher than that of SNU387 cells. These results suggest that HBx may play a role in the process of hcc by activating IGF-I receptor gene expression.

Activation of the insulin-like growth factor II transcription by aflatoxin B1 induced p53 mutant 249 is caused by activation of transcription complexes; implications for a gain-of-function during the formation of hepatocellular carcinoma.

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.

Hepatitis B virus-X protein upregulates the expression of p21waf1/cip1 and prolongs G1-->S transition via a p53-independent pathway in human hepatoma cells.

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.

The human hepatitis B virus transactivator X gene product regulates Sp1 mediated transcription of an insulin-like growth factor II promoter 4.

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 Spl 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.

Cloning and characterization of cDNAs coding for heavy and light chains of agglutinating monoclonal antibody (HAG12islrh) specific for human red blood cells.

Complementary DNAs encoding the heavy and light chains of the Fab fragment of mouse agglutinating monoclonal antibody against human red blood cells were cloned by polymerase chain reaction and their nucleotide sequences were determined. The sequence analysis showed that the variable regions of the heavy and light chains were the members of mouse heavy-chain subgroup IIa and kappa light-chain subgroup I, respectively. A few unusual amino acids in the constant regions of the heavy chain were also recognized.

Human interleukin 6 gene is activated by hepatitis B virus-X protein in human hepatoma cells.

Interleukin 6 (IL-6) is a pleiotropic cytokine that induces many biological activities, including some aspects of the immune reaction and inflammatory responses. In the liver, IL-6 regulates the synthesis of a broad spectrum of acute-phase proteins. IL-6 is also known to be a factor involved in the immunoregulatory perturbations in patients with chronic liver diseases (CLDs). Here, we report that IL-6 can be induced by hepatitis B virus (HBV)-X protein, as evidenced by high levels of serum IL-6 in patients with CLD with HBV infection, IL-6 productions observed in HBV-X-transfected cells, and transcriptional transactivations of the IL-6 gene by HBV-X. We determined serum levels of IL-6 in patients with chronic hepatitis B (CH-B), chronic hepatitis C (CH-C), liver cirrhosis (LC) caused by hepatitis B, and LC with hepatocellular carcinoma (HCC) caused by hepatitis B (LC+HCC). Mean serum levels of IL-6 in all CLD patients were higher than those in normal controls, and the difference was statistically significant (P < 0.05). Mean IL-6 levels of LC and LC+HCC patients were significantly higher than those of CH-B patients (P < 0.05). Because the etiological factor in all cases except CH-C (CH-B, LC, and LC+HCC) was HBV, we checked the possibility of HBV-transactivator-X activation of IL-6 promoter. Using deletion constructs of 5'-flanking regulatory regions of the IL-6 gene linked to the chloramphenicol acetyltransferase gene as a reporter, we found that the binding of nuclear factor-kappaB to a cis element is essential and sufficient for the induction of the IL-6 gene by HBV-X. We also found that HBV-X enhances the binding of two subunits of nuclear factor-kappaB (p65 and p52) to their target DNA binding sequences. These observations are relevant, in that HBV-X might play an important role in hepatic inflammation and diseases by up-regulating IL-6 production, which can eventually lead to LC and HCC.

Stage-dependent regulation of ovarian pituitary adenylate cyclase-activating polypeptide mRNA levels by GnRH in cultured rat granulosa cells.

The present study was designed to test whether GnRH regulates pituitary adenylate cyclase-activating polypeptide mRNA levels in a stage-dependent manner during follicle development in the rat ovary. The granulosa cells of preovulatory and immature follicles obtained from PMSG- and estrogen-treated rats, respectively, were cultured in serum-free conditions in the presence of various hormones. GnRH receptor mRNA expression was detected in both preovulatory and immature granulosa cells and was down-regulated by gonadotropins. Treatment of preovulatory granulosa cells with GnRH agonist stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner. In situ hybridization analysis of cultured preovulatory follicles revealed that GnRH-induced pituitary adenylate cyclase- activating polypeptide signals were detected in granulosa cells, but not thecal cells. In immature granulosa cells, cotreatment with GnRH agonist suppressed FSH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner, whereas treatment with GnRH alone had no effect. Furthermore, treatment with GnRH antagonist inhibited LH-induced pituitary adenylate cyclase-activating polypeptide gene expression in preovulatory granulosa cells, whereas it stimulated FSH-induced pituitary adenylate cyclase-activating polypeptide gene expression in immature granulosa cells. Interestingly, GnRH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in preovulatory granulosa cells was inhibited by arachidonyltri fluoromethyl ketone, an inhibitor of phospholipase A(2), but not by an inhibitor of protein kinase A or C. Lastly, treatment of preovulatory follicles with pituitary adenylate cyclase-activating polypeptide antagonist suppressed GnRH-stimulated progesterone production during 6--9 h of culture. Taken together, these results demonstrate the stage-dependent regulation of pituitary adenylate cyclase-activating polypeptide mRNA levels by GnRH, the stimulatory and inhibitory effect in granulosa cells of preovulatory and immature follicles, respectively.

Mutations in the p53 tumor suppressor gene in tree shrew hepatocellular carcinoma associated with hepatitis B virus infection and intake of aflatoxin B1.

Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered to be major risk factors in the development of hepatocellular carcinoma (HCC) in humans. A high rate of p53 mutations at codon 249 has been reported in these tumors. The tree shrew (Tupaia belangeri chinensis) is a useful animal model for the development of HCC after human hepatitis B virus (HBV) infection or AFB1 treatment. Therefore, it was of particular interest to determine whether the p53 gene in tree shrew HCCs associated with HBV infection and/or with exposure to AFB1 is affected in the same manner as in human HCCs. We determined the tree shrew p53 wild-type nucleotide sequences by RT-PCR and automatic DNA-sequencing. Tree shrew wild-type p53 sequence showed 91.7 and 93.4% homologies with human p53 nucleotide and amino acids sequences, respectively, while it showed 77.2 and 73.7% homologies in mice. One HCC and normal liver tissue from AFB1 treated and one HCC from AFB1- and HBV-treated tree shrew showed no change in p53 sequences, while three HCCs from AFB1- and HBV-treated tree shrews showed point mutations in p53 sequences. One HCC showed point mutations at codon 275, which is on the DNA-binding domain of p53 gene, which might be a cause of gain-of-function during the development of HCC. As a result, our finding indicates that tree shrews exposed to AFB1 and/or HBV had neither codon 249 mutations nor significant levels of other mutations in the p53 gene, as is the case with humans.

Characterization of the P4 promoter region of the human insulin-like growth factor II gene.

The human insulin-like growth factor II (IGF-II) gene contains four promoters (P1, P2, P3 and P4). In order to determine the mechanism by which the P4 promoter is controlled, the human IGF-II P4 promoter was analyzed in cell lines. DNA sequence analysis of the human IGF-II P4 promoter gene showed that the P4 promoter region contains a TATA-like sequence and several G+C rich regions which are essential for transcription. Analysis of the transcription initiation site by S1 nuclease mapping revealed two transcription start sites; both are located immediately behind TATA-like sequence. To determine the location of sites that may be important for the function of the human IGF-II P4 promoter, we constructed chimeric genes of the human IGF-II P4 promoter fused to the coding region for chloramphenicol acetyltransferase (CAT). These constructs were transfected into HepG2, PLC/PRF/5, G401 and A549 cells, and were examined for CAT activity. All transfected cells showed a similar profile of CAT activity. Sequences responsible for putative enhancer and silencer regions were identified and the 5' flanking sequences of the human IGF-II P4 promoter contain negative regulatory regions (-213 to -174). The 53-base pair fragment located between 111 and 59 base pairs upstream of the start site contains positive regulatory activity. Gel mobility shift assay showed that Sp1 and another proteins might be involved in positive regulation of the human IGF-II P4 promoter.

Detection of extrapulmonary tuberculosis with gallium-67 scan and computed tomography.

We evaluated 23 patients with extrapulmonary tuberculosis (TB) with 67Ga imaging to assess its usefulness in the diagnosis of this condition. We performed computed tomography (CT) in 17 patients to assess CT features of extrapulmonary TB in comparison with findings from 67Ga scans. Nineteen of 23 patients (83%) had positive findings on 67Ga scans. One of five patients with tuberculous mediastinal lymphadenopathy, two patients with cervical lymphadenitis and a patient with renal TB had negative 67Ga scans. It was observed that the detection of previously unrecognized primary foci of TB, without concomitant pulmonary TB, was possible using 67Ga imaging in five patients (22%). The 67Ga scan was relatively sensitive for the localization of extrapulmonary TB. It is suggested that the 67Ga scan could serve as a screening method, when followed by CT and ultrasonography, for the initial detection of occult tuberculous lesions, especially in patients with prolonged fever.

Biallelic expression of the H19 and IGF2 genes in hepatocellular carcinoma.

The imprinted genes, H19 and insulin-like growth factor II (IGF2), have been demonstrated to be necessary for embryonal development in humans. Both genes are reciprocally imprinted, with expression of the maternal H19 and paternal IGF2 alleles, and are normally characterized by monoallelic expression. Recently, loss of imprinting of these genes producing biallelic expression has been observed in childhood tumors including Wilms' tumors (WT), embryonal rhabdomyosarcoma, and adulthood tumors such as lung cancer. To test the existence of loss of imprinting in hepatocellular carcinoma (HCC), we analyzed the status of imprinting of H19 and IGF2 genes in three independent tumors, three HCC and one hepatoblastoma cell lines using AluI and ApaI polymorphisms of these genes, respectively. In contrast to the previous report, all the cases except one tumor and one HCC cell line showed biallelic expression of both H19 and IGF2 genes. Unlike WT, loss of imprinting (LOI) of IGF2 in HCC was not linked to down-regulation of H19 expression, but rather associated with coexpression for H19 and IGF2. Thus, Hl9 and IGF2 expression can be uncoupled in tumors with LOI. The frequent biallelic expression of H19 and IGF2 in hepatocellular carcinoma might play a causal role in the epigenetic mechanism involved in tumor development and/or process.

Transcriptional repression of human insulin-like growth factor-II P4 promoter by Wilms' tumor suppressor WT1.

The Wilms' tumor suppressor gene wt1 encodes a zinc finger-containing protein that binds to the same DNA sequence as Egr-1, a mitogen-inducible immediate-early gene product that activates transcription. In this study, we investigated whether the human insulin-like growth factor-II (IGF-II) P4 promoter might be a target for transcriptional repression mediated by WT1. Using constructs of the IGF-II P4 promoter linked to the chloramphenicol acetyltransferase gene, we have demonstrated that the WT1 protein represses expression of the IGF-II gene through a GCGGGGGAG response element spanning nucleotides -87 to -65 of the IGF-II P4 promoter. Conversely, we have shown that the Egr-1 activates transcription of the IGF-II gene through the same response element. WT1 and Egr-1 proteins interact directly with the WT1/Egr-1 response element of the IGF-II promoter 4 in gel mobility-shift assays. These findings demonstrate the importance of the WT1/Egr-1 consensus element for the expression of the IGF-II gene in response to positive or negative transcription signals.

Attenuation of c-Fos basal expression in the cerebral cortex of aged rat.

Cellular immediate early genes (IEG) such as c-fos were originally defined as rapid and transient inducible gene, but their products show a varying degree of basal expression in the brain of normal animals, suggesting that they also play a role in the transcriptional control under physiological conditions. In this study, we used an immunohistochemical method to investigate changes in the number of c-Fos-immunoreactive cells in the cerebral cortex and hippocampal formation of the aged rat. There was a remarkable decrease in the number of c-Fos-immunoreactive cells in the piriform and temporal cortex of aged rats compared with young adult rats. There was a slight decrease in the number of c-Fos-immunoreactive cells in the parietal cortex of aged rat. In the hippocampal complex, there were also decreases in the number of c-Fos-immunoreactive cells in aged rat; the degree of decrease was most prominent in the dentate gyrus. This report provides the first morphological evidence for decreased levels of basal c-Fos expression in some cerebral cortical areas and in the hippocampal complex of aged rats.

Phosphorylation of purified recombinant hepatitis B virus-X protein by mitogen-activated protein kinase and protein kinase C in vitro.

The recombinant human hepatitis B virus-X protein (rhHBx) has been expressed as inclusion bodies in Escherichia coli and purified. By sequential dialysis of urea, rhHBx was folded into the native structure, which was demonstrated by both the efficacy of its transcriptional activation of the adenovirus major late promoter, fluorescence and circular dichroism (CD) analysis. The increase in CD values at 220 nm and a corresponding blue shift of the intrinsic fluorescence emission confirmed the ability of HBx to refold in lower concentrations of urea to produce the active protein. After purification and renaturation, the rhHBx protein was found to be phosphorylated by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). In vivo phosphorylation of HBx was also demonstrated. Although PKC and MAPK enhance the HBx phosphorylation in vitro, neither protein kinase A nor caseine kinase II (CKII) phosphorylate HBx protein, though there are possible substrate residues of both kinases in HBx protein. Phosphoamino acid analysis of the total acid hydrolyzed HBx showed that serine residues can be phosphorylated by PKC or MAPK.