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.

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).

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.

Development of AFLP-derived STS markers for the selection of 5-methyltryptophan-resistant rice mutants.

To increase the specific free amino acid content in the japonica rice (Oryza sativa L.) cultivar Donganbyeo, mutant cell lines resistant to growth inhibition by 5-methyltryptophan (5MT) were selected from embryo-cultured callus irradiated with 50 Gy gamma-rays. Four 5MT-resistant homozygous M4 lines, MRI-40, MRI-116, MRII-8, and MRII-12, were obtained. The mean content of nine free essential amino acids were 70.1, 72.5, 31.7, and 35.4% greater than the original variety in these four mutant lines, respectively. For AFLP analysis, 8 EcoRI (+2) and 8 MseI (+3) primers used in 45 primer combinations generated a total of 3,684 bands with a mean of 82 bands, of which 361 (9.8%) were clearly polymorphic with the control cultivar, the four 5MT-resistant mutants, and five sensitive lines. The lines were grouped into three clusters through cluster analysis using unweighted pair grouping method of averages. The 36 polymorphic PCR products present only in the four homozygous 5MT-resistant lines were cloned and sequenced, and 10 of these sequenced products were converted into sequence tagged site (STS) markers. These STS primer sets were designated OSMR1-OSMR10. Six STS primer sets (OSMR1, OSMR2, OSMR3, OSMR4, OSMR5, and OSMR6) generated a single monomorphic PCR product identical in size to the original AFLP fragments. The broad applicability of these STS markers for the screening of 5MT resistance was evaluated with seven putative 5MT-resistant M2 plants (PM-1 to PM-7). Four STS markers (OSMR1, OSMR2, OSMR4, and OSMR5) out of six STS primer sets were revealed as polymorphic products between the control cultivar and the seven M2 plants. These markers can be utilized for the fine selection of 5MT resistance in rice, and this PCR-screening technique is less time-consuming, less labor-intensive, and more accurate and reliable than selection based solely on phenotypic evaluation involving soaking in 5MT solutions.

Suppression of glomerulosclerosis by adenovirus-mediated IL-10 expression in the kidney.

Glomerulosclerosis is a common morphologic result seen in almost all progressed renal diseases, and is the characteristic change in focal segmental glomerulosclerosis (FSGS). The most convincing hypothesis for glomerulosclerosis is cytokine-mediated injury by infiltrating immune cells in the glomerulus and tubulointerstitial area. This study investigated whether the anti-inflammatory effect of interleukin-10 (IL-10) when expressed by a recombinant adenoviral vector can prevent the onset of glomerulosclerosis in FGS/Kist mice (an animal model with naturally occurring renal failure initiated by FSGS). Each group of mice received recombinant adenoviruses encoding human IL-10 (Ad:hIL-10) by intraparenchymal injection at 6 weeks and were examined for cytokine expression, glomerular sclerotic index, and proteinuria. After injection of Ad:hIL-10 to the kidney, IL-10 expression was found to last over 20 days. Mice treated with Ad:hIL-10 were shown to have a significant reduction in the glomerular sclerotic index at 10 weeks when compared to control groups. The level of proteinuria in Ad:hIL-10-treated mice was also significantly reduced. About 50% of the urine samples of naive and Ad:LacZ-treated groups had severe levels of proteinuria. By contrast, at 10 weeks the group treated with Ad:hIL-10 had lower levels of proteinuria and transforming growth factor-beta1 (TGF-beta1) expression. These results demonstrate that IL-10 effectively prevents the development of glomerulosclerosis in FGS/Kist mice, and IL-10 gene therapy may be of use for the treatment of renal failure.

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.

Activation of the IGF-II gene by HBV-X protein requires PKC and p44/p42 map kinase signalings.

We have recently shown that HBx protein, one of the causative agents of hepatocellular carcinomas, regulates Sp1 mediated transcription of insulin-like growth factor II promoter 4 (Lee et al. (1998) Oncogene 16, 2367-2380). Here we show that PKC and p44/p42MAPK signalings are required for the HBx-induced Sp1-mediated IGF-II P4 transcriptional activity since (i) PKC activation by PMA or PKC expression vector increases Sp1 phosphorylation and P4 activity in HBx-transfected HepG2 cells; (ii) PKC inhibition by PKC inhibitor Gö6976 reduces Sp1 phosphorylation, P4 activity, and IGF-II mRNA in HBx-transfected HepG2 cells; and (iii) the inhibition of MEK activation by U0126 reduces Sp1 phosphorylation, P4 activity and IGF-II mRNA in HBx-transfected HepG2 cells. These results demonstrate that PKC and p44/p42 MAPK cascades are the essential signaling pathways in Sp1-mediated IGF-II gene activation by HBx.

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.

Hepatitis C virus core protein transactivates insulin-like growth factor II gene transcription through acting concurrently on Egr1 and Sp1 sites.

The possibility that hepatitis C virus core gene product (HCV-core) acts as a transactivator in insulin-like growth factor II (IGF-II) gene transcription was tested. HCV-core protein increases endogenous IGF-II expression from promoter 4 (P4) of the IGF-II gene through two cis-acting elements: Sp1 and Egr1 binding sites. Sp1 and Egr1 both bind to IGF-II P4 and functionally cooperate in mediating the maximal activity of IGF-II P4. HCV-core protein induced the binding of Sp1 and Egr1 on its binding sites on IGF-II P4. In addition, Sp1 and Egr1 were stimulated to phosphorylate by HCV-core, and its DNA binding activity was up-regulated upon HCV-core transfection. Transfection with HCV-core in HepG2 cells stimulated the membrane translocation of protein kinase C (PKC) and the treatment of HCV-core transfected cells with calphostin C, a PKC inhibitor, blocked induction of Sp1 and Egr1 DNA binding activity, and eventually transcriptional transactivations of the IGF-II gene. Increasing the DNA binding activity of the phosphorylated form of Sp1 and Egr1 might be an important mechanism for regulating IGF-II gene expression and for promoting cell division during hepatic carcinogenesis. These results indicate that HCV-core functions as a positive regulator of IGF-II transcription through the PKC pathway and that Sp1 and Egr1 are direct targets of the transcriptional regulation of the IGF-II gene which plays an important role in hepatitis C virus pathogenesis during the formation of hepatocellular carcinoma (HCC).

Metal-assisted esterification: glutaric acid-iron(II) complexes in the gas phase.

Transition metal ions are routinely used to assist organic reactions; however, direct detection of the intermediates in such reactions is uncommon. Here, we demonstrate a transition metal ion-assisted reaction between glutaric acid (L) and methanol, using electrospray ionization mass spectrometry (ESI-MS). Esterification of glutaric acid does not occur in aqueous methanol solution under ESI conditions, but the FeII-bound acid cluster, [FeII L2 - H]+, adds methanol and dehydrates to give rise to an abundant product ion with a 14 Da increased mass. The occurrence of methyl esterification is supported by collision-induced dissociation and isotopic labeling data, which indicate that the sequence by which the product ion is generated is loss of water, followed by the addition of methanol. Electrospray ionization conditions, specifically the tube lens offset voltage, strongly affect the reaction efficiency, presumably through control of the dehydration process. Other transition metal ions, such as NiII, ZnII, CoII and CuII, also show distinctive metal-assisted reactions.

The hepatitis B virus-X protein activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade.

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.

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.

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.

TGF-beta-induced cell-cycle arrest through the p21(WAF1/CIP1)-G1 cyclin/Cdks-p130 pathway in gastric-carcinoma cells.

Transforming growth factor-beta1 (TGF-beta) inhibits cell-cycle progression of many types of cells by arresting them in G(1)/S phase through inhibition of the active cyclin-Cdk complexes that lead to inhibition of Rb phosphorylation. In gastric-cancer cells, SNU16, TGF-beta treatment induced enhanced expression of p21(WAF1/CIP1) (p21), which inhibited the kinase activity of cyclin-D- and cyclin-E-associated Cdks and blocked p130 phosphorylation. TGF-beta also enhanced the stability of p130, suggesting that hypophosphorylation of p130 and increased stability of p130 contribute to p130-mediated G(1) arrest in gastric-cancer cells. Our results demonstrate that p21 and p130 are major downstream targets of TGF-beta in gastric-cancer cells and that a p21-G(1) cyclin/Cdks-p130/E2F pathway mediates growth inhibition by TGF-beta in these cells.

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.

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.

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.

In vitro stimulation of cumulus-cell expansion by human cord serum in mouse oocyte-cumulus complexes.

PURPOSE: The aim of this study was to test whether human cord serum (HCS) containing gonadotropins has an effect on the expansion of oocyte-cumulus complexes (OCCs). METHODS: The concentration of follicle stimulating hormone (FSH) and luteinizing hormone (LH) was measured in HCS by radioimmunoassay (RIA). After short-term culture (4 hr) with or without OCCs, medium containing 0.4% bovine serum albumin (BSA) as control or 10% HCS was collected and analyzed for its concentration of estradiol, progesterone, and testosterone. RESULTS: The FSH concentration was at the basal level, but the LH level was as high as 142.4 mIU/ml in both natured and denatured serum. Undetectable levels of steroids were observed in control media with or without OCCs. In contrast, a moderate amount of steroid hormones was detected in culture medium containing HCS. OCCs secreted a minute amount of steroid hormones in response to HCS. Similar patterns of cumulus expansion were observable by treatment with HCS, human chorionic gonadotropin (hCG), or HCS plus hCG after 4, 8, or 22 hr of culture. However, no cumulus expansion was observed in controls. CONCLUSION: These results suggest that LH in HCS induces cumulus expansion but does not affect the secretion of steroid hormones by OCCs during culture.