Influence of anti-HBc seropositivity on the risk of hepatocellular carcinoma in HCV-infected patients after adjusting for confounding factors.

It is controversial whether past hepatitis B virus infection constitutes an additional risk of hepatocellular carcinoma (HCC) among patients with hepatitis C virus (HCV). The incidence of HCC between 1994 and 2004 was analysed among 1262 patients who were only positive for HCV. The cumulative incidence of HCC was assessed by Kaplan-Meier analysis and the difference between two groups was assessed by the log-rank test. The effect of anti-HBc positivity on the risk of HCC was assessed with multivariate Cox proportional analysis. Anti-HBc was positive in 522 (41.4%) patients. The proportion of male patients (56.7 vs 46.8%, P < 0.001) and mean age (60.8 vs 56.9 years, P < 0.001) were significantly higher in the anti-HBc positive group. HCC developed in 339 patients (mean follow-up 7.0 years), with cumulative incidence rates at 3, 5 and 10 years of 12.7, 24.5 and 41.9% in the anti-HBc positive group and 10.6, 17.7 and 33.4% in the negative group, respectively (P = 0.005). However, anti-HBc seropositivity did not reach statistical significance in multivariate analysis including age and gender (hazard ratio, 1.06; 95% CI, 0.85-1.31; P = 0.63). Anti-HBc positivity and HCC incidence were confounded by male gender and older age.

The hepatitis B virus X protein enhances AP-1 activation through interaction with Jab1.

Hepatitis B virus X protein (HBx) has many cellular functions and is a major factor in hepatitis and hepatocellular carcinoma caused by HBV infection. A proteomic approach was used to search for HBx-interacting proteins in order to elucidate the molecular mechanism of hepatocarcinogenesis. HBx was attached to myc and flag tags (MEF tags) and expressed in 293T cells; the protein complex formed within the cells was purified and characterized by mass spectrometry. COP9 signalosome (CSN) subunits 3 and 4 were subsequently identified as HBx-interacting proteins. In addition, CSN subunit 5, Jun activation domain-binding protein 1 (Jab1), was shown to be a novel cellular target of HBx. In vivo and in vitro interactions between HBx and Jab1 were confirmed by standard immunoprecipitation and GST pull-down assays. An analysis of HBx deletion constructs showed that amino acids 30-125 of HBx were responsible for binding to Jab1. Confocal laser microscopy demonstrated that HBx was mainly localized in the cytoplasm, while Jab1 was found mainly in the nucleus and partially in the cytoplasm, and that the two proteins colocalized in the cytoplasm. The cotransfection of HBx and Jab1 resulted in substantial activator protein 1 (AP-1) activation and knockdown of endogenous Jab1 attenuated AP-1 activation caused by HBx. In addition, the coexpression of HBx and Jab1 potentiated phosphorylation of JNK, leading to the subsequent phosphorylation of c-Jun, whereas the level of c-Jun and JNK phosphorylation induced by HBx was decreased in Jab1 knockdown cells. These results suggest that the interaction between HBx and Jab1 enhances HBx-mediated AP-1 activation.

Delimitation of the fertility restorer locus Rfk1 to a 43-kb contig in Kosena radish (Raphanus sativus L.).

We are pursuing a positional cloning strategy to isolate the fertility restoration gene Rfk1 from radish. Random polymorphic DNA-sequence-tagged site (RAPD-STS) markers tightly linked to the gene in radish were isolated, and a RAPD map surrounding the Rfk1 locus was constructed. We surveyed 948 F2 plants with adjacent RAPD-STS markers to isolate recombinants for bulk segregant analysis. This analysis was effective in isolating tightly linked amplification fragment length polymorphism (AFLP) markers surrounding the gene of interest. Ten tightly linked AFLP markers were obtained and used to construct a high-resolution map of the region. The closest AFLP-STS markers flanking Rfk1 were 0.1 cM and 0.2 cM away. Using the four adjacent AFLP markers, we screened lambda and cosmid libraries. The lambda and cosmid clones were aligned by examination of end sequences and restriction fragment length polymorphism (RFLP) patterns for each clone, and by hybridization to the DNA isolated from recombinants. Finally, we constructed a 198-kb contig encompassing the Rfk1 gene and comprising 20 lambda and two cosmid clones. By analysis of the breakpoints in recombinants with the rfk1/rfk1 or Rfk1/- genotype, the Rfk1 locus could be assigned to a 43-kb region comprising four lambda clones and one cosmid clone. This pinpoint localization in the radish genome has made it possible for us to identify the gene by sequence analysis and genetic transformation of cytoplasmic male-sterile Brassica napus plants.

Transoral transclival approach for intradural lesions using a protective bone baffle to block cerebrospinal fluid pulse energy--two case reports.

The transoral transclival approach for the treatment of intradural lesions of the clivus is often associated with serious complications such as cerebrospinal fluid (CSF) leakage and meningitis. CSF pulse energy may be the most significant factor in CSF leakage and meningitis, but a bone baffle can block such CSF pulse energy. A 64-year-old female presented with sudden onset of severe headache. She had subarachnoidal hemorrhage due to a rupture of the vertebral-posterior inferior cerebellar artery aneurysm. A 66-year-old female complaining of occipitalgia and numbness of the extremities had a foramen magnum meningioma. Both patients were treated via the transoral transclival route with a protective bone baffle, obtained from the iliac bone, securely fixed in the bone window to protect the repaired dura from injury by CSF pulse energy. Neither patient showed CSF leakage or meningitis, and the period of continuous lumbar CSF drainage was only 7 days. The transoral transclival approach with a bone baffle is still very effective in selected cases.

Identification and expression of the kosena radish (Raphanus sativus cv. Kosena) homologue of the ogura radish CMS-associated gene, orf138.

A CMS-associated gene, orf125, present in the Japanese radish cultivar Kosena, has a sequence homologous to that of the ogura CMS-associated gene, orf138, except for two amino acid substitutions and a 39 bp deletion in the orf138 coding region. In Kosena radish, orf125 is linked with orfB, whereas the orf125 locus differs in a Brassica napus CMS cybrid derived from protoplast fusion between Kosena radish and B. napus. A novel mtDNA sequence is present in the 3'-flanking region of orf125 in the B. napus kosena CMS cybrid. The orf125 is expressed both in the radish and the B. napus kosena CMS cybrid. Its accumulation is strongly associated with the CMS phenotype in B. napus. Fertility restoration was accompanied by a decrease in the amount of ORF125 in B. napus.

Fatal epistaxis caused by rupture of an intratumoral aneurysm enclosed by a large prolactinoma--case report.

A 72-year-old female presented with episodes of epistaxis. Neuroimaging demonstrated a large prolactinoma totally enclosing a large intracavernous aneurysm of the internal carotid artery. Adjacent bony structures were eroded and destroyed by tumor invasion and extension. Rupture of the intratumoral aneurysm caused fatal epistaxis rather than subarachnoid hemorrhage before surgery. Intratumoral aneurysm is rare and epistaxis caused by rupture of it is extremely rare. Lack of bony protection apparently have contributed to the aneurysmal growth and rupture.

[Perforation of the intestine by a peritoneal tube 10 years after a ventriculo-peritoneal shunt].

A case is reported of intestinal perforation by a ventriculoperitoneal shunt (V-P shunt) tube 10 years after V-P shunt. A 49-year-old male received V-P shunt for normal pressure hydrocephalus following subarachnoid hemorrhage. Ten years later he was admitted to our department with an abscess on the anterior chest and on the abdominal wall along the shunt tube. When CT scan revealed that the peritoneal tube had perforated the bowel, the shunt was removed. During the operation it was found that the peritoneal tube was wrapped with fibrous tissue and that it had perforated the intestine. The subcutaneous abscess healed after the patient received systemic antibiotics. He was discharged and returned to work. We discussed the mechanism of bowel perforation in this case. It is assumed that bowel perforation occurred because of continuous friction at the same site of the bowel wall after the peritoneal tube received fibrous encasement in the abdominal cavity. Bowel perforation was diagnosed ten years after the V-P shunt in this case. To our knowledge, this is the longest period amongst reported cases.

[Simplification of on-line connection of analyzers and standardization problems].

On-line connection of automated analyzers to laboratory information system (LIS) reduces mistakes in inputing data for each samples. It also makes reporting faster in clinical laboratory. Moreover, connection of these instruments with sample transporting system enables analyses without touching samples directly. It, however, costs extremely high to construct such a system. It is because every automated analyzer uses different connecting protocol, so that we have to make a different program for each machine. For solving this problem, we have to make a standard for connecting protocol. It is very difficult to make a standard protocol fitting on all of the analyzers, considering its cost and other things. Furthermore, it must take a few decades to spreading the standard through the end users. ICCLS and NCCLS has been taking a central role for this problem since 1996, with a five-year plan to make an international standard. Until the standard will be laid, each clinical laboratories have to pay high costs to construct their systems, connecting different manufacturers' analyzers each other. Thus, we have developed a novel system which enables us to construct a laboratory automation system in a shorter time. For realizing this new system, we have reduced the number of connecting protocols for analyzers. Moreover, we have corrected the flow of laboratory works in order. Each programs are put together into a system as parts, or modules. This software system is now in operation in the clinical laboratory of Kochi Medical School. In this report, we describe the construction of this novel software system, and the effect obtained by using this system. Furthermore, we would show problems and things to be improved for making international standards for communication protocols between host and analyzing instruments.

Polymerase chain reaction amplification of Asp f I and alkaline protease genes from fungus balls: clinical application in pulmonary aspergillosis.

Asp fI(18 kDa) and alkaline protease (33 kDa) are the 2 major antigens which are derived from Aspergillus (A.) fumigatus and have been implicated as possible virulence factors in the pathogenesis of Aspergillus-induced diseases. We attempted to detect fragments of genes encoding both proteins from fungus balls obtained at surgery or autopsy by polymerase chain reaction (PCR) amplification and then used PCR to test clinical samples. Frozen-stored fungus ball samples from a patient with acute myeloid leukemia complicated by Aspergillus pneumonia and from a patient with pulmonary aspergilloma were studied. We successfully amplified a 315 bp PCR product, the target sequence for Asp f I, and a 747 bp PCR product as a target sequence for alkaline protease (ALP) in both cases. In addition, 13 clinical samples including sputum specimens from patients with pulmonary aspergillosis were also examined. PCR analysis for the Asp f I (ALP) gene in clinical samples showed positive results in 5/10 (6/10) patients with pulmonary aspergilloma and in 3/3 (1/ 3) patients with invasive pulmonary aspergillosis. Culture data on A. fumigatus revealed positive results in 3/9 patients with pulmonary aspergilloma and in 2/3 patients with invasive pulmonary aspergillosis. This method can be used to recognize the involvement of A. fumigatus in various clinical settings where conventional culture results are not readily available.

Sphenoethmoidal mucoceles with intracranial extension--three case reports.

Three unusual cases of sphenoethmoidal mucoceles with rare intracranial extension are reported. A 64-year-old female presented with a 7-month history of right visual disturbance. Computed tomography (CT) and magnetic resonance (MR) imaging demonstrated a huge mass in the right middle fossa. She underwent right frontotemporal craniotomy. Postoperatively, her proptosis and cranial nerve dysfunction had improved markedly. A 53-year-old female complained of headache, nausea, and dizziness. CT and MR imaging revealed a cystic mass filling the right sphenoid sinus. The cystic lesion was evacuated through the transnasal approach. She was doing well postoperatively and has been asymptomatic. A 39-year-old male complained of headache, vomiting, and right visual disturbance. CT and MR imaging demonstrated a homogeneous mass occupying the sphenoid sinus. Sphenoidotomy exposed the cyst extending superiorly into the anterior cranial fossa. He recovered from the visual disturbances and has been asymptomatic since. MR imaging provides confirmation of the diagnosis of sphenoethmoidal mucocele and is important for preoperative evaluation.

Establishment of a clonal cell line producing granulocyte colony-stimulating factor and parathyroid hormone-related protein from a lung cancer patient with leukocytosis and hypercalcemia.

Squamous cell lung carcinoma cells obtained from a patient who presented with leukocytosis and hypercalcemia were transplanted into nude mice and a serially transplantable cell line, OKa-N-1, was established. The nude mice transplanted with OKa-N-1 cells displayed leukocytosis and hypercalcemia. Serum levels of granulocyte colony-stimulating factor (G-CSF) and parathyroid hormone-related protein (PTHrP) were both elevated in these mice. In vitro cultivation of this tumor cell line gave rise to a clonal cell line, OKa-C-1. Nude mice transplanted with the OKa-C-1 cell line also showed leukocytosis and hypercalcemia with high serum G-CSF and PTHrP levels. The culture supernatant of OKa-C-1 contained high levels of G-CSF and PTHrP. Immunohistochemical studies showed the expression of PTHrP in OKa-C-1 cells. Reverse transcription polymerase chain reaction revealed the presence of G-CSF and PTHrP mRNA in this cell line. Dexamethasone treatment inhibited the transcription of G-CSF and PTHrP genes. This new human squamous carcinoma cell line, OKa-C-1, would be useful for studying the mechanism of simultaneous production of G-CSF and PTHrP and their control in cancer patients with leukocytosis and hypercalcemia.

Introduction of a gene from fertility restored radish (Raphanus sativus) into Brassica napus by fusion of X-irradiated protoplasts from a radish restorer line and iodacetoamide-treated protoplasts from a cytoplasmic male-sterile cybrid of B. napus.

To establish a cytoplasmic male-sterile/restored fertility (cms-Rf) system for F1 seed production in Brassica napus, we transferred a gene from fertillity restored radish to B. napus by protoplast fusion. X-irradiated protoplasts, isolated from shoots of Raphanus sativus cv Kosena (Rf line), were fused with iodoacetamide-treated protoplasts of a B. napus cms cybrid. Among 300 regenerated plants, six were male-fertile. The fertile plants were characterized for petal color, chromosome number and the percentage of viable pollen grains. Three fertile plants had aneuploid chromosome numbers and white or cream petals, which is a dominant marker in radish. Of these three plants, one which had 2n = 47 chromosomes and white petals was used for further backcrosses. After two backcrosses, chromosome number and petal color became identical to that of B. napus. No female sterility was observed in the BC3 generations.

Characterization of adenosine-uridine-rich RNA binding factors.

The adenosine-uridine (AU)-rich sequences within the 3' untranslated region (UTR) of many short-lived mRNAs are important in their rapid degradation. We present evidence that human embryonic lung fibroblasts (W138) contain five major proteins of 70, 45, 40, 38, 32.5 kd, which specifically bind the AU-rich region of human granulocyte-macrophage colony-stimulating factor (GM-CSF) 3'UTR containing 7 x AUUUA motifs. The 40 and 38 kd proteins also bound the 3x and 5 x AUUUA cassettes and even more strongly bound to the AUUUUUUUA motif. All five of these proteins showed more abundant localization in the nucleus than the cytoplasm. The 32.5 kd protein was the major cytoplasmic AU-binding protein. Incubation with actinomycin D resulted in a marked increase in binding activity of 45, 40, 38, and 32.5 kd proteins in the cytoplasm, accompanied by decreased binding activity of the 32.5 kd protein in the nucleus. Antibody against heterogeneous nuclear ribonucleoprotein C (hnRNP C) immunoprecipitated the 40 and 38 kd proteins, and antibody against the AU-rich element RNA-binding protein (AUF1) immunoprecipitated the 45, 40, and 38 kd proteins. The present results not only demonstrated that hnRNP C are AU-binding proteins which are present in the cytoplasm as well as the nucleus, but another group of AU-binding proteins (AUF1 [45, 40, 38 kd], and 32.5 kd), which are not hnRNP, have characteristics related to those of hnRNPs. Taken together with our previous results (Akashi et al., 1994, Blood, 83:3182-3187), AU-binding factors including hnRNP C and AUF1, which bind more than 3 x AUUUA motifs, may be involved in rapid degradation of these transcripts. No significant quantitative changes of these proteins in their binding activity to AU-rich sequences occurred in response to several stimuli that stabilize GM-CSF mRNA, indicating that binding of these proteins to their cognate RNA is not responsible for the stabilization of these transcripts.

Mutations of p53 in Wilms' tumors.

Mutations of p53 frequently occur in a wide variety of cancers including lung, breast, gastrointestinal, brain, and hematologic malignancies. These alterations apparently contributed to development of the malignant phenotype. Wilms' tumor is one of the most common solid tumors in childhood. The frequency of p53 alterations in this tumor is unknown. We analyzed 66 Wilms' tumor samples for p53 mutations by single-stand conformational polymorphism (SSCP) following polymerase chain reaction (PCR). Samples with an abnormal SSCP pattern were reamplified and analyzed by direct sequencing method. Mutations of p53 were found in three (5%) of 66 Wilms' tumors within the coding region (exons 2-11), showing that the frequency of p53 mutations was low. Two mutations substituted amino acids residues and one encoded a stop codon. Two of the mutations were located in the mutational hotspots (exons 5 and 6); the other was in exon 10. These data suggest that p53 mutations are infrequent in the development of Wilms' tumors.

Improvement in the quality of seed storage protein by transformation of Brassica napus with an antisense gene for cruciferin.

The levels of certain essential amino acids, in particular cysteine, lysine and methionine, in the seed storage protein of a commercial spring variety of rape, Brassica napus, have been increased by the introduction of an antisense gene for cruciferin, which is the most abundant storage protein in rapeseed. The antisense construct contained part of the cruA gene in an inverted orientation, and the gene was driven by the 5' flanking region of the gene for napin such that antisense RNA was expressed in a seed-specific manner. The construct was introduced by Agrobacterium-mediated gene transfer. In self-pollinated seeds (T1 seeds) of transgenic plants there was a reduction in the levels of the α1ß1 and α2/3ß2/3 subunits of cruciferin, whereas the level of the α4ß4 subunit was unchanged. The total protein and lipid contents of transgenic seeds did not differ significantly from that of normal seeds. Seeds with reduced amounts of cruciferin accumulated higher amounts of napin than non-transformed seeds, but the level of oleosin was unaffected. Amino-acid analysis of the seed storage protein revealed that T1 seeds with reduced amounts of cruciferin contained higher relative levels of three essential amino acids, namely, lysine, methionine and cysteine, with increases of 10%, 8% and 32% over the respective levels in non-transgenic seeds (B. napus cv Westar).

Effects of an antisense napin gene on seed storage compounds in transgenic Brassica napus seeds.

To manipulate the quantity and quality of storage components in Brassica napus seeds, we have constructed an antisense gene for the storage protein napin. The antisense gene was driven by the 5'-flanking region of the B. napus napin gene to express antisense RNA in a seed-specific manner. Seeds of transgenic plants with antisense genes often contained reduced amounts of napin. In some transgenic plants, no accumulation of napin was observed. However, the total protein content of transgenic and wild-type seeds did not differ significantly. Seeds lacking napin accumulated 1.4 to 1.5 times more cruciferin than untransformed seeds, although the oleosin content was not affected. Fatty acid content and composition in the seeds of transgenic plants were also analyzed by gas chromatography. Though the total fatty acid content of the transformants was the same as that of non-transformants, there was a reduction in 18:1 contents and a concomitant increase of 18:2 in seeds with reduced napin levels. This observed change in fatty acid composition was inherited in the next generation.

Mutation of the p53 gene in neuroblastoma and its relationship with N-myc amplification.

Mutation of the p53 tumor suppressor gene frequently occurs in a variety of tumors including lung, breast, gastrointestinal, and brain, as well as lymphomas-leukemias. Neuroblastoma, one of the most common solid tumors in childhood, often has amplification of the N-myc gene. We examined for mutations of the p53 tumor suppressor gene by single-strand conformational polymorphism using polymerase chain reaction products and direct sequencing method in neuroblastoma; in addition, we assessed the relationship between p53 mutation and N-myc gene amplification in the disease. Of 86 DNA samples from patients with neuroblastoma, two mutations (2%) were found in the coding region of the p53 gene. Each mutation caused a substitution of amino acid residues. One mutation was located in exon 5, and another was in exon 6. N-myc gene was amplified in 26% of the samples. No p53 mutations were found in neuroblastoma samples with N-myc amplification. In the two individuals, p53 mutations appeared as their disease became more progressive. The neurofibromatosis 1 (NF1) gene is frequently abnormal in another neural disorder, neurofibromatosis type 1; in addition, a potential mutational hot spot of NF1 at lysine at codon 1423 has been identified in several types of tumors. Using single-strand conformational polymorphism, we were unable to detect an abnormality in this region of NF1 in 50 samples of neuroblastoma. The data suggest that p53 mutations occasionally are associated with progression of neuroblastomas, and tumorigenetic influences of mutant p53 may differ from those of N-myc.

Absence of HTLV-I infection among seronegative subjects in an endemic area of Japan.

To examine the prevalence of infection by human T-cell leukemia virus type I (HTLV-I) among seronegative subjects, healthy subjects on Tsushima Island, Japan, where the infection is endemic, were evaluated. A total of 209 healthy adults were examined for HTLV-I provirus in peripheral blood mononuclear cells by the polymerase chain reaction (PCR), as well as for anti-HTLV-I antibodies by the particle agglutination (PA) method, the enzyme-linked immunosorbent assay (ELISA) and by immunofluorescence analysis (IF). A total of 76 subjects were positive and 133 were negative for the provirus, showing a close correlation with the results of 3 assays for anti-HTLV-I serum antibodies. None of the seronegative subjects reacted positively on PCR analysis. These observations indicate that seronegative HTLV-I carriers are rare in an area of Japan in which this viral infection is endemic.