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.

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.

Projections of cervical nerves to the rat medulla.

The central course of dorsal root ganglia (DRG) fibers from C1, C2 and C3, and particularly, their brainstem terminations were studied in rats using anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). WGA-HRP was injected into the exposed DRG, and after 3 days the animals were sacrificed and sections of spinal cord and brainstem were processed with tetramethylbenzidine and examined for anterograde transport. Labeled fiber terminals were identified in the dorsal horn and the central cervical nucleus in the spinal cord, and in the intermediate nucleus, cuneate nucleus, external cuneate nucleus and the caudal portion of the nucleus of the solitary tract (NTS) in the brainstem. The projection of primary sensory fibers to the visceral NTS is suggestive of a functional relationship between upper cervical and vagal nerve afferents. The potential association of these nerves with clinical problems of headache and other cephalgias is of interest.

Multiple brain abscesses associated with a mycotic aneurysm of the left common carotid artery. Case report.

A case is reported of multiple microabscesses confined to the left cerebral hemisphere and an aneurysm of the left common carotid artery. The aneurysm was presumed to be mycotic, secondary to extension of a tonsillar and pharyngeal infection. Infected microemboli dislodged from the aneurysmal sac were presumed to be the cause of the multiple microabscesses.

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.

An application of the microcomputer as a generator of target movements for the eye tracking test (ETT).

We have developed a new system to produce target movements for our particular purposes using a microcomputer and 9-bit digital-to-analog converter. A continuously changing sinusoidal wave was produced to examine the relationship between the frequencies of target movements and smooth pursuit. A random sinusoidal wave was produced to make clear the predictive function of smooth pursuit.

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.

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.

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.

[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.

Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (cms line) and Brassica napus through cytoplast-protoplast fusion.

Cytoplasts isolated from hypocotyl protoplasts of Raphanus sativus cv Kosena (cms line) by ultracentrifugation through Percoll/mannitol discontinuous gradient were fused with iodoacetamide(IOA)-treated protoplasts of Brassica napus cv Westar. Seventeen randomly selected regenerated plants were characterized for morphology and chromosome numbers. All of the regenerated plants had morphology identical to B. napus and 10 of them possessed the diploid chromosome number of B. napus. The remaining plants had chimeric or aneuploid chromosome numbers. The mitochondrial genomes in the 10 fusion products possessing the diploid chromosome numbers of B. napus were examined by Southern hybridization analysis. Four of the 10 plants contained mitochondrial DNA showing novel hybridization patterns. Of these 4 plants, 1 was male sterile, and 3 were male fertile. The remaining plants showed mitochondrial DNA patterns identical to B. napus and were male fertile.

Alteration of mitochondrial genomes containing atpA genes in the sexual progeny of cybrids between Raphanus sativus cms line and Brassica napus cv. Westar.

We have investigated the fate of the mitochondrial genomes of cybrids derived from "donor-recipient" protoplast fusion between X-irradiated Raphanus sativus (cms line) and iodoacetamide-treated Brassica napus cv. Westar. Two out of ten fusion products were male-sterile with the diploid chromosome number of B. napus. The mitochondrial (mt) genomes of the cybrids and their progeny were further analyzed by DNA-DNA hybridizaion using the pea mitochondrial ATPase subunit gene (atpA) as a probe. One cybrid, 18-3, had a 3.0 kb fragment characteristic of B. napus and a 2.0 kb non-parental fragment when the BamHI-digested DNA was hybridized with the probe. In the first-backcrossed progeny of this cybrid, the hybridization pattern was not stably inherited. A 4.0 kb radish fragment, not detectable in the cybrid, appeared in one of the BC1 generation siblings, and the 2.0 kb non-parental fragment was lost in another. The hybridization patterns in BC1 progeny siblings of cybrid 12-9 were also varied. The alteration of mtDNA in the cybrid progeny continued to the BC2 generation. There was no clear evidence of a heteroplasmic state or of sub-stoichiometric molecules in the mt genome of cybrid 18-3. A possible cause of the observed alteration in the mt genome is discussed.

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.

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).

Agrobacterium-mediated transformation and regeneration of kiwi fruit.

Genetically transformed kiwi fruit (Actinidia deliciosa) plants were obtained from hypocotyl and stem segments co-cultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector, pLAN411 or pLAN421, which contained the neomycin phosphotransferase II (nptII) gene and the ß-glucuronidase (GUS) gene. After co-culturing with the A. tumefaciens, the hypocotyl or stem segments were cultured on a selection medium containing 25µg/ml kanamycin and 500µg/ml Claforan. After one month in culture, shoots had regenerated from the cuttings. Green shoots were analyzed for NPTII activity and GUS activity. Eighty-five percent of the green shoots examined expressed the nptII and GUS genes. GUS histochemical assays revealed strong GUS expression in guard cells, mesophyll cells, and trichomes.