Role of aquaporin-5 in gallbladder carcinoma.

BACKGROUND/PURPOSE: Aquaporins (AQPs) are important in controlling bile formation. However, the exact role in human gallbladder carcinogenesis has not yet been defined. METHODS: AQP-5-expressing gallbladder carcinoma (GBC) cell lines (NOZ) were transfected with anti-AQP-5 small interfering RNA (siRNA). Growth, migration, invasion assay, and drug susceptibility tests were performed. Next, microRNA (miRNA) expression was analyzed by miRNA oligo chip (3D-Gene®). AQP-5 and AQP-5-related miRNA target gene expressions were also analyzed using tissue microarray (TMA) in 44 GBC samples. RESULTS: Treatment with AQP-5 siRNA decreased cell proliferation, migration, and invasion. On the other hand, those cells increased IC50 of gemcitabine. By performing miRNA assays, miR-29b, -200a, and -21 were shown to be highly overexpressed in cells treated with AQP-5 siRNA NOZ. When focusing on miR-21, phosphatase and tensin homolog (PTEN) was found to be a target of miR-21. In the TMA, AQP-5/PTEN coexpression was significantly associated with the depth of invasion and MIB-1 index (p = 0.003, 0.010). Survival of patients with a high AQP-5/PTEN coexpression was longer than that of patients with a low coexpression (p = 0.003). CONCLUSIONS: Our result suggested that miR-21 and PTEN may contribute to the role of AQP-5 in GBC. AQP-5 and PTEN cascades are favorable biomarkers of GBC.

Ion-production efficiency of a singly charged ion source developed toward a 11C irradiation facility for cancer therapy.

The ion-production efficiency of a newly developed singly charged ion source (SCIS) has been investigated to discuss the possibility of it being used in an isotope separation on-line system that provides 11C ions for heavy-ion cancer therapy with simultaneous verification of the irradiation field using positron emission tomography. The SCIS uses a low-energy hollow electron beam to produce singly charged carbon ions efficiently. To deliver sufficient 11C ions to the treatment room from a limited amount of 11C molecules, which are produced from a boron compound target and proton-beam irradiation via the 11B(p,n)11C reaction, the SCIS must have high ion-production efficiency. To realize this high efficiency, the SCIS was designed using a three-dimensional particle-in-cell code in previous work. With the fabricated SCIS, we performed experiments to measure the efficiency of producing CO2 + ions from nonradioactive 12CO2 molecules and C+ ions from nonradioactive 12CH4 molecules. We found that the SCIS achieved efficiencies of εC+ =4×10-3 (0.4%) for C+ production and εCO2 + =0.107 (10.7%) for CO2 + production.

Development of gas pulsing system for electron cyclotron resonance ion source.

A gas-pulsing system for an electron cyclotron resonance ion source with all permanent magnets (Kei2 source) at NIRS has been developed and tested. The system consists of a small vessel (30 ml) to reserve CH(4) gas and two fast solenoid valves that are installed at both sides of the vessel. They are connected to each other and to the Kei2 source by using a stainless-steel pipe (4 mm inner diameter), where the length of the pipe from the valve to the source is 60 cm and the conductance is 1.2 l/s. From the results of the test, almost 300 e microA for a pulsed (12)C(4+) beam was obtained at a Faraday cup in an extraction-beam channel with a pressure range of 4000 Pa in the vessel. At this time, the valve has an open time of 10 ms and the delay time between the valve open time and the application of microwave power is 100 ms. In experiments, the conversion efficiency for input CH(4) molecules to the quantity of extracted (12)C(4+) ions in one beam pulse was found to be around 3% and the ratio of the total amount of the gas requirement was only 10% compared with the case of continuous gas provided in 3.3 s of repetition in HIMAC.

Application of compact electron cyclotron resonance ion source.

The compact electron cyclotron resonance (ECR) ion source with a permanent magnet configuration (Kei2 source) has been developed at National Institute of Radiological Sciences for a new carbon therapy facility. The Kei2 source was designed for production of C(4+) ions; its performance such as beam intensity and stability has already reached the medical requirements. Therefore, the prototype development of the source for medical use is essentially finished. Recently, we have started a few studies on other applications of the source. One is the production of fullerenes in the ECR plasma and modified fullerenes with various atoms for new materials. A second application is the production of multiply charged ions (not only carbon) for ion implantation. In this paper, some basic experiments for these applications are reported.

Singly charged ion source designed using three-dimensional particle-in-cell method.

A singly charged ion source (SCIS) has been designed using a newly developed three-dimensional particle-in-cell (PIC) code. The SCIS is to be used in an isotope separation on-line (ISOL) system that provides 11C ions for heavy-ion cancer therapy with simultaneous verification of the dose distribution using positron emission tomography. The SCIS uses low-energy electron beams to produce singly charged carbon ions efficiently and maintain a high vacuum in the ISOL system. Because the SCIS has to realize a production efficiency of 1% if its carbon ions are to be used in the ISOL system, a suitable design for the SCIS was investigated by using the developed PIC code to study the beam trajectories of the electrons and extracted ions. The simulation results show that hollow electron beams are produced in the designed SCIS resulting in a high effective electron current. The results also predict that the designed SCIS would realize ion-production efficiencies (IPEs) of ε SCIS ≃ 6.7% for C O 2 + production from CO2 gas and ε SCIS ≃ 0.1% for C+ production from CH4 gas. Moreover, to examine the validity of the developed code and confirm that the SCIS was able to be designed appropriately, the space-charge-limited current of the electron gun and the total IPE obtained by adding the IPEs of each ion were compared between the experiment and the simulation.

A singly charged ion source for radioactive ¹¹C ion acceleration.

A new singly charged ion source using electron impact ionization has been developed to realize an isotope separation on-line system for simultaneous positron emission tomography imaging and heavy-ion cancer therapy using radioactive (11)C ion beams. Low-energy electron beams are used in the electron impact ion source to produce singly charged ions. Ionization efficiency was calculated in order to decide the geometric parameters of the ion source and to determine the required electron emission current for obtaining high ionization efficiency. Based on these considerations, the singly charged ion source was designed and fabricated. In testing, the fabricated ion source was found to have favorable performance as a singly charged ion source.

Development of a compact ECR ion source for various ion production.

There is a desire that a carbon-ion radiotherapy facility will produce various ion species for fundamental research. Although the present Kei2-type ion sources are dedicated for the carbon-ion production, a future ion source is expected that could provide: (1) carbon-ion production for medical use, (2) various ions with a charge-to-mass ratio of 1/3 for the existing Linac injector, and (3) low cost for modification. A prototype compact electron cyclotron resonance (ECR) ion source, named Kei3, based on the Kei series has been developed to correspond to the Kei2 type and to produce these various ions at the National Institute of Radiological Sciences (NIRS). The Kei3 has an outer diameter of 280 mm and a length of 1120 mm. The magnetic field is formed by the same permanent magnet as Kei2. The movable extraction electrode has been installed in order to optimize the beam extraction with various current densities. The gas-injection side of the vacuum chamber has enough space for an oven system. We measured dependence of microwave frequency, extraction voltage, and puller position. Charge state distributions of helium, carbon, nitrogen, oxygen, and neon were also measured.

Cryogenic molecular separation system for radioactive (11)C ion acceleration.

A (11)C molecular production/separation system (CMPS) has been developed as part of an isotope separation on line system for simultaneous positron emission tomography imaging and heavy-ion cancer therapy using radioactive (11)C ion beams. In the ISOL system, (11)CH4 molecules will be produced by proton irradiation and separated from residual air impurities and impurities produced during the irradiation. The CMPS includes two cryogenic traps to separate specific molecules selectively from impurities by using vapor pressure differences among the molecular species. To investigate the fundamental performance of the CMPS, we performed separation experiments with non-radioactive (12)CH4 gases, which can simulate the chemical characteristics of (11)CH4 gases. We investigated the separation of CH4 molecules from impurities, which will be present as residual gases and are expected to be difficult to separate because the vapor pressure of air molecules is close to that of CH4. We determined the collection/separation efficiencies of the CMPS for various amounts of air impurities and found desirable operating conditions for the CMPS to be used as a molecular separation device in our ISOL system.

Effect of JTP-2942, a novel thyrotropin-releasing hormone analog, on motor deficits after chronic focal cerebral ischemia in rats.

To investigate the chronic effects of a novel thyrotropin-releasing hormone analog, JTP-2942 (N(alpha)-[(1S, 2R)-2-methyl-4-oxocyclopentylcarbonyl]-L-histidyl-L-prolinamide monohydrate), on behavioral changes after stroke, the authors examined its effects on motor and neurologic deficits using a middle cerebral artery (MCA) occlusion model in rats. A left MCA was permanently occluded at a proximal site. From 1 week after occlusion, JTP-2942 was intravenously administered once a day for 4 weeks. Sensorimotor performance was evaluated weekly for 10 weeks after the occlusion. The ability of the rat to maintain its body position on an inclined plane and neurologic examination based on hemiparesis and abnormal posture were examined. After all behavioral examinations were completed, the degree of shrinkage of the left hemisphere was measured. The ability of MCA-occluded rats to maintain body position on an inclined plane in the left-headed position was significantly lower than that of sham-operated rats throughout the test period. JTP-2942 gradually improved this deficit dose dependently, and a dose of 0.03 mg/kg of JTP-2942 significantly improved performance to the levels of the sham-operated rats. Neurologic deficits were also observed in MCA-occluded rats. JTP-2942 also significantly improved these deficits dose dependently. On the other hand, CDP-choline (500 mg/kg, administered intravenously), a therapeutic agent for the disturbance of consciousness and hemiparesis after stroke, improved neurologic deficits but did not affect the motor deficits measured using the inclined plane. It is noteworthy that the effects of JTP-2942 on these deficits were observed 4 weeks after cessation of drug administration. Furthermore, there was no difference in the degree of shrinkage of the cerebrum among the MCA-occluded groups. In the present study, long-lasting improving effects of JTP-2942 on the impairment of motor and neurologic functions were observed in rats with MCA occlusion, which continued after cessation of drug administration and which were not attributable to a reduction in ipsilateral cerebral shrinkage. It is considered that the effect of JTP-2942 on functional recovery is attributable to the activation of substitutive functions such as neuronal reconstruction. These pharmacologic properties of JTP-2942 may be of interest for the treatment of patients with motor and neurologic deficits during the chronic or subacute phase of stroke.

Secretion of laminin alpha 2 chain in cerebrospinal fluid.

The absence of laminin alpha 2 chain causes muscle cell degeneration and peripheral dysmyelination in congenital muscular dystrophy patients and dy mice, suggesting its role in the maintenance of sarcolemmal architecture and peripheral myelinogenesis. Here we demonstrate the secretion of laminin alpha 2 chain in cerebrospinal fluid (CSF). Laminin alpha 2 chain was detected as a minor component of the total CSF proteins or glycoproteins. Laminin alpha 2 chain was localized in the cytoplasm of epithelial cells of choroid plexus, suggesting active secretion. Our results suggest that immunochemical analysis of CSF laminin alpha 2 chain could be useful as an aid for the diagnosis of congenital muscular dystrophy.

Mechanisms of the release of CYFRA21-1 in human lung cancer cell lines.

The CYFRA 21-1 assay which detects cytokeratin 19 (CK19) fragment is widely used as a tumor marker for lung cancer. However, the reason why some lung cancer cell lines release CK19 fragment in culture supernatants and others do not, remains unclear. It was hypothesized that the release of CK19 fragment may be elucidated by the expression of mRNA for CK19. In order to prove this, the mRNA for CK19 was quantitatively evaluated by the competitive reverse transcriptase-polymerase chain reaction (competitive RT-PCR). The level of CYFRA 21-1 in the culture supernatant was measured by an immunoradiometric assay. CK19 protein synthesis was evaluated by a Western blotting and immunohistochemistry. Fourteen lung cancer cell lines were evaluated, and the amount of mRNA correlated well with the level of CYFRA 21-1 in culture supernatants. Analysis of genomic DNA for CK19 demonstrated that three cell lines which could not produce CYFRA 21-1, conjectured that some abnormalities in exon 1 or the 5'-region upstream from exon 1. In conclusion, it was demonstrated that the release of CK19 fragment was closely related to the expression of mRNA for CK19, and the possibility that genomic change of CK19 DNA down-regulated the expression of mRNA for CK19 was suggested.

Increased intensity of lung infiltrates at the side of lung cancer in patients with lung cancer associated with pulmonary fibrosis.

It has been reported that lung cancer is frequently associated with idiopathic pulmonary fibrosis (IPF). The purpose of this study was to compare the intensity of lung infiltrates between the side associated with lung cancer and the side without lung cancer. Twenty-three patients (24 lung cancers) with primary lung cancer associated with pulmonary fibrosis were retrospectively evaluated. Chest CT findings were evaluated by three expert radiologists using the intensity scores. In 16 of the 23 patients, it was possible to compare the intensity of lung infiltrates between both sides of the lungs. As a result, increased intensity at the side in which lung cancer developed was demonstrated in 12 of 16 patients (75%). In the remaining four patients, intensity of lung infiltrates was the same in both lungs. In operated patients as well as autopsied patients, it was possible to evaluate the pathological findings of lung tissues around cancer cells. This study clearly demonstrates that the intensity of lung infiltrates increased at the side in which lung cancer developed.

The point mutation in the promoter region and the single nucleotide polymorphism in exon 1 of the cytokeratin 19 gene in human lung cancer cell lines.

The CYFRA 21-1 assay which detects the cytokeratin 19 (CK19) fragment is widely used as a tumor marker for lung cancer. We previously suggested that the failure of PCR amplification of exon 1 is closely related to the inability of the expression of mRNA for CK19, and hypothesized that point mutations might exist within exon 1. In order to prove this, sequence analysis of the promoter region and exon 1 was performed in 14 human lung cancer cell lines. Among the 14 lung cancer cell lines evaluated, point mutations within the promoter region (at -99, G-->C) of the CK19 gene were demonstrated in two cell lines (Lu135 and HI1017). In addition, point mutations within exon 1 (at 90, T-->C, Ala-->Ala and at 179, G-->C, Gly-->Ala) were also demonstrated in three cell lines (LU135, HI1017, and LC2/AD). Point mutations within the promoter region of CK19 (at -99) and within exon 1 (at 179) were confirmed by analysis of digestion by specific restriction enzymes. Since the same point mutation within exon 1 (at 179) was observed in genomes of normal volunteers, this mutation was considered as a single nucleotide polymorphism. In contrast, there were no mutations within the promoter region of exon 1 in genomes of normal volunteers. After a computer search, it was demonstrated that several transcription factors bind to the sense primer sequence which was designed for amplification of exon 1. In addition, after point mutations within the promoter region occurred (at -99), new sequences appeared to which known transcription factors (AP2) bind. In conclusion, analysis of genomic DNA for CK19 suggested that expression of mRNA for CK19 was regulated by several transcription factors which bound to the specific sequence with the promoter region of the CK19 gene. It was also suggested that the mutation in the promoter region of the CK19 gene down-regulated the expression of mRNA for CK19.

Investigation of nosocomial infection caused by arbekacin-resistant, methicillin-resistant Staphylococcus aureus.

An outbreak of coagulase VII-producing, arbekacin (ABK)-resistant, methicillin-resistant Staphylococcus aureus (MRSA) occurred between September 1994 and December 1995, involving five different wards. Twenty-one patients developed skin, wound, drainage, or respiratory tract colonization with coagulase VII-producing, (ABK)-resistant MRSA. Phenotypic characteristics (production of enterotoxin and TSST-1, antimicrobial susceptibility) and molecular-typing procedure (plasmid DNA profile, pulsed-field gel electrophoresis [PFGE] and arbitrarily primed polymerase chain reaction [AP-PCR] of chromosomal DNA) in isolated strains were compared. Plasmid analysis identified four different profiles and 19 of 22 strains recovered had identical patterns. PFGE of chromosomal DNA identified three different subtypes and 18 (81.8%) isolates shared the same subtype. AP-PCR also demonstrated that most strains had the same phenotypic characteristics. Although traditional epidemiological methods; for example, coagulase typing, plays a central role in hospital infection control, combination of plasmid DNA profile, AP-PCR, and PFGE may prove to be a particularly informative means of tracking the nosocomial spread of MRSA.

Acid profile in carious dentin.

Organic acids in carious dentin from 69 permanent teeth were analyzed by gas chromatography. Lactate, acetate, propionate, and butyrate were detected in most samples, and limited amounts of isobutyrate, valerate, isovalerate, caproate, and isocaproate were occasionally detected. Lactate, acetate, and propionate were major acids and altogether accounted for about 90% of total acid in most samples of carious dentin. However, the proportion of these three acids varied among the samples. Some samples contained over 85% lactate, while others contained mainly acetate and propionate. A high percentage of acetate was usually accompanied by an appreciable amount of propionate. All seven samples in carious dentin under fillings or restorations had little lactate, but a high percentage of acetate plus propionate. The differences in acid profiles of carious dentin may reflect differences in the microbial ecology of carious dentin, and a stage of progress of dentin caries or a type of dentin caries.

Acid profiles and pH of carious dentin in active and arrested lesions.

Organic acids in caries lesions play important roles in initiation and progress of dental caries. We investigated relationships between clinical types of dentin caries and acid profile or pH in the lesions. Caries lesions in dentin from 76 permanent teeth were classified into active, arrested, situated beneath a restoration, and unclassified types. The pH of carious dentin was distinctly lower than that of sound dentin (p < 0.001). Carious dentin with a high percentage of lactate had a lower pH than that with a high percentage of acetate and propionate (p < 0.001). Dentin from active lesions showed a mean pH of 4.9, and the dominant acid was lactate (mean percentage, 88.2). In contrast, carious dentin from arrested lesions showed a higher pH, 5.7, with acetate and propionate as the dominant acids (mean percentages of acetate and propionate, 64.0 and 18.2, respectively). The acid profile (mean percentages of acetate and propionate, 54.0 and 27.7, respectively) and pH (mean 5.8) of carious dentin sampled from lesions beneath a restoration were similar to those of dentin from arrested lesions. This study showed a clear relationship between clinical classification of dentin caries and acid profile and pH, suggesting that both factors are important in dentin caries etiology.

Elevation of cytokeratin 19 fragment in serum in patients with hepatoma: its clinical significance.

OBJECTIVES: Cytokeratin 19 fragment (CK19) levels in serum have already been documented as a useful tumour marker for lung cancer. In the present study, we hypothesize that CK19 may be increased in serum from patients with hepatoma. METHODS: We measured the CK19 levels in serum from patients with hepatoma and evaluated the correlation between CK19 level and each clinical parameter. We studied 70 patients diagnosed with hepatoma, and used 14 patients with chronic hepatitis C and 45 patients with liver cirrhosis as controls. RESULTS: In 33 of 70 patients (47.1%) with hepatoma, the serum CK19 level was elevated to above the normal range. CK19 levels in serum from patients with hepatoma were significantly correlated with levels of alpha-fetoprotein and prothrombin induced by vitamin K absence for factor II (PIVKA-II). In 57 patients with hepatoma in whom both CK19 and alpha-fetoprotein were measured, only CK19 was elevated in seven patients (12.3%). Immunohistochemical studies using hepatoma tissues demonstrated that hepatoma cells were stained by anti-human CK19 antibody. We also demonstrated that the HepG2 cell line expressed CK1 9. CONCLUSIONS: Our data demonstrate that hepatomas aberrantly express CK19, and that measurement of CK19 might be a useful tumour marker in diagnosing hepatoma.

Clinical features of Stenotrophomonas maltophilia pneumonia in immunocompromised patients.

Between January 1988 and December 1992, 68 patients admitted to our Department of Internal Medicine with haematological malignancies or solid tumours showed colonization of the respiratory tract with Stenotrophomonas maltophilia. To characterize the significance of respiratory tract colonization by S. maltophilia, we retrospectively reviewed the medical records of the 68 patients colonized with this organism. Twenty-nine of these 68 patients developed pneumonia, with S. maltophilia being implicated in 10 cases. The majority of these 10 patients showed lobular infiltration on chest X-ray. Pleural effusion was observed in two (20%) of the 10 patients. All 68 strains of S. maltophilia were resistant to imipenem. Latamoxef was effective against 98 center dot 5% of strains, while minocycline was effective against 100% of strains. This report describes the clinical features of nosocomial S. maltophilia pneumonia in immunocompromised patients.

Overexpression of p53 protein in interstitial lung diseases.

The p53 gene is well known as a tumour suppressor gene. In addition, the mutated p53 gene is detected in a variety of human cancers including lung cancer, and is considered as an oncogene. Lung cancer is also frequently associated with interstitial lung diseases. Therefore, it may be possible to hypothesize that there might be some abnormality of p53 gene in interstitial lung diseases. This work examined the relationship between the p53 protein and gene in lung tissues of 28 patients with interstitial lung diseases. Among 28 patients, 13 cases were pathologically diagnosed to have usual interstitial pneumonia (UIP), 12 cases were diagnosed as having collagen vascular lung diseases, and three cases were diagnosed to have a non-specific interstitial pneumonia. Twenty-three tissue samples were obtained by open lung biopsy and five samples were taken by autopsy. Paraffin-embedded tissues were treated by microwave, and stained with an anti-p53 antibody (DO7) by the Avidin-Biotin-Peroxidase (ABC) method. In selected patients, mutations in exons 5-8 of the p53 gene were also examined by single-strand conformation polymorphism (SSCP) analysis and DNA sequence. In addition, the presence of anti-p53 antibodies in patients' sera was screened for by ELISA. Fifteen samples (53.6%) revealed overexpression of the p53 protein in the nuclei of alveolar epithelial cells. However, SSCP or sequence analysis, which was performed in 13 tissues, showed no mutations in exons 5-8 of the p53 gene. In conclusion, p53 proteins were overexpressed in interstitial lung diseases, and the expressed p53 protein was considered to be wild-type. This wild-type p53 protein may play a role in blocking the transformation of proliferative epithelial cells.

Detection of Pneumocystis carinii sequences in serum by polymerase chain reaction.

The clinical significance of the detection of Pneumocystis carinii DNA was evaluated, as well as the detection of circulating P. carinii antigen from serum using previously collected samples. Fourteen serum samples from 13 patients were diagnosed positively for P. carinii DNA by polymerase chain reaction (PCR). Ten of 14 episodes (71.4%) of pulmonary complications were compatible with P. carinii pneumonia. Two patients were definitely diagnosed as having had P. carinii pneumonia at autopsy. All patients positive for circulating antigens were also positive for P. carinii DNA, suggesting that the detection of P. carinii DNA by PCR is more sensitive compared to the detection of circulating antigens by the Ouchterlony method. It is concluded that the detection of P. carinii DNA in serum by PCR provides useful information for identifying P. carinii pneumonia.