RESUMO
Based on physical and chemical determinations, the mnolecular weight of the type 4 adeno-satellite virus is 5.4 X 10(6) daltons, and the virion contains 1.4 X 10(6) daltons of DNA. Denaturation and renaturation studies indicate that the viral genome is a single-stranded DNA molecule and that each virion contains either a minus or a plus strand. Upon extraction, the minus and plus strands unite to form double-stranded DNA molecules with no obvious excess of unpaired strands.
Assuntos
Adenoviridae/análise , DNA Viral/isolamento & purificação , Centrifugação com Gradiente de Concentração , Microscopia Eletrônica , Peso Molecular , Desnaturação de Ácido Nucleico , Vírus Satélites , Análise EspectralRESUMO
Double applications of phorbol esters trigger excessive reactive oxygen species (ROS) production in mouse skin. Previously reported data suggest that the two applications induce distinguishable biochemical events, namely, priming and activation. The former is characterized as a recruitment of inflammatory cells, such as neutrophils, by chemotactic factors to inflammatory regions and edema formation. The latter is responsible for ROS generation. Thus, inhibitory effects of 1'-acetoxychavicol acetate (ACA), previously reported to be a superoxide generation inhibitor in vitro, on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative stress and inflammatory responses in mouse skin model were examined using a double application of ACA. We demonstrated that two pretreatments and pretreatment with ACA (810 nmol) in the activation phase suppressed double TPA application-induced H2O2 formation in mouse skin. ACA exhibited no inhibitory effects on edema formation and the enhancement of myeloperoxidase activity during the first TPA treatment, whereas the anti-inflammatory agent genistein administered at the same dose inhibited both biomarkers. No inhibitory potential of ACA for TPA-induced H2O2 formation in the priming phase was confirmed. On the other hand, in the in vitro study, ACA inhibited ROS generation in differentiated HL-60 cells more strongly than did 1'-hydroxychavicol, which showed no inhibition by pretreatment in the activation phase. In addition, allopurinol did not inhibit double TPA application-induced H2O2 formation in mouse skin. These findings suggest that the NADPH oxidase system of neutrophils rather than the epithelial xanthine oxidase system is dominant for the O2--generating potential in double TPA-treated mouse skin. ACA significantly inhibited mouse epidermis thiobarbituric acid-reacting substance formation, known as an overall oxidative damage biomarker. Moreover, histological studies demonstrated that ACA inhibited double TPA treatment-induced morphological changes reflecting inflammatory response, such as edema formation, leukocyte infiltration, hyperplasia, and cell proliferation. Furthermore, pretreatment with ACA but not 1'-hydroxychavicol in the activation phase inhibits double TPA application-induced increases in both number of leukocytes and proliferating cell nuclear antigen index. These results suggested that ROS from leukocytes including O2- plays an important role for continuous and excessive production of chemotactic factors, leading to chronic inflammation and hyperplasia, which are inhibitable by ACA. Thus, we concluded that O2- generation inhibitors are agents that effectively inhibit oxidative stress and inflammatory responses in mouse skin.
Assuntos
Anticarcinógenos/farmacologia , Inibidores Enzimáticos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Superóxidos/metabolismo , Terpenos/farmacologia , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Xantina Oxidase/antagonistas & inibidores , Animais , Álcoois Benzílicos , Feminino , Células HL-60 , Humanos , Peróxido de Hidrogênio/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Antígeno Nuclear de Célula em Proliferação/análise , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologiaRESUMO
The intake of citrus fruits has been suggested as a way to prevent the development of some types of human cancer. Nitric oxide (NO) is closely associated with the processes of epithelial carcinogenesis. We attempted a search for NO generation inhibitors in Citrus unshiu. The active constituent was traced by an activity-guiding separation. NO and superoxide (O2-) generation was induced by a combination of lipopolysaccharide and IFN-gamma in mouse macrophage RAW 264.7 cells, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated human promyelocyte HL-60, respectively. Expression of inducible NO synthase and cyclooxygenase 2 proteins were detected by Western blotting. The in vivo anti-inflammatory and antitumor promoting activities were evaluated by topical TPA application to ICR mouse skin with measurement of edema formation, epidermal thickness, leukocyte infiltration, hydrogen peroxide production, and the rate of proliferating cell nuclear antigen-stained cells. As a result, nobiletin, a polymethoxyflavonoid, was identified as an inhibitor of both NO and O2- generation. Nobiletin significantly inhibited two distinct stages of skin inflammation induced by double TPA application [first stage priming (leukocyte infiltration) and second stage activation (oxidative insult by leukocytes)] by decreasing the inflammatory parameters. It also suppressed the expression of cyclooxygenase-2 and inducible NO synthase proteins and prostaglandin E2 release. Nobiletin inhibited dimethylbenz[a]anthracene (0.19 micromol)/TPA (1.6 nmol)-induced skin tumor formation at doses of 160 and 320 nmol by reducing the number of tumors per mouse by 61.2% (P < 0.001) and 75.7% (P < 0.001), respectively. The present study suggests that nobiletin is a functionally novel and possible chemopreventive agent in inflammation-associated tumorigenesis.
Assuntos
Anticarcinógenos/uso terapêutico , Citrus/química , Toxidermias/prevenção & controle , Flavonas , Flavonoides/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Neoplasias Cutâneas/prevenção & controle , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Antioxidantes/farmacologia , Carcinógenos , Linhagem Celular , Ciclo-Oxigenase 2 , Dinoprostona/biossíntese , Toxidermias/metabolismo , Feminino , Flavonoides/isolamento & purificação , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Humanos , Interferon gama/farmacologia , Isoenzimas/biossíntese , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos ICR , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Prostaglandina-Endoperóxido Sintases/biossíntese , Pele/efeitos dos fármacos , Pele/metabolismo , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/metabolismo , Superóxidos/antagonistas & inibidores , Superóxidos/metabolismo , Acetato de TetradecanoilforbolRESUMO
The aim of this study was to investigate the contribution of various tobacco components to the generation of smoke constituents using a tobacco pyrolysis model. We analyzed the amounts of primary tobacco components (sugars, protein, polyphenols, alkaloids, organic acids, inorganics etc.) in flue-cured and burley tobacco leaves. Each of the components was added to the tobacco leaves at the 0.5-fold and 1.0-fold amount naturally present in the leaves. The treated tobacco samples were pyrolyzed at 800 degrees C in a nitrogen atmosphere with an infrared image furnace, and the selected smoke constituents (benzo[a]pyrene, hydrogen cyanide, carbonyl compounds, aromatic amines, volatile organic compounds and phenolics) were quantitatively analyzed by several methods, including high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC-MS). The contribution of each tobacco component to the generation of selected smoke constituents was estimated from a regression line determined by the three yields (no addition, 0.5-fold addition, and 1.0-fold addition). The results of this study can provide useful and comprehensive information on the relationship between tobacco components and selected smoke constituents during pyrolysis.
Assuntos
Modelos Teóricos , Nicotiana/química , Fumaça , Cromatografia Gasosa-Espectrometria de Massas , Incineração , Compostos Orgânicos/análise , Folhas de Planta/químicaRESUMO
The modifying effects of topical application of a catechol antioxidant protocatechuic acid (PA) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory responses in mouse skin were investigated. Treatment with a high dose (20,000 nmol) of PA, based on time of application, modifies inflammatory responses in the skin of the B6C3F(1) mouse, a resistant strain to inflammatory response induction by TPA, but shows much higher tyrosinase expression than that of an albino mouse. The responsibility of a large amount of PA-induced leukocyte infiltration to an inflamed region in a B6C3F(1) mouse is more sensitive than that of an ICR albino mouse. When ICR mice were treated with TPA (1.6 nmol) twice weekly for 5 weeks to induce chronic inflammatory responses, pretreatment with 1600 nmol PA 30 min prior to each TPA treatment significantly enhanced the inflammatory responses including edema formation, leukocyte infiltration, and the level of thiobarbituric acid-reacting substances. The dose-dependency was closely parallel to the results of a tumor promotion study of PA previously reported. Further, the treatment of PA alone resulted in tyrosinase-dependent contact hypersensitivity in ICR mouse skin. In addition, the in vitro study of cytotoxicity demonstrated that bioactivation by tyrosinase but not myeloperoxidase of PA significantly enhanced cytotoxicity and intracellular glutathione consumption. We conclude that the tyrosinase-derived reactive quinone intermediate(s) of PA, which binds nucleophilic residues of proteins including sulfhydryl group and conjugates of which are recognized as haptens, was partially involved in alteration of the cellular immune functions including oxygen radical-generating leukocytes migration to inflamed regions.
Assuntos
Antioxidantes/toxicidade , Hidroxibenzoatos/toxicidade , Pele/efeitos dos fármacos , Pele/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Dermatite de Contato/etiologia , Sinergismo Farmacológico , Feminino , Células HL-60 , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Modelos Biológicos , Monofenol Mono-Oxigenase/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pele/citologia , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
The inhibitory effects of the diacetylenic spiroketal enol ether epoxide AL-1 from Artemisia lactiflora on a variety of tumor promoter-induced biological responses such as oxidative stress as well as tumor promotion in ICR mouse skin were investigated. AL-1 inhibited TPA-induced intracellular peroxide formation in differentiated HL-60 cells, suggesting that this suppression might be attributable to the inhibition of O2- generation. In a double TPA application system in mouse skin, double pretreatments of AL-1 (810 nmol) significantly suppressed double TPA application-induced H2O2 generation. Pretreatment of AL-1 only before the second TPA treatment was sufficient to inhibit, while only with first treatment was not. From these results we concluded that AL-1 is a specific inhibitor of the activation phase in H2O2 production induced by double TPA treatments. In addition, AL-1 strongly inhibited tumor promoter-induced Epstein-Barr virus (EBV) activation in Raji cells (IC50 = 0.5 microM), which was comparable to or even stronger than that of curcumin, a well-known antioxidative chemopreventer from turmeric. In a two-stage carcinogenesis experiment with TPA (topical application at 1.6 nmol) and 7,12-dimethylbenz[a]anthracene (DMBA, at 0.19 micromol) in ICR mouse skin, topical application of AL-1 (at 160 nmol) significantly reduced tumor incidence, the numbers of tumors per mouse, and edema formation by 58% (P < 0.01 in t-test), 20% (P < 0.005 in chi2-test) and 42% (P < 0.01), respectively. These results together indicate that an inhibitor of O2 generation is an effective chemopreventer of mouse skin carcinogenesis by their antioxidative property.
Assuntos
Anticarcinógenos/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Piranos/uso terapêutico , Neoplasias Cutâneas/prevenção & controle , Compostos de Espiro/uso terapêutico , 9,10-Dimetil-1,2-benzantraceno , Animais , Anticarcinógenos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Testes de Carcinogenicidade , Relação Dose-Resposta a Droga , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Feminino , Células HL-60 , Herpesvirus Humano 4/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Piranos/farmacologia , Neoplasias Cutâneas/induzido quimicamente , Compostos de Espiro/farmacologia , Acetato de Tetradecanoilforbol , Células Tumorais Cultivadas , Ativação Viral/efeitos dos fármacosRESUMO
Collagenase-like (CL) peptidase activity in serum, which was measured using a newly synthesized substrate, (succinyl-Gly-Pro-Leu-Gly-Pro)-4-methylcoumaryl-7-amide, was significantly lower in patients with advanced rheumatoid arthritis or with systemic lupus erythematosus than that in normal controls. Decrease of the serum enzyme activity was more pronounced in systemic lupus erythematosus. No significant change in serum CL-peptidase activity was found in other connective tissue diseases such as mixed connective tissue disease and Sjögren's syndrome.
Assuntos
Artrite Reumatoide/enzimologia , Lúpus Eritematoso Sistêmico/enzimologia , Colagenase Microbiana/sangue , Peptídeo Hidrolases/metabolismo , Humanos , Hepatopatias/enzimologia , Doença Mista do Tecido Conjuntivo/enzimologia , Síndrome de Sjogren/enzimologiaRESUMO
A case of cloverleaf skull (CLS) syndrome with Chiari malformation was reported. The patient developed congenital hydrocephalus, upper airway obstruction and breath holding spells. Ventriculo-peritoneal shunt improved the hydrocephalus, but the patient progressively developed apneic episodes in spite of intubation. Brain magnetic resonance imaging (MRI) disclosed severe Chiari malformation. Laminectomy of the cervical vertebrae and craniectomy at the occipital bone were performed to relieve the brain stem compression. Postoperatively, the respiratory symptoms improved slightly, but 8 weeks later the patient suddenly died. Literature survey revealed that this is the second case report of CLS associated with Chiari malformation. It was assumed that the respiratory problems in our patient are ascribable to the Chiari malformation as well as the upper airway obstruction. The patient here reported seems to be a case of Beare-Stevenson cutis gyrata syndrome, in view of an association of multiple characteristic dysmorphic features, in addition to CLS.
Assuntos
Malformação de Arnold-Chiari/complicações , Craniossinostoses/complicações , Anormalidades Múltiplas/diagnóstico , Apneia , Malformação de Arnold-Chiari/diagnóstico , Craniossinostoses/diagnóstico , Evolução Fatal , Feminino , Humanos , Hidrocefalia/complicações , Hidrocefalia/diagnóstico , Recém-Nascido , Imageamento por Ressonância MagnéticaRESUMO
It has previously been reported that a toxic dose of protocatechuic acid (PA), a naturally occurring simple phenolic antioxidant in dietary plant foodstuff, has a potential to enhance tumorigenesis and induce contact hypersensitivity in mouse skin. In this study, the modifying effect of a toxic dose of PA on the glutathione (GSH) level in mouse liver and kidney was examined. Intraperitoneal administration of PA (500 mg/kg) caused significant hepatic and nephrotic GSH depletion. Interestingly, slight but significant hepatotoxicity and nephrotoxicity, characterized by the enhancement of plasmic alanine aminotrasferase (ALT) activity and urea level, respectively, were also observed. The subchronic administration of PA (0.1% in drinking water) for 60 days showed not only a significant decrease in the GSH level in kidney but also a significant enhancement of ALT activity in plasma. The protective role of GSH for acute hepatotoxicity using GSH-depleted mice administered a GSH synthesis inhibitor buthionine sulfoximine was also demonstrated. Thus, it is suggested that overdoses of PA can disturb the detoxification of other electrophilic toxicants including ultimate carcinogens.
Assuntos
Antioxidantes/toxicidade , Glutationa/metabolismo , Hidroxibenzoatos/toxicidade , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Animais , Feminino , Rim/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos ICRRESUMO
The pyrolysis of tobacco under a variety of conditions was performed to examine the generation profiles of 29 known toxic compounds in tobacco smoke (hydrogen cyanide, benzo[a]pyrene, aldehydes, volatile organic compounds, phenolics, aromatic amines, etc.). The generation profiles of smoke compounds varied according to three conditions: the pyrolysis temperature (300-1000 degrees C), the pyrolysis atmosphere (in nitrogen and air) and pH of the tobacco leaf (2.89-7.07). Most of the smoke compounds (28 compounds) were generated primarily at temperatures less than 800 degrees C. More than half of the smoke compounds (17 compounds) were unaffected by the type of atmosphere, and seven compounds were significantly affected by pH. These results can provide basic and useful information to further study on the formation mechanisms and the technology involved in the control of smoke generation.
Assuntos
Temperatura Alta , Nicotiana/química , Nitrogênio , Oxigênio , Fumaça/análise , Concentração de Íons de Hidrogênio , Incineração , Xenobióticos/análiseRESUMO
We compared early and delayed Tc-99m ECD SPECT scans in 32 SLE patients (Group 1, definite neuropsychiatric disorders; Group 2, minor neurologic symptoms or normal) with those of normal controls by visual inspection and semi-quantitative evaluation. With visual interpretation, 13 out of 14 patients in Group 1 (93%) and 7 out of 18 patients in Group 2 (39%) had diffuse uneven decrease in early scans. Seven patients in Group 2 (39%) who had normal early scans demonstrated focal decrease in the medial frontal lobe in delayed scans. With cerebral region to cerebellar ratios, in early scans, the medial frontal lobe in Group 1 and Group 2 was significantly lower than in normal controls, and lateral frontal lobe and occipital lobes in Group 1 were significantly lower than in normal controls. Nevertheless, in delayed scans, every cortical region except for the parietal lobe in Groups 1 and 2 was significantly lower than in normal controls. The retention rates in all regions in SLE patients were significantly lower than in normal controls. No case showed SPECT improvement on follow-up studies in either group in spite of clinical improvement. Delayed Tc-99m ECD brain SPECT of high sensitivity might be useful in detecting CNS involvement. Although the SPECT findings did not correlate with the neuropsychiatric symptoms, early and delayed Tc-99m ECD SPECT seems to provide useful objective diagnostic information in SLE patients.
Assuntos
Encéfalo/diagnóstico por imagem , Cisteína/análogos & derivados , Lúpus Eritematoso Sistêmico/diagnóstico por imagem , Vasculite Associada ao Lúpus do Sistema Nervoso Central/diagnóstico por imagem , Compostos de Organotecnécio , Compostos Radiofarmacêuticos , Adulto , Cerebelo/diagnóstico por imagem , Feminino , Lobo Frontal/diagnóstico por imagem , Humanos , Vasculite Associada ao Lúpus do Sistema Nervoso Central/psicologia , Masculino , Valores de Referência , Fatores de Tempo , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
We report a case of fulminant pneumonia that was due to aspiration of contaminated hot spring water and was not affected by beta-lactam antibiotics. We suspected that the patient had Legionnaires' disease and treated the clinical symptoms with erythromycin. Legionellaceae could not be isolated from sputum or lung biopsy material, but an elevated titer to Legionella pneumophila serogroup 4 was found by indirect immunofluorescence test. We diagnosed the patent as having Legionnaires' disease with improved clinical symptoms. Furthermore, we went to the hot spring that he visited and tried to isolate Legionellaceae. Hot spring water was collected from the bathroom and water, hot water, and shower water from the guest-room. After using a low-pH method, samples were cultured on BCYE alpha medium. Serogroups are classified by agglutination method with immune rabbit serum. As a result, we successfully isolated Legionella pneumophila serogroup 4 from hot spring water (42 degrees C) from the bath. No bacteria could be isolated from the other samples. Therefore, we believe that this case of Legionnaires' disease was caused by aspiration of contaminated hot spring water. The infection route of Legionnaires' disease is unclear. There are no previous reports of isolation of Legionellaceae from Japanese hot springs. This case would provide important information when considering the infection route of Legionnaires' disease in Japan.
Assuntos
Doença dos Legionários/transmissão , Microbiologia da Água , Acidentes , Estâncias para Tratamento de Saúde , Humanos , Inalação , Legionella pneumophila/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Águas Minerais , Pneumonia/etiologiaRESUMO
T-2588 was used on 55 patients with respiratory tract infections and 44 cases were evaluated; 23 patients with pneumonia, 12 patients with acute bronchitis, 2 patients with chronic bronchitis, 1 patient with diffuse panbronchiolitis and 6 patients with bronchiectasis with infection. Clinical effects of T-2588 were as follows; excellent in 6 and good in 28 patients. The efficacy rate was 77.3% (34/44). Bacteriological effects of T-2588 were prominent in 8 patients infected with B. catarrhalis, H. influenzae, K. pneumoniae and E. coli, but not in a patient infected with P. putida. The elimination rate was 90.0% (9/10 strains). As side effects, stomatitis, anorexia, diarrhea X vomiting and pruritus were observed in one patient each. Abnormal laboratory findings were observed in 4 patients with elevated GOT and/or GPT. These side effects and abnormal laboratory findings were not serious. The usefulness of T-2588 was 68.2% (30/44). Therefore, T-2588 is a useful drug and its effects are promising in clinical management of respiratory tract infections.
Assuntos
Cefmenoxima/análogos & derivados , Cefalosporinas/uso terapêutico , Infecções Respiratórias/tratamento farmacológico , Administração Oral , Adulto , Idoso , Idoso de 80 Anos ou mais , Bronquite/tratamento farmacológico , Cefalosporinas/administração & dosagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia/tratamento farmacológicoRESUMO
Infectious complications are important factors for poor prognosis of connective tissue diseases (CTD). 33% of the fatal cases with CTD were caused by infectious complications in our study. It was characteristic of infectious complications with CTD, that pneumonia was recognized in all CTD, especially, commoner in PSS, PM and OL, and frequently caused a fatal outcome. Gram negative bacillus, for instance E. coli and K. pneumoniae were frequently detected as pathogens. On the other hand, methicillin resistant Staphylococcus aureus (MRSA) was increased recently, and tuberculosis, especially atypical tuberculosis, such as, miliary and cutaneous forms were also recognized. The risk factors in the development of bacterial infections is considered to be intricately correlated with cellular and humoral immunodeficiency due to CTD or steroid hormones.
Assuntos
Infecções Bacterianas/complicações , Doenças do Tecido Conjuntivo/complicações , Infecções Oportunistas/complicações , Formação de Anticorpos , Humanos , Imunidade Celular , Imunossupressores/efeitos adversos , PrognósticoRESUMO
Spread-out Bragg peaks made by ridge filters or wheel range modulators are used in charged particle therapy with passive methods to achieve uniform biological responses in irradiated tumors. Following the biological responses needed to design the ridge filters, which were developed at the National Institute of Radiological Sciences in Japan, new ridge filters were designed using recent developments in heavy-ion reactions and dosimetry. The Monte Carlo code of Geant4 was used to calculate the qualities of carbon ion beams in a water phantom. The results obtained from the simulation were corrected so that they agreed with the measurements of depth dose distributions. The calculations of biological responses to fragments other than carbon ions were assumed to be for helium ions. The measured dose distributions with the designed ridge filters were compared to the calculated distributions. A beam modifying system using this adaptable method was successively applied to carbon ion therapy at Gunma University.
Assuntos
Radioterapia com Íons Pesados/métodos , Método de Monte Carlo , Planejamento da Radioterapia Assistida por Computador/métodos , Radiometria , Dosagem RadioterapêuticaRESUMO
A high-energy carbon-ion radiotherapy facility is under construction at Gunma University Heavy Ion Medical Centre (GHMC). Its design was based on a study of the heavy ion radiotherapy at the National Institute of Radiological Sciences (NIRS) in order to reduce the size and construction cost of the facility. A compact electron cyclotron resonance ion source (ECRIS) for Gunma University, called KeiGM, was installed in 2008. It is almost a copy of the prototype ECRIS Kei2 which was developed by NIRS; meanwhile this prototype produced over 1 e mA of C(4+) using C(2)H(2) gas (660 W and 40 kV). The beam intensity of C(4+) was 600 e microA with CH(4) gas (250 W and 30 kV). The beam intensity satisfies the required value of 300 e microA.