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Two methods for the feed of vapor from solid materials in the Center for Nuclear Study ECR ion source are described. A rod placed near the wall of the plasma chamber, operating up to a melting point of 2600 °C, has been used for CaO, SiO2, and FeO. An oven with a number of openings, operating up to 800 °C, has been used for P2O5, Li, and S. Typical ion beam intensities of (7)Li(2+), (6)Li(3+), (40)Ca(12+), and (56)Fe(15+) are achieved 280, 75, 28, and 7 eµA, respectively. High intensity heavy ion beams are stably supplied into the azimuthally varying field cyclotron.

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Activated interleukin (IL)-4Ralpha stimulates production of IgE through signal transducer and activator of transcription-6 (Stat6) activation in lymphocytes. Genetic studies have shown an association between polymorphisms in the genes encoding IL-4Ralpha and Stat6 and elevated serum IgE in patients with atopic disease. Some authors, including us, have reported an association of Graves' disease and elevated serum IgE. To analyse the relationship between IL-4Ralpha and Stat6 polymorphisms and elevated serum IgE in patients with Graves' disease, 169 patients with Graves' disease were studied. We investigated whether these polymorphisms affect IL-4Ralpha-Stat6 signalling in cultured human lymphocytes. A high frequency of both the Ile50 polymorphism in IL-4Ralpha and 13GT repeat variants of the Stat6 gene was observed in patients with Graves' disease and elevated serum IgE (Ile50 allele; P < 0.05, 13GT allele; P < 0.01 versus controls) but not in subjects with normal IgE. Cultured human lymphocytes with the Ile50 IL-4Ralpha polymorphism and the 13GT repeat variant of Stat6 showed increased IL-4 (and/or IL-13)-induced Stat6 activation (2.7-fold; P < 0.05 and 2.2-fold; P < 0.05, respectively). These findings suggest that polymorphisms in the IL-4Ralpha and Stat6 genes play an important role in elevation of serum IgE through increased Stat6 action in patients with Graves' disease.

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Three euthyroid patients with Hashimoto's thyroiditis developed hypothyroidism after the administration of rifampin. We studied 67 patients with tuberculosis. All of them were treated with rifampin. Of the 67 patients, 42 had negative tests for anti-thyroid antibodies (ATA) and 25 had positive tests for ATA. The diagnosis of Hashimoto's thyroiditis was made on the basis of positive tests for ATA. After the administration of rifampin, TSH levels were not significantly altered in all of the former 42 ATA-negative patients and in 22 of the latter 25 ATA-positives, but TSH levels increased in the other three (Patients 1, 2 and 3) of the latter 25 ATA-positives. Three euthyroid Hashimoto's patients (Patients 1, 2 and 3) developed hypothyroidism after the administration of rifampin. This rifampin-induced hypothyroidism resolved in each, once rifampin was discontinued. A) Patient 1: a 62-yr-old man with lymphoma had pulmonary tuberculosis. After the administration of rifampin, serum TSH increased to 170 mU/l; B) Patient 2: a peritoneal-biopsy specimen containing Langhans' giant cells led to a diagnosis of tuberculous peritonitis in a 66-yr-old woman with ascites. After the administration of rifampin, TSH increased to 12.4 mU/l; C) Patient 3: a 56-yr-old woman with a liver abscess and lymphadenopathy underwent lymph-node biopsy that showed Mycobacterium tuberculosis with caseating granulomas. After the administration of rifampin, TSH increased to 21.3 mU/l. After its administration, Patients 1, 2 and 3 developed hypothyroidism, and received T4. When rifampin was discontinued, the hypothyroidism resolved. After the course of rifampin-therapy had been completed, T4 was discontinued. At-risk patients who receive rifampin may become hypothyroid.

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BACKGROUND: The development of hemiplegia as a result of hypoglycaemia was first described in 1928. However, the mechanism remains unclear. CASE REPORT: We report a case of a 58-year-old male with diabetes, who developed left hemiplegia during a severe hypoglycaemic event. Results Diffusion-weighted magnetic resonance imaging detected an increased signal intensity in the pons, indicating that the patient's hemiplegia resulted from acute brain injury. CONCLUSIONS: This report provides evidence that acute brain injury may be a cause of the neurological deficit.

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Pro-opiomelanocortin (POMC)-derived peptides play a critical role in body weight regulation in the central nervous system. Mice deficient in POMC developed obesity. We sought mutations in the POMC gene in 50 morbidly obese (body mass index 35-60 kg/m(2)) Japanese subjects with diabetes by direct sequencing. Apart from two silent mutations (C6982T and C7285T), no other mutations were detected. Frequencies of these mutations were not significantly different between 100 obese subjects and 100 controls. Also, the frequencies did not differ in the subjects with or without diabetes. These results suggest that mutations in the POMC gene are unlikely to be a major factor of obesity or diabetes in Japanese subjects.

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The GNB3 825C/T polymorphism, which is common worldwide, is associated with enhanced G-protein activation. The frequency of 825-T allele was increased with body mass index (BMI) and finally had a high frequency in relatively mild obese (BMI >27 kg/m(2)) subjects in some populations. In the present study, we investigated 208 severely obese [BMI >or=30 kg/m(2) (97th percentile)] Japanese subjects including 146 probands with diabetes. No increase in the 825-T allele frequency was observed in the 208 severely obese and even in a subgroup of the 55 most obese [BMI >or=35 kg/m(2) (99.7th percentile)] subjects compared with that in 150 controls (BMI <25 kg/m(2)) (0.48 and 0.48 vs 0.51, respectively). Also, the frequency was not increased in the 146 obese subjects with diabetes (0.48). We concluded that the 825-T allele is not associated with obesity or diabetes associated with obesity at least in the Japanese population.

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Plant nitrate reductase (NR) produces nitric oxide (NO) when nitrite is provided as the substrate in the presence of NADH [H. Yamasaki and Y. Sakihama (2000) FEBS Lett. 468, 89-92]. Using a NR-dependent NO producing system, we investigated the effects of NO on the energy transduction system in plant mitochondria isolated from mung bean (Vigna radiata). Plant mitochondria are known to possess two respiratory electron transport pathways-the cytochrome and alternative pathways. When the alternative pathway was inhibited by n-propyl gallate, the addition of NR strongly suppressed respiratory O(2) consumption driven by the cytochrome pathway. In contrast, the alternative pathway measured in the presence of antimycin A was not affected by NO. The extent of the steady-state membrane potential (Deltapsi) generated by respiratory electron transport rapidly declined in response to NO production. The addition of bovine hemoglobin, a quencher of NO, resulted in the recovery of Deltapsi to the uninhibited level. Consistent with its inhibition of Deltapsi, NO produced by NR strongly suppressed ATP synthesis in the mitochondria. These results provide substantial evidence to confirm that the plant alternative pathway is resistant to NO and support the idea that the alternative pathway may lower respiration-dependent production of active oxygens under conditions where NO is overproduced.

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Gasless hand-assisted surgery, an alternative technique for colorectal cancer, is described. The abdomen is lifted by two metal disks especially designed for this procedure. The gasless condition evokes no hazard from pneumoperitoneum, and the procedure is greatly simplified by the hand assistance.

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The prevaleance of morbid obesity (body mass index of 35.0 or greater) is low in Japan (0.2-0.3%), and little systematic investigation of its cause in this population has been carried out. Leptin plays a central role in regulation of body weight; mice deficient in leptin develop marked obesity. We sought mutations in the leptin gene in 53 morbidly obese Japanese (maximum body mass index 35-60) including 46 with type 2 diabetes. Direct DNA sequencing was performed following polymerase chain reaction amplification. Apart from a silent mutation at codon 25 (CAA/CAG, glutamine) detected in eight subjects, no mutations were detected. We found a significantly higher prevalence of the variant leptin 25CAG allele among the 53 obese subjects (0.085) studied than in 132 nonobese control subjects (0.011, P<0.001). In Japanese populations mutations in the protein coding sequence of the leptin gene are unlikely to be a major cause of morbid obesity. However, the leptin 25CAG allele may be linked to morbid obesity in this population. Specifically, genetic variation located near the leptin gene may be involved in pathogenesis. The leptin polymorphism 25CAG appears to be a new genetic marker for obesity susceptibility, at least in Japanese.

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DegR is a positive regulator for degradative enzyme synthesis in Bacillus subtilis. The degR gene is transcribed by RNA polymerase containing delta D, and the level of its expression is low in a mecA-deficient mutant. In a search for suppressors of the mecA effect through mini-Tn10 transposon mutagenesis, a lacR mutation designated lacR288 was discovered. The B. subtilis lacR gene encodes the repressor for lacA which specifies beta-galactosidase, and therefore, inactivation of the lacR gene results in overproduction of the enzyme. In the lacR288 mutant, however, the expression of lacA was at a negligible level, indicating that the repressor activity was not destroyed by the mutation. The putative gene product of the lacR288-containing gene is a 288-amino acid protein lacking the C-terminal 42 amino acids of intact LacR and carries no extra amino acids derived from the transposon sequence. The suppression by lacR288 of the decreased degR expression in the mecA background was found to be caused by an increase in the delta D level as shown by Western blot analysis. Furthermore, the increase was due to post-transcriptional regulation of sigD, the gene encoding delta D, as revealed by using both transcriptional and translational sigD-lacZ fusions. The lacR288 mutation had no effect on the stability of the delta D protein. Based on these results we conclude that the lacR288 mutation stimulates sigD expression at the translational level.

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Decreased function of the melanocortin-4 receptor (MC4R) was reported to cause late-onset obesity and insulin resistance in rodents. Thus mutations in the MC4R gene drew strong attention as a possible cause of obesity and diabetes. We screened for mutations in the MC4R gene in extremely obese [body mass index (BMI) > or = 35 kg/m2] Japanese with diabetes by direct sequencing. A heterozygous mutation (V103I) was detected in one case (2.0 %), however the frequency was not significantly different from that in non-obese (BMI < or = 24 kg/m2) and non-diabetic subjects (2.7 %). No other mutations were detected. These results suggest that mutations including V103I in the MC4R gene are not a major cause of obesity or diabetes in Japanese.

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Bacillus subtilis degR, a positive regulator of the production of degradative enzymes, is negatively regulated by the competence transcription factor ComK which is overproduced in mecA null mutants. We used transposon Tn10 to search for a mutation that reduced the repression level of degR caused by a mecA mutation. A new gene exerting positive regulation on comK was obtained and designated med (suppressor of mecA effect on degR). Sequence determination, Northern analysis, and primer extension analyses revealed that the med gene contained an open reading frame (ORF) composed of 317 codons and was transcribed into an approximately 1,250-nucleotide mRNA together with its short downstream gene. The expression of comK is positively regulated by factors such as ComK itself, ComS (SrfA)-MecA, DegU, SinR, and AbrB. Quantitative analyses using comK'-'lacZ, srfA-lacZ, degU'-'lacZ, and sinR'-'lacZ fusions showed that disruption of med caused a significant decrease in comK expression in both mecA+ and mecA strains, while expression of srfA, sinR, and degU was not affected by the mutation. An epistatic analysis revealed that overproduction of ComK resulted in alteration of med expression, suggesting a regulatory loop between comK and med. Several possible mechanisms for positive regulation of comK by Med are discussed.

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To investigate the role of cell surface glycosaminoglycans (GAGs), including heparan sulfate (HS), on HIV-1 infection in human T cells, HIV-1 binding and infection were determined after treatment of T-cell lines and CD4+ T cells from normal peripheral blood mononuclear cells (PBMC) with GAG-degrading enzyme or a GAG metabolic sulfation inhibitor. Heparitinase I (hep I) and sodium chlorate prevented binding of HIV-1/IIIB to MT-4 cells as revealed by indirect immunofluorescence procedures, thereby inhibiting infection. Hep I was less effective in the binding inhibition of the macrophage-tropic strain HIV-1/SF162 than that of the T-cell line-tropic strain HIV-1/IIIB. The binding of HIV-1/SF162 was about 100-fold less dependent on cell surface HS than HIV-1/IIIB. Human HTLV-I positive T-cell lines expressed more HS than HTLV-I negative T-cell lines or normal CD4+ T cells when stained with anti-HS mAbs against either native or heparitinase-treated HS. With the exception of endo-beta-galactosidase (endo-beta-gal), GAG-degrading enzymes, including hep I, chondroitinase ABC (chon ABC), chondroitinase AC II (chon AC II) and keratanase, did not prevent the binding of HIV-1/IIIB to CD4+ T cells from normal PBMC. These results indicate that the cell surface HS of human T cells participates in HIV-1 infection by facilitating HIV-1/IIIB binding to MT-4 cells. In particular, the sulfation of HS chains is critical. Since the expression of cell surface HS varies among T cells, which are not consistently sensitive to hep I treatment in HIV-1 binding inhibition, other GAG-like molecules may also be involved.

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Bacterial chromosomes, mostly of circular form, have an unique primary structure that are stably maintained. We initiated a systematic study to induce changes of the structure of the Bacillus subtilis chromosome. There are two main goals: (i) to obtain general concepts for possible plasticity of the bacterial genome and (ii) to apply the proposed genome technology to bacteria.

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The crystal structure of azurin from a denitrifying bacterium, Achromobacter xylosoxidans NCIB11015, has been refined at 2.5 A resolution using diffraction data obtained by means of synchrotron radiation at KEK. Crystals suitable for X-ray experiment were obtained by the macro-seeding method and an intensity data were obtained on imaging plates mounted on a Weissenberg camera (Rmerge = 0.09). The initial model was obtained by the molecular replacement method using the structure of azurin from Alcaligenes denitrificans NCTC8582 as a starting model. The structure was refined by molecular dynamics optimization and the restrained least-squares method to a crystallographic R-value of 0.205. However, the current model gave an electron-density of the side-chain regions of several residues close to the N-terminus quite different from those expected from the amino acid sequences reported. Very recently, two kinds of azurins (Az-I and Az-II) were isolated from this bacterium by a slightly modified purification method and have been characterized and found to have different CD spectra. On analysis of amino acid sequences around the N-terminus, the second azurin (Az-II) was proved to be a new type of azurin in this bacterium. It was consequently revealed that the current model corresponds to a new type of azurin because of the complete agreement between the electron-density and the amino acid sequence of the newly determined 20 residues from the N-terminus. Determination of the whole amino acid sequence of this azurin and further refinement are in progress.

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Nucleosides and nucleotides as a precursor for nucleic acid synthesis may be essential for rapidly growing cells, since intestinal epithelial cells have limited capacity for the de novo purine and pyrimidine synthesis. The present study was undertaken to determine the effect of intraperitoneal administration of nucleoside-nucleotide mixture (NNM) or saline on endotoxin-induced bacterial translocation, ileal histology, and cecal population levels in protein-deficient mice. Intraperitoneal administration of NNM for 14 days was associated with reduced translocation of gram-negative enterics to the mesenteric lymph node and spleen in comparison to saline. Histologically, the extent of the damage to the gut mucosa was greater in the saline group. This was confirmed by the profound diminution of the villous height, crypt depth, and the intestinal wall in the saline treated group as compared to the NNM treated group, suggestive of the efficacy of NNM in improving the gut and epithelial mucosal cells. However, the cecal population levels in both groups were not different. Additionally, the mice in the saline group were more susceptible to the lethal effects of endotoxin as compared to the NNM group suggesting that NNM may be essential for the enhancement of the host defense system. These results suggest that NNM may be used to an advantage to inhibit or reduce the incidence of endotoxin-induced bacterial translocation and improved survival in protein-deficient mice.

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The crystal structure of pseudoazurin from Methylobacterium extorquens AM1 (PAZAM1) has been solved by the molecular replacement method using copper-copper distances as translation parameters, which were obtained from difference Patterson maps calculated with the synchrotron radiation data containing the multiwavelength anomalous-dispersion effect. The structure refinement was carried out by the use of molecular dynamics optimization and the restrained least-squares method. The final crystallographic R factor was 19.9% for the 14 365 reflections greater than 3sigma between 1.5 and 8.0 A resolution. This report describes the characteristic features of the structure of PAZAM 1 as well as the effectiveness of synchrotron radiation for structure analysis of metalloproteins. The environment of the metal active site and the structural differences among blue-copper proteins are discussed.

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A subclonal cl.1-14 cell was established from a monocytic cell line U937 by a limiting dilution method. The anti-HIV-1 activity of some antiviral compounds was evaluated in HIV-1-infected cl.1-14 cells. The results demonstrated that although AZT was a potent inhibitor of HIV-1 replication in cl.1-14 cells, its 50% effective concentration (EC50) values was 80 times higher than that in HIV-1 infected MT-4 cells; the EC50 of AZT was 0.16 microM and 0.002 microM in cl.1-14 and MT-4 cells, respectively. In contrast, the anti-HIV-1 activity of ddA, ddI and ddC in cl.1-14 cells was comparable to that in MT-4 cells. The antiviral activity of nevirapine, dextran sulfate, curdlan sulfate and T22 did not differ significantly between the cl.1-14 and MT-4 cells. The antiviral activity of several compounds in the HIV-1-infected cl.1-14 cells was similar to that in the HIV-1JR-FL-infected human peripheral macrophages. Our results suggest that cl.1-14 cell cultures are very useful for estimating antiviral activity and more advantageous than the use of peripheral blood macrophages.

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