Role of peroxynitrite and recombinant human manganese superoxide dismutase in reducing ischemia-reperfusion renal tissue injury.

BACKGROUND: In an acute kidney transplant rejection rat model, we demonstrated that manganese superoxide dismutase (MnSOD) activity was significantly reduced and MnSOD was nitrated by peroxynitrite (ONOO(-)), resulting in tissue injury. We examined whether tissue injury was reduced after external supplementation of recombinant human MnSOD in a rat renal ischemia-reperfusion injury model. METHODS: Male Brown-Norway rats underwent dissection of the right kidney. The animals were divided into 3 groups. The controls had the left renal blood vessels clamped for 90 minutes to induce ischemia, followed by reperfusion for 16 hours. In the intraperitoneal administration group, MnSOD was administered 30 minutes before ischemia and immediately before reperfusion. In the sham group, neither ischemia nor reperfusion was performed. After reperfusion, blood was collected, the left kidney was dissected and renal function and tissue injury were evaluated. RESULTS: Serum creatinine and K(+), blood urea nitrogen, and aspartate aminotransferase activity decreased significantly, whereas serum Na(+) and renal function improved in the MnSOD group compared with the control and sham groups. On hematoxylin and eosin staining, the histological score indicated that acute tubular necrosis was significantly reduced by MnSOD administration. Periodic acid-Schiff staining was absent in the nonadministration group, whereas it persisted in the MnSOD group. In the proximal renal tubules a large proportion of anti-nitrotyrosine staining was present before but absent after MnSOD administration. CONCLUSIONS: MnSOD administration improved renal function and reduced tissue injury. It may also reduce tissue injury in acute kidney transplant rejection and other tissue injuries caused by similar molecular mechanisms.

Use of multiplex PCR and real-time PCR to detect human herpes virus genome in ocular fluids of patients with uveitis.

AIM: To measure the genomic DNA of human herpes viruses (HHV) in the ocular fluids and to analyse the clinical relevance of HHV in uveitis. METHODS: After informed consent was obtained, a total of 111 ocular fluid samples (68 aqueous humour and 43 vitreous fluid samples) were collected from 100 patients with uveitis. The samples were assayed for HHV-DNA (HHV1-8) by using two different polymerase chain reaction (PCR) assays, qualitative PCR (multiplex PCR) and quantitative PCR (real-time PCR). RESULTS: In all of the patients with acute retinal necrosis (n = 16) that were tested, either the HSV1 (n = 2), HSV2 (n = 3), or VZV (n = 11) genome was detected. In all patients, high copy numbers of the viral DNA were also noted, indicating the presence of viral replication. In another 10 patients with anterior uveitis with iris atrophy, the VZV genome was detected. When using multiplex PCR, EBV-DNA was detected in 19 of 111 samples (17%). However, real-time PCR analysis of EBV-DNA indicated that there were only six of the 19 samples that had significantly high copy numbers. The cytomegalovirus (CMV) genome was detected in three patients with anterior uveitis of immunocompetent patients and in one immunocompromised CMV retinitis patient. In addition, one patient with severe unilateral panuveitis had a high copy number of HHV6-DNA. There was no HHV7- or HHV8-DNA detected in any of the samples. CONCLUSIONS: A qualitative multiplex PCR is useful in the screening of viral infections. However, the clinical relevance of the virus infection needs to be evaluated by quantitative real-time PCR.

Residue level of polycyclic aromatic hydrocarbons in Japanese paddy soils from 1959 to 2002.

The residue level of 21 polycyclic aromatic hydrocarbons (PAHs) and the temporal changes in this level were investigated in paddy soils collected from particular experimental sites in Japan from 1959 to 2002. The average total PAH concentration in all the samples was 496 microg kg(-1), and it ranged from 52.9 to 2180 microg kg(-1). The residue level of the PAHs was the highest during the 1960s, rapidly decreased during the 1970s, and remained almost constant thereafter. Relatively high PAH concentrations were observed in soils from areas that experienced heavy snowfall and that had relatively low air temperature. The predominant PAHs were phenanthrene, fluoranthene, naphthalene, and pyrene, and their concentration overall and in relation to that of the total PAHs decreased each year since the 1960s. Similarities in the PAH profiles among the locations were determined using the concentration correlation matrix and cluster analysis, and ratios of the levels of specific PAH pairs were also calculated to determine their origin. The collected data suggested that the origins of soil PAHs changed chronologically from the burning of agricultural wastes such as stubble before the mid-1970s to the combustion of fossil fuel and its secondary products after the mid-1970s.

Reactive oxygen species in mechanical stress-induced cardiac hypertrophy.

Mechanical stress induces various hypertrophic responses including activation of mitogen-activated protein kinases (MAPKs) in cardiac myocytes. Here we examined the role of the small GTP-binding proteins of Rho family and reactive oxygen species (ROS) in stretch-induced activation of p38MAPK in cardiomyocytes. Overexpression of dominant-negative mutants of Rac1 (D.N. Rac1), D.N.RhoA and D.N.Cdc42 suppressed stretch-induced activation of p38MAPK. Overexpression of constitutively active mutants of Rac1 (C.A.Rac1) and C.A.Cdc42 increased the p38MAPK activity in the absence of mechanical stress. Pretreatment with N-acetyl-L-cysteine and N-(2-mercaptopropionyl)-glycine (NAC) suppressed stretch-induced activation of p38MAPK. Mechanical stretch increased intracellular ROS generation, which was abrogated by overexpression of D.N.Rac1 and attenuated by overexpression of D.N.RhoA and D.N.Cdc42. An increase in protein synthesis evoked by mechanical stretch was suppressed by overexpression of D.N.Rac1 and pretreatment with NAC. These results suggest that mechanical stress induces cardiac hypertrophy through the Rac1-ROS-p38MAPK pathway in cardiac myocytes.

Molecular biological markers and micrometastasis in resected non-small-cell lung cancer. Prognostic implications.

OBJECTIVES: Recent advances in molecular biology and genetics have created new diagnostic and treatment possibilities in clinical oncology. We evaluated the usefulness of molecular biological factors in primary tumor and micrometastasis in the bone marrow and pathological negative (pN0) lymph nodes as prognostic parameters in non-small-cell lung cancer (NSCLC) patients. METHODS: Pathological specimens were collected from 129 NSCLC patients to analyze molecular biological markers, including K-ras, p53, Rb, p16, loss of heterozygosity (LOH) at 3p, vascular endothelial growth factor (VEGF), and telomerase activity. Bone marrow samples from 250 NSCLC patients and pN0 lymph nodes from 85 of these patients were collected for micrometastasis detection by immunohistochemistry against cytokeratin. RESULTS: p53 abnormalities and 3p LOH were significantly associated with reduced patient survival in adenocarcinoma, whereas VEGF expression was significantly associated with reduced survival in a squamous cell carcinoma histological subtype by univariate or multivariate analysis. We identified micrometastatic tumor cells in bone marrow of 78 (31.2%) of 250 patients and in pN0 lymph nodes of 26 (30.6%) of 85 patients. Both bone marrow and lymph nodal micrometastases were associated with decreased survival among patients with stage I, however, only lymph nodal micrometastasis had a significant impact on survival. CONCLUSIONS: Molecular biological features of primary tumor and micrometastatic status appear useful in defining groups of patients with a poor prognosis who could benefit from adjuvant systemic treatment.

Molecular cloning of groESL locus, and purification and characterization of chaperonins, GroEL and GroES, from Bacillus brevis.

The groESL locus of a protein-hypersecreting bacterium, Bacillus brevis, was cloned by PCR using primers designed based on the DNA sequence of a B. subtilis homolog. GroEL protein was purified to apparent homogeneity and its ATPase activity was characterized: it hydrolyzed ATP, CTP, and TTP in this order of reaction rate, and its specific activity for ATP was 0.1 micromole/min/mg protein. Purified GroEL forms a tetradecamer. GroEL was estimated to contain 22% alpha-helix, 24% beta-sheet, and 19% turn structures, by CD measurement. GroES protein was also highly purified to examine its chaperonin activity. GroEL protected from thermal inactivation of and showed refolding-promoting activity for malate dehydrogenase, strictly depending on the presence of ATP and GroES.

Calcineurin plays a critical role in the development of pressure overload-induced cardiac hypertrophy.

BACKGROUND: Although activation of the Ca(2+)-dependent phosphatase calcineurin has been reported to induce cardiomyocyte hypertrophy, whether calcineurin is involved in pressure overload-induced cardiac hypertrophy remains controversial. METHODS AND RESULTS: We examined in the present study the role of calcineurin in pressure overload-induced cardiac hypertrophy using transgenic mice that overexpress the dominant negative mutant of calcineurin specifically in the heart. There were no significant differences in body weight, blood pressure, heart rate, heart weight, and the cardiac calcineurin activity between the transgenic mice and their littermate wild-type mice at basal state. The activity of calcineurin was markedly increased by pressure overload produced by constriction of the abdominal aorta in the heart of wild-type mice but less increased in the heart of the transgenic mice. Pressure overload induced increases in heart weight, wall thickness of the left ventricle, and diameter of cardiomyocytes; reprogramming of expressions of immediate early response genes and fetal-type genes; activation of extracellular signal-regulated protein kinases; and fibrosis. All these hypertrophic responses were more prominent in the wild-type mice than in the transgenic mice. CONCLUSIONS: These results suggest that calcineurin plays a critical role in the development of pressure overload-induced cardiac hypertrophy.

Pentalenolactone I and hygromycin A, immunosuppressants produced by Streptomyces filipinensis and Streptomyces hygroscopicus.

Immunosuppressants isolated from Streptomyces filipinensis and S. hygroscopicus were identified with pentalenolactone I and hygromycin A, respectively. The compounds as well as cyclosporin A showed immunosuppressant activity in the mixed lymphocyte reaction, and pentalenolactone I and cyclosporin A suppressed IL-2 production, however, hygromycin A did not. Hygromycin A may have immunosuppressant activity by a different mechanism from pentalenolactone I, cyclosporin A and tacrolimus.

[Calcineurin inhibitor attenuates the development and induces the regression of cardiac hypertrophy in rats with salt-sensitive hypertension].

BACKGROUND: It remains unclear how hemodynamic overload induces cardiac hypertrophy. Recently, activation of calcium-dependent phosphatase, calcineurin, has been elucidated to induce cardiac hypertrophy. In the present study, we examined the role of calcineurin in load-induced cardiac hypertrophy by using Dahl salt-sensitive (DS) rats, which develop both pressure and volume overload when fed a high salt diet. METHODS AND RESULTS: In the DS rat heart, the activity of calcineurin was increased and cardiac hypertrophy was induced by high salt diet. Treatment of DS rats with the calcineurin inhibitor FK506 (0.1 or 0.01 mg/kg every second day) from the age of 6 weeks to 12 weeks inhibited the activation of calcineurin in the heart in a dose-dependent manner and attenuated the development of load-induced cardiac hypertrophy and fibrosis without change of hemodynamic parameters. Additionally, treatment with 0.1 mg/kg every second day but not with 0.01 mg/kg every second day of FK506 from the age of 12 weeks to 16 weeks induced regression of cardiac hypertrophy in DS rats. Load-induced reprogramming of gene expression was also suppressed by the FK506 treatment. CONCLUSIONS: These results suggest that calcineurin is involved in the development of cardiac hypertrophy in rats with salt-sensitive hypertension and that inhibition of calcineurin could induce regression of cardiac hypertrophy.

Calcineurin inhibitor attenuates the development and induces the regression of cardiac hypertrophy in rats with salt-sensitive hypertension.

BACKGROUND: It remains unclear how hemodynamic overload induces cardiac hypertrophy. Recently, activation of calcium-dependent phosphatase, calcineurin, has been elucidated to induce cardiac hypertrophy. In the present study, we examined the role of calcineurin in load-induced cardiac hypertrophy by using Dahl salt-sensitive (DS) rats, which develop both pressure and volume overload when fed a high salt diet. METHODS AND RESULTS: In the DS rat heart, the activity of calcineurin was increased and cardiac hypertrophy was induced by high salt diet. Treatment of DS rats with the calcineurin inhibitor FK506 (0.1 or 0.01 mg/kg twice daily) from the age of 6 weeks to 12 weeks inhibited the activation of calcineurin in the heart in a dose-dependent manner and attenuated the development of load-induced cardiac hypertrophy and fibrosis without change of hemodynamic parameters. Additionally, treatment with 0.1 mg/kg twice daily but not with 0.01 mg/kg twice daily of FK506 from the age of 12 weeks to 16 weeks induced regression of cardiac hypertrophy in DS rats. Load-induced reprogramming of gene expression was also suppressed by the FK506 treatment. CONCLUSIONS: These results suggest that calcineurin is involved in the development of cardiac hypertrophy in rats with salt-sensitive hypertension and that inhibition of calcineurin could induce regression of cardiac hypertrophy.

Functional analyses of three Csx/Nkx-2.5 mutations that cause human congenital heart disease.

A homeodomain-containing transcription factor Csx/Nkx-2.5 is an important regulator of cardiogenesis in mammals. Three different mutants, Gln170ter (designated A) and Thr178Met (designated B) in the helix 2 of the homeodomain and Gln198ter mutation (designated C) just after homeodomain, have been reported to cause atrial septal defect with atrial ventricular block. We here examined the functions of these three mutants of Csx/Nkx-2.5. The atrial natriuretic peptide (ANP) promoter was activated by wild type Csx/Nkx-2.5 (WT, approximately 8-fold), B ( approximately 2-fold), and C ( approximately 6-fold) but not by A. When A, B, or C was cotransfected into COS-7 cells with the same amount of WT, WT-induced activation of the ANP promoter was attenuated by A and B (A > B), whereas C further enhanced the activation. Immunocytochemical analysis using anti-Myc tag antibody indicated that transfected Myc-tagged WT, B, and C were localized in the nucleus of both COS-7 cells and cardiomyocytes of neonatal rats, whereas A was distributed diffusely in the cytoplasm and nucleus in COS-7 cells. Electrophoretic mobility shift assay showed that Csx/Nkx-2.5-binding sequences were bound strongly by WT and C, weakly by B, but not by A. Immunoprecipitation and GST pull-down assay revealed that WT and all mutants interacted with GATA-4. The synergistic activation of the ANP promoter by WT and GATA-4 was further enhanced by C but was inhibited by A and B. In the cultured cardiomyocytes, overexpression of C but not WT, A, or B, induced apoptosis. These results suggest that although the three mutants induce the same cardiac phenotype, transactivation ability and DNA binding ability are different among the three mutants and that apoptosis may be a cause for C-induced cardiac defect.

Ca2+/calmodulin-dependent kinase II and calcineurin play critical roles in endothelin-1-induced cardiomyocyte hypertrophy.

Endothelin-1 (ET-1) induces cardiac hypertrophy. Because Ca(2+) is a major second messenger of ET-1, the role of Ca(2+) in ET-1-induced hypertrophic responses in cultured cardiac myocytes of neonatal rats was examined. ET-1 activated the promoter of the beta-type myosin heavy chain gene (beta-MHC) (-354 to +34 base pairs) by about 4-fold. This activation was inhibited by chelation of Ca(2+) and the blocking of protein kinase C activity. Similarly, the beta-MHC promoter was activated by Ca(2+) ionophores and a protein kinase C activator. beta-MHC promoter activation induced by ET-1 was suppressed by pretreatment with the calmodulin inhibitor, W7, the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitor, KN62, and the calcineurin inhibitor, cyclosporin A. beta-MHC promoter activation by ET-1 was also attenuated by overexpression of dominant-negative mutants of CaMKII and calcineurin. ET-1 increased the activity of CaMKII and calcineurin in cardiac myocytes. Pretreatment with KN62 and cyclosporin A strongly suppressed ET-1-induced increases in [(3)H]phenylalanine uptake and in cell size. These results suggest that Ca(2+) plays a critical role in ET-1-induced cardiomyocyte hypertrophy by activating CaMKII- and calcineurin-dependent pathways.

Removal of cadmium from scallop hepatopancreas by microbial processes.

A microbial process for removing cadmium from a homogenate of hepatopancreas, a waste of scallop processing, was devised to use this waste for value-added protein resources. Microorganisms were screened on the basis of the ability to remove cadmium from a medium with the initial concentration of 10 mg/l of cadmium. One soil isolate, identified as Xanthomonas sp. UR No. 2 by its taxonomical characteristics, removed 98% of the cadmium in the medium in 2 d. During cultivation of this strain in the homogenates of hepatopancreas digested by endopeptidases, 90% of cadmium was removed, while this strain had little effect on the simple non-digested homogenates. The mass balance of cadmium during homogenizations of the hepatopancreas tissues and cultivations in the protease-treated homogenate were examined. The content of crude proteins of culture supernatant treated by Xanthomonas sp. UR No. 2 was equivalent to those of various feedstuffs on the market.

Structural conversion from non-native to native form of recombinant human epidermal growth factor by Brevibacillus choshinensis.

Brevibacillus choshinensis (Bacillus brevis) HPD31 is a very efficient producer of recombinant human epidermal growth factor (EGF). The produced EGF is secreted into the medium with high efficiency. However part of the EGF that accumulates in the medium, exists as multimeric forms which are biologically inactive. We found the bacterium has the activity to structurally convert multimeric forms to the monomeric, native ones. Optimal temperature and pH for the conversion were 40 degrees C and pH 9, respectively. The reaction was promoted in the presence of reduced glutathione or cysteine. But the cells which had been sonicated or exposed to moderate heat treatment completely lost the activity. Thus, it was presumed that the activity might be due to the enzyme(s) that catalyze the protein disulfide exchanging reaction, and that they resides on the surface of viable cells.

[Analysis of abnormal findings observed on brain MRI T 2 weighted image in a system for the detection of asymptomatic brain disease in 1,200 cases].

In this study we described the significance of asymptomatic cerebral infarction (ACI) and periventricular hyperintensity (PVH) observed on brain MRI in a system for detection of asymptomatic brain disease with 1,200 cases. The risk factors (RF), population in each age bracket of ACI and PVH, among groups with hypertension (HTG) and without RF (no-RFG), were investigated. The RF of ACI were hypertension (HT), diabetes mellitus (DM), and aging. Without DM, those are common RF of PVH. The population of PVH and ACI with PVH increased with aging in no -RFG. On the other hand, only the population of ACI with PVH increased with aging in HTG. The rate of these abnormal findings in HTG was significantly higher than that in no-RFG. In addition, HT accelerated the occurrence of these findings by 10-20 years. When patients were over 60 years old, ACI increased rapidly. Accordingly, we concluded that (1) PVH and ACI had a common background. (2) Long term follow up concerning the incidence of ACI in the group with only PVH was necessary. (3) It was desirable that treatment for RF should be effected before the age of sixty.

Molecular cloning of the dnaK locus, and purification and characterization of a DnaK protein from Bacillus brevis HPD31.

Using part of the dnaK gene from Bacillus subtilis as a probe, a 4. 4-kbp SacI-BglII fragment of chromosomal DNA of Bacillus brevis, a protein-hypersecreting bacterium, was cloned. Nucleotide sequencing revealed 3 open reading frames in the order of grpE-dnaK-dnaJ homologues. We purified DnaK protein to homogeneity from B. brevis HPD31 harboring a multi-copy dnaK expression plasmid. Purified DnaK showed ATPase activity which was synergistically stimulated 14-fold by the addition of glutathione S-transferase-DnaJ and glutathione S-transferase-GrpE fusion proteins. DnaK hydrolyzed not only ATP but also CTP, UTP, and GTP at about 40% of the efficiency of ATP. The specific activity of DnaK-ATPase was 7.25x10-3 unit/mg protein (the turnover number against ATP was 0.47 min-1) under our assay conditions. The DnaK dimers dissociated into monomers on addition of ATP, GTP, CTP, UTP and ATPgammaS, but not ADP or AMP. DnaK formed a stable complex with permanently unfolded carboxymethylated alpha-lactalbumin but not with native alpha-lactalbumin, and this complex was dissociated by addition of ATP/Mg. Formation of this complex was inhibited in the presence of inorganic phosphate.

Rehabilitation of spinal cord injury in the national rehabilitation center for the disabled of Japan: profile of a spinal service.

The National Rehabilitation Center for the Disabled (hereunder abbreviated NRC) in Japan was established in 1979. It consists of four divisions: the hospital, the rehabilitation training center, the research institute, and the information service section. The spinal unit has been functioning and cooperating corelatively with all of these divisions. There were 1047 patients with a spinal cord injury treated in the 15 years from September, 1980 to August, 1994; consisting of 924 males (88.3%), and 123 females (11.7%). The breakdown of causes of injury were traffic accidents 44.9%, having a fall 14.7%, sports accidents 6.7%, and other causes of spinal paralysis 10.5%. The sites of the spinal cord lesions were cervical 372 (35.5%), thoracic 547 (52.5%), and lumbar spinal cord 128 patients (12.3%). The incidence of complete paralysis in those with a cervical spinal cord injury (SCI) was 68.8%, and for thoracic and lumbar SCI 79% each. The time for completion of activities of daily living (ADL) was 12.0 +/- 1.54 months in those with tetraplegia, and 5.6 +/- 1.71 months for those with paraplegia. The rate of employment for reentry into society was 59% in those with a cervical spinal cord injury, and 74% each in those with a thoracic or lumbar spinal cord injury.

Simultaneous alignment of optical axes by monitoring reflected light from core and cladding.

We propose a method of aligning optical axes by monitoring reflected light from the core and the cladding at the coupling end. This method enables the simultaneous alignment of optical axes at multiple coupling ends because it is not necessary to monitor the transmitted light through the couplings. Experimental results show that this method can be used for coupling between cleaved single-mode fibers and between fibers with slant ends. The simultaneous alignment of two coupling ends is also achieved, and it is shown to obtain the optical power transmitted through the coupling, which is enough to begin the precise alignment. Moreover, we confirm fundamentally that one can use this method for precise alignment of optical axes by monitoring the optical beam profiles of the reflected light.

Relationship between functional levels and movement in tetraplegic patients. A retrospective study.

The aim of the present study was to set targets for each spinal lesion level after determining the relationship between the spinal lesion level and movement abilities in patients with tetraplegia following injury to the cervical cord. A total of 109 patients, 96 men and 13 women were included in the study. We mainly examined locomotion and transfer capabilities according to Zancolli's classification. The results of this study showed that 50% of the patients classified as C6A, 75% of C6B1 and 96% of the patients classified C6B2 accomplished bed transfer. The number of patients who could manage toilet transfer was 53% in the group classified as C6B1 and 85% in group C6B2. These results suggest that achievement of those classified as C6B2 is a clue to the assumption that the patient will achieve toilet transfer capability and can perform ADL independently.

Free-space optical module configuration using a guide-frame assembly method.

A new assembly method is described for easy construction of optical modules consisting of guide frames, spacer frames, and a housing frame. This method is used to assemble a two-dimensional optical-fiber collimator and a digital discrete correlator, which are fundamental parts of free-space optical computing systems. We show that a multistage optical system can be constructed simply by stacking of several optical functional blocks. Moreover, these compact modules do not need a conventional optical bench, they are compact, and assembly time is reduced. We demonstrated by experiment that the accuracy of optical modules assembled with this method is within the specifications of the optical system.