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Biomedical subjects

N Watanabe

Publications and source records attributed to N Watanabe.

At least 181 records · Page 10Linked to original sources
PubMed2000

A highly sensitive immuno-polymerase chain reaction assay for human angiotensinogen using the identical first and second polyclonal antibodies.

We describe an immuno-polymerase chain reaction (immuno-PCR) assay for the detection of human angiotensinogen using identical first and second polyclonal antibodies. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and was bound with streptavidin to biotinylated second antibody. Human recombinant angiotensinogen sandwiched by antibodies was detected by amplifying the reporter DNA using PCR. To reduce the effect of nonspecific amplification, the optimal concentrations of streptavidin and DNA label were determined to be 0.1 mg/l and 0.5 ng/l, respectively. The detection limit of the immuno-PCR assay was 0.1 ng/l, an approximately 2.5x10(5)-fold improvement compared with a conventional enzyme-linked immunosorbent assay. These results indicate that a highly sensitive immuno-PCR for human angiotensinogen can be developed even with identical first and second polyclonal antibodies.

Angiotensinogen↗
PubMed2000

Photooxidation mechanism of nitrogen-containing compounds at TiO2/H2O interfaces: an experimental and theoretical examination of hydrazine derivatives.

The photocatalytic oxidation of oxalyldihydrazide, N,N'-bis(hydrazocarbonyl)hydrazide, N,N'-bis(ethoxycarbonyl)hydrazide, malonyldihydrazide, N-malonyl-bis[(N'-ethoxycarbonyl)hydrazide] was examined in aqueous TiO2 dispersions under UV illumination. The photomineralization of nitrogen and carbon atoms in the substrates into N2 gas, NH4+ (and/or NO3-) ions, and CO2 gas was determined by HPLC and GC analysis. The formation of carboxylic acid intermediates also occurred in the photooxidation process. The photocatalytic mechanism is discussed on the basis of the experimental results, and with molecular orbital (MO) simulation of frontier electron density and point charge. Substrate carbonyl groups readily adsorb on the TiO2 surface, and the bonds between carbonyl group carbon atoms and adjacent hydrazo group nitrogen atoms are cleaved predominantly in the initial photooxidation process. The hydrazo groups were photoconverted mainly into N2 gas (in mineralization yields above 70%) and partially to NH4 ions (below 10%). The formation of NO3- ions was scarcely recognized.

Catalysis↗
PubMed2000

Induction of apoptosis of activated murine splenic T cells by cycloprodigiosin hydrochloride, a novel immunosuppressant.

Two types of immunosuppressants, cycloprodigiosin hydrochloride (cPrG) and L-leucyl-L-leucine methyl ester (LeuLeuOMe), both have the ability to selectively inhibit the lysosomal function, and a related compound to cPrG, prodigiosin 25-C, and LeuLeuOMe have been reported to selectively inhibit the T cell function in vitro. We therefore examined the cell-type specificity of cPrG and LeuLeuOMe using murine splenocytes. Concanavalin A (Con A)- and lentil lectin-induced proliferation was suppressed by cPrG more profoundly than lipopolysaccharide-induced proliferation. At the optimal concentration, Con A induced the proliferation of both CD4+ and CD8+ cells, whereas at a supra-optimal concentration Con A induced rather selective proliferation of CD8+ cells. Irrespective of the dose of Con A, CD4+ and CD8+ cells were equally affected by cPrG. In contrast, LeuLeuOMe induced the selective loss of CD8+ cells. cPrG enhanced the apoptosis of murine splenocytes and nylon fiber column-purified T cells cultured in the presence of Con A, as shown by the decrease in cell size and/or DNA fragmentation. Overall, this study revealed that the cell-type specificity of cPrG is different from that of LeuLeuOMe, and that the immunosuppression by cPrG is associated with apoptosis.

Animals↗
PubMed2000

Tumor uptake of radioiodinated anti-human pulmonary surfactant-associated protein monoclonal antibody PE10 in nude mice bearing human pulmonary adenocarcinoma in combination with an unlabeled preload.

This study assessed the potential use of radioimmunoscintigraphy of pulmonary alveolar Type II cells tumor with the radiolabeled anti-human surfactant-associated protein (SP) monoclonal antibody (MAb) PE 10 in combination with preloads of unlabeled MAb. The in vitro binding of iodine-125 ((125)I)-labeled MAb PE 10 (1 microg), which had a specific radioactivity of 400 MBq/mg, on human pulmonary papillary adenocarcinoma NCI-H441 cells that produced SP was investigated. In NCI-H441 tumor-bearing nude mice, the tumor uptake of (125)I-MAb PE 10 (5 microg) was examined in combination with preloads of unlabeled MAb PE 10 (0, 5, 10, and 50 microg). An isotype-matched unassociated murine MAb was used as a control both in vitro and in vivo. (125)I-MAb PE 10 showed specific cell binding compared with (125)I-control MAb. Tumor uptake of (125)I-MAb PE 10 in vivo reached a peak of 4.97+/-0.33% injected dose per gram (%ID/g) at 48 h postinjection. Preloads of 5 and 10 microg unlabeled MAb PE 10 significantly enhanced tumor uptake at 48 h postinjection ( 5.94+/-0.29% ID/g and 5.72+/-0.29% ID/g, respectively), whereas preload of 50 microg unlabeled MAb PE 10 significantly decreased tumor uptake ( 2.75+/-0.32% ID/g) at 48 h. Preload of 5 microg unlabeled MAb PE 10 significantly increased the tumor-to-blood radioactivity ratio at 48 h ( 2.39+/-0.16). Preloads of unlabeled control MAb did not cause any significant change in tumor uptake. Immunohistochemistry showed the intracellular and pericellular patterns of SP expression in tumor cells. In conclusion, radioimmunoscintigraphy with MAb PE 10 labeled with a gamma-emitting radioiodine such as (123)I might be a useful means of targeting pulmonary alveolar Type II tumor cells in combination with preloading with an optimal dose of the unlabeled MAb.

Adenocarcinoma↗
PubMed2000

Generation, expansion, migration and activation of mouse B1 cells.

We have studied the expansion, activation, homing and antibody production of B1 cells in two different mouse models. One is the HL transgenic mouse, which carries Ig genes encoding the anti-red blood cell autoantibody (4C8) and develops autoimmune hemolytic anemia by the activation of autoreactive B1 cells that escape from clonal deletion and expand in the peritoneal cavity (PEC). The other model is represented by alymphoplasia (aly) mice, which carry a point mutation in the gene encoding NF-kappaB-inducing kinase (NIK) and have drastically reduced immunoglobulin serum levels, in spite of their peritoneal cavity containing a large number of B1 cells. We have found that a) expression levels of the B-cell antigen receptor (BCR) influence the size of the B1 -cell compartment and efficiency of allelic exclusion and B2-cell deletion; b) antibody production of B1 cells is closely related with their migration from PEC to other lymphoid organs and is dependent on NIK; and c) infection, lipopolysaccharide stimulation, cytokine administration or T-cell activation by noncanonical antigens induces migration and differentiation of peritoneal B1 cells into antibody-producing cells. We describe a scenario where most of B1 and B2 differences are due to a distinct activation threshold of BCR and antigen repertoire.

Anemia, Hemolytic, Autoimmune↗
PubMed2000

N-acetylcysteine replenishes glutathione in HIV infection.

BACKGROUND: Glutathione (GSH) deficiency is common in HIV-infected individuals and is associated with impaired T cell function and impaired survival. N-acetylcysteine (NAC) is used to replenish GSH that has been depleted by acetaminophen overdose. Studies here test oral administration of NAC for safe and effective GSH replenishment in HIV infection. DESIGN: Oral NAC administration in a randomized, 8-week double-blind, placebo-controlled trial followed by optional open-label drug for up to 24 weeks. SUBJECTS: HIV-infected, low GSH, CD4 T cells < 500 micro L(-1), no active opportunistic infections or other debilitation; n = 81. Study conducted prior to introduction of protease inhibitors. RESULTS: Whole blood GSH levels in NAC arm subjects significantly increased from 0.88 mM to 0.98 mM, bringing GSH levels in NAC-treated subjects to 89% of uninfected controls (P = 0.03). Baseline GSH levels in the placebo group (0.91) remained essentially the same during the 8 week placebo-controlled trial. T cell GSH, adjusted for CD4 T cell count and beta2-microglobulin levels, also increased in the NAC-treated subjects (P = 0.04). Adverse effects were minimal and not significantly associated with NAC ingestion. CONCLUSION: NAC treatment for 8 weeks safely replenishes whole blood GSH and T cell GSH in HIV-infected individuals. Thus, NAC offers useful adjunct therapy to increase protection against oxidative stress, improve immune system function and increase detoxification of acetaminophen and other drugs. These findings suggest that NAC therapy could be valuable in other clinical situations in which GSH deficiency or oxidative stress plays a role in disease pathology, e.g. rheumatoid arthritis, Parkinson's disease, hepatitis, liver cirrhosis, septic shock and diabetes.

Acetylcysteine↗
PubMed2000

Identification and characterization of human Wee1B, a new member of the Wee1 family of Cdk-inhibitory kinases.

BACKGROUND: In eukaryotic cells, the kinase activity of the mitosis-promoting complex composed of cyclin B and Cdc2 (Cdk1) is negatively regulated by the phosphorylation of Cdk1 on threonine or tyrosine residues within its ATP binding domain. RESULTS: We identified human Wee1B by searching a sequence database. The predicted human Wee1B protein comprises 561 amino acids. Northern blot analysis revealed that human Wee1B mRNA is particularly abundant in testis. Interestingly, RT-PCR using early embryos revealed that the Wee1B product was readily detectable at the mature oocyte, but abruptly disappeared at embryonic day 2.5, suggesting that the amount of Wee1B mRNA is dependent on the maternal expression. GFP-Wee1B showed a predominantly nuclear localization in HeLa cells. Human Wee1B was able to rescue the lethal phenotype of the fission yeast wee1-50Deltamik1 mutant, and over-expression of the human protein in these cells resulted in cell elongation as a result of arrest of the cell cycle at the G2-M transition. Recombinant Wee1B effectively phosphorylated cyclin B-associated Cdk1 on tyrosine-15, resulting in an inactivation of the kinase activity of Cdk1. CONCLUSION: We identified human Wee1B as a novel Cdk1-inhibitory kinase. The identification of this new member of the Wee1 family suggests that inhibition of Cdk1 is mediated at multiple levels in mammals.

Amino Acid Sequence↗
PubMed2000

Effects of heart rate and right ventricular pressure on right coronary arterial flow and its systolic versus diastolic distribution in a variety of congenital heart diseases in children.

BACKGROUND AND METHODS: In order to elucidate the underlying adjusting mechanism of human right coronary arterial (RCA) flow to increased right ventricular pressure (RVP) in children, we recorded RCA flow velocity in 24 pediatric cardiac patients at the orifice of its main trunk at the time of heart catheterization using the Doppler guidewire. RESULTS: The ratio of diastolic flow (DF)/total flow (TF), or the proportion of the DF time integral over a total of one cardiac cycle, had a negative correlation with heart rate (HR; r = -0.58, n = 11) in children with normal right ventricular systolic pressure (RVSP; RVSP < 35 mmHg). In contrast, the DF/TF ratio had a good correlation (r = 0.88, n = 24) with RVSP in all patients under study. The ratio of diastolic area (DA)/total area (TA), defined as the ratio of an area encircled by the aortic pressure curve above and the RVP curve below for diastole, over a total of one cardiac cycle, representing the overall effect of both HR and transcoronary pressure difference, also correlated well (r = 0.89, n = 24) with DF/TF. Total volume flow of the RCA also increased (r= 0.76, n = 24) with increases in RVSP, first by an increase in flow velocity through the RCA, during both systole and diastole, then by widening of the RCA lumen at very high pressures. These changes were initially more dependent on diastole with increasing RVSP because: (i) of a more marked augmentation of flow velocity in diastole compared with systole; and then (ii) of a significant decrease in flow velocity in systole at very high pressures. CONCLUSIONS: We clarify how the RCA manages to increase flow through it at different HR as a function of chronic RVP overload in pediatric cardiac patients.

Adolescent↗
PubMed2000

A patient with improvement of ulcerative colitis after appendectomy.

Recently, several retrospective studies have shown an inverse association between appendectomy and development of ulcerative colitis. We describe a 21-year-old man with distal ulcerative colitis and appendiceal involvement. The patient passed bloody stools continually during the 3 years before admission. Macroscopic and microscopic findings showed chronic moderate inflammation of the appendix and rectum. The ratio of CD4 to CD8 lymphocytes isolated from rectal and appendiceal mucosa was increased (4.3 and 3.8, respectively) compared with controls (n = 11; 1.0 in the rectum and 1.4 in the appendix). Clinical symptoms and colonoscopic and microscopic findings improved significantly after appendectomy. In addition, the amount of interferon gamma secreted from rectal lymphocytes was reduced to 89 pg/mL after surgery (before appendectomy, 254 pg/mL). However, interleukin 4 production was below detectable levels both before and after appendectomy. These findings suggest that appendectomy resulted in altered T-helper (Th)1/Th2 balance in this patient. In the 3 years since surgery, the patient has been in good condition without recurrence of symptoms. This is the first report demonstrating therapeutic benefit of appendectomy in a patient with ulcerative colitis and potential mechanistic relationship.

Adult↗
PubMed2000

Functional evaluation of hydronephrosis by diffusion-weighted MR imaging. Relationship between apparent diffusion coefficient and split glomerular filtration rate.

PURPOSE: To determine the relationship between apparent diffusion coefficient (ADC) values measured by diffusion-weighted MR imaging and split renal function determined by renal scintigraphy in patients with hydronephrosis. MATERIAL AND METHODS: Diffusion-weighted imaging on a 1.5 T MR unit and renal scintigraphy were performed in 36 patients with hydronephrosis (45 hydronephrotic kidneys, 21 non-hydronephrotic kidneys). ADC values of the individual kidneys were measured by diffusion-weighted MR imaging. Split renal function (glomerular filtration rate (GFR)) was determined by renal scintigraphy using 99mTc-DTPA. The relationship between ADC values and split GFR was examined in 66 kidneys. The hydronephrotic kidneys were further classified into three groups (severe renal dysfunction, GFR <10 ml/min, n=7; moderate renal dysfunction, GFR 10-25 ml/min, n= 10; normal renal function, GFR >25 ml/ min, n=28), and mean values for ADCs were calculated. RESULTS: In hydronephrotic kidneys, there was a moderate positive correlation between ADC values and split GFR (R2=0.56). On the other hand, in nonhydronephrotic kidneys, poor correlation between ADC values and split GFR was observed (R2=0.08). The mean values for ADCs of the dysfunctioning hydronephrotic kidneys (severe renal dysfunction, 1.32 x 10(-3) +/- 0.18 x 10(-3) mm2/s; moderate renal dysfunction, 1.38 x 10(-3) +/- 0.10 x 10(-3) mm2/s) were significantly lower than that of the normal functioning hydronephrotic kidneys (1.63 x 10(-3) +/- 0.12 +/- 10(-3) mm2/s). CONCLUSION: These results indicated that measurement of ADC values by diffusion-weighted MR imaging has a potential value in the evaluation of the functional status of hydronephrotic kidneys.

Adult↗
PubMed2000

Optimal timing of administration of hyperthermia in combined radioimmunotherapy.

Local hyperthermia (HT) may enhance the efficacy of radioimmunotherapy (RIT). However, the optimal timing of HT relative to administration of antibody is unknown. Human colon cancer xenografts (290 +/- 26 mm3) were treated with 4.63 MBq 131I-A7 monoclonal antibody (MAb) anti-Mr 45,000 glycoprotein antigen on colorectal cancer, and HT at 43 degrees C for 1 h was administered at: (A), 2 days after the 131I-A7 injection at the maximum 131I-A7 tumor accumulation (radiation); (B), soon after the 131I-A7 injection aiming to increase the tumor accumulation of 131I-A7 due to HT vascular effects; or (C), 2 days before the 131I-A7 injection in an attempt at injecting 131I-A7 when increased antigen expression could be expected. Specific growth delay (SGD) of tumors was calculated as (Tqtreat-Tqcontrol)/Tqcontrol where Tq was tumor quadrupling time. The biodistribution and intratumoral distribution of 131I-A7 were investigated to explore the mechanism of tumor response among the different HT regimens. HT alone produced some antitumor effect (SGD 1.90 +/- 0.26), which was less effective than RIT (3.11 +/- 0.50). HT soon after 131I-A7 RIT (B) significantly enhanced RIT efficacy (6.57 +/- 0.51, p < 0.0001) whereas neither HT at 2 days after RIT (A) nor at 2 days before RIT (C) did so. Biodistribution study revealed that HT soon after RIT (B) increased the tumor radiation absorbed dose by a factor of 2.4, while HT after RIT (A) did not increase radiation dose and HT before RIT (C) decreased it. Radioluminograms of tumor sections indicated that HT soon after RIT (B) improved the uniformity of 131I-A7 distribution whereas HT after RIT (A) did not and HT before RIT (C) diminished the uniformity of A7 distribution. In conclusion, the best therapeutic efficacy was obtained when HT was combined soon after the initiation of RIT with 131I-A7. The increased tumor radiation absorbed dose and the uniform intratumoral distribution of 131I-A7 were important factors underlying this improvement, and the additive cytotoxicity of HT is suspected to some extent. HT-induced radiosensitization of tumor was not apparent in this model when HT was given 2 days after 131I-A7 MAb.

Animals↗
PubMed2000

Purification and properties of protoporphyrinogen oxidase from spinach chloroplasts.

Protoporphyrinogen oxidase (Protox), an enzyme that catalyzes the common step of chlorophyll and heme biosynthetic pathways, was purified from spinach chloroplasts. The molecular weight of purified protein was estimated to be approximately 60,000 by SDS-PAGE. Protox activity was stimulated by addition of FAD, suggesting that chloroplast Protox requires FAD as a cofactor. Furthermore, the Protox-inhibiting herbicide, S23142, specifically inhibited the purified Protox activity at an IC50 value of 1 nM.

Chromatography, Gel↗