Tetsuo Sasabe

Sodium and chloride transport across the isolated rabbit ciliary body

Unidirectional Na+ and Cl- fluxes were determined in the isolated rabbit ciliary body under short-circuited conditions. A statistically significant net Cl- flux towards aqueous was detected in the standard bathing solution (HCO3- = 115 mM), but no net Na+ flux was demonstrated in this solution. The net Cl- flux exceeded the short-circuit current, suggesting the existence of a net flux of other ion(s). In a bathing solution containing 5 mM HCO3-, the net Cl- flux was abolished, suggesting that HCO3- in the bathing solution enhances the net Cl- flux.

Detection of gangliosides as N-glycolylneuraminic acid-specific tumor-associated Hanganutziu-Deicher antigen in human retinoblastoma cells

Gangliosides were shown to bear the tumor‐associated N‐glycolylneuraminic acid (NeuGc)‐specific Hanganutziu‐Deicher (HD) antigen expressed in human retinoblastoma cells. HD antigenie gangliosides were detected by thin‐layer chromatography/enzyme‐immunostaining using affinity‐purified chicken antibody against GM3 containing NeuGc and horseradish peroxidase‐conjugated anti‐chicken IgG. One to four species of the antigenic gangliosides were detected from all of 4 cell lines, Y79, WERI‐Rb1, TOTL1, and YK, as well as freshly cultured retinoblastoma cells and isolated tumor tissue. All cases contained GMS(NeuGc) as an HD antigen. No HD antigenic ganglioside was detected in normal retinal tissues by the same procedure.

Differential effects of fibronectin-derived oligopeptides on the attachment of rabbit lens epithelial cells in vitro

Among the Arg-Gly-Asp (RGD)-containing sequences that are known to be cell-binding domains of fibronectin, 500 migrograms/ml of Arg-Gly-Asp-Ser (RGDS) and Gly-Arg-Gly-Asp-Ser showed 100% inhibition of the attachment of cultured lens epithelial cells (TOTL-86 cells), when they were added to culture medium and coincubated with the cells for 24 h whereas RGD at concentrations of 500, 1,000 and 2,000 micrograms/ml had no such activity. After 48 h of cocultivation of 800, 400 or 200 micrograms/ml of RGDS with TOTL-86 cells, the percentage of floating cells was 100, 30.1 or 11.1%, respectively. After 144 h of cocultivation with RGDS, the percentage of floating cells was 1.6, 2.4 or 1.9%, respectively, indicating that RGDS was not cytotoxic to lens epithelial cells. However, replacing the medium with fresh medium containing new RGDS peptide resulted in floating of cells. We also studied the inhibitory effect of two other amino acid sequences that are found in cell-binding sites of the fibronectin molecule: Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr (EILDVPST) and Arg-Glu-Asp-Val (REDV). At 500 and 1,000 micrograms/ml, respectively, neither EILDVPST nor REDV has an inhibitory effect on the attachment of TOTL-86 cells, while RGDS at a concentration of 500 micrograms/ml completely inhibited the attachment of the cells in 24 h of incubation.

Cysteine conjugate of methazolamide is metabolized by β‐lyase

Abstract Bovine kidney and liver homogenates degraded a cysteine conjugate of methazolamide, S ‐(5‐acetylimino‐4‐methyl‐Δ 2 ‐1,3,4‐thiadiazolin‐2‐yl)cysteine. We isolated the degradation product following incubation with kidney homogenate by high‐performance liquid chromatography on reversed‐phase columns. The chemical structure was confirmed by proton and carbon‐13 nuclear magnetic resonance spectroscopy ( 1 H NMR and 13 C NMR, respectively), and elemental analysis by high‐resolution mass spectrometry to be N ‐(3‐methyl‐5‐mercapto‐Δ 4 ‐1,3,4‐thiadiazol‐2‐yl)acetamide, a thiol compound. The reaction is thought to be catalyzed by a pyridoxal‐dependent enzyme(s) as indicated by an inhibition study using aminooxyacetic acid. Possible involvement of the thiol compound in the development of an adverse effect is discussed.

Interferon enhances the natural killer cell activity of the retinoblastoma patients to autologous retinoblastoma cells

The cytotoxic activities of the peripheral lymphocytes prepared from two retinoblastoma patients to freshly isolated autologous tumor cells were measured using 51Cr-release assay. The retinoblastoma cells of the two patients resisted lysis by unstimulated effectors, whereas interferon(IFN)-stimulated effector cells gave a significantly higher cytotoxicity. This result implies that IFN therapy may be potent in treating high-risk retinoblastoma patients.

The Metabolism of Methazolamide - Identification of Metabolites in Guinea Pig Urine

The in vivo metabolism of methazolamide, a carbonic anhydrase inhibitor, was studied using guinea pigs as the animals. (14)C-Labeled methazolamide was synthesized. Eighty percent of intraperitoneally injected radioactivity was recovered from urine and feces within 24 hours. HPLC analysis on a C(18) column detected 2 radioactive metabolites (Peaks A and B). The Peaks A and B were isolated from the urine of the animals dosed with non-radioactive methazolamide.They were purified on the C(18) column. Their chemical structure was revealed by UV-absorbance spect a and LC/MS, and confirmed by comparing it with that of chemically synthesized compound. They were a glucuronide, (2-acetylimino-3-methyl-Δ(4)-1,3,4-thiadiazol-5-yl)-1-thio-β-D-glucopyranosiduronic acid, and a sulfonic acid, N-[3-methyl-5-sulfo-1,3,4-thiadiazol-2(3H)-ylidene]acetamide.

Cataracts occur in patients with atopic dermatitis when the serum IgE increases

The serum-IgE (sIgE) levels of eight patients with cataract complicated by atopic dermatitis (atopic cataract) who had undergone cataract surgery were reported. Five of them were diagnosed as having cataract the first visit to our eye clinic. The other three cases did not show cataract at the first visit but developed it later. The average sIgE of the five cases with cataract was 25,478 +/- 15,936 (mean +/- standard deviation) iu/ml whereas that of the latter three cases without cataract, at the first visit was 4,638 +/- 1,810 iu/ml. The average sIgE of these three cases when their cataracts were diagnosed first went up to 13,210 +/- 5,574 iu/ml. These results suggested that a high level of sIgE may be a warning of the appearance of atopic cataract.

The Metabolism of Methazolamide in Immortalized Human Keratinocytes, HaCaT Cells

Objective: Drug therapy is occasionally accompanied by an idiosyncratic severe toxicity, which occurs very rarely, but can lead to patient mortality. Methazolamide, an anti-glaucomatous agent, could cause severe skin eruptions called Stevens-Johnson syndrome/toxic epidermal necrolyis (SJS/TEN). Its precise etiology is still uncertain. In this study, the metabolism of methazolamide was investigated in immortalized human keratinocytes to reveal the possible mechanism which causes SJS/TEN. Methods: The metabolism of methazolamide was studied using immortalized human keratinocytes, HaCaT cells. HPLC was used to isolate a metabolite from the culture medium. Mass spectrometry (LC-MS/MS) was employed for its characterization. Three typical chemical inducers were assessed for the inducibility of cytochrome P450, and methimazole was used as the inhibitor of flavin-containing monooxygenase (FMO). Results: A sulfonic acid, N-[3-methyl-5-sulfo-1,3,4-thiadiazol-2(3H)-ylidene]acetamide (MSO) was identified as the final metabolite. Dexamethasone and β-naphthoflavone behaved as an inducer of cytochrome P450 in the metabolism, but isoniazid did not. The effect of methimazole was not consistent. We did not detect any glucuronide nor any mercapturic acid (N-acetylcysteine conjugate). Conclusion: N-[3-methyl-5-sulfo-1,3,4-thiadiazol-2(3H)-ylidene]acetamide (MSO) is not considered to be a direct product of an enzymatic reaction, but rather an auto-oxidation product of N-[3-methyl-5-sulfe-1,3,4-thiadiazol-2(3H)-ylidene]acetamide, a chemically unstable sulfenic acid, which is produced by cytochrome P450 from the β-lyase product of cysteine conjugate of methazolamide. MSO is considered to be susceptible to glutathione and to return to glutathione conjugate of methazolamide, forming a futile cycle. A hypothetical scenario is presented as to the onset of the disease.