Experimental studies on cryopreservation combined with the cultural process of isolated rat pancreatic islets

Recently many investigators have attempted transplantation of isolated islets of Langerhans in an expectation of therapeutic effects in patients with diabetes. In the course of time, preservation of the islets was found to be a prerequisite for successful transplantation. In the present studies, we used our newly designed method of preserving islets, i.e., cryopreservation combined with the cultural process before and after freezing, and we examined the insulin-releasing activity of the islets and attempted transplantation of the islets so-obtained. As to the insulin-releasing activity, the islets cryopreserved after short-term culture increased insulin output as compared with those cryopreserved without culture. Damage to Langerhans’ islets resulting from isolation and cryopreservation was overcome during the culture. Transplantation of Langerhans’ islets cultured for 3–4 days before and after the cryopreserving process produced favorable results.     

Palm temperature monitoring during exercise in patients with heart disease

The quantitative use of palm temperature changes during a fixed-load treadmill exercise was evaluated in normal subjects and patients with various degrees of cardiac disability. Treadmill exercise revealed different temperature patterns between subject groups. Normal subjects showed an initial transient decrease to a plateau phase, followed by a prompt return to the control level after cessation of exercise. Cardiac patients with severe disabilities showed a progressive decrease during and even after exercise, and the return to the control level was delayed. Patients with less severe cardiac disabilities showed an intermediate pattern. A significant correlation was observed between the temperature pattern and the plasma catecholamine concentration. The simultaneous measurement of forearm and hand blood flow, and palm temperature during a supine ergometer exercise showed that the temperature change reflected the blood flow changes. In conclusion, palm temperature monitoring during treadmill exercise is a simple and useful method for assessment of the vasoconstrictor response to exercise and, therefore, the pattern of temperature changes indicates indirectly the exercise capacity in heart disease.     

Studies on the nephrotoxicity of aminoglycoside antibiotics and protection from these effects (8): Protective effect of pyridoxal-5'-phosphate against tobramycin nephrotoxicity

We investigated the effect of pyridoxal-5'-phosphate (PALP) on tobramycin (TOB)-induced nephrotoxicity in rats. Paper electrophoretic analysis showed that in the mixture of TOB and PALP, the spot corresponding to TOB alone almost disappeared and the spot associated with TOB overlapped with that associated with PALP, although the spots of TOB alone and PALP alone were observed as single spots on the cathode and anode sides, respectively. The overlapping of both compounds indicated that TOB could directly interact with PALP in vitro. In the assay of TOB binding to renal brush border membranes (BBMs), PALP significantly inhibited the binding of TOB to BBMs by interacting with TOB outside of BBMs vesicles. Intrarenal TOB levels in rats receiving TOB and PALP were lower than those in rats given TOB alone. Combination with PALP markedly suppressed the urinary protein content, urinary N-acetyl-beta-D-glucosaminidase activity and blood urea nitrogen content elevated by TOB, and also reduced the degree of TOB-induced renal tubular cell necrosis. These results indicate that PALP protects the rat kidneys from TOB-induced nephrotoxicity and that the protective effect of PALP may be due to the reduced intrarenal TOB concentration and less binding to TOB to BBMs induced by the interaction of PALP with TOB.     

Establishment and characterization of resistant cells to etoposide (VP16) from a mouse breast cancer cell line, FM3A

We established 4 cell lines resistant to VP16 from a mouse breast cancer cell line, FM3A. The IC50 values of all 4 resistant strains were approximately 2 micrograms/ml as measured by colony formation in soft agar; about 40 times higher than that of parent cell (0.05 microgram/ml). These cells showed a cross-resistance to VM26, a compound related to VP16, but not to a variety of other antitumor drugs including adriamycin, mitomycin C, cis-platinum, 5-fluorouracil, bleomycin, vincristine, 4-hydroperoxycyclophosphamide, methotrexate and cytosine arabinoside. Topoisomerase II, the putative target of VP16, was partially purified from cells, and was assayed using knotted P4 phage DNA as a substrate. However, no significant difference was observed between enzymes from resistant cells and from the parent cells in either activity per cell or sensitivity to VP16. On the other hand, the resistance of these cell lines to VP16 was greatly reduced by adding a calcium antagonist, verapamil, to the soft agar at a concentration as low as 5 microM, at which the viability of cells was hardly affected. A similar verapamil-induced reduction in the resistance of the cells to VM26 was also observed. These results suggest that the acquired resistance may be largely due to an altered membrane permeability to drugs, which may be overcome by verapamil, rather than to an altered topoisomerase II.     

Demonstration of intracytoplasmic immunoglobulin in non-T-acute lymphocytic leukemia cases of children with immunoelectron microscopic examination

Leukemic cells from 25 cases of non-T-acute lymphocytic leukemia (ALL) in children were investigated for intracellular immunoglobulin by the use of immunoelectron microscopic study (immuno-EM). As a result, 12 cases were negative for both heavy and light chains of the immunoglobulin in the neoplastic cells. Of these, one case that was also negative for common ALL antigen (CALLA) was defined as null cell type. The remaining 11 cases were defined as common ALL type. Six cases were defined as pre-B-cell type because they were positive for cytoplasmic mu chain but negative for cytoplasmic light chains. Seven cases were defined as B-cell type, all of which possessed lambda light chains in the neoplastic cells. Immunohistologic studies using monoclonal antibodies showed that most non-T-ALL cases were positive for CALLA and B4, whereas about 50% of them were positive for B1. Our immuno-EM study suggests that it is one of the effective ways of subclassifying non-T-ALL in children.