[Suppression of corneal graft rejection in rabbits by mizoribine].
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Biomedical subjects
Publications and source records attributed to T Awata.
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A polymorphic locus flanking the 5' end of the insulin gene was studied in 154 unrelated Japanese diabetic and nondiabetic subjects. A predominance of the small allele was found with the following frequency: of 64 nondiabetic subjects, only 3 of 128 alleles were of the large class (2%); none of 78 alleles were of the large class in 39 Type 1 (insulin-dependent) diabetic subjects, and 4 of 102 alleles (4%) were of the large class in 51 Type 2 (non-insulin-dependent) diabetic subjects. The very low frequency of large allele may relate to the lower prevalence of atherosclerosis in Japanese. However, this possibility requires further examination.
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To assess the effects of metabolic control upon beta cell function in diabetes, pro-insulin and insulin were determined following gel-filtration of plasma at two time points in each three diabetic patients, once when the metabolic state was severely deranged and again after the metabolic state had improved after therapy. Before therapy, proinsulin concentration were 30 pM (as IRI), both fasting and at 2 hours after an oral glucose load. These values did not change with treatment. Insulin concentrations were 22 pM at both time points before therapy. With treatment, plasma insulin increased 2-fold at fasting and 7-fold at 2 hours after oral glucose. These results suggest an exhaustion of the insulin pool in beta cells during severe metabolic decompensation of diabetes, a condition which may be reversed by correction of the metabolic states of the patients with proper therapy.
An experiment was conducted in 76 mice (3 to 9 weeks of age) to investigate the characteristics in growth of major skeletal bones and their sexual differences. The length of ulna, radius, femur and tibia attained their maximum growth rate at earlier age, and showed no sexual difference in their growth patterns. Scapula length and width, humerus length and os coxae width reached the maximum growth rate at a later age than the forearm and shank, and showed no sexual difference in their growth, although those bones presented larger adult sizes for male than female. The length of vertebrae showed significant sexual differences in their growth patterns and adult sizes, except for lumber vertebrae. A significant sex difference in body weight caused the sexual differences in relative growth of bones to body weight. According to their relative growth to the length of humerus, the skeletal bones could be well grouped into three categories. A significant sexual difference was seen in the relative growth of scapula length. In general, it seemed that the bones growing in a vertical direction grew earlier than the bones growing in a horizontal direction, especially those of the forearm and shank. The results obtained in mice were in good agreement with the findings in pigs.
We investigated whether the cornea can synthesize fibronectin which participates in corneal wound-healing. We also examined the effects of epidermal growth factor (EGF) and cyclic AMP analogs on fibronectin synthesis by the cornea. Rabbit corneal blocks were cultured in medium without serum, and the amount of fibronectin in culture medium was determined by ELISA assay. When rabbit corneal blocks were cultured in the medium alone, fibronectin contents in the medium increased with increase in the incubation period. This increase was enhanced by the addition of EGF to the culture medium, and was inhibited by addition of a protein synthesis inhibitor, cycloheximide. The addition of cAMP analogs, 8-bromo cyclic-AMP and dibutyryl cAMP, to the culture medium also increased the rate of fibronectin production by the cornea. These results show that the cornea can synthesize fibronectin and its synthesis is stimulated by EGF and cAMP analogs.
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Fibronectin, a glycoprotein, is present in plasma and extracellular matrix and is responsible for cellular adhesion. With the use of affinity chromatography, we purified plasma fibronectin from two patients with trophic corneal ulcer and persistent epithelial defect after conventional therapy was ineffective. Reepithelialization began three days after initiation of treatment with autologous purified fibronectin eyedrops, and the epithelial defects disappeared completely within three weeks. No recurrence was observed after treatment ended. By modifying the original chromatographic procedure, we were able to obtain purified fibronectin in two hours, which allowed us to treat outpatients as well as inpatients.
We investigated the effect of fibronectin on epithelial migration onto the stroma in cultured rabbit cornea. Rabbit plasma fibronectin was purified by affinity chromatography using gelatin-Sepharose 4B, and its purity was confirmed by SDS polyacrylamide slab gel electrophoresis. Antibody against rabbit plasma fibronectin raised in guinea pigs formed a single precipitin line against rabbit plasma and purified rabbit plasma fibronectin by Ouchterlony double diffusion test. When rabbit cornea was cut into small blocks and cultured in TCM-199 medium alone, corneal epithelial cells began to migrate on the cut edge of the corneal stroma. The addition of purified rabbit plasma fibronectin to the culture medium significantly enhanced epithelial migration. The degree of enhancement depended on the amount of fibronectin added. When guinea pig IgG anti-rabbit plasma fibronectin was added, epithelial migration was significantly inhibited when compared with that in control cultured corneal blocks. The results demonstrate that fibronectin promotes epithelial migration in the cornea and thus plays an important role in corneal wound healing.
An experiment was conducted with 236 mice (3-9 weeks of age) to determine the bias in taking soft-X-ray photograph, and systematic and random errors of the values measured with a picture analyzer. Sites measured are length of scapula (SCAL), humerus (HUML), ulna (ULNL), coxae (COXL), femur (FEML), tibia (TIBL), thoracic vertebrae (VTL), lumbar vertebrae (VLL) and sacral vertebrae (VSL), and width of scapula (SCAW) and coxae (COXW). Systematic bias peculiar to the procedure was found in the values of bones measured. The X-ray photograph caused the downward bias to the length of sacral vertebrae alone, but did not to others. The standard errors of measurements (squared root of error variance) with picture analyzer ranged between 0.12 and 0.24 mm and had no apparent relationship to the size of bone.
Fibronectin (FN) has been detected at the site of corneal wounds. To investigate whether FN is supplied by the cornea itself or by extracorneal sources, we cultured small blocks of rabbit cornea in vitro with and without autologous serum (5%). The sliding rate of epithelial cells was determined and localization of FN in the corneal block examined by indirect immunofluorescent microscopy. Epithelial cells slid more rapidly in the presence of serum and covered most of the stromal surface in about 24 hours. FN was detected at the surface edge of the cut stroma shortly after culturing began. During epithelial cell sliding, FN-specific fluorescence was noted beneath the migrating cells. The location of FN was essentially the same in cultures with and without autologous serum. These results suggest that FN might be supplied by the cornea itself and that it may play a role in epithelial cell sliding.
Fibronectin (FN), a glycoprotein present in plasma and the extracellular matrix, has been reported to be effective on corneal trophic ulcer. This study reported a rapid method for preparing purified FN eyedrops from a patient's own plasma. Plasma FN concentration in healthy Japanese was found to be 235 +/- 62 micrograms per ml(age 21-31 years old). Men had a higher plasma FN level than women (264 +/- 62 vs. 206 +/- 44 micrograms/ml). The FN eyedrops were prepared by gelatin-coupled Sepharose 4B affinity chromatography and gel filtration by Sephadex G-25 (PD-10). Biochemical and immunochemical analyses revealed that the prepared FN eyedrops contained electrophoretically pure FN and no other plasma proteins were detected. Average FN concentration in the eyedrops was 425 micrograms per ml and recovery rate was 54%. The FN eyedrops were found to be stable at room temperature up to 10 days in biological activity as far as we examined. The total time required to prepare the FN eyedrops was within 2 hours. Thus it is possible to apply them to outpatients as well as inpatients.
This study aimed to compare the effects of reduced glutathione (GSH) with those of S-(1,2-dicarboxyethyl)glutathione (DCE-GS) (in rabbits and humans), and different concentrations of the latter (in humans), on corneal endothelial permeability when added to solutions bathing the isolated cornea. Inulin/dextran permeability was determined from stromal- to endothelial-facing surfaces of de-epithelialized corneas. The bathing solution was modified Opeguard(R)-MA (MOMA), an ocular irrigating solution, to which either GSH or another intrinsic tripeptide, DCE-GS, was added. Paired corneas were used to compare either different combinations of GSH with DCE-GS (rabbit or human) or various concentrations of DCE-GS from 0.25 to 2.0 mM (human). Endothelial cyclic AMP levels were determined in cultured rabbit cells. MOMA alone resulted in approximately the same permeability as MOMA + 0.3 mM GSH while the use of 2 mM DCE-GS significantly reduced rabbit (40% maximum, p < 0.00001) and human (30% maximum, p < 0.01) corneal permeability. Human corneal endothelial permeability remained reduced through a range of concentrations of DCE-GS from 0.25 to 2.0 mM DCE-GS. Tissue-cultured rabbit corneal endothelium showed an increase in cyclic AMP after DCE-GS or GSH. DCE-GS potentially offers a viable alternative to GSH for inclusion in ocular irrigating or corneal preservative solutions since it maintains human corneal endothelial permeability at a lower, stable value relative to non-DCE-GS-containing solutions.