We investigated the roles of Rho-associated protein kinase (ROCK) in regulating activities such as adhesion, contraction and migration in cultured human trabecular meshwork (TM) cells. Human TM cells in culture were treated with Y-27632, a specific ROCK inhibitor. Trypan blue exclusion test and TUNEL staining showed little or no direct toxicity of Y-27632 on TM cells. By MTT assay, Y-27632 did not significantly affect the proliferation of TM cells. The cell adhesion assay showed that Y-27632 promoted the cell adhesiveness to both fibronectin and collagen type I in a dose-dependent manner. Collagen gel contraction activity of TM cells was significantly inhibited by the treatment of Y-27632 in a dose-dependent manner. The addition of Y-27632 accelerated motility of TM cells in wound healing assay. Phosphorylated LIM kinase 2 and cofilin, related to actin bundling and integrin clustering, were dephosphorylated (activated) by Y-27632. In conclusion, Y-27632 elicits profound effects on TM cell activities including adhesion, gel contraction, and cell motility. These Y-27632-induced changes of TM cells may be relevance to the physiology of the aqueous outflow system.

PURPOSE To investigate ultrastructural changes in the aqueous outflow route and discuss the mechanisms associated with intraocular pressure (IOP) elevation in a patient with presumably early stage Chandler's syndrome. METHODS A 47-year-old man underwent trabeculectomy because of elevated IOP. A specimen obtained during surgery was studied by transmission electron microscopy. RESULTS Electron microscopy showed the presence of a monolayer composed of corneal endothelium-like cells and thick basement membrane-like material. Neovascularization was also observed in the corneoscleral trabeculum. CONCLUSIONS Our results indicate that several mechanisms, including the formation of basement membrane-like tissue, infiltration of inflammatory cells and neovascularization, might contribute to the elevation of IOP in Chandler's syndrome. These may occur even when there is no history of conspicuous inflammatory reaction in the anterior ocular segments.

We investigated the role of an endoplasmic reticulum stress-associated protein, CHOP/GADD153, after NMDA-induced mouse retinal damage. After injection of NMDA into the vitreous, TUNEL-positive cells were detected in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) at 6 h after NMDA injection, and these gradually increased in number up to 24 h. Analysis by real-time RT-PCR revealed that CHOP mRNA was induced by about 3-fold, at 2 h after NMDA injection. Immunoreactivity for the CHOP protein was intense in cells of the GCL following NMDA treatment. Immunoblot analysis showed that NMDA injection increased the expression of CHOP protein in the retina. Compared with wild-type mice, CHOP-/- mice were more resistant to NMDA-induced retinal cell death as determined by TUNEL assay. At 7 days after NMDA treatment, the thickness of the inner plexiform layer and INL were larger in CHOP-/- mice than in wild-type mice. The number of residual cells in the GCL following NMDA treatment was significantly higher in CHOP-/- mice than in wild-type mice. In conclusion, CHOP is induced in mouse retina by NMDA treatment, and CHOP-/- mice are more resistant to NMDA-induced retinal damage, suggesting that CHOP plays an important role in NMDA-induced retinal cell death.

We used lipopolysaccharide (LPS) to provoke immune responses and observed the changes in the localization of iron and iron-related proteins, such as transferrin receptor, ferritin and hemosiderin in the rat spleen. After intravenous injection of 250 micrograms LPS (salmonella minnesota R595), spleen weight and serum IgM levels increased, cells incorporating 5-bromo-2'-deoxyuridine (BrdU), and transferrin receptor positive cells increased in the peripheral portion of the periarterial lymphoid sheath (PALS), the marginal zone (MZ) and the follicles. Ferritin positive cells increased markedly in the white pulp and stainable iron increased in the marginal metallophils (MM) and in the macrophages in the MZ and the outer PALS. Even in iron deficient rats, a similar change was observed for the localization of iron and iron-related proteins after injection of LPS. After injection of 0.4 mg keyhole limpet hemocyanin (KLH), changes similar to but less pronounced than that in the LPS injected rats were observed for serum IgM levels and for the localization of iron and iron-related proteins. These results showed that the iron in the MM and the macrophages in the white pulp have a dynamic response to immunological challenges and suggested that they play some role in immune responses.

The effects of three isomers of pyridinecarboxamide (picolinamide (2-pyridinecarboxamide), nicotinamide (3-pyridinecarboxamide) and isonicotinamide (4-pyridinecarboxamide)) on iron-induced renal damage were studied. Pyridinecarboxamide (250 mg/kg body weight, ip) was administered 10 min before injection of ferric nitrilotriacetate (Fe(III)-NTA) (7.5 mgFe/kg body weight, ip). In picolinamide-treated rats, the renal tubular necrosis induced by Fe(III)-NTA was attenuated and serum creatinine did not increase. Picolinamide most efficiently suppressed renal lipid peroxidation in vivo-induced by Fe(III)-NTA. Non-heme iron levels in the kidneys after Fe(III)-NTA injection did not differ in groups to which pyridinecarboxamides were administered. To elucidate the protective effects of picolinamide, we studied the action of pyridinecarboxamides on lipid peroxidation and DNA damage in vitro. These isomers inhibited iron-induced lipid peroxidation of linolenic acid. Picolinamide had no effect on DNA damage, but nicotinamide and isonicotinamide promoted DNA damage by iron, especially when ascorbate was used as a reductant. None of these pyridinecarboxamide isomers changed the chlelate structure of Fe(III)-NTA as shown by electronic absorption spectra. Among the three isomers, picolinamide most effectively protected the kidneys against iron-induced renal damage, since it not only inhibited iron-induced lipid peroxidation, but also had little enhancing action on DNA damage by iron.

Iron overload was produced in Wistar rats by repeated intraperitoneal injections of ferric nitrilotriacetate (Fe3+‐NTA) for one to six months. Pancreatic tissues from these iron‐overloaded rats and untreated controls were examined for insulin (for B cells), glucagon (for A cells), transferrin receptor (TfR), transferrin (Tf) and ferritin (Ft) using immunohistochemical methods, and for iron by histochemical Berlin blue staining. In the islets of iron‐overloaded rats, increased Ft staining appeared prior to deposition of Berlin blue‐stainable iron, and the staining intensity of Ft and iron was stronger in B cells than in A cells. In the islets of untreated control rats, the staining intensity of TfR was stronger in B cells than in A cells. TfR staining of the islets was weaker in iron‐overloaded rats than in the controls. These findings suggest that 1) iron uptake by islet cells in vivo is regulated and mediated by TfR, 2) intracytoplasmic Ft transforms into stainable iron in iron‐overloaded rats, and 3) predominance of TfR expression in B cells may result in selective deposition of iron and predispose B cells to damage and diabetes mellitus in iron‐overloaded rats. Acta Pathol Jpn 41: 647–652, 1991.

We have previously found toxic effects of iron chelate, Fe-NTA on cultured normal rat liver epithelial cells (RL34). In the present study, when RL34 cells were exposed to 50 μg/ml iron of Fe-NTA for 15 days, besides the expected cytolytic effects in most cells, the appearance of resistant cells was observed. The resistant cells showed drastic morphological transformation, grew in soft agar, and induced hepatocellular carcinomas when transplanted into syngeneic newborn rats in a short period of time. Since DNA instability in the transformed cells was ascertained by differential AO staining, it is suggested that DNA damage by Fe-NTA is of a critical importance for extremely rapid neoplastic transformation of normal epithelial cells.

This experiment was undertaken to study the possible difference in the intestinal iron absorption efficiency among iron compounds with different electric charges. Observation of rats given oral administration of 59Fe-labeled cationic cacodylate ferric (59Fe-Cac) colloid, anionic citrate ferric (59Fe-Cit) colloid, cationic 59Fe-Cac complex and anionic 59Fe-Cit complex revealed that iron absorption was more efficient in the 59Fe-Cac colloid, moderate in the 59Fe-Cac complex, low in the 59Fe-Cit colloid, and lowest in the 59Fe- cases given 59Fe-Cac colloid and 59Fe-Cac complex, a very high ratio activity was found in the liver and in the erythrocyte or hemoglobin in circulating blood, while the blood plasma, bone marrow, and spleen were low in activity. Histochemical observations of rat jejunal mucosa exposed independently for 10 min to the Fe-Cac colloid, anionic Fe-Cit colloid, and Fe-Cac and Fe-Cit complexes revealed that the cationic Fe-Cac colloid and Fe-Cac complex adhered to the luminal surface of the mucosa covering the apical area of villi with some ferric iron in the capillaries, while the anionic Fe-Cit colloid and complex did not adhere to the epithelial cells and were found free in the jejunal lumen. Electron microscopy revealed that Fe-Cac colloid particles were taken into epithelial cells by pinocytosis at the webs of microvilli, moved to the Golgi area, exocytosed to the intercellular spaces, and then translocated into the basement membrane toward blood capillaries.

ABSTRACT Macrophages in mouse bone marrow cultures were investigated with macrophage-specific monoclonal antibody F4/80 and anti-Forssman glycosphingolipid (GSL) antibody, which was specific for macrophages in hematopoietic foci. Antibody F4/80 stained two types of cells, small macrophages and large flat macrophages associated with hematopoietic cells. The cytochemical and phagocytotic characteristics were similar between these two types of cells, but Forssman GSL was positive only for the large flat macrophages associated with hematopoietic cells. The data suggest that Forssman GSL positive macrophages, derived from resident bone marrow macrophages, play an important role in hematopoiesis and are clearly distinguished from small macrophages in vitro.

The immunoreactivity of chondrocytes for glial fibrillary acidic protein (GFAP), other intermediate filament proteins and S-100 protein was studied in formalin-fixed paraffin-embedded sections. A total of 95 cartilage specimens were examined from five immature teratomas, 12 mature teratomas, and a teratocarcinoma. GFAP-immunoreactive chondrocytes were abundant in immature cartilages, and as the cartilages maturated, these chondrocytes decreased and became distributed peripherally. Elastic cartilage had more GFAP-immunoreactive chondrocytes than non-elastic cartilage. GFAP-immunoreactive cartilage was often located close to central nervous tissue. Immunostaining for vimentin and S-100 protein revealed extensive distribution of immunoreactive chondrocytes in immature and mature cartilages, but in mature cartilage, chondrocytes at the center had less vimentin immunoreactivity. GFAP-immunoreactive chondrocytes also showed apparent immunostaining for vimentin. There was no difference in immunohistochemical staining for the alpha and beta subunits of S-100 protein. The immunoreactivities of teratoma cartilage specimens were quite similar to those of respiratory tract cartilage.

Three types of traditional Chinese herb medicine were used to treat 98 patients with advanced esophageal squamous cell carcinoma prior to surgical treatment. Forty-two patients with the same diagnosis were treated with these herbs plus cyclophosphamide (endoxan). One hundred similar patients received surgical treatment without herbs or endoxan treatment as controls. Histologic examinations of surgical specimens were made on all of these patients. Stromal lymphoid-cell infiltration and cancer tissue degeneration were more prominent in Menispernum dehuricum DC- or Chelidonium majus L-treated patients, and were less clear in patients treated with herbs plus endoxan and the controls. The antitumor action of herbs is thought to be brought about by the activation of an immunological rejection mechanism. Herbs plus endoxan may result in the masking of the immunological response of hosts without obviously damaging cancer tissues.

To clarify the initiation, development and recovery processes of disseminated intravascular coagulation (DIC), rat glomerular capillaries and fibrin thrombi were examined under transmission and scanning electron microscopes. DIC was induced in rats by a single intraperitoneal injection of endotoxin (Et., 7.5 mg/kg lipopolysaccharide:B, E. coli 026:B6). At 2 h after Et. injection, the endothelial surface of the glomerular capillary became irregular with projections like a sea anemone. At 4 h after Et. injection, agglomerated fibrin thrombi composed of fibrin fiber bundles with fine cross-striated fibriform structures were observed in the capillary lumen. The fibrin thrombi gradually changed into fine reticular systems suggesting a degradation process by 6 h after Et. injection, and formed a coarse granular agglomerate by 8 h after Et. injection. These fibrin thrombi disappeared within 12 h of Et. injection, but the endothelial surface remained edematous. At 24 h after Et. injection, the microstructure of the glomerular capillaries returned normal. Based on these observations, we concluded that DIC was primarily initiated by injury to the capillary endothelium, and that changes on the endothelial surface contributed to the development of DIC.

The process of angiogenesis was studied under serum-free conditions using rat aortic segments in three-dimensional collagen gel. In serum-free and growth-factor free conditions, the capillaries formed networks and tube-like structures, and the endothelial cells produced von Willebrand factor, laminin and type IV collagen, but the number of capillaries was lower and their growth was slower than in medium containing 20% fetal calf serum (FCS). Incorporation of bromodeoxyuridine (BrdU) and inhibition of growth by hydroxyurea suggested that capillary growth depended mainly on cell proliferation and not on migration. Capillary growth in PRMI 1640 or DMEM was similar and more efficient than in MEM. Only slight growth was seen in Medium 199 and HAM-F12. The addition of serum to the medium accelerated capillary growth in proportion to the amount added. In serum-free conditions, ITS(+) and fibroblast growth factor (FGF) promoted capillary growth, but not to a significant extent. There ware no differences in capillary growth among the gel matrices used (type I collagen, type I + II collagen, type I + IV collagen, fibrin and plasma clot).

Pancreatic islet cells were examined ultrastructurally in rats after repeated intraperitoneal injections of ferric nitrilotriacetate (Fe3+‐ NTA) to produce a model of bronze diabetes. Despite diabetic signs such as glycosuria and ketouria, no ultrastructural alterations were found in islet cells up to 90 days after the beginning of the Fe3+‐NTA injections. After 120 days, however, degenerative changes appeared, with most B cells of the islets of Langerhans showing clumped nuclear chromatin, a dilated nuclear envelope, vacuolated and dilated endoplasmic reticulum, and a loss of cell polarization toward the capillary lumen. The cells contained a number of light secretory granules with an electron‐lucent core and a narrow halo. Numerous electron‐dense ferritin‐like particles were also found in the cytoplasmic matrix, and A and D cells were almost intact. Repeated venesection therapy of rats injected with Fe3+‐NTA for 120 days resulted in an increase of morphologically normal B cells with a smaller number of necrotizing cells. This process was accompanied by recovery from diabetic symptoms. The toxic effect of injected iron on B cells was thus clarified.

We have previously reported that the iron-loading of mice, by feeding them carbonyl iron, caused an elevation of hepatic glutathione concentration and an increase in glutathione excretion from the liver (Kawabata, T., Ogino, T. and Awai, M. (1989) Biochim. Biophys. Acta 1004, 89–94). To elucidate the mechanism of glutathione elevation, hepatic cysteine concentration and γ-glutamylcysteine synthetase (l-glutamate:l-cysteine γ-ligase (ADP-forming), EC 6.3.2.2) activity were measured and possible changes in cysteine metabolism were also compared between iron-loaded and control mice. Hepatic cysteine concentration was higher in iron-loaded mice (185 ± 12nmol/g wet wt.) than in the controls (164 ± 8nmol/g wet wt.), and γ-glutamylcysteine synthetase activity was also elevated in iron-loaded mice (34.3 ± 3.2nmol/mg protein per min) compared with the controls (28.6 ± 3.8nmol/mg protein per min). A comparison of the metabolic pathways with intravenously injected [35S]cysteine showed that organ distribution of the isotope was not significantly different, and also the rate of [35S]cysteine uptake into the hepatic glutathione fraction exhibited no difference between the two groups of mice. This shows that hepatic cysteine turnover may not be different between the two groups of mice. Since hepatic cysteine concentration was higher in iron-loaded mice, the apparently equal turnover of hepatic cysteine suggests that GSH synthesis may be elevated in iron-loaded mice. The high γ-glutamylcysteine synthetase activity is suggested to stimulate GSH synthesis in iron-loaded mice.