We supported most of the revision, such as dividing stage I, dividing stage II, and putting T3 N0 M0 to stage IIB. Furthermore, we found some candidates for a subsequent revision, such as putting T3 N1 M0 to stage IIB, putting T2 N0 M0 and T1 N1 M0 together, regarding diaphragm invasion as T4, and putting T3 N2 M0 to stage IIIB.

Hypoxia and inflammation often occur simultaneously due to prevention of adequate gas exchange. Understanding the influence of hypoxia on the inflammatory response is important because hypoxia directly regulates expression of many genes, including those regulating inflammation, and plays a role in modulating the resolution of an inflammatory response. LPS is a major mediator of cellular injury and inflammation that induces its effects through Toll-like receptor 4 (TLR4). The aim of this study was to evaluate the effect of hypoxia on TLR4 expression. Hypoxia decreased TLR4 expression on cultured endothelial cells. Furthermore, LPS-induced ICAM-1 up-regulation was decreased by hypoxia. Because reactive oxygen species (ROS) generated from mitochondria are one of the signaling molecules induced by hypoxia, the role of ROS in hypoxia-induced TLR4 down-regulation was evaluated. Our data showed that hypoxia increased ROS generation and that hypoxia-induced TLR4 down-regulation was inhibited by myxothiazol, a mitochondrial site III electron transport inhibitor. Hypoxia also inhibited AP-1 translocation. Since the TLR4 promoter has a binding site for AP-1, hypoxia-induced TLR4 down-regulation may be due to an ROS-mediated decrease in AP-1-binding activity. We conclude that hypoxia decreases TLR4 expression in endothelial cells and that this change is mediated by mitochondrial ROS leading to attenuation of AP-1 transcriptional activity.

No abstract

Osteogenic disorder shionogi (ODS) rats carry a hereditary defect in ascorbic acid synthesis, mimicking human scurvy when fed with an ascorbic acid-deficient (aa-def) diet. As aa-def ODS rats were shown to feature disordered bone formation, we have examined the bone mineralization in this rat model. A fibrous tissue layer surrounding the trabeculae of tibial metaphyses was found in aa-def ODS rats, and this layer showed intense alkaline phosphatase activity and proliferating cell nuclear antigen-immunopositivity. Many osteoblasts detached from the bone surfaces and were characterized by round-shaped rough endoplasmic reticulum (rER), suggesting accumulation of malformed collagen inside the rER. Accordingly, fine, fragile fibrillar collagenous structures without evident striation were found in aa-def bones, which may result from misassembling of the triple helices of collagenous α-chains. Despite a marked reduction in bone formation, ascorbic acid deprivation seemed to have no effect on mineralization: while reduced in number, normal matrix vesicles and mineralized nodules could be seen in aa-def bones. Fine needle-like mineral crystals extended from these mineralized nodules, and were apparently bound to collagenous fibrillar structures. In summary, collagen mineralization seems unaffected by ascorbic acid deficiency in spite of the fine, fragile collagenous fibrils identified in the bones of our animal model. Ascorbic acid, i.e., vitamin C, is essential for the hydroxylation of proline and lysine in the α-chains that occurs in the rough endoplasmic reticulum (rER) during the process of collagen synthesis. Shortage of ascorbic acid may disrupt the process of crosslinking through which collagenous α-chains are bundled so as to form the stable helical structure known as superhelix (9, 28). Unlike humans, wild-type rats can synthesize ascorbic acid; osteogenic disorder shionogi (ODS) rats, however, carry a hereditary defect in ascorbic acid synthesis caused by the absence of l-gulonolactone oxidase, which catalyzes the conversion of l-gulono-lactone into ascorbic acid (18,19). ODS rats, when fed with an ascorbic acidfree diet, show low body weight, small craniofacial size, low concentrations of plasma alkaline phosphatase (ALPase) and defective bone formation (23,35,30,36). Accordingly, the hydroxyproline level in collagen synthesis (20) was also decreased (18). Since type I collagen accounts for approximately 90% of the organic fraction of bone (15), the deficiency of ascorbic acid in ODS rats results in reduced bone formation accompanied by deleterious effects in bone mechanical properties (14,30,36).

Parotid glands of experimental animals fed a liquid diet are reported to show atrophy (Hall and Schneyer 1964;Wilborn and Schneyer 1970;Hand and Ho 1981;Scott et al. 1990;Scott and Gunn 1991). To clarify whether apoptosis and proliferation of acinar cells participate in atrophy of rat parotid glands induced by liquid diet, rats were fed a liquid diet and compared to pellet-fed controls. Parotid glands were removed at 3, 7, 14 or 21 days, weighed, and examined using transmission electron microscopy (TEM), and studied immunohistochemically for cleaved-caspase-3 (Casp-3), a marker of apoptotic cells, and 5-bromo-2`-deoxyuridine (BrdU), a marker for proliferating cells. Body weights of experimental rats fed liquid diets were not significantly different from controls fed pellet diets; however weights of experimental parotid glands were smaller than those of controls. In the experimental parotid glands, structures like apoptotic bodies were histologically observed in acini at each time point; more Casp-3-positive acinar cells were identified in experimental parotid glands than in the controls on days 3, 7, and 14. Experimental glands showed fewer BrdU-positive acinar cells at each time point. TEM confirmed typical apoptotic acinar cells in the atrophic glands. These findings suggest that increased acinar cell apoptosis and reduced acinar cell proliferation occur in atrophic parotid glands of rats fed a liquid diet.