Different Roles of 8-Hydroxyguanine Formation and 2-Thiobarbituric Acid-reacting Substance Generation in the Early Phase of Liver Carcinogenesis Induced by a Choline-deficient, l-Amino Acid-defined Diet in Rats

The present study was performed to assess the roles of hepatocellular oxidative damage to DNA and constituents other than DNA in rat liver carcinogenesis caused by a choline-deficient, l -amino acid-defined (CDAA) diet by examining the effects of the antioxidant N, N' -diphenyl- p -phenylenediamine (DPPD). The parameters used for cellular oxidative damage were the level of 8-hydroxyguanine (8-OHGua) for DNA and that of 2-thiobarbituric acid-reacting substance (TBARS) for constituents other than DNA. A total of 40 male Fischer 344 rats, 6 weeks old, were fed the CDAA diet for 12 weeks with or without DPPD (0.05, 0.10 or 0.20%) or butylated hydroxytoluene (BHT, 0.25%). In the livers of the rats, the numbers and sizes of glutathione S -transferasc (EC 2.5.1.18) placental form (GSTP)- and/or γ-glutamyltransferase (GGT, EC 2.3.2.2)-positive lesions and levels of 8-OHGua and TBARS were determined. The GSTP-positive lesions of 0.08 mm² or larger were all stained positively for GGT as well in cross-sectional area, whereas the smaller lesions were generally negative for GGT. DPPD and BHT reduced the size of the GSTP-positive lesions without affecting their total numbers. At the same time, they reduced TBARS generation without affecting 8-OHGua formation in DNA. The present results indicate that oxidative DNA damage (represented by 8-OHGua formation) and damage to constituents other than DNA (represented by TBARS generation) may play different roles in rat liver carcinogenesis caused by the CDAA diet; the former appears to be involved in the induction of phenotypically altered hepatocyte populations while the latter may be related to the growth of such populations.     

Immunohistological investigation of type IV collagen in the basement membrane surrounding the cancer nest (cancer nest membrane) of head and neck squamous cell carcinoma--its relation to frequency of cervical lymph node metastasis]

Lymphoducts and blood vessels exist in the stroma, while none can be detected in the cancer nest itself within cancerous tissue. This explains why metastasis of carcinoma cannot occur without the escape of tumor cells through the basement membrane surrounding the cancer nest into the stroma. Accordingly, observation of the continuity of the basement membrane, what we call the cancer nest membrane, is essential for elucidating the first step of metastasis. Since type IV collagen is the most important structure composing the basement membrane, investigation of the immunohistological localization and continuity of type IV collagen is of value in predicting the metastatic aggressiveness of squamous cell carcinoma. We therefore studied biopsy tissues from the advancing lesion of head and neck squamous cell carcinoma in 95 untreated patients. The tissues were fixed in 85% ethanol and embedded in paraffin, and 5-um thin sections prepared were then immunohistochemically stained for type IV collagen by the ABC method for observation of the continuity status of the cancer nest membrane in relation to metastasis. The basement membranes of normal mucosal epithelium and normal interstitial capillaries were utilized as positive controls, and negative controls were obtained by using PBS in place of the primary antibodies for the immunohistochemical reaction. Membrane discontinuity (breaks or absence) correlated significantly with cervical lymph node metastasis, while intact membrane was associated with a low frequency of cervical lymph node metastasis. There was no obvious relation between the clinical T category and the continuity of the membrane; pN (+) carcinomas with membrane discontinuity included even T1 supraglottic and hypopharyngeal carcinomas, as well as T2 or higher oral mucosal carcinomas and T3 or higher glottic carcinomas, suggesting variation with tumor site. Hypopharyngeal and supraglottic carcinoma was associated with membrane discontinuity and a high incidence of cervical lymph node metastasis. On the other hand, glottic and oral carcinoma more often presented with intact membranes and had a lower incidence of metastasis, although carcinomas in these sites that did present with discontinuity of the membrane were associated with a high incidence of cervical metastasis. Therefore, observation of the continuity of the cancer nest membrane by the expression of type IV collagen may be useful in selecting better specific therapies and determining the necessity of prophylactic neck dissection. A correlation between the degree of tumor differentiation and the continuity of the membrane was also found; well-differentiated tumors with discontinuity of the membrane were frequently associated with cervical lymph node metastasis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Graded contusion model of the mouse spinal cord using a pneumatic impact device

OBJECTIVE: This study examined the effects of varying magnitudes of controlled spinal cord impact to the mouse spinal cord on neurological and histopathological variables to obtain a mouse model of spinal cord injury (SCI). METHODS: A laminectomy of the T10 vertebra was performed on anesthetized C57BL/6 mice. A pneumatic pressure-driven impact was performed on the spinal cord through the dura mater. Experimental groups were subdivided according to the energy of impact (0.25-mm-deep deformations): Group 1 (n = 5), impact velocity at 1 m/s; Group 2 (n = 5), impact velocity at 2 m/s; and Group 3 (n = 5), impact velocity at 3 m/s. Functional deficits over time were evaluated up to 28 days after SCI by testing hindlimb reflex and coordinated motor function. The extent of the lesions was analyzed histopathologically and quantified by a morphometric measurement. RESULTS: Mice of all groups exhibited profound functional deficits immediately after injury and subsequent gradual symptomatic recovery. The degrees of recovery were precisely correlated with the magnitudes of impact. The extent of resultant cord lesions was highly reproducible among animals, with little variance: means +/- standard deviation, 0.86 +/- 0.06/100 mm3 in Group 1; 2.4 +/- 0.28/100 mm3 in Group 2; and 11.0 +/- 1.0/100 mm3 in Group 3. CONCLUSION: Our results indicate that this model provides constant functional and histopathological lesions according to impact energy. This new mouse model of SCI opens a new avenue for studies investigating roles and/or effects of specific genes in the recovery process of SCI.     

Cyclotheonamide E4 and E5, new potent tryptase inhibitors from an Ircinia species of sponge

Tryptase is a protease released from mast cells and is believed to contribute to the inflammatory process in allergic diseases including asthma. In the course of screening to find tryptase inhibitors, we isolated two new tryptase inhibitors, cyclotheonamide E4 (3) and E5 (4), from a marine sponge of the genus Ircinia. The structures of these molecules were determined by interpretation of 1H and 13C NMR spectra, and they were shown to be closely related to the previously reported cyclotheonamides E (1), E2, and E3 (2). These molecules contain two unusual amino acids, vinylogous tyrosine and alpha-ketohomoarginine, which are involved in strong activities against serine proteases. Cyclotheonamide E4 showed potent inhibitory activity against human tryptase (IC50 5.1 nM). Therefore, cyclotheonamide E4 may be useful as a therapeutic agent in the treatment of allergic diseases including asthma.     

Eosinophilic myelitis associated with atopic diathesis: a combined neuroimaging and histopathological study

Histologically proven eosinophilic myelitis has rarely been reported except in connection with parasitism. To clarify its clinicopathological features, we conducted a nationwide survey of biopsy-proven eosinophilic myelitis of unknown cause throughout Japan. Six such cases were collected and studied immunologically and pathologically. All were young to middle-aged men. All showed a protracted and fluctuating course with mild disability for 3-25 (mean 12.5) months before biopsy. Magnetic resonance imaging revealed localized lesions of T2-high and T1-iso signal intensity with a partial gadolinium enhancement in all cases. Cerebrospinal fluid (CSF) examinations were completely normal except for modest pleocytosis in two cases. Eosinophilia was present in the peripheral blood in two cases but was absent from the CSF of all cases. In spite of the chronic nature of the disease, spinal cord pathology revealed very active lesions with marked cell infiltration consisting mainly of CD8(+) T cells and varying numbers of eosinophils in the perivascular areas and the parenchyma. Both the myelin and axons were severely disrupted in all cases. Moreover, eosinophil cationic protein (ECP), an activated eosinophil product, was heavily deposited in the tissues. All but one case had hyperIgEemia and mite antigen-specific IgE in the sera, and two had accompanying atopic disorders. The present study thus revealed idiopathic eosinophilic myelitis to be a localized and persistent inflammation of the spinal cord, with distinct clinicopathological features, that has a possible link to atopic diathesis.     

Mitogenic effect of glial cell line-derived neurotrophic factor is dependent on the activation of p70S6 kinase,but independent of the activation of ERK and up-regulation of Ret in SH-SY5Y cells

Glial cell line-derived neurotrophic factor (GDNF) activates c-Ret tyrosine kinase and several downstream intracellular pathways; the biological effects caused by the activation of each of these pathways, however, remain to be elucidated. Here we report the ability of GDNF to induce proliferation, rather than differentiation, of neuroblastoma cells (SH-SY5Y) by targeting the signaling pathway responsible for mediating this proliferative effect. GDNF induces the phosphorylation of Akt and p70S6 kinase (p70S6K) in SH-SY5Y cells in which Ret protein expression is relatively low. Interestingly, treating SH-SY5Y cells with retinoic acid greatly increases Ret protein levels and GDNF-induced Ret tyrosine phosphorylation, but does not affect the mitogenic action of GDNF and the activation of the Akt/p70S6K pathway. In contrast, the activation of the ERK pathway and the resulting induction of immediate-early genes parallel the increases in Ret protein levels. Rapamycin, a specific inhibitor of p70S6K activation by the mammalian target of rapamycin, completely prevents GDNF-induced proliferation and activation of p70S6K. These results suggest that GDNF promotes cell proliferation via the activation of p70S6K, independent of the ERK signaling pathway, and that GDNF activates the Akt/p70S6K pathway more efficiently than the ERK pathway in the cells in which Ret expression is low.     

Discrimination between composite resin and teeth using fluorescence properties

The differentiation of composite resin from teeth using fluorescence emission was investigated as basic research for the visual detection of resin filled teeth in mass dental health examinations. Fluorescence spectra were taken from extracted human maxillary central incisors and 12 types of light-cured composite resins with a maximum of 15 shades via excitation using light with wavelengths of 400-500 nm. The fluorescence intensity ratio of resin to tooth was lowest around 500 nm for all the resins. The fluorescent images were taken based on spectroscopic results, which confirmed discrimination between the resin part and the tooth in the resin filled tooth.     

Heparin facilitates glial cell line-derived neurotrophic factor signal transduction

Glial cell-line neurotrophic factor (GDNF), a neurotrophic factor with heparin binding affinity, promotes the survival and differentiation of a variety of neuronal cells including dopaminergic neuron. The effect of heparin on GDNF signaling was investigated based on the expression of the tyrosine hydroxyrase (TH) gene in neurobalstoma cells. Up-regulation of TH gene mRNA by GDNF was enhanced by co-administration of heparin. This facilitation by heparin was particularly evident at suboptimal levels of GDNF, which was consistent with the luciferase assay using TH gene promoter. Pretreatment with heparitinase decreased TH promoter activity in the absence of heparin. Phosphorylation of extracellular regulated kinase was increased in the presence of heparin, although tyrosine phosphorylation of Ret receptor tyrosine kinase was not affected by heparin. Expression of early response genes such as c-fos or Egr1 increased and sustained in the presence of heparin more than that without heparin. These results indicate that interaction with glycosaminoglycans such as heparin affects GDNF signal transduction positively.