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Immunostaining depending on antigen-antibody specificity is the commonest approach for determining the localization of specific antigens in tissue sections. This procedure is applicable not only with frozen or specially fixed samples, but also has proved reliable with formalin-fixed paraffin-embedded tissue sections through improvement of antigen-retrieval. Immunostaining is thus firmly established as a tool for diagnostic pathology and in our institute multiple antibodies are applied for 13-15% of the cases examined, as well as H and E staining. With the standard approach, approximately 3 h is necessary from the beginning of deparaffinization till covering sections with the Envision system. We utilized intermittent microwave irradiation for 10 min during hybridization with primary and secondary antibodies in a special moist-chamber, to achieve all immunostaining steps within 1 h in 178 primary antibodies frequently used for diagnostic pathology. According to our 5 years experience, such microwave irradiation not only obtained significant specific staining for enhancing the specificity of antigen-antibody reactions, but also inhibited nonspecific binding. We present herein the details of the methodology and recommendations for its application with particular primary antibodies. This method can contribute to savings in time and energy, allowing pathologists to rapidly obtain diagnostic information.

To elucidate compositional changes of the tendons and ligaments with aging, the authors investigated age-related changes of element contents in the insertion tendons of the biceps brachii muscle, central tendons of the diaphragma, Achilles' tendons, posterior longitudinal ligaments (PLLs) of the cervical spine, ligamenta capitum femorum, and anterior cruciate ligaments. After ordinary dissections by medical students, the three tendons and three ligaments were resected and element contents were determined by inductively coupled plasma-atomic emission spectrometry. It was found that the elements, such as Ca, P, S, Mg, Na, Zn, and Fe, did not change significantly in the three tendons and two ligaments with aging, except for the PLLs where Ca and Mg increased significantly with aging and Fe decreased significantly with aging. With regard to the relationships among elements, the common finding that there were significant correlations between Ca and P contents and between Ca and Mg contents was obtained in the three ligaments. Likewise, the common finding that there was a significant correlation between Ca and Mg contents was obtained in the three tendons. Regarding the relationship between Ca and P contents, the three tendons were different from the three ligaments.

A neonate had a globular mass bulging through the eyelids of the left eye. Computed tomography revealed a large ectatic corneal lesion and the absence of a lens. The enucleated globe revealed that the posterior surface of the ectatic cornea was lined by iris tissue, indicating corneal staphyloma.

Cyclooxygenase-2 (COX-2) is known as one of the critical prognostic factors in carcinomas of the various organs. However, the importance of COX-2 overexpression in oral squamous cell carcinomas has not been fully described yet. We investigated overexpression of COX-2 by immunohistochemistry in 72 surgical specimens from patients with squamous cell carcinoma of the oral cavity, and evaluated correlations between COX-2 overexpression and clinicopathologic variables. The immunoreactivity of COX-2 was cytoplasmic. COX-2 overexpression was observed in 10 (13.9%) of 72 tumours and it was well correlated with lymph node involvement at the time of surgical treatment (P=0.011) and postoperative recurrence (P=0.025), but not with the other clinicopathologic variables including age, gender, tumour stage and histological grade. In addition, COX-2 overexpression showed a close association with postoperatively disease-free survival (P=0.039) and overall survival as well (P=0.043), and multivariate analyses revealed that COX-2 overexpression was an independent predictor for disease-free survival but not for overall survival. The current study suggests that overexpression of COX-2 could impact on disease-free survival for patients with oral squamous cell carcinoma and that selective inhibition of COX-2 is a possible target for the therapeutic strategies.

To elucidate compositional changes of the cartilage with aging, the authors investigated age-related changes of element contents in the xiphoid process and costal cartilage by inductively coupled plasma-atomic emission spectrometry. After the ordinary dissection by medical students was finished, the xiphoid processes and bilateral the fourth costal cartilages were resected and element contents were determined. The subjects consisted of 17 men and 9 women, ranging in age from 52 to 96 yr. No significant correlations were found between age and the content of elements, such as Ca, P, S, Mg, Na, Zn, Fe, and Si in the xiphoid process and costal cartilage, except for S in the costal cartilage, in which an extremely significant correlation was found. With regard to the relationships among element contents, the common finding that there were significant direct correlations both between Ca and P contents and between Ca and Mg contents, whereas there were significant inverse correlations both between Ca and S contents and between P and S contents obtained in the xiphoid process and costal cartilage.

The synthesis and biological properties of a novel class of 7-heterocycle-substituted quinoxalinecarboxylic acids, which bear a substituted phenyl group through a urethane linkage at the C-7 position, are described. One of the synthesized compounds, 15l, which has a 4-carboxyphenyl carbamoyloxymethyl imidazole group at the C-7 position and is water-soluble, was found to possess high potency in vitro and showed excellent neuroprotective efficacy in vivo.

Solubilized membrane proteins from HL-60 cells were separated by two-step affinity chromatography. Proteins eluted with MgCl2 in the first heparin-gel were applied to the second heparin-gel and eluted with CaCl2. The eluted proteins were analysed and purified by electrophoresis. N-terminal amino acid sequences of eight proteins on the characteristic bands were determined. Homology search for the sequences indicated that three microsomal proteins, two nuclear proteins and a glycolytic enzyme were eluted with divalent cations, whereas a nuclear ribonucleoprotein and a membrane-cytoskelton linker protein were not dissociated with divalent cations, but with 2 M NaCl. Heparin affinity chromatography combined with differential elution with divalent cations can be a useful method for separation of membrane proteins.

To elucidate compositional changes of elements in the meniscus with aging, the authors investigated the relationships among elements in the medial meniscus by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of 16 men and 7 women, ranging in age from 65 to 93 yr. In the left medial meniscus, extremely significant correlations were found both between calcium and phosphorus contents and between sulfur and magnesium contents, whereas significant correlations were found between calcium and either iron or zinc contents, between phosphorus and either iron or zinc contents, and between iron and zinc contents. On the other hand, in the right medial meniscus, there were only an extremely significant correlation between calcium and phosphorus contents and a significant correlation between sulfur and magnesium contents. The common finding between the left and right medial menisci was an extremely significant correlation between calcium and phosphorus contents and a significant correlation between sulfur and magnesium contents.

To elucidate the characteristics of calcium accumulation of human plantar and palmar aponeuroses, the authors determined the calcium content of human plantar and palmar aponeuroses by atomic absorption flame emission spectrophotometry. The subjects consisted of 9 men and 14 women, ranging in age from 61 to 93 yr. In the plantar aponeurosis, the calcium content was significantly higher in the anterior and posterior parts than in the middle part. It is known that pressure distribution under the sole of a foot is higher in the anterior and posterior parts than in the middle part. The present study suggests that the accumulation of calcium in the plantar aponeurosis is related with the pressure distribution under the sole of a foot. The calcium content increased progressively with aging in the anterior part of the plantar aponeurosis, but not in the middle and posterior parts. Regarding the palmar aponeurosis, the calcium content was significantly higher in the anterior and posterior parts in comparison with the middle part. It was found that the calcium content increased progressively with aging in the posterior part of the palmar aponeurosis, whereas it did not increase significantly with aging in the anterior and middle parts. Regarding the relationship between the calcium content of the aponeuroses and the bone mineral density, a significant correlation was found between the calcium content in the anterior part of the palmar aponeurosis and the bone mineral density of the scaphoid bone.

The lung is the primary target organ of airborne heavy metal-induced toxicity. The aims of this study were to investigate differential acute lung cytotoxicity caused by heavy metals using a primary culture of alveolar type II cells and to establish an in vitro assessment model of lung toxicity. The cytotoxicity of heavy metals was determined by measuring the lactate dehydrogenase release and (51)chromium release from lyzed cells. With respect to the LC(50) values, drug concentrations causing a 50% loss in cell viability, the mean value of Hg was 110 microM and that of Cd was 220 to 250 microM. Cytotoxicity was graded high for Hg and Cd, moderate for Pb and Ni, and negligible for Mn. Additional morphological observations of cell membrane integrity by scanning electron microscopy were compatible with the results of biochemical measurements. In conclusion, we have presented an in vitro assessment model of lung toxicity, which can be used effectively to assess the differential effects of heavy metals on alveolar type II cells. The findings suggests that the potential mechanisms of cytotoxicity are dependent on both the nature and the concentration of the metals.

A modified western blotting protocol was developed to increase the binding specificity of antigens and antibodies, using intermittent microwave irradiation (IMWI) with seven antibodies and two cell lines. The method was based on IMWI of the blotting membrane in the immunoblotting step using 5% skim milk as the diluting buffer. For some antibodies against p53, CDK4 and cyclinE, there were no distinct differences between the IMWI(+) and IMWI(-) counterparts; but improvement over the standard protocol was noted in both. For some antibodies, such as the polyclonal antibody against tubulin and the monoclonal antibodies against beta-tubulin, cyclinA and cyclinB1 (which were otherwise difficult to obtain good results with), IMWI was extremely effective, resulting in clear, specifically binding bands and a clean background. Moreover, the times were reduced from 8 to 3 h. Both the IMWI(+) and IMWI(-) protocols can be applied as simple, rapid and highly specific detection techniques for applications with various antigens, reducing background 'noise' to a minimum.

The matrix metalloproteinases (MMPs) and endothelin-1, a potent vasoconstrictor and mitogen for smooth muscle cells, have been shown to be involved in the pathogenesis of various vascular disorders. However, the expression of endothelin-1 and the activation of MMPs have not been fully evaluated in plexogenic pulmonary arteriopathy (PPA). Immunohistochemical and confocal microscopic studies were conducted to evaluate the reactivity of lung tissue from six patients with pulmonary hypertension for alpha-smooth muscle actin (alpha-SMA), desmin, vimentin, factor VIII, endothelin-1, various types of MMPs (MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9), membrane type-MMPs (MT-MMPs), tissue inhibitors of MMPs (TIMPs), and type IV collagen. Four major arterial morphological abnormalities were recognized in PPA: muscularization of pulmonary arterioles, onion-skin lesions, cellular and mature plexiform lesions, and atheromas in elastic pulmonary arteries. Reactivity for MMP-2 and MT-1-MMP was found in endothelial cells and, to a lesser extent, in myofibroblasts proliferating in various lesions of PPA. Increased expression of endothelin-1 was observed in the latter cells and in endothelial cells. Some myofibroblasts were positive for MMP-3 and MMP-7 in the vascular lesions except for mature plexiform lesions. MMP-1, MMP-9 and TIMP-2 tended to be positive only in the atheromatous lesions. Staining for type IV collagen showed focal thinning and discontinuities of the endothelial basement membrane in plexiform lesions. This study demonstrates colocalization of MMP-2 with MT-1-MMP and increased expression of endothelin-1 in various arterial lesions of PPA. These changes may play important roles in the remodeling of arterial structures, particularly of basement membranes, in this disorder.