Lectin histochemistry in rat thyroid tumours

The thyroid tumours and background goiterous and adenomatous lesions induced in rats with diisopropanolnitrosamine (DIPN) plus methylthiouracil (MTU), and regenerative thyroid tissues after wounding were studied by lectin histochemistry. Ten weeks after cessation of the carcinogen treatments, carcinomas invading the surrounding tissues and blood vessels (13/20) and papillary micronodules (11/20) were formed in the thyroid tissues. In general, the carcinoma lesion was solitary, and the papillary micronodules were multiple in a single thyroid gland. Among the lectins tested, Maclura pomifera (MPA) and Solanum tuberosum (STA) showed specific binding with both carcinoma and papillary micronodule lesions, but not with the background goiterous and adenomatous lesions and regenerative thyroid tissues. The former both lesions showed higher labelling indices with BUdR or 3H-thymidine and poorer thyroglobulin accumulation than the latters, thereby indicating their enhanced proliferative capability and depressed potency of cyto-differentiation. The common cytological and histochemical properties of carcinoma lesions and papillary micronodules allow us to regard the latter as pre-invading carcinoma lesions. The lectins MPA and STA may be, therefore, used as the specific markers of malignancy in rat thyroid carcinogenesis.     

Apoptosis inhibitor of macrophage (AIM) expression in alveolar macrophages in COPD

Background

Marked accumulation of alveolar macrophages (AM) conferred by apoptosis resistance has been implicated in pathogenesis of chronic obstructive pulmonary disease (COPD). Apoptosis inhibitor of macrophage (AIM), has been shown to be produced by mature tissue macrophages and AIM demonstrates anti-apoptotic property against multiple apoptosis-inducing stimuli. Accordingly, we attempt to determine if AIM is expressed in AM and whether AIM is involved in the regulation of apoptosis in the setting of cigarette smoke extract (CSE) exposure.

Methods

Immunohistochemical evaluations of AIM were performed. Immunostaining was assessed by counting total and positively staining AM numbers in each case (n = 5 in control, n = 5 in non-COPD smoker, n = 5 in COPD). AM were isolated from bronchoalveolar lavage fluid (BALF). The changes of AIM expression levels in response to CSE exposure in AM were evaluated. Knock-down of anti-apoptotic Bcl-xL was mediated by siRNA transfection. U937 monocyte-macrophage cell line was used to explore the anti-apoptotic properties of AIM.

Results

The numbers of AM and AIM-positive AM were significantly increased in COPD lungs. AIM expression was demonstrated at both mRNA and protein levels in isolated AM, which was enhanced in response to CSE exposure. AIM significantly increased Bcl-xL expression levels in AM and Bcl-xL was involved in a part of anti-apoptotic mechanisms of AIM in U937 cells in the setting of CSE exposure.

Conclusions

These results suggest that AIM expression in association with cigarette smoking may be involved in accumulation of AM in COPD.     

Essential structure of orexin 1 receptor antagonist YNT-707, part III: Role of the 14-hydroxy and the 3-methoxy groups in antagonistic activity toward the orexin 1 receptor in YNT-707 derivatives lacking the 4,5-epoxy ring

Morphinan derivatives lacking the 4,5-epoxy ring were synthesized to examine the participation of the 14-OH group, the 3-OMe group, and the aromaticity of the A-ring in the activity and selectivity for the orexin 1 receptor (OX₁R). The assay results and the conformational analyses of the 14-dehydrated and 14-H derivatives suggested that the orientations of the 6-amide side chain and the 17-benzenesulfonyl group would play important roles in the activity for OX₁R. In the 6β-derivatives, removal of the 3-OMe group and the reduction of the A-ring significantly decreased the activity toward the OX₁R, but these changes did not affect the 6α-derivatives. These results indicate that the 3-OMe group and the A-ring would be essential structural moieties for the 6β-derivatives.     

Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626

Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, alpha-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM alpha-ketobutyrate, and 100 mM L-glutamate with a molar yield to alpha-ketobutyrate of 4.3% in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM MnCl(2) x 4H(2)O at 28 degrees C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and alpha-ketobutyrate was purified from A. simplex AKU 626. Mn(2+) and pyridoxal 5'-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.     

Cardiac overexpression of hormone-sensitive lipase inhibits myocardial steatosis and fibrosis in streptozotocin diabetic mice

Intracellular lipid accumulation (steatosis) and resultant lipotoxicity are key features of diabetic cardiomyopathy. Since cardiac hormone-sensitive lipase (HSL) is activated in diabetic mice, we sought to explore a pathophysiological function of cardiac HSL in the development of diabetic cardiomyopathy. Transgenic (Tg) mice with heart-specific HSL overexpression were generated, and cardiac histology, function, lipid profile, and gene expressions were analyzed after induction of diabetes by streptozotocin. Electron microscopy showed numerous lipid droplets in wild-type (Wt) hearts after 3 wk of diabetes, whereas Tg mice showed no lipid droplet accumulation. Cardiac content of acylglycerides was increased approximately 50% with diabetes in Wt mice, whereas this was blunted in Tg hearts. Cardiac lipid peroxide content was twofold lower in Tg hearts than in Wt hearts. The mRNA expressions for peroxisome proliferator-activated receptor-alpha, genes for triacylglycerol synthesis, and lipoprotein lipase were increased with diabetes in Wt hearts, whereas this induction was absent in Tg hearts. Expression of genes associated with lipoapoptosis was decreased, whereas antioxidant protein metallothioneins were increased in diabetic Tg hearts. Diabetic Wt hearts showed interstitial fibrosis and increased collagen content. However, Tg hearts displayed no overt fibrosis, concomitant with decreased expression of collagens, transforming growth factor-beta, and matrix metalloproteinase 2. Notably, mortality during the experimental period was approximately twofold lower in diabetic Tg mice compared with Wt mice. In conclusion, since HSL overexpression inhibits cardiac steatosis and fibrosis by apparently hydrolyzing toxic lipid metabolites, cardiac HSL could be a therapeutic target for regulating diabetic cardiomyopathy.     

Isotopic composition and morphology of living Globorotalia scitula: a new proxy of sub-intermediate ocean carbonate chemistry?

Abundance, isotopic composition and morphological imprints of the planktonic foraminifera Globorotalia scitula (Brady) were closely examined for possible use as a novel reconstruction tool of chemical environments in sub-intermediate depth seawater in the past. Based on the MOCNES plankton tow observation of dwelling depths of G. scitula and the isotopic compositions together with hydrochemistry data, the empirical relations between isotopic disequilibria in carbon (Δδ¹³C=δ¹³C_{G. scitula}−δ¹³C_DIC) and oxygen (Δδ¹⁸O=δ¹⁸O_{G. scitula}−δ¹⁸O_w) isotopes in the carbonate tests and the seawater δ¹⁸O and δ¹³C of dissolved inorganic carbon (DIC), respectively, are introduced. The morphological information such as pore density and porosity is also examined for significant relations to carbonate chemistry. Shell porosity is strongly correlated saturation state of calcite. The dissolution of living G. scitula tests may promote the observed isotopic differences as well as the increases in porosity. Δδ¹⁸O of G. scitula is found effectively to be linear function of both water temperature and calcite saturation state (Ω), and thereby temperature equation for G. scitula is provided, while Δδ¹³C of G. scitula is a linear function of only calcite saturation state.The equation was validated by using Globorotalia scitula collected by a sediment trap in intermediate water depths. Satisfactory agreements were found between observed and calculated Δδ¹⁸O from the empirical equations based on temperature and hydrochemistry data at sediment trap deployment site, indicating that the equation may be useful in paleo-environmental reconstruction of sub-intermediate water. The sediment trap observation further suggests that the abundance of G. scitula does not necessarily correspond to surface water productivity and to POC flux, but instead, it correlates well with the supply of fine organic matter, which appears to be a result of water convection. Thus, G. scitula may be an unambiguous and excellent paleo-environmental recorder for carbonate chemistry and for fine organic matter transport to the depths, if isotopic and morphological observations are combined.     

Qualitative and quantitative changes in nuclear DNA and phenotypic gene expression in human malignant skin tumors during their progression.

Qualitative and quantitative changes in nuclear DNA and phenotypic expression of human malignant skin tumors were examined during the course of progression. The numerical abnormalities of chromosomes demonstrated by interphase cytogenetics using the chromosome-specific in situ hybridization technique, were also used to reveal qualitative DNA changes in malignant tumor cells. For the analysis of the quantitative changes in nuclear DNA, fluorescence cytophotometry was used on the DAPI-stained tumor cells isolated from the paraffin-embedded sections. To survey abnormal gene expression in malignant tumor cells, lectin histochemistry for different sugar residues, immunohistochemical staining of HLA-DR, and in situ hybridization for H-ras, c-myc, N-myc or v-fos were used. The results showed that: 1) in one case of squamous cell carcinoma with invasion, the number of chromosomal abnormalities was much greater in the invasive than in non-invasive parts, with marked topographical heterogeneities; 2) the DNA-ploidies were largely shifted to the higher side with aneuploid stem-lines and polyploid cells in the invasive parts of all malignant tumors; 3) the expression of HLA-DR was induced at the invasive fronts of malignant melanomas; 4) the GS-I specific sugar residue(D-galactose) appeared in all extra-mammary Paget's cells; and 5) expression of "oncogenes" was found in about 60% of all malignant tumors examined. Thus, the progression of malignancy is accompanied by both qualitative and quantitative changes in nuclear DNA, resulting in abnormal gene expression.     

Essential structure of orexin 1 receptor antagonist YNT-707, Part II: Drastic effect of the 14-hydroxy group on the orexin 1 receptor antagonistic activity

The 14-dehydration- and 14-H derivatives of the orexin 1 receptor (OX₁R) antagonist YNT-707 (2) were synthesized. The obtained derivatives showed higher affinities for OX₁R than the corresponding 14-hydroxy derivatives. The conformational analysis suggested that the 17-sulfonamide groups in the derivatives without the 14-hydroxy group have a greater tendency to be oriented toward the upper side of the D-ring compared with the 14-hydroxy derivatives. Additionally, the 14-dehydration-derivative with 6α-amide side chain showed significantly higher affinity than the 14-hydroxy derivative, while the corresponding 14-H derivative showed only slightly higher affinity. Thus, the 14-hydroxy group strongly affects the affinity of the antagonist for the OX₁R.