Cell type-specific expression of beta-carotene 15,15'-mono-oxygenase in human tissues.

We studied the cell type-specific expression of human beta-carotene 15,15'-mono-oxygenase (BCO1), an enzyme that catalyzes the first step in the conversion of dietary provitamin A carotenoids to vitamin A. Immunohistochemical analysis using two monoclonal antibodies against different epitopes of the protein revealed that BCO1 is expressed in epithelial cells in a variety of human tissues, including mucosa and glandular cells of stomach, small intestine, and colon, parenchymal cells in liver, cells that make up the exocrine glands in pancreas, glandular cells in prostate, endometrium, and mammary tissue, kidney tubules, and in keratinocytes of the squamous epithelium of skin. Furthermore, BCO1 is detected in steroidogenic cells in testis, ovary, and adrenal gland, as well as skeletal muscle cells. Epithelia in general are structures that are very sensitive to vitamin A deficiency, and although the extraintestinal function of BCO1 is unclear, the finding that the enzyme is expressed in all epithelia examined thus far leads us to suggest that BCO1 may be important for local synthesis of vitamin A, constituting a back-up pathway of vitamin A synthesis during times of insufficient dietary intake of vitamin A.     

Cell type-specific localization of sphingosine kinase 1a in human tissues.

Cell type-specific localization of sphingosine kinase 1a (SPHK1a) in tissues was analyzed with a rabbit polyclonal antibody against the 16 C-terminal amino acids derived from the recently reported mouse cDNA sequence of SPHK1a. This antibody (anti-SPHK1a antibody) can react specifically with SPHK1a of mouse, rat, and human tissues. Utilizing its crossreactivity to human SPHK1a, the cell-specific localization of SPHK1a in human tissues was histochemically examined. Strong positive staining for SPHK1a was observed in the white matter in the cerebrum and cerebellum, the red nucleus and cerebral peduncle in the midbrain, the uriniferous tubules in the kidney, the endothelial cells in vessels of various organs, and in megakaryocytes and platelets. The lining cells of sinusoids in the liver and splenic cords in the spleen showed moderate staining. Columnar epithelia in the intestine and Leydig's cells in the testis showed weak staining patterns. In addition, TPA-treated HEL cells, a human leukemia cell line, showed a megakaryocytic phenotype accompanied with increases in immunostaining of both SPHK1a and SPHK enzyme activity, suggesting that SPHK1a may be a novel marker of megakaryocytic differentiation and that this antibody is also useful for in vitro study of differentiation models.(J Histochem Cytochem 49:845-855, 2001)     

Studies on human monocytes with a multiparameter cell sorter.

Suspensions of human lymphocytes and monocytes separated by the Ficoll-hypaque method from the peripheral blood show a Coulter volume distribution, measured with a multiparameter cell sorter, characterized by a minor peak at 500 mu3, containing 5-15% of the cells, and a major peak at 200 mu3. Using fluorescent latex particles we have found that the monocytes, the cells that ingest the latex particles, all lie in the 500 mu3 peak; conversely, all of the cells in the 500 mu3 peak are monocytes. When the cell suspensions are incubated, the monocytes increase both the average volume and in absolute numbers. The number of monocytes approximately doubles during 3 days of incubation, when it reaches its maximum value. At that time we have found that all of the monocytes lack receptors for sheep red blood cells and all possess receptors for human gamma-globulin. The increase in monocyte number appears, therefore, to arise from the enlargement of "monocyte presursors" that resemble lymphocytes in volume and resemble both the monocytes and the B lyphocytes with respect to surface sheep red blood cell and human gamma-globulin receptors.     

32P-postlabelling analysis of DNA from human tissues.

DNA extracted from human lung, bladder, liver, pancreas, cervix and breast tissue samples taken at autopsy (37 sample sets) was analysed by the nuclease P1 enhancement modification of the 32P-postlabelling assay for levels of aromatic carcinogen DNA adducts. Results were combined with those from a previous study for statistical analysis of 56 sample sets (32 male+24 female). A strong trend was seen for increased adduct levels in the lung DNA of smokers and a weak association for the bladder DNA of smokers compared to non-smokers. Aromatic adducts were also detected in other tissues.     

Cell type-specific expression of beta-carotene 9',10'-monooxygenase in human tissues.

The symmetrically cleaving beta-carotene 15,15'-monooxygenase (BCO1) catalyzes the first step in the conversion of provitamin A carotenoids to vitamin A in the mucosa of the small intestine. This enzyme is also expressed in epithelia in a variety of extraintestinal tissues. The newly discovered beta-carotene 9',10'-monooxygenase (BCO2) catalyzes asymmetric cleavage of carotenoids. To gain some insight into the physiological role of BCO2, we determined the expression pattern of BCO2 mRNA and protein in human tissues. By immunohistochemical analysis it was revealed that BCO2 was detected in cell types that are known to express BCO1, such as epithelial cells in the mucosa of small intestine and stomach, parenchymal cells in liver, Leydig and Sertoli cells in testis, kidney tubules, adrenal gland, exocrine pancreas, and retinal pigment epithelium and ciliary body pigment epithelia in the eye. BCO2 was uniquely detected in cardiac and skeletal muscle cells, prostate and endometrial connective tissue, and endocrine pancreas. The finding that the BCO2 enzyme was expressed in some tissues and cell types that are not sensitive to vitamin A deficiency and where no BCO1 has been detected suggests that BCO2 may also be involved in biological processes other than vitamin A synthesis.     

Quantitative analysis of volatile halothane metabolites in biological tissues by gas chromatography

A simple and sensitive gas chromatographic method for the determination of 2-chloro-1, 1-difluoroethylene (CDE) and 2-chloro-1,1,1-trifluoroethane (CTE), two highly volatile metabolites of halothane, in blood, liver and isolated hepatic microscomes is described. The entire head-space in equilibrium with a known volume or weight of the sample is injected into the gas chromatograph equipped with a flame ionization detector. Quantification is accomplished with standards prepared by fortifying blank samples with known concentrations of CDE and CTE which are treated under the same conditions as the samples. Detection limits for CDE and CTE were 2 pmole/ml in blood and 10 pmole/g in liver and the mean relative standard deviations are no greater than ± 6% except for CTE in hepatic microsomes (± 9%). A preliminary study of blood CDE and CTE levels in humans anesthetized with halothane is reported.     

The dynamics of chromatin carcinogen interactions in the human cell.

Human lung epithelioid cells were treated with Benzo (a) pyrene diol epoxide (anti) in order to establish the binding and removal of covalent adducts in chromosomal components. Isolating two different classes of mononucleosomes, it was found that their DNA contained different concentrations of B(a)PDE-DNA adducts, while in both these mononucleosomal preparations only histones H2A and H3 contained detectable amounts of the carcinogen. Further analysis showed that in the intact human cell the carcinogen-DNA adduct distribution is constantly changing as a function of differential accessibility and repair. These results emphasize the dynamics of chromatin-carcinogen modifications.     

Immunohistochemical analysis of steroid sulfatase in human tissues

Steroid sulfatase (EC 3.1.6.2) is an enzyme that removes the sulfate group from 3β-hydroxysteroid sulfates. This enzyme is best known for its role in estrogen production via the fetal adrenal–placental pathway during pregnancy; however, it also has important functions in other physiological and pathological steroid pathways. The objective of this study was to examine the distribution of steroid sulfatase in normal human tissues and in breast cancers using immunohistochemistry, employing a newly developed steroid sulfatase antibody. A rabbit polyclonal antiserum was generated against a peptide representing a conserved region of the steroid sulfatase protein. In Western blotting experiments using human placental microsomes, this antiserum crossreacted with a 65 kDa protein, the reported size of steroid sulfatase. The antiserum also crossreacted with single protein bands in Western blots of microsomes from two human breast cancer cell lines (MDA-MB-231 and MCF-7) and from rat liver; however, there were some size differences in the immunoreactive bands among tissues. The steroid sulfatase antibody was used in immunohistochemical analyses of individual human tissue slides as well as a human tissue microarray. For single tissues, human placenta and liver showed strong positive staining against the steroid sulfatase antibody. ER+/PR+ breast cancers also showed relatively strong levels of steroid sulfatase immunoreactivity. Normal human breast showed moderate levels of steroid sulfatase immunoreactivity, while ER−/PR− breast cancer showed weak immunoreactivity. This confirms previous reports that steroid sulfatase is higher in hormone-dependent breast cancers. For the tissue microarray, most tissues showed some detectable level of steroid sulfatase immunoreactivity, but there were considerable differences among tissues, with skin, liver and lymph nodes having the highest immunoreactivity and brain tissues having the lowest. These data reveal the utility of immunohistochemistry in evaluation of steroid sulfatase activity among tissues. The newly developed antibody should be useful in studies of both humans and rats.     

Direct analysis and stability of methylated trivalent arsenic metabolites in cells and tissues

Chronic ingestion of water containing inorganic arsenic (iAs) has been linked to a variety of adverse health effects, including cancer, hypertension and diabetes. Current evidence suggests that the toxic methylated trivalent metabolites of iAs, methylarsonous acid (MAs(III)) and dimethylarsinous acid (DMAs(III)) play a key role in the etiology of these diseases. Both MAs(III) and DMAs(III) have been detected in urine of subjects exposed to iAs. However, the rapid oxidation of DMAs(III) and, to a lesser extent, MAs(III) in oxygen-rich environments leads to difficulties in the analysis of these metabolites in samples of urine collected in population studies. Results of our previous work indicate that MAs(III) and DMAs(III) are relatively stable in a reducing cellular environment and can be quantified in cells and tissues. In the present study, we used the oxidation state-specific hydride generation-cryotrapping-atomic absorption spectroscopy (HG-CT-AAS) to examine the presence and stability of these trivalent metabolites in the liver of mice and in UROtsa/F35 cells exposed to iAs. Tri- and pentavalent metabolites of iAs were analyzed directly (without chemical extraction or digestion). Liver homogenates prepared in cold deionized water and cell culture medium and lysates were stored at either 0 °C or -80 °C for up to 22 days. Both MAs(III) and DMAs(III) were stable in homogenates stored at -80 °C. In contrast, DMAs(III) in homogenates stored at 0 °C began to oxidize to its pentavalent counterpart after 1 day; MAs(III) remained stable for at least 3 weeks under these conditions. MAs(III) and DMAs(III) generated in UROtsa/F35 cultures were stable for 3 weeks when culture media and cell lysates were stored at -80 °C. These results suggest that samples of cells and tissues represent suitable material for the quantitative, oxidation state-specific analysis of As in laboratory and population studies examining the metabolism or toxic effects of this metalloid.     

Lysine-ketoglutarate reductase in human tissues.

Lysine-ketoglutarate reductase (saccharopine dehydrogenase (NADP+, lysine-forming) EC 1.5.1.8) from human liver has been partially purified and characterized. A spectrophotometric assay is described. The Michaelis constants have been determined for lysine (1.5-10-3 M), alpha-ketoglutarate (1-10-3 M) and NADPH (8-10-5 M). The pH optimum is 7.8. The enzyme is product inhibited. The specificity of the enzyme, response to inhibitors, pH and thermal stability are reported. Lysine-ketoglutarate reductase is present in high concentration in liver and heart, to a lesser degree in kidney and skin and in trace amounts in several other tissues. Saccharopine dehydrogenase (saccharopine dehydrogenase (NAD+, L-glutamate-forming) EC 1.5.1.9) was demonstrable only in liver and kidney. Lysine-ketoglutarate reductase reacts effectively with delta-hydroxylysine.     

Global analysis of G-protein-coupled receptor signaling in human tissues

The G-protein-coupled receptor (GPCR) family mediates a host of cell–cell communications upon activation by diverse ligands. Numerous GPCRs have been shown to display anatomically selective patterns of gene expression, however, our understanding of the complexity of GPCR signaling within human tissues remains unclear. In an effort to characterize global patterns of GPCR signaling in the human body, microarray analysis was performed on a large panel of tissues to monitor the gene expression levels of the receptors as well as related signaling and regulatory molecules. Analysis of the data revealed complex signaling networks in many tissue types, with tissue-specific patterns of gene expression observed for the majority of the receptors and a number of components and regulators of GPCR signaling.     

Immunocytochemical analysis of apoprotein E distribution in human tissues

Localization of apoprotein E (apo E) has been studied in different human tissues. For this aim the immunoperoxidase method and peroxidase-antiperoxidase complex, polyclonal and monoclonal antibodies, to apo E have been used. In every human tissue analysed apo E-containing cells have been revealed. To the latter the following cell types belong: hepatocytes and hepatic sinusoidal cells, macrophages of the spleen, lymph nodes and pulmonary tissues, glial cells and cells in all layers of the adrenals, skin keratinocytes, cells of the glomerular capsule and convoluted tubules of the kidney, spermatocytes and smooth muscle cells of the testis. Besides, apo E is revealed in endothelium of some vessels. As demonstrate the results obtained, apo E is found practically in all human tissues. A suggestion is made that besides its participation in reverse cholesterol transport, this protein performs a number of additional functions, such as regulation of local hormonal homeostasis of an organ.     

Activation of extracellular signal-regulated kinase in lung tissues of mice treated with carcinogen

The activation of extracellular signal-regulated kinase (ERK) in lung tissues of mice, as determined by the appearance of phosphorylated form, was observed on day 30 after urethane injection, and the activation also occurred in urethane-induced lung tumors. Immunohistochemical analysis using anti-phosphorylated ERK antibody indicated that the active form of ERK localized in alveolar epithelial cells. Furthermore, we confirmed by immunoprecipitation and immunoblot analysis that other essential components of the ERK cascade, that is, Ras, Raf and MEK (known as ERK kinase) were activated. These results indicate that the activation of the ERK signal in alveolar epithelial cells at the early stage of urethane-induced lung carcinogenesis is an important factor to develop lung tumors.