Constitutive expression and localization of cytochrome P-450 1A1 in rat and human brain: presence of a splice variant form in human brain1

Cytochrome P-450 was purified to apparent homogeneity from the brain microsomes of phenobarbital-treated rats. The specific content of the purified P-450 was 12.7 nmol/mg of protein. NADPH-cytochrome P-450 reductase (reductase) was also purified to apparent homogeneity from brain microsomes. The specific content was 34.7 mumol of cytochrome c reduced/min per mg of protein. The reduced carbon monoxide spectrum of purified P-450 exhibited a peak at 450 nm. Both the P-450 and the reductase moved as single bands on SDS/PAGE. The molecular masses of the purified P-450 and the reductase were determined to be 53.3 and 72.0 kDa respectively. The purified brain P-450 cross-reacted with antibodies to rat liver P-450IIB1/IIB2 when examined by Western immunoblotting, but no immunological similarity was observed with rat liver P-450IA1/IA2 or P-450IIE1. Purified rat brain reductase cross-reacted with antibodies to rat liver reductase. Further, immunoblot experiments with untreated rat and human brain microsomes using antisera to the purified rat brain P-450 and reductase indicated that these forms of P-450 and NADPH-cytochrome P-450 reductase exist constitutively in rat and human brain. Purified rat brain P-450 was reconstituted with purified NADPH-cytochrome P-450 reductase, deoxycholate and dilauroyl glyceryl 3-phosphocholine. NADPH-dependent N-demethylation of aminopyrine and morphine was observed in the reconstituted system. The catalytic-centre activities were 80.25 and 38.2 nmol of formaldehyde formed/min per nmol of P-450 respectively. The reconstituted system had a comparatively lower catalytic-centre activity for 7-ethoxycoumarin O-de-ethylase (10.5 nmol of product formed/min per nmol of P-450).

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Differential regulation of cytochrome P-450 genes along rat intestinal crypt-villus axis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1992.263.2.g215 ◽

1992 ◽

Vol 263 (2) ◽

pp. G215-G223 ◽

Cited By ~ 2

Author(s):

P. G. Traber ◽

W. Wang ◽

L. Yu

Keyword(s):

Small Intestine ◽

Epithelial Cells ◽

Constitutive Expression ◽

Specific Pattern ◽

Monooxygenase System ◽

Intestinal Crypt ◽

Chemical Inducers ◽

Microsomal Monooxygenase ◽

Cytochrome P 450

Mammalian small intestine contains cytochrome P-450-dependent monooxygenase enzymes that are capable of metabolizing a wide variety of xenobiotics and activating procarcinogens to mutagenic compounds. The epithelial cells lining the small intestine are separated into a proliferating undifferentiated compartment located in crypts and a nonproliferating differentiated compartment located on villi. The constitutive expression and induction by xenobiotics of genes that encode components of the cytochrome P-450-dependent mono-oxygenase system along the rat intestinal crypt-villus axis were investigated using isolated epithelial cells and in situ hybridization. For each gene examined, hybridization analysis of RNA obtained from isolated epithelial cells correlated with findings on in situ RNA hybridization. Cytochrome P-450IA1 mRNA (CYP1A1), the major aromatic hydrocarbon-inducible P-450, and cytochrome P-450IIB1 mRNA (CYP2B1), the major phenobarbital-inducible P-450, were constitutively expressed in villus cells with no detectable mRNA present in crypts. Treatment with several chemical inducers resulted in a marked increase in CYP1A1 mRNA in both crypt and villus cells. In contrast, although CYP2B1 mRNA was inducible in villus cells, CYP2B1 mRNA was not detected in crypts after treatment with chemical inducers. NADPH cytochrome P-450 reductase, a necessary component for the activity of all P-450 enzymes, was expressed constitutively at low levels only in villus cells. Treatment with dexamethasone induced reductase mRNA in both crypt and villus cells. Taken together, these results demonstrate that there is a complex gene-specific pattern of expression of the microsomal monooxygenase system along the crypt-villus axis of rat small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

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Molecular Cloning and Expression of a Functionally Different Alternative Splice Variant of Prointerleukin-1α from the Rat Testis*

Endocrinology ◽

10.1210/endo.141.12.7824 ◽

2000 ◽

Vol 141 (12) ◽

pp. 4413-4418 ◽

Cited By ~ 17

Author(s):

Taranum Sultana ◽

Konstantin Svechnikov ◽

Günther Weber ◽

Olle Söder

Keyword(s):

Alternative Splice ◽

Splice Variant ◽

Messenger Rna ◽

Constitutive Expression ◽

Cloning And Expression ◽

Seminiferous Tubules ◽

Alternative Splice Variant ◽

Rat Testis ◽

Dose Dependent

Abstract We report here the characterization of an alternative splice variant of prointerleukin-1α (proIL-1α), constitutively expressed by the normal adult rat testis. In addition to the classical 32K proIL-1α (32proIL-1α) messenger RNA, the testis produced a shorter variant encoding a putative protein of 24K (24proIL-1α). In situ hybridization demonstrated constitutive expression of the splice transcript in the seminiferous tubules. This alternative complementary DNA lacked the fifth exon, harboring the calpain cleavage site essential for generation of mature 17K IL-1α. This was verified by calpain treatment, producing the expected cleavage products of recombinant 32proIL-1α, but not of 24proIL-1α. Similarly, expression in COS-7 cells demonstrated processing of 32proIL-1α to the mature 17K form and secretion, whereas 24proIL-1α remained unprocessed. Both 32proIL-1α and 24proIL-1α showed a dose-dependent stimulatory effect in a thymocyte proliferation assay, although at lower potency than mature 17K IL-1α. In contrast, when tested on hCG-stimulated Leydig cells in vitro, a dose-dependent inhibition of testosterone production was obtained with mature 17K IL-1α and at a lower potency with 32proIL-1α, whereas 24proIL-1α was inactive. In conclusion, the three IL-1 bioactive proteins described here contribute to IL-1 protein heterogeneity and may serve as constitutive paracrine mediators in the testis.

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Expression of a novel secreted splice variant of the receptor for advanced glycation end products (RAGE) in human brain astrocytes and peripheral blood mononuclear cells

Molecular Immunology ◽

10.1016/j.molimm.2003.11.027 ◽

2004 ◽

Vol 40 (16) ◽

pp. 1203-1211 ◽

Cited By ~ 96

Author(s):

I Park

Keyword(s):

Human Brain ◽

Advanced Glycation End Products ◽

Peripheral Blood Mononuclear Cells ◽

Splice Variant ◽

Mononuclear Cells ◽

Advanced Glycation ◽

Peripheral Blood Mononuclear ◽

Glycation End Products ◽

End Products ◽

Blood Mononuclear Cells

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The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00453.2003 ◽

2004 ◽

Vol 287 (3) ◽

pp. L584-L591 ◽

Cited By ~ 46

Author(s):

Steven G. Kelsen ◽

Mark O. Aksoy ◽

Yi Yang ◽

Syed Shahabuddin ◽

Judith Litvin ◽

...

Keyword(s):

Epithelial Cells ◽

Airway Inflammation ◽

Chemokine Receptor ◽

Splice Variant ◽

Constitutive Expression ◽

Airway Epithelial Cells ◽

Mrna Levels ◽

Airway Epithelial ◽

Human Airway ◽

Human Airway Epithelial Cells

Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were ∼35% of β-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% β-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was ∼78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.

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