17 beta-estradiol hydroxylation catalyzed by human cytochrome P450 1B1.

The 4-hydroxy metabolite of 17 beta-estradiol (E2) has been implicated in the carcinogenicity of this hormone. Previous studies showed that aryl hydrocarbon-receptor agonists induced a cytochrome P450 that catalyzed the 4-hydroxylation of E2. This activity was associated with human P450 1B1. To determine the relationship of the human P450 1B1 gene product and E2 4-hydroxylation, the protein was expressed in Saccharomyces cerevisiae. Microsomes from the transformed yeast catalyzed the 4- and 2-hydroxylation of E2 with Km values of 0.71 and 0.78 microM and turnover numbers of 1.39 and 0.27 nmol product min-1.nmol P450-1, respectively. Treatment of MCF-7 human breast cancer cells with the aryl hydrocarbon-receptor ligand indolo[3,2-b]carbazole resulted in a concentration-dependent increase in P450 1B1 and P450 1A1 mRNA levels, and caused increased rates of 2-, 4-, 6 alpha-, and 15 alpha-hydroxylation of E2. At an E2 concentration of 10 nM, the increased rates of 2- and 4-hydroxylation were approximately equal, emphasizing the significance of the low Km P450 1B1-component of E2 metabolism. These studies demonstrate that human P450 1B1 is a catalytically efficient E2 4-hydroxylase that is likely to participate in endocrine regulation and the toxicity of estrogens.

Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.

gamma-aminobutyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are constructed from four subunit classes with multiple members: alpha (1-6), beta (1-4), gamma (1-4), and delta (1). The contribution of subunit diversity in determining receptor subcellular targeting was examined in polarized Madin-Darby canine kidney (MDCK) cells. Significant detection of cell surface homomeric receptor expression by a combination of both immunological and electrophysiological methodologies was only found for the beta 3 subunit. Expression of alpha/beta binary combinations resulted in a nonpolarized distribution for alpha 1 beta 1 complexes, but specific basolateral targeting of both alpha 1 beta 2 and alpha 1 beta 3 complexes. The polarized distribution of these alpha/beta complexes was unaffected by the presence of the gamma 2S subunit. Interestingly, delivery of receptors containing the beta 3 subunit to the basolateral domain occurs via the apical surface. These results show that beta subunits can selectively target GABAA receptors to distinct cellular locations. Changes in the spatial and temporal expression of beta-subunit isoforms may therefore provide a mechanism for relocating GABAA receptor function between distinct neuronal domains. Given the critical role of these receptors in mediating synaptic inhibition, the contribution of different beta subunits in GABAA receptor function, may have implications in neuronal development and for receptor localization/clustering.

Cloning, expression, and distribution of a Ca(2+)-activated K+ channel beta-subunit from human brain.

We have cloned and expressed a Ca(2+)-activated K+ channel beta-subunit from human brain. The open reading frame encodes a 191-amino acid protein possessing significant homology to a previously described subunit cloned from bovine muscle. The gene for this subunit is located on chromosome 5 at band q34 (hslo-beta). There is no evidence for alternative RNA splicing of this gene product. hslo-beta mRNA is abundantly expressed in smooth muscle, but expression levels are low in most other tissues, including brain. Brain subregions in which beta-subunit mRNA expression is relatively high are the hippocampus and corpus callosum. The coexpression of hslo-beta mRNA together with hslo-alpha subunits in either Xenopus oocytes or stably transfected HEK 293 cells give rise to Ca(2+)-activated potassium currents with a much increased calcium and/or voltage sensitivity. These data indicate that the beta-subunit shows a tissue distribution different to that of the alpha-subunit, and in many tissues there may be no association of alpha-subunits with beta-subunits. These beta-subunits can play a functional role in the regulation of neuronal excitability by tuning the Ca2+ and/or the voltage dependence of alpha-subunits.

Involvement of Asn-293 in stereospecific agonist recognition and in activation of the beta 2-adrenergic receptor.

To investigate the molecular mechanism for stereospecific binding of agonists to beta 2-adrenergic receptors we used receptor models to identify potential binding sites for the beta-OH-group of the ligand, which defines the chiral center. Ser-165, located in transmembrane helix IV, and Asn-293, situated in the upper half of transmembrane helix VI, were identified as potential binding sites. Mutation of Ser-165 to Ala did not change the binding of either isoproterenol isomer as revealed after transient expression in human embryonic kidney (HEK)-293 cells. In contrast, a receptor mutant in which Asn-293 was replaced by Leu showed substantial loss of stereospecific isoproterenol binding. Adenylyl cyclase stimulation by this mutant after stable expression in CHO cells confirmed the substantial loss of stereospecificity for isoproterenol. In a series of agonists the loss of affinity in the Leu-293 mutant receptor was strongly correlated with the intrinsic activity of the compounds. Full agonists showed a 10-30-fold affinity loss, whereas partial agonists had almost the same affinity for both receptors. Stereospecific recognition of antagonists was unaltered in the Leu-293 mutant receptor. These data indicate a relationship between stereospecificity and intrinsic activity of agonists and suggest that Asn-293 is important for both properties of the agonist-receptor interaction.

Active heterodimers are formed from human DNA topoisomerase II alpha and II beta isoforms.

DNA topoisomerase II is a nuclear enzyme essential for chromosome dynamics and DNA metabolism. In mammalian cells, two genetically and biochemically distinct topoisomerase II forms exist, which are designated topoisomerase II alpha and topoisomerase II beta. In our studies of human topoisomerase II, we have found that a substantial fraction of the enzyme exists as alpha/beta heterodimers in HeLa cells. The ability to form heterodimers was verified when human topoisomerases II alpha and II beta were coexpressed in yeast and investigated in a dimerization assay. Analysis of purified heterodimers shows that these enzymes maintain topoisomerase II specific catalytic activities. The natural existence of an active heterodimeric subclass of topoisomerase II merits attention whenever topoisomerases II alpha and II beta function, localization, and cell cycle regulation are investigated.

Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

Antibody-induced engagement of beta 2 integrins on adherent human neutrophils triggers activation of p21ras through tyrosine phosphorylation of the protooncogene product Vav.

It is known that beta 2 integrins are crucial for leukocyte cell-cell and cell-matrix interactions, and accumulating evidence now suggests that integrins serve not only as a structural link but also as a signal-transducing unit that controls adhesion-induced changes in cell functions. In the present study, we plated human neutrophils on surface-bound anti-beta 2 (CD18) antibodies and found that the small GTP-binding protein p21ras is activated by beta 2 integrins. Pretreatment of the cells with genistein, a tyrosine kinase inhibitor, led to a complete block of p21ras activation, an effect that was not achieved with either U73122, which abolishes the beta 2 integrin-induced Ca2+ signal, or wortmannin, which totally inhibits the phosphatidylinositol 3-kinase activity. Western blot analysis revealed that antibody-induced engagement of beta 2 integrins causes tyrosine phosphorylation of several proteins in the cells. One of these tyrosine-phosphorylated proteins had an apparent molecular mass of 95 kDa and was identified as the protooncogene product Vav, a p21ras guanine nucleotide exchange factor that is specifically expressed in cells of hematopoietic lineage. A role for Vav in the activation of p21ras is supported by the observations that antibody-induced engagement of beta 2 integrins causes an association of Vav with p21ras and that the effect of genistein on p21ras activation coincided with its ability to inhibit both the tyrosine phosphorylation of Vav and the Vav-p21ras association. Taken together, these results indicate that antibody-induced engagement of beta 2 integrins on neutrophils triggers tyrosine phosphorylation of Vav and, possibly through its association, a downstream activation of p21ras.

Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L.

Proliferation of dispersed plant cells in culture is strictly dependent on cell density, and cells in a low-density culture can only grow in the presence of conditioned medium (CM). No known plant hormones have been able to substitute for CM. To quantify the mitogenic activity of CM, we examined conditions for the assay system using mechanically dispersed mesophyll cells of Asparagus officinalis L. and established a highly sensitive bioassay method. By use of this method, the mitogenic activity of CM prepared from asparagus cells was characterized: it was heat-stable, susceptible to pronase digestion, and resistant to glycosidase treatment. On the basis of these results, the mitogenic activity in CM was purified 10(7)-fold by column chromatography, and two factors named phytosulfokine-alpha and -beta (PSK-alpha and PSK-beta) were obtained. By amino acid sequence analysis and mass spectrometry, the structures of these two factors were determined to be sulfated pentapeptide (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH) and sulfated tetrapeptide (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-OH). PSK-alpha and PSK-beta were prepared by chemical synthesis and enzymatic sulfation. The synthetic peptides exhibited the same activity as the natural factors, confirming the structure for PSK-alpha and PSK-beta mentioned above. This is the first elucidation of the structure of a conditioned medium factor required for the growth of low-density plant cell cultures.

Isolation and characterization of mutant cell lines defective in transforming growth factor beta signaling.

To isolate and characterize effector molecules of the transforming growth factor beta (TGFbeta) signaling pathway we have used a genetic approach involving the generation of stable recessive mutants, defective in their TGFbeta signaling, which can subsequently be functionally complemented to clone the affected genes. We have generated a cell line derived from a hypoxanthine-guanine phosphoribosyltransferase negative (HPRT-) HT1080 clone that contains the selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) linked to a TGFbeta-responsive promoter. This cell line proliferates or dies in the appropriate selection medium in response to TGFbeta. We have isolated three distinct TGFbeta-unresponsive mutants following chemical mutagenesis. Somatic cell hybrids between pairs of individual TGFbeta-unresponsive clones reveal that each is in a distinct complementation group. Each mutant clone retains all three TGFbeta receptors yet fails to induce a TGFbeta-inducible luciferase reporter construct or TGFbeta-mediated plasminogen activator inhibitor-1 (PAI-1) expression. Two of the three have an attenuated TGFbeta-induced fibronectin response, whereas in the other mutant the fibronectin response is intact. These TGFbeta-unresponsive cells should allow selection and identification of signaling molecules through functional complementation.

Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.

Deletion of the mouse T-cell receptor beta gene enhancer blocks alphabeta T-cell development.

Intrathymic T-cell development requires temporally regulated rearrangement and expression of T-cell receptor (TCR) genes. To assess the role of the TCR beta gene transcriptional enhancer (Ebeta) in this process, mouse strains in which Ebeta is deleted were generated using homologous recombination techniques. We report that mice homozygous for the Ebeta deletion, whether a selectable marker gene is present or not, show a block in alphabeta T-cell development at the CD4-CD8- double-negative cell stage, whereas the number of gammadelta+ T cells is normal, few CD4+CD8+ double-positive thymocytes and no alphabeta+ T cells are produced. DNA-PCR and RNA-PCR analyses of thymic cells from homozygous mutants showed no evidence of TCR beta gene rearrangement although germ-line Vbeta transcripts were detected at a low level, in heterozygous T cells, the targeted allele is not rearranged. Thus, deletion of Ebeta totally prevents rearrangement, but not transcription, of the targeted beta locus. These data formally establish the critical role played by Ebeta in cis-activation of the TCR beta locus for V(D)J recombination during alphabeta T-cell development.

Amyloid beta-protein reduces acetylcholine synthesis in a cell line derived from cholinergic neurons of the basal forebrain.

The characteristic features of a brain with Alzheimer disease (AD) include the presence of neuritic plaques composed of amyloid beta-protein (Abeta) and reductions in the levels of cholinergic markers. Neurotoxic responses to Abeta have been reported in vivo and in vitro, suggesting that the cholinergic deficit in AD brain may be secondary to the degeneration of cholinergic neurons caused by Abeta. However, it remains to be determined if Abeta contributes to the cholinergic deficit in AD brain by nontoxic effects. We examined the effects of synthetic Abeta peptides on the cholinergic properties of a mouse cell line, SN56, derived from basal forebrain cholinergic neurons. Abeta 1-42 and Abeta 1-28 reduced the acetylcholine (AcCho) content of the cells in a concentration-dependent fashion, whereas Abeta 1-16 was inactive. Maximal reductions of 43% and 33% were observed after a 48-h treatment with 100 nM of Abeta 1-42 and 50 pM of Abeta 1-28, respectively. Neither Abeta 1-28 nor Abeta 1-42 at a concentration of 100 nM and a treatment period of 2 weeks was toxic to the cells. Treatment of the cells with Abeta 25-28 (48 h; 100 nM) significantly decreased AcCho levels, suggesting that the sequence GSNK (aa 25-28) is responsible for the AcCho-reducing effect of Abeta. The reductions in AcCho levels caused by Abeta 1-42 and Abeta 1-28 were accompanied by proportional decreases in choline acetyltransferase activity. In contrast, acetylcholinesterase activity was unaltered, indicating that Abeta specifically reduces the synthesis of AcCho in SN56 cells. The reductions in AcCho content caused by Abeta 1-42 could be prevented by a cotreatment with all-trans-retinoic acid (10 nM), a compound previously shown to increase choline acetyltransferase mRNA expression in SN56 cells. These results demonstrate a nontoxic, suppressive effect of Abeta on AcCho synthesis, an action that may contribute to the cholinergic deficit in AD brain.

Critical contribution of beta chain residue 57 in peptide binding ability of both HLA-DR and -DQ molecules.

Position 57 in the beta chain of HLA class II molecules maintains an Asp/non-Asp dimorphism that has been conserved through evolution and is implicated in susceptibility to some autoimmune diseases. The latter effect may be due to the influence of this residue on the ability of class II alleles to bind specific pathogenic peptides. We utilized highly homologous pairs of both DR and DQ alleles that varied at residue 57 to investigate the impact of this dimorphism on binding of model peptides. Using a direct binding assay of biotinylated peptides on whole cells expressing the desired alleles, we report several peptides that bind differentially to the allele pairs depending on the presence or absence of Asp at position 57. Peptides with negatively charged residues at anchor position 9 bind well to alleles not containing Asp at position 57 in the beta chain but cannot bind well to homologous Asp-positive alleles. By changing the peptides at the single residue predicted to interact with this position 57, we demonstrate a drastically altered or reversed pattern of binding. Ala analog peptides confirm these interactions and identify a limited set of interaction sites between the bound peptides and the class II molecules. Clarification of the impact of specific class II polymorphisms on generating unique allele-specific peptide binding "repertoires" will aid in our understanding of the development of specific immune responses and HLA-associated diseases.

Development of Valpha4+ NK T cells in the early stages of embryogenesis.

The majority of T lymphocytes start to develop at around day 15 of gestation (d15)-d17 in the thymus and comprise the peripheral repertoire characterized by the expression of polymorphic T-cell antigen receptors (TCRs). Contrary to these conventional T cells, a subset of T cells, called natural killer (NK) T cells (most of them expressing an invariant TCR encoded by the Valpha14Jalpha281 gene with a 1-nt N-region), preferentially differentiates extrathymically and dominates the peripheral T-cell population at a high frequency (5% in splenic T cells and 40% in bone marrow T cells). Here, we investigated the development of NK T cells and found that the invariant Valpha14+ TCR transcripts and the circular DNA created by Valpha14 and Jalpha281 gene rearrangements can be detected in the embryo body at d9.5 of gestation and in the yolk sac and the fetal liver at d11.5-d13.5 of gestation, but not in the thymus, whereas T cells with Valpha1+ TCR expression, a major population in the thymus, were not observed at these early stages of gestation. Fluorescence-activated cell sorter analysis also demonstrated that there exist CD3+ alpha beta+ T cells, almost all of which are Valpha14/Vbeta8+ NK+ T cells, during early embryogenesis. To our knowledge, this demonstrates for the first time that a T lymphocyte subset develops in extrathymic tissues during the early stages of embryogenesis.

Effect of deletion of 5'HS3 or 5'HS2 of the human beta-globin locus control region on the developmental regulation of globin gene expression in beta-globin locus yeast artificial chromosome transgenic mice.

To analyze the function of the 5' DNase I hypersensitive sites (HSs) of the locus control region (LCR) on beta-like globin gene expression, a 2.3-kb deletion of 5'HS3 or a 1.9-kb deletion of 5'HS2 was recombined into a beta-globin locus yeast artificial chromosome, and transgenic mice were produced. Deletion of 5'HS3 resulted in a significant decrease of epsilon-globin gene expression and an increase of gamma-globin gene expression in embryonic cells. Deletion of 5'HS2 resulted in only a small decrease in expression of epsilon-, gamma-, and beta-globin mRNA at all stages of development. Neither deletion affected the temporal pattern of globin gene switching. These results suggest that the LCR contains functionally redundant elements and that LCR complex formation does not require the presence of all DNase I hypersensitive sites. The phenotype of the 5'HS3 deletion suggests that individual HSs may influence the interaction of the LCR with specific globin gene promoters during the course of ontogeny.