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Biomedical subjects

G A Michelotti

Publications and source records attributed to G A Michelotti.

11 recordsLinked to original sources
PubMed2000

Alpha 1-adrenergic receptor regulation: basic science and clinical implications.

Adrenergic receptors (ARs) are members of the G-protein-coupled receptor family, which includes alpha 1ARs, alpha 2ARs, beta 1ARs, beta 2ARs, beta 3ARs, adenosine, muscarinic, angiotensin, endothelin receptors, and many others that are responsible for a large variety of physiologic effects through G-protein coupling. This review focuses on alpha 1ARs and their regulation at both the mRNA and protein levels. Currently, three alpha 1AR subtypes have been characterized both pharmacologically and at the gene level: alpha 1aAR, alpha 1bAR, and alpha 1dAR. These are expressed in a species- and tissue-dependent manner. Mutagenesis approaches have been extremely valuable in the identification of key residues that govern alpha 1AR ligand binding and signaling. These studies reveal that alpha 1ARs have evolved an exquisitely sensitive regulation of their activity in which any disruption of the native structure has profound effects on subsequent function and effector coupling. Significant advances have also been made in the elucidation of signaling pathway components, resulting in the identification of novel pathways that can lead to pathologic conditions. Specific topics include mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and G-protein-coupled receptor cross-talk pathways. Within this context, recent studies identifying underlying transcriptional mechanisms involved in the regulation of the alpha 1AR subtypes are also discussed. Finally, given the potentially important role of alpha 1ARs in the vasculature, as well as in the pathology of many diseases, such as myocardial hypertrophy and benign prostatic hyperplasia, the clinical relevance of alpha 1AR distribution, pharmacology, and therapeutic intervention is reviewed.

Cardiomyopathy, Hypertrophic↗
PubMed2000

Immortalization of a human prostate stromal cell line using a recombinant retroviral approach.

PURPOSE: We established an immortalized human prostate stromal cell line with retained markers of cell differentiation and alpha1-adrenergic receptor expression. MATERIALS AND METHODS: Primary human prostate stromal explants were infected with an amphotrophic retrovirus encoding the E6/E7 open reading frame of the human papillomavirus type 16. Immunohistochemistry was used to verify the expression of prostate stromal markers. alpha1-Adrenergic receptor expression was investigated using ribonuclease protection assays and radioligand binding. Cell proliferation was measured by the WST-1 assay and cell counting. RESULTS: Clonal isolates of individual prostate stromal cells were isolated and passed in selection media. E6 and E7 expression was verified using reverse transcriptase polymerase chain reaction in the selected cell line. The new prostate stromal cell line PS30 was established which maintains the expression of alpha-smooth muscle actin and expresses 22 fmol./mg. of protein of alpha 1-adrenergic receptors, approximately equal to native human prostate alpha 1-adrenergic receptor expression. However, at a subtype level alpha 1a-adrenergic receptor expression is down-regulated and not detectable by ribonuclease protection assays or radioligand binding, while alpha 1b and alpha 1d-adrenergic receptor expression is enhanced. From a physiological prospective PS30 cells do not form tumors in nude mice and stimulation with phenylephrine does not increase cell proliferation. CONCLUSIONS: We successfully established and characterized an in vitro human prostate stromal cell line. This cell line should facilitate studies designed to characterize the role of the adrenergic nervous system in the regulation of prostate growth.

Actins↗
PubMed1999

Characterization of alpha-adrenoceptor subtypes in the corpus cavernosum of patients undergoing sex change surgery.

PURPOSE: To characterize the subtypes of alpha1- and alpha2-adrenoceptors in the human corpus cavernosum from patients undergoing sex change surgery. MATERIALS AND METHODS: Saturation and competition radioligand binding studies were performed for characterization at the protein level. Alpha1-adrenoceptors were labeled with [3H]prazosin and [3H]tamsulosin, while alpha2-adrenoceptors were labeled with [3H]RX 821002. Alpha1-adrenoceptor subtype mRNA was additionally determined by reverse-transcriptase polymerase chain reaction and RNase protection assays. RESULTS: Human corpus cavernosum expressed approximately 32 and approximately 22 fmol./mg. protein alpha1- and alpha2-adrenoceptors, respectively. Competition studies with the alpha1A-selective antagonists 5-methylurapidil and (+)-niguldipine and the alpha1D-selective BMY 7378 revealed a mixed alpha1A/alpha1B-adrenoceptor population with no evidence for alpha1D-adrenoceptor protein. In contrast alpha1D-adrenoceptors were readily detected at the mRNA level. Competition binding studies with the alpha2A-selective oxymetazoline and the alpha2B-selective prazosin and ARC 239 revealed a homogeneous population of alpha2A-adrenoceptors. CONCLUSIONS: We conclude that human corpus cavernosum expresses predominantly alpha1A-, alpha1B- and alpha2A-adrenoceptor protein; additionally the alpha1D-adrenoceptor is present at the mRNA level.

Adrenergic alpha-Antagonists↗
PubMed1998

Expression and characterization of recombinant subunits of human complement component C8: further analysis of the function of C8 alpha and C8 gamma.

Human C8 is composed of three nonidentical subunits (C8 alpha, C8 beta, and C8 gamma) that are encoded in separate genes. In C8 isolated from serum, these are arranged as a disulfide-linked C8 alpha-gamma dimer that is noncovalently associated with C8 beta. In this study, a recombinant form of C8 alpha-gamma was expressed independently of C8 beta in insect cells and COS-7 cells and was shown to be equivalent to serum-derived C8 alpha-gamma with respect to its ability to combine with C8 beta and form functional C8. Also expressed separately were mutant (mut) forms of C8 alpha and C8 gamma in which the single interchain disulfide bond was eliminated. MutC8 alpha exhibited the ability to combine with C8 beta and express hemolytic activity, although at a lower level than human C8. Addition of purified mutC8 gamma increased this activity, presumably by binding to mutC8 alpha. A possible role for C8 gamma as a retinol binding protein was also investigated. Absorbance spectroscopy and fluorescence emission and quenching revealed no specific binding of retinol to mutC8 gamma. Together, these results indicate that 1) the biosynthesis and secretion of C8 alpha-gamma is not dependent on C8 beta, which is consistent with in vivo observations in C8 beta-deficient humans; 2) C8 alpha can be synthesized independently of C8 gamma; therefore, protection of C8 alpha from premature membrane interactions during biosynthetic processing is not a likely function of C8 gamma; 3) C8 gamma enhances but is not required for expression of C8 activity; and 4) C8 gamma does not bind retinol; therefore, it cannot function as a retinol transport protein.

Amino Acid Substitution↗
PubMed1998

Unrestraining genetic processes with a protein-DNA hinge.

Genetic processes require direct interactions between proteins bound at nonadjacent cis elements. Because duplex DNA is rigid, either the protein-protein interactions are strong enough to deform the double helix or some feature of the intervening DNA must encourage juxtaposition of separated sites. For example, bent DNA can bring together only certain precisely positioned cis elements with the same helical phase. Interposing a DNA segment that both bends and twists easily to create a universal joint would provide an even more general mechanism to promote the association of separated sites regardless of position. A cis element of the human c-myc gene, known to be melted in vivo, and its associated single-strand DNA binding protein were examined and found to comprise just such a protein-DNA hinge.

DNA, Single-Stranded↗
PubMed1996

Heterogeneous nuclear ribonucleoprotein K is a transcription factor.

The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Spl and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind. The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro. The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence. Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing single-stranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted. Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA-binding protein (TBP)-TBP-associated factor complex. Coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K. Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor.

Base Sequence↗
PubMed1996

Multiple single-stranded cis elements are associated with activated chromatin of the human c-myc gene in vivo.

Transcription activation and repression of eukaryotic genes are associated with conformational and topological changes of the DNA and chromatin, altering the spectrum of proteins associated with an active gene. Segments of the human c-myc gene possessing non-B structure in vivo located with enzymatic and chemical probes. Sites hypertensive to cleavage with single-strand-specific S1 nuclease or the single-strand-selective agent potassium permanganate included the major promoters P1 and P2 as well as the far upstream sequence element (FUSE) and CT elements, which bind, respectively, the single-strand-specific factors FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K in vitro. Active and inactive c-myc genes yielded different patterns of S1 nuclease and permanganate sensitivity, indicating alternative chromatin configurations of active and silent genes. The melting of specific cis elements of active c-myc genes in vivo suggested that transcriptionally associated torsional strain might assist strand separation and facilitate factor binding. Therefore, the interaction of FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K with supercoiled DNA was studied. Remarkably, both proteins recognize their respective elements torsionally strained but not as liner duplexes. Single-strand- or supercoil-dependent gene regulatory proteins may directly link alterations in DNA conformation and topology with changes in gene expression.

Base Sequence↗
PubMed1995

Genomic organization of human complement protein C8 alpha and further examination of its linkage to C8 beta.

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target membranes. It is composed of three nonidentical subunits (C8 alpha, C8 beta, C8 gamma) encoded by separate genes. C8 alpha and C8 beta are linked on chromosome 1p32, whereas C8 gamma is located on 9q22.3-q32. In this study, overlapping genomic clones were isolated and used to decipher the organization of the human C8 alpha gene. The gene contains at least 11 exons spanning approximately 70 kb of DNA. When compared to C6, C8 beta and C9, there is a remarkable similarity in genomic organization, consistent with amino acid sequence comparisons that suggest these proteins are ancestrally related. Regions of each protein that are structurally similar are encoded in exons of correspondingly similar lengths with highly conserved boundaries and phases. Availability of genomic sequence also facilitated a more detailed analysis of C8 alpha and C8 beta linkage. Based on analysis of genomic digests with cDNA probes, the loci were previously reported to be physically linked (< 2.5 kb) and in a 5' alpha-beta 3' orientation. In the present study, results obtained using exon-specific probes indicate the loci are not as closely linked as initially believed. Furthermore, they suggest that cDNA probes used earlier yielded misleading information because they encode exons that are distributed across large segments of genomic DNA.

Amino Acid Sequence↗
PubMed1987

Characterization of a slow folding reaction for the alpha subunit of tryptophan synthase.

The equilibria and kinetics of urea-induced unfolding and refolding of the alpha subunit of tryptophan synthase of E. coli have been examined for their dependences on viscosity, pH, and temperature in order to investigate the properties of one of the rate-limiting steps, domain association. A viscosity enhancer, 0.58 M sucrose, was found to slow unfolding and accelerate refolding. This apparently anomalous result was shown to be due to the stabilizing effect of sucrose on the folding reaction. After accounting for this stabilization effect by using linear free-energy plots, the unfolding and refolding kinetics were found to have a viscosity dependence. A decrease in pH was found to stabilize the domain association reaction by increasing the refolding rate and decreasing the unfolding rate. This effect was accounted for by protonation of a single residue with a pK value of 8.8 in the native state and 7.1 in the intermediate, in which the two domains are not yet associated. The activation energy of unfolding is 4.8 kcal/mol, close to the diffusion limit. The negative activation entropy of unfolding, -47 cal/deg-mol, which controls this reaction, may result from ordering of solvent about the newly exposed domain interface of the transition state. These results may provide information on the types of noncovalent interactions involved in domain association and improve the ability to interpret the folding of mutants with single amino-acid substitutions at the interface.

Escherichia coli↗