Transthoracic decompression of emphysematous bulla: a novel experience.

Emphysematous bullae are closed air containing spaces in lung parenchyma that may severely compromise lung function in patients of chronic obstructive pulmonary disease (COPD). We describe a simple and minimally invasive procedure to decompress a large emphysematous bullae in a patient with advanced COPD and high surgical risk. Transthoracic decompression of the bulla was accomplished under short-acting anaesthesia and muscle relaxation resulting in significant symptomatic, radiological and functional improvement.

Cloning of the cDNA and the genomic DNA of the mouse angiotensin II type 2 receptor.

Of the two major isoforms of the angiotensin II receptors, type 1 (AT1) and type 2 (AT2), little is known about the structure and features of AT2. We cloned a mouse AT2 cDNA from a mouse fetus cDNA library and an AT2 genomic DNA from a 129SV mouse genomic DNA library. The amino acid sequence of the mouse AT2 (363 residues) deduced from a mouse cDNA clone showed seven membrane-spanning domains. Amino acid identity of the mouse AT2 with mouse AT1 is 37%, and 98% with rat AT2. The genomic DNA (4.4 kb) contained three exons and two introns and the entire coding region was contained in the third exon.

Identification of a novel membrane protein, HP59, with therapeutic potential as a target of tumor angiogenesis.

CM101, a polysaccharide isolated from the culture medium of Group B streptococcus, a neonatal pathogen, targets pathological angiogenesis and inhibits tumor growth in mice and humans. CM101 also targets neonatal lung and adult sheep lung endothelial cells. A gene encoding a transmembrane protein that interacts with CM101 was isolated from a sheep lung endothelial cell cDNA library. The gene, termed sp55, encodes a 495-amino acid polypeptide. COS-7 cells transfected with a vector containing sp55 express the SP55 protein-bound CM101 in a concentration-dependent manner. Stably transfected CHO cells also bound CM101. The corresponding human gene, hp59, was isolated from a human fetal lung cDNA library and had a predicted identity to SP55 of 86% over 495 amino acids. HP59 protein was shown by immunohistochemistry to be present in the pathological tumor vasculature of the lung, breast, colon, and ovary, but not in the normal vasculature, suggesting that the protein may be critical to pathological angiogenesis. The hp59 gene and/or the HP59 protein was not expressed in a variety of normal tissues, but was significantly expressed in human fetal lung, consistent with the pathophysiology of Group B streptococcus infections in neonates. Mice immunized with HP59 and SP55 peptides showed significant attenuation of tumor growth. Immunization effectively inhibited both the tumor angiogenesis and vasculogenesis processes, as evidenced by lack of both HP59- and CD34-positive vessels. These results and the immunohistochemistry data suggest a therapeutic potential for the CM101 target protein HP59 both as a drug target and as a vaccine against pathoangiogenesis.

Angiotensin II receptors: cloning and expression.

To identify the mechanisms of action of isoforms angiotensin II receptors (AT1A, AT1B, and AT2) and to overcome the difficulties encountered in attempts to purify the receptors, we have expression-cloned their cDNAs from bovine and rat sources and isolated human cDNA and rat and human genomic DNA. The AT1A and AT1B cDNAs were found to encode respective receptor proteins with 359 amino acid residues, whereas, AT2 encodes a 363 amino acid residue receptor protein. Both AT1 and AT2 were found to conform with the seven transmembrane receptor structural motif, but showed only 32% amino acid residue identity to each other. The AT1 receptor was shown to be coupled to, at least, three different G proteins activating phospholipase C, inhibiting adenylyl cyclase and opening an L-type Ca(2+)-channel, whereas, AT2 was found to inhibit a phosphotyrosine phosphatase activity without affecting guanylyl cyclase by a pertussis-toxin-sensitive, presumably G-protein-mediated mechanism.

Cloning, expression and regulation of angiotensin II receptors.

Complementary DNAs for angiotensin II type 1 receptor isoforms AT1A and AT1B were cloned by expression cloning from bovine adrenal and rat vascular smooth muscles. Human AT1 receptor was also cloned. Seven transmembrane structures emerged. The AT1 type receptor interacted with more than one type of G-proteins. The ligand binding site of AT1 involving Arg167, Lys199, and Asp263 has been identified by site directed mutagenesis. The regulation of the receptors occur at many stages. The isoform, AT2, was also expression cloned from rat pheochromocytoma cells. Although its ligand binding is not affected by stable GTP analogs, it is a seven transmembrane domain receptor. It mediates the modulations of phosphotyrosine phosphatase by angiotensin II and AT2 specific CGP42112A. The modulation was abolished by pertussis toxin. Thus, AT2 belongs to a new class of angiotensin receptors with unique signalling and regulatory mechanisms. AT1 mediates cellular growth. Interestingly, AT2 expression is inversely related to the mitogenic activity of cells.

Peptide growth factors markedly decrease the ligand binding of angiotensin II type 2 receptor in rat cultured vascular smooth muscle cells.

Of two major isoforms of angiotensin II receptors, AT1 and AT2, biological roles of AT2 remain unclear. Using vascular smooth muscle cells, we investigated the regulation of expression of AT2 by growth factors in comparison with that of AT1. The cultured rat aorta smooth muscle cells had detectable AT2 binding sites, which were reduced significantly by treatment with platelet derived growth factor-BB. On the other hand, AT1 binding sites were increased under the same conditions. Other growth factors, such as epidermal growth factor and endothelin-1, also suppressed AT2 receptors to varying extents. A negative correlation between DNA synthesis promoted by these growth factors and the binding capacity of AT2 sites was observed. This study indicated that the expression of AT2 is downregulated in cultured vascular smooth muscle cells by growth factors in contrast to that of AT1, which was slightly upregulated.

Chdromosome aberrations in mice by the antifungal antibiotic, nystatin.

Nystatin, a fungicide of current medical use, was tested in mice for its effect on chromosomes of bone marrow cells. A significant increase of aberrations, mostly of chromatid type, was observed over a period of from 15 min to 15 days following the application of the drug. Our data indicate a non-random distribution of the breaks.

Cloning and expression of protein tyrosine phosphatase-like protein derived from a rat pheochromocytoma cell line.

A novel protein [designated protein tyrosine phosphatase-like protein (PTPLP)] which is distantly related to receptor-type protein tyrosine phosphatases (PTPases) was cloned from a rat pheochromocytoma cell line. The PTPLP was detected exclusively in the brain. Overexpression of the PTPLP decreased the basal PTPase activity of COS-7 cells for Raytide. These results suggest that PTPLP may function as a negative regulator of PTPases in neuronal tissues.

Molecular structure and function of angiotensin type 2 receptor.

More than two isoforms have been identified for angiotensin receptors based on their ligand selectivity. The objective of this study is to determine the molecular structure of angiotensin type 2 receptor (AT2), whose physiological functions are still an enigma despite extensive studies on its distribution in fetal tissues. We expression-cloned a cDNA of an affinity-purified AT2 from rat pheochromocytoma cells (PC12w). The AT2 cDNA clone comprises 2,868 nucleotides and encodes a 363 amino acid protein with seven putative transmembrane domains. The dissociation constant for its binding to 125I-CGP42112A, an AT2-specific ligand, was 0.11 +/- 0.01 nM. Its binding to 0.5 nM 125I-[Sar1,Ile8]-Ang II was not inhibited by Dup 753 but by PD123319 (IC50 = 1.7 +/- 0.2 nM). These binding features are characteristic of angiotensin type 2 receptor. The amino acid sequence analysis of the purified AT2 corroborated the amino terminus of the deduced primary structure of AT2. Angiotensin type 1 receptor (AT1) is the most closely related to AT2 but with only 32% amino acid sequence identity. Angiotensin II attenuated membrane-associated protein tyrosine phosphatase activity in the COS-7 cells stably expressing AT2 through a pertussis toxin-sensitive G protein. However, the physiological function of AT2 in the fetal kidney is still unresolved.

Polymorphism in chromosome 4.

Chromosomal analyses were performed on 302 individuals, using QFQ banding techniques to determine the occurrence of heteromorphism of chromosome 4. Two type of heteromorphism were observed, one showing an intensely fluorescent band in the centromere region and the other an intensely fluorescent band in the proximal area of the short arm. No one individual possessed both type of heteromorphism. A higher frequency of chromosome 4 heteromorphism was found in patients with schizophrenia, mental retardation, hyperactivity, developmental delay and speech impediments than in normal individuals.

Catenated dimers and knotted DNA structures: putative intermediates in the replication of T. cruzi kinetoplast minicircle DNA.

Upon centrifugation of gently lysed T. cruzi cells through a sucrose gradient, a free DNA fraction was shown to contain catenated dimers and knotted DNA structures. Southern hybridization and electron microscopic studies indicated that both of these structures derived from minicircle DNA, the major component of T. cruzi kinetoplast DNA. Partial denaturation analysis of a random population of catenated dimers suggests that these structures may have arisen from a late stage in the replication of minicircle DNA. On the other hand, the T. cruzi knotted minicircles we have isolated appear to be very similar to trefoil structures recently reported and implicated as replicative intermediates in two other trypanosoma species.

Protein tyrosine phosphatase inhibition by angiotensin II in rat pheochromocytoma cells through type 2 receptor, AT2.

Two major isoforms of angiotensin II receptors, AT1 and AT2, have been defined on the basis of their ligand selectivity. While AT1 is known to mediate typical biological actions of angiotensin II as a cardiovascular regulator, the biological function of AT2 has not yet been established. In the present study using a rat pheochromocytoma cell line, which expresses AT2 exclusively, we found that angiotensin II inhibits phosphotyrosine phosphatase activity in vivo as measured by the inhibition of hydrolysis of [32P]-phosphate from the 32P-labeled synthetic peptide substrate, Raytide. This phosphotyrosine phosphatase inhibition was completely reversed by pertussis toxin, which indicates a G-protein coupled mechanism. In SDS-polyacrylamide gel electrophoresis we found that the phosphotyrosine group of an 85 kDa protein was a substrate mainly preserved, presumably as a consequence of the plausible intracellular phosphotyrosine phosphatase inhibition by angiotensin II.

Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition.

There are two major isoforms of the angiotensin II receptor, type 1 (AT1) and type 2 (AT2). AT2 is distinguished from AT1 with respect to its ligand selectivity, its insensitivity to non-hydrolyzable GTP analogues, and its as yet unidentified biological functions. In the present study we have expression-cloned AT2 cDNA from a cDNA library of a rat pheochromocytoma cell line (PC12w). Rat AT2 cDNA encodes a 363-amino acid protein that has seven transmembrane domains. AT1 is the closest in homology to AT2 but with only a 32% identity of amino acid sequence. Stably expressed in COS-7 cells, the receptor showed selective binding to AT2-specific ligands PD123319 and CGP42112A but not to the AT1-specific ligand, losartan. Northern blot analysis revealed that the mRNA of rat AT2 was expressed not only in PC12w cells but also in the adrenal glands and in the inferior olive of the brain, both of which are known to contain AT2 type binding sites. The expressed AT2 receptor mediated angiotensin II-induced inhibition of protein tyrosine phosphatase, an action that was dependent on a pertussis toxin-sensitive G-protein-coupled mechanism in COS-7 cells. The AT2-specific ligand CGP42112A was an agonist rather than antagonist in the inhibition of phosphotyrosine phosphatase. AT2 did not cause a decrease in cGMP in PC12w or COS-7 cells expressing AT2 stably. These results indicate that the AT2 receptor is structurally and functionally different from AT1 and suggest novel functional roles of the renin-angiotensin system in cross-talk with phosphotyrosine signaling by modulating protein phosphotyrosine levels.

Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor.

Angiotensin II elicits different responses which affect cardiovascular, neuronal and electrolyte transport regulation. To understand the mechanisms responsible for its various actions, the receptor for angiotensin II has long been sought, but numerous attempts to purify the receptor have been unsuccessful owing to its instability and low concentration. We report here the expression cloning of a complementary DNA encoding a bovine angiotensin II receptor to overcome these difficulties. The receptor cDNA encodes a protein of 359 amino-acid residues with a transmembrane topology similar to that of other G protein-coupled receptors. COS-7 cells transfected with the cDNA expressed specific and high-affinity binding sites for angiotensin II, angiotensin II antagonist and a non-peptide specific antagonist for type-1 receptor. Dithiothreitol inhibited ligand binding. The concentration of intracellular Ca2+ and of inositol-1,4,5-trisphosphate increased in the transfected COS-7 cells in response to angiotensin II or angiotensin III, indicating that this receptor is the type-1 receptor for angiotensin II. Northern blot analysis revealed that the messenger RNA for this receptor is expressed in bovine adrenal medulla, cortex and kidney.

Rat angiotensin II receptor: cDNA sequence and regulation of the gene expression.

The nucleotide and amino acid sequences for rat type I angiotensin II receptor were deduced through molecular cloning and sequence analysis of its complementary DNAs. The rat angiotensin II receptor consists of 359 amino acid residues and has a sequence similar to G protein-coupled receptors. The expression of this receptor gene was detected in the adrenal, liver and kidney by Northern blotting. Sodium deprivation positively modulated the expression of the receptor gene in the adrenal. No detectable change was observed in the expression levels of this receptor gene between spontaneously hypertensive rats and Wistar-Kyoto rats in the tissues examined including the adrenal, brain, kidney and liver. Interestingly the expression of this receptor gene was developmentally regulated.

Cloning, expression and regulation of angiotensin II receptors.

Angiotensin II isoform 1 (AT1) receptor cDNAs were cloned by expression cloning from bovine adrenal and rat vascular smooth muscles. Human AT1 receptor was also cloned. Seven transmembrane structures emerged. A single type of receptor seems to interact with more than one type of G-protein. AT1 consists of subtypes AT1A and AT1B, and the regulation of the receptors occurs at many stages. The isoform AT2 was also expression cloned from rat pheochromocytoma cells. Although its ligand binding is not affected by GTP analogs, it is a seven transmembrane domain receptor. It mediates the inhibition of phosphotyrosine phosphatase by angiotensin II and AT2 specific CGP42112A; the inhibition was abolished by pertussis toxin. Thus, AT2 belongs to a new class of angiotensin receptors with unique signalling and regulatory mechanisms.

Angiotensin II receptors: cloning and regulation.

Angiotensin II subtype 1 (AT1) receptor cDNA was cloned by expression cloning from bovine adrenal cortical cells. Human AT1 receptor was also cloned. These receptors were found to have a seven transmembrane structure. The receptor seems to interact with more than one type of G-proteins. AT1 consists of subtypes. cDNA for AT1A was cloned from rat kidney and that for AT1B was cloned from rat adrenal by plaque hybridization. They have similar base sequences in the coding region but are different in non-coding regions. Their functional implication is not clear. The regulation of the receptors occur at many stages. Expression of mRNA is studied in cultured rat mesangial cells. It was down regulated by angiotensin II and cAMP. On the other hand in whole body experiments, chronic infusion of angiotensin II was shown to upregulate adrenal AT1, and bilateral nephrectomy or losartan (CAS 124750-99-8) administration reduced AT1 mRNA expression. In addition to AT1 and AT2 the presence of a new subtype AT3 has been shown.