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Query: UNIPROT:P06889 (Mol)
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Transforming growth factor-beta 1 (TGF-beta 1) has been shown to induce squamous differentiation of cultured airway epithelial cells. It has also been shown to increase expression of matrix proteins and integrin receptors in cell culture of these and other cells. However, it is unknown if TGF-beta 1 affects expression of genes encoding intercellular junctional proteins. Therefore, we have investigated the effect of TGF-beta 1 on the expression of proteins and mRNAs for desmoplakins (DPs) I and II, desmosomal plaque proteins. Fibronectin, known to be induced by TGF-beta 1 was used as a positive control and tubulin as a negative control. Twenty-four hours after TGF-beta 1 stimulation, DP I and II mRNA levels assessed by Northern blotting analysis had increased significantly (DP I mRNA, 1.8-fold, P less than 0.05; DP II mRNA, 2.4-fold, P less than 0.04), thereby indicating pretranslational regulation of DP expression. By comparison, mRNA for fibronectin increased 8.1-fold whereas mRNA for tubulin was unchanged. Immunofluorescence using the monoclonal anti-DP I and II antibodies revealed dramatic increased expression of punctate DP structures after exposure to TGF-beta 1. Immunoblot analyses with polyclonal anti-DP I antibodies showed increased levels of both DP I (250 kD) and DP II (215 kD), with the DP I increase being more pronounced (DP I, 2.5-fold; DP II, 1.4-fold at 48 h relative to controls), suggesting translational regulation by TGF-beta 1. This study therefore demonstrates the ability of TGF-beta 1 to alter cellular phenotype by altering expression of proteins involved in intercellular junctions.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1992 Apr
PMID:Transforming growth factor-beta stimulates the expression of desmosomal proteins in bronchial epithelial cells. 155 Jun 89

The Brevibacterium ammoniagenes fatty acid synthetase (FAS) gene was isolated from a series of overlapping clones by both immunological and plaque hybridization screening of two independent gene libraries. From the isolated DNA a contiguous segment of 10,549 bp was sequenced in both directions. The sequenced DNA contained a very long (9312 nucleotides) open reading frame coding for a protein of 3104 amino acids and with a molecular mass of 327,466 daltons. Based on characteristic sequence motifs known from other FAS systems, seven different FAS active centres were identified at distinct locations within the polypeptide chain. Only one component enzyme, the 3-hydroxydecanoyl beta, gamma-dehydratase, has not yet been localized definitively within the gene. Translation is presumed to start from a GUG triplet located 25 nucleotides downstream of the transcriptional initiation site. There is a canonical Shine-Dalgarno sequence just before this start codon. Comparison of the B. ammoniagenes FAS sequence with those of other known fatty acid synthetases revealed a particularly high degree of similarity to the products of the two yeast genes, FAS1 and FAS2 (30% identical and 46% identical plus closely related amino acids). This similarity extends over the entire length of the genes and involves not only the primary sequences of individual component enzymes but also their sequential order within the multifunctional proteins. These data, together with those on the structure of other fatty acid synthetases, are interpreted in terms of a contribution of both primary structure and subunit cooperation to a conserved topology of functional domains common to all type I FAS complexes.
Mol Gen Genet 1992 Mar
PMID:Molecular structure of the multifunctional fatty acid synthetase gene of Brevibacterium ammoniagenes: its sequence of catalytic domains is formally consistent with a head-to-tail fusion of the two yeast genes FAS1 and FAS2. 155 98

We used the cDNA of human retinal arrestin as a probe to screen a human thyroid cDNA library. We isolated and plaque-purified one clone (hTHY-ARRX). The nucleotide sequence of the 1.8 kb cDNA insert had an open reading frame of 1227 bp coding for a protein of 409 amino acids. Northern blot analysis revealed a single transcript of 1.7 kb in human thyroid cells. There is significant homology between amino acid sequences of human thyroid arrestin and human retinal arrestin (63%) and bovine beta-arrestin (74%), respectively. The hTHY-ARRX cDNA was stably transfected into Chinese hamster ovary cells already expressing a functional human thyrotropin (TSH) receptor. The cAMP response to TSH stimulation was unaltered in these cells, and homologous desensitization to TSH stimulation was not restored. It is not presently known whether hTHY-ARRX is human beta-adrenergic arrestin or a new member of the arrestin family.
Mol Cell Endocrinol 1992 Apr
PMID:Cloning of a member of the arrestin family from a human thyroid cDNA library. 158 86

Incorporation of the available data on rac in neutrophils, CDC42 in yeast, and rho in fibroblasts suggests a general model for the function of rho-like GTPase (Figure 1). Conversion of an inactive cytoplasmic rho-related p21GDP/GDI complex to active p21. GTP occurs by inhibition of GAP and/or stimulation of exchange factors in response to cell signals. p21.GTP is then able to interact with its target at the plasma membrane. This could result in a conformational change in the target, enabling it to bind cytosolic protein(s). Alternatively, p21.GTP could be actively involved in transporting cytosolic protein(s) to the target. A GAP protein, perhaps intrinsic to the complex, would stimulate GTP hydrolysis allowing p21.GDP to dissociate. Solubilization of p21GDP by interaction with GDI would complete a cycle. What about the nature of the final complex? The rac-regulated NADPH oxidase complex in neutrophils is currently the best understood and most amenable to further biochemical analysis. Two plasma-membrane bound subunits encode the catalytic function necessary for producing superoxide, but the two cytosolic proteins, p47 and p67, are essential for activity. Why the complexity? Production of superoxide is tightly coordinated with phagocytosis, a membrane process driven by rearrangement of cortical actin. This is not unrelated to the membrane ruffling and macropinocytosis that we observe in fibroblasts microinjected with p21rac. It is tempting to speculate, therefore, that in neutrophils rac is involved not only in promoting the assembly of the NADPH oxidase but also in the coordinate reorganization of cortical actin leading to phagocytosis. For CDC42 controlled bud assembly in yeast, the components of the plasma-membrane complex are not so clear. By analogy with rac in neutrophils, it seems likely that CDC42 is involved in promoting the assembly of cytosolic components at the bud site on the plasma membrane. These putative cytosolic proteins have not yet been identified, but BEM1 and ABP1 are two possible candidates. The biochemical basis for the stimulation of adhesion plaques and actin stress fibers by p21rho in fibroblasts is also unclear. However, components of the adhesion plaque such as vinculin and talin are known to be cytosolic when not complexed with integrin receptors, and rho could be involved in regulating their assembly into the adhesion plaque. Several things are still difficult to incorporate into this model. First the target for CDC42, the bud site, although not yet structurally defined requires the activity of another small GTPase, BUD1. Similarly, in activated neutrophils, the NADPH oxidase is found in a complex with rap1, the mammalian homologue of BUD1 (BoKoch et al., 1989). It seems likely, therefore, that the target is not simply a plasma-membrane protein but may be a complex of proteins whose formation is under the control of the rap1/BUD1 GTPase. The other black box in this model is the actin connection: activation of bud assembly by CDC42 is followed by actin polymerization, activation of NADPH oxidase in neutrophils occurs concomitantly with phagocytosis, a cortical actin-dependent process, and p21rho in fibroblasts couples the formation of adhesion plaques to actin stress fibers. One possible link between the GTPase-driven assembly of a plasma-membrane complex and actin polymerization could involve the SH3 domain. Interestingly, both p47 and p67 and yeast ABP1 and BEM1 have SH3 domain. If rho-like GTPases recognize plasma-membrane targets already associated with cortical actin, then this could promote an interaction with a subset of SH3-containing proteins. The result of this would be a GTPase-regulated aggregation of a group of proteins at a single site in the plasma membrane. It is not too difficult to imagine biological processes where such a spatial integration of different biochemical activities would be essential: coupling the assembly of bud components to the formation of actin fibers in yeast; or the activation of NADPH oxidase to phagocytosis in neutrophils; or the assembly of adhesion plaques and the formation of actin stress fibers in fibroblasts are just three examples that have emerged so far. In conclusion, although rho-like GTPases clearly have distinct roles in different mammalian cell types and in yeast, their underlying mechanism of action appears to be strikingly similar. Whether this will remain so when there are some biochemical data to back up these initial observations, time will tell.
Mol Biol Cell 1992 May
PMID:Ras-related GTPases and the cytoskeleton. 161 Nov 53

Neuritic plaque and cerebrovascular amyloid deposits have been detected in the aged monkey, dog, and polar bear and have rarely been found in aged rodents (Biochem. Biophy. Res. Commun., 12 (1984) 885-890; Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 4245-4249). To determine if the primary structure of the 42-43 residue amyloid peptide is conserved in species that accumulate plaques, the region of the amyloid precursor protein (APP) cDNA that encodes the peptide region was amplified by the polymerase chain reaction and sequenced. The deduced amino acid sequence was compared to those species where amyloid accumulation has not been detected. The DNA sequences of dog, polar bear, rabbit, cow, sheep, pig and guinea pig were compared and a phylogenetic tree was generated. We conclude that the amino acid sequence of dog and polar bear and other mammals which may form amyloid plaques is conserved and the species where amyloid has not been detected (mouse, rat) may be evolutionarily a distinct group. In addition, the predicted secondary structure of mouse and rat amyloid that differs from that of amyloid bearing species is its lack of propensity to form a beta sheeted structure. Thus, a cross-species examination of the amyloid peptide may suggest what is essential for amyloid deposition.
Brain Res Mol Brain Res 1991 Jul
PMID:Conservation of the sequence of the Alzheimer's disease amyloid peptide in dog, polar bear and five other mammals by cross-species polymerase chain reaction analysis. 165 57

alpha 1-Adrenergic receptor (alpha 1-AR) subtypes (alpha 1A and alpha 1B) play a critical role in vascular smooth muscle contraction and circulatory homeostasis. Transcripts for these guanine nucleotide-binding protein-coupled receptors are extremely low in abundance, however, and isolation of their cDNAs is difficult. We have developed a novel technique for identifying rare clones in a cDNA library, which has been used successfully to isolate a cDNA clone encoding an alpha 1D-AR. A 564-bp polymerase chain reaction product encoding a region between the third and sixth transmembrane domains of the alpha 1D-AR was first generated using rat brain mRNA as template and highly degenerate primers. The primers corresponded to those domains but contained mismatches to the alpha 1B-AR sequences. A 3-kb transcript was identified with this polymerase chain reaction probe, by Northern analysis of rat hippocampus. However, traditional plaque hybridization failed to identify a cDNA in a rat hippocampus lambda gt10 library. By solution-phase screening of virtually the entire library, a cDNA containing a 3-kb insert was identified, amplified, and purified. This insert encodes a 560-amino acid protein corresponding to the topology of guanine nucleotide-binding protein-coupled receptors. This receptor has approximately 71% amino acid identity, in the transmembrane regions, to the hamster and rat alpha 1B-ARs. Characterization of the receptor expressed in COS-7 cells, by ligand binding and photoaffinity labeling, revealed some of the characteristics of an alpha 1A-AR. However, unlike alpha 1A-ARs characterized previously in membrane preparations or in solubilized partially purified preparations, the expressed receptor could be extensively inactivated by chlorethylclonidine. In addition, it displays ligand-binding properties that are not consistent with an alpha 1A-AR. This indicates that the cDNA clone that we have isolated encodes a novel alpha 1-AR subtype, which we classify as the alpha 1D-AR.
Mol Pharmacol 1991 Dec
PMID:Solution-phase library screening for the identification of rare clones: isolation of an alpha 1D-adrenergic receptor cDNA. 166 38

The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross-linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.
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PMID:Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis. 168 40

Two tomato cDNA libraries were synthesized from poly(A)+ RNAs isolated from unwounded and wounded tomato stems. These cDNA libraries were packaged in lambda gt10 and screened by in situ plaque hybridization with a tomato extensin gene clone (pTom 5.10). Several cDNA clones were identified and isolated from both libraries in this manner and subjected to restriction enzyme digestion. Southern gel blot hybridization, RNA gel blot hybridization, and DNA sequence analyses. From these analyses, the various cDNA clones were found to fall into one of five distinct classes (classes I-V). Class I clones hybridized to a 4.0 kb mRNA which accumulated markedly after wounding and encoded an extensin characterized largely by Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-(Tyr)3-Lys repeats. Class II clones hybridized to a 2.6 kb mRNA which showed no accumulation following wounding and encoded an extensin containing Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-Thr-(Tyr)1-3-Ser repeats. Class III clones hybridized to a 0.6 kb mRNA which greatly accumulated in response to wounding and encoded a glycine-rich protein (GRP) with (Gly)2-6-Tyr-Pro and (Gly)2-6-Arg repeats. Class IV clones contained both class I and class III DNA sequences and consequently hybridized to both the 4.0 kb and the 0.6 kb wound-accumulating mRNAs; these clones encoded a portion of a GRP sequence on one DNA strand and encoded a portion of an extensin sequence on the other DNA strand. Class V clones hybridized to a 2.3 kb mRNA which decreased following wounding and encoded a GRP sequence characterized by (Gly)2-5-Arg repeats.
Plant Mol Biol 1991 Apr
PMID:Tomato extensin and extensin-like cDNAs: structure and expression in response to wounding. 171 16

The estradiol-dominated baboon oviduct synthesizes and secretes a glycoprotein (mol wt, 120,000) that binds to oocyte zona pellucida in vivo. This glycoprotein separates into two major isoelectric variants (pl 8.0 and 4.5) on two-dimensional electrophoretic gels. This study was undertaken to further characterize the steroid-controlled gene expression of this glycoprotein. A recombinant cDNA library was prepared to poly(A)+ RNA isolated from oviducts obtained from estradiol-treated baboons. The library was screened with a polyclonal antibody prepared against the acidic component (pl 4.5) of the glycoprotein. A single positive plaque was purified. Digestion of the recombinant plasmid with EcoRI revealed fragments of 230 and 1000 basepairs. The antibody used to screen the library was epitope selected against the fusion protein produced by the purified recombinant phage. The epitope-selected antibody, when incubated with Western blots of oviductal explant culture medium obtained from estradiol-treated baboons, recognized both the acidic and the basic variants of the glycoprotein. Northern blot hybridization with a 32P-labeled insert indicated that a single message of 2.8 kilobases was present in oviducts obtained from estradiol-treated baboons; no hybridization was detectable to RNA from oviducts obtained from progesterone-treated baboons. A mRNA of comparable size was also found in human, hamster, rabbit, and mouse oviduct tissue. In vitro translation of oviductal poly(A)+ RNA from an estradiol-treated baboon followed by immunoprecipitation with the polyclonal antibody against the acidic component indicated that the protein component of the oviductal glycoprotein had a mol wt of 66,000.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1991 Mar
PMID:Cloning of a recombinant complementary DNA to a baboon (Papio anubis) estradiol-dependent oviduct-specific glycoprotein. 171 45

The donor specific bacteriophage PRDI has been shown to mediate the genes transfer into Escherichia coli and Francisella tularensis cell under certain conditions. It is necessary for the process that the recipient cells inherit the plasmids determining absorbtion of bacteriophages on the cellular surface while the transferred genes are able to be expressed. The frequencies of the tet-gene transfer from the plasmid pSKFT5 into Escherichia coli and Francisella tularensis 15 cells inheriting the plasmid Sa are, correspondingly, 10(-6) and 10(-7) clones per bacteriophage plaque.
Mol Gen Mikrobiol Virusol 1991 Sep
PMID:[Transfer of bacteriophage PRD1 genes into modified plasmid Sa of Escherichia coli and Francisella tularensis cells]. 174 72


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