Protein gene product 9.5 expression in the human fetal cochlea.

The distribution of protein gene product (PGP) 9.5 was analyzed in the human fetal cochlea using the indirect immunofluorescence method. In the 12- and 14-week-old human fetuses, the cells of the greater epithelial ridge and the lesser epithelial ridge were overall labelled with PGP 9.5, while the stria vascularis and the Reissner's membrane did not exhibit any staining. Spiral ganglion cells and cochlear nerve fibers were labelled with PGP 9.5 and PGP 9.5-positive nerve fibers made contact with the basement membrane of the Corti primordium in the 12-week-old human fetus. These results suggest that PGP 9.5 might be used as a histological marker of maturation and innervation in the human cochlea.     

Protein phosphatase activity is required for light-inducible gene expression in maize.

Chlorophyll accumulation and photosynthetic gene activation are two hallmarks of greening process in etiolated maize leaves in response to light signals. However, very little is known about the relevant signal transduction pathways mediating these essential processes that lead to photosynthetic competence. It is shown here that a potent and specific protein phosphatase 1 (PP1) and PP2A inhibitor, okadaic acid, efficiently blocks chlorophyll accumulation induced by light in etiolated maize leaves. In addition, the light-inducible expression of two photosynthetic fusion genes can be specifically suppressed by the structurally unrelated PP1 and PP2A inhibitors, okadaic acid and calyculin A, using a sensitive and physiological maize protoplast transient assay. The specificity and effective concentration of the inhibitors in vivo and in vitro strongly suggest that PP1 is required for transmitting light signals. Intriguingly, several partial cDNAs encoding novel as well as conserved PP1 can be identified in maize leaves using the polymerase chain reaction. Studies of chimeric promoters indicate that PP1 activity is essential for the interaction of multiple regulatory elements. Although PP1 and PP2A have been implicated in the suppression of gene activity in yeast and animals, the present data indicate that PP1 appears to be essential for light-dependent gene activation in plants.     

Analysis of the ACP1 gene product: classification as an FMN phosphatase.

The relationship between the ACP1 gene product, an 18kDa acid phosphatase (E.C. 3.1.3.2) postulated to function as a protein tyrosyl phosphatase, and the cellular flavin mononucleotide (FMN) phosphatase has been examined in vitro and by using cultured Chinese hamster ovary (CHO) cells. Kinetic analysis indicated that at pH 6 the acid phosphatase utilized a variety of phosphate monoesters as substrates. While small molecules such as FMN were effectively utilized as substrates (kcat/Km = 7.3 x 10(3) s-1M-1), the tyrosyl phosphorylated form of the adipocyte lipid binding protein was a relatively poor substrate (kcat/Km = 1.7 x 10(-1) s-1M-1) suggesting a role for the phosphatase in flavin metabolism. Fractionation of CHO cell extracts revealed that 90% of the FMN phosphatase activity was soluble and that all of the soluble activity eluted from a Sephadex G-75 column with the acid phosphatase. All of the soluble FMN phosphatase activity was inhibited by immunospecific antibodies directed against the bovine heart ACP1 gene product. These results suggest that the ACP1 gene product functions cellularly not as a protein tyrosyl phosphatase but as a soluble FMN phosphatase.     

Influence of sustained mechanical stress on Egr-1 mRNA expression in cultured human endothelial cells

Restenosis after initially successful balloon angioplasty of coronary artery stenosis remains a major problem in clinical cardiology. Previous studies have identified pathogenetic factors which trigger cell proliferation and vascular remodeling ultimately leading to restenosis. Since there is evidence that endothelial cells adjacent to the angioplasty wound area synthesize factors which may initiate this process, we investigated the effects of mechanical stimulation on endothelial gene expression in vitro and focussed on the influence of sustained mechanical stress on expression of immediate early genes which have previously been shown to be induced in the vascular wall in vivo. Primary cultured human umbilical vein endothelial cells (HUVEC) and the human endothelial cell line EA.hy 926 were plated on collagen-coated silicone membranes and subjected to constant longitudinal stress of approximately 20% for 10 min to 6 h. Total RNA was isolated and the expression of the immediate early genes c-Fos and Egr-1 was studied by Northern blot analysis. We found a rapid upregulation c-Fos and Egr-1 mRNA which started at 10 min and reached its maxima at 30 min. HUVEC lost most of their stretch response after the third passage whereas immediate early gene expression was constantly in EA.hy 926 cells. Using specific inhibitors we investigated the contribution of several signal transduction pathways to stretch-activated Egr-1 mRNA expression. We found significant suppression of stretch-induced Egr-1 mRNA expression by protein kinase C (PKC) inhibition (p < 0.05) and by calcium depletion (EA.hy926, p < 0. 05; HUVEC, p = 0.063). No effect on stretch-activated Egr-1 mRNA expression was detected by inhibition of protein kinase A, blockade of stretch-activated cation channels or inhibition of microtubule synthesis. We conclude that sustained mechanical strain induces Egr-1 mRNA expression by PKC- and calcium-dependent mechanisms.     

Protein gene product 9.5 expression in the human fetal cochlea

Summary The distribution of protein gene product (PGP) 9.5 was analyzed in the human fetal cochlea using the indirect immunofluorescence method. In the 12- and 14-week-old human fetuses, the cells of the greater epithelial ridge and the lesser epithelial ridge were overall labelled with PGP 9.5, while the stria vascularis and the Reissner's membrane did not exhibit any staining. Spiral ganglion cells and cochlear nerve fibers were labelled with PGP 9.5 and PGP 9.5-positive nerve fibers made contact with the basement membrane of the Corti primordium in the 12-week-old human fetus. These results suggest that PGP 9.5 might be used as a histological marker of maturation and innervation in the human cochlea.     

Protein tyrosine phosphatase 1B inhibitors isolated from Artemisia roxburghiana

Artemisia roxburghiana is used in traditional medicine for treating various diseases including diabetes. The present study was designed to evaluate the antidiabetic potential of active constituents by using protein tyrosine phosphatase 1B (PTP1B) as a validated target for management of diabetes. Various compounds were isolated as active principles from the crude methanolic extract of aerial parts of A. roxburghiana. All compounds were screened for PTP1B inhibitory activity. Molecular docking simulations were performed to investigate the mechanism behind PTP1B inhibition of the isolated compound and positive control, ursolic acid. Betulinic acid, betulin and taraxeryl acetate were the active PTP1B principles with IC₅₀ values 3.49?±?0.02, 4.17?±?0.03 and 87.52?±?0.03?µM, respectively. Molecular docking studies showed significant molecular interactions of the triterpene inhibitors with Gly220, Cys215, Gly218 and Asp48 inside the active site of PTP1B. The antidiabetic activity of A. roxburghiana could be attributed due to PTP1B inhibition by its triterpene constituents, betulin, betulinic acid and taraxeryl acetate. Computational insights of this study revealed that the C-3 and C-17 positions of the compounds needs extensive optimization for the development of new lead compounds.     

Protein tyrosine phosphatase 1B inhibitors from Morus root bark

An organic layer prepared from the Chinese crude drug 'Sang-Bai-Pi' (Morus root bark) was studied in order to identify the inhibitory compounds for protein tyrosine phosphatase 1B (PTP1B). Bioassay-guided fractionation resulted in the isolation of sanggenon C (1), sanggenon G (2), mulberrofuran C (3) and kuwanon L (4) as PTP1B inhibitors, along with moracin O (5) and moracin P (6). Compounds 1-4 inhibited PTP1B with IC(50) values ranging from 1.6+/-0.3 microM to 16.9+/-1.1 microM.     

Protein tyrosine phosphatase 1B inhibitors isolated from Morus bombycis

Bioassay-guided fractionation of the chloroform-soluble fraction of Morus bombycis, using an in vitro PTP1B inhibitory assay led to the identification of three 2-arylbenzofurans, albafuran A (1), mulberrofuran W (2) and mulberrofuran D (6), along with three chalcone-derived Diels–Alder products, kuwanon J (3), kuwanon R (4), and kuwanon V (5). Compounds 1–6 showed remarkable inhibitory activity against PTP1B with IC₅₀ values ranging from 2.7 to 13.8 μM. Inhibition kinetics were analyzed by Lineweaver–Burk plots, which suggested that compounds 1–6 inhibited PTP1B in a mixed-type manner. The present results indicate that the respective lipophilic and hydroxyl groups of 2-arylbenzofurans and chalcone-derived Diels–Alder products play an important role in inhibition of PTP1B.     

Sequence and expression of the Escherichia coli K1 neuC gene product.

The nucleotide sequence of the neuC gene of the Escherichia coli K1 capsule gene cluster encodes a protein with a predicted molecular weight of 44,210 containing 391 amino acids. A chimeric protein with beta-galactosidase fused to the carboxy terminus of the neuC gene product (P7) was constructed and purified. Its amino-terminal sequence confirmed the prediction from the nucleotide sequence that the neuC gene overlaps the distal end of the neuA gene by a single base pair. Both the neuA and neuC genes are coexpressed under the control of a single upstream T7 or tac promoter, suggesting that neuA and neuC are part of an operon.     

Identification and characterization of the Egr-1 gene product, a DNA-binding zinc finger protein induced by differentiation and growth signals.

Egr-1 is an immediate-early response gene induced by diverse signals that initiate growth and differentiation. Its cDNA sequence predicts a protein with zinc fingers. We have generated an antiserum to the Egr-1 gene product and identified it as an 80-kilodalton short-lived protein in serum-stimulated mouse fibroblasts. The rat Egr-1 product has also been identified in nerve growth factor-induced PC12 cells. In addition, we show by cell fractionation and immunocytochemistry that the Egr-1 protein is located in the nucleus. We also demonstrate that it is phosphorylated. In vitro-generated Egr-1 protein binds with high affinity to the sequence CGCCCCCGC in a zinc-dependent manner.     

The immediate-early gene product Egr-1 regulates the human interleukin-2 receptor beta-chain promoter through noncanonical Egr and Sp1 binding sites.

The interleukin-2 IL-2 receptor beta-chain (IL-2Rbeta) is an essential component of the receptors for IL-2 and IL-15. Although IL-2Rbeta is constitutively expressed by lymphocytes, its expression can be further induced by a number of stimuli, including phorbol 12-myristate 13-acetate (PMA). We have now characterized factors that bind to an enhancer region located between nucleotides -170 and -139 of the human IL-2Rbeta promoter. Both Sp1 and Sp3 bound to the 5' portion of this region, whereas a PMA-inducible factor (PIF) mainly bound to its 3' portion and bound to the Sp binding motifs as well. In Jurkat T cells, induction of PIF DNA binding activity was rapidly induced, required de novo protein synthesis, and was sustained at a high level for at least 23 h. Interestingly, PIF was constitutively activated in human T-cell leukemia virus type 1-transformed MT-2 cells. In this paper, we demonstrate that PIF is Egr-1 based on its recognition by anti-Egr-1 antisera in gel mobility shift assays, even though the IL-2Rbeta DNA binding motif differed substantially from the canonical Egr-1 binding site. In addition, Egr-1 bound to the Sp binding site. In Jurkat cells, both sites were required for maximal IL-2Rbeta promoter activity, and in HeLaS3 cells, transfection of Egr-1 could drive activity of a reporter construct containing both sites. Moreover, Sp1 and Egr-1 could form a complex with kinetics that correlated with the production of Egr-1 in Jurkat cells upon PMA stimulation. Thus, Sp1 and Egr-1 physically and functionally cooperate to mediate maximal IL-2Rbeta promoter activity.     

Cloning and expression of the cry1Ea4 gene of Bacillus thuringiensis and the comparative toxicity of its gene product

A new cry gene (cry1Ea4) was cloned and sequenced from a Bacillus thuringiensis isolate native to Mexico (LBIT-147). The gene coded for a 133kDa protoxin which had greater than 99% homology with the holotype Cry1Ea1, as only four mismatches were found between the two amino acid sequences. When the Cry1Ea4 toxin was expressed in a crystal-negative strain of B. thuringiensis, bipyramidal crystals were produced. Purified crystals from this recombinant strain and from the holotype (Cry1Ea1) were bioassayed against first instar larvae of the tobacco hornworm. Statistically different mean LC₅₀ values indicated that Cry1Ea4 was more toxic than its holotype. This increase in toxicity may be attributed to the three amino acids which differ from the holotype sequence in the toxic fragment.     

Developmental expression of the murine Prl-1 protein tyrosine phosphatase gene

The expression of the murine Prl-1 protein tyrosine phosphatase gene was examined in normal embryos from E10.5 through E18.5. Prl-1 mRNA was detected in the brain, neural tube, and dorsal root ganglia, and in several non-neuronal tissues, including the skeletal system. Heart and skeletal muscle were consistently negative. At E13.5, Prl-1 was expressed in the condensing prechondrogenic cells of the vertebrae, whereas at E18.5, Prl-1 mRNA was localized to the hypertrophic chondrocytes. The dynamic expression of Prl-1 during cartilage differentiation may suggest a functional role in skeletal development.