Alternative splicing of human prostaglandin G/H synthase mRNA and evidence of differential regulation of the resulting transcripts by transforming growth factor beta 1, interleukin 1 beta, and tumor necrosis factor alpha.

Prostaglandin G/H synthase (PGG/HS) is the rate-limiting enzyme in the conversion of arachidonic acid to prostaglandins and thromboxanes. We screened a human lung fibroblast cDNA library with an ovine PGG/HS cDNA and isolated a 2.3-kilobase clone (HCO-T9). Sequence analysis of this clone showed that (a) it contained the entire translated region of PGG/HS and (b) it displayed an in-frame splicing of the last 111 base pairs encoded by exon 9, which resulted in the elimination of the N-glycosylation site at residue 409. Polymerase chain reaction amplification with specific oligonucleotides of reverse-transcribed mRNA from diverse human tissues and cultured cells yielded 400- and 300-base pair fragments that corresponded, respectively, to the intact and spliced transcripts. The expression of these two transcripts in cultured human lung fibroblasts was differentially regulated by serum, transforming growth factor beta 1, interleukin 1 beta, tumor necrosis factor alpha, and phorbol 12-myristate 13-acetate, as each of these conditions stimulated preferentially the expression of the unspliced transcripts. The elimination of one of the four N-glycosylation sites by the alternative splicing of exon 9 and the differential regulation of this process by relevant cytokines and growth factors may represent a mechanism for the regulation of PGG/HS enzymatic activity under physiological or pathological conditions

Powering AGNs with super-critical black holes

We propose a novel mechanism for powering the central engines of Active Galactic Nuclei through super-critical (type II) black hole collapse. In this picture, ~$10^3 M_\odot$ of material collapsing at relativistic speeds can trigger a gravitational shock, which can eject a large percentage of the collapsing matter at relativistic speeds, leaving behind a "light" black hole. In the presence of a poloidal magnetic field, the plasma collimates along two jets, and the associated electron synchrotron radiation can easily account for the observed radio luminosities, sizes and durations of AGN jets. For Lorentz factors of order 100 and magnetic fields of a few hundred $\mu G$, synchrotron electrons can shine for $10^6$ yrs, producing jets of sizes of order 100 kpc. This mechanism may also be relevant for Gamma Ray Bursts and, in the absence of magnetic field, supernova explosions.Comment: 4 pages, 1 figur

Detection and characterization of Sp1 binding activity in human chondrocytes and its alterations during chondrocyte dedifferentiation.

We have detected DNA binding activity for a synthetic oligonucleotide containing an Sp1 consensus sequence in nuclear extracts from human chondrocytes. Changes in the levels of Sp1 oligonucleotide binding activity were examined in nuclear extracts from freshly isolated human chondrocytes, from chondrocytes that had been cultured under conditions that allowed the maintenance of a chondrocyte-specific phenotype on plastic dishes coated with the hydrogel poly(2-hydroxyethyl methacrylate), and from chondrocytes induced to dedifferentiate into fibroblast-like cells by passage in monolayer culture on plastic substrata. It was observed that Sp1 binding was 2-3-fold greater in nuclear extracts from dedifferentiated chondrocytes than in nuclear extracts from either freshly isolated chondrocytes or from cells cultured in suspension. The Sp1 binding activity was specific, since it was competed by unlabeled Sp1 but not by AP1 or AP2. The addition of a polyclonal antibody against Sp1 to nuclear extracts from freshly isolated chondrocytes or to extracts isolated from chondrocytes cultured in monolayer decreased the binding of Sp1 by approximately 85%. However, when the same experiment was carried out with nuclear extracts prepared from cells cultured on poly(2-hydroxyethyl methacrylate)-coated plates, only a very slight inhibition of Sp1 binding was observed. When fragments of the COL2A1 promoter containing putative Sp1 binding sites amplified by polymerase chain reaction were examined, it was found that the amounts of DNA-protein complex formed with nuclear extracts from dedifferentiated chondrocytes were 2-3-fold greater than the amounts formed with nuclear extracts from freshly isolated chondrocytes or from cells cultured in suspension. Quantitation of DNA binding activity by titration experiments demonstrated that nuclear extracts from fibroblast-like cells contained approximately 2-fold greater Sp-1 specific binding activity than nuclear extracts from chondrocytes. The direct role of Sp1 in type II collagen gene transcription was demonstrated by co-transfection experiments of COL2A1 promoter-CAT constructs in Drosophila Schneider line L2 cells that lack Sp1 homologs. This is the first demonstration of Sp1 binding activity in human chondrocytes and of differences in Sp1 DNA binding activity between differentiated and dedifferentiated chondrocytes