Stephen L. Phillips

Nitric Oxide Inhibition of Transforming Growth Factor-β and Collagen Synthesis in Mesangial Cells

Culture of mesangial cells (MCs) in 5.6 vs. 30.0 mmol/1 glucose for 3 weeks induced a sustained increase in protein kinase C (PKC) activity, transforming growth factor (TGF)-β1 mRNA, bioactive TGF-β, and collagen synthesis. Nitric oxide (NO), generated exogenously by the NO donor S-nitroso-N-acetyl, D,L-penicillamine (SNAP) or endogenously after the exposure of MC to interleukin-1β (IL-1β), suppressed bioactive TGF-β in MCs cultured in 5.6 or 30.0 mmol/1 glucose and suppressed or abolished increases in TGF-β1 mRNA and collagen synthesis induced by high concentrations of glucose or phorbol 12,13-dibutyrate without altering values obtained with normal glucose concentrations. SNAP had a transient suppressive effect on PKC activity, which may explain at least in part some of the actions of SNAP. The selective inhibitor of PKC, bisindolylmaleimide (GFX), mimicked NO action. The ability of SNAP and IL-lβ to suppress TGF-β and collagen synthesis was not mediated by cGMP, since the cGMP analog, 8-Br-PET-cGMP, did not mimic NO action and an antagonist of cGMP-dependent protein kinase, Rp-8-pCPT-cGMPs, did not prevent the inhibitory actions of SNAP. N-ω-L-arginine methyl ester (NMMA) increased TGF-β in glomerular capillary endothelial cells (GCECs) and stimulated collagen synthesis by MC in a co-culture with GCECs. Captopril inhibited TGF-β and collagen synthesis and increased cGMP in co-cultures of GCECs and MCs. These effects of captopril were abolished by NMMA, implying mediation by NO. Thus, endogenous NO produced by GCECs may modulate TGF-β production by both GCECs and MCs and act to suppress matrix protein synthesis by MCs.

Assignment of the perlecan (heparan sulfate proteoglycan) gene to mouse Chromosome 4

Basement membranes are sheets of extracellular matrix produced by epithelial and endothelial cells which separate these cells from underlying connective tissue. The primary constituents of basement membranes are collagen type IV, laminin and entactin (reviewed by Timpl 1987) and perlecan, a heparan sulfate proteoglycan (Ledbetter et al. 1985). These components interact with each other to maintain the structural integrity of the matrix. Perlecan (Plc) has been shown to modify cell attachment (Clement et al. 1989) and is likely to be involved in the ionic filtration performed by the glomerular basement membrane (Kanwar et al. 1980). The core protein of perlecan has an approximate size of Mr = 400,000 (Ledbetter et al. 1985). Recently, the mouse perlecan cDNA has been partially cloned and sequenced (Noonan et al. 1989). The perlecan gene has been localized to human chromosome (chr) lp36. l by in situ hybridization using the mouse perlecan cDNA probe BPG-7 [a 2193 base pair (bp) mouse perlecan cDNA insert] (Wintle et al. 1990). In this report we demonstrate the presence of DNA sequence variants in the mouse perlecan (Plc) gene. In addition, we localized perlecan to mouse Chr 4 by segregation analysis of restr ic t ion fragment length variants (RFLVs) in recombinant inbred (RI) strains of mice. EcoR I and B a m H I restriction endonuclease digests of DNA from five progenitor mice strains (AKR/ J, DBA/2J, C57L/J, C57BL/6J and C3H/HeJ) were tested for RFLVs in the Ptc gene by Southern blot hybridization using the BPG-7 probe (Fig. 1). Two alleles were evident. In the DNA digests of AKR/J, DBA/2J and C3H/HeJ strains, two DNA fragments, approximately 7.4 kb (faint band) and 6.4 kb (intense band) in size, were detected. The alternative pattern seen in C57L/J and C57BL/6J consisted of 5.8 kb (intense band), 10.4 kb and 1.9 kb (faint bands) DNA fragments. A 2.2 kb DNA fragment was common to all

Lomofungin inhibition of allophanate hydrolase synthesis in Saccharomyces cerevisiae

SummaryThe RNA polymerase inhibitor, lomofungin has been used to determine the half life of specific synthetic capacities (invertase and α-glucosidase) as well as that for gross protein synthesis. In both cases the studies conclude that cognate messenger RNAs decay with a half life of approximately 20 minutes. This antibiotic has been used to determine the half life of allophanate hydrolase specific synthetic capacity. We find that it decays with a half life of about three minutes; a value that agrees with the decay rates of allophanate hydrolase synthetic capacity following removal of inducer. These observations argue that mRNA may be metabolized by two separate routes in Saccharomyces.

Gene mapping of mouse laminin A and B2 subunits using mouse-Chinese hamster somatic cell hybrids.

Laminin is a multichain extracellular matrix glycoprotein found primarily in basement membranes. The molecule is made up of three subunits, designated as A, B1, and B2. Using an 850-base cDNA probe for the mouse laminin A-chain and a 1000-base cDNA probe for the mouse laminin B2 chain, we have screened mouse—Chinese hamster somatic cell hybrids in order to assign the genes for each of these polypeptides to their respective mouse chromosome. We have determined that the mouse laminin B2-chain gene is located on chromosome 1 (confirming this assignment) and the laminin A-chain gene is located on chromosome 17.