Incidence of multinucleated and polyploid aortic smooth muscle cells cultured from different age groups of spontaneously hypertensive rats.

Cell size and incidence of multinucleated, polyploid cells in cultured aortic smooth muscle cells from different age groups of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were compared. Smooth muscle cells from SHR were generally larger than those from WKY, and the percentage of multinucleated smooth muscle cells was always higher in SHR than WKY in the three age groups of rats studied (3-4, 10-12, and 28-30 weeks). In smooth muscle cells from the 3- to 4-week group, there was a positive correlation between cell diameter and the percentage of multinucleated smooth muscle cells. Microdensitometric measurements also showed that the incidence of polyploid smooth muscle cells was always higher in SHR than WKY in the three age groups. There was a positive correlation between DNA density and nuclear area measurements in all the age groups of SHR and WKY. We conclude that cultured aortic smooth muscle cells from different age groups of SHR and WKY contained heterogeneous populations of cells and that, under our culture conditions, the polyploidy of the smooth muscle cells found in vivo was maintained in the SHR and WKY.     

The role of the basement membrane in differential expression of keratin proteins in epithelial cells

Extracellular matrix is considered to play an important role in determining the phenotype of cells with which it interacts. Here we have investigated the possibility that extracellular matrix is involved in specifying the pattern of keratin expression in epithelial cells. For these studies, we have developed an explant system in which epithelial cells from one type of stratified epithelial tissue, namely conjunctiva, are maintained on an extracellular matrix substrate derived from a different tissue, namely cornea. These ocular tissues are ideal for such analyses since they express distinct sets of keratins. For example, bovine conjunctival epithelium processed for immunofluorescence is not recognized by antibody preparations against keratin K3 or K12. In contrast, K3 and K12 antibodies generate intense staining in bovine corneal epithelium. At the immunochemical level, conjunctival cells in situ appear to possess no K12 and only trace amounts of K3, whereas corneal epithelial cells in situ possess both K3 and K12. When conjunctival cells are maintained on a corneal substrate with an intact basement membrane for 10 days in vitro they begin to express keratin K12 as determined by immunofluorescence. On the other hand, conjunctival cells that are maintained on a corneal substrate lacking a basement membrane fail to show staining with K12 antibodies. Conjunctival cells begin to show intense staining using K3 antibodies within about 10 days of being placed in culture regardless of their substrate. These results indicate that basement membrane can play a positive role in determining cell-specific expression of certain keratins such as K12. However, other keratins such as K3 may be "unmasked" and/or their expression may be upregulated simply by placing conjunctival epithelial cells in culture. We speculate that in conjunctiva K3 expression is influenced by certain negative exogenous factors. We discuss the possible means of regulation of keratin expression in our model system.     

Structural and functional relationships of human DNA polymerases

A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during the cell cycle.     

32P-postlabeling of acrolein-deoxyguanosine adducts in DNA after nuclease P1 digestion.

In order to study the relationship between the level of acrolein-DNA adducts and their biological effects, sensitive methods are needed to quantitate DNA adducts. 32P-postlabeling is one such method that has been widely used and we have adapted the technique to detect acrolein-deoxyguanosine adducts. Adducts formed by the reaction of acrolein and deoxyguanosine-3'-monophosphate were isolated by HPLC. Based on their UV spectra and cochromatography with standards after dephosphorylation with acid phosphatase, these adducts were identified as the nucleotide equivalents of cyclic 1,N2-propanodeoxyguanosine adducts formed by acrolein that have been described by Chung et al. [15]. As nucleotides, the adducts were good substrates for polynucleotide kinase-mediated transfer of phosphate from ATP and were able to be detected by 32P-postlabeling. These adducts were resistant to the activity of nuclease P1 and dinucleoside monophosphates in the form d(G*pN) where G* is the acrolein-guanine adduct also resisted digestion by nuclease P1. Digestion of DNA by nuclease P1 and acid phosphatase resulted in the conversion of normal nucleotides to nucleosides and selective enrichment of the adducts as dinucleoside monophosphates. Using nuclease P1/acid phosphatase digestion, followed by 32P-postlabeling and TLC separation, levels of the two adducts in acrolein-treated DNA were found to be about 6185 and 19,222 nmol/mol.     

Purification, characterization, and molecular cloning of a 60-kDa phosphoprotein in rabbit skeletal sarcoplasmic reticulum which is an isoform of phosphoglucomutase.

A 60-kDa substrate of calmodulin-dependent protein kinase in rabbit "heavy" skeletal sarcoplasmic reticulum (SR) was characterized by purification and cDNA cloning. Purification was achieved by column chromatography using DEAE-Sephacel, heparin-agarose, and hydroxylapatite in 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS). Analyses of amino acid sequence and composition indicated that the CHAPS-soluble 60-kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding two isoforms of PGM were isolated from rabbit skeletal muscles. The translated amino acid sequences show that the isoforms, PGM1 and PGM2, differ in the N-terminal 77 amino acids and that PGM2 is identical to the 60-kDa protein in the SR. Northern blot analysis showed that the size of the mRNA encoding PGM2 is 2.4 kilobases. The PGM enzyme activity was markedly inhibited in SR membranes, while perturbation of the membranes with CHAPS or guanidine-HCl recovered the enzyme activity. KCl (0.15-1 M) led to a partial recovery of the enzyme activity suggesting that the charge interaction is not the primary force for PGM-SR interaction. PGM is localized in the heavy fraction of SR, where calsequestrin and Ca2+ release channel are enriched. Our results demonstrate that an isoform of PGM localized in junctional skeletal SR is the 60-kDa substrate of calmodulin-dependent protein kinase.     

Studies of the association and conformational properties of metal-free insulin in alkaline sodium chloride solutions by one- and two-dimensional 1H NMR.

One- and two-dimensional 1H NMR spectroscopy have been employed to probe the association and subsequent conformational changes of metal-free insulin in sodium chloride solution at pH 9 and 9.4. These studies establish that the proton resonances of His(B5) and His(B10) are useful signatures of aggregation and conformation. Changes in chemical shifts and areas of resonances due to the C2 protons of His(B10) and His(B5) and transfer of magnetization experiments served to identify the association as the assembly of tetramer from dimers under our experimental conditions (pH 9.4, [insulin] greater than 1 mM, [NaCl] = 0.1 M). Sodium chloride also alters the equilibrium distribution of species in favor of a tetrameric species. The association equilibrium constant was estimated from area measurements to be approximately 5 x 10(3) M-1 at pH 9.4, 26 +/- 0.1 degrees C, and 0.1 M sodium chloride. Under conditions of 0.1 M sodium chloride concentration, nuclear Overhauser effect experiments in the one- and two-dimensional modes revealed an operative nuclear Overhauser effect between the His(B5) C2 protons and the 2,6 ring protons of a Tyr residue provisionally assigned as Tyr(B16). We conclude that this interaction is a diagnostic signature of a conformational transition whereupon an extended chain from residues B1 to B9 (T-state) is transformed into an alpha-helix (R-state) thus bringing the rings of His(B5) and Tyr(B16) from adjacent subunits across the monomer-monomer interface into van der Waals contact. This conformational flexibility is an added consideration to the discussion of the relevant structure of insulin for receptor binding.     

A novel CoA-independent transacetylase produces the ethanolamine plasmalogen and acyl analogs of platelet-activating factor (PAF) with PAF as the acetate donor in HL-60 cells.

In this study, we demonstrate the presence of a unique membrane-associated transacetylase that transfers the acetate group from platelet-activating factor (PAF) to lysoplasmalogen (in the presence of EDTA and sodium acetate) with the formation of 1-alk-1-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alk-1-enylacetyl-GPE). The identity of alk-1-enylacetyl-GPE was confirmed by acid hydrolysis, phospholipases A2 or C treatment and derivatization by fluorodinitrobenzene. The transacetylase has no requirement for Ca2+, Mg2+, or CoA and a broad pH optimum (7.0-8.0) with Km values of 12.0 microM for PAF and 106.4 microM for lysoplasmalogens. The enzyme activity from the isolated membrane fraction is not changed when whole cells are supplemented with 20:4, induced to differentiate into granulocytes, or treated with ionophore A23187. Radyllyso-sn-glycero-3-phosphocholine (GPC), radyllyso-GPE, acyllyso-sn-glycero-3-phosphoserine (GPS), acyllyso-sn-glycero-3-phosphoinositol (GPI), alkyllyso-sn-glycero-3-phosphate (GP), acyllyso-GP, or cis-9-octadecen-1-ol can also serve as acetate acceptors, whereas alkylglycerol, acylglycerol, or cholesterol are inactive. Differences in substrate acceptor specificity, sensitivity toward phenylmethylsulfonyl fluoride, and response to temperature suggest that the CoA-independent transacetylase and the CoA-independent transacylase that transfers long-chain acyl moieties are two separate enzymes. With intact differentiated HL-60 cells, [3H]acetate from [3H]PAF can be incorporated into alk-1-enylacetyl-GPE in the presence of ionophore A23187, but not in its absence. Moreover, phospholipase A2 inhibitors (p-bromophenacyl bromide and mepacrine) block the transacetylation process in whole cell system. These results indicate the production of alk-1-enyllyso-GPE is a rate-limiting factor for the subsequent transacetylation step during cell activation. We conclude that the transacetylase may participate in the biosynthesis of ethanolamine plasmalogen and acyl analogs of PAF, in vivo, fine-tuning of PAF biological responses, and cross-talk between de novo and remodeling pathways of PAF biosynthesis.     

Location of a Bombyx mori receptor binding region on a Bacillus thuringiensis delta-endotoxin.

Receptor binding studies were performed with 125I-labeled trypsin-activated insecticidal toxins, CryIA(a) and CryIA(c), from Bacillus thuringiensis on brush-border membrane vesicles (BBMV) prepared from Bombyx mori larval midgut. Bioassays were performed by gently force feeding B. mori with diluted toxins. CryIA(a) toxin (LD50; 0.002 micrograms) was 200 times more active against B. mori larvae than CryIA(c) toxin (LD50; 0.421 micrograms) and showed high-affinity saturable binding. The Kd and the binding site concentration for CryIA(a) toxin were 3.5 nM and 7.95 pmol/mg, respectively. CryIA(c) toxin (Kd, 50.35 nM; Bmax, 2.85 pmol/mg) did not demonstrate high-affinity binding to B. mori BBMV. Control experiments with CryIA(a) and CryIA(c) toxins revealed no binding to mouse small intestine BBMV and nonspecific binding to pig kidney BBMV. These data provide evidence that binding to a specific receptor on the membrane of midgut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of insecticidal crystal proteins. To locate a B. mori receptor binding region on the CryIA(a) toxin, homologous and heterologous competition binding studies were performed with a set of mutant proteins which had previously been used to define the B. mori "specificity domain" on this toxin (Ge, A. Z., Shivarova, N. I., and Dean, D. H. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4037-4041). These mutant proteins have had regions of their genes reciprocally exchanged with the cryIA(c) gene. A B. mori receptor binding region on CryIA(a) toxin includes the amino-terminal portion of the hypervariable region, amino acids 332-450, which is identical to the previously described B. mori specificity determining region. These data provide direct evidence that delta-endotoxins contain a tract of amino acids that comprise a binding region and as a results determines the specificity of a toxin.     

Purification and identification of the heat-stable factor required for pregnenolone-binding protein activity. Evidence that the factor is adenosine 3',5'-diphosphate.

This paper presents data identifying adenosine 3',5'-diphosphate (3',5'-ADP) as the small heat-stable factor essential for the active steroid binding complex of the adrenocortical pregnenolone-binding protein (PBP). Factor activity obtained from the boiled supernatant of partially purified PBP was isolated by high performance liquid chromatography using weak anion-exchange and hydrophobic (C18) chromatography sequentially. The purified material retained characteristic factor activity and presented a UV spectrum identical to that for authentic 3',5'-ADP. Mass spectroscopic analysis of the isolated factor revealed an M-H ion of appropriate mass (m/z = 426) and a decomposition pattern for the M-H ion that was consistent with the structure of 3',5'-ADP. The studies presented here demonstrate that authentic 3',5'-ADP can categorically substitute for factor prepared from the soluble fraction of the guinea pig adrenal. Specifically, 3',5'-ADP potentiated ligand binding of partially purified native PBP and restored binding capacity to alkaline phosphatase-inactivated PBP in a dose-dependent manner. As is the case for adrenocortical factor activity, these effects were negated by pretreating the 3',5'-ADP with calf intestinal alkaline phosphatase. Other nucleotides similarly tested, including ADP isomers, were ineffective as factor substitutes. The sulfated form of 3',5'-ADP (i.e. 3'-phosphoadenosine 5'-phosphosulfate) demonstrated some potential for restoring binding capacity to phosphatase-inactivated PBP; however, this compound was clearly inhibitory rather than stimulatory for native PBP activity. Taken collectively, the data overwhelmingly demonstrate that 3',5'-ADP is in fact the molecule required by the PBP for high affinity steroid binding complex formation. It is not yet known whether 3',5'-ADP acts allosterically or contributes directly to the structure of the steroid binding site.     

On the location and function of tyrosine beta 331 in the catalytic site of Escherichia coli F1-ATPase.

1) Using a combination of site-directed mutagenesis and fluorescence spectroscopy we have studied the location and function of residue beta Y331 in the catalytic site of Escherichia coli F1-ATPase. The fluorescent analog lin-benzo-ADP was used as a catalytic-site probe, and was found to bind to three sites in normal F1, with Kd1 = 0.20 microM and Kd2,3 = 5.5 microM. lin-Benzo-ATP was a good substrate for hydrolysis. 2) The mutants investigated were beta Y331F, L, A and E. kcat/KM for ATP hydrolysis in purified F1 was reduced according to the series Y greater than or equal to F greater than L greater than A greater than E, with E being severely impaired; concomitant decreases in binding affinity for lin-benzo-ADP were seen. 3) Fluorescence properties of lin-benzo-ADP bound to F1 differed widely, depending on the residue present at position beta 331. Red shifts of excitation and emission spectra occurred with F and L residues, but not with Y, A, or E. There was strong quenching of fluorescence with wild-type (Y), partial quenching with A, and no quenching with F, L, or E. 4) We conclude that (a) the environment around the bound adenine moiety in the catalytic site is nonpolar, (b) residue beta 331 is part of the adenine-binding subdomain and when tyrosine is the residue, the phenolic hydroxyl makes direct interaction with the fluorophore, (c) an aromatic residue is not absolutely required at position beta 331 for catalytic function, but an increase in polarity leads to functional impairment, and (d) in terms of fluorescence response of bound lin-benzo-ADP all three catalytic sites behaved the same. 5) F1 from mutant beta Y297F bound lin-benzo-ADP with the same fluorescence and binding characteristics as normal F1, and catalytic properties were similar to normal. Therefore, there was no reason to conclude that residue beta Y297 is involved in binding the adenine moiety of ATP.