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D A Leonard

Publications and source records attributed to D A Leonard.

At least 19 recordsLinked to original sources
PubMed1997

Interaction between Cdc42Hs and RhoGDI is mediated through the Rho insert region.

Members of the Rho subfamily of GTP-binding proteins contain a region of amino acid sequence (residues 122-134) that is absent from other Ras-like proteins and is termed the Rho insert region. To address the functional role of this domain, we have constructed a Cdc42Hs/Ras chimera in which loop 8 from Ha-Ras was substituted for the region in Cdc42Hs that contains the 13-amino acid insert region. Our data indicate that the insert region of Cdc42Hs is not essential for its interactions with various target/effector molecules or for interactions with the guanine nucleotide exchange factor, Dbl, or the Cdc42 GTPase-activating protein (GAP). However, the regulation of GDP dissociation and GTP hydrolysis on Cdc42Hs by the Rho GDP-dissociation inhibitor (GDI) is extremely sensitive to changes in the insert region, such that a Cdc42Hs/Ha-Ras chimera that lacks this insert is no longer susceptible to a GDI-induced inhibition of GDP dissociation and GTP hydrolysis. The insensitivity to GDI activity is not due to the inability of the GDI molecule to bind to the Cdc42Hs/Ha-Ras chimera, and in fact, the GDI is fully capable of stimulating the release of this chimera from membranes.

Amino Acid Sequence↗
PubMed1997

Structural basis of DNA bending and oriented heterodimer binding by the basic leucine zipper domains of Fos and Jun.

Interactions among transcription factors that bind to separate sequence elements require bending of the intervening DNA and juxtaposition of interacting molecular surfaces in an appropriate orientation. Here, we examine the effects of single amino acid substitutions adjacent to the basic regions of Fos and Jun as well as changes in sequences flanking the AP-1 site on DNA bending. Substitution of charged amino acid residues at positions adjacent to the basic DNA-binding domains of Fos and Jun altered DNA bending. The change in DNA bending was directly proportional to the change in net charge for all heterodimeric combinations between these proteins. Fos and Jun induced distinct DNA bends at different binding sites. Exchange of a single base pair outside of the region contacted in the x-ray crystal structure altered DNA bending. Substitution of base pairs flanking the AP-1 site had converse effects on the opposite directions of DNA bending induced by homodimers and heterodimers. These results suggest that Fos and Jun induce DNA bending in part through electrostatic interactions between amino acid residues adjacent to the basic region and base pairs flanking the AP-1 site. DNA bending by Fos and Jun at inverted binding sites indicated that heterodimers bind to the AP-1 site in a preferred orientation. Mutation of a conserved arginine within the basic regions of Fos and transversion of the central C:G base pair in the AP-1 site to G:C had complementary effects on the orientation of heterodimer binding and DNA bending. The conformational variability of the Fos-Jun-AP-1 complex may contribute to its functional versatility at different promoters.

Amino Acid Sequence↗
PubMed1997

Use of a fluorescence spectroscopic readout to characterize the interactions of Cdc42Hs with its target/effector, mPAK-3.

The family of p21-activated kinases (PAKs) has been shown to contain a domain that can independently bind to the Ras-like proteins Cdc42Hs and Rac. We have expressed a 72 amino acid recombinant form of this p21-binding domain (PBD) from mPAK-3 in Escherichia Coli for use in structure-function studies. The protein can be purified on a nickel affinity resin due to a hexa-His tag that is incorporated onto the amino terminus of the domain. PBD binds to Cdc42Hs in a guanine nucleotide-dependent manner as demonstrated by a novel fluorescence assay that takes advantage of the spectroscopic properties of N-methylanthraniloyl (Mant)-guanine nucleotides. Ionic strength has little effect on the affinity of PBD for Cdc42Hs, but alkaline pH values tend to weaken the interaction. We have shown that the inhibition of the GTPase activity of Cdc42Hs, as well as a previously undescribed inhibition of guanine nucleotide dissociation, is mediated by the PBD portion of the mPAK-3 molecule. These findings suggest that PBD binding alters the geometry of the guanine nucleotide binding site on Cdc42Hs, perhaps as an outcome of the target/effector molecule binding in close proximity to the nucleotide domain. We therefore tested if mutations in the effector region of Cdc42Hs (32-40), which in Ras are very close to the guanine nucleotide binding site, had any effect on PBD binding. Changing tyrosine 32 to lysine (Y32K) resulted in a small (5-fold) inhibition of PBD binding, but the very conservative mutation D38E yielded at least a 50-fold decrease in affinity. Finally, the catalytic domain of the GTPase activating protein, Cdc42-GAP, was shown to inhibit PBD binding in a competitive manner, indicating that this target molecule and the negative regulator (GAP) bind to overlapping sites on the Cdc42Hs molecule.

Animals↗
PubMed1996

Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using extrinsic reporter group fluorescence.

The overall goal of these studies was to examine the applicability of extrinsic reporter group fluorescence in monitoring the GTP-binding/GTPase cycle of a Ras-like GTP-binding protein. Toward this end, we have labeled the GTP-binding protein Cdc42Hs with the environmentally sensitive fluorophore succinimidyl 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoate (sNBD) at a single reactive lysine residue. We find that the sNBD-labeled Cdc42Hs undergoes a fluorescence enhancement at 545 nm when Cdc42Hs exchanges bound GDP for GTP. This enhancement is then fully reversed upon GTP hydrolysis. The specific GTPase-activating protein for Cdc42Hs, the Cdc42Hs-GAP, strongly stimulates the rate of reversal of the fluorescence enhancement at 545 nm, consistent with its ability to fully catalyze the GTPase reaction of Cdc42Hs. Conversely, the specific guanine nucleotide exchange factor (GEF), Cdc24, strongly stimulates the fluorescence enhancement that accompanies GTP binding, consistent with its ability to stimulate the GDP-GTP exchange reaction on Cdc42Hs. Resonance energy transfer measurements yielded a distance of approximately 32 A for the sNBD moiety and the guanine nucleotide binding site occupied with either N-methylanthraniloyl- (Mant) dGDP or MantdGTP. Taken together, these results identify a conformationally sensitive reporter site on the Cdc42Hs molecule that is located some distance away from the guanine nucleotide binding site but nonetheless provides a highly sensitive monitor for GTP-binding, GTPase activity, and the interactions of key regulatory proteins.

Animals↗
PubMed1995

A single residue can modify target-binding affinity and activity of the functional domain of the Rho-subfamily GDP dissociation inhibitors.

The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins for the Rho- and Rab-subtype GTP-binding proteins. As a first step toward identifying the key functional domain(s) on the Rho-subtype GDI, truncations of the amino and carboxyl termini were performed. Deletion of the final four amino acids from the carboxyl terminus of Rho GDI or the removal of 25 amino acids from the amino terminus had no significant effect on the ability of the GDI to inhibit GDP dissociation from the Rho-like protein Cdc42Hs or on its ability to release Cdc42Hs from membrane bilayers. However, the deletion of 8 amino acids from the carboxyl terminus of Rho GDI eliminated both activities. To further test the importance of the carboxyl-terminal domain of the Rho GDI molecule, chimeras were constructed between this GDI and a related protein designated LD4, which is 67% identical to Rho GDI but is less potent by a factor of 10-20 than Rho GDI in functional assays with the Cdc42Hs protein. Two sets of chimeras were constructed that together indicated that as few as 6 amino acids near the carboxyl terminus of Rho GDI could impart full GDP dissociation inhibition and membrane dissociation activities on the LD4 molecule. Further analysis of this region by site-directed mutagenesis showed that a single change at residue 174 of LD4 to the corresponding residue of Rho GDI (i.e., Asp-174-->Ile) could impart nearly full (70%) Rho GDI activity on the LD4 molecule.

Amino Acid Sequence↗
PubMed1995

15-substituted lanosterols: post-transcriptional suppressors of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The oxolanosterol oxime 3 beta-hydroxylanost-7-en-15-one 15-oxime and the structurally similar 3 beta-hydroxylanost-7-en-15-one are dual-action inhibitors of cholesterol synthesis which cause both inhibition of lanosterol 14 alpha-methyl demethylase and suppression of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). This report examines the mechanism by which these compounds lower HMGR protein levels in Chinese hamster ovary fibroblasts. Data are presented which suggest that both sterols reduce the translational efficiency of the HMGR mRNA as well as increase the rate of enzyme degradation. The effect of these sterols on the concentration of the low-density lipoprotein receptor (LDLR) in normal human fibroblasts was also determined. In these cells, both lanosterol analogs lowered HMGR protein levels without affecting LDLR concentration. This in contrast to the previously reported coordinate transcriptional regulation of these two genes by the C27-sterol 25-hydroxy-cholesterol. These findings are consistent with the hypothesis that different sterols regulate HMGR activity through distinct mechanisms.

Animals↗
PubMed1994

Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy.

We have developed several high-resolution assays for the nucleotide state of a rho-subfamily low molecular weight GTP-binding protein, Cdc42Hs. The first involves the use of the fluorescent N-methylanthraniloyl derivative of GDP (mant-GDP). As has been shown for the ras protein, mant-dGDP fluorescence is significantly enhanced (approximately 20%) upon binding to Cdc42Hs. It was further found that the binding of mant-nucleotides results in an efficient energy transfer between the single tryptophan residue of Cdc42Hs and the mant moiety. The exchange of mant-dGDP for GDP bound to Cdc42Hs, as read-out either by the enhancement of the mant fluorescence or by energy transfer, is inhibited by physiological (mM) Mg2+ concentrations and correlates exactly to the rate of [3H]GDP exchange observed in filter-binding assays. Moreover, changes in the fluorescence of mant-dGDP are also sensitive to nucleotide dissociation induced by the dbl-oncogene product, a known nucleotide exchange factor for Cdc42Hs. A second fluorescence read-out for the nucleotide-bound state of Cdc42Hs involves the measurements of intrinsic fluorescence of a single tryptophan residue (W97) which is highly sensitive to whether GDP or GTP is bound in the nucleotide pocket. The hydrolysis of GTP to GDP by Cdc42Hs results in an approximately 30% enhancement of the protein fluorescence. The rate of this fluorescence change corresponds well to the rate of conversion of [gamma-32P]GTP to GDP plus [32P]Pi as measured by filter-binding assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed1994

Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by 3 beta-hydroxy-lanost-8-en-32-al, an intermediate in the conversion of lanosterol to cholesterol.

The lanosterol demethylation intermediate 3 beta-hydroxylanost-8-en-32-al is a known suppressor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate-limiting enzyme of cholesterol biosynthesis. Studies on the mechanism of action of this compound have been hampered by its rapid metabolism. As one approach to this problem, the effects of 3 beta-hydroxy-lanost-8-en-32-al on HMGR gene expression were examined using a mutant cell line which lacks lanosterol 14 alpha-methyl demethylase activity. Data are presented which suggest that 3 beta-hydroxy-lanost-8-en-32-al inhibits HMGR gene expression by reducing the translational efficiency of the HMGR mRNA. We have recently reported that 15 alpha-fluoro-3 beta-hydroxy-lanost-7-en-32-aldehyde, a compound which is structurally similar to 3 beta-hydroxy-lanost-8-en-32-aldehyde, suppresses HMGR activity in cultured Chinese hamster ovary cells by a posttranscriptional process, inhibiting translation without affecting either transcription or enzyme degradation (Trzaskos et al., 1993, J. Biol. Chem. 268, 22591-22599). In contrast to the results obtained with the 15 alpha-fluorolanostenol, the lanostenol 32-aldehyde increased the rate of degradation of HMGR in a manner similar to that reported for oxycholesterols. These data suggest that 15 alpha-fluoro-3 beta-hydroxy-lanost-7-en-32-aldehyde and 3 beta-hydroxy-lanost-8-en-32-aldehyde, although structurally similar posttranscriptional regulators of HMGR suppress enzyme activity, at least in part, by different mechanisms.

Animals↗
PubMed1994

Tissue repair: a critical determinant in CCl4 hepatotoxicity.

A single antimitotic dose of colchicine administered 24 hr after a single ip dose of CCl4 profoundly enhanced lethality in the adult Sprague-Dawley male rat from < 10% to over > or = 70% within the next 24 hr. These findings suggest that disruption of normal hepatic cellular repair processes is a critical factor affecting the outcome of CCl4 intoxication.

Animals↗
PubMed1994

Evaluation of a rodent peroxisome proliferator in two species of freshwater fish: rainbow trout (Onchorynchus mykiss) and Japanese medaka (Oryzias latipes)

Rainbow trout (Onchorynchus mykiss) and Japanese medaka (Oryzias latipes) were exposed to the hypolipidemic drug gemfibrozil, a known rodent peroxisome proliferator. Trout were injected (i.p.) daily for 2 weeks at doses of 0, 46, 87, or 152 mg/kg/day. Medaka were exposed to the nominal concentrations of 0, 1.25, 2.5, or 5 ppm in water for 2 weeks in a static-renewal system. Peroxisome proliferation was assessed by measuring fatty acyl-CoA oxidase (FAO) activity, peroxisomal bifunctional enzyme (PBE) quantity, and changes in liver-to-body weight ratios (LWR). Results indicate that a mild peroxisome proliferative response was observed in rainbow trout (significant increases in FAO activity at all dose levels and in LWR at the highest dose level). Medaka demonstrated a significant increase in PBE at the highest dose level, while nonsignificant increases in FAO activity were observed at the mid- and high-dose levels.

Animals↗
PubMed1994

Oxolanosterol oximes: dual-action inhibitors of cholesterol biosynthesis.

A series of oxolanosterol oximes and oxime ethers have been prepared as potential dual-action inhibitors of cholesterol biosynthesis. The synthesis of these oximes along with the evaluation of their ability to inhibit lanosterol 14 alpha-methyl demethylase (P450DM) and to suppress 3-hydroxy-3-methylglutaryl coenzyme. A reductase (HMGR) activity is presented. 3 beta-Hydroxylanost-7-en-15-one 15-oxime XIX was found to be an effective inhibitor of P450DM in rat liver microsomal preparations. In [14C]acetate incorporation studies using Chinese hamster ovary (CHO) cells, compound XIX was found to cause a dramatic reduction in the incorporation of acetate into C27 sterols with a concomitant increase in radiolabeled C30 sterols which is consistent with the inhibition of P450DM. In addition, 15-oxime XIX was shown to suppress HMGR activity in both wild-type CHO and P450DM-deficient (AR45) cells, indicating that suppression of HMGR is independent of any effects of this oxime on P450DM. In both cell lines, parallel declines in HMGR activity and HMGR protein levels were observed suggesting that compound XIX suppresses HMGR activity by regulation of gene expression. These results demonstrate that, as predicted, 15-oxime XIX is indeed a dual-action inhibitor of cholesterol biosynthesis which causes both the inhibition of P450DM and a reduction in HMGR activity.

Animals↗
PubMed1993

Modulation of 3-hydroxy-3-methylglutaryl-CoA reductase by 15 alpha-fluorolanost-7-en-3 beta-ol. A mechanism-based inhibitor of cholesterol biosynthesis.

The chemical synthesis and metabolic characteristics of the lanosterol analogue, 15 alpha-fluorolanost-7-en-3 beta-ol, are described. The 15 alpha-fluorosterol is shown to be a competitive inhibitor of the lanosterol 14 alpha-methyl demethylase (Ki = 315 microM), as well as substrate for the demethylase enzyme. Metabolic studies show that the 15 alpha-fluorosterol is converted to the corresponding 15 alpha-fluoro-3 beta-hydroxylanost-7-en-32-aldehyde by hepatic microsomal lanosterol 14 alpha-methyl demethylase but that further metabolic conversion to cholesterol biosynthetic intermediates is blocked by virtue of the 15 alpha-fluoro substitution. When cultured cells are treated with the fluorinated lanosterol analogue, a decrease in 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and immunoreactive protein was observed. However, when the lanosterol 14 alpha-methyl demethylase-deficient mutant cell line, AR45, is treated with the fluorosterol, no effect upon HMG-CoA reductase is observed. Thus, metabolic conversion of the sterol to its 32-carboxaldehyde analogue by the lanosterol 14 alpha-methyl demethylase is required for HMG-CoA reductase suppressor activity. Measurement of HMG-CoA reductase mRNA levels in 15 alpha-fluorosterol-treated Chinese hamster ovary (CHO) cells reveals that mRNA levels are not decreased by the sterol as would be expected for a sterol regulator of HMG-CoA reductase activity. The decrease in HMG-CoA reductase protein is due to inhibition of enzyme synthesis, suggesting that the 15 alpha-fluorosterol reduces the translational efficiency of the reductase mRNA. Measurements of the half-life of HMG-CoA reductase show that, in contrast to other oxysterols, the 15 alpha-fluorolanostenol does not increase the rate of degradation of the enzyme. Collectively, these data support the premise that oxylanosterols regulate HMG-CoA reductase expression through a post-transcriptional process which may be distinct from other previously described sterol regulatory mechanisms.

Animals↗
PubMed1993

32-Methyl-32-oxylanosterols: dual-action inhibitors of cholesterol biosynthesis.

Lanosterol 14 alpha-methyl demethylase (P-450DM) is the cytochrome P-450 monooxygenase which oxidatively removes the 14 alpha-methyl group of lanosterol. This demethylation is considered to be a rate-limiting step in the conversion of lanosterol to cholesterol. The intermediates in this transformation are known to bind very tightly to P-450DM and have been implicated in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity, the rate-limiting enzyme in overall cholesterol biosynthesis. Three 32-methylated analogs of the intermediates generated during the removal of the 14 alpha-methyl group by P-450DM, compounds 17a, 17b, and 18, have been prepared and their biochemical activities assessed. All three compounds were found to be direct inhibitors of P-450DM. These compounds were also shown to suppress HMGR activity by reducing the level of enzyme protein.

Animals↗
PubMed1993

Ornithine decarboxylase (ODC) activity in the liver of individual medaka (Oryzias latipes) of both sexes.

Ornithine decarboxylase (ODC) activity has been determined in the liver of individual medaka (N = 48). The results indicate a mean ODC activity of 1.69 +/- 1.77 nmol 14CO2/hr/mg protein. The fish displayed a large interindividual variation of normal ODC activity with a range of 140fold. However, no sex differences were observed. The findings also indicate that the mean ODC activity in this fish model is approximately 100fold greater than observations in the published literature as well as in our laboratory for commonly employed rodent models.

Animals↗
PubMed1993

Effect of hypolipidemic drugs gemfibrozil, ciprofibrate, and clofibric acid on peroxisomal beta-oxidation in primary cultures of rainbow trout hepatocytes.

Primary cultures of hepatocytes were established from sexually mature male rainbow trout (Oncorhyncus mykiss) and treated with the hypolipidemic drugs gemfibrozil (0.25-1.25 mM), clofibric acid (2.25-3.00 mM), or ciprofibrate (0.25-1.00 mM). Significant dose-related increases in peroxisomal fatty acyl-CoA oxidase (FACO) were seen after exposure for 48 hr to clofibric acid (P < 0.01) and ciprofibrate (P < 0.05) but not gemfibrozil (P = 0.08). Strong correlation was obtained between increased acyl-CoA oxidase activity and the relative amount of peroxisomal bifunctional enzyme (PBE), further supporting evidence of a proliferative effect. These preliminary studies demonstrate that peroxisomal beta-oxidation can be induced in vitro in a primary rainbow trout hepatocyte system.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1993

Interactive potential of omega-3 fatty acids with clofibrate or DEHP on hepatic peroxisome proliferation in male Wistar rats.

The interactive potential of three known peroxisome proliferators, omega-3 fatty acids, clofibrate and di(2-ethylhexylphthalate (DEHP), was evaluated in male weanling Wistar rats for the effect on peroxisomal beta-oxidation. Omega-3 fatty acids were supplied by menhaden oil which was fed in six regimens: low fat (5% w/w), low fat and clofibrate (0.3% w/w) or DEHP (0.25% w/w), high fat (20% w/w), high fat and clofibrate or DEHP in the aforementioned concentrations. Induction of peroxisomal beta-oxidation was measured by changes in liver-to-body weight ratio, fatty acyl-CoA oxidase (FAO) activity, and peroxisomal bifunctional enzyme (PBE) quantity. Analysis of transformed data indicated a less than additive response in FAO activity with no deviation from additivity seen with liver-to-body ratios and PBE.

3-Hydroxyacyl CoA Dehydrogenases↗