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M F Taylor

Publications and source records attributed to M F Taylor.

36 records · Page 2Linked to original sources
PubMed1996

In vitro efficacy of morpholino-modified antisense oligomers directed against tumor necrosis factor-alpha mRNA.

Chemical modification of antisense oligonucleotides to increase nuclease resistance may improve their efficacy within enzyme-rich cellular targets (e.g. macrophages). We evaluated a panel of morpholino antisense oligomers (M-AS) for their ability to inhibit macrophage tumor necrosis factor-alpha (TNF-alpha) release and compared them to phosphodiester (O-AS) and phosphorothioate (S-AS) types of oligonucleotides. M-AS inhibited translation in vitro (rabbit reticulocyte lysate) of target mRNA at concentrations as low as 200 nM (e.g. percent inhibition by M-AS 2 at 0.2, 1.0, and 2.0 microM was 40.9 +/- 5.3%, 50.2 +/- 4.6%, and 57.7 +/- 3.6%, respectively, n = 4, p </= 0.002 versus control). Similarly, M-AS 2 effectively, albeit partially, inhibited TNF-alpha production by LPS-stimulated macrophages (RAW 264.7 cells). Incubation of cells with 25 microM M-AS 2 resulted in 32.6 +/- 2.6% (n = 3, p = 0.002 versus control) decrease in TNF-alpha release. In contrast, S-AS inhibited translation of the target mRNA in the rabbit reticulocyte lysate assay, but not in the cell-based assay. In fact, S-AS nonspecifically augmented TNF-alpha release. O-AS were without effect in either system. Uptake studies with fluorescent M-AS revealed that inhibitory effects were seen despite relatively low cellular uptake (intracellular concentration 30.5 +/- 6.7 nM; efficiency of uptake 0.1%). In contrast, flow cytometric and confocal analysis revealed that S-AS were avidly taken up by RAW 264. 7 cells, confirming that their lack of efficacy was not due to lack of uptake. With improved methods of delivery, M-AS may represent an important therapeutic modality.

Animals↗
PubMed1996

Photoaffinity labeling of rat steroid 5 alpha-reductase (isozyme-1) by a benzophenone derivative of a 4-methyl-4-azasteroid.

[1,2-3H]N-4(Benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha-androstane-17 beta-carboxamide ([3H]-4MABP) has been synthesized as a photoaffinity probe of the steroid-binding domain of rat steroid 5 alpha-reductase isozyme-1 (5 alpha R-1). Reversible binding of the probe to 5 alpha R-1 in microsomal preparations yielded a reversible dissociation constant (Kd) of -3 nM, whereas inhibition experiments indicated that the probe had a 50% inhibition concentration of 4.4 nM and was a competitive inhibitor of the enzyme (Ki approximately 3 nM) with respect to testosterone. SDS-PAGE analysis of microsomal, detergent-solubilized, and (6.5%) polyethylene glycol-precipitated fractions of 5 alpha R-I photolyzed with [3H]4MABP in the presence of NADPH showed that the radioactivity was incorporated into a single protein band with a mass of 26 kDa (apparent molecular weight of 5 alpha R-1). UV photolysis was accompanied by an irreversible loss in enzyme activity, consistent with its covalent modification. Increasing the time of UV irradiation and concentration of [3H]4MABP indicated that the half-life and apparent Kd for its photo insertion were approximately 3 min and 7.5 nM, respectively. Photolysis in the presence of a 20-fold excess of N,N-diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide or the 3-carboxysteroid SKF-105111 resulted in partial protection of 5 alpha R-1 from the probe, whereas minimal incorporation of radioactivity was observed in the absence of NADPH or in the presence of NADP+. The results indicate that [3H]4MABP is an effective probe of the steroid (D-ring) binding domain of 5 alpha R-1.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1995

A population genetic test of selection at the molecular level.

The role of natural selection in molecular evolution has been inferred primarily by rejection of null hypotheses based on neutral theory, rather than by acceptance of specific predictions based on selection. In this report, a population genetic test of a specific prediction for selection on DNA polymorphism is presented. Pyrethroid insecticide use constitutes an experiment for which form of selection and molecular target (voltage-gated sodium channels) are both known. As predicted, differential pyrethroid selection on tobacco budworm populations generated significant geographic heterogeneity in sodium channel marker allele frequencies, compared with arbitrary loci.

Alleles↗
PubMed1995

Identification of the NADP(H) binding site of rat liver microsomal 5 alpha-reductase (isozyme-1): purification of a photolabeled peptide corresponding to the adenine binding domain.

We have previously shown that [2'-32P]-2-azido-NADP+ is an effective probe of the NADP-(H) binding site of rat liver microsomal 5 alpha-reductase (5 alpha R-1) [Bhattacharyya et al. (1994) Steroids 59, 634-641]. PEG-fractionated (6.5%) detergent-solubilized preparations (40 mg) containing 5 alpha R-1 activity were UV-photolyzed with [32P]-2-azido-NADP+ and subjected to preparative gel electrophoresis on 8% SDS-PAGE. Fractions corresponding to the second major [32P]-labeled peak following the dye-front were analyzed by 10% SDS-PAGE and showed a single [32P]-labeled species with an apparent molecular mass of approximately 26 kDa (5 alpha R-1). TCA precipitation (13.6%) of the labeled fractions resulted in recovery of > 70% of the total radioactivity in the protein pellet. Trypsin digestion of the resuspended pellet followed by immobilized-Al3+ affinity chromatography indicated that > 90% of the radioactivity remained bound to the affinity column. The [32P]-2N3-NADP(+)-labeled peptide was eluted with potassium phosphate, concentrated, and further purified by reverse-phase (C8) HPLC. Sequence analysis of the purified peptide indicated that it consisted of 11 amino acids with the sequence N-L-R-K-P-G-E-T-G-Y-K, corresponding to residues 170-180 of the rat 5 alpha R-1 sequence [Andersson et al. (1989) J. Biol. Chem. 264, 16249-16255].

Adenine↗
PubMed1995

Inhibition of rat steroid 5 alpha-reductase (isozyme 1) by suramin.

In this study, we show the inhibition of rat steroid 5 alpha-reductase (isozyme 1) by suramin. The enzyme activity decreased in a dose-dependent manner as suramin concentrations increased with the calculated drug dose required for 50% inhibition (at 5 microM testosterone and 200 microM NADPH) being 13 microM. Suramin showed non-competitive inhibition of 5 alpha-reductase with respect to testosterone (KT1 = 2.4 microM) and competitive inhibition with respect to NADPH (KiNADPH = 220 nM). Furthermore, suramin and NADP+, but not NAD+, protected 5 alpha-reductase from labeling by 2-azido-NADP+, a photoactive probe which has recently been used to identify the NADPH binding domain of 5 alpha-reductase. These results suggest that suramin inhibits rat steroid 5 alpha-reductase (isozyme 1) at the level of NADPH binding to the enzyme.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1994

Complications of the use of the Harrison-Nicolle Intramedullary Peg in digital arthrodesis.

The Harrison-Nicolle Intramedullary Peg is used for the arthrodesis of digital joints. We report our experience of complications found using this peg in a series of 16 arthrodeses. At review after an average of 2.9 years, three cases had been revised. Only five of the remaining 13 cases showed a bony fusion and in only two of these five was the fusion in an acceptable position. In nine cases the joint became straight or extended; the preset angle of the peg did not determine the final angle of the arthrodesis. In five cases the tips of the peg appeared to be eroding through bone.

Aged↗
PubMed1994

Effect of a peripheral and a central acting opioid antagonist on the testicular response to stress in rats.

The possible involvement of opioid receptors in mediating the inhibitory effects of immobilization stress on testicular steroidogenesis was determined in adult male rats. Unstressed controls and animals exposed to 3 h of immobilization stress were injected subcutaneously with either vehicle or 1 or 10 mg/kg body weight (BW) of naloxone or naltrexone methobromide (NMB; an opioid receptor antagonist that does not cross the blood-brain barrier) at the beginning of and at 1.5 h of the stress period. Animals were sacrificed at 2 h (30 min after the second injection of antagonist) or 3 h (90 min after the second injection of antagonist) of stress. Plasma LH was not affected by stress, but 30 min after naloxone (1 or 10 mg/kg BW) injection, LH was elevated in both control and stressed rats above levels in vehicle-injected animals. By 90 min after naloxone injection, plasma LH had declined to levels comparable to those in vehicle-injected animals. NMB had no effect on plasma LH concentrations in either control or stressed rats. Three hours of stress reduced plasma testosterone (T) levels by 60% in vehicle-injected animals. This effect of stress on plasma T levels was antagonized by the 10 mg/kg BW dose of naloxone and 1 or 10 mg/kg BW of NMB. The ability of naloxone to reverse the effect of stress on plasma T levels was likely related to its ability to stimulate LH secretion, but NMB normalized plasma T values in stressed animals without altering plasma LH concentrations. Only the highest dose of NMB increased plasma T levels in unstressed control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde-Lyases↗
PubMed1993

Identification and characterisation of a type-1 protein phosphatase from the okadaic acid-producing marine dinoflagellate Prorocentrum lima.

The unicellular marine dinoflagellate, Prorocentrum lima, an established producer of okadaic acid (OA), was shown to contain a type-1 protein phosphatase (PP-1) the biochemical profile of which on Mono-Q and Superdex-75 fast protein liquid chromatography was identical to the catalytic subunit of PP-1 from rabbit skeletal muscle. Purified P. lima PP-1 (apparent molecular mass 37.5 kDa) was highly sensitive to inhibition by mammalian protein phosphatase inhibitor-1 and inhibitor-2, and to OA itself. A 6-7-fold increase in OA production by P. lima, when grown under controlled conditions, correlated with an up to 300-fold increase in P. lima PP-1 activity. Furthermore, P. lima did not contain any detectable type-2A protein phosphatase activity. This study represents the first identification of a serine/threonine protein phosphatase in a dinoflagellate.

Animals↗
PubMed1993

Linkage of pyrethroid insecticide resistance to a sodium channel locus in the tobacco budworm.

Pyrethroids, with their lack of persistence and low mammalian toxicity, have been important insecticides since the early 1970s. However, heavy use has selected for resistance to pyrethroids in populations of many insects, in particular the tobacco budworm Heliothis virescens, a major cotton pest in the Americas. Several studies have identified the voltage-gated sodium channel as the principal target of pyrethroid action, and the sodium channel has been implicated in pyrethroid resistance in Musca domestica and Drosophila melanogaster. We present molecular genetic evidence that pyrethroid resistance is linked to a sodium channel locus in a strain of H. virescens. This is the first such evidence for any major agricultural pest, and is an important step towards understanding the molecular basis of resistance. This in turn will facilitate assessment, modeling, and control of resistance in pest populations, and increase our understanding of sodium channel function.

Alleles↗
PubMed1993

The lepidopteran mitochondrial control region: structure and evolution.

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies--although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.

Animals↗
PubMed1993

Effect of immobilization stress on plasma luteinizing hormone, testosterone, and corticosterone concentrations and on 3 beta-hydroxysteroid dehydrogenase activity in the testes of adult rats.

We have examined the effect of 3 hr of immobilization stress on plasma luteinizing hormone, testosterone, and corticosterone levels, and on the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in microsomal and mitochondrial fractions of the testis from adult rats. Immobilization for 3 hr increased plasma corticosterone and reduced plasma testosterone concentrations by 57%. Plasma luteinizing hormone levels were lower, although not significantly (P = 0.093) so, in stressed animals. Immobilization (3 hr) reduced the Vmax values of 3 beta-HSD in the mitochondria and in the microsomal fraction of the testis by 40% and 34%, respectively, but had no effect on the Km values of 3 beta-HSD in the two cellular compartments. These results suggest that the inhibition of the activity of 3 beta-HSD may be partially responsible for the disruption of testicular steroidogenesis during immobilization stress.

3-Hydroxysteroid Dehydrogenases↗
PubMed1993

The British household panel study.

"With the release of the first wave, The British Household Panel Survey is now available from the Archive. The article below is intended to provide an overview of the subject coverage of the British Household Panel Survey, give some idea of the analysis potential of the data, and outline the further dissemination plans for the data."

Communication↗
PubMed1990

Azotobacter vinelandii flavodoxin: purification and properties of the recombinant, dephospho form expressed in Escherichia coli.

The nifF gene coding for the flavodoxin from the nitrogen-fixing bacterium Azotobacter vinelandii (strain OP) was cloned into the plasmid vector pUC7 [Bennett, L. T., Jacobsen, M. R., & Dean, D. R. (1988) J. Biol. Chem. 263 1364-1369] and the resulting plasmid transformed and expressed in Escherichia coli strain DH5. Recombinant Azotobacter flavodoxin is expressed at levels 5-6-fold higher in E. coli than in comparable yields of Azotobacter cultures grown under nitrogen-fixing conditions. Even higher levels were observed with flavodoxin expressed in E. coli under control of a tac promoter. Electron spin resonance spectroscopy on whole cells and in cell-free extracts showed the flavodoxin to be largely in the semiquinone form. The flavodoxin purified from E. coli exhibited the same molecular weight, isoelectric point, flavin mononucleotide (FMN) content, N-terminal sequence, and carboxyl-terminal amino acids as for the wild-type Azotobacter protein. The recombinant flavodoxin differed from native flavodoxin in that it exhibited an increased antigenicity to flavodoxin antibody and did not contain a covalently bound phosphate. Small differences are also observed in circular dichroism spectral properties in the visible and ultraviolet spectral regions. The recombinant, dephospho flavodoxin exhibits an oxidized/semiquinone potential (pH 8.0) of -224 mV and a semiquinone/hydroquinone couple (pH 8.0) of -458 mV. This latter couple is 50-60 mV higher than that exhibited by the native flavodoxin. Resolution of recombinant dephospho flavodoxin resulted in an apoflavodoxin that was much less stable than that prepared from the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Acute immobilization stress disrupts testicular steroidogenesis in adult male rats by inhibiting the activities of 17 alpha-hydroxylase and 17,20-lyase without affecting the binding of LH/hCG receptors.

We have investigated the effect of acute immobilization (3 hours) stress on testicular steroidogenesis in the adult rat. Immobilization did not alter plasma luteizing hormone (LH) levels, but plasma testosterone (T) levels were reduced by 82%. Plasma levels of corticosterone in stressed rats were elevated more than ninefold over control levels. After 3 hours of stress, testicular levels of progesterone were elevated 33%, and levels of 17 alpha-hydroxyprogesterone and T were reduced 47% and 37%, respectively, compared to controls. Immobilization for 3 hours had no effect on the association or dissociation rate constants of LH/human chorionic gonadotropin (hCG) receptors of testicular interstitial cells and did not alter specific hCG binding. The effect of 3 hours of immobilization on testicular 17 alpha-hydroxylase and 17,20-lyase was assessed by incubating testicular microsomes from stressed and control animals in the presence of 21[14C]progesterone and [3H]17 alpha-hydroxyprogesterone. Immobilization of rats reduced the Vmax values of 17 alpha-hydroxylase and 17,20-lyase by 47% and 48%, respectively, but had no effect on the Km values. These results support the hypothesis that stress for 3 hours disrupts rat testicular steroidogenesis via a mechanism that is independent of changes in circulating levels of LH and the binding characteristics of LH/hCG receptors. The effects of immobilization on the content of testicular steroids and on the activities of 17 alpha-hydroxylase and 17,20-lyase suggest that stress inhibits the activities of both 17 alpha-hydroxylase and 17,20-lyase.

Aldehyde-Lyases↗
PubMedDate not supplied

Leydig cell apoptosis in response to ethane dimethanesulphonate after both in vivo and in vitro treatment.

The biological effects of ethane dimethanesulphonate (EDS) are unique since cytotoxicity in the adult rat is almost exclusively confined to the Leydig cells. For this reason, EDS has been used extensively to investigate the physiological role of the Leydig cell and its products. Experiments were conducted to determine whether the Leydig cell will undergo apoptosis in response to EDS or methylprednisolone (MP), a glucocorticoid known to cause apoptosis in a number of other cell types. Percoll-purified Leydig cells were incubated for 24 hours with EDS (750 micrograms/ml), at which time the cells attached to the culture plate became rounded up while control cells were flattened and polyhedral. Following incubation with EDS or MP (10 microM), cells that became detached from the plate were characteristically apoptotic when stained with the fluorescent DNA dye, acridine orange. These cells had shrunk and the nuclear chromatin had become condensed, which is an early characteristic of apoptosis in other cells; eventually, apoptotic bodies formed, reflecting a later apoptotic stage. Electrophoresis of DNA extracted from the treated Leydig cells exhibited the characteristic ladder of the apoptotic process. Increasing the concentration of EDS or MP resulted in a dose-dependent increase in the incidence of apoptosis that reached a maximum of 25% (EDS) or 12% (MP) of detached cells. Administration of EDS in vivo caused a 20-fold increase in the number of apoptotic cells observed in interstitial cell preparations. In conclusion, the data indicates that programmed cell death, apoptosis, can occur in the Leydig cell and that this is the likely mechanism by which EDS kills the cells in vivo and in vitro.

Animals↗