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M D Davis

Publications and source records attributed to M D Davis.

At least 73 records · Page 4Linked to original sources
PubMed1995

Permeability of the blood-brain barrier to the neurotensin8-13 analog NT1.

Neurotensin (NT) has been suggested to be a neuropeptide with therapeutic potential. We used multiple-time regression analysis to measure the unidirectional influx constant (Ki) of a tritiated analog of NT8-13, NT1, with improved metabolic stability. The Ki of [3H]NT1 across the blood-brain barrier (BBB) was 5.12(10(-4)) ml/g-min and was decreased 66% by unlabeled NT1 system. The amount of NT1 crossing the BBB, 0.087% of the injected dose per gram of brain, is consistent with its exerting central effects after peripheral administration. The stable [3H]NT1 crossed the BBB in intact form as assessed by HPLC and completely crossed the endothelial cells that comprise the BBB as assessed by the capillary depletion method. The presence of a transport system could be important for the development of NT analogs.

Animals↗
PubMed1995

Induction of tissue plasminogen activator mRNA and activity by structurally altered estrogens.

The effect of structure of the estrogen ligand on the accumulation of tPA mRNA and the activity of extracellular fibrinolytic enzyme has been examined in cultures of MCF-7 cells. Estradiol(E2)-stimulated fibrinolytic activity was preceded by an increase in actinomycin D sensitive tPA mRNA synthesis which peaked at 18 h. Ten A- and D-ring structural analogs of E2 affected tPA mRNA accumulation and extracellular fibrinolytic activity. Only in the case of two A-ring isomers (2- and 4-hydroxyestratrien-17 beta-ol) was the decreased effect of the ligand's structural change on tPA mRNA accumulation and fibrinolysis not explained by a comparable decline in affinity of the ligand for estrogen receptor. Both of these analogs functioned as antiestrogens. The stimulatory capacity of androstanediols on the tPA gene required that the 3-hydroxyl group be positioned in the beta-configuration. Absence of the 17 beta-hydroxy group was beneficial to the maximum accumulation of tPA mRNA. As has been reported for other estrogen responsive genes (progesterone receptor, cathepsin D and pS2), regulation by estrogens is not related directly to the affinity of the ligand for ER, but this activity may be determined by the location of the electronegative isopotential above the A-ring of estrogenic ligands.

Dactinomycin↗
PubMed1995

CI-1007, a dopamine partial agonist and potential antipsychotic agent. I. Neurochemical effects.

The receptor binding and biochemical effects of the putative dopamine (DA) partial agonist CI-1007 ([R(+)-1,2,3,6-tetrahydro-4-phenyl- 1-[(3-phenyl-3-cyclohexen-1-yl)methyl]pyridine] maleate) and potential antipsychotic were evaluated with a variety of biochemical methods. In receptor binding studies, CI-1007 bound to rat striatal DA receptors exhibiting a Ki of 3 nM as assessed by inhibition of [3H]N-propylnorapomorphine binding. CI-1007 also exhibited high affinity for cloned human D2L (Ki = 25.5 nM) and D3 (Ki = 16.6 nM) receptors with less affinity for D4.2 receptors (Ki = 90.9 nM). The affinity for serotonin-1A (5-HT-1A), alpha-2 adrenergic and 5-HT-2 receptors was moderate (submicromolar range) and slight or negligible for alpha-1, DA D1 and various other receptors. Unlike dopamine, the inhibition of [3H]spiperone binding was monophasic for CI-1007 and only slightly affected by the addition of Gpp-(NH)p. In vitro CI-1007 antagonized the forskolin-induced increases in cyclic AMP levels in GH4C1 cells expressing the human D2L receptor, having an intrinsic activity of 53% of that seen with the full agonist quinpirole. In vivo CI-1007 antagonized the gamma-butyrolactone (GBL)-induced accumulation of L-3,4-dihydroxyphenylalanine in striatum and mesolimbic regions of rat brain, causing a maximal 64% reversal in striatum, consistent with a partial agonist profile. In microdialysis studies it decreased DA overflow in both striatum and nucleus accumbens, indicating decreased release of DA. CI-1007 also reduced brain DA synthesis (DOPA accumulation), metabolism (DOPAC and HVA) and utilization (after tyrosine hydroxylase inhibition with alpha-methyl-p-tyrosine). CI-1007 did not affect striatal acetylcholine levels indicating lack of potent postsynaptic DA actions. CI-1007 seemed to be selective for DA neurons as it did not alter rat brain norepinephrine (NE) synthesis in the NE-enriched brainstem or NE utilization in the mesolimbic region. In addition, it did not affect in general 5-HT synthesis and metabolism in striatum and mesolimbic regions. These neurochemical results demonstrate that CI-1007 is a selective potent brain dopamine partial agonist with limited agonist activity at postsynaptic DA receptors.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1995

Neurochemical and functional characterization of the preferentially selective dopamine D3 agonist PD 128907.

The present study determined the biochemical and pharmacological effects of PD 128907 [R-(+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H- [1]benzopyrano[4,3-b]-1,4-oxazin-9-ol], a dopamine (DA) receptor agonist that shows a preference for the human D3 receptor. In transfected Chinese hamster ovary cells (CHO K1), PD 128907 displaced [3H]spiperone in a biphasic fashion which fit best to a two-site model, generating Ki values of 20 and 6964 nM for the high- and low-affinity sites for the D2L receptors and 1.43 and 413 nM for the corresponding sites for the D3 receptors. Addition of sodium and the GTP analog Gpp(NH)p to both the D2L and D3 caused a modest reduction in the affinity of the compound suggestive of an agonist type action. In agonist binding ([3H]N-0437), PD 128907 exhibited an 18-fold selectivity for D3 versus D2L, a selectivity similar to that found with antagonist binding to the high-affinity sites. PD 128907 exhibited only weak affinity for D4.2 receptors (Ki = 169 nM). No significant affinity for a variety of other receptors was observed. PD 128907 stimulated cell division (measured by [3H]thymidine uptake) in CHO p-5 cells transfected with either D2L or D3 receptors exhibiting about a 6.3-fold greater potency in activating D3 as compared to D2L receptors. In vivo the compound was active in reducing DA synthesis both in normal and gamma-butyrolactone (GBL) treated rats; in the GBL model, the decrease was greater in the higher D3-expressing mesolimbic region as compared with striatum which has a lower expression of D3 receptors. PD 128907 decreased DA release (as measured by brain microdialysis) both in rat striatum, nucleus accumbens and medial frontal cortex, as well as in monkey putamen. Behaviorally PD 128907 decreased spontaneous locomotor activity (LMA) in rats at low doses, whereas at higher doses stimulatory effects were observed. PD 128907 at high doses reversed the reserpine-induced decrease in LMA and induced stereotypy in combination with the D1 agonist SKF 38393 indicating postsynaptic DA agonist actions. It is unclear which of the subtypes of DA receptors might be mediating the pharmacological effects of PD 128907. However, the present findings indicating that PD 128907 shows a preference for DA D3 over D2L and D4.2 receptors indicates that its action at low doses may be due to interaction with D3 receptors and at higher doses, with both D2 and D3 receptors.

Animals↗
PubMed1994

Electrostatic activation of rat phenylalanine hydroxylase.

The conversion of phenylalanine to tyrosine is accelerated approximately five fold by phosphorylation of the enzyme which catalyzes this step, phenylalanine hydroxylase. To gain a clearer understanding of the mechanism of this activation, we have applied site-directed mutagenesis to specifically modify a clone of the hydroxylase at the phosphorylation site, the serine at position 16. We converted this serine residue to alanine and to glutamic acid. The wild-type and mutant proteins were purified and the activation states of the enzymes were examined with respect to the single phosphorylation site at position 16. Substitution of Ser16 with a negatively charged Glu residue resulted in activation of the enzyme, whereas substitution with an uncharged Ala residue did not. These results indicate that activation of the native enzyme by phosphorylation is due to the introduction of a negative charge, and suggest involvement of electrostatic interactions.

Alanine↗
PubMed1994

Treatment of cytomegalovirus retinitis with an intraocular sustained-release ganciclovir implant. A randomized controlled clinical trial.

BACKGROUND AND METHODS: We performed a randomized controlled clinical trial to assess the safety and efficacy of a 1 microgram/h ganciclovir implant for the treatment of newly diagnosed cytomegalovirus (CMV) retinitis in patients with the acquired immunodeficiency syndrome (AIDS). Patients with previously untreated peripheral CMV retinitis were randomly assigned either to immediate treatment with the ganciclovir implant or to deferred treatment. Standardized fundus photographs were taken at 2-week intervals and analyzed in a masked fashion. The study end point was progression of retinitis based on the photographic assessment. RESULTS: Twenty-six patients (30 eyes) were enrolled. The median time to progression of retinitis was 15 days in the deferred treatment group (n = 16) vs 226 days in the immediate treatment group (n = 14) (P < .00001, log-rank test). During the study, 39 primary implants and 12 exchange implants were placed in immediate-treatment eyes, deferred-treatment eyes that progressed, or contralateral eyes that developed CMV retinitis. Postoperative complications in the total series included seven late retinal detachments and one retinal tear without detachment. Final visual acuity was 20/25 or better in 34 of 39 eyes. The estimated risk of developing CMV retinitis in the fellow eye was 50% at 6 months. Biopsy-proven visceral CMV disease developed in eight (31%) of 26 patients. The median survival was 295 days. CONCLUSION: The ganciclovir implant is effective for the treatment of CMV retinitis. Patients with unilateral CMV retinitis treated with the implant are likely to develop CMV retinitis in the fellow eye, and some patients will develop visceral CMV disease.

AIDS-Related Opportunistic Infections↗
PubMed1994

Structure-activity and conformational studies of a series of modified C-terminal hexapeptide neurotensin analogues.

Neurotensin (NT), is a linear tetradecapeptide (pGlu1-Leu2-Tyr3-Glu4-Asn5- Lys6-Pro7-Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13) that has been found in the central nervous system and peripheral tissues and appears to have a variety of physiological properties. A C-terminal hexapeptide analogue [N alpha Me-Arg-Lys-Pro-Trp-Tle-Leu, (1) Tle = tert-leucine] has recently been reported to have high affinity for the NT receptor and appears to possess central activity after systemic administration. In an effort to probe the structure-activity and conformational properties of the dipeptide, Pro-Trp for binding and functional activity, these residues have been substituted with several natural and unnatural amino acids. Some of these analogues have binding affinities similar to compound 1, while in other cases, such as D-amino acid substitutions, the peptides had negligible binding affinity. In general, the Pro10 position seems more tolerant of substitution by amino acids that favor a reverse turn, rather than those that favor an extended conformation. The Trp11 position accepted extra steric bulk more readily than conformational constraints.

Animals↗
PubMed1993

Products of the tyrosine-dependent oxidation of tetrahydrobiopterin by rat liver phenylalanine hydroxylase.

Phenylalanine hydroxylase, a tetrahydrobiopterin (BH4)-dependent oxygenase, catalyzes the conversion of phenylalanine to tyrosine. During this physiological reaction, the oxidation of BH4 is tightly coupled to the hydroxylation of the amino acid substrate with a stoichiometry of 1 mol of BH4 oxidized for every mole of tyrosine formed. In the presence of an activator and certain analogues of either the amino acid or the pterin coenzyme, the enzyme also catalyzes a partially uncoupled reaction in which the amount of tetrahydropterin oxidized exceeds the amount of amino acid hydroxylated. With tyrosine as the amino acid analogue, the reaction is completely uncoupled, i.e., the enzyme functions as a tetrahydropterin oxidase with no net hydroxylation of the amino acid. We have investigated whether the normal pterin intermediate, a 4a-hydroxytetrahydropterin, is formed during the completely uncoupled reaction and whether oxygen is reduced exclusively to the level of water, as in the tightly coupled reaction, or whether some reduction to hydrogen peroxide occurs. We present direct evidence that both the normal hydroxytetrahydropterin intermediate and hydrogen peroxide are formed. The amount of hydrogen peroxide formed, however, is not stoichiometric with the amount of oxygen reduced, an indication that there are at least two pathways for this reduction, one leading to the formation of water and one to hydrogen peroxide. We also postulate that hydrogen peroxide and the hydroxytetrahydropterin are both derived from alternate routes of breakdown of a common precursor, the corresponding 4a-hydroperoxytetrahydropterin. By contrast, in the normal tightly coupled reaction, we suggest that this peroxy compound participates in the hydroxylation of the amino acid substrate.

Animals↗
PubMed1993

Mutation in the 4a-carbinolamine dehydratase gene leads to mild hyperphenylalaninemia with defective cofactor metabolism.

Hyperphenylalaninemias represent a major class of inherited metabolic disorders. They are most often caused by mutations in the phenylalanine hydroxylase gene and, less frequently but with usually more serious consequences, in genes necessary for the synthesis and regeneration of the cofactor, tetrahydrobiopterin. This cofactor is absolutely required for all aromatic amino acid hydroxylations, and, recently, nitric oxide production from L-arginine has also been found to be dependent on tetrahydrobiopterin. Phenylalanine hydroxylase catalyzes a coupled reaction in which phenylalanine is converted to tyrosine and in which tetrahydrobiopterin is converted to the unstable carbinolamine, 4a-hydroxytetrahydrobiopterin. The enzyme, carbinolamine dehydratase, catalyzes the dehydration of the carbinolamine to quinonoid dihydropterin. A decreased rate of dehydration of this compound has been hypothesized to be responsible for the production of 7-biopterin found in certain mildly hyperphenylalaninemic individuals. We have now identified nonsense and missense mutations in the 4a-carbinolamine dehydratase gene in a hyperphenylalaninemic child who excretes large amounts of 7-biopterin. This finding is consistent with the role of the carbinolamine dehydratase in the phenylalanine hydroxylation reaction. Together with previously identified inherited disorders in phenylalanine hydroxylase and dihydropteridine reductase, there are now identified mutations in the three enzymes involved in the phenylalanine hydroxylation system. In addition, the genetics of this system may have broader implications, since the product of the dehydratase gene has previously been shown to play an additional role (as dimerization cofactor for hepatocyte nuclear factor-1 alpha) in the regulation of transcription, through interaction with hepatocyte nuclear factor-1 alpha.

Amino Acid Metabolism, Inborn Errors↗
PubMed1992

Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins.

The principal pathway for the metabolism of phenylalanine in mammals is via conversion to tyrosine in a tetrahydrobiopterin-dependent hydroxylation reaction occurring predominantly in the liver. Recently, the proposal that certain hyperphenylalaninemic children may have a deficiency of carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, has widened the interest in this area of metabolism. Upon cloning and sequencing the dehydratase, we discovered that this protein is identical to DCoH, the cofactor which regulates the dimerization of hepatic nuclear factor 1 alpha, a homeodomain transcription factor. The identity of the nuclear and cytoplasmic proteins is demonstrated by size, immunoblotting, stimulation of phenylalanine hydroxylase, and dehydratase activity. The evolution of the dual functions of regulation of phenylalanine hydroxylation activity and transcription activation in a single polypeptide is unprecedented.

Amino Acid Sequence↗
PubMed1992

"7-tetrahydrobiopterin," a naturally occurring analogue of tetrahydrobiopterin, is a cofactor for and a potential inhibitor of the aromatic amino acid hydroxylases.

The ability of 2-amino-4-hydroxy-7-[dihydroxylpropyl-(L-erythro)-5,6,7,8-tetrahyd ropterin] ("7-tetrahydrobiopterin" or 7-BH4) to substitute for the natural cofactor tetrahydrobiopterin (BH4) has been studied in vitro in the reactions of the three mammalian aromatic amino acid hydroxylases. With rat liver phenylalanine hydroxylase, the apparent Km for 7-BH4 is 160 microM, a value that is approximately 60-fold greater than that for the natural cofactor. In contrast, the hydroxylase reaction is severely inhibited by as little as 1 microM 7-BH4 when assayed in the presence of physiological concentrations of BH4. This inhibition can be overcome either by an increase in the concentration of BH4 or a decrease in the concentration of phenylalanine. With both rat brain tryptophan hydroxylase and rat pheochromocytoma tyrosine hydroxylase, the Km value for 7-BH4 is about one order of magnitude greater than the Km for BH4. Accordingly, 7-BH4 is a poor competitive inhibitor of both tryptophan and tyrosine hydroxylase. Thus, our results suggest that the observed hyperphenylalaninemia in patients who excrete 7-BH4 in their urine may arise directly from the inhibition of phenylalanine hydroxylase by low levels of this pterin. On the other hand, it is less likely that low levels of 7-BH4 would affect the activity of tyrosine or tryptophan hydroxylase in vivo.

Animals↗
PubMed1992

Distribution of 4a-hydroxytetrahydropterin dehydratase in rat tissues. Comparison with the aromatic amino acid hydroxylases.

A 4a-carbinolamine intermediate is generated stoichiometrically during the tetrahydrobiopterin-dependent phenylalanine hydroxylation reaction catalyzed by phenylalanine hydroxylase. The dehydration of the carbinolamine is catalyzed by the enzyme, 4a-hydroxytetrahydropterin dehydratase. We have now examined the distribution of the dehydratase activity in various rat tissues by activity measurements and by immunoblot analysis to explore the possibility that the dehydratase may also play a role in tyrosine and tryptophan hydroxylation. The only two tissues that express relatively high dehydratase activity are liver and kidney, which are also the only two tissues that express phenylalanine hydroxylase activity. The dehydratase activity was generally very low in those tissues which contain high levels of tyrosine and tryptophan hydroxylase activity, except for the pineal gland. These results suggest that the dehydratase may not play an important role in the regulation of the synthesis of those neurotransmitters which are derived from the hydroxylated aromatic amino acids.

Animals↗
PubMed1992

Reversibility of the effects of normothermic global ischemia on the ryanodine-sensitive and ryanodine-insensitive calcium uptake of cardiac sarcoplasmic reticulum.

The effect of normothermic ischemia and ischemia/reperfusion on the function of cardiac sarcoplasmic reticulum (CSR) was investigated using a modified Langendorff perfusion of isolated rat hearts. The function of the CSR was assessed by the oxalate-supported Ca2+ uptake rate of ventricular homogenates. The contribution of the ryanodine-sensitive portion of the CSR was determined by using 20 microM ruthenium red or 625 microM ryanodine to close the CSR Ca2+ release channel. The Ca2+ uptake rate of the CSR decreased progressively with increasing duration of ischemia, but this depression was much less when uptake was assayed in the presence of ryanodine. The depression in CSR Ca2+ uptake preceded ischemic contracture. Ryanodine and ruthenium red stimulated uptake almost equally in control hearts, but ruthenium red was much less effective than ryanodine after ischemia. This difference could not be overcome by increasing the ruthenium red concentration. These results confirm the suggestion that the Ca2+ release channel is inappropriately opened after ischemia. The CSR uptake rates were almost completely restored at 15 minutes of reperfusion after 5 and 10 minutes of ischemia but were only partially restored after 15 minutes of ischemia. At reperfusion, mechanical function (end-diastolic pressure and peak systolic developed pressure) was markedly depressed after only 15 minutes of ischemia. The degree of "stunning" correlated well with the depression of CSR function in individual hearts. The decreased Ca2+ uptake of the CSR was not due to a buildup of ADP in the homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Diabetic retinopathy. A clinical overview.

After an introductory summary, this review presents a description of the natural course of diabetic retinopathy, then briefly considers selected pathogenetic and epidemiological concepts, and concludes with a discussion of current treatment.

Aneurysm↗
PubMed1992

Purification and biochemical characterization of recombinant rat liver phenylalanine hydroxylase produced in Escherichia coli.

Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase. Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli. The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized. The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme. The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme. The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme. The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme. We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase.

Animals↗
PubMed1991

7-Tetrahydrobiopterin is an uncoupled cofactor for rat hepatic phenylalanine hydroxylase.

Rat hepatic phenylalanine hydroxylase requires both a tetrahydropterin cofactor and molecular oxygen to convert phenylalanine to tyrosine. During the physiological hydroxylation, a single mol of the natural cofactor, tetrahydrobiopterin, is oxidized for each mol of phenylalanine converted to tyrosine. Artificial conditions have been devised in which the oxidation of the tetrahydropterin is uncoupled from the hydroxylation of the aromatic amino acid substrate. Recently, an isomer of tetrahydrobiopterin, 7-tetrahydrobiopterin, has been isolated from the urine of certain mildly hyperphenylalaninemic children. We report in this communication that 7-tetrahydrobiopterin may be an inefficient cofactor for phenylalanine hydroxylase because, in vitro, the phenylalanine-dependent oxidation of 7-tetrahydrobiopterin is accompanied by the hydroxylation of the aromatic amino acid substrate only about 15% of the time, i.e. the enzymatic oxidation of 7-tetrahydrobiopterin is about 85% uncoupled from the hydroxylation of the amino acid substrate.

Animals↗