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Biomedical subjects

M S Wolfe

Publications and source records attributed to M S Wolfe.

At least 37 records · Page 2Linked to original sources
PubMed2000

Toward the characterization and identification of gamma-secretases using transition-state analogue inhibitors.

The amyloid-beta protein (A beta), strongly implicated in the etiology of Alzheimer's disease (AD), is formed from the amyloid-beta precursor protein (APP) through sequential proteolysis by beta- and gamma-secretases. Cleavage by gamma-secretase takes place within the middle of the single transmembrane region of APP and results primarily in 40- and 42-amino acid A beta C-terminal variants, A beta 40 and A beta 42. The latter form of A beta is highly fibrillogenic, is invariably elevated in autosomal-dominant forms of AD, and is the major A beta component found presymptomatically in cerebral deposits. Thus, blocking production of A beta in general and A beta 42 in particular is considered an important therapeutic goal. We have developed transition-state analogue inhibitors of gamma-secretase as molecular probes for characterizing the active site of this enzyme, as pharmacological tools for understanding its role in biology, and as affinity labels toward its definitive identification. Specifically, we found that: (1) difluoro ketone and difluoro alcohol peptidomimetics are effective inhibitors of gamma-secretase activity in APP-transfected cells, strongly suggesting an aspartyl protease mechanism; (2) gamma-secretases that form A beta 40 and A beta 42 are pharmacologically distinct but are nevertheless closely similar; (3) large hydrophobic P1 substituents increase the inhibitory potency of these peptidomimetics, suggesting a large complementary S1 pocket for gamma-secretases; (4) A beta 42 production is increased several fold over control by these gamma-secretase inhibitors after replacement with inhibitor-free media; (5) a bromoacetamide derivative of one of these analogues continues to inhibit total A beta and A beta 42 production hours after replacement with compound-free media and should help identify the target(s) of these protease transition-state mimics.

Alzheimer Disease↗
PubMed2000

Rapid Notch1 nuclear translocation after ligand binding depends on presenilin-associated gamma-secretase activity.

Recent data suggest an intimate relationship between the familial Alzheimer disease gene presenilin 1 (PS1) and proteolytic processing of both the amyloid precursor protein (APP) and the important cell signaling molecule, Notch1. We now show, using mammalian cells transfected with full-length Notch1, that the C terminal domain of Notch1 rapidly translocates to the nucleus upon stimulation with the physiologic ligand Delta and initiates a CBF1-dependent signal transduction cascade. Using this assay, we demonstrate that the same aspartate mutations in PS1 that block APP processing also prevent Notch1 cleavage and translocation to the nucleus. Moreover, we show that two APP gamma-secretase inhibitors also diminish Notch1 nuclear translocation in a dose-dependent fashion. However, Notch1 signaling, assessed by measuring the activity of CBF1, a downstream gene, was reduced but not completely abolished in the presence of either aspartate mutations or gamma-secretase inhibitors. Our results support the hypothesis that similar PS1-related enzymatic activity is necessary for both APP and Notch1 processing, yet suggest that Notch signaling may remain relatively preserved with moderate levels of gamma-secretase inhibition.

Alzheimer Disease↗
PubMed2000

Gender differences in labetalol kinetics: importance of determining stereoisomer kinetics for racemic drugs.

STUDY OBJECTIVE: To evaluate the impact of gender on labetalol kinetics. DESIGN: Part of a randomized, crossover study. SETTING: Academic medical center. PATIENTS: Nineteen hypertensive patients (14 men, 5 women; 6 blacks, 13 whites). INTERVENTIONS: Participants had labetalol dosages titrated to a specific antihypertensive response, then underwent ambulatory blood pressure monitoring (ABPM) and a pharmacokinetic study. Labetalol plasma concentrations were measured by high-performance liquid chromatography (HPLC) and labetalol stereoisomer ratios were determined in a single plasma sample by chiral HPLC, both with fluorescence detection. MEASUREMENTS AND MAIN RESULTS: Labetalol concentrations were 80% higher in women (area under the concentration-time curve [AUC]/dose x 1000: 6.79 +/- 2.11 in women vs 3.82 +/- 1.37 hr/L in men, p<0.05), yet both genders had a similar antihypertensive response by 24-hour ABPM. Dose-corrected AUC (AUC/dose x 1000) for labetalol's stereoisomers in women and men, respectively, were S,R-labetalol 7.55 +/- 1.47 and 4.83 +/- 1.54 hr/L (p<0.05), S,S-labetalol 8.23 +/- 2.93 and 4.65 +/- 1.78 hr/L (p<0.05), R,S-labetalol 6.99 +/- 3.30 and 4.25 +/- 2.35 hr/L (p=0.11), and R,R-labetalol 3.91 +/- 2.57 and 3.55 +/- 3.08 hr/L (NS). CONCLUSION: The higher labetalol concentration in women than in men was explained largely by differences in inactive and alpha1-blocking stereoisomers. However, concentrations were similar between genders for the beta-blocking stereoisomer (R,R-labetalol), possibly explaining the similarity in antihypertensive response to the drug. This study highlights the importance of determining stereoisomer kinetics for agents administered as racemates, particularly when relating concentrations to pharmacologic response.

Adult↗
PubMed2000

CYP1A2 and CYP2D6 4-hydroxylate propranolol and both reactions exhibit racial differences.

We have previously shown racial differences in propranolol kinetics, with the largest differences appearing to be in its 4-hydroxylation. The purpose of this study was to identify and confirm the cytochrome P450 enzymes (CYP) with propranolol 4-hydroxylase activity, describe their enzyme kinetics, and determine whether there were racial differences in their catalytic activity. Eleven human recombinant, expressed CYPs were screened, but only CYP1A2 and CYP2D6 possessed propranolol 4-hydroxylase activity. Subsequent studies were conducted in human liver microsomes, including correlation, inhibition, enzyme kinetics, and racial comparison studies. Significant correlations were noted between propranolol 4-hydroxylation and ethoxyresorufin-O-deethylation (marker of CYP1A2 activity), with marked improvement in the correlations when CYP2D6-mediated propranolol 4-hydroxylation was inhibited with quinidine. Inhibition studies showed that quinidine inhibited approximately 55% of propranolol 4-hydroxylation and furaphylline (CYP1A2-selective inhibitor) inhibited about 45% of propranolol 4-hydroxylation. Median (range) parameter estimates of (S)-4-hydroxypropranolol [(S)-HOP] formation were a V(max) value of 307 (165-2397) and 721 (84-1975) pmol/mg of protein/60 min for CYP1A2 and CYP2D6, respectively, and a K(m) value of 21.2 (8.9-77.5) and 8.5 (5.9-31.9) microM for CYP1A2 and CYP2D6, respectively. CYP1A2- and CYP2D6-mediated propranolol 4-hydroxylation was about 70 and 100% higher (P <.05 for both), respectively, in African-Americans compared with Caucasians. In summary, we found that both CYP1A2 and CYP2D6 catalyze formation of 4-hydroxypropranolol and that both enzymes exhibited racial differences in this reaction. The observed racial differences in drug metabolism may have relevance to drug efficacy, toxicity, or carcinogen activation for CYP1A2 or CYP2D6 substrates.

Aryl Hydrocarbon Hydroxylases↗
PubMed1999

Are presenilins intramembrane-cleaving proteases? Implications for the molecular mechanism of Alzheimer's disease.

The amyloid-beta protein (Abeta) is strongly implicated in the pathogenesis of Alzheimer's disease. The final step in the production of Abeta from the amyloid precursor protein (APP) is proteolysis by the unidentified gamma-secretases. This cleavage event is unusual in that it apparently occurs within the transmembrane region of the substrate. Studies with substrate-based inhibitors together with molecular modeling and mutagenesis of the gamma-secretase cleavage site of APP suggest that gamma-secretases are aspartyl proteases that catalyze a novel intramembranous proteolysis. This proteolysis requires the presenilins, proteins with eight transmembrane domains that are mutated in most cases of autosomal dominant familial Alzheimer's disease. Two conserved transmembrane aspartates in presenilins are essential for gamma-secretase activity, suggesting that presenilins themselves are gamma-secretases. Moreover, presenilins also mediate the apparently intramembranous cleavage of the Notch receptor, an event critical for Notch signaling and embryonic development. Thus, if presenilins are gamma-secretases, then they are also likely the proteases that cleave Notch within its transmembrane domain. Another protease, S2P, involved in the processing of the sterol regulatory element binding protein, is also a multipass integral membrane protein which cleaves within or very close to the transmembrane region of its substrate. Thus, presenilins and S2P appear to be members of a new type of polytopic protease with an intramembranous active site.

Alzheimer Disease↗
PubMed1999

Peptidomimetic probes and molecular modeling suggest that Alzheimer's gamma-secretase is an intramembrane-cleaving aspartyl protease.

The amyloid beta-protein (Abeta), implicated in the pathogenesis of Alzheimer's disease (AD), is a proteolytic metabolite generated by the sequential action of beta- and gamma-secretases on the amyloid precursor protein (APP). The two main forms of Abeta are 40- and 42-amino acid C-terminal variants, Abeta40 and Abeta42. We recently described a difluoro ketone peptidomimetic (1) that blocks Abeta production at the gamma-secretase level [Wolfe, M. S., et al. (1998) J. Med. Chem. 41, 6-9]. Although designed to inhibit Abeta42 production, 1 also effectively blocked Abeta40 formation. Various amino acid changes in 1 still resulted in inhibition of Abeta40 and Abeta42 production, suggesting relatively loose sequence specificity by gamma-secretase. The alcohol counterparts of selected difluoro ketones also lowered Abeta levels, indicating that the ketone carbonyl is not essential for activity and suggesting that these compounds inhibit an aspartyl protease. Selected compounds inhibited the aspartyl protease cathepsin D but not the cysteine protease calpain, corroborating previous suggestions that gamma-secretase is an aspartyl protease with some properties similar to those of cathepsin D. Also, since the gamma-secretase cleavage sites on APP are within the transmembrane region, we consider the hypothesis that this region binds to gamma-secretase as an alpha-helix and discuss the implications of this model for the mechanism of certain forms of hereditary AD.

Alzheimer Disease↗
PubMed1999

Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity.

Accumulation of the amyloid-beta protein (Abeta) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer's disease. The final step in the generation of Abeta from the beta-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive gamma-secretase(s). The most common cause of familial Alzheimer's disease is mutation of the genes encoding presenilins 1 and 2, which alters gamma-secretase activity to increase the production of the highly amyloidogenic Abeta42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces gamma-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Abeta production and increases the amounts of the carboxy-terminal fragments of beta-amyloid precursor protein that are the substrates of gamma-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp --> Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 --> TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer's disease and which does not require this cleavage, the Asp 385 --> Ala mutation still inhibited gamma-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and gamma-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for gamma-secretase or is itself gamma-secretase, an autoactivated intramembranous aspartyl protease.

Alzheimer Disease↗
PubMed1999

A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain.

Signalling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD), and is reminiscent of gamma-secretase-mediated cleavage of beta-amyloid precursor protein (APP), a critical event in the pathogenesis of Alzheimer's disease. A deficiency in presenilin-1 (PS1) inhibits processing of APP by gamma-secretase in mammalian cells, and genetic interactions between Notch and PS1 homologues in Caenorhabditis elegans indicate that the presenilins may modulate the Notch signalling pathway. Here we report that, in mammalian cells, PS1 deficiency also reduces the proteolytic release of NICD from a truncated Notch construct, thus identifying the specific biochemical step of the Notch signalling pathway that is affected by PS1. Moreover, several gamma-secretase inhibitors block this same step in Notch processing, indicating that related protease activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus the targeting of gamma-secretase for the treatment of Alzheimer's disease may risk toxicity caused by reduced Notch signalling.

Alzheimer Disease↗
PubMed1999

Travel medicine.

This review covers significant recent developments in the field of travel medicine. New vaccines related to travel are discussed: cholera, Lyme disease, and rotavirus. Pertinent travel vaccine-related issues with varicella, polio, meningitis, Japanese encephalitis, and tick-borne encephalitis are described. New developments in malaria prophylaxis, diagnosis and treatment are discussed. Imported cases of African tick bite fever, arboviruses, African trypanosomiasis, and Helicobacter pylori, and diarrheal illness are reviewed.

Journal Article↗
PubMed1999

Eosinophilia in the returning traveler.

The most common cause of eosinophilia in the traveler returning from the developing world is from infection with a helminth parasite. Protozoal, bacterial, and fungal infections are uncommon causes of eosinophilia. Allergic disorders and drug reactions must also be considered, as well as numerous uncommon miscellaneous disorders. This article reviews the diagnostic features of the more common causes of eosinophilia in the returned traveler and suggested drug treatment.

Eosinophilia↗
PubMed1996

Sex differences in absolute rates of bone resorption in young rats: appendicular versus axial bones.

This study compares absolute rates of bone resorption and formation at the organ level in adolescent Sprague-Dawley rats as a function of sex and type of bone. Bone resorption and formation were quantified in rapidly growing male and female rats (4-7 weeks of age) who were multiply prelabeled with [3H]tetracycline. Ten different whole bones were compared: four cranial or appendicular bones and six axial bones. Absolure rate of bone resorption was measured isotopically by the loss of 3H-tetracycline from each whole bone. Bone growth was quantified in terms of relative and absolute increase in bone calcium mass. When the rates of bone resorption (loss of [3H]-tetracycline as percent of whole bone per 3 weeks) were compared between sexes, the six axial bones showed significantly higher rates (P < 0.05-0.001) in males (64-73) than in females (37-66). No significant sex differences were observed in rate for the two cranial and two appendicular bones. During 4-7 weeks of age, a comparison of bone masses showed that only one bone (calvaria) gained more mass in the male and two bones (mandible and humerus) gained more mass in the female. In contrast, five of six axial bones gained more mass in the female. Thus, 7 out of 10 bones were larger in the female. In growing male and female rats, an inverse relationship appears between rate of bone resorption and mass for most of the axial bones; this relationship was not apparent for cranial or appendicular bones. Sexual dimorphism was consistently seen by greater axial bone mass in females. However, greater rates of bone resorption were seen in male axial bones but not in cranial or appendicular bones. It is apparent that the different types of bones are heterogeneous in their rates of resorption and formation during this period of growth.

Animals↗
PubMed1995

Properties of clock-controlled and constitutive N-acetyltransferases from chick pineal cells.

The pineal gland synthesizes its hormone melatonin (O-methyl-N-acetylserotonin) from serotonin. Acetyl-CoA: serotonin N-acetyltransferase (SNAT), the enzyme that catalyzes the committed step in this biosynthesis, is largely restricted to the pineal gland and is regulated by adrenergic and circadian mechanisms. Another enzyme, acetyl-CoA: arylamine N-acetyltransferase (ANAT), having an apparently similar activity, is also present in the pineal. This enzyme, however, is not rhythmically regulated. SNAT activity of cultured chick pineal cells was obtained without ANAT after ammonium sulfate precipitation. ANAT activity was retained without SNAT activity after pre-incubation at 37 degrees C. Thus, each enzyme could be examined independently. Overlap in substrate specificity between the two enzymes was minimal. Kinetic analysis of the separated enzyme activities revealed that while SNAT operates via a random or ordered bi bi mechanism, ANAT catalysis occurs through a ping pong bi bi mechanism with substrate inhibition by acetyl-CoA. By size-exclusion chromatography, ANAT was confirmed to be 30-35 kDa, and SNAT was estimated at 15-20 kDa. Taken together, these results indicate that the two enzymes differ in their structure, reactivity, stability, and mechanism of catalysis.

Acetyl Coenzyme A↗
PubMed1995

Hepatitis A and the American traveler.

Morbidity from hepatitis A in American travelers may be greater than currently recorded. Risks for acquiring hepatitis A are widespread in developing countries, and a single lapse from appropriate hygiene for food or water is sufficient to cause infection. In Peace Corps personnel, a strictly regulated policy of immune serum globulin (ISG) at 4-month intervals reduced the rate of hepatitis A to 0.1-0.3 cases (from 1.6-2.1 cases)/100 persons/year. Data from United States Foreign Service personnel, for whom there is no mandated ISG policy, show hepatitis A to be the most commonly reported immunization-preventable infection; rates are 0.06-0.16/100 person-years, depending on the area of the world. With the availability of a hepatitis A vaccine, frequent travelers or those who stay long-term in hepatitis-endemic areas can attain higher antibody concentrations and much longer protection than is currently available from ISG.

Government Agencies↗
PubMed1995

Solubilization of receptors for pancreatic polypeptide from rat liver membranes.

We have previously identified, on rat liver microsomes and plasma membranes, proteins that bind pancreatic polypeptide (PP) with high affinity and specificity and that may serve as receptors for a hepatic effect of PP (J. Biol. Chem. 267: 9416-9421, 1992). Further characterization of these proteins requires the solubilization of receptors with conserved ability to bind PP selectively and efficiently. In this report, using 6 mM of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), we solubilized, from liver microsomes, receptors that bound PP with high affinity (dissociation constant 6.15 +/- 1.6 nM) and specificity (no interaction with the homologous peptides neuropeptide Y and peptide YY). Gel filtration chromatography showed different degrees of receptor aggregation related to different concentrations of CHAPS in the eluent. To characterize the structure of these solubilized receptors, the chemical cross-linker N-(5-azido-2-nitrobenzoyloxy)succinimide was used to covalently bind these receptors to radiolabeled PP, and the resulting PP-receptor complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A radioactive band with an apparent molecular weight (M(r)) of 46,000 was detected that was inhibited by unlabeled PP with a half-maximal inhibitory concentration of approximately 10(-8) M. It most likely reflected a PP receptor with an estimated M(r) of 42,000, excluding the molecular weight of PP. The migration of this complex was not affected by the reducing agent dithiothreitol, suggesting the absence of disulfide bonding. The solubilization and identification of a bioactive hepatic PP receptor will allow further characterization and purification of this receptor and will lead to the clarification of the interaction between PP and the digestive system.

Animals↗
PubMed1995

Risk factors for malaria among expatriates living in Kampala, Uganda: the need for adherence to chemoprophylactic regimens.

This investigation was conducted in response to a report of an increased number of malaria cases among United States Embassy personnel in Kampala, Uganda in the spring of 1992. The objectives of the investigation were to determine if an outbreak had occurred, to identify potential risk factors for malaria in this population, and to assess the effectiveness of various chemoprophylactic regimens. The risk of developing malaria during the first half of 1992 was more than six times greater than during the same time period in 1991 (relative risk [RR] = 6.6, 95% confidence interval [CI] = 1.6-27.8) and almost seven times greater than all the previous six years combined (RR = 6.8, 95% CI = 2.9-15.9). In this outbreak, children and young adults less than 20 years of age had more than a three-fold increase in risk (RR = 3.7, 95% CI = 0.7-19.8) than those in the 20-39-year-old age group. African-Americans had a six-fold increased risk compared with Caucasians (RR = 6.0, 95% CI = 1.6-22.7). Those who did not take any drug prophylaxis were 10 times more likely to develop malaria (RR = 10.0, 95% CI = 2.7-37.0) than those who took mefloquine, doxycycline, or chloroquine plus proguanil. In this setting, weekly mefloquine was 82% more effective, and chloroquine plus proguanil was 92% more effective than weekly chloroquine alone. This outbreak underscores the need for compliance with appropriate chemoprophylactic regimens in preventing malaria infection.

Adolescent↗