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Biomedical subjects

C L Moore

Publications and source records attributed to C L Moore.

At least 19 recordsLinked to original sources
PubMed2001

Gamma -secretase inhibitors repress thymocyte development.

A major therapeutic target in the search for a cure to the devastating Alzheimer's disease is gamma-secretase. This activity resides in a multiprotein enzyme complex responsible for the generation of Abeta42 peptides, precipitates of which are thought to cause the disease. Gamma-secretase is also a critical component of the Notch signal transduction pathway; Notch signals regulate development and differentiation of adult self-renewing cells. This has led to the hypothesis that therapeutic inhibition of gamma-secretase may interfere with Notch-related processes in adults, most alarmingly in hematopoiesis. Here, we show that application of gamma-secretase inhibitors to fetal thymus organ cultures interferes with T cell development in a manner consistent with loss or reduction of Notch1 function. Progression from an immature CD4-/CD8- state to an intermediate CD4+/CD8+ double-positive state was repressed. Furthermore, treatment beginning later at the double-positive stage specifically inhibited CD8+ single-positive maturation but did not affect CD4+ single-positive cells. These results demonstrate that pharmacological gamma-secretase inhibition recapitulates Notch1 loss in a vertebrate tissue and present a system in which rapid evaluation of gamma-secretase-targeted pharmaceuticals for their ability to inhibit Notch activity can be performed in a relevant context.

Amyloid Precursor Protein Secretases↗
PubMed2001

Prenatal diagnosis of mosaicism for triploidy and trisomy 13.

Mosaicism for trisomy 13 and triploidy was detected by amniocentesis performed at 18 weeks' gestation because of fetal anomalies. Pregnancy continued and a live-born male was delivered vaginally at 37 weeks. The infant had features common to both trisomy 13 and triploidy: intrauterine growth retardation (IUGR), small abnormal ears, cleft palate, and a small jaw. In addition, he had complete cutaneous syndactyly of fingers 3 and 4 and partial syndactyly of the toes, as seen in triploidy. Mixoploidy for trisomy 13 and triploidy was confirmed postnatally in blood, skin, and placenta. Examination of chromosome heteromorphisms and DNA markers suggested the presence of two maternal contributions in the triploid cell line. In addition, the extra chromosome 13 in the trisomic cell line was derived from the mother.

Adult↗
PubMed2001

Rna15 interaction with the A-rich yeast polyadenylation signal is an essential step in mRNA 3'-end formation.

In Saccharomyces cerevisiae, four factors [cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP)] are required for maturation of the 3' end of the mRNA. CF I and CF II are required for cleavage; a complex of PAP and PF I, which includes CF II subunits, participates in polyadenylation, along with CF I. These factors are directed to the appropriate site on the mRNA by two sequences: one A-rich and one UA-rich. CF I contains five proteins, two of which, Rna15 and Hrp1, interact with the mRNA through RNA recognition motif-type RNA binding motifs. Previous work demonstrated that the UV cross-linking of purified Hrp1 to RNA required the UA-rich element, but the contact point of Rna15 was not known. We show here that Rna15 does not recognize a particular sequence in the absence of other proteins. However, in complex with Hrp1 and Rna14, Rna15 specifically interacts with the A-rich element. The Pcf11 and Clp1 subunits of CF I are not needed to position Rna15 at this site. This interaction is essential to the function of CF I. A mutant Rna15 with decreased affinity for RNA is defective for in vitro RNA processing and lethal in vivo, while an RNA with a mutation in the A-rich element is not processed in vitro and can no longer be UV cross-linked to the Rna15 subunit assembled into CF I. Thus, the recognition of the A-rich element depends on the tethering of Rna15 through an Rna14 bridge to Hrp1 bound to the UA-rich motif. These results illustrate that the yeast 3' end is defined and processed by a mechanism surprisingly different from that used by the mammalian system.

3' Untranslated Regions↗
PubMed2001

Fip1 regulates the activity of Poly(A) polymerase through multiple interactions.

Fip1 is an essential component of the Saccharomyces cerevisiae polyadenylation machinery and the only protein known to interact directly with poly(A) polymerase (Pap1). Its association with Pap1 inhibits the extension of an oligo(A) primer by limiting access of the RNA substrate to the C-terminal RNA binding domain (C-RBD) of Pap1. We present here the identification of separate functional domains of Fip1. Amino acids 80 to 105 are required for binding to Pap1 and for the inhibition of Pap1 activity. This region is also essential for viability, suggesting that Fip1-mediated repression of Pap1 has a crucial physiological function. Amino acids 206 to 220 of Fip1 are needed for the interaction with the Yth1 subunit of the complex and for specific polyadenylation of the cleaved mRNA precursor. A third domain within amino acids 105 to 206 helps to limit RNA binding at the C-RBD of Pap1. Our data demonstrate that the C terminus of Fip1 is required to relieve the Fip1-mediated repression of Pap1 in specific polyadenylation. In the absence of this domain, Pap1 remains in an inhibited state. These findings show that Fip1 has a crucial regulatory function in the polyadenylation reaction by controlling the activity of poly(A) tail synthesis through multiple interactions within the polyadenylation complex.

Amino Acid Transport Systems↗
PubMed2000

Difluoro ketone peptidomimetics suggest a large S1 pocket for Alzheimer's gamma-secretase: implications for inhibitor design.

The final step in the generation of the amyloid-beta protein (Abeta), implicated in the etiology of Alzheimer's disease, is proteolysis within the transmembrane region of the amyloid precursor protein (APP) by gamma-secretase. Although considered an important target for therapeutic design, gamma-secretase has been neither well-characterized nor definitively identified. Previous studies in our laboratory using substrate-based difluoro ketone and difluoro alcohol transition-state analogue inhibitors suggest that gamma-secretase is an aspartyl protease with loose sequence specificity. To further characterize the active site of gamma-secretase, we prepared a series of difluoro ketone peptide analogues with varying steric bulkiness in the P1 position and tested the ability of these compounds to inhibit Abeta production in APP-transfected cells. Incorporation of bulky, aliphatic P1 side chains, such as sec-butyl or cyclohexylmethyl, led to increased gamma-secretase inhibitory potency, suggesting a large S1 pocket to accommodate these substituents and providing further evidence for loose sequence specificity. The cyclohexylmethyl P1 substituent allowed N-terminal truncation to a low-molecular-weight compound (<600 Da) that effectively blocked Abeta production (IC(50) approximately 5 microM). This finding suggests that optimal S1 binding may allow the development of potent inhibitors with ideal pharmaceutical properties. Moreover, a difluoro alcohol analogue with a cyclohexylmethyl P1 substituent was equipotent with its difluoro ketone counterpart, providing strong evidence that gamma-secretase is an aspartyl protease. All new analogues inhibited total Abeta and Abeta(42) production with the same rank order of potency and increased Abeta(42) production at low concentrations, providing further evidence for distinct gamma-secretases that are nevertheless closely similar with respect to active site topology and mechanism.

Alzheimer Disease↗
PubMed2000

Structure of yeast poly(A) polymerase alone and in complex with 3'-dATP.

Polyadenylate [poly(A)] polymerase (PAP) catalyzes the addition of a polyadenosine tail to almost all eukaryotic messenger RNAs (mRNAs). The crystal structure of the PAP from Saccharomyces cerevisiae (Pap1) has been solved to 2.6 angstroms, both alone and in complex with 3'-deoxyadenosine triphosphate (3'-dATP). Like other nucleic acid polymerases, Pap1 is composed of three domains that encircle the active site. The arrangement of these domains, however, is quite different from that seen in polymerases that use a template to select and position their incoming nucleotides. The first two domains are functionally analogous to polymerase palm and fingers domains. The third domain is attached to the fingers domain and is known to interact with the single-stranded RNA primer. In the nucleotide complex, two molecules of 3'-dATP are bound to Pap1. One occupies the position of the incoming base, prior to its addition to the mRNA chain. The other is believed to occupy the position of the 3' end of the mRNA primer.

Binding Sites↗
PubMed2000

FAD mutations in presenilin-1 or amyloid precursor protein decrease the efficacy of a gamma-secretase inhibitor: evidence for direct involvement of PS1 in the gamma-secretase cleavage complex.

To investigate the mechanism of regulation of Ass production by familial Alzheimer's disease (FAD)-linked presenilin 1 (PS1), we used a cell-free system that allows de novo Ass generation to examine whether PS1 participates directly in the gamma-secretase reaction. Optimal Ass generation in vitro was achieved at mildly acidic pH and could be inhibited by the aspartyl protease inhibitor pepstatin A, consistent with the suggestion that gamma-secretase is an aspartyl protease. Dominant negative mutations of the critical transmembrane aspartates in PS1 or full deletion of PS1 did not alter the maturation of APP in the secretory pathway. Instead, PS1 had a direct effect on the inhibition of Ass production by a designed peptidomimetic inhibitor: the inhibition was significantly less effective in cells expressing FAD-causing mutations in either APP or PS1 than in cells expressing the wild-type proteins. Taken together, these findings suggest that PS1 participates physically in a complex with APP during the gamma-secretase cleavage event.

Alzheimer Disease↗
PubMed2000

Transition-state analogue inhibitors of gamma-secretase bind directly to presenilin-1.

The beta-amyloid precursor protein (beta-APP), which is involved in the pathogenesis of Alzheimer's disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown gamma-secretases. Here we show that an affinity reagent designed to interact with the active site of gamma-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer's and are known mediators of gamma-secretase cleavage of both beta-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of gamma-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer's disease.

Affinity Labels↗
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

Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II.

The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

Cyclin-Dependent Kinases↗
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

Quality of care for Medicare patients hospitalized with heart failure in rural Georgia.

BACKGROUND: It is not known whether quality of care for congestive heart failure (CHF) at rural hospitals is similar to that in larger, urban hospitals. METHODS: We reviewed hospital charts for 310 Medicare patients hospitalized with CHF at six hospitals in rural Georgia. RESULTS: Of the 310 patients, 101 (33%) had left ventricular systolic dysfunction, and 60 (19%) had preserved systolic function. Information on left ventricular function was not available for 48% (range, 29% to 87% across the six hospitals). Among patients with systolic dysfunction, 77% were prescribed an angiotensin converting enzyme (ACE) inhibitor at discharge, and 73% were prescribed digoxin. However, the mean daily ACE inhibitor dose was only 48% of the recommended target dose. Only 30% of all patients with atrial fibrillation were prescribed warfarin. CONCLUSIONS: Overall quality of care for CHF at rural hospitals appears similar to that in other settings, though many patients may not receive evaluation of ventricular function.

Aged↗
PubMed1998

Effects of lactation on sodium intake in Fischer-344 and Long-Evans rats.

Rats of most strains are attracted to salt in low concentrations, and this attraction is increased by pregnancy and lactation. Nonreproducing Fischer-344 (F344) rats are unusual in that they avoid saline at all concentrations, raising the question of whether lactation alters their sodium appetite. Therefore, lactating and cycling Long-Evans and F344 rats were compared in their relative consumption of water and several concentrations of saline. We found that Long-Evans rats preferred saline, but F344 rats preferred plain water. In comparison with cycling rats, lactating Long-Evans rats markedly increased saline intake whereas F344 rats exhibited only a modest increase. Lactating rats of both strains increased total fluid intake, but this increase was primarily from water in F344 rats and from saline in Long-Evans rats. It was concluded that the aversion to saline characteristic of nonreproducing adult F344 rats continues during lactation. Their aversion to sodium in pup urine may account for the low levels of maternal anogenital licking observed in the F344 strain.

Analysis of Variance↗
PubMed1998

Cholesterol concentrations and cardiovascular reactivity to stress in African American college volunteers.

Cholesterol levels and cardiovascular responses to emotionally arousing stimuli were examined in 60 healthy African American males and females. Cardiac output, stroke volume, contractile force, heart rate, and blood pressure were measured as the participants viewed two racially noxious scenes on videotape. Total serum cholesterol, high-density lipoproteins (HDL), low-density lipoproteins (LDL), and triglycerides were measured within 2 weeks of viewing the scenes. Multiple regression analysis showed that LDL and HDL were significant predictors of blood pressure responses. A correlation analysis revealed that total serum cholesterol and LDL were positively correlated with stroke volume, contractile force, and blood pressure reactivity. A possible relationship among stress, beta-adrenergic activity, and nonmetabolized free fatty acids is discussed. These findings suggest that cardiovascular reactivity to stress may be a new risk factor for heart and vascular diseases.

Adolescent↗
PubMed1998

Photoprotection of mammalian acid-soluble collagen by cuttlefish sepia melanin in vitro.

Several important clinical conditions can result in close association between the pigment melanin and dermal collagen. Because melanin and its precursors can be chemically reactive in ground and excited states, it is important to know whether the resulting melanin-collagen interaction results in photoprotection or photoaggression. Acidic and neutral air-saturated collagen suspensions (0.033%) were irradiated with 0-2.6 x 10(4) J/m2 UVC or with 0-83 x 10(4) J/m2 solar-simulating UV radiation (SSR). Photochemical destruction of a photolabile collagen fluorophore (lambda em 360 nm) and collagen chain degradation were monitored as functions of irradiation time in the presence and absence of added (0-100 micrograms) sepia eumelanin. Melanin retarded collagen photodamage but did not qualitatively alter the fluorescence fading kinetics. Both H2O2 and O2-. can be produced by UV irradiation of eumelanin. Added H2O2 and KO2 destroyed collagen fluorescence and caused 50% chain degradation at ca 10-20-fold molar excess. Previous studies have demonstrated that eumelanins efficiently scavenge O2-.. We demonstrated that eumelanin also efficiently scavenges H2O2 as evidenced by its ability to (a) compete with scopoletin for peroxide uptake and (b) directly take up H2O2 through a dialysis bag. The latter observation suggests that peroxide scavenging could occur in vivo by melanin sequestered in melanophages. Thus, neither UV-generated O2-. nor H2O2 are likely to be present in concentrations high enough to cause measurable collagen damage. Absorption and/or scattering of excitation radiation away from the target chromophore appears to be the primary photoprotection mechanism, although scavenging of active O2 intermediates may play an important, if subtle role.

Animals↗
PubMed1997

Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3'-end formation in yeast.

In yeast, four factors (CF I, CF II, PF I, and PAP) are required for accurate pre-mRNA cleavage and polyadenylation in vitro. CF I can be separated further into CF IA and CF IB. Here we show that CF IB is the 73-kD Hrp1 protein. Recombinant Hrp1p made in Escherichia coli provides full CF IB function in both cleavage and poly(A) addition assays. Consistent with the presence of two RRM-type motifs, Hrp1p can be UV cross-linked to RNA, and this specific interaction requires the (UA)6 polyadenylation efficiency element. Furthermore, the CF II factor enhances the binding of Hrp1p to the RNA precursor. A temperature-sensitive mutant in HRP1 yields mRNAs with shorter poly(A) tails when grown at the nonpermissive temperature. Genetic analyses indicate that Hrp1p interacts with Rna15p and Rna14p, two components of CF 1A. The HRP1 gene was originally isolated as a suppressor of a temperature-sensitive npl3 allele, a gene encoding a protein involved in mRNA export. Like Npl3p, Hrp1p shuttles between the nucleus and cytoplasm, providing a potential link between 3'-end processing and mRNA export from the nucleus.

Amino Acid Sequence↗