Striatal 6-[18F]fluorodopa accumulation after combined inhibition of peripheral catechol-O-methyltransferase and monoamine oxidase type B: differing response in relation to presynaptic dopaminergic dysfunction

The aim was to investigate the effects of inhibition of monoamine oxidase type B (MAO-B) with selegiline alone and the combined inhibition of peripheral catechol-O-methyltransferase (COMT) with entacapone and MAO-B with selegiline on striatal 6-[18F]fluorodopa (FDOPA) accumulation, and whether the effect of entacapone + selegiline on FDOPA uptake differed depending on the severity of the presynaptic dopaminergic dysfunction. Thus, eight healthy controls, eight de novo patients with Parkinson's disease (PD), and 18 levodopa-treated PD patients were investigated with positron emission tomography (PET). Half of the subjects in each population belonged to the selegiline group and half to the entacapone + selegiline group. Both groups were studied twice with PET using FDOPA. After the first (baseline) FDOPA PET investigation, both groups were on 2 weeks of selegiline treatment, 10 mg daily. Thereafter, the second FDOPA PET was performed for all subjects with a premedication administered 60 min before the PET imaging; one group received 10 mg of selegiline, and the other group received a single 400 mg dose of entacapone coadministered with 10 mg of selegiline. Selegiline treatment alone had no significant influence on striatal FDOPA metabolism. The FDOPA accumulation, expressed as striatal-to-occipital ratios and modified decarboxylation coefficients (k3R0), increased significantly after entacapone + selegiline administration in all subject populations. The FDOPA uptake rate constant (Ki) remained virtually unchanged in controls and in de novo patients but decreased significantly in levodopa-treated PD patients after entacapone + selegiline intake. Entacapone + selegiline administration did not influence significantly the unidirectional blood-to-brain clearance for FDOPA (K1D) or the relative dopadecarboxylase activity (k3D). The changes in the studied parameters after entacapone + selegiline administration probably reflect the effects of entacapone, since entacapone alone has caused similar changes in previous PET studies. Response in FDOPA accumulation to entacapone + selegiline was higher in controls and de novo patients compared with levodopa-treated PD patients. The milder response in levodopa-treated patients might reflect the reduced ability of the degenerated dopaminergic neurons to utilize the prolonged FDOPA availability, produced by entacapone.     

Exploratory studies on azole carboxamides as nucleobase analogs: thermal denaturation studies on oligodeoxyribonucleotide duplexes containing pyrrole-3-carboxamide

In order to study base pairing properties of the amide group in DNA duplexes, a nucleoside analog, 1-(2'-deoxy-beta-D-ribofuranosyl)pyrrole-3-carboxamide, was synthesized by a new route from the ester, methyl 1-(2'-deoxy-3',5'-di-O-p -toluoyl-beta-D-erythro-pentofuranosyl)pyrrole-3-carboxylate, obtained from the coupling reaction between 1-chloro-2-deoxy-3,5-di-O -toluoyl-d-erythropentofuranose and methyl pyrrole-3-carboxylate by treatment with dimethylaluminum amide. 1-(2'-Deoxy-beta-D-ribofuranosyl)pyrrole-3-carboxamide was incorporated into a series of oligodeoxyribonucleotides by solid-phase phosphoramidite technology. The corresponding oligodeoxyribonucleotides with 3-nitropyrrole in the same position in the sequence were synthesized for UV comparison of helix-coil transitions. The thermal melting studies indicate that pyrrole-3-carboxamide, which could conceptually adopt either a dA-like or a dI-like hydrogen bond conformation, pairs with significantly higher affinity to T than to dC. Pyrrole-3-carboxamide further resembles dA in the relative order of its base pairing preferences (T >dG >dA >dC). Theoretical calculations on the model compound N-methylpyrrole-3-carboxamide using density functional theory show little difference in the preference for a syntau versus anti conformation about the bond from pyrrole C3 to the amide carbonyl. The amide groups in both the minimized antitau and syntau conformations are twisted out of the plane of the pyrrole ring by 6-14 degrees. This twist may be one source of destabilization when the amide group is placed in the helix. Another contribution to the difference in stability between the base pairs of pyrrole-3-carboxamide with T and pyrrole-3-carboxamide with C may be the presence of a hydrogen bond in the former involving an acidic proton (N3-H of T).     

Characterization of a point mutation in the hormone binding domain of the estrogen receptor from an breast tumor

It is clear that growth of the MCF-7 breast cancer cell line is stimulated by estrogen and when estrogen is removed, growth slows. We have observed a tumor derived from MCF-7 cells that grows in athymic mice in the absence of estrogen stimulation. We hypothesized that a mutation in the estrogen receptor (ER) could be responsible for this constitutive growth. Using single stranded conformational polymorphism (SSCP) and DNA sequencing analysis, we have identified an ER containing a point mutation at position 415 (gly to val) within the hormone binding domain. The functional activity of this mutant was assessed in vitro and in vivo. Using transient transfection into an ER negative breast cancer cell with an ERE luciferase reporter gene, we found that both the wild-type and mutant receptors have similar efficacy. Additionally, the estrogenic responses were blocked by antiestrogens in a concentration related manner. We also found that tumors with the mutant receptor show similar growth response in athymic mice as wild-type: stimulation with estradiol and inhibition with antiestrogens. We conclude that the point mutation at position 415 (gly to val) is not responsible for constitutive growth.     

The antiproliferative and cell cycle effects of 5,6,7, 8-tetrahydro-N5,N10-carbonylfolic acid, an inhibitor of methylenetetrahydrofolate dehydrogenase, are potentiated by hypoxanthine

5,6,7,8-Tetrahydro-N5,N10-carbonylfolic acid (LY354899) has been demonstrated to inhibit the dehydrogenase activity of C1-tetrahydrofolate synthase. This compound was only moderately antiproliferative toward CCRF-CEM lymphocytic leukemia cells in culture, but induced apoptosis after long incubation times. Slightly greater potency was observed in CEM cells adapted to grow in low folate media. Cell cycle alterations induced by LY354899 were unique relative to antifolates that inhibit either the purine or thymidine de novo biosynthetic pathways. Based on the observed changes in DNA content, we hypothesized that inhibition of the dehydrogenase resulted in two temporally distinct events: the first was a purineless-like effect and the second was a thymineless-like effect that resulted in apoptosis. To test this hypothesis, we combined LY354899 with the purine salvage metabolite, hypoxanthine. This combination resulted in an earlier and more dramatic apoptotic response, indicating that the thymineless effect had been potentiated. Biochemical analysis of ribo- and deoxyribonucleoside triphosphates confirmed that inhibition of the dehydrogenase activity initially resulted in decreased pools of deoxypurines and deoxypyrimidines, followed 16 hr later by an increase in deoxyadenosine triphosphate (dATP) and a further decrease in deoxythymidine triphosphate (dTTP). These studies demonstrate that the inhibition of the dehydrogenase activity of C1-tetrahydrofolate synthase may represent a viable target for the development of novel antifolates. The results are discussed in terms of deoxypurine and deoxypyrimidine biosynthesis.     

Merlin, the neurofibromatosis type 2 gene product, and beta1 integrin associate in isolated and differentiating Schwann cells

Neurofibromatosis type 2, a disease characterized by the formation of multiple nervous system tumors, especially schwannomas, is caused by mutation in the gene-encoding merlin/schwannomin. The molecular mechanism by which merlin functions as a tumor suppressor is unknown, but is hypothesized to involve plasma membrane and cytoskeleton interaction. Several merlin antibodies were used to study merlin expression, localization, and protein association in primary cultures of rat sensory neurons, Schwann cells (SCs), and SCs grown with neurons (SC/N cultures) before and during differentiation into myelinating cells. Western blot analysis revealed that neurons predominantly expressed a 68-kD protein, but SCs expressed two additional 88- and 120-kD related proteins. Extensive immunological characterization demonstrated that the 88-kD protein shared three domains with the 68-kD merlin protein. Western blot analysis of soluble and insoluble culture fractions demonstrated that the majority of merlin and related proteins were soluble in isolated SCs and undifferentiated SC/N cultures, but became insoluble in myelinating SC/N cultures. Double immunofluorescence staining suggested that merlin translocated from the perinuclear cytoplasm in undifferentiated SCs to the subplasmalemma in differentiating SCs and partially colocalized with beta1 integrin. Finally, beta1 integrin antibody coimmunoprecipitated 68-kD merlin from isolated SC and undifferentiated SC/N cultures, but predominantly the 88-kD protein from differentiating SC/N cultures. Together, these results provide evidence that merlin interacts with beta1 integrin and that merlin localization changes from a cytosolic to cytoskeletal compartment during SC differentiation.     

Tuberculin skin testing of hospitalized patients

OBJECTIVES: We sought to define the prevalence of tuberculin skin test (TST) positivity in a group of newly hospitalized patients, to identify risk factors for positive tests, and to examine the impact of testing on infection control practices. DESIGN: Unblinded cohort study over 5 days in July 1992. SETTING: A 1,000-bed university-affiliated hospital. PATIENTS: All patients admitted (excluding obstetric patients and newborns) were interviewed. Patients without a history of tuberculosis (TB) or a positive TST were offered a TST with Candida and tetanus controls. RESULTS: Of 346 patients offered the test, 21 (6%) had a prior history of TB or a positive TST, and 36 (10%) declined to participate; 279 of the remaining 289 completed the study. Anergy was demonstrated in 94 (33.7%) of 279 patients. New positive TSTs were identified in 19 (10.3%) of 185 nonanergic patients. Of the 19 TST-positive patients, 6 (32%) had infiltrates on chest radiographs and were evaluated for active TB. One patient was treated empirically for active TB, and five received isoniazid prophylaxis. Risk factors for a new positive TST included age (odds ratio [OR], 1.56 per decade of life; P = .021), African American race (OR, 4.81; P = .008), alcohol abuse (OR, 5.53; P = .005), and peptic ulcer disease (OR, 4.53; P = .017). Risk factors for anergy included admission to a surgical service (OR, 2.1; P = .006), current use of steroids (OR, 2.65; P = .005), and human immunodeficiency virus (HIV) infection (OR, undefined; P = .034). CONCLUSIONS: Despite a high rate of anergy, routine tuberculin skin testing identified a substantial number of patients with TB infection who might otherwise have gone unrecognized.     

Attentional processing of "unattended" flankers: evidence for a failure of selective attention

The objective of this study was to investigate the interaction between sulfogalactosylceramide (SGC) and dimyristoylphosphatidylcholine (DMPC) in a mixed model liposomal system (molar ratio SGC:DMPC, 2:3). Structural and dynamic changes of the liposome components were monitored by Fourier-transform infrared spectroscopy (FTIR). Thermotropic FTIR analysis of the mixed liposomes showed a single gel/liquid crystalline phase transition, centered at approximately 42 degrees C. Spectral changes of the amide and ester C = O bands arising from functional groups at the interfacial region indicated a reduced hydrogen bonding of these groups in the mixed liposomes. Pressure-tuning FTIR of mixed liposomes showed that the methylene chains of SGC and DMPC were more orientationally disordered than those of the individual lipid SGC liposomes or DMPC liposomes. These results suggest that the mixed liposomes (molar ratio SGC:DMPC, 2:3) consisted of a homogeneous mixture of SGC and DMPC molecules in which mutual shielding reduced hydrogen bonding in the interfacial region, with a concurrent increase in the orientational disorder of the hydrocarbon chains of both SGC and DMPC.     

A pilot study of soluble adhesion molecules as surrogate markers for acute liver allograft rejection

OBJECTIVE: The purpose of this study was to determine the degree to which the number and angular disparity of component projections influence depth discrimination with tuned-aperture computed tomography. STUDY DESIGN: Groups of three tiny steel spheres served as fiducial references on and in four partially edentulous mandibles. Two spheres were attached to the facial and lingual surfaces of each mandible, and the third was fixed in the apical region of an open tooth socket. Errors in estimates of the depth of the apically positioned sphere relative to the other two spheres were determined from three-dimensional tuned-aperture computed tomography reconstructions. These data were compared with actual measurements produced independently with an optical micrometer. Multiple projections required by the tuned-aperture computed tomography reconstruction algorithm were produced from radially symmetric exposures bearing angular disparities of 5, 15, 30, and 45 degrees. The number of symmetrically dispersed projections per tuned-aperture computed tomography reconstruction likewise was varied systematically (2, 4, 8, 12, and 16 projections). These variables were manipulated through the use of a balanced factorial design. Depth estimates were performed by trained observers; the estimates were based on the determination of tuned-aperture computed tomography slices perceived as imaging the respective apical spheres in sharpest focus. Specimen and observer effects were also considered as independent variables. Resulting data were normalized by logarithmic transformation and analyzed statistically by analysis of variance. RESULTS: Significant differences (p < 0.005) were demonstrated for angular disparity and specimen effects, but the number of projections and the effect of the observer were not found to be statistically significant. CONCLUSIONS: In dentistry, angular disparities of 15 degrees or greater should be used when tuned-aperture computed tomography is being applied to diagnostic tasks requiring maximal depth discrimination accuracy.     

Application of FISH for in situ detection and quantification of DNA breakage

(TTAGGG)n sequence repeats in human telomeres and in Chinese hamster interstitial centromeric areas were digested in situ with exonuclease III (ExoIII) and exonuclease Bal 31. Incubation with AluI was performed beforehand to increase DNA breaks near telomere sequence areas. DNA removal at these specific regions was quantified by digital image analysis of the fluorescence in situ hybridization signal produced by a telomeric probe. Exonuclease III was 2.6 times more active in interstitial than in terminal telomeric sequence areas. Exonuclease Bal 31 was 2.3 times more effective in terminal than in interstitial telomeric sequence regions. These results support the hypothesis that chromatin is differentially organized in both telomeric sequence areas, despite their similar DNA composition.