Conformational preference for the binding of biaryl substrates and inhibitors to the active site of phenylethanolamine N-methyltransferase

We have previously described regions of steric bulk tolerance in the aromatic-ring binding site of phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) for phenylethanolamine substrates and alpha-methylbenzylamine inhibitors. For bound substrates, this region is located in the vicinity of the para position of the aromatic ring, while for bound alpha-methylbenzylamine inhibitors, it is located in the region complementary to the meta position. In the present study, we sought to determine the preferred conformation of the biaryl portion of (m-phenylphenyl)- and (p-phenylphenyl)ethanolamine (4 and 5, respectively) as well as for m-phenyl- and p-phenyl-alpha-methylbenzylamine (7 and 8, respectively) for PNMT active site interactions. Planar derivatives of 4, 5, 7, and 8 were obtained through the synthesis of 2-(1-fluorenyl)-2-hydroxyethylamine (9), 2-(2-fluorenyl)-2-hydroxyethylamine (10), 1-(1-fluorenyl)ethylamine (11), and 1-(2-fluorenyl)ethylamine (12). The four fluorene derivatives were examined for in vitro activity as substrates and inhibitors of the PNMT-catalyzed reaction. As in the case of 4, 5, 7, and 8, we have observed a positional preference for the alkylamine side chain with respect to the biphenyl skeleton present in 9-12. Thus, fluorenylethanolamine 10 ("p-biphenyl") displays a Michaelis constant (Km = 26 microM) that is approximately 10 times lower than that for 9 ("m-biphenyl", Km = 297 microM); in the alpha-methylbenzylamine inhibitors, fluorenyl derivative 11 ("m-biphenyl", Ki = 4.14 microM) is approximately 40 times better than 12 ("p-biphenyl", Ki = 185 microM) for in vitro inhibition of PNMT. In each case, conformational restriction of the biaryl system present in 4, 5, 7, and 8, such that the aromatic rings are coplanar, resulted in enhanced affinity for the PNMT active site. Thus, conformational restriction of ethanolamine 5 (Km = 82 microM) as in 10 (Km = 26 microM) and alpha-methylbenzylamine 7 (Ki = 89 microM) as in 11 (Ki = 4.14 microM) leads, in each case, to a stronger enzyme-ligand dissociable complex. These results, in conjunction with others from these laboratories, indicate that the PNMT active site beyond the zone that interacts with the central aromatic ring portion of phenylethanolamine substrates and alpha-methylbenzylamine inhibitors is essentially a flat, hydrophobic pocket.     

Amount and distribution of dietary protein affects clinical response to levodopa in Parkinson's disease

Reducing dietary protein improves the effectiveness of levodopa (LD) but the most effective distribution of a low-protein diet (0.8 g/kg) is unclear. We compared a 1.6 g/kg protein diet, a 0.8 g/kg diet with protein evenly distributed between meals, and a 0.8 g/kg diet with protein restricted to the evening meal in 5 parkinsonian patients with motor fluctuations. We monitored clinical response, plasma LD, and plasma large amino acids (LNAAs) hourly throughout the day. Mean "on" times were 51% (1.6 g/kg diet), 67% (0.8 g/kg evenly distributed), and 77% (0.8 g/kg restricted). Hourly averages of plasma LD did not differ between the diets. The mean plasma LNAAs were 732 nmol/ml (1.6 g/kg diet), 640 (0.8 g/kg distributed), and 542 (0.8 g/kg restricted), and the diurnal pattern reflected the distribution of protein intake. In conclusion, the amount and distribution of dietary protein affect clinical response to LD. These effects are not related to LD absorption but are explained by the variation in plasma LNAAs.     

Image-directed percutaneous biopsies with a biopsy gun

Core tissue for histologic study is believed by many pathologists to be more diagnostic than material from needle aspiration. Recently, a biopsy "gun" has been introduced, which simplifies core biopsies. With this device, 182 biopsies of multiple anatomic sites were performed with ultrasonic, computed tomographic, and fluoroscopic guidance and 18-gauge needles. High-quality histopathologic specimens were obtained in 177 of the biopsies, and diagnostic target tissue was obtained in 167. Only three significant complications occurred: one bleeding complication that required transfusion and two cases of pneumothorax that necessitated placement of chest tubes. The biopsy gun eliminated the disjointed movements of conventional "skinny" needle biopsies, and none of the samples demonstrated significant "crush" artifact or obscuring blood, problems that are commonly associated with manual biopsy techniques. Patient discomfort was decreased with this system compared with that of manual biopsies, and the total procedure time was reduced. Because of these distinct advantages, the authors now use the biopsy gun exclusively for all percutaneous biopsies and recommend that other institutions consider the use of this biopsy method.     

Polymorphisms of the equine major histocompatibility complex class II DRA locus

The full extent of the polymorphism of ELA-DRA in Equidae is not yet known. Given the apparent differences in DRA polymorphisms between Equidae and other species, the aims of this study were to more fully characterize ELA-DRA, determine the extent of gene polymorphism and establish the allele-frequency distribution. An allele reference panel for the second exon of ELA-DRA was established by sequence-based typing of 69 equine DNA samples consisting of various breeds of domestic horse (Equus caballus), together with donkeys (Equus asinus), Grant's zebras (Equus boehmi) and one onager (Equus hemionus). Five of the six previously reported alleles detected using single-strand conformation polymorphism were found: ELA-DRA*0101, ELA-DRA*0201, ELA-DRA*0301, ELA-DRA*0501 (Albright-Fraser DG et al. Polymorphism of DRA among equids. Immunogenetics 1996: 43: 315-7) and ELA-DRA*0601 (GenBank accession number AF5419361). In addition to the previously reported alleles, five novel ELA-DRA alleles were detected within the ELA-DRA allele reference panel. One of these was identified in E. caballus (ELA-DRA*JBH11), one in E. boehmi and E. hemionus (ELA-DRA*JBZ185) and three in E. asinus (ELA-DRA*JBD3, ELA-DRA*JBD17 and ELA-DRA*JBH45). A total of 565 equine DNA samples were screened using reference-strand-mediated conformation analysis, a double-stranded conformation-based mutation detection system that can be used to type existing ELA-DRA alleles and identify new variants. Based on our findings, at least 11 ELA-DRA alleles are now known to exist, and this level of polymorphism at the DRA locus appears to be unique to the genus Equus. Both the previously reported alleles and the new alleles displayed a species-specific distribution.     

Early diagnosis of relapse in acute myeloblastic leukemia: Serologic detection of leukemia-associated antigens in human marrow.

We tested serial bone-marrow samples from 47 adults with acute myeloblastic leukemia in remission for reactivity with heteroantiserums to leukemia-associated antigens, to determine whether imminent relapse could be detected in patients with acute leukemia. Of 26 patients who relapsed by standard morphologic criteria, 21 had increased immunoreactivity of bone marrow for one to six months (mean, 3.7 months) before relapse. High concordance was observed between a positive test and relapse during the period of study (chi-square = 27.53, P less than 0.001). The median time to relapse after a positive test was four months, as compared with the median remission duration of 19 months for the whole group (P less than 0.02, Peto's log-rank analysis). Serologic detection of leukemia-associated antigens in marrow may be a reliable indicator of imminent relapse in acute myeloblastic leukemia.     

Isolation of the etiologic agent of human granulocytic ehrlichiosis from the white-footed mouse (Peromyscus leucopus)

We examined white-footed mice (Peromyscus leucopus) from Minnesota for infection with the etiologic agent of human granulocytic ehrlichiosis (HGE). From April to September 1997, we collected P. leucopus from Washington County, Minnesota, an area enzootic for HGE. Blood was cultivated in HL60 cells for isolation of the HGE agent. Of 59 mice examined, only a single mouse was culture positive for the HGE agent. The 16S ribosomal DNA sequence of the isolate was determined to be identical to that of the HGE agent. The isolate was reactive with monoclonal antibodies to the 44-kDa antigen of the HGE agent and was infectious for laboratory mice.     

Self-reported urogenital symptoms in postmenopausal women: Women's Health Initiative

OBJECTIVE: To examine the prevalence and correlates of self-reported urogenital symptoms (dryness, irritation or itching, discharge, dysuria) among postmenopausal women aged 50-79. DESIGN: A cross-sectional analysis based on n=98,705 women enrolled in the US-based Women's Health Initiative observational study and clinical trials. Urogenital symptoms, symptom severity (mild, moderate, severe), and all covariates were self-reported through questionnaires at enrollment. Prevalence rates of each urogenital symptom were examined and logistic regression was used to identify potential correlates. RESULTS: Prevalence rates for each symptom were: dryness, 27.0%; irritation or itching, 18.6%; discharge, 11.1%; and dysuria, 5.2%. Four factors were correlated with two or more symptoms: Hispanic ethnicity (adjusted odds ratio (AOR)=2.1-3.1 versus white women across all symptoms), obesity (AOR=2.2 severe discharge versus none, AOR=3.6 severe irritation/itching versus none), treated diabetes (pills or shots) compared to no diabetes (AOR=2.4 severe dysuria versus none, AOR=3.2 severe irritation/itching versus none), and vaginal cream HRT/ERT compared to those who never used HRT/ERT (AOR=4.4 severe dryness versus none, AOR=4.6 severe irritation/itching versus none). Factors not associated with the symptoms included sexual activity, age, years since menopause, current smoking, marital status, gravidity, and natural versus surgical menopause. CONCLUSIONS: This is the first report to document urogenital symptoms by race/ethnicity among an exclusively postmenopausal population. We found an elevated prevalence of urogenital symptoms among women who are Hispanic, obese, and/or diabetic. Confirmation of our findings in these subgroups, and, if confirmed, analysis on why these populations are at greater risk, are areas for future research.     

Fatal cerebral phaeohyphomycosis due to Curvularia lunata in an immunocompetent patient

Curvularia infections in humans are relatively uncommon despite the ubiquitous presence of this soil-dwelling dematiaceous fungus in the environment. Originally thought to be solely a pathogen of plants, Curvularia has been described as a pathogen of humans and animals in the last half-century, causing respiratory tract, cutaneous, and corneal infections. Only three previous cases of central nervous system involvement by Curvularia have been documented in the medical literature. We report a fatal case of cerebral Curvularia infection in which there was no known history of immunocompromise or prior respiratory tract or sinus infection in the patient.     

A family study of hydrocephalus resulting from aqueduct stenosis.

Stenosis of the aqueduct of Sylvius accounts for about one third of cases of congenital hydrocephalus. At least 32 families have been reported in which the aqueduct stenosis is inherited in an X linked fashion. In half of these families, flexed adducted thumbs were noted in some affected family members. Occasionally other male members were mentally retarded, suggesting limited expression of the gene. The problem of giving genetic advice to an isolated, clinically unremarkable, case of aqueduct stenosis remains, so a family study was undertaken based on 24 such cases seen at The Hospital for Sick Children over a 19-year period. There were 15 male and nine female index patients. The diagnosis was confirmed in all cases by air encephalogram. One boy had a radial club hand and another developed clasped thumbs secondary to spasticity. No cases had hyaloidoretinal dysplasia. The 15 boys had 18 brothers and 19 sisters, of whom one sister was similarly affected. The nine girls had 12 sibs, none of whom was affected. This study, combined with a similar study in the USA, suggests that the empirical risk of recurrence of a sporadic case of aqueduct stenosis is about 4.5%.