Genomic organization of a human 5beta-reductase and its pseudogene and substrate selectivity of the expressed enzyme

The enzyme 5beta-reductase catalyzes the reduction of the 4-ene of 3-ketosteroids, converting them into 5beta-dihydro-3-ketosteroids and, thus, could be involved in the metabolism of 4-cholestene-3-one, progesterone, 17~-hydroxyprogesterone, aldosterone, corticosterone, cortisol, 4-androstenedione, and testosterone. In this study, we report the genomic structure of a human 5beta-reductase gene, its tissue distribution, the characterization of an intronless pseudogene and the substrate selectivity of the enzyme. The gene coding for the active 5beta-reductase contains nine exons like most members of the aldo-keto reductase family, but the sequence covered by the gene, more than 42 kb, is much longer than the sequence of other members of this family. There are many large introns, especially introns 3, 4 and 7 that span approx. 7 kb, and intron 1 that contains more than 10 kb. Northern blot analysis showed three band sizes of 1.3, 2.2 and 2.7 kb. The 1.3 and 2.7 kb bands are highly expressed in the liver while weaker 2.2 and 1.3 kb bands have been observed in the testis and colon, respectively. We also identified an intronless gene having 86% homology with the 5beta-reductase cDNA sequence. Since its sequence contains many stop codons, this gene is most probably a pseudogene. To determine more precisely the substrate selectivity of the enzyme, we established a stable cell line expressing human 5beta-reductase in transformed embryonic kidney (HEK-293) cells. The transfected cells efficiently catalyze the transformation of progesterone, androstenedione, 17alpha-hydroxyprogesterone and testosterone. However, they catalyze much less efficiently the transformation of compounds containing an 11beta-hydroxy group, such as aldosterone, corticosterone and cortisol. In addition to its role in cholesterol catabolism, it is well recognized that 5beta-reductase inactivates active androgens. Indeed, 5beta-dihydrotestosterone (5beta-DHT), the product of the reduction of testosterone by 5beta-reductase, is not active while its 5~-isomer (DHT) is the most potent natural androgen. Recent findings show that 5beta-pregnanes are active ligands in the induction of CYP3A through the orphan receptor hPAR. Our results thus open an opportunity for studying the new role of 5beta-reductase in the formation of a new type of active steroids.     

Kinetochores prevent repair of UV damage in Saccharomyces cerevisiae centromeres

Centromeres form specialized chromatin structures termed kinetochores which are required for accurate segregation of chromosomes. DNA lesions might disrupt protein-DNA interactions, thereby compromising segregation and genome stability. We show that yeast centromeres are heavily resistant to removal of UV-induced DNA lesions by two different repair systems, photolyase and nucleotide excision repair. Repair resistance persists in G(1)- and G(2)/M-arrested cells. Efficient repair was obtained only by disruption of the kinetochore structure in a ndc10-1 mutant, but not in cse4-1 and cbf1 Delta mutants. Moreover, UV photofootprinting and DNA repair footprinting showed that centromere proteins cover about 120 bp of the centromere elements CDEII and CDEIII, including 20 bp of flanking CDEIII. Thus, DNA lesions do not appear to disrupt protein-DNA interactions in the centromere. Maintaining a stable kinetochore structure seems to be more important for the cell than immediate removal of DNA lesions. It is conceivable that centromeres are repaired by postreplication repair pathways.     

Optimal search strategies for detecting health services research studies in MEDLINE

Background

Evidence from health services research (HSR) is currently thinly spread through many journals, making it difficult for health services researchers, managers and policy-makers to find research on clinical practice guidelines and the appropriateness, process, outcomes, cost and economics of health care services. We undertook to develop and test search terms to retrieve from the MEDLINE database HSR articles meeting minimum quality standards.

Methods

The retrieval performance of 7445 methodologic search terms and phrases in MEDLINE (the test) were compared with a hand search of the literature (the gold standard) for each issue of 68 journal titles for the year 2000 (a total of 25 936 articles). We determined sensitivity, specificity and precision (the positive predictive value) of the MEDLINE search strategies.

Results

A majority of the articles that were classified as outcome assessment, but fewer than half of those in the other categories, were considered methodologically acceptable (no methodologic criteria were applied for cost studies). Combining individual search terms to maximize sensitivity, while keeping specificity at 50% or more, led to sensitivities in the range of 88.1% to 100% for several categories (specificities ranged from 52.9% to 97.4%). When terms were combined to maximize specificity while keeping sensitivity at 50% or more, specificities of 88.8% to 99.8% were achieved. When terms were combined to maximize sensitivity and specificity while minimizing the differences between the 2 measurements, most strategies for HSR categories achieved sensitivity and specificity of at least 80%.

Interpretation

Sensitive and specific search strategies were validated for retrieval of HSR literature from MEDLINE. These strategies have been made available for public use by the US National Library of Medicine at www.nlm.nih.gov/nichsr/hedges/search.html.With the increasing emphasis on “using evidence” and “value for money” in health services, it is essential that researchers, clinicians, health system managers and public policy-makers be able to retrieve relevant, high-quality reports of health services research (HSR). Efficiently retrieved research evidence can aid in decision-making about which services to provide and in the resource allocation decisions to support those services, reducing the need for arbitrary decisions and aiding collaboration with clinicians and consumers.¹ MEDLINE is a huge and expanding bibliographic resource that is freely available to all with Internet access. Yet the volume of the literature often overwhelms both clinicians and health system decision-makers.^2,3 End-users of MEDLINE and other large bibliographic databases have difficulty executing precise searches^2,3 and are often unaware of what kind of information to seek, where to find it^3,4 and how to judge its quality.³HSR has been defined as the scientific study of the effect of health care delivery; the organization and management of health care access, quality, cost and financing; and the evaluation of the impact of health services and technology (Allmang NA, Koonce TY. Health services research topic searches. Bethesda [MD]: National Library of Medicine; 2000. Unpublished report). More recently, HSR has been defined as the multidisciplinary field of scientific investigation that studies how social factors, financing systems, organizational structures and processes, health technologies and personal behaviours affect access to health care, the quality and cost of health care and, ultimately, health and well-being.⁵ HSR articles constitute only a tiny fraction of the MEDLINE database and are spread through a large number of journals; hence, MEDLINE searching is challenging. Conversely, journal browsing is impractical as a means of retrieving all relevant studies for a given question or staying abreast of the literature. Our aim was to develop methodologic search filters for MEDLINE to enable end-users to efficiently retrieve articles of relevance to clinical practice guidelines (CPGs) and the appropriateness, process, outcomes, cost and economics of health services.     

The law and the local fairness of care practice. Reflections on the ethnographic field investigation concerning the failure of the Bopz Act in psychogeriatric nursing home care

Ten years after its introduction the Special Admissions to Psychiatric Hospitals Act (Bopz) still awaits full implementation in the field of psychogeriatric nursing home care. Ethnographic research into moral problems related to care giving in dementia patients in Dutch nursing homes yielded several reasons for this discrepancy between law and care practice. Firstly, practical effectuation of this law rests predominantly on the shoulders of nurses and nurse assistants who are mainly inspired by moral motives such as carefulness and providing safety and who are ill informed about the law and its prerequisites. Secondly, there is the problem of the loss of a common shared world of meaning, which in relational terms is typical for the process of dementia. As a result of this, crucial concepts of the Bopz loose their applicability as the disease progresses. Finally, there is an immanent tension between the anthropology of care and care giving and the anthropological presuppositions of the law, health law included, which on a fundamental level contributes to the resistance of the care practice to the legal procedures of the Bopz. These factors must be accounted for in new legislation or revision of the current law. In the mean time we urge the necessity to develop a moral code concerning how to deal with resistive behaviour and opposition to care of demented nursing home patients.     

Risk of Disease Transmission between Conservation Personnel and the Mountain Gorillas: Results from an Employee Health Program in Rwanda

Humans and gorillas share 97% of their genetic makeup which means the risk of disease transmission between the two is potentially high. Humans with high exposure and whose exposure-related activity can most easily be managed are park conservation personnel. In June 2001, the Morris Animal Foundations Mountain Gorilla Veterinary Project initiated a health program for all employees working in Rwandas Parc National des Volcans in collaboration with in-country government and nongovernmental agencies. The goal is to improve the health of conservation personnel and reduce the risk of zoonotic disease transmission between employees and the parks mountain gorillas. Employees annually receive a clinical examination and laboratory testing, and provide a clinical history, In 2002, analyses were performed on the dataset of 127 employees to identify potential risk factors associated with positive laboratory tests. Considering all fecal tests combined, 70.1% were positive for one or more pathogenic organisms. A high percentage (> 80%) tested positive on viral antibody titer testing for various communicable diseases including measles, chickenpox, and hepatitis. On multivariate analysis, the main risk factor for testing positive for any pathogenic organism was use of a pit latrine at home. Vaccination against childhood communicable diseases and improved human waste disposal could be critical control points for preventing disease transmission to mountain gorillas. Program results have been shared with local health officials to aid in their efforts to improve village health and sanitation standards, and with park employers as a basis for ongoing employee health education.The Mountain Gorilla Veterinary Project 2002 Employee Health Group members are listed in Appendix 1. by area of contribution.     

Dissemination of rat cytomegalovirus through infected granulocytes and monocytes in vitro and in vivo

The role of leukocytes in the in vivo dissemination of cytomegalovirus was studied in this experiment. Rat cytomegalovirus (RCMV) could be transferred to rat granulocytes and monocytes by cocultivation with RCMV-infected fibroblasts in vitro. Intravenous injection of purified infected granulocytes or monocytes resulted in a systemic infection in rats, indicating that our model is a powerful tool to gain further insight into CMV dissemination and the development of new antivirals.     

Variation in mitochondrial DNA of Vietnamese pigs: relationships with Asian domestic pigs and Ryukyu wild boars

Mitochondrial DNA (mtDNA) sequences (574 bp) of 30 Vietnamese pigs (large and small) were examined and compared with those of 61 haplotypes from wild boars and domestic pigs from various locations in Asia. The large Vietnamese pigs had genetic links to Ryukyu wild boars in southern Japan. The small Vietnamese pigs were closely related to other East Asian domestic pigs. These results indicate that Vietnamese pigs are genetically diverse and may be descendents of wild and domestic pigs from other regions of Asia.     

The intercellular adhesion molecule-1 K469E polymorphism in type 1 diabetes

Type 1 (insulin-dependent) diabetes is a complex trait. The region harboring the ICAM1 gene on 19p13 links to type 1 diabetes, and a growing body of evidence indicates that intercellular adhesion molecule-1 (ICAM-1) could play a role in type 1 diabetes development. Recently, association studies of an ICAM-1 K469E polymorphism in type 1 diabetes populations have reported conflicting results. Hence, we performed a transmission disequilibrium test analysis of the ICAM-1 K469E variations in 253 Danish type 1 diabetes families. Linkage and association was not found between the ICAM-1 K469E variation and type 1 diabetes in Danish patients (P(tdt)> or =0.48), and our data did not indicate an interaction between ICAM1 and IDDM1 in predisposition to type 1 diabetes in Danes (P=0.78). We did not observe significant association with late-onset type 1 diabetes (P(tdt)> or =0.12) or differences in transmission patterns between groups of affected offspring stratified for age at onset (P> or =0.19), as suggested in Japanese patients. Combined analysis of the present and previously reported transmission data comprising 728 affected offspring of Romanian, Finnish, and Danish ancestry suggested association between the ICAM-1 E469 allele and type 1 diabetes (P(tdt)=0.013), but association was not found in the combined Scandinavian material. In conclusion, we found no association of the ICAM-1 K469E polymorphism with type 1 diabetes or its subsets stratified for age at onset and HLA risk in Danish patients. Analysis of ICAM-1 K469E transmissions reported in three populations suggested association to type 1 diabetes, but also demonstrated heterogeneity between populations.