Expression, purification, and initial kinetic characterization of the large subunit of the human mitochondrial DNA polymerase

Faulty replication of the human mitochondrial genome is thought to be the cause of many diseases; moreover, the low selectivity of the mitochondrial DNA polymerase has been implicated as the cause of many side effects observed in the treatment of viral infections such as HIV. To better understand how the mitochondrial genome is replicated, we cloned a cDNA encoding the large subunit of human DNA polymerase gamma, the enzyme that replicates the mitochondrial genome. The large subunit was recombinantly expressed and purified to near homogeneity. The purified enzyme demonstrated both polymerase and 3'-5' exonuclease activity. The purified protein was examined in single nucleotide incorporation assays, demonstrating that the enzyme had a maximum polymerization rate of 3.5 s-1 and a dissociation rate from the DNA substrate of 0.03 s-1, affording a calculated processivity of 116. The dissociation constants for the enzyme binding to DNA and nucleoside triphosphate were 39 nM and 14 microM, respectively. The 3'-5' exonuclease rate was measured at 0. 18 s-1. Though the slow rate of polymerization suggests that the large subunit of human DNA polymerase gamma may require accessory factors to increase its processivity of polymerization, the kinetic parameters indicate that the large subunit of DNA polymerase gamma could replicate the mitochondrial genome in a physiologically relevant time frame. This study provides the initial characterization of the large subunit of DNA polymerase gamma and establishes the baseline for examination of the effects of accessory proteins such as the putative small subunit.     

A school-based schistosomiasis and intestinal helminthiasis control programme in Nigeria: acceptability to community members

In a population with high prevalences in schoolchildren of infection with hookworm (32.4%), Ascaris (22.9%) and Trichuris (2.5%), visible haematuria (17.9%), micro-haematuria (17%) and proteinuria (47.3%), the knowledge about transmission of schistosomiasis and acceptability of a school-based control programme were assessed. The community perceived schistosomiasis (80.6%) and intestinal helminthiasis (66.5%) as important health problems in school-age children and most people would prefer placement of the control programme in school because it would eliminate transportation cost to the health facility. They welcomed the idea of using teachers for detection of infection and drug administration. The health staff, on the other hand, were willing to work with teachers, but emphasized that teachers should be limited to organizational and supervisory roles while they do tests and administer the drug. This view was also shared by the officials in the state ministries of health and education.     

Developments in UK service provision for people with epilepsy: the impact of the NHS Executive Letter 95/120

The author describes the operating course of the Occupational Medicine Operating Units (OMOU) in our region. (Lombardia) These Units, born as a second level support to the Basal Occupational Medicine Units, greatly enlarged, as time passed, the limits of their users together with the charge of preventive duties ordered to the employers by the recent laws regarding Occupational Hygiene. (DLgs 277/91-DLgs 626/94) The author also illustrates the variety of the diagnostic demands to the OMOU according to the enlargement of the legal protection of occupational diseases and to the numerous aspects of specific job fitness. He underlines the need of multiple specialistic skillfulnesses achievable by means of a complete integration of OMOU in to the hospital to which they belong. In conclusion the author illustrates the new tasks of the OMOU in the field of the therapy and rehabilitation.     

Allelotype analysis of uterine leiomyoma: localization of a potential tumor suppressor gene to a 4-cM region of chromosome 7q

The degree and nature of patient involvement in consultations with health professionals influences problem and needs recognition and management, and public accountability. This paper suggests a framework for understanding the scope for patient involvement in such consultations. Patients are defined as co-producers of formal health services, whose potential for involvement in consultations depends on their personal rights, responsibilities and preferences. Patients' rights in consultations are poorly defined and, in the National Health Service (NHS), not legally enforceable. The responsibilities of patients are also undefined. I suggest that these are not to deny, of their own volition, the rights of others, which in consultations necessitate mutuality of involvement through information-exchange and shared decision-making. Preferences should be met insofar as they do not militate against responsibilities and rights.     

Crystal structure of the spliceosomal U2B"-U2A' protein complex bound to a fragment of U2 small nuclear RNA

We have determined the crystal structure at 2.4 A resolution of a ternary complex between the spliceosomal U2B"/U2A' protein complex and hairpin-loop IV of U2 small nuclear RNA. Unlike its close homologue the U1A protein, U2B" binds to its cognate RNA only in the presence of U2A', which contains leucine-rich repeats in its sequence. The concave surface of a parallel beta-sheet within the leucine-rich-repeat region of U2A' interacts with the ribonucleoprotein domain of U2B" on the surface opposite its RNA-binding surface. The basic carboxy-terminal region of U2A' interacts with the RNA stem. The crystal structure reveals how protein-protein interaction regulates RNA-binding specificity, and how replacing only a few key residues allows the U2B" and U1A proteins to discriminate between their cognate RNA hairpins by forming alternative networks of interactions.     

Nickel biochemistry

We present a method for the determination of the lignan enterolactone in plasma (serum). This compound, produced by intestinal bacteria from matairesinol and secoisolariciresinol in fiber-rich food, is a biomarker related to the intake of a healthy diet. The method is based on time-resolved fluoroimmunoassay using a europium chelate as a label. After synthesis of 5'-O-carboxymethoxyenterolactone the compound is coupled to bovine serum albumin and then used as antigen in immunization of rabbits. The tracer with the europium chelate is synthesized using the same 5'-derivative of enterolactone. After enzymatic hydrolysis and ether extraction the immunoassay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Turku, Finland). No antiserum cross-reactivity with available lignans, isoflavonoids, or flavonoids could be detected. The intraassay and interassay coefficients of variation at different concentrations vary 4.6-6.0 and 5.5-9.9, respectively. The working range of the assay is 1.5-540 nmol/liter. We measured enterolactone in serum/plasma of 224 Finnish subjects: 98.8% of the subjects had values <100 nmol/liter, 38.0% had 20-39.9 nmol/liter, and 34.4% had <20 nmol/liter.     

Reduction of triceps muscle force after shortening of the distal humerus: a computational model

A retrospective case reference study examining the use of alcohol and tobacco in 303 women aged 40 or over suffering from oral or oropharyngeal cancer was conducted in the south-west Netherlands. Both alcohol and tobacco consumption are important in the development of oral and oropharyngeal cancer with increased consumption of both markedly increasing the risks of cancer, but alcohol having the greater effect.     

Pharmacokinetics of nicotine carbomer enemas: a new treatment modality for ulcerative colitis

BACKGROUND: Ulcerative colitis is largely a disease of nonsmokers, and transdermal nicotine is of therapeutic value in the active disease. Because side effects are common, we developed a topical enema formulation of nicotine. OBJECTIVE: To study the pharmacokinetics of nicotine complexed with a polyacrylic carbomer and administered by enema to eight healthy volunteers and to eight patients with active ulcerative colitis, verified sigmoidoscopically. PATIENTS AND METHODS: All 16 subjects were nonsmokers. The mean age for normal subjects was 33 years; the mean for patients with ulcerative colitis was 60 years. Median stool frequency for patients with ulcerative colitis was four daily. Patients were taking 5-amino salicylic acid compounds and five were taking oral prednisolone (median dose, 12 mg daily). Nicotine, 6 mg, complexed with carbomer 974P, 400 mg, was administered in a 100 ml enema after an overnight fast, with serial blood measurements taken over 8 hours. Serum nicotine and cotinine were measured by gas liquid chromatography. Area under the concentration-time curves were calculated by the trapezoidal method, and the terminal elimination half-life was derived by extrapolation of the log-linear terminal phase. RESULTS: With the exception of nicotine time to reach peak concentration, which was longer in patients (median of 60 minutes compared with 45 minutes; p < 0.005), other comparisons between normal subjects and patients showed no statistically significant difference, although there was considerable inter-subject variation. Maximum concentration of nicotine, 8.1 +/- 3.5 ng/ml, in the 16 subjects occurred after a median of 60 minutes (range, 30 to 180 minutes); maximum cotinine concentrations of 60.4 +/- 11.5 ng/ml occurred after 4 hours. Side effects in five subjects were mild (four subjects) or moderate (one subject) and included lightheadedness, nausea, and headache; these five subjects were female lifelong nonsmokers of low body weight. CONCLUSION: Because most of the active ingredient of nicotine is converted to continine on the first pass through the liver, substantial concentrations can be achieved at the site of disease with only modest rises in serum nicotine, which are responsible for side effects; cotinine has low pharmacologic activity. Topical administration of nicotine may be useful treatment for distal ulcerative colitis.     

Clinical relevance of parvovirus B19 as a cause of anemia in patients with human immunodeficiency virus infection

Parvovirus B19 (B19) DNA was detected by dot blot hybridization in sera from 5 (17%) of 30 human immunodeficiency virus (HIV)-infected patients with hematocrits (HCT) of < or =24 and 4 (31%) of 13 HRV-infected patients with HCT of < or =20, suggesting that B19 is a reasonably common cause of severe anemia in HIV infection. The anemia promptly remitted after immunoglobulin therapy in 3 of 4 treated patients. The presence of IgM to B19, the clinical circumstance in which anemia developed, and the marrow morphology were poor predictors of chronic B19 infection. DNA hybridization studies of sera from 191 HIV-infected and 117 HIV-seronegative homosexual males attending a clinic in the Seattle area revealed that 1 (0.5%) and 2 (2%) samples, respectively, from the 2 groups contained B19. However, when assayed by polymerase chain reaction (PCR), 5% of the serum samples from HIV-infected persons and 9% from uninfected persons contained B19, although each had an HCT of > or =40. The data argue that anemia results from chronic high-titer B19 infection. Although a negative PCR assay excludes this diagnosis, DNA hybridization may be the more specific serum test.     

Carcinogenic polycyclic aromatic hydrocarbons increase intracellular Ca2+ and cell proliferation in primary human mammary epithelial cells

A method was developed to determine in eggs 2 components [4,6-dimethyl-2-hydroxypyrimidine and 1,3-bis(4-nitrophenyl)urea] of the anticoccidial drug nicarbazin, used to treat poultry. Samples were extracted with acetonitrile, and the extracts were washed with hexane and evaporated to dryness before analysis by liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization. By switching from positive to negative ion monitoring and using gradient elution, both components were measured within one run. The limit of quantitation of the assay was 10 ng/g for each component. The results of a preliminary feeding trial in which chickens were fed contamination levels of the drug are also reported.