Crystal structures of thrombin complexed to a novel series of synthetic inhibitors containing a 5,5-trans-lactone template.

The binding modes of four active site-directed, acylating inhibitors of human alpha-thrombin have been determined using X-ray crystallography. These inhibitors (GR157368, GR166081, GR167088, and GR179849) are representatives of a series utilizing a novel 5, 5-trans-lactone template to specifically acylate Ser195 of thrombin, resulting in an acyl complex. In each case the crystal structure of the complex reveals a binding mode which is consistent with the formation of a covalent bond between the ring-opened lactone of the inhibitor and residue Ser195. Improvements in potency and selectivity of these inhibitors for thrombin are rationalized on the basis of the observed protein/inhibitor interactions identified in these complexes. Occupation of the thrombin S2 and S3 pockets is shown to be directly correlated with improved binding and a degree of selectivity. The binding mode of GR179849 to thrombin is compared with the thrombin/PPACK complex [Bode, W., Turk, D., and Karshikov, A. (1992) Protein Sci. 1, 426-471] as this represents the archetypal binding mode for a thrombin inhibitor. This series of crystal structures is the first to be reported of synthetic, nonpeptidic acylating inhibitors bound to thrombin and provides details of the molecular recognition features that resulted in nanomolar potency.     

Carriage of Neisseria meningitidis and Neisseria lactamica among ethnic Greek school children from Russian immigrant families in Athens

During February and March 1995, a survey of meningococcal carriage in 625 school children was carried out in a suburb of Athens in which there was a large number of ethnic Greeks who had immigrated from Russia beginning in the early 1990s. The objectives of the study were: (1) to determine if factors associated with carriage of meningococci observed in a previous study of Greek school children were similar for the immigrant population; (2) to compare phenotypic characteristics of meningococci from the immigrant population with those isolated from children in Athens. Overall isolation rate for meningococci was 82/625 (13.1%), significantly higher than that found for school children in Athens (5.8%) during the winter of 1990 1991 (5.8%) (chi=25.98, P=0.0000003). By univariate analysis, carriage was not associated with sex, number of individuals per household, blood group, secretor status, socioeconomic level or maternal smoking; however, it was associated with fathers' smoking. The high proportion of men who smoked compared with the low proportion of women smokers might contribute to this finding. The main serogroup of meningococci isolated from this population was A (28%). While serogroup A appears to be more prevalent among Russian and Kurdish immigrants (14%) than among Greek school children or military recruits (4%), there has not been an increase in group A meningococcal disease in Greece. The isolation rate for N. lactamica was high 105/625 (17.3%). A few of these strains bound some of the monoclonal antibodies used for meningococcal serotyping and subtyping, and they are being examined in greater detail.     

Relationship between fertilization success and the number of milt donors in rainbow smelt Osmerus mordax (Mitchell): implications for population growth rates

A series of artificial fertilizations using rainbow smelt Osmerus mordax were conducted, each designed to assess the fertilization success of a female's eggs with milt from one to five males. Mean egg fertilization success increased and variance in fertilization success decreased when more males contributed to a constant volume of milt. This may indicate the presence of an Allee effect. The results suggest that potential changes in egg fertilization success due to a decrease in the number of males, in addition to changes in egg production due to a decrease in the number of females, can affect estimates of maximum sustainable harvest rates, and recovery of depleted populations.     

Fedbatch design for periplasmic product retention in Escherichia coli

The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.3, 0.2 and 0.1 h(-1) where studied and resulted in 20, 9 and 6%, respectively, of the total ZZ-cutinase accumulating in the medium. It was also shown that leakage during fedbatch production of a Fab fragment was also influenced by the feed rate in a similar manner to ZZ-cutinase. If intracellular product accumulation is desired the advantage of a high productivity, resulting from a high substrate feed rate, is diminished because of a reduced product retention. Biochemical analysis revealed that the growth rate, resulting from a glucose limited feed, influenced the outer membrane protein compositions with respect to OmpF and LamB, whilst OmpA was largely unaffected. As the feed rate increased the amount of total outer membrane protein decreased. When ZZ-cutinase was produced there were further reductions in outer membrane protein accumulation, by 82, 100 and 22% for OmpF, LamB and OmpA, respectively, and the total reduction was almost 60% with a high product formation rate. We suggest that the reduced titre of the outer membrane proteins, OmpF and LamB, may have contributed to a reduced ability for the cell to retain recombinant protein secreted to the periplasm.     

Calibrating the avian molecular clock

Molecular clocks are widely used to date phylogenetic events, yet evidence supporting the rate constancy of molecular clocks through time and across taxonomic lineages is weak. Here, we present 90 candidate avian clock calibrations obtained from fossils and biogeographical events. Cross-validation techniques were used to identify and discard 16 inconsistent calibration points. Molecular evolution occurred in an approximately clock-like manner through time for the remaining 74 calibrations of the mitochondrial gene, cytochrome b . A molecular rate of approximately 2.1% (± 0.1%, 95% confidence interval) was maintained over a 12-million-year interval and across most of 12 taxonomic orders. Minor but significant variance in rates occurred across lineages but was not explained by differences in generation time, body size or latitudinal distribution as previously suggested.     

Genotypic probabilities for pairs of inbred relatives

Expressions for the joint genotypic probabilities of two related individuals are used in many population and quantitative genetic analyses. These expressions, resting on a set of 15 probabilities of patterns of identity by descent among the four alleles at a locus carried by the relatives, are generally well known. There has been recent interest in special cases where the two individuals are both related and inbred, although there have been differences among published results. Here, we return to the original 15-probability treatment and show appropriate reductions for relatives when they are drawn from a population that itself is inbred or when the relatives have parents who are related. These results have application in affected-relative tests for linkage, and in methods for interpreting forensic genetic profiles.     

Identifying and managing adverse environmental health effects: 3. Lead exposure

LEAD LEVELS IN NORTH AMERICAN CHILDREN AND ADULTS have declined in the past 3 decades, but lead persists in the environment in lead paint, old plumbing and contaminated soil. There are also a number of occupations and hobbies that carry a high risk of lead exposure. There is no evidence for a threshold below which lead has no adverse health effects. Blood lead levels previously considered safe are now known to cause subtle, chronic health effects. The health effects of lead exposure include developmental neurotoxicity, reproductive dysfunction and toxicity to the kidneys, blood and endocrine systems. Most lead exposures are preventable, and diagnosing lead poisoning is relatively simple compared with diagnosing health effects of exposures to other environmental toxins. Accurate assessment of lead poisoning requires specific knowledge of the sources, high-risk groups and relevant laboratory tests. In this article we review the multiple, systemic toxic effects of lead and provide current information on groups at risk, prevention, diagnosis and clinical treatment. We illustrate how the CH²OPD² mnemonic (Community, Home, Hobbies, Occupation, Personal habits, Diet and Drugs) and specific screening questions are useful tools for physicians to quickly obtain an environmental exposure history and identify patients at high risk of lead exposure. By applying effective primary prevention, case-finding and treatment interventions for lead exposure, both the individual patient and the larger community reap the benefits of better health.CaseA previously healthy 2-year-old girl and her mother visit their family physician because of the daughter''s 2-month history of intermittent complaints of a mild “tummy ache.” There is no associated vomiting, weight loss, or change in appetite, bowels or diet. There are no abnormal findings on physical examination. When asked about symptom onset the mother reports that it began shortly after the family started to renovate their kitchen. They live in an old farmhouse on the outskirts of town and drink water from a drilled well on the property. The physician decides to take an environmental exposure history using the CH²OPD² mnemonic (Community, Home, Hobbies, Occupation, Personal habits, Diet and Drugs; for children, the occupation question refers to workplace contaminants brought into the child''s environment).¹ The child''s exposure history (Open in a separate windowQuestions surrounding this case: Is the family at risk of health effects from lead exposure? Who else might be at risk? Are other laboratory tests indicated? Where can the physician get advice on the significance of the family''s blood lead levels? How should this case of lead exposure be treated?To some extent lead is one of the small success stories of environmental health. The association of lead poisoning with cognitive impairment is well established² and has resulted in the removal of lead from gasoline, paint and food cans. Despite these preventive measures, however, silent, low-level lead exposure continues to present a problem for many communities and populations. In 1997, data from the US National Health and Nutrition Examination Surveys showed that 4.4% of children in the United States had elevated blood lead levels.³ Black children living in older housing, children living in metropolitan areas with populations of 1 million or more and poor children living in older housing were at highest risk of exposure.³In Canada children living near a point-source smelter in the South Riverdale area of Toronto were tested in 1973 and found to have an unusually high mean lead level (1.34 μmol/L).⁴ Canada''s Federal–Provincial Committee on Environmental and Occupational Health suggested in 1994 that 5%–10% of Canadian children living in urban areas have blood lead levels exceeding 0.48 μmol/L, even though they are not exposed to point sources.⁵ The Ontario government estimated in 1994 that 4% of children in the province still had blood lead levels above 0.48 μmol/L;⁶ a 1992 study found that the mean level in Ontario children had fallen from 0.91 μmol/L in 1972 to 0.29 μmol/L in 1988.⁷ A study of Vancouver children using blood lead levels collected in 1989 found that 8% had elevated levels (mean 0.29 μmol/L).⁸ A later study of the children living in Trail, BC, the site of a lead and zinc smelter, demonstrated that 50% had an elevated blood lead level.⁹