General considerations for proteolytic cascades.

Proteases are involved in the regulation of a wide variety of essential physiological processes, often by participating in a highly orchestrated sequence of events termed a 'proteolytic cascade'. Four major proteolytic cascades with disease relevance are candidates for therapeutic intervention, namely caspase-mediated apoptosis, blood coagulation, the matrix metalloproteinase cascade and the complement cascade. Understanding the various steps involved in the functioning of a cascade is key to deciding possible points of intervention for the design of potential drug molecules. This brief review illustrates some of the common features of proteolytic cascades using the blood coagulation pathway as an example.

Individual estimation of exposures to extremely low frequency magnetic fields in jobs commonly held by women.

Exposures to extremely low frequency (ELF) magnetic fields have not been documented extensively in occupations besides the work environments of electric or telephone utilities. A 1980-1993 study of childhood acute lymphoblastic leukemia (ALL) in Québec, Canada, gathered detailed information about the occupations of 491 mothers of ALL cases and mothers of a similar number of healthy controls. This information was combined with published data on the intensities of ELF magnetic fields associated with sources or work environments to estimate ELF magnetic field exposures for a wide range of jobs commonly held by women. Estimated exposures for 61 job categories ranged from 0.03 to 0.68 microT; the 25th, 50th, and 75th percentiles were 0.135, 0.17, and 0.23 microT, respectively. By job category, the most highly exposed jobs (>0.23 microT) included bakery worker, cashier, cook and kitchen worker, electronics worker, residential and industrial sewing machine operator, and textile machine operator. By work environment, the most highly exposed job categories were electronics worker in an assembly plant (0.70 microT) and sewing machine operators in a textile factory (0.68 microT) and shoe factory (0.66 microT). These results provide new information on expected levels of exposure in a wide range of jobs commonly held by women.

Inhibition of human alpha-thrombin by a phosphonate tripeptide proceeds via a metastable pentacoordinated phosphorus intermediate.

X-ray crystallographic studies of human alpha-thrombin with a novel synthetic inhibitor, an acyl (alpha-aminoalkyl)phosphonate, reveal the existence of a pentacovalent phosphorus intermediate state. Crystal structures of the complex of alpha-thrombin with the phosphonate compound were determined independently using crystals of different ages. The first structure, solved from a crystal less than seven days old, showed a pentacoordinated phosphorus moiety. The second structure, determined from a crystal that was 12 weeks old, showed a tetracoordinated phosphorus moiety. In the first structure, a water molecule, made nucleophilic by coordination to His57 of alpha-thrombin, is bonded to the pentacoordinated phosphorus atom. Its position is approximately equivalent to that occupied by the water molecule responsible for hydrolytic deacylation during normal hydrolysis. The pentacoordinated phosphorus adduct collapses to give the expected pseudo tetrahedral complex, where the phosphorus atom is covalently bonded to Ser195 O(gamma). The crystallographic data presented here therefore suggest that the covalent bond formed between the inhibitor's phosphorus atom and O(gamma) of Ser195 proceeds via an addition-elimination mechanism, which involves the formation of a pentacoordinate intermediate.

Taking responsibility for cancer treatment.

One hundred and fourteen consecutive patients with early breast cancer were entered into a study on the psychological effects of involvement in treatment choice. All women were offered counselling throughout. One group of women (n = 34), were advised to undergo mastectomy, due to the nature or position of the tumour. These women fared less well psychologically when compared on a battery of measures, before and after surgery, with women who were involved in choosing their own treatment (n = 80). The latter group itself was randomly allocated into two groups for taking explicit responsibility for treatment choice, using a double-blind procedure. These were a Patient Decision Group (n = 41) and a Surgeon Decision Group (n = 39). Results support the hypothesis that over and above the benefits of receiving their preferred treatment, women can further benefit from taking explicit responsibility for their treatment choice.

The determinants of Canadian children's personal exposures to magnetic fields.

Study of the health effects of magnetic fields often depends on identifying determinants and hence indicators of personal exposure. This study identified determinants of children's exposure to magnetic fields and constructed a prediction model for them. For 632 children participating in a case-control study of childhood leukemia, we made direct measures of exposure over 48 h using a portable device, together with observations on candidate determinants. A child's age and sex, the proportion of time spent in the home, and their parents' education or income were very weak predictors of (logged) mean 48 h magnetic field (R(2) < 1%). More important were province (R(2) = 8.0%) and type of residence (R(2) = 11.3%). Low temperatures at the time of measurement were associated with high fields (about 20% increase for each 10 degrees C below 14, R(2) = 4.9%). Several visible attributes of wiring around residences predicted exposure, mostly captured in the Wertheimer-Leeper wire code (R(2) = 13.5%). Stationary 24 h measurement in the bedroom (R(2) = 63.3%) and spot measurements outside the house (R(2) = 40.7%) predicted personal exposures best. Adding other minor predictors increased only slightly variance explained by 24 h stationary (R(2) = 66.2%) and spot (R(2) = 46.8%) measurements. Without spot or stationary measurements, the best model was much less powerful (R(2) = 29.0%). We conclude that spot measurements outside the residence provide a moderately effective basis for estimating exposure for children living there, but do not perform as well as 24 h stationary measurements in the child's bedroom. Although several other easily-observed variables were associated with personal exposure, they were weak determinants, either individually or in combination.

Proteinase inhibitors and activators strategic targets for therapeutic intervention.

The contributions in this meeting represent the state of the art in study of proteinases and their biological and therapeutic regulation. The near future of proteinase research will be fashioned by the new horizons of proteomics research [61], adding substance to the genomic data. Even new approaches in drug discovery, such as combinatorial chemistry, impact upon the understanding of the proteinase function, as with the discovery of a novel allosteric exosite in FVIIa by probing with libraries of ligands [62]. What is without question is that proteinases will remain at the forefront of understanding and intervention in human biochemistry and human disease pathology.

Exposures of children in Canada to 60-Hz magnetic and electric fields.

OBJECTIVES: This study sought to characterize personal exposures of Canadian children to 60-Hz magnetic and electric fields and explain the variability. METHODS: Altogether 382 Canadian children up to 15 years of age wore meters recording 60-Hz electric and magnetic fields over 2 days. Meter location was noted. Thereafter, meters measured fields in the center of the children's bedrooms for 24 hours. Personal exposures were calculated for home, school or day care, outside the home, bedroom at night, and all categories combined (total). RESULTS: The arithmetic mean (AM) was 0.121 microT [geometric mean (GM): 0.085 microT), range 0.01-0.8 microT] for total magnetic fields. Fifteen percent of the total exposures exceeded 0.2 microT. The AM of the total electric fields was 14.4 (GM 12.3, range 0.82-64.7) V/m. By location category, the highest and lowest magnetic fields occurred at home during the day (0.142 microT) and during the night (0.112 microT), respectively. Measurements during sleep provided the highest correlation with total magnetic field exposure. Province of measurement explained 14.7% of the variation in the logarithms of total magnetic fields, and season accounted for an additional 1.5%. CONCLUSIONS: This study has identified differences in children's magnetic field exposures between provinces. Measurements at night provided the best surrogate for predicting total magnetic field exposure, followed by at-home exposure and 24-hour bedroom measurements. Electrical heating and air conditioning, wiring type, and type of housing appear to be promising indicators of magnetic field levels.

Power-frequency electric and magnetic fields and risk of childhood leukemia in Canada.

In a case-control study of childhood leukemia in relation to exposure to power-frequency electric and magnetic fields (EMF), 399 children resident in five Canadian provinces who were diagnosed at ages 0-14 years between 1990 and 1994 (June 1995 in British Columbia and Quebec) were enrolled, along with 399 controls. Exposure assessment included 48-hour personal EMF measurement, wire coding and magnetic field measurements for subjects' residences from conception to diagnosis/reference date, and a 24-hour magnetic field bedroom measurement. Personal magnetic fields were not related to risk of leukemia (adjusted odds ratio (OR) = 0.95, p for trend = 0.73) or acute lymphatic leukemia (OR = 0.93, p for trend = 0.64). There were no clear associations with predicted magnetic field exposure 2 years before the diagnosis/reference date or over the subject's lifetime or with personal electric field exposure. A statistically nonsignificant elevated risk of acute lymphatic leukemia was observed with very high wiring configurations among residences of subjects 2 years before the diagnosis/reference date (OR = 1.72 compared with underground wiring, 95% confidence interval 0.54-5.45). These results provide little support for a relation between power-frequency EMF exposure and risk of childhood leukemia.

A cyclic peptide analogue of the loop III region of platelet-derived growth factor-BB is a synthetic antigen for the native protein.

We report the synthesis and characterization of a cyclic peptide analogue of the loop III region of platelet-derived growth factor (PDGF) B-chain sequence, cyclo(73Arg-Lys-Ile-Glu-Ile-Val-Arg-Lys-Lys81-Cys), incorporating a C-terminus cysteine residue for the conjugation to a carrier protein. The synthesis involved solid-phase chemistry, utilizing Fmoc-tBu chemistry and acid labile side-chain protecting groups, followed by 'head-to-tail' cyclization using the allyl-protected glutamic acid anchored on its side chain to the solid support with HATU/HOAt as the coupling agent. Conformational differences between the cyclic and its linear counterpart PDGF peptides were determined by circular dichroism measurements in aqueous media. High titre antisera were raised to both cyclic and linear peptide immunogens. Antisera raised to the cyclic peptide cross-reacted with PDGF-BB in both Western blot and ELISA, whereas antisera raised to the linear peptide had no reactivity with PDGF-BB. The cyclic peptide (conformational design analogue) produces an immunogen which is able to antigenically mimic the secondary structure of loop III of PDGF-BB and forms a basis from which further small molecular mimetics of PDGF may be designed for use as both immunogens and also potential agonists/antagonists of PDGF. Similarly constructed immunogens may also be useful in the design of vaccines which direct responses to loop regions in other target proteins.

Assembly of urokinase receptor-mediated plasminogen activation complexes involves direct, non-active-site interactions between urokinase and plasminogen.

The binding of the zymogenic form of urokinase-type plasminogen activator (pro-uPA) to its specific cellular receptor, uPAR, leads to a large potentiation of plasmin generation. This is dependent on the concurrent cellular binding of plasminogen, and is completely abrogated by the plasminogen lysine-binding site ligand, 6-aminohexanoic acid. Previous data have provided circumstantial evidence for the formation of specific complexes to mediate the kinetically favorable reciprocal interactions between the protease and zymogen components [Ellis, V., and Dano, K. (1993) J. Biol. Chem. 268, 4806-4813]. To further investigate the formation of these putative complexes, we have studied the effect of various lysine-binding site ligands on the binding and activation of plasminogen on U937 cells. Lysine-binding site ligands resembling internal lysine residues, such as Nalpha-acetyl-L-lysine methyl ester, were found to specifically inhibit uPAR-mediated cell-surface plasminogen activation at concentrations up to 40-fold lower than those inhibiting the cellular binding of 125I-labeled plasminogen (IC50s 300 microM vs 8.5 mM). By contrast, 6-aminohexanoic acid, resembling a C-terminal lysine residue, did not display this disparity (IC50s 25 vs 30 microM). These lysine analogues were also found to compete a non-active-site interaction between uPA and plasminogen, detected by surface plasmon resonance (Kd 50 nM), at concentrations correlating with their effect on cell-surface plasminogen activation, suggesting that this interaction is part of the kinetic mechanism. Consistent with this, synthetic peptides corresponding to the sequence uPA149-158 (GQKTLRPRFK) and uPA149-157 (GQKTLRPRF) specifically abolished the amplification of cell-surface plasminogen activation. These data demonstrate that a novel non-active-site interaction between uPA and plasminogen is necessary for the assembly and efficiency of cell-surface plasminogen activation complexes.

Bifunctional peptide boronate inhibitors of thrombin: crystallographic analysis of inhibition enhanced by linkage to an exosite 1 binding peptide.

The affinity of the hirudin49-64 segment for exosite 1 of thrombin has been used previously to enhance the potency of simple competitive inhibitors [DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J. , Ni, F., Konishi, Y. (1990) J. Biol. Chem. 265, 21698-21703., and Maraganore, J. M., Bourdon, P., Jablonski, J., Ramachandran, K. L., and Fenton, J. W., II (1990) Biochemistry 29, 7095-7087.]. Using a similar approach, we have enhanced the activity of two active site directed thrombin inhibitors by attaching this segment via a novel reverse oriented linker to each of two tripeptide boronate inhibitors. At P1, compound 1 contains an arginine-like, isothiouronium, side chain, while compound 2 contains an uncharged, bromopropyl residue. Inhibition of human alpha-thrombin by compound 1 shows slow, tight-binding competitive kinetics (final Ki of 2.2 pM, k1 of 3.51 x 10(7) M-1 s-1, and k-1 of 1.81 x 10(-)4 s-1). The addition of hirugen peptide (20 microM) competes for exosite 1 binding and restores the k1 and k-1 to that of the analogous tripeptide, 0.29 x 10(7) M-1 s-1 and 0.13 x 10(-)4 s-1, respectively. Compound 1 has enhanced specificity for thrombin over trypsin with KiTry/KiThr of approximately 900 compared to the analogous tripeptide, with KiTry/KiThr of approximately 4. Compound 2 acts as a competitive inhibitor (KiThr of 0.6 nM) and is highly selective with no effect on trypsin. Crystallographic analysis of complexes of human alpha-thrombin with compound 1 (1.8 A) and compound 2 (1.85 A) shows a covalent bond between the boron of the inhibitor and Ser195 (bond lengths B-O of 1.55 and 1.61 A, respectively). The isothiouronium group of compound 1 forms bidentate interactions with Asp189. The P2 and P3 residues of the inhibitors form interactions with the S2 and S3 sites of thrombin similar to other D-Phe-Pro based inhibitors [Bode, W., Turk, D., and Karshikov, A. (1992) Protein Sci. 1, 426-471.]. The linker exits the active site cleft of thrombin forming no interactions, while the binding of Hir49-64 segment to exosite 1 is similar to that previously described for hirudin [Rydel, T. J., Tulinsky, A., and Bode, W. (1991) J. Mol. Biol. 221, 583-601.]. Because of the similarity of binding at each of these sites to that of the analogous peptides added alone, this approach may be used to improve the inhibitory activity of all types of active site directed thrombin inhibitors and may also be applicable to the design of inhibitors of other proteases.

Task-based estimation of past exposures to 60-hertz magnetic and electric fields at an electrical utility.

OBJECTIVES: Past exposures of electric utility workers to extremely low-frequency (ELF) magnetic (B) and electric fields (E) in Quebec were estimated. METHODS: The current intensities were measured and durations of exposures determined for tasks or work locations in 14 job categories. Past task or location intensities were extrapolated from the present on the basis of interviews with long-term workers and utility personnel. Past task or location durations were estimated for the long-term workers. Time-weighted average (TWA) exposures for past periods were reconstructed for jobs from the intensity and duration estimates. RESULTS: Magnetic fields were estimated to have increased the most over time for substation and distribution-line jobs. Magnetic field exposures for jobs in the generation and transmission of electricity were estimated to have increased very little. For substation jobs, the ratios of magnetic fields in 1945 to those in 1990 ranged from 0.42 to 0.69; the corresponding figures for distribution-line jobs ranged from 0.36 to 0.94. For electric fields in substations, the estimated increase over time was less than for magnetic fields, the 1945:1990 ratios ranging from 0.59 to 0.88. For the distribution line jobs, the 1945:1990 ratios for electric fields were less than 1.0 in 4 cases (0.6 to 0.89), more than 1.0 in 3 others (1.13 to 2.01) and unchanged in 1. CONCLUSIONS: Reconstruction of TWA exposures allowed changes in the intensity and the duration of exposures to be considered separately. Documentation of the intensity and duration of exposures for different tasks allows exposure reconstruction for jobs that have ceased to exist. The method is applicable elsewhere if exposure-monitoring records allow the level and duration of exposures for tasks or locations to be calculated and if estimates of past durations and intensities of exposures can be reliably obtained.

Exposure to 60-Hz magnetic and electric fields at a Canadian electric utility.

OBJECTIVES: The purpose of this study was to estimate exposure to extremely low frequency (ELF) magnetic and electric fields in the Québec electrical utility Hydro-Quebec. METHODS: Personal exposures to ELF magnetic and electric fields were measured for workers randomly selected from 32 job categories at Hydro-Québec. Weekly arithmetic and geometric means, and other indices of exposure were estimated from 465 worker-weeks of data. RESULTS: By job category, the arithmetic means of the ELF magnetic field ranged from 0.09 to 2.36 microT. Those of the ELF electric field exposures ranged from 2.5 to 400 V.m-1. Within each field, correlations of either the arithmetic or geometric means with alternative indices, including an index of the time rate of change, were generally high (r > or = 0.8). Exceptions were the 20th percentile of the electric means and the proportion of time above 12.4 and 100 microT. The day-to-day variation of exposure was greater than the variation between workers. The median between-day and between-worker components of variance (as geometric standard deviations) by job category were 2.13 and 1.71 for magnetic fields (2.24 and 1.81 for electric fields). CONCLUSIONS: Substation workers, hydroelectric generating station operators, and cable splicers showed the highest arithmetic means for 60-Hz magnetic fields above 1 microT. For 60-Hz electric fields, forestry workers, equipment electricians in 735 kV substations, and distribution linemen (contact method) had arithmetic mean exposures greater than 100 W.m-1. Of the total variance in the logarithms of the weekly magnetic and electric field means, job category explained 49.6% and 59.5%, respectively.

Heparin enhances the catalytic activity of des-ETW-thrombin.

The thrombin mutant, des-ETW-thrombin, lacking Glu(146), Thr(147), and Trp(148) within a unique insertion loop located at the extreme end of the primary specificity pocket, has been shown previously to exhibit reduced catalytic activity with respect to macromolecular and synthetic thrombin substrates and reduced or enhanced susceptibility to inhibition. Investigation of the hydrolysis of peptidyl p-nitroanilide substrates by des-ETW-thrombin showed increased activity in the presence of heparin and other sulphated glycosaminoglycans. No effect was observed upon the activity of wild-type thrombin. Heparin was found to decrease the K(m) for cleavage of four thrombin-specific substrates by des-ETW-thrombin by 3-4-fold. Similarly, pentosan polysulphate (PPS) decreased the K(m) with these substrates by 8-10-fold. Heparin also increased the rate of inhibition of des-ETW-thrombin by antithrombin III and D-phenylalanyl-prolyl-arginylchloromethane (PPACK). The inhibition of des-ETW-thrombin by a number of thrombin-specific peptide boronic acids also showed significant reduction in the final K(i) in the presence of heparin, due to reduction in the off-rate. A peptide analogue of a sequence of hirudin which binds thrombin tightly to exosite I (fibrinogen recognition site) potentiated the activity of des-ETW-thrombin against peptide p-nitroanilide substrates in a manner similar to heparin. The K(i) for the inhibition of des-ETW-thrombin by p-aminobenzamidine was decreased by these ligands from 9.7 mM to 7.5 mM, 5.1 mM, and 2.5 mM in the presence of heparin, hirudin peptide and PPS respectively, suggesting the increased catalytic activity is due to enhanced access to the primary specificity pocket. The positive influence of these ligands on des-ETW-thrombin was reversed in the presence of ATP or ADP; the latter has previously been shown to inhibit thrombin activity by blocking initial interaction with fibrinogen at exosite 1. Because the effect of heparin and PPS is similar to that of hirudin peptide, it is proposed that the most likely mechanism is that binding at the heparin-binding site (thrombin exosite 2) facilitates interaction at exosite 1 causing a conformational change which partially corrects the defective ground-state binding of the mutant thrombin. Although no effect was observed upon the activity of wild-type thrombin, our findings do provide further evidence of an allosteric property of thrombin which may control the geometry of, and access to, the primary specificity pocket.

A case cohort study of suicide in relation to exposure to electric and magnetic fields among electrical utility workers.

OBJECTIVES: This case cohort study examines whether there is an association between exposure to electric and magnetic fields and suicide in a population of 21,744 male electrical utility workers from the Canadian Province of Québec. METHODS: 49 deaths from suicide were identified between 1970 and 1988 and a subcohort was selected comprising a 1% random sample from this cohort as a basis for risk estimation. Cumulative and current exposures to electric fields, magnetic fields, and pulsed electromagnetic fields (as recorded by the POSITRON meter) were estimated for the subcohort and cases through a job exposure matrix. Two versions of each of these six indices were calculated, one based on the arithmetic mean (AM), and one on the geometric mean (GM) of field strengths. RESULTS: For cumulative exposure, rate ratios (RR) for all three fields showed mostly small non-significant increases in the medium and high exposure groups. The most increased risk was found in the medium exposure group for the GM of the electric field (RR = 2.76, 95% CI 1.15-6.62). The results did not differ after adjustment for socioeconomic state, alcohol use, marital state, and mental disorders. There was a little evidence for an association of risk with exposure immediately before the suicide. CONCLUSION: Some evidence for an association between suicide and cumulative exposure to the GM of the electric fields was found. This specific index was not initially identified as the most relevant index, but rather emerged afterwards as showing the most positive association with suicide among the 10 indices studied. Thus the evidence from this study for a causal association between exposure to electric fields and suicide is weak. Small sample size (deaths from suicide) and inability to control for all potential confounding factors were the main limitations of this study.

A mortality study of electrical utility workers in Québec.

OBJECTIVES: The objective of this study was to investigate the mortality of electrical utility workers exposed to electric and magnetic fields. METHODS: A historical cohort mortality study was carried out on 21,744 workers who were employed in an electrical company in the province of Québec between 1970 and 1988. The last job held by each study subject was coded. A job exposure matrix (JEM) was used to estimate the exposure to 60 Hz electric and magnetic fields, and pulsed electromagnetic fields (as recorded by the PEMF (POSITRON) meter) in this job. Standardised mortality ratios (SMRs) were calculated relative to Québec men. Because the exposure was exclusively among blue collar workers, the remainder of the analyses by exposure were restricted to them. Rate ratios (RRs) in the exposed groups relative to the background groups were estimated with Poisson regression. There were 1582 deaths by the end of follow up. RESULTS: SMRs were almost all below one and never substantially increased, although there were a few increased rate ratios (RRs). There was a significant RR of 2.00 (95% confidence interval (95% CI) 1.37-2.93) for deaths caused by accidents and violence in workers exposed to magnetic fields, RR of 1.82 (95% CI 1.25-2.65) for electric fields, and RR of 1.62 (95% CI 1.13-2.32) for pulsed electromagnetic fields. Occupational accidents related to power lines explain some of the excess of deaths from accidents and violence. Some association was found between magnetic fields and leukaemia, brain cancer, and suicide, between electric fields and brain cancer and suicide, and between pulsed electromagnetic fields and lung cancer, but these were not significant. CONCLUSION: These results are broadly reassuring that major causes of death are not strongly associated with exposure to electric and magnetic fields, but small numbers and approximate exposure assessments preclude the denial of any risk, in particular if it were to affect a rare cause of death.

Thrombin receptor activating peptide (TRAP) stimulates mitogenesis, c-fos and PDGF-A gene expression in human vascular smooth muscle cells.

The synthetic peptide SFLLRNPNDKYEPF, identical in sequence to the new amino-terminus of the thrombin receptor generated following cleavage of thrombin, acts a thrombin receptor agonist/activating peptide (TRAP). In this study, Northern blot analysis showed that cultured human vascular smooth muscle cells (HVSMC) express a thrombin receptor transcript. TRAP, in contrast to thrombin was shown to be a weak mitogen for HVSMC. A combination of TRAP and enzymatically-inactivated thrombin (PPACK-thrombin) which provides receptor occupancy, did not potentiate TRAP-induced mitogenesis, indicating that TRAP and PPACK-thrombin do not reproduce the mitogenic effect of enzymatically-active thrombin. Both thrombin and TRAP, induced the expression of c-fos and the PDGF-A gene in a pertussis toxin (PTX)-insensitive manner. Examination of thrombin and TRAP-treated cells by immunofluorescence staining followed by computer assisted image analysis revealed that thrombin and to a lesser extent TRAP induced PDGF-AA protein expression. Antibodies to PDGF-AA partially inhibited thrombin but not TRAP-induced mitogenesis in HVSMC. This study indicates that in addition to the common signalling pathways utilised by thrombin and TRAP, enzymatically-active thrombin activates other signalling pathways and hence TRAP does not mimic fully the biological effect of thrombin on HVSMC.

Electric and magnetic field exposures for people living near a 735-kilovolt power line.

The purpose of this study was to assess the effect of a 735-kV transmission line on the electric and magnetic field exposures of people living at the edge of the line's right of way. Exposure of 18 adults, mostly white-collar workers, living in different bungalows located 190-240 feet from the line (exposed subjects) was compared to that of 17 adults living in similar residences far away from any transmission line. Each subject carried a Positron meter for 24 hr during 1 workday, which measured 60-Hz electric and magnetic fields every minute. All measurements were carried out in parallel for exposed and unexposed subjects during the same weeks between September and December. During measurements the average loading on the line varied between 600 and 1100 A. The average magnetic field intensity while at home was 4.4 times higher among exposed subjects than unexposed (7.1 versus 1.6 mG, p = 0.0001) and 6.2 times higher when considering only the sleeping period (6.8 versus 1.1 mG, p = 0.0001). Based on the 24-hr measurement, average magnetic field exposure was three times higher among the exposed. Electric field intensity was also higher among the exposed while at home (26.3 versus 14.0 V/m, p = 0.03). Magnetic field intensity among the exposed was positively correlated with the loading on the line (r = 0.8, p = 0.001). Percentage of time above a magnetic field threshold (2 mG or 7.8 mG) was a good indicator to distinguish the two types of exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a class of peptide boronates with neutral P1 side chains as highly selective inhibitors of thrombin.

Z-D-Phe-Pro-boroMpg-OPin (9a)1,2 has been shown previously to be a highly specific inhibitor of thrombin in spite of lacking an arginine-like guanidino group at the P1 site. A range of compounds have been synthesized based upon this lead compound, varying the neutral side chain at the P1 site. Of the 20 examples based upon the structures at P2 and P3 of Z-D-X-Pro (X being Phe or beta,beta-diphenylalanine), all were found to be effective inhibitors of thrombin (Ki's between 10 and 100 nM). Furthermore all exhibited a high specificity toward thrombin having values for a Ki(trypsin)/Ki(thrombin) ratio of between 10- and 100-fold. High ratio values were found for a number of the compounds tested against a range of serine proteinases (plasmin, factor Xa, kallikrein, urokinase, protein Ca, chymotrypsin, elastase, and cathepsin G). As far as potency toward thrombin, compounds containing the methoxypropyl group at P1 were favored over those with a methoxy grouping on a shorter alkyl chain (8) or without the methoxy group (1-5). The compounds display potent anticoagulant activity with values for 18 in thrombin time of 0.63 microM and in activated partial thromboplastin time of 2.0 microM. 11B NMR has been used to confirm interaction of the boron atom with the active site. From the high specificity shown with all the compounds we propose that the compounds, constitute a new class of thrombin inhibitors.