Assessing Public Opinion Regarding Appropriate Responses to Would-be Mass Shooters: Applying a Balanced Justice Framework.

The unpredictable and widespread threat of mass shootings make them a concern that could affect anyone, anywhere. As such, being able to interrupt the process of planning and conducting a mass shooting represents a matter of public safety. Willingness to report, particularly on loved ones or associates, ultimately requires the public to be supportive of the interventions they think will be applied. In this study, we analyzed responses to an online "opt-in" survey (n = 274) that measured public opinion regarding how punitive (or therapeutic) the public at large suppose the sanctions for planning (but not [yet] conducting) a mass shooting should be. Our findings suggest the public is supportive of a balanced justice approach for both juveniles and adults, with and without mental illness, who plan a mass shooting when given the option.

Biotic differentiation in headwater creeks after the massive introduction of non-native freshwater aquarium fish in the Paraíba do Sul River basin, Brazil

This study evaluated fish beta diversity in six headwater creeks located in the area affected by the largest ornamental aquaculture center implemented in the Minas Gerais State, southeastern Brazil. We sampled fish assemblages in 2017 and 2018 to investigate changes in assemblage structure (species richness and beta diversity), comparing these data with the historic species pool. We recorded 60 fish species, of which 16 were native and 44 non-native with 19 translocated, and 25 exotic. The exotics Poecilia reticulata, Xiphophorus maculatus, X. variatus, Danio rerio, and Misgurnus anguillicaudatus were the most widely distributed in the headwater creeks. The Contamination Index showed that most creeks had high proportional contamination by exotic species (above 60%). Beta diversity increased from historical to contemporary periods in all creeks due to the introduction and differential colonization pressure of several non-native translocated and exotic species, indicating biotic differentiation. Temperature and number of ponds were the main preditors of change in beta diversity in the headwater creeks during the contemporary period. In summary, we observed that invaders have induced substantial changes to fish communities under influence of environmental filters. Our results support the hipothesis that aquaculture is a main driver of fish non-native fish introduction and native biodiversity loss in the Neotropics.(AU)

Este estudo avaliou a diversidade beta de peixes em seis riachos de cabeceira localizados em uma área afetada pelo maior centro de aqüicultura ornamental do Brasil, localizado em Minas Gerais. Amostramos assembleias de peixes em 2017 e 2018 para investigar mudanças na estrutura (riqueza de espécies e diversidade beta), comparando esses dados com tendências históricas de composição de comunidades. Registramos 60 espécies de peixes, sendo 16 nativos e 44 não nativos: 19 translocados e 25 exóticos. Os exóticos Poecilia reticulata, Xiphophorus maculatus, X. variatus, Danio rerio e Misgurnus anguillicaudatus foram os mais distribuídos nos riachos. O Índice de Contaminação mostrou que a maioria dos riachos apresentou alta contaminação proporcional por espécies exóticas (acima de 60%). A diversidade beta aumentou do período histórico para o contemporâneo em todos os riachos devido à introdução e pressão de colonização de várias espécies não-nativas translocadas e exóticas, indicando diferenciação biótica. Temperatura da água e número de tanques de piscicultura foram os principais fatores de mudança na diversidade beta dos riachos no período contemporâneo. Os não-nativos induziram mudanças em nível de comunidades e sob influência de variáveis ambientais. Os resultados mostram que a aquicultura é um dos principais vetores da introdução de peixes não-nativos e perda de biodiversidade nativa nos Neotrópicos.(AU)

Relação entre a progressão do escore do functional movement screen e lesão em atletas futebol / Relationship between progression of functional movement screen test and injury in athletes of football

O futebol é uma modalidade de grande exigência física com movimentos de potência e velocidade com mudança de direção. Devido à alta intensidade, ocorre uma maior predisposição às lesões. O Functional Movement Screen (FMS) é um modelo de avaliação para identificar assimetrias e desequilíbrios musculoesqueléticos. O objetivo deste trabalho foi comparar os efeitos de do is m eses de pré-temporada na progressão do escore do teste FMS e sua relação com as lesões ao longo da competição. Participaram deste estudo 28 atletas profissionais de futebol, com idade média de 25,1 ± 6,5. Foi realizado o FMS antes e após a pré-temporada e durante o período competitivo foi verificada a ocorrência de lesões. Houve uma melhora (p<0,01) dos escores do FMS da avaliação pré (15,61 ± 1,39) para a avaliação pós pré-temporada (17,29 ± 1,24) entre todo o elenco, porém, quando considerado o tamanho do efeito, os atletas que não se lesionaram tiveram um efeito maior na progressão do escore (d=1,17) do que os que se lesionaram (d=0,35). Conclui-se que os atletas que não se lesionaram durante a competição, foram os que mais progrediram no FMS durante a pré-temporada, sendo, provavelmente, a progressão do escore do s atletas mais interessante do que o atendimento ao ponto de corte sugerido pela literatura...(AU)

Football is a modality that demands high physical fitness, with intense movements and rapid direction changing. Due to the high intensity demanded, athletes are more susceptible t o in juries. The Functional Movement Screen (FMS) test it is a method to evaluate asymmetries and muscle im balance. The objective was to compared the effects of two months of preseason, on the score of the FMS t est and their relationship with the injuries resulting from the competitive period. Participated o f th is study 28 professional football players (25.1 ± 6.5 years). FMS was performed before and after the p reseason and during the competitive period the occurrence of injuries was verified. There was a significant improvement of the FMS score after the preseason (p<0.01) for the all athletes. When considering the size effect, athletes who were not injured during the competitive period had a greater effect (d=1.17) than those who injured (d=0.35). The athletes who progressed the most during the evaluation were the athletes who were exempted from injury during the competitive period. Perhaps the progression of the athlete's score was more interesting than the attendance to the cut-off point suggested by the literature...(AU)

Brazil's vulnerability to COVID-19 quantified by a spatial metric.

•An easy-to-apply index that can support decision-makers to identify the most vulnerable regions to COVID-19.•The impact of the lack of resources in the country-side to fight SARS-CoV-2 can be assuaged by shielding selected regions.•IndCo can support countries to prepare the reopening process while controlling COVID-19 in their regions.

Influence of Shoulder Position on Functional Control Ratio During Isokinetic Assessment.

CONTEXT: One of the possible mechanisms leading to secondary impingement syndrome may be the strength imbalance of shoulder rotators which is known as functional control ratio (FCR). The FCR is a ratio dividing the eccentric peak torque of the external rotators by the concentric peak torque of the internal rotators. Previous studies have focused on the reproducibility and reliability of isokinetic assessment, but there is little information on the influence of variable shoulder positions on FCR. OBJECTIVE: To compare shoulder FCR across 3 different shoulder abduction positions during isokinetic assessment. DESIGN: Cross-sectional study. SETTING: Biomechanics laboratory. PARTICIPANTS: Thirty-one healthy young university students (age 22.35 [0.95] y, weight 60.52 [9.31] kg, height 168.23 [9.47] cm). INTERVENTIONS: The concentric peak torque of internal rotators and eccentric peak torque of external rotators of right shoulder were measured on an isokinetic dynamometer. MAIN OUTCOME MEASURES: Concentric peak torque of the internal rotators and eccentric peak torque of the external rotators, measured using an isokinetic dynamometer. RESULTS: The concentric peak torque of internal rotators was significantly lower at 120° shoulder abduction compared with other positions (P < .001). The FCR was significantly higher at 120° shoulder abduction than 90° (P = .002) or 60° (P < .001) shoulder abduction because of the lower concentric peak torque. No significant difference was found in the FCR between the other 2 shoulder positions (P = .14). CONCLUSIONS: Shoulder position variations may influence FCR because of weakness of the internal rotators. Rehabilitation and injury prevention training programs should specifically focus on strengthening the internal rotators at more elevated angles of shoulder abduction.

Is Treatment Readiness Associated With Substance Use Treatment Engagement? An Exploratory Study.

With nearly 8.2% of Americans experiencing substance use disorders (SUDs), a need exists for effective SUD treatment and for strategies to assist treatment participants to complete treatment programs (Chandler, Fletcher, & Volkow, 2009). The purpose of the current research is to contribute to an emerging knowledge base about treatment readiness and its utility for predicting substance use treatment process performance measures. The study examines the relative salience of treatment readiness as a predictor of treatment engagement. Data are derived from adult cases included in the 2012 Global Appraisal of Individual Needs-Intake data set ( n = 5,443). Binary logistic regression was used to identify if treatment readiness predicts substance use treatment engagement. The findings of this study do not provide support for treatment readiness significantly predicting substance use treatment engagement. Further research is needed to better understand treatment engagement.

Motivational tools to improve probationer treatment outcomes.

BACKGROUND: Motivational interviewing (MI) is a promising practice to increase motivation, treatment retention, and reducing recidivism among offender populations. Computer-delivered interventions have grown in popularity as a way to change behaviors associated with drug and alcohol use. METHODS/DESIGN: Motivational Assistance Program to Initiate Treatment (MAPIT) is a three arm, multisite, randomized controlled trial, which examines the impact of Motivational interviewing (MI), a motivational computer program (MC), and supervision as usual (SAU) on addiction treatment initiation, engagement, and retention. Secondary outcomes include drug/alcohol use, probation progress, recidivism (i.e., criminal behavior) and HIV/AIDS testing and treatment among probationers. Participant characteristics are measured at baseline, 2, and 6 months after assignment. The entire study will include 600 offenders, with each site recruiting 300 offenders (Baltimore City, Maryland and Dallas, Texas). All participants will go through standard intake procedures for probation and participate in probation requirements as usual. After standard intake, participants will be recruited and screened for eligibility. DISCUSSION: The results of this clinical trial will fill a gap in knowledge about ways to motivate probationers to participate in addiction treatment and HIV care. This randomized clinical trial is innovative in the way it examines the use of in-person vs. technological approaches to improve probationer success. TRIAL REGISTRATION: NCT01891656.

Kinetic studies on the reaction of chlorosulfonyl isocyanate with monofluoroalkenes: experimental evidence for both stepwise and concerted mechanisms and a pre-equilibrium complex on the reaction pathway.

Chlorosulfonyl isocyanate (CSI) is reported to react with hydrocarbon alkenes by a stepwise dipolar pathway to give N-chlorosulfonyl-ß-lactams that are readily reduced to ß-lactams. Substitution of a vinyl hydrogen for a vinyl fluorine changes the dynamics for reaction with CSI so that a concerted pathway is favored. Rate constants were measured for reactions of CSI with monofluoroalkenes and some hydrocarbon alkenes. Activation parameters for two hydrocarbon alkenes and two monofluoroalkenes support this change in mechanism. A plot generated from the natural log of rate constants vs ionization potentials (IP) indicates that fluoroalkenes with IP values >8.9 eV react by a concerted process. Electron-rich monofluoroalkenes with IP values <8.5 eV were found to react by a single-electron transfer (SET) pathway. Hydrocarbon alkenes were also found to react by this dipolar stepwise SET intermediate rather than the previously accepted stepwise dipolar pathway. Data support a pre-equilibrium complex on the reaction pathway just before the rate-determining step of the concerted pathway and a SET intermediate for the stepwise reactions. When the reactions are carried out at lower temperatures, the equilibrium shifts toward the complex or SET intermediate enhancing the synthetic utility of these reactions. Kinetic data also support formation of a planar transition state rather than the orthogonal geometry as reported for ketene [2 + 2] cycloadditions.

Incursion of epizootic hemorrhagic disease into the Okanagan Valley, British Columbia in 1999.

In September 1999, unusually high mortality rates in white-tailed deer and California bighorn sheep occurred in the southern Okanagan Valley. Necropsy and histopathologic findings were compatible with epizootic hemorrhagic disease (EHD); the presence of virus was not demonstrated. Subsequent serologic and polymerase chain reaction assays on sentinel cattle suggested an EHD virus incursion.

Flow and pattern formation in a binary mixture of rotating granular materials

In a horizontally rotating cylinder, size segregation, pattern formation, and its time development are studied for a binary mixture of rodlike and disklike materials at various rotational frequencies. The rodlike particles formed a network that influenced their mobility and the shape of the avalanching surface. Windows installed on the cylinder enabled us to examine and control the distribution of the components of the mixture throughout the bulk. This has allowed us to study the evolution of naturally occurring and artificially created patterns. All observed patterns had a degree of asymmetry and were unstable. The stability of a band pattern is shown to depend on its symmetry. Qualitatively, the time for the transition from one set of bands to another was inversely related to the degree of asymmetry of the pattern. In addition, we propose that the parameter D/d (diameter of the cylinder over the diameter of the grains) plays a significant role in the functional dependence of the avalanching surface current on the dynamical angle of repose, and in the segregation process itself.

Ca2+ regulation of gelsolin activity: binding and severing of F-actin.

Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.

Severing of F-actin by the amino-terminal half of gelsolin suggests internal cooperativity in gelsolin.

Gelsolin is a Ca2+-regulated actin-binding protein that can sever, cap, and nucleate growth from the pointed ends of actin filaments. In this study we have measured the binding of the amino-terminal half of gelsolin, G1-3, to pyrene-labeled F-actin as a function of Ca2+ concentration. The rate of binding is shown to be dependent on micromolar concentrations of Ca2+. Independent experiments demonstrate that conformational changes in G1-3 are induced by micromolar concentrations of Ca2+. Titrations of pyrene-F-actin with G1-3 and gelsolin show that the quenching of pyrene fluorescence is identical in extent and stoichiometry for both G1-3 and gelsolin. In contrast, severing of F-actin by G1-3 is found to be much less efficient than is severing by gelsolin. In experiments in which F-actin severing is quantitatively measured, the filament number is found to be proportional to the 1.35 power of the G1-3 concentration. This deviation from linearity may be explained by cooperativity; the binding of two G1-3 molecules in close proximity may lead to cooperative severing of the polymer, thus increasing the severing efficiency. This model is supported by experiments that show that the efficiency of G1-3 severing of F-actin increases with increasing G1-3:F-actin ratios. Extrapolating from these results, we conclude that G4-6, the carboxyl-terminal half of gelsolin, has an active role in the severing of F-actin by intact gelsolin. Whereas F-actin severing by G1-3 is enhanced by cooperative binding of two separate G1-3 molecules, cooperativity is inherent to intact gelsolin because the cooperative partners are covalently linked.

A Kex2-related endopeptidase activity present in rat liver specifically processes the insulin proreceptor.

The insulin proreceptor is cleaved by limited proteolysis post-translationally at an Arg-Lys-Arg-Arg site to generate its mature alpha- and beta-subunit form. An 35S-labelled insulin proreceptor substrate preparation and a 15-mer peptide substrate that mimics the amino acid sequence around and including the insulin proreceptor processing site (IRP-peptide) has revealed an endopeptidase activity that catalyses insulin proreceptor cleavage in a rat liver subcellular fraction. Under optimal conditions, normal 35S-labelled insulin proreceptor substrate processing by this fraction was quantitative. This fraction was not able to process an 35S-labelled insulin proreceptor variant substrate (where the Arg-1 of the tetrabasic cleavage site had been replaced by Ala-1), similarly to previous in vivo observations, suggesting that this endopeptidase activity has physiological relevance. Biochemical characterization of the insulin proreceptor/IRP-peptide processing revealed this rat liver endopeptidase activity to have a broad pH range (> 70% maximal activity between pH 5.5 and 10.0) and a pH optimum of pH 8-10. It was Ca(2+)-dependent activity, maximally active between 0.5 and 5 mM Ca2+ and half-maximally activated between 50 and 90 microM Ca2+. Endoproteolytic activity was not inhibited by group-specific inhibitors of serine-, cysteinyl or aspartyl proteinases or by 1,10-phenanthroline; however, EDTA and 1,2-cyclohexanediaminetetraacetic acid did inhibit the activity, but this was accounted for by Ca2+ chelation. The IRP-peptide substrate assay enabled measurement of an apparent Km of 22 microM and a Vmax of 18.6 pmol/min for this endopeptidase activity. These biochemical characteristics suggest that insulin proreceptor processing endopeptidase activity to be a legitimate member of the Kex2-related proprotein convertase family. Immunoblotting detected furin and PACE4 proteins (both members of this family) to be present in the rat liver subcellular fraction containing insulin proreceptor processing activity. Since the biochemical characteristics of the insulin proreceptor processing endopeptidase activity mostly resembled those of furin activity, it is likely that insulin proreceptor proteolytic maturation can be catalysed by furin in the liver.

Processing of proopiomelanocortin by insulin secretory granule proinsulin processing endopeptidases.

A lysed preparation of isolated insulin secretory granules efficiently cleaved murine proopiomelanocortin (mPOMC) at physiologically important Lys-Arg processing sites. This processing was mostly attributed to an activity that co-eluted with the proinsulin processing type-II endopeptidase from anion exchange chromatography (Lys-Arg-directed; Davidson, H. W., Rhodes, C. J., and Hutton, J. C. (1988) Nature 333, 93-96). The principal peptide hormone products generated by the insulin secretory granule lysate were identified by specific radioimmunoassay and NH2-terminal microsequencing analysis of high performance liquid chromatography-separated products as alpha-melanocyte-stimulating hormone, corticotropin-like intermediate, gamma-lipotropin, beta-endorphin-(1-31), 18-kDa NH2-terminal fragment and, to a lesser extent, adrenocorticotrophin and beta-lipotropin. This processing had an acidic pH optimum (pH 5-5.5) and was Ca(2+)-dependent (K0.5 activation = 5-80 microM). With increasing Ca2+ concentrations there was an increase in the extent to which mPOMC was processed. The in vitro processing of mPOMC by the insulin secretory granule endopeptidase activity reported here is in excellent agreement with the in vivo processing of this prohormone by a combination of PC2 and PC3, candidates of prohormone endpeptidase, in gene transfer studies with cells that express the regulated secretory pathway (Thomas, L., Leduc, R., Thorne, B. A., Smeekens, S. S., Steiner, D. F., and Thomas, G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5297-5301).

The biosynthesis of the subtilisin-related proprotein convertase PC3, but no that of the PC2 convertase, is regulated by glucose in parallel to proinsulin biosynthesis in rat pancreatic islets.

The biosynthesis of proinsulin is specifically stimulated by glucose in the pancreatic beta-cell, and this, in turn, places an increased demand on the mechanism for proinsulin to insulin conversion. Proteolytic proinsulin processing is catalyzed by two endopeptidases putatively identified as the subtilisin-related PC2 and PC3 convertases (Bennett, D. L., Bailyes, E. M., Nielson, E., Guest, P. C., Rutherford, N. G., Arden, S. D., and Hutton, J. C. (1992) J. Biol. Chem. 267, 15229-15236; Bailyes, E. M., Shennan, K. I. J., Seal, A. J., Smeekens, S. P., Steiner, D. F., Hutton, J. C., and Docherty, K. (1992) Biochem. J. 285, 391-394). In this study, we demonstrate in isolated rat pancreatic islets that the biosynthesis of PC3 was specifically stimulated by glucose relatively parallel to that of proinsulin. In contrast, however, PC2 biosynthesis was not glucose-regulated. The stimulation of PC3 and proinsulin biosynthesis was observed above a threshold of 4 mM glucose and reached a maximum (about 7-10-fold) above 10 mM glucose concentrations. Glucose stimulation for PC3 and proinsulin biosynthesis was rapid (occurring within 20 min and reaching a maximum by 60 min) and was not affected by the additional presence of actinomycin D, suggesting regulation predominantly at the translational level. Moreover, the intracellular signals for glucose-stimulated PC3 and proinsulin biosynthesis appeared to be similar, requiring the metabolism of glucose. PC3 has been implicated as the key endopeptidase in proinsulin to insulin conversion, in that it is the enzyme which preferentially initiates the process (Rhodes, C. J., Lincoln, B., and Shoelson, S. E. (1992) J. Biol. Chem. 267, 22719-22727). We suggest that co-ordinate stimulation of PC3 biosynthesis, along with that of its proinsulin substrate, elucidates an additional control point by which the mechanism of proprotein processing might be regulated.

Preferential cleavage of des-31,32-proinsulin over intact proinsulin by the insulin secretory granule type II endopeptidase. Implication of a favored route for prohormone processing.

Two Ca(2+)-dependent endopeptidase activities are involved in proinsulin to insulin conversion: type I cleaves COOH-terminal to proinsulin Arg31-Arg32 (B-chain/C-peptide junction); and type II preferentially cleaves at the Lys64-Arg65 site (C-peptide/A-chain junction). To further understand the mechanism of proinsulin processing, we have investigated types I and II endopeptidase processing of intact proinsulin in parallel to that of the conversion intermediates, des-31,32-proinsulin and des-64,65-proinsulin. The type I processed des-64,65-proinsulin and proinsulin at the same rate. In contrast, the type II endopeptidase processed des-31,32-proinsulin at a much faster rate (> 19-fold; p < 0.001) than it did intact proinsulin. Furthermore, unlabeled proinsulin concentrations required for competitive inhibition of 125I-labeled des-64,65-proinsulin and 125I-proinsulin processing by a purified insulin secretory granule lysate were similar (ID50 = 14-16 microM), whereas inhibition of 125I-labeled des-31,32-proinsulin processing required a higher nonradiolabeled proinsulin concentration (ID50 = 197 microM). Synthetic peptides corresponding to the sequences surrounding Lys64-Arg65 (AC-peptide/substrate) and Arg31-Arg32 (BC-peptide/substrate) of human proinsulin were synthesized for use as specific substrates or competitive inhibitors. Cleavage of the BC-substrate by type I and AC-substrate by type II was COOH-terminal of the dibasic sequence, with similar Ca(2+)-and pH requirements previously observed for proinsulin cleavage. Apparent Km and Vmax for type I processing of the BC-substrate was Km = 20 microM; Vmax = 22.8 pmol/min, and for type II processing of the AC-substrate was Km = 68 microM; Vmax = 97 pmol/min. In competitive inhibition assays, the BC-peptide similarly blocked insulin secretory granule lysate processing of des-64,65-proinsulin and proinsulin (ID50 = 45-55 microM), but did not inhibit des-31,32-proinsulin processing. However, the AC-peptide preferentially inhibited insulin secretory granule lysate processing of des-31,32-proinsulin (ID50 = microM) compared to proinsulin (ID50 = 330 microM), and not des-64,65-proinsulin. We conclude that the type I endopeptidase recognized des-64,65-proinsulin and proinsulin as similar substrates, whereas the type II endopeptidase has a stronger preference for des-31,32-proinsulin compared to intact proinsulin. Furthermore, we suggest that in intact proinsulin there exists a constraint to efficient processing that is relieved following type I processing. Structural flexibility, in addition to the presence of Lys64-Arg65, therefore appears to be important for type II endopeptidase specificity and may provide a molecular basis for a preferential route of proinsulin conversion via des-31,32-proinsulin.

Fluorometric assay of a calcium-dependent, paired-basic processing endopeptidase present in insulinoma granules.

A novel fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC (RSKR-AMC) was used to characterize Ca(++)-activated proteolytic activity present in purified insulinoma secretory granules. Secretory granules efficiently cleaved this substrate in a time- and protein-dependent manner; the hydrolysis rate was between 2 and 4 pmol/min/ug of protein, with an apparent Km of 55 microM. Greater than 90% of the activity against this substrate was dependent on the presence of Ca++, with half-maximal stimulation obtained at 100 microM Ca++. The pH optimum of enzymatic activity was 5.5-6, and the profile of inhibition by various proteinase inhibitors was similar to that previously described for the type I and II proinsulin processing enzymes. These biochemical characteristics and co-elution of the RSKR-AMC processing activity with the type II endopeptidase activity on anion-exchange chromatography suggest that the new assay selectively detects the Lys-Arg-directed, or type II, proinsulin processing endopeptidase. This fluorogenic assay is more quantitative, sensitive and rapid than methods previously used, and therefore presents a significant improvement for the study of similar Ca(++)-activated processing endopeptidases.

Quantitation of 1,3-butanediol and its acidic metabolites by gas chromatography-mass spectrometry.

A number of problems present themselves during the gas chromatographic-mass spectrometric assay of R,S-1,3-butanediol as its bis-tert-butyldimethylsilyl ether. To circumvent these problems, three labeled internal standards were synthesized: (i) R,S-1,3-[3,4-13C2]-butanediol, (ii) R,S-1,3-[1,1,3-2H3]butanediol, and (iii) R,S-1,3-[1,1,3-2H3,3,4-13C2]butanediol. The availability of internal standards with different degrees of labeling allows (i) assaying of either unlabeled or 13C-labeled R,S-1,3-butanediol and (ii) analysis of 1,3-butanediol in either blood or urine samples. Reproducible standard curves were obtained using both electron impact and ammonia chemical ionization modes. The latter provides greater sensitivity and a lower limit of detection (5 microM). We have also designed an indirect assay of S-3-hydroxybutyrate, a catabolite of R,S-1,3-butanediol, which is difficult to analyze by conventional methods. This assay relies on the difference between (i) the concentration of R,S-3-hydroxybutyrate assayed by gas chromatography-mass spectrometry and (ii) the concentration of R-3-hydroxybutyrate assayed enzymatically.

Heme catabolism in cultured hepatocytes: evidence that heme oxygenase is the predominant pathway and that a proportion of synthesized heme is converted rapidly to biliverdin.

Heme oxygenase has been considered to be involved in the predominant pathway of heme degradation in vivo. However, alternative pathways involving cytochrome P-450 reductase, and lipid peroxidation, have previously been demonstrated in vitro, and studies with cultured rat hepatocytes were interpreted to show a majority of endogenous hepatic heme breakdown by non-heme oxygenase pathways. To clarify the pathway of heme breakdown in hepatocytes and the role of heme oxygenase in this process, cultured hepatocytes were pre-labelled with 5-[5-14C]aminolevulinate [( 14C]ALA). Radioactivity in heme, carbon monoxide, and bile pigments was measured for 8-24 h after the removal of [14C]ALA. In cultured chick embryo hepatocytes, which lack biliverdin reductase, the rate of production of biliverdin IXa was closely similar to the rate of catabolism of exogenous heme and radioactivity in carbon monoxide and biliverdin IXa was similar to the loss of radioactivity from endogenous heme. These results support the conclusion that heme breakdown occurred predominantly, if not solely, by heme oxygenase. Also, no evidence of non-heme oxygenase pathways was found in the presence of tin protoporphyrin, an inhibitor of heme oxygenase or mephenytoin, an inducer of both cytochrome P-450 and heme oxygenase. Similarly, in untreated cultured rat hepatocytes, radioactivity in carbon monoxide corresponded with loss of radioactivity in endogenous heme. In other experiments with chick hepatocyte cultures, rates of heme synthesis and breakdown were measured, and data were fitted to various models of hepatic heme metabolism. The results observed were consistent only with models in which an appreciable fraction (control cells, 17%, mephenytoin treated cells, 41%) of the newly synthesized heme was degraded rapidly to biliverdin.