Private groundwater contamination and risk management: A comparative scoping review of similarities, drivers and challenges across two socio-economically developed regions.

Consolidation of multi-domain risk management research is essential for strategies facilitating the concerted government (educational) and population-level (behavioural) actions required to reduce microbial private groundwater contamination. However, few studies to date have synthesised this literature or sought to ascertain the causal generality and extent of supply contamination and preventive responses. In light of the Republic of Ireland (ROI) and Ontario's high reliance and research focus on private wells and consequent utility for empirical comparison, a scoping review of pertinent literature (1990-2022) from both regions was undertaken. The SPICE (Setting, Perspective, Intervention, Comparison, Evaluation) method was employed to inform literature searches, with Scopus and Web of Science selected as primary databases for article identification. The review identified 65 relevant articles (Ontario = 34, ROI = 31), with those investigating well user actions (n = 22) and groundwater quality (n = 28) the most frequent. A markedly higher pooled proportion of private supplies in the ROI exhibited microbial contamination (38.3 % vs. 4.1 %), despite interregional similarities in contamination drivers (e.g., weather, physical supply characteristics). While Ontarian well users demonstrated higher rates of historical (≥ 1) and annual well testing (90.6 % vs. 71.1 %; 39.1 % vs. 8.6 %) and higher rates of historical well treatment (42.3 % vs. 24.3 %), interregional levels of general supply knowledge were analogous (70.7 % vs. 71.0 %). Financial cost, organoleptic properties and residence on property during supply construction emerged as predictors of cognition and behaviour in both regions. Review findings suggest broad interregional similarities in drivers of supply contamination and individual-level risk mitigation, indicating that divergence in contamination rates may be attributable to policy discrepancies - particularly well testing incentivisation. The paucity of identified intervention-oriented studies further highlights the importance of renewed research and policy agendas for improved, targeted well user outreach and incentivised, convenience-based services promoting routine supply maintenance.

Impacts of COVID-19 lockdown on private domestic groundwater sample numbers, E. coli presence and E. coli concentration across Ontario, January 2020-March 2021: An interrupted time-series analysis.

Approximately 1.5 million individuals in Ontario are supplied by private water wells (private groundwater supplies). Unlike municipal supplies, private well water quality remains unregulated, with owners responsible for testing, treating, and maintaining their own water supplies. The COVID-19 global pandemic and associated non-pharmaceutical interventions (NPIs) have impacted many environmental (e.g., surface water and air quality) and human (e.g., healthcare, transportation) systems over the past 15-months (January 2020 to March 2021). To date, the impact of these interventions on private groundwater systems remains largely unknown. Accordingly, the current study aimed to investigate the impact of a province-wide COVID-19 lockdown (late-March 2020) on health behaviours (i.e., private domestic groundwater sampling) and groundwater quality (via Escherichia coli (E. coli) detection and concentration) in private well water in Ontario, using time-series analyses (seasonal decomposition, interrupted time-series) of a large-spatio-temporal dataset (January 2016 to March 2021; N = 743,200 samples). Findings indicate that lockdown concurred with an immediate (p = 0.015) and sustained (p < 0.001) decrease in sampling rates, equating to approximately 2200 fewer samples received per week post-interruption. Likewise, a slightly decreased E. coli detection rate was observed approximately one month after lockdowns began (p = 0.003), while the proportion of "highly contaminated" samples (i.e., E. coli > 10 CFU/100 mL) was shown to increase within one month (p = 0.02), followed by a sustained decrease for the remainder of the year (May 2020-December 2020). Analyses strongly suggest that COVID-19 interventions resulted in discernible impacts on both well user behaviours and hydrogeological mechanisms. Findings may be used as an evidence-base for assisting policy makers, public health practitioners and private well owners in developing recommendations and mitigation strategies to manage public health risks during extreme and/or unprecedented future events.

Psychological impairment and extreme weather event (EWE) exposure, 1980-2020: A global pooled analysis integrating mental health and well-being metrics.

Extreme Weather Events (EWEs) impose a substantial health and socio-economic burden on exposed populations. Projected impacts on public health, based on increasing EWE frequencies since the 1950s, alongside evidence of human-mediated climatic change represents a growing concern. To date, the impacts of EWEs on mental health remain ambiguous, largely due to the inherent complexities in linking extreme weather phenomena with psychological status. This exploratory investigation provides a new empirical and global perspective on the psychological toll of EWEs by exclusively focusing on psychological morbidity among individuals exposed to such events. Morbidity data collated from a range of existing psychological and well-being measures have been integrated to develop a single ("holistic") metric, namely, psychological impairment. Morbidity, and impairment, were subsequently pooled for key disorders-, specifically PTSD, anxiety and depression. A "composite" (any impairment) post-exposure pooled-prevalence rate of 23% was estimated, with values of 24% calculated for depression and ⁓17% for both PTSD and anxiety. Notably, calculated pooled odds ratios (pOR = 1.9) indicate a high likelihood of any negative psychological outcome (+90%) following EWE exposure. Pooled analyses of reported risk factors (p < 0.05) highlight the pronounced impacts of EWEs among individuals with higher levels of event exposure or experienced stressors (14.5%) and socio-demographic traits traditionally linked to vulnerable sub-populations, including female gender (10%), previous history (i.e., pre-event) of psychological impairment (5.5%), lower socio-economic status (5.5%), and a lower education level (5.2%). Inherent limitations associated with collating mental health data from populations exposed to EWEs, and key knowledge gaps in the field are highlighted. Study findings provide a robust evidence base for developing and implementing public health intervention strategies aimed at ameliorating the psychological impacts of extreme weather among exposed populations.

Modelling COVID-19 severity in the Republic of Ireland using patient co-morbidities, socioeconomic profile and geographic location, February to November 2020.

Understanding patient progression from symptomatic COVID-19 infection to a severe outcome represents an important tool for improved diagnoses, surveillance, and triage. A series of models have been developed and validated to elucidate hospitalization, admission to an intensive care unit (ICU) and mortality in patients from the Republic of Ireland. This retrospective cohort study of patients with laboratory-confirmed symptomatic COVID-19 infection included data extracted from national COVID-19 surveillance forms (i.e., age, gender, underlying health conditions, occupation) and geographically-referenced potential predictors (i.e., urban/rural classification, socio-economic profile). Generalised linear models and recursive partitioning and regression trees were used to elucidate COVID-19 progression. The incidence of symptomatic infection over the study-period was 0.96% (n = 47,265), of whom 3781 (8%) required hospitalisation, 615 (1.3%) were admitted to ICU and 1326 (2.8%) died. Models demonstrated an increasingly efficacious fit for predicting hospitalization [AUC 0.816 (95% CI 0.809, 0.822)], admission to ICU [AUC 0.885 (95% CI 0.88 0.89)] and death [AUC of 0.955 (95% CI 0.951 0.959)]. Severe obesity (BMI ≥ 40) was identified as a risk factor across all prognostic models; severely obese patients were substantially more likely to receive ICU treatment [OR 19.630] or die [OR 10.802]. Rural living was associated with an increased risk of hospitalization (OR 1.200 (95% CI 1.143-1.261)]. Urban living was associated with ICU admission [OR 1.533 (95% CI 1.606-1.682)]. Models provide approaches for predicting COVID-19 prognoses, allowing for evidence-based decision-making pertaining to targeted non-pharmaceutical interventions, risk-based vaccination priorities and improved patient triage.

Private groundwater management and risk awareness: A cross-sectional analysis of two age-related subsets in the Republic of Ireland.

Risk communication represents the optimal instrument for decreasing the incidence of private groundwater contamination and associated waterborne illnesses. However, despite attempts to promote voluntary well maintenance in high groundwater-reliant regions such as the Republic of Ireland, awareness levels of supply status (e.g. structural integrity) have remained low. As investigations of supply awareness are often thematically narrow and homogeneous with respect to sub-population, revised analyses of awareness among both current and future supply owners (i.e. adults of typical well owner and student age) are necessary. Accordingly, the current study utilised a national survey of well users and an age-based comparison of supply awareness. Awareness was measured among 560 Irish private well users using a multi-domain scoring framework and analysed in conjunction with experiential variables including experience of extreme weather events and previous household infections, and perceived self-efficacy in maintaining supply. Respondents displayed a median overall awareness score of 66.7%, with supply owners (n = 399) and students (n = 161) exhibiting median scores of 75% and 58.3%. Awareness among both combined respondent subsets and well owners was significantly related to gender, well use factors and self-perceived behavioural efficacy while awareness among students was not correlated with any independent variable. Cluster analysis identified three distinct respondent groups characterised by awareness score and gender in both current and future well owner subsets. Male well owners and students displayed higher perceived self-efficacy irrespective of awareness score while female well owners that demonstrated high awareness were significantly more likely to report postgraduate educational (p < 0.001). Findings suggest that recent experience of extreme weather events does not significantly influence supply awareness and mirror previously identified knowledge differences between well owners and young adults. Age, gender, supply use and perceived self-efficacy emerge as recurring focal points and accordingly merit consideration from groundwater and health communication practitioners for future risk interventions.

Private groundwater contamination and extreme weather events: The role of demographics, experience and cognitive factors on risk perceptions of Irish private well users.

Extreme weather events (EWEs) may significantly increase pathogenic contamination of private (unregulated) groundwater supplies. However, due to the paucity of protective guidance, private well users may be ill-equipped to undertake adaptive actions. With rising instances of waterborne illness documented in groundwater-dependent, developed regions such as the Republic of Ireland, a better understanding of well user risk perceptions pertaining to EWEs is required to establish appropriate educational interventions. To this end, the current study employed an online and physical questionnaire to identify current risk perceptions and correspondent predictors among Irish private well users concerning extreme weather. Respondents were elicited via purposive sampling, with 515 private well users elucidating perceived supply contamination risk in the wake of five EWEs between the years 2013-2018 including drought and pluvial flooding. A novel scoring protocol was devised to quantify overall risk perception (i.e. perceived likelihood, severity and consequences) of extreme weather impacts. Overall risk perception of EWEs was found to demonstrate a significant relationship with gender (p = 0.017) and event experience (p < 0.001), with female respondents and those reporting prior event experience exhibiting higher median risk perception scores. Risk perception was additionally mediated by perceived self-efficacy in undertaking supply maintenance (p = 0.001), as well users citing confidence in ability scored significantly lower than those citing no confidence. Two-step cluster analysis identified three distinct respondent subsets based on risk perception of EWEs (high, moderate and low perception), with female respondents and those with a third-level education significantly more likely to fall within the high perception cluster. Study findings affirm that certain demographic, experiential and cognitive factors exert a significant influence on private well user risk perceptions of EWE impacts and highlight potential focal points for future educational interventions seeking to reduce the risk of human infection associated with groundwater and extreme weather.

Contamination of domestic groundwater systems by verotoxigenic escherichia coli (VTEC), 2003-2019: A global scoping review.

Verocytotoxin-producing E. coli (VTEC) are important agents of diarrhoeal disease in humans globally. As a noted waterborne disease, emphasis has been given to the study VTEC in surface waters, readily susceptible to microbial contamination. Conversely, the status of VTEC in potable groundwater sources, generally regarded as a "safe" drinking-water supply remains largely understudied. As such, this investigation presents the first scoping review seeking to determine the global prevalence of VTEC in groundwater supply sources intended for human consumption. Twenty-three peer-reviewed studies were identified and included for data extraction. Groundwater sample and supply detection rates (estimated 0.6 and 1.3%, respectively) indicate VTEC is infrequently present in domestic groundwater sources. However, where generic (fecal indicator) E. coli are present, the VTEC to E. coli ratio was found to be 9.9%, representing a latent health concern for groundwater consumers. Geographically, extracted data indicates higher VTEC detection rates in urban (5.4%) and peri­urban (4.9%) environments than in rural areas (0.9%); however, this finding is confounded by the predominance of research studies in lower income regions. Climate trends indicate local environments classified as 'temperate' (14/554; 2.5%) and 'cold' (8/392; 2%) accounted for a majority of supply sources with VTEC present, with similar detection rates encountered among supplies sampled during periods typically characterized by 'high' precipitation (15/649; 2.3%). Proposed prevalence figures may find application in preventive risk-based catchment and groundwater quality management including development of Quantitative Microbial Risk Assessments (QMRA). Notwithstanding, to an extent, a large geographical disparity in available investigations, lack of standardized reporting, and bias in source selection, restrict the transferability of research findings. Overall, the mechanisms responsible for VTEC transport and ingress into groundwater supplies remain ambiguous, representing a critical knowledge gap, and denoting a distinctive lack of integration between hydrogeological and public health research. Key recommendations and guidelines are provided for prospective studies directed at increasingly integrative and multi-disciplinary research.

Cryptosporidium spp. in groundwater supplies intended for human consumption - A descriptive review of global prevalence, risk factors and knowledge gaps.

Cryptosporidiosis is one of the leading causes of diarrhoeal illness and mortality induced by protozoan pathogens worldwide. As a largely waterborne disease, emphasis has been given to the study of Cryptosporidium spp. in surface waters, readily susceptible to pathogenic contamination. Conversely, the status of Cryptosporidium in potable groundwater sources, generally regarded as a pristine and "safe" drinking-water supply owing to (sub)-soil protection, remains largely unknown. As such, this investigation presents the first literature review aimed to ascertain the global prevalence of Cryptosporidium in groundwater supply sources intended for human consumption. Thirty-seven peer-reviewed studies were identified and included in the review. Groundwater sample and supply detection rates (estimated 10-20%) indicate Cryptosporidium is frequently present in domestic groundwater sources, representing a latent health concern for groundwater consumers. Specifically, sample (10.4%) and source (19.1%) detection rates deriving from comprehensive "temporal" investigations are put forward as representative of a contamination 'baseline' for Cryptosporidium in 'domestic' groundwater supplies. Proposed 'baseline' prevalence figures are largely applicable in preventive risk-based catchment and groundwater quality management including the formulation of Quantitative Microbial Risk Assessment (QMRA). Notwithstanding, a large geographical disparity in available investigations and lack of standardized reporting restrict the transferability of research findings. Overall, the mechanisms responsible for Cryptosporidium transport and ingress into groundwater supplies remain ambiguous, representing a critical knowledge gap, and denoting a distinctive lack of integration between groundwater and public-health sub-disciplines among investigations. Key recommendations and guidelines are provided for prospective studies directed at more integrative and multi-disciplinary research.

Knowledge and behavioural interventions to reduce human health risk from private groundwater systems: A global review and pooled analysis based on development status.

Groundwater contamination constitutes a significant health risk for private well users residing in rural areas. As the responsibility to safeguard rural private domestic groundwater typically rests with non-expert homeowners, interventions promoting risk mitigation and awareness represent the most viable means of preventing supply contamination. However, no global review or pooled analyses of these interventions has been undertaken to date. The current study sought to identify and quantify the performance of private well interventions from 1990 to 2018 via a global systematised review and pooled analysis. The PICO (Population-Intervention-Comparison-Outcome) approach was employed for literature identification. Relevant studies were statistically analysed across two quantitative outcome (performance) types, namely knowledge and behaviour, controlling for intervention characteristics and country development status. Mean behavioural and knowledge attainment across interventions was 53% and 48%, respectively, with interventions in economically developed regions exhibiting higher behavioural outcomes (56% vs. 45%) than those in developing regions. Geographically, interventions were located in southern or southeast Asia (n = 23), North America (n = 15), Central America (n = 1) and Africa (n = 1), with none identified in Australia/Oceania, Europe, or South America. Behavioural outcomes were significantly associated with presence of educational/research coordinator (p = 0.023), with these interventions attaining higher levels of efficacy (+74%) than those implemented by other coordinator types. Findings indicate that instructor-led, practical interventions allied with both large- and local-scale awareness-raising campaigns represent an optimum approach for future private well risk interventions. Subsequent adoption of such interventions may lead to increased levels of private well maintenance and provide a point of reference for myriad water and health communication contexts.

Assessment of two behavioural models (HBM and RANAS) for predicting health behaviours in response to environmental threats: Surface water flooding as a source of groundwater contamination and subsequent waterborne infection in the Republic of Ireland.

Extreme weather events (EWEs) are increasing in frequency, posing a greater risk of adverse human health effects. As such, developing sociological and psychological based interventions is paramount to empowering individuals and communities to actively protect their own health. Accordingly, this study compared the efficacy of two established social-cognitive models, namely the Health Beliefs Model (HBM) and Risks-Attitudes-Norms-Abilities-Self-regulation (RANAS) framework, in predicting health behaviours following EWEs. Surface water flooding was used as the exemplar EWE in the current study, due to the increasing incidence of these events in the Republic of Ireland over the past decade. Levels of prior experience with flooding were considered for analyses and comparative tools included a number of variables predicting health behaviours and intervention potential scores (i.e. measure of impact of targeting each model element). Results suggest that the RANAS model provides a robust foundation for designing interventions for any level of experience with an extreme weather event, however, use of the simpler HBM may be more cost-effective among participants unacquainted with an EWE and in relatively infrequent health threat scenarios. Results provide an evidence base for researchers and policymakers to appropriately engage with populations about such threats and successfully promote spatiotemporally appropriate health behaviours in a changing climate.

Surface water flooding, groundwater contamination, and enteric disease in developed countries: A scoping review of connections and consequences.

Significant volumes of research over the past four decades has sought to elucidate the social, infrastructural, economic, and human health effects of climate change induced surface flooding. To date, epidemiological and public health studies of flooding events have focused on mental health effects, vector-borne diseases, and infectious enteric disease due to floodwater contact (i.e. typically low consumption rates). The inherent nature of groundwater (i.e. out of sight, out of mind) and the widely held belief that aquifers represent a pristine source of drinking water due to natural attenuation may represent the "perfect storm" causing direct consumption of relatively large volumes of surface flood-contaminated groundwater. Accordingly, the current study sought to systematically identify and synthesize all available peer-reviewed literature pertaining to the nexus between surface flooding, groundwater contamination and human gastroenteric outcomes. Just 14 relevant studies were found to have been published during the period 1980-2017, thus highlighting the fact that this potentially significant source of climate-related exposure to environmental infection has remained understudied to date. Studies differed significantly in terms of type and data reporting procedures, making it difficult to discern clear trends and patterns. Approximately 945 confirmed cases of flood-related enteric disease were examined across studies; these concurred with almost 10,000 suspected cases, equating to approximately 20 suspected cases per confirmed case. As such, no regional, national or global estimates are available for the human gastrointestinal health burden of flood-related groundwater contamination. In light of the demonstrable public health significance of the concurrent impacts of groundwater susceptibility and climate change exacerbation, strategies to increase awareness about potential sources of contamination and motivate precautionary behaviour (e.g. drinking water testing and treatment, supply interruptions) are necessary. Mainstreaming climate adaptation concerns into planning policies will also be necessary to reduce human exposure to waterborne sources of enteric infection.

A geostatistical investigation of agricultural and infrastructural risk factors associated with primary verotoxigenic E. coli (VTEC) infection in the Republic of Ireland, 2008-2013.

Ireland reports the highest incidence of verotoxigenic Escherichia coli (VTEC) infection in Europe. This study investigated potential risk factors for confirmed sporadic and outbreak primary VTEC infections during 2008-2013. Overall, 989 VTEC infections including 521 serogroup O157 and 233 serogroup O26 were geo-referenced to 931 of 18 488 census enumeration areas. The geographical distribution of human population, livestock, unregulated groundwater sources, domestic wastewater treatment systems (DWWTS) and a deprivation index were examined relative to notification of VTEC events in 524 of 6242 rural areas. Multivariate modelling identified three spatially derived variables associated with VTEC notification: private well usage [odds ratio (OR) 6·896, P < 0·001], cattle density (OR 1·002, P < 0·001) and DWWTS density (OR 0·978, P = 0·002). Private well usage (OR 18·727, P < 0·001) and cattle density (OR 1·001, P = 0·007) were both associated with VTEC O157 infection, while DWWTS density (OR 0·987, P = 0·028) was significant within the VTEC O26 model. Findings indicate that VTEC infection in the Republic of Ireland is particularly associated with rural areas, which are associated with a ubiquity of pathogen sources (cattle) and pathways (unregulated groundwater supplies).

A peroxidase homologue and novel plastocyanin located by proteomics to the Arabidopsis chloroplast thylakoid lumen.

A study by two-dimensional electrophoresis showed that the soluble, lumenal fraction of Arabidopsis thaliana thylakoids can be resolved into 300 protein spots. After subtraction of low-intensity spots and accounting for low-level stromal contamination, the number of more abundant, lumenal proteins was estimated to be between 30 and 60. Two of these proteins have been identified: a novel plastocyanin that also was the predominant component of the total plastocyanin pool, and a putative ascorbate peroxidase. Import studies showed that these proteins are routed to the thylakoid lumen by the Sec- and delta pH-dependent translocation pathways, respectively. In addition, novel isoforms of PsbO and PsbQ were identified.

Different lumen-targeting pathways for nuclear-encoded versus cyanobacterial/plastid-encoded Hcf136 proteins.

Lumenal proteins are transported across the thylakoid membrane by two very different pathways: Sec-dependent or twin-arginine translocase (Tat)-dependent, where the substrate protein can be transported in a folded state. We present the first evidence that a given protein can be targeted by different pathways in different organisms. Arabidopsis Hcf136 is targeted exclusively by the Tat pathway in pea chloroplasts and no Sec-dependent transport is evident even when the twin-arginine is replaced by twin-lysine. However, twin-arginine motifs are absent from the presequences of Hcf136 proteins encoded by plastid or cyanobacterial genomes, strongly implying translocation by another pathway (presumably Sec). We suggest that the Hcf136 protein was transferred to the Tat pathway when the gene became incorporated into the nuclear genome, possibly due to the tighter folding associated with the more involved, post-translational targeting pathway.

The sec-independent twin-arginine translocation system can transport both tightly folded and malfolded proteins across the thylakoid membrane.

A subset of lumen proteins is transported across the thylakoid membrane by a Sec-independent translocase that recognizes a twin-arginine motif in the targeting signal. A related system operates in bacteria, apparently for the export of redox cofactor-containing proteins. In this report we describe a key feature of this system, the ability to transport folded proteins. The thylakoidal system is able to transport dihydrofolate reductase (DHFR) when an appropriate signal is attached, and the transport efficiency is almost undiminished by the binding of folate analogs such as methotrexate that cause the protein to fold very tightly. The system is moreover able to transport DHFR into the lumen with methotrexate bound in the active site, demonstrating that the DeltapH-driven transport of large, native structures is possible by this pathway. However, correct folding is not a prerequisite for transport. Truncated, malfolded DHFR can be translocated by this system, as can physiological substrates that are severely malfolded by the incorporation of amino acid analogs.

Targeting of thylakoid proteins by the delta pH-driven twin-arginine translocation pathway requires a specific signal in the hydrophobic domain in conjunction with the twin-arginine motif.

Superficially similar cleavable targeting signals specify whether lumenal proteins are transported across the thylakoid membrane by a Sec- or delta pH-dependent pathway. A twin-arginine motif is essential but not sufficient to direct delta pH-dependent targeting; here we show that a second determinant is located in the hydrophobic region. A highly hydrophobic amino acid is found either two or three residues C-terminal to the twin-arginine in all known transfer peptides for the delta pH-dependent system, and substitution of this residue in the 23-kDa (23K) peptide markedly inhibits translocation. Further, whereas the insertion of twin-arginine in a Sec-dependent precursor does not permit efficient delta pH-dependent targeting, the simultaneous presence of a leucine at the +3 position (relative to the RR) enables the peptide to function as efficiently as an authentic transfer peptide. RRNVL, RRAAL and RRALA within a Sec targeting signal all support efficient delta pH-dependent targeting, RRNVA is less effective and RRNAA/RRNAG are totally ineffective. We conclude that the core signal for this pathway is a twin-arginine together with an adjacent hydrophobic determinant.

Multiple pathways for the targeting of thylakoid proteins in chloroplasts.

The assembly of the photosynthetic apparatus requires the import of numerous cytosolically synthesised proteins and their correct targeting into or across the thylakoid membrane. Biochemical and genetic studies have revealed the operation of several targeting pathways for these proteins, some of which are used for thylakoid lumen proteins whereas others are utilised by membrane proteins. Some pathways can be traced back to the prokaryotic ancestors of chloroplasts but at least one pathway appears to have arisen in response to the transfer of genes from the organelle to the nucleus. In this article we review recent findings in this field that point to the operation of a mechanistically unique protein translocase in both plastids and bacteria, and we discuss emerging data that reconcile the remarkable variety of targeting pathways with the natures of the substrate precursor proteins.

Biochemical characterization of matrilysin. Activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases.

The latent precursor of matrilysin (EC 3.4.24.23; punctuated metalloproteinase (PUMP) was purified from transfected mouse myeloma cell conditioned medium and was found to contain one zinc atom per molecule which was essential for catalytic activity. Promatrilysin could be activated to the same specific activity by (4-aminophenyl)mercuric acetate, trypsin, and incubation at elevated temperatures (heat activation). Active matrilysin hydrolyzed the fluorescent substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond with a maximum value for kcat/Km of 1.3 x 10(4) M-1 s-1 at the pH optimum of 6.5 and pKa values of 4.60 and 8.65. Activity is inhibited by the tissue inhibitor of metalloproteinases-1 in a 1:1 stoichiometric interaction. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with N-terminal sequencing revealed that, as with all other matrix metalloproteinases similarly studied, promatrilysin activation was accompanied by the stepwise proteolytic removal of an M(r) 9000 propeptide from the N-terminus. The intermediates generated were dependent on the mode of activation used but, in all cases studied, activation terminated with an autocatalytic cleavage at E77-Y78 to yield the final M(r) 19,000 active matrilysin. From an analysis of the stability of the various intermediates, we propose that the sequence L13-K33 is particularly important in protecting the E77-Y78 site from autocatalytic cleavage, thereby maintaining the latency of the proenzyme.

A standardized procedure for quantification of the Ames Salmonella/mammalian-microsome mutagenicity test.

Factors determining the precision and variability of the Ames Salmonella test for mutagenicity were investigated. The most important source of variability in the agar-overlay method was nonuniformity in the soft-agar layer thickness. Solution of this problem resulted from application of an agar-leveling table described in this paper. Several other procedural elements also contribute to improved precision, including temperature uniformity during incubation, incubation interval, consistency of plate agar volume, completeness of mixing the soft-agar overlay, peculiarities in the interaction of mutagens and mammalian liver microsomal extract (S9), and methods of storage and controls for tester strains. When these and other effects were well-controlled, variability of the test results was reduced from 200 or 300% to only +/- 10% or less. The significance of the factors affecting precision are discussed and an improved experimental protocol is presented.