Loop-mediated isothermal amplification: Development, validation and application of simple and rapid assays for quantitative detection of species of Arcobacteraceae family- and species-specific Aliarcobacter faecis and Aliarcobacter lanthieri.

AIM: The family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis and Aliarcobacter lanthieri have been identified as emerging pathogens potentially cause health risks to humans and animals. This study was designed to develop/optimize, validate and apply Arcobacteraceae family- and two species-specific (A. faecis and A. lanthieri) loop-mediated isothermal amplification (LAMP) assays to rapidly detect and quantify total number of cells in various environmental niches. METHODS AND RESULTS: Three sets of LAMP primers were designed from conserved and variable regions of 16S rRNA (family-specific) and gyrB (species-specific) genes. Optimized Arcobacteraceae family-specific LAMP assay correctly amplified and detected 24 species, whereas species-specific LAMP assays detected A. faecis and A. lanthieri reference strains as well as 91 pure and mixed culture isolates recovered from aquatic and faecal sources. The specificity of LAMP amplification of A. faecis and A. lanthieri was further confirmed by restriction fragment length polymorphism analysis. Assay sensitivities were tested using variable DNA concentrations extracted from simulated target species cells in an autoclaved agricultural water sample by achieving a minimum detection limit of 10 cells mL-1 (10 fg). Direct DNA-based quantitative detection, from agricultural surface water, identified A. faecis (17%) and A. lanthieri (1%) at a low frequency compared to family-level (93%) with the concentration ranging from 2·1 × 101 to 2·2 × 105 cells 100 mL-1 . CONCLUSIONS: Overall, these three DNA-based rapid and cost-effective novel LAMP assays are sensitive and can be completed in less than 40 min. They have potential for on-site quantitative detection of species of family Arcobacteraceae, A. faecis and A. lanthieri in food, environmental and clinical matrices. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed LAMP assays are specific, sensitive, accurate with higher reproducibility that have potential to facilitate in a less equipped lab setting and can help in early quantitative detection and rate of prevalence in environmental niches. The assays can be adopted in the diagnostic labs and epidemiological studies.

Enhanced Single-tube Multiplex PCR Assay for Detection and Identification of Six Arcobacter Species.

AIM: A single-tube multiplex PCR (mPCR) assay was developed for rapid, sensitive and simultaneous detection and identification of six Arcobacter species including two new species, A. lanthieri and A. faecis, along with A. butzleri, A. cibarius, A. cryaerophilus and A. skirrowii on the basis of differences in the lengths of their PCR products. Previously designed monoplex, mPCR and RFLP assays do not detect or differentiate A. faecis and A. lanthieri from other closely related known Arcobacter spp. METHODS AND RESULTS: Primer pairs for each target species (except A. skirrowii) and mPCR protocol were newly designed and optimized using variable regions of housekeeping including cpn60, gyrA, gyrB and rpoB genes. The accuracy and specificity of the mPCR assay was assessed using DNA templates from six targets and 11 other Arcobacter spp. as well as 50 other bacterial reference species and strains. Tests on the DNA templates of target Arcobacter spp. were appropriately identified, whereas all 61 other DNA templates from other bacterial species and strains were not amplified. Sensitivity and specificity of the mPCR assay was 10 pg µl-1 of DNA concentration per target species. The optimized assay was further evaluated, validated and compared with other mPCR assays by testing Arcobacter cultures isolated from various faecal and water sources. CONCLUSIONS: Study results confirm that the newly developed mPCR assay is rapid, accurate, reliable, simple, and valuable for the simultaneous detection and routine diagnosis of six human- and animal-associated Arcobacter spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The new mPCR assay is useful not only for pure but also mixed cultures. Moreover, it has the ability to rapidly detect six species which enhances the value of this technology for aetiological and epidemiological studies.

Blood donation clusters in Québec, Canada (2003-2008): spatial variations according to sex and age.

BACKGROUND AND OBJECTIVES: The detection of spatial clusters of blood donation rate is an important issue, especially for targeting spatial units with significantly low rates, where it could be possible to increase the numbers of donors. The objective of this study is to detect spatial clusters of high or low blood donation rate in Québec according to sex and age of the donors. MATERIALS AND METHODS: Blood donation data were obtained from Héma-Québec over a period of 5 years. We aggregated these data for each of 101 municipalités regionales de comté (i.e. counties) for men, women and four age groups. To detect spatial high/low donation rate areas, we used the Kulldorff's scan statistics. Kappa coefficient was used to assess discordance between clusters obtained for the different groups (18-29, 30-39, 40-49, 50-59, 60-69 years old). T-test analyses were conducted to identify significant associations between spatial clusters and socio-economic variables. RESULTS: The results indicate the presence of several geographical areas with high or low blood donation rates for each group. The size, the location and the socio-demographic profiles of low/high clusters vary according to sex and age categories. CONCLUSION: The Kulldorff's scan statistics are an efficient tool to assess the blood donation performance across a country or even a specific region over a period of several years. In terms of strategic planning and monitoring, it can be used as a fully operational tool to target areas with significantly low rates (for all donors or specific demographic groups) in future blood donation campaigns.

Genome-wide association study of body mass index in 23 000 individuals with and without asthma.

BACKGROUND: Both asthma and obesity are complex disorders that are influenced by environmental and genetic factors. Shared genetic factors between asthma and obesity have been proposed to partly explain epidemiological findings of co-morbidity between these conditions. OBJECTIVE: To identify genetic variants that are associated with body mass index (BMI) in asthmatic children and adults, and to evaluate if there are differences between the genetics of BMI in asthmatics and healthy individuals. METHODS: In total, 19 studies contributed with genome-wide analysis study (GWAS) data from more than 23 000 individuals with predominantly European descent, of whom 8165 are asthmatics. RESULTS: We report associations between several DENND1B variants (P = 2.2 × 10(-7) for rs4915551) on chromosome 1q31 and BMI from a meta-analysis of GWAS data using 2691 asthmatic children (screening data). The top DENND1B single nucleotide polymorphisms(SNPs) were next evaluated in seven independent replication data sets comprising 2014 asthmatics, and rs4915551 was nominally replicated (P < 0.05) in two of the seven studies and of borderline significance in one (P = 0.059). However, strong evidence of effect heterogeneity was observed and overall, the association between rs4915551 and BMI was not significant in the total replication data set, P = 0.71. Using a random effects model, BMI was overall estimated to increase by 0.30 kg/m(2) (P = 0.01 for combined screening and replication data sets, N = 4705) per additional G allele of this DENND1BSNP. FTO was confirmed as an important gene for adult and childhood BMI regardless of asthma status. CONCLUSIONS AND CLINICAL RELEVANCE: DENND1B was recently identified as an asthma susceptibility gene in a GWAS on children, and here, we find evidence that DENND1B variants may also be associated with BMI in asthmatic children. However, the association was overall not replicated in the independent data sets and the heterogeneous effect of DENND1B points to complex associations with the studied diseases that deserve further study.

Parametric sensitivity analysis and reduction of a detailed nutritional model of plant cell cultures.

A kinetic model of plant nutrition described by Cloutier et al. (Cloutier et al., 2008. Biotechnol Bioeng 99:189-200) is progressively simplified so as to obtain a predictive model that describes the evolution of the biomass and the extracellular and intracellular concentrations of three determining nutrients, that is, free intracellular nitrogen, phosphate, and carbohydrate compounds. Three techniques of global sensitivity analysis are successively applied to assess the model parameter influence and potential correlation. The resulting dynamic model is able to predict plant growth for the two most encountered plant bioprocesses, namely suspension cells and hairy roots.

Distribution of selected virulence genes and antibiotic resistance in Enterococcus species isolated from the South Nation River drainage basin, Ontario, Canada.

AIMS: Isolate and characterize water enterococci from the South Nation River drainage basin, an area dominated by agriculture. METHODS AND RESULTS: A total of 1558 enterococci were isolated from 204 water samples from the South Nation River obtained over a 3-year period. PCR was used to identify isolates to the species level and characterize them for carriage of 12 virulence determinants. Antibiotic resistance was evaluated phenotypically. Enterococcus faecalis (36·4%), Enterococcus faecium (9·3%) and Enterococcus durans (8·5%) were the major enterococci species isolated. Enterococci carrying more than two virulence determinants were more frequently detected in the summer (59·6%) than in other seasons (≤ 37·6%). Very few isolates (≤ 2·0%) were resistant to category I antibiotics ciprofloxacin and vancomycin. CONCLUSIONS: Comparison of major water enterococci species with major faecal enterococci species obtained from various host groups (human, domesticated mammals and birds, wildlife) in this drainage basin suggest that water enterococci may have varied faecal origins. The low level of antibiotic resistance among enterococci suggests that dispersion of antibiotic resistance via waterborne enterococci in this watershed is not significant. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study suggests that water enterococci in the SNR have a faecal origin and that their potential impact on public health regarding antibiotic resistance and virulence determinants is minimal.

Mapping responsible conduct in the uncharted field of research-creation: A scoping review.

This scoping review addresses the issues of responsible conduct of research (RCR) that can arise in the practice of research-creation (RC), an emergent, interdisciplinary, and heterogeneous field at the interface of academic research and creative activities. Little is yet known about the nature and scope of RCR issues in RC, so our study examined three questions: (1) What are the specific issues in RC in relation to RCR? (2) How does the specificity of RC influence the understanding and practice of RCR? (3) What recommendations could help address the issues highlighted in the literature? To answer these questions, we conducted a scoping review of the academic literature (n = 181 texts) dealing with RCR in RC. We found that researcher-creators faced some very different RCR challenges in comparison with their colleagues in the rest of academia. Addressing these issues is important for both the RCR and RC communities in order to ensure that the rapid development of this field occurs in line with the norms of RCR which, nonetheless, should be adapted to respect the particularities of RC and allow its contributions to the academic world.

A predictive nutritional model for plant cells and hairy roots.

A structured nutritional model is proposed to describe growth and nutritional behavior of Eschscholtzia californica suspension cells and Catharanthus roseus and Daucus carota hairy roots in in vitro culture. The model describes the cells specific growth rate from concentration of intracellular nutrients such as inorganic phosphate (Pi), nitrogen sources (NO(3) (-) and NH(4) (+)) and sugars. Two-level Michaelis-Menten kinetics are used to describe Pi and NO(3) (-) uptake and simple Michaelis-Menten kinetics for description of sugars uptake. Model parameters for each cell line were calibrated using data from batch cultures. The predictive capacity of the model was tested using data from medium exchange hairy root cultures. The model describes growth and nutritional behavior for the cell and hairy root lines. A sensitivity analysis was performed to identify critical model parameters and effect of initial conditions. The cell and hairy roots lines are also compared from their kinetic parameters. The kinetic model is efficient for describing and predicting growth and nutritional behaviors of suspension cells and hairy roots.

Outcomes of an early childhood obesity prevention program in a low-income community: a pilot, randomized trial.

BACKGROUND: Obesity is a major and worldwide health problem in children. OBJECTIVES: The Early Childhood Obesity Prevention Program is a multi-component, randomized, controlled trial of a pilot community-focused obesity prevention program for mother/newborn dyads. METHODS: Underserved, mother/newborn dyads were recruited to receive a standard home visitation program (Nurturing Families Network, NFN) or an enhanced program (NFN+) that incorporated behavioural change strategies (e.g., goal-setting, problem-solving) and focused on six obesity-associated behaviours (breastfeeding, juice/sugar-sweetened beverages, solids, infant sleep, TV/screen time and soothability) with linkages to community resources. Weight-for-length (WFL) z-score and maternal diet were secondary outcomes. RESULTS: Fifty-seven dyads were recruited and 47 fully eligible dyads were enrolled (NFN = 21, NFN+ = 26). Forty-one (87.2%) were assessed at 6 months and 34 (72.3%) at 12 months. Retention at 12 months was higher for NFN+ dyads (84.6% vs. 56.1%, p = 0.04). NFN+ mothers were more likely to continue breastfeeding at 6 and 12 months vs. NFN mothers (p = 0.03 and 0.003, respectively), and at 12 months, NFN+ infants had fewer nocturnal awakenings (p = 0.04). There were no differences in other primary outcome measures or in WFL z-score at 6 or 12 months. CONCLUSIONS: A multi-component behavioural intervention increased breastfeeding duration and decreased nocturnal awakenings in infants of low-income families.

Dynamic flux cartography of hairy roots primary metabolism.

A dynamic model for plant cell and hairy root primary metabolism is presented. The model includes nutrient uptake (Pi, sugars, nitrogen sources), the glycolysis and pentose phosphate pathways, the TCA cycle, amino acid biosynthesis, respiratory chain, biosynthesis of cell building blocks (structural hexoses, organic acids, lipids, and organic phosphated molecules). The energy shuttles (ATP, ADP) and cofactors (NAD/H, NADP/H) are also included. The model describes the kinetics of 44 biochemical reactions (fluxes) of the primary metabolism of plant cells and includes 41 biochemical species (metabolites, nutrients, biomass components). Multiple Michaelis-Menten type kinetics are used to describe biochemical reaction rates. Known regulatory phenomena on metabolic pathways are included using sigmoid switch functions. A visualization framework showing fluxes and metabolite concentrations over time is presented. The visualization of fluxes and metabolites is used to analyze simulation results from Catharanthus roseus hairy root 50 d batch cultures. The visualization of the metabolic system allows analyzing split ratios between pathways and flux time-variations. For carbon metabolism, the cells were observed to have relatively high and stable fluxes for the central carbon metabolism and low and variable fluxes for anabolic pathways. For phosphate metabolism, a very high free intracellular Pi turnover rate was observed with higher flux variations than for the carbon metabolism. Nitrogen metabolism also exhibited large flux variations. The potential uses of the model are also discussed.

Relationship between antioxidant intakes and class I sarcopenia in elderly men and women.

BACKGROUND: The effect of nutritional intake on sarcopenia has been mostly examined in class II sarcopenia, i.e. when muscle mass has sufficiently decreased to induce a loss in physical capacity. Although this provides important information regarding the treatment of sarcopenia, it may not help highlight mechanisms involved at the very beginning of its development. HYPOTHESIS: We hypothesized that class I sarcopenia is associated with differences in antioxidant intakes (vitamins A, C, E and selenium) and status in healthy, older white men and women when physical activity and protein intake are taken into account. DESIGN: Fat-free mass and total appendicular skeletal muscle mass was determined by dual-energy X-ray absorptiometry in 50 healthy, older white men (n = 16) and women (n = 34) aged 60-75 yrs. Physical activity energy expenditure (PAEE) was determined using a Caltrac accelerometer over a 3-d period. Dietary protein and antioxidant intakes were estimated from a 3-d food record and serum total antioxidant activity (TAA) was measured by a ferrylmyoglobin- ABTS assay. RESULTS: The prevalence of class I sarcopenia was 23.5 % in women and 25.0 % in men; 12 participants were thus considered sarcopenic (4 men and 8 women) and 38 participants were considered nonsarcopenic (12 men and 26 women). Our results showed that PAEE, serum albumin concentrations, TAA, and the four antioxidants intake levels were similar between groups. On the other hand, our results showed that total protein intake was significantly higher (P < 0.01) in the non-sarcopenic group than in the sarcopenic group. Also, the number of Recommended Dietary Allowances (RDAs) reached for the antioxidant nutrients and protein intakes by the non-sarcopenic group was significantly higher (P < 0.01) than in the sarcopenic group. CONCLUSIONS: Although there were no significant differences between the sarcopenic and the non-sarcopenic group when antioxidant intakes were considered individually, we observed that the number of RDAs reached for antioxidant micronutrients and protein in healthy, older white men and women was lower in sarcopenic than nonsarcopenic individuals. Our results also suggest that a higher total dietary protein intake is associated with the preservation of muscle mass loss although both groups displayed values above actual RDAs. Obviously, prospective studies are needed to determine the minimum amount of protein in the diet needed to prevent class I sarcopenia and to examine the utility of antioxidant intake to combat the age-related loss in skeletal muscle mass.

Implementing a routine flow cytometry assay for nucleated red blood cell counts in cord blood units.

INTRODUCTION: As required by standards organizations, Héma-Québec Cord Blood Bank performs enumeration of nucleated red blood cells (NRBCs) in cord blood units (CBUs). This study presents the validation and implementation approaches developed to transfer the routine NRBC enumeration from the manual blood film method to a flow cytometric assay. METHODS: The flow cytometry method was adapted from Tsuji (Cytometry, 37, 1999, 291). This assay was validated to assess the specificity, detection limit, repeatability, and reproducibility of the method, including interoperator and interlaboratory testing. Finally, postimplementation follow-up and adjustments were performed for CBU over a 7-month period. RESULTS: Blood film and flow cytometry NRBC enumerations showed a strong correlation (n = 40; Pearson's r correlation = 0.90). Validation was successful as exemplified by the correlation in interlaboratory testing (n = 30; r = 0.98). During implementation, our routine laboratory analyses revealed that CBU with low NRBC content (≤2%), representing 26% of all CBU tested, resulted in 15% of repeated reading and/or staining and was the principal source of nonconformity. Small adjustments in the standard operating procedures (SOPs), including a fixed 200-event setting in the NRBC gate for the second reading of the replicates, have completely solved this issue. CONCLUSION: Flow cytometric NRBC enumerations, now implemented in Héma-Québec Public Cord Blood Bank, is an improvement in the efficiency of our operations by integrating the count for NRBC into our flow cytometry platform.

Controlled Distribution and Clustering of Silver in Ag-DLC Nanocomposite Coatings Using a Hybrid Plasma Approach.

Incorporation of selected metallic elements into diamond-like carbon (DLC) has emerged as an innovative approach to add unique functional properties to DLC coatings, thus opening up a range of new potential applications in fields as diverse as sensors, tribology, and biomaterials. However, deposition by plasma techniques of metal-containing DLC coatings with well-defined structural properties and metal distribution is currently hindered by the limited understanding of their growth mechanisms. We report here a silver-incorporated diamond-like carbon coating (Ag-DLC) prepared in a hybrid plasma reactor which allowed independent control of the metal content and the carbon film structure and morphology. Morphological and chemical analyses of Ag-DLC films were performed by atomic force microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. The vertical distribution of silver from the surface toward the coating bulk was found to be highly inhomogeneous due to top surface segregation and clustering of silver nanoparticles. Two plasma parameters, the sputtered Ag flux and ion energy, were shown to influence the spatial distribution of silver particles. On the basis of these findings, a mechanism for Ag-DLC growth by plasma was proposed.

Antibacterial Coatings: Challenges, Perspectives, and Opportunities.

Antibacterial coatings are rapidly emerging as a primary component of the global mitigation strategy of bacterial pathogens. Thanks to recent concurrent advances in materials science and biotechnology methodologies, and a growing understanding of environmental microbiology, an extensive variety of options are now available to design surfaces with antibacterial properties. However, progress towards a more widespread use in clinical settings crucially depends on addressing the key outstanding issues. We review release-based antibacterial coatings and focus on the challenges and opportunities presented by the latest generation of these materials. In particular, we highlight recent approaches aimed at controlling the release of antibacterial agents, imparting multi-functionality, and enhancing long-term stability.

Red light violations by adult pedestrians and other safety-related behaviors at signalized crosswalks.

To study human factors linked to red light violations, and more generally to safety-related behaviors at signalized crosswalks, the present study combines the collection of observational data with questionnaires answered by 422 French adult pedestrians. Thirteen behavioral indicators were extracted (12 before and while crossing, and red light violation), and the roles of several demographical, contextual and mobility-associated variables were examined. The results of the stepwise logistic regression analyses carried out on each of the 12 behavioral indicators observed before and while crossing revealed that gender had no major impact, but age did, with more cautious behaviors as pedestrians were older. The three contextual variables (group size, parked vehicles, and traffic density), as four mobility-associated variables (driving and walking experiences, self-reported crossing difficulties and falls in the street) were also found to be important factors in safety-related crossing behaviors. A wider logistic regression analysis, made specifically on red light violations with all behavioral indicators observed before and while crossings and the several demographical, contextual and mobility-associated variables put together, showed that red light violations were mostly affected by current situational factors (group size, parked vehicles) and particularly associated with some behavioral patterns (looking toward the traffic, the ground, the light, running and crossing diagonally). The overall results encourage the development of safer pedestrian infrastructures and engineering countermeasures.

Chronic adaptation of dog parathyroid function to a low-calcium-high-sodium-vitamin D-deficient diet.

The development of secondary hyperparathyroidism was studied in relation to changes in serum ionized Ca (Ca2+), 25-OHD, and 1,25-(OH)2D concentrations in six dogs maintained on a low-Ca (0.05%), high-Na (1.6%), and vitamin D-deficient diet for 91 weeks. Blood samples and evaluations of the parathyroid function were obtained before and after 3, 12, 24, 36, and 91 weeks of diet. Serum iPTH was measured by an intact hormone (I) and a carboxy-terminal (C) assay. The sigmoidal relationship between ionized Ca and iPTH values was evaluated mathematically. Results are means +/- SD. Statistically significant changes over a time period were evaluated by an ANOVA for repeated measurements. Over the first 3 weeks, serum Ca2+, 25-OHD, and 1,25-(OH)2D did not change but stimulated I-iPTH increased 84.3 +/- 39.9% (p less than 0.005) and C-iPTH only 25.3 +/- 12.2% (p less than 0.01), a significant difference (p less than 0.02). The increase in stimulated I-iPTH reached 487.4 +/- 139.6% (p less than 0.0001) and 418.4 +/- 76.9% (p less than 0.0001) for C-iPTH by the end of the study. Similar significant increases were seen in basal and nonsuppressible iPTH at or after week 12.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of involution of hyperplastic parathyroid glands in dogs: adaptation via a decrease in the calcium stimulation set point and a change in secretion profile.

This study analyzes the parathyroid function in four dogs before and after 2 years of a low-calcium, high-sodium, vitamin D-deficient diet and the involution of the same function following (1) correction of dietary calcium deficiency and administration of i.v. 1,25-(OH)2D (0.25 micrograms twice per day) during 1 month, (2) after an additional month of normal dog chow supplemented with oral vitamin D (25 micrograms per day), and, finally, (3) after 5 and 17 months of a diet with normal levels of calcium and vitamin D. The parathyroid function was evaluated through i.v. infusion of CaCl2 and Na2 EDTA with measurement of intact (I) and carboxyl-terminal (C) immunoreactive parathyroid hormone (iPTH). The C-iPTH/I-iPTH ratio was calculated to assess the modulation of molecular forms of iPTH induced by the various treatments. The 2 years of calcium and vitamin D deprivation lowered ionized calcium (1.23 +/- 0.04, p < 0.05) and 25-OHD (4.02 +/- 2.06 nM, p < 0.005) and tended to decrease 1,25-(OH)2D (80.8 +/- 8.6 pM); it increased basal I- and C-iPTH levels approximately eightfold (I-iPTH, 40.2 +/- 20.7, p < 0.05; C-iPTH, 185.4 +/- 94.9, p < 0.05) and stimulated I-iPTH (60.2 +/- 23.0 pM, p < 0.05) and C-iPTH (239.6 +/- 80.7 pM, p < 0.05) fivefold. A greater rise in nonsuppressible I-iPTH levels than in C-iPTH levels led to a decreased C-iPTH/I-iPTH ratio in hypercalcemia (12.5 +/- 2.8 versus 27.8 +/- 6.05 pM, p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of a non-(1-84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values.

A molecular form of PTH different from PTH-(1-84) and present in normal serum is recognized by two-site intact (I-) PTH assays; it responds to Ca2+ changes in the same way that PTH carboxyl-terminal fragments do. To evaluate the impact of this finding, we have compared basal, stimulated, and nonsuppressible I-PTH values in 14 normal subjects and 15 renal failure patients, subdivided into 8 patients with low (< 12 pmol/L; LBI) and 7 with high (> 12 pmol/L; HBI) basal I-PTH. Samples obtained under various calcemic conditions in these 3 groups were further fractionated by high performance liquid chromatography (HPLC) and assayed for I-PTH, and the various peaks observed were quantitated by planimetry. Differences among the 3 groups were reinterpreted knowing the exact composition of I-PTH. Basal I-PTH was greatly increased in HBI (mean +/- SD, 44.1 +/- 38.6 pmol/L) compared to that in normal subjects (2.5 +/- 0.8 pmol/L; P < 0.001) or LBI (6.1 +/- 2.4 pmol/L; P < 0.001); the difference was less in these last 2 groups (P < 0.01). Similar differences were observed for stimulated and nonsuppressible I-PTH, except for stimulated I-PTH, which was similar in normal and LBI subjects. Two I-PTH HPLC molecular forms accounted for I-PTH immunoreactivity in the 3 groups. In normal subjects, PTH-(1-84) accounted for 74.9 +/- 4.3%, 79.0 +/- 3.0%, and 87.2 +/- 1.0% of I-PTH in hyper-, normo-, and hypocalcemia, respectively, but only for 44.6 +/- 2.5%, 50.5 +/- 0.7%, and 63.6 +/- 0.1% in renal failure patients, with similar results in HBI and LBI. The accumulation of a non-(1-84) PTH peak accounted for the difference between normal subjects and renal failure patients. When basal, stimulated, and nonsuppressible I-PTH values were separated into their 2 components, prior differences between HBI and LBI or normal subjects remained unchanged because of very high I-PTH values in HBI, but differences between normal and LBI subjects were entirely explained by the accumulation of the non-(1-84) PTH peak [basal, 3.0 +/- 1.2 vs. 0.5 +/- 0.2 pmol/L (P < 0.001); stimulated, 6.8 +/- 2.3 vs. 2.3 +/- 1.0 pmol/L (P < 0.001); nonsuppressible, 1.3 +/- 0.7 vs. 0.2 +/- 0.08 pmol/L (P < 0.001)]; PTH-(1-84) values were similar (basal, 3.1 +/- 1.2 vs. 2.0 +/- 0.6 pmol/L; stimulated, 12.0 +/- 3.9 vs. 15.5 +/- 6.6 pmol/L; nonsuppressible, 1.1 +/- 0.6 vs. 0.52 +/- 0.22 pmol/L). Thus, a non-(1-84) PTH molecular form detected by two-site I-PTH assays accumulates in renal failure and accounts for a larger proportion of I-PTH than that in normal subjects. Levels of I-PTH 1.57 times higher than those in normocalcemic subjects are thus required in renal failure to achieve similar PTH-(1-84) concentrations. The composition of I-PTH is also identical in all hemodialyzed patients.

Immunolocalization and biochemical determination of cytochrome P450C17 in adrenals of hamsters treated with ACTH.

We used an anti-rat adrenal cytochrome P450C17 (P450C17) antibody to perform immunofluorescence and also immunogold electron microscopic studies to determine the zonal and intracellular distribution of P450C17 in hamster adrenals. Because P450C17 activity is regulated mainly by adrenocorticotropin (ACTH), its zonal and intracellular localization was also analyzed after ACTH treatment. The effect of ACTH treatment on protein concentration was also investigated by Western blotting analysis. By immunofluorescence, we found P450C17 to be confined to the zona fasciculata (ZF) in the hamster, in contrast to other small rodents, which do not express P450C17 in their adrenals. After treatment with ACTH, the thickness of the ZF remained unchanged compared to that of control animals, whereas a marked increase in fluorescence intensity was observed. In addition, dispersed cells in the zona reticularis (ZR) showed positive staining after ACTH treatment. Immunocytochemistry with colloidal gold showed P450C17 to be localized and importantly increased only in the cytoplasmic areas between the mitochondria of ZF cells of ACTH-treated animals. These areas are predominantly occupied by elements of the endoplasmic reticulum and other unidentified organelles. Immunoblotting analysis of whole glands revealed a single protein band at approximately 55 kD, which reacted with the 450C17 antibody. After stimulation with ACTH injected at 5-hr intervals over a period of 20 hr, P450C17 protein concentrations were considerably greater than in control animals. In conclusion, P450C17 is located not over mitochondria but probably in the endoplasmic reticulum of the ZF cells in hamster adrenals. Treatment with ACTH induced expression of cytochrome P450C17 in ZF cells, increasing its production in these cells without stimulating cell proliferation.