Search

Abstract Background The human Prader-Willi syndrome (PWS) domain and its mouse orthologue include a cluster of paternally expressed genes which imprinted expression is co-ordinately regulated by an imprinting center (IC) closely associated to the Snurf-Snrpn gene. Besides their co-regulated imprinted expression, two observations suggest that the spatio-temporal expression of these genes could also be co-regulated. First, the PWS genes have all been reported to be expressed in the mouse nervous system. Second, Snurf-Snrpn and its associated IC are the most ancient elements of the domain which later acquired additional functional genes by retrotransposition. Although located at least 1.5 megabases from the IC, these retroposons acquired the same imprinted regulation as Snurf-Snrpn. In this study, we ask whether the IC, in addition to its function in imprinting, could also be involved in the spatio-temporal regulation of genes in the PWS domain. Results We compared the expression pattern of Snurf-Snrpn and C/D-box small nucleolar RNAs (snoRNAs) MBII-85 and MBII-52 to the expression pattern of the two evolutionary related retroposons Ndn and Magel2, in the developing mouse embryo. We show that these genes have highly similar expression patterns in the central nervous system, suggesting that they share a common central nervous system-specific regulatory element. Among these genes, Ndn and Magel2 display the most similar expression patterns. Using transgenic mice containing the Ndn and Magel2 genes, we show that the transgenic Ndn gene whereas not imprinted is correctly expressed. Search for DNase I hypersensitive sites in the Ndn-Magel2 genomic region and comparative genomic analyses were performed in order to identify potential transcriptional cis-regulatory elements. Conclusions These results strongly suggest that paternally expressed genes of the PWS domain share a common central nervous system-specific regulatory element. We proposed that this regulatory element could co-localize with the IC. However, we demonstrate that the IC, if required for imprinted regulation, is not involved in the spatio-temporal regulation of distantly located retrotransposed genes such as the Ndn gene in the PWS domain.

Context: Environmental changes will foster the spread of Ixodes scapularis ticks and increase the incidence of Lyme disease in Québec in the coming years. The objective of this study is to estimate the epidemiological and clinical burden and part of the current economic burden of Lyme disease in Québec and to estimate the number of cases expected by 2050., Methods: Cases of Lyme disease reported in Québec from 2015 to 2019 were used to describe their demographic, geographical and clinical characteristics and the cost of their initial care. Three incidence rate scenarios were then developed to estimate the number of cases expected by 2050, based on demographic and climate projections., Results: From 2016 to 2019, 1,473 cases of Lyme disease were reported in Québec. Over 90% of those cases were acquired in two regions of southern Québec (Estrie and Montérégie), while the individuals infected were residents from all over Québec. The average age of cases is 44 years and 66% of infections were at the localized stage, the first stage of Lyme disease. The cost of initial care is estimated at an average of $182 CAN per patient ($47 CAN at the localized stage and $443 CAN at the disseminated stage). According to projections, over 95% of the Québec population will live in a climate zone conducive to the establishment of ticks by 2050, with a number of cases acquired in Québec being 1.3 to 14.5 times higher than in 2019, depending on the incidence rate scenario used., Conclusion: The epidemiological burden is concentrated primarily in southern Québec, but the clinical and economic burden is already distributed throughout the province. The projections for 2050 should help the regions of Québec adapt and optimize public health protection measures., Competing Interests: Competing interests None.

Background: Ixodes scapularis and Ixodes pacificus ticks are the principal vectors of the agent of Lyme disease and several other tick-borne diseases in Canada. Tick surveillance data can be used to identify local tick-borne disease risk areas and direct public health interventions. The objective of this article is to describe the seasonal and spatial characteristics of the main Lyme disease vectors in Canada, and the tick-borne pathogens they carry, using passive and active surveillance data from 2020., Methods: Passive and active surveillance data were compiled from the National Microbiology Laboratory Branch (Public Health Agency of Canada), provincial and local public health authorities, and eTick (an online, image-based platform). Seasonal and spatial analyses of ticks and their associated pathogens are presented, including infection prevalence estimates., Results: In passive surveillance, I. scapularis (n=7,534) were submitted from all provinces except Manitoba and British Columbia, while I. pacificus (n=718) were submitted only from British Columbia. No ticks were submitted from the Territories. The seasonal distribution of I. scapularis submissions was bimodal, but unimodal for I. pacificus . Four tick-borne pathogens were identified in I. scapularis ( Borrelia burgdorferi , Anaplasma phagocytophilum , Babesia microti and Borrelia miyamotoi ) and one in I. pacificus ( B. miyamotoi ). In active surveillance, I. scapularis (n=688) were collected in Ontario, Québec and New Brunswick. Five tick-borne pathogens were identified: B. burgdorferi , A. phagocytophilum , B. microti, B. miyamotoi and Powassan virus., Conclusion: This article provides a snapshot of the distribution of I. scapularis and I. pacificus and their associated human pathogens in Canada in 2020, which can help assess the risk of exposure to tick-borne pathogens in different provinces., Competing Interests: Competing interests None.

Lyme disease (LD) is a tick-borne disease which has been emerging in temperate areas in North America, Europe, and Asia. In Quebec, Canada, the number of human LD cases is increasing rapidly and thus surveillance of LD risk is a public health priority. In this study, we aimed to evaluate the ability of active sentinel surveillance to track spatiotemporal trends in LD risk. Using drag flannel data from 2015-2019, we calculated density of nymphal ticks (DON), an index of enzootic hazard, across the study region (southern Quebec). A Poisson regression model was used to explore the association between the enzootic hazard and LD risk (annual number of human cases) at the municipal level. Predictions from models were able to track both spatial and interannual variation in risk. Furthermore, a risk map produced by using model predictions closely matched the official risk map published by provincial public health authorities, which requires the use of complex criteria-based risk assessment. Our study shows that active sentinel surveillance in Quebec provides a sustainable system to follow spatiotemporal trends in LD risk. Such a network can support public health authorities in informing the public about LD risk within their region or municipality and this method could be extended to support Lyme disease risk assessment at the national level in Canada.

The incidence of Lyme disease is increasing in Québec and is closely linked to the distribution of Ixodes scapularis ticks. A time-to-establishment model developed in 2012 by Leighton and colleagues predicted the year of tick population establishment for each municipality in eastern Canada. To validate if this model correctly predicted tick distribution in Québec, predicted tick establishment was compared to field data from active tick surveillance (2010-2018) using two criteria: i) the detection of at least one tick and ii) the detection of the three questing stages of the tick. The speed of tick establishment and the increase in the exposed human population by 2100 were predicted with the time-to-establishment model. Field observations were consistent with model predictions. Ticks were detected on average 3 years after the predicted year. The probability of tick detection is significantly higher after the predicted year than before (61% vs 27% of collections). The trend was similar for the detection of three tick stages (16% vs 9% of collections). The average speed of tick range expansion was estimated by the model to be 18 km/year in Québec, with 90% of the human population exposed by 2027. The validation of the time-to-establishment model using field data confirmed that it could be used to project I. scapularis range expansion in Québec, and consequently the increase in Lyme disease risk over the coming decades. This will help public health authorities anticipate and adapt preventive measures, especially in areas not yet affected by Lyme disease., Competing Interests: The authors have declared that no competing interests exist.

Lyme disease (LD) is an emerging public health threat in Canada, associated with the northward range expansion of the black-legged tick (Ixodes scapularis). To address this, public health authorities have been carrying out surveillance activities and awareness campaigns targeting vulnerable populations such as outdoor workers. Implementing these measures is time-consuming and resource-intensive, prompting the assessment of alternatives. Our goal was to evaluate the feasibility and implementation of a training-of-trainers-inspired approach in raising awareness about LD risk and prevention among workers and general population, as well as to evaluate its potential to contribute to provincial LD surveillance efforts. We trained a group of workers from publicly-accessible outdoor parks of the province of Québec to become "LD education ambassadors". Ambassadors were trained to raise tick and LD awareness, share information on preventive measures in their respective communities, and lead tick sampling activities using a standardised protocol similar to that used by Public Health authorities. Ambassador-led outreach activities, public reach, sampling activities and collected ticks were documented, as well as ambassadors' satisfaction with the training using forms and semi-structured interviews. In total, 18 ambassadors from 12 organizations were trained. Between June and September 2019, they led 28 independent outreach activities, reaching over 1 860 individuals (from occupational and general public settings) in seven public health units. Ambassadors led 28 tick samplings, together collecting 11 I. scapularis ticks. This study suggests that an adapted training-of-trainers is a feasible approach to raising tick and LD risk awareness among Québec outdoor workers and public. Trained ambassadors have the potential of reaching a large portion of the population visiting or working in outdoor parks while also providing much-needed outreach regarding risk and prevention. Pushing this concept further to include other types of workers and jurisdictions may contribute to national LD surveillance efforts., Competing Interests: The authors have declared that no competing interests exist.

Background: Lyme disease is an emerging vector-borne zoonotic disease of increasing public health importance in Canada. As part of its mandate, the Canadian Lyme Disease Research Network (CLyDRN) launched a pan-Canadian sentinel surveillance initiative, the Canadian Lyme Sentinel Network (CaLSeN), in 2019., Objectives: To create a standardized, national sentinel surveillance network providing a real-time portrait of the evolving environmental risk of Lyme disease in each province., Methods: A multicriteria decision analysis (MCDA) approach was used in the selection of sentinel regions. Within each sentinel region, a systematic drag sampling protocol was performed in selected sampling sites. Ticks collected during these active surveillance visits were identified to species, and Ixodes spp. ticks were tested for infection with Borrelia burgdorferi , Borrelia miyamotoi , Anaplasma phagocytophilum , Babesia microti and Powassan virus., Results: In 2019, a total of 567 Ixodes spp. ticks ( I. scapularis [n=550]; I. pacificus [n=10]; and I. angustus [n=7]) were collected in seven provinces: British Columbia, Manitoba, Ontario, Québec, New Brunswick, Nova Scotia and Prince Edward Island. The highest mean tick densities (nymphs/100 m 2 ) were found in sentinel regions of Lunenburg (0.45), Montréal (0.43) and Granby (0.38). Overall, the Borrelia burgdorferi prevalence in ticks was 25.2% (0%-45.0%). One I. angustus nymph from British Columbia was positive for Babesia microti , a first for the province. The deer tick lineage of Powassan virus was detected in one adult I. scapularis in Nova Scotia., Conclusion: CaLSeN provides the first coordinated national active surveillance initiative for tick-borne disease in Canada. Through multidisciplinary collaborations between experts in each province, the pilot year was successful in establishing a baseline for Lyme disease risk across the country, allowing future trends to be detected and studied., Competing Interests: Competing interests: None.

Background: Patients with lower extremity peripheral artery disease (PAD) frequently experience claudication, a clinical symptom indicative of reduced walking capacity. Recommended care consists of exercise rehabilitation combined with optimal medical treatment and surgery. The effects of a single oral dose of sildenafil, a phosphodiesterase type-5 inhibitor, on patients with claudication are discussed. The aim of this study was to test the efficacy of a single 100 mg dose of sildenafil compared to placebo in terms of maximal walking time (MWT) in patients with claudication., Methods: The ARTERIOFIL study is a crossover, double-blind, prospective, randomized, single-center study conducted at Angers University Hospital in France. MWT (primary endpoint) was assessed using a treadmill test (10% incline; 3.2 km/h). Secondary endpoints (pain-free walking time (PFWT), transcutaneous oximetry during exercise and redox cycle parameters and safety) were also studied., Results: Fourteen patients were included of whom two were ultimately excluded. In the 12 remaining patients, the MWT was significantly improved during the sildenafil period compared with the placebo period (300 s [95% CI 172 s-428 s] vs 402 s [95% CI 274 s-529 s] p < 0.01). Sildenafil had no significant effect on pain-free walking time or skin tissue oxygenation during exercise. According to redox cycle parameters, sildenafil significantly reduced blood glucose and pyruvate levels and the 3-hydroxybutyrate/acetoacetate ratio, while there was no significant effect on lactate, 3-hydroxybutyrate, acetoacetate and free fatty acid levels. Symptomatic transient hypotension was observed in two women., Conclusions: The ARTERIOFIL study has shown that a single 100 mg oral dose of sildenafil had a significant effect on increase in MWT but had no significant effects on PFWT and oxygenation parameters in patients with claudication. A double-blind, prospective, randomized, multicenter study (VIRTUOSE©) is ongoing to evaluate the chronic effect of six month-long sildenafil treatment on MWT in PAD patients with claudication., Clinical Trial Registration: This clinical trial was registered at clinicaltrials.gov, registration. number: NCT02832570, (https://clinicaltrials.gov/ct2/show/NCT02832570)., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Since 2002, human cases of West Nile virus (WNV) have occurred every year in southern Canada, but WNV risk remains challenging to predict. Here, we explored the ability of weather-based forecasting models to predict the seasonal abundance of two WNV vector species (Culex pipiens-restuans and Aedes vexans) in Québec, Canada, and explored the importance of accounting for larvicide use and local habitat (forest park vs residential garden). A gamma-generalized linear model predicting mosquito abundance was developed based on an approach previously used in Ontario combining temperature and precipitation during the days preceding mosquito captures. This model was calibrated and validated for each species with independent entomological datasets from the Montréal region collected in 2013 and 2014. Culex pipiens-restuans abundance was associated with mean degree days (dd; >9°C) over the 22 d before mosquito capture and with mean precipitation over the 71 d before capture; Ae. vexans abundance with the mean dd (>12°C) over the 24 d before capture and mean precipitation over the 30 d before capture. These results are consistent with temperature effects on immature development rates and adult activity, and effects of precipitation on the abundance and suitability of breeding sites. Taking into account larvicide use and habitat significantly improved the predictions. This study provides evidence that weather conditions can yield robust short-term predictions of the regional daily mosquito abundance, particularly when accounting for local variation in habitat or mosquito control efforts, and may provide real-time indicators of WNV or other mosquito-borne disease risks during the summer., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Lyme disease, the most commonly reported vector-borne disease in North America, is caused by the spirochete Borrelia burgdorferi sensu stricto, which is transmitted by Ixodes scapularis in eastern Canada and Ixodes pacificus in western Canada. Recently, the northward range expansion of I. scapularis ticks, in south-eastern Canada, has resulted in a dramatic increase in the incidence of human Lyme disease. Detecting emerging areas of Lyme disease risk allows public health to target disease prevention efforts. We analysed passive tick surveillance data from Ontario and Manitoba to i) assess the relationship between the total numbers of I. scapularis submissions in passive surveillance from humans, and the number of human Lyme disease cases, and ii) develop province-specific acarological indicators of risk that can be used to generate surveillance-based risk maps. We also assessed associations between numbers of nymphal I. scapularis tick submissions only and Lyme disease case incidence. Using General Estimating Equation regression, the relationship between I. scapularis submissions (total numbers and numbers of nymphs only) in each census sub-division (CSD) and the number of reported Lyme disease cases was positively correlated and highly significant in the two provinces (P ≤ 0.001). The numbers of I. scapularis submissions over five years discriminated CSDs with ≥ 3 Lyme disease cases from those with < 3 cases with high accuracy when using total numbers of tick submission (Receiver Operating Characteristics area under the curve [AUC] = 0.89) and moderate accuracy (AUC = 0.78) when using nymphal tick submissions only. In Ontario the optimal cut-off point was a total 12 tick submissions from a CSD over five years (Sensitivity = 0.82, Specificity = 0.84), while in Manitoba the cut-off point was five ticks (Sensitivity = 0.71, Specificity = 0.79) suggesting regional variability of the risk of acquiring Lyme disease from an I. scapularis bite. The performances of the acarological indicators developed in this study for Ontario and Manitoba support the ability of passive tick surveillance to provide an early signal of the existence Lyme disease risk areas in regions where ticks and the pathogens they transmit are expanding their range., Competing Interests: The authors have declared that no competing interests exist.

Lyme disease is an emerging public health threat in Canada. In this context, rapid detection of new risk areas is essential for timely application of prevention and control measures. In Canada, information on Lyme disease risk is collected through three surveillance activities: active tick surveillance, passive tick surveillance, and reported human cases. However, each method has shortcomings that limit its ability to rapidly and reliably identify new risk areas. We investigated the relationships between risk signals provided by human cases, passive and active tick surveillance to assess the performance of tick surveillance for early detection of emerging risk areas. We used regression models to investigate the relationships between the reported human cases, Ixodes scapularis (Say; Acari: Ixodidae) ticks collected on humans through passive surveillance and the density of nymphs collected by active surveillance from 2009 to 2014 in the province of Quebec. We then developed new risk indicators and validated their ability to discriminate risk levels used by provincial public health authorities. While there was a significant positive relationship between the risk signals provided all three surveillance methods, the strongest association was between passive tick surveillance and reported human cases. Passive tick submissions were a reasonable indicator of the abundance of ticks in the environment (sensitivity and specificity [Se and Sp] < 0.70), but were a much better indicator of municipalities with more than three human cases reported over 5 yr (Se = 0.88; Sp = 0.90). These results suggest that passive tick surveillance provides a timely and reliable signal of emerging risk areas for Lyme disease in Canada.

Background: The risk of contracting Lyme disease (LD) can vary spatially because of spatial heterogeneity in risk factors such as social-behavior and exposure to ecological risk factors. Integrating these risk factors to inform decision-making should therefore increase the effectiveness of mitigation interventions., Objectives: The objective of this study was to develop an integrated social-behavioral and ecological risk-mapping approach to identify priority areas for LD interventions., Methods: The study was conducted in the Montérégie region of Southern Quebec, Canada, where LD is a newly endemic disease. Spatial variation in LD knowledge, risk perceptions, and behaviors in the population were measured using web survey data collected in 2012. These data were used as a proxy for the social-behavioral component of risk. Tick vector population densities were measured in the environment during field surveillance from 2007 to 2012 to provide an index of the ecological component of risk. Social-behavioral and ecological components of risk were combined with human population density to create integrated risk maps. Map predictions were validated by testing the association between high-risk areas and the current spatial distribution of human LD cases., Results: Social-behavioral and ecological components of LD risk had markedly different distributions within the study region, suggesting that both factors should be considered for locally adapted interventions. The occurrence of human LD cases in a municipality was positively associated with tick density ( p <0.01) but was not significantly associated with social-behavioral risk., Conclusion: This study is an applied demonstration of how integrated social-behavioral and ecological risk maps can be created to assist decision-making. Social survey data are a valuable but underutilized source of information for understanding regional variation in LD exposure, and integrating this information into risk maps provides a novel approach for prioritizing and adapting interventions to the local characteristics of target populations. https://doi.org/10.1289/EHP1943.

Since its detection in Canada in the early 1990s, Ixodes scapularis , the primary tick vector of Lyme disease in eastern North America, has continued to expand northward. Estimates of the tick's broad-scale distribution are useful for tracking the extent of the Lyme disease risk zone; however, tick distribution may vary widely within this zone. Here, we investigated I. scapularis nymph distribution at three spatial scales across the Lyme disease emergence zone in southern Quebec, Canada. We collected ticks and compared the nymph densities among different woodlands and different plots and transects within the same woodland. Hot spot analysis highlighted significant nymph clustering at each spatial scale. In regression models, nymph abundance was associated with litter depth, humidity, and elevation, which contribute to a suitable habitat for ticks, but also with the distance from the trail and the type of trail, which could be linked to host distribution and human disturbance. Accounting for this heterogeneous nymph distribution at a fine spatial scale could help improve Lyme disease management strategies but also help people to understand the risk variation around them and to adopt appropriate behaviors, such as staying on the trail in infested parks to limit their exposure to the vector and associated pathogens., Competing Interests: The authors declare no conflict of interest.

Galectin 1 and galectin 3 are first expressed in the trophectoderm cells of the implanting embryo and have been implicated in the process of implantation. However, we had previously shown that the lack of galectin 1 in galectin 1 null mutant mice is compatible with implantation. In this study, we describe the generation of galectin 3 null mutant mice and show that they are viable and have no overt abnormalities. The importance of galectin 1 and galectin 3 in implantation was assessed by obtaining double mutant mice [gal1 -/-; gal3 -/-]. We find that implantation can still occur in the absence of both galectin 1 and galectin 3. However, we show that galectin 5, a third member of this gene family, is also present in the blastocyst at the time of implantation.

The galectins are a family of low molecular weight, calcium-independent mammalian carbohydrate binding proteins that exhibit specificity for beta-galactoside derivatives. We have examined the expression pattern of galectin-3 in the developing mouse embryo by in situ hybridisation and immunohistochemistry. In the embryo proper, galectin-3 message and protein are first detected in notochord, starting from 8.5 days post coitum (dpc), and persist until this structure disappears. Galectin-3 is later found in cartilage primordia and in developing skin from 13.5 dpc. This very restricted and dynamic pattern suggests that galectin-3 may participate in the establishment and/or maintenance of notochord as well as the formation of cartilage and differentiation of skin. Finally, we find that galectin-3, which is identical to the macrophage marker Mac-2, is also expressed in embryonic macrophages.

The rat L-type pyruvate kinase gene possesses two promoters located 500 bp apart. The L' promoter is specific to erythroid cells. The L promoter is specific to liver and is regulated by diet and hormones; positively by glucose and insulin and negatively by glucagon via cAMP. The DNA elements involved in this tissue-specific and hormone-regulated gene expression are located within 3.2 kbp of 5' flanking region as previously demonstrated by transgenic mice analysis [Tremp, G. L., Boquet, D., Ripoche, M. A., Cognet, M., Yu-Chun, L., Jami, J., Kahn, A. and Daegelen, D. (1989) J. Biol. Chem. 264, 19,904-19,910]. Moreover, we have observed in these mice that gene expression was dependent on the transgene copy number and independent of the integration site. We present here DNase-I-hypersensitivity analysis of the endogenous rat L-type pyruvate kinase gene and of two transgene constructs in relation to development, tissue differentiation, nutritional and hormonal status. In rats, two groups of proximal sites were detected on the endogenous gene; hypersensitive site (HSS) HSS-1 in adult liver and HSS-A in fetal liver (a major erythropoietic tissue). Both groups are probably related to the transcriptional initiation complexes at either the L or L' promoter. Two other distal groups were detected; HSS-2 at -3 kbp (with respect to the liver-specific cap site) in adult liver and HSS-B around -4 kbp in fetal liver. These sites are thought to correspond to activating sequences; in adult liver, deletion of a fragment encompassing HSS-2 provokes a dramatic reduction of transcription starting at the L promoter of the transgene. In adult liver, HSS-1 appears to be a transcription-associated site, being greatly weakened in fasted rats, while HSS-2 is transcription independent. The pattern of DNase-I hypersensitivity is similar for the rat endogenous gene and for the complete rat transgene; the liver-specific HSS-1 and HSS-2 are present and the intensity of the sites is correlated to the number of integrated copies. Interestingly, HSS-1 is still detectable and its intensity remains proportional to the number of integrated copies in a truncated transgene with HSS-2 deletion, while this transgene is very weakly (but nevertheless tissue-specifically) expressed. These results strongly suggest that each transgene copy possesses a complete set of specific nucleoprotein complexes and that, with or without HSS-2, the DNA is in a potentially active configuration.

Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific transcripts in pancreas, but not in other tissues, 3) the specific immunofluorescence staining of pancreatic islets for human C-peptide, and 4) the synthesis and accumulation of human (pro)insulin in isolated islets. Deletions in the injected DNA fragment of sequences upstream from positions -353, -258, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50% of the total insulin does not alter the normal proportion of mice insulins I and II. These results suggest that expression of the human insulin gene in vivo results from the cooperation of several cis-regulatory elements present in the various deleted fragments. With none of the deletions used, expression of the transgene was observed in cell types other than beta-islet cells.

A calcium-binding protein (CaBP) similar to rat duodenal vitamin D-dependent CaBP was identified in rat uterus. Uterine CaBP and duodenal CaBP had the same mol wt (9,000-10,000), exhibited the same calcium-dependent electrophoretic mobility, and were immunologically identical. The localization of CaBP in the rat uterus was explored using indirect immunoperoxidase methods, and by CaBP RIA in the endometrium and myometrium after enzyme separation. In the endometrium CaBP was found in the cytoplasm of the stroma cells but not in the epithelium or in the glandular cells. In the myometrium, it was located inside the smooth myometrial fibers. Hormonal regulation of CaBP was shown to differ in the uterus and duodenum. Duodenal CaBP concentrations increased in response to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and were not influenced by ovariectomy or sex steroids administration. By contrast, CaBP synthesis fell drastically in the uterus of ovariectomized rats, but was greatly enhanced by low physiological doses of 17 beta-estradiol. This effect of 17 beta-estradiol on uterine CaBP was dose dependent. Medroxyprogesterone and more especially 1,25(OH)2D3 exerted no such stimulating effect on uterine CaBP. In vitamin D-deficient ovariectomized rats, administration of 17 beta-estradiol alone restored the uterine CaBP concentrations to normal and this potency contrasted with the apparent inability of 1,25(OH)2D3 to affect the uterine CaBP concentrations. Our data suggest that, unlike duodenal CaBP regulation, the expression of the CaBP gene in rat uterus is predominantly controlled by 17 beta-estradiol.

Three transgenic mouse lines, Tg 74, Tg 174, and Tg 171, were obtained by microinjection of an 11-kb human DNA fragment carrying the insulin gene into pronuclei of fertilized mouse eggs. The human insulin gene was expressed in all three transgenic mouse lines as shown by the presence of human C peptide in serum and urine and of human insulin transcripts in RNA prepared from pancreas. Several copies of the human DNA fragment arranged in head-to-tail arrays were present in each line. The human DNA insert was transmitted to the progeny as a single genetic locus. The chromosomal integration of the human insulin transgene was directly demonstrated by in situ hybridization to metaphase chromosomes of mitotic cells prepared from spleen and bone marrow. The insert appeared unique and located on a different chromosome in each line, namely 7 for Tg 74, 13 for Tg 174, and 18 for TG 171. Separation of DNA fragments larger than 20 kb by pulse-field electrophoresis showed that several insertion sites were present in each chromosome locus. This is the first direct evidence in transgenic mice that a gene located at various chromosome loci can be correctly expressed.