Comparative efficacy of epidural clonidine versus epidural fentanyl for treating breakthrough pain during labor: a randomized double-blind clinical trial.

BACKGROUND: Breakthrough pain during neuraxial labor analgesia is typically alleviated with additional administration of epidural local anesthetics, with or without adjuvants. Sometimes avoiding neuraxial opioids may be warranted and clonidine is an alternative. In a randomized double-blind trial we compared the efficacy of clonidine versus fentanyl, added to bupivacaine, for the management of breakthrough pain. METHODS: Term parturients (n=98) receiving bupivacaine 0.0625% with fentanyl 2 µg/mL at 12 mL/h, a patient-administered bolus of 5 mL at lockout 6-10 min and a maximum of four boluses per hour, and experiencing breakthrough pain ≥5/10, were randomized to receive a 10 mL bolus containing 12.5 mg bupivacaine and either clonidine 100 µg or fentanyl 100 µg. The primary outcome was 'success' of study drug treatment, defined as a pain score reduction ≥4/10 within 15 min of administration. Maternal hemodynamics and fetal heart rate were documented for two hours after treatment. RESULTS: There was no significant difference between groups in success rates (66.0% after clonidine (n=47) vs 74.5% after fentanyl (n=51), P=0.48) or in the incidence of hypotension (systolic blood pressure ≤80% of baseline or <90 mmHg) or sedation at 15 min, with 2/51 and 1/47 subjects in the fentanyl and clonidine groups, respectively, receiving phenylephrine. CONCLUSION: Epidural clonidine 100 µg was not superior to fentanyl 100 µg for decreasing pain scores within 15 min of co-administration with bupivacaine 0.125% for intrapartum breakthrough pain. The analgesic efficacy and hemodynamic side effects did not significantly differ.

Routine blood monitoring in maintenance immunoglobulin treatment of inflammatory neuropathy: Is it clinically relevant?

BACKGROUND: Pre-treatment screening for IgA deficiency and close monitoring of full blood count(FBC) and renal function is recommended with intravenous immunoglobulin(IVIg) therapy in neurological diseases. AIMS: To examine the frequency of biochemically defined and clinically significant episodes of treatment associated haemolysis, neutropenia, thrombocytopenia and acute kidney injury(AKI) in a cohort of patients on maintenance Immunoglobulin(Ig) therapy for inflammatory neuropathy. METHODS: A retrospective review of routine blood monitoring in patients from two UK specialist peripheral nerve centres. Accepted definitions for clinically and biochemically significant haemolysis, neutropenia, thrombocytopenia and AKI were used. RESULTS: 1919 infusion episodes in 90 patients were analysed. Age(mean(S.D)) = 58.09(14.4)years, 63% male, 72% CIDP(28% MMN), 97% IVIg(3% SCIg). Dose = 1.57(0.79)g/kg/month or 97.1(37.3)g/infusion, frequency:3.9(1.4) weeks. Relative IgA deficiency was noted in 2 individuals (prevalence:2.2%, 95%C.I.:0-5.2) who received a combined total of 38 infusions(3800 g IVIg) without adverse event. No clinically significant episodes of haemolysis, neutropenia, thrombocytopenia or AKI occurred in relation to treatment. An asymptomatic drop>10 g/L haemoglobin(Hb) occurred in 3.5%(95%CI:2.7-4.3) of treatment episodes in 38 individuals, mean reduction:17.7(7.4)g/L; lowest Hb:86 g/L. Lower pre-treatment haemoglobin correlated with risk of recurrent Ig-related drop(p:0.007). Two patients with chronic renal failure(stage 1 and 3) received 28(IV) and 104(SC) infusions respectively(6416 g) without impact on estimated glomerular filtration rate(eGFR). CONCLUSIONS: No clinically significant Ig-related episodes of haemolysis or AKI were identified in this representative cohort. This suggests that routine monitoring is not essential in long-term Ig use but should be considered when clinically indicated.

Stillbirth risk across pregnancy by size for gestational age in Western Cape Province, South Africa: Application of the fetuses-at-risk approach using perinatal audit data.

BACKGROUND: There is little published work on the risk of stillbirth across pregnancy for small-for-gestational-age (SGA) and large-for-gestational (LGA) pregnancies in low-resource settings. OBJECTIVES: To compare stillbirth risk across pregnancy between SGA and appropriate-for-gestational-age (AGA) pregnancies in Western Cape Province, South Africa (SA). METHODS: A retrospective audit of perinatal mortality data using data from the SA Perinatal Problem Identification Program was conducted. All audited stillbirths with information on size for gestational age (N=677) in the Western Cape between October 2013 and August 2015 were included in the study. The Western Cape has antenatal care (ANC) appointments at booking and at 20, 26, 32, 34, 36, 38 and 41 (if required) weeks' gestation. A fetuses-at-risk approach was adopted to examine stillbirth risk (28 - 42 weeks' gestation, ≥1 000 g) across gestation by size for gestational age (SGA <10th centile Theron growth curves, LGA >90th centile). Stillbirth risk was compared between SGA/LGA and AGA pregnancies. RESULTS: SGA pregnancies were at an increased risk of stillbirth compared with AGA pregnancies between 30 and 40 weeks' gestation, with the relative risk (RR) ranging from 3.5 (95% confidence interval (CI) 1.6 - 7.6) at 30 weeks' gestation to 15.3 (95% CI 8.8 - 26.4) at 33 weeks' gestation (p<0.001). The risk for LGA babies increased by at least 3.5-fold in the later stages of pregnancy (from 37 weeks) (p<0.001). At 38  weeks, the greatest increased risk was seen for LGA pregnancies (RR 6.6, 95% CI 3.1 - 14.2; p<0.001). CONCLUSIONS: There is an increased risk of stillbirth for SGA pregnancies, specifically between 33 and 40 weeks' gestation, despite fortnightly ANC visits during this time. LGA pregnancies are at an increased risk of stillbirth after 37 weeks' gestation. This high-risk period highlights potential issues with the detection of fetuses at risk of stillbirth even when ANC is frequent.

INTERGROWTH-21st v. local South African growth standards (Theron-Thompson) for identification of small-for-gestational-age fetuses in stillbirths: A closer look at variation across pregnancy.

BACKGROUND: Global growth standards for fetuses were recently developed (INTERGROWTH-21st). It has been advocated that professional bodies should adopt these global standards. OBJECTIVES: To compare the ability of INTERGROWTH-21st with local standards (Theron-Thompson) to identify small-for-gestational-age (SGA) fetuses in stillbirths in the South African (SA) setting. METHODS: Stillbirths across SA were investigated (>500 g, 28 - 40 weeks) between October 2013 and December 2016 (N=14 776). The study applied the INTERGROWTH-21st standards to classify stillbirths as <10th centile (SGA) compared with Theron-Thompson growth charts, across pregnancy overall and at specific gestational ages. RESULTS: The prevalence of SGA was estimated at 32.2% and 31.1% by INTERGROWTH-21st and Theron-Thompson, respectively. INTERGROWTH-21st captured 13.8% more stillbirths as SGA in the earlier gestations (28 - 30 weeks, p<0.001), but 4.0% (n=315) fewer between 33 and 38 weeks (p<0.001). Observed agreement and the Kappa coefficient were lower at earlier gestations and at 34 - 36 weeks. CONCLUSIONS: Our findings demonstrated differences in the proportion of stillbirths considered SGA at each gestational age between the INTERGROWTH-21st and the local SA standard, which have not been considered previously by other studies.

Does antenatal care timing influence stillbirth risk in the third trimester? A secondary analysis of perinatal death audit data in South Africa.

OBJECTIVE: To explore stillbirth risk across gestation in three provinces of South Africa with different antenatal care schedules. DESIGN: Retrospective audit of perinatal death data using South Africa's Perinatal Problem Identification Programme. SETTING: In 2008, the Basic Antenatal Care Programme was introduced in Limpopo and Mpumalanga provinces, reducing appointments to five visits at booking, 20, 26, 32, 38 weeks and 41 weeks if required. In the Western Cape province seven appointments remained at booking, 20, 26, 32, 34, 36, 38 and 41 weeks if required. POPULATION: All audited stillbirths (n = 4211) between October 2013 to August 2015 in Limpopo, Mpumalanga and Western Cape. METHODS: Stillbirth risk (26-42 weeks of gestation, >1000 g) across gestation was calculated using Yudkin's method. Stillbirth risk was compared between provinces and relative risks were calculated between Limpopo/ Mpumalanga and Western Cape. MAIN OUTCOME MEASURES: Stillbirth risk across gestation. RESULTS: Stillbirth risk peaked at 38 weeks of gestation in Limpopo (relative risk [RR] 3.11, 95% CI 2.40-4.03, P < 0.001)and Mpumalanga (RR 3.09, 95% CI 2.37-4.02, P < 0.001) compared with Western Cape, where no peak was observed. Stillbirth risk at 38 weeks gestation in Limpopo and Mpumalanga were statistically greater than both the 37 and 39 weeks gestation within provinces (P < 0.001). As expected, a peak at 41 weeks of gestation was observed in all provinces. CONCLUSIONS: The increased period of stillbirth risk occurs after a 6-week absence of antenatal care. This calls for a refocus on the impact of reduced antenatal care visits during the third trimester. TWEETABLE ABSTRACT: Reduced antenatal care in the third trimester may increase stillbirth risk.

Risk of stillbirth, preterm delivery, and fetal growth restriction following exposure in a previous birth: systematic review and meta-analysis.

BACKGROUND: Little is known about the risk of non-recurrent adverse birth outcomes. OBJECTIVES: To evaluate the risk of stillbirth, preterm birth (PTB), and small for gestational age (SGA) as a proxy for fetal growth restriction (FGR) following exposure to one or more of these factors in a previous birth. SEARCH STRATEGY: We searched MEDLINE, EMBASE, Maternity and Infant Care, and Global Health from inception to 30 November 2016. SELECTION CRITERIA: Studies were included if they investigated the association between stillbirth, PTB, or SGA (as a proxy for FGR) in two subsequent births. DATA COLLECTION AND ANALYSIS: Meta-analysis and pooled association presented as odds ratios (ORs) and adjusted odds ratios (aORs). MAIN RESULTS: Of the 3399 studies identified, 17 met the inclusion criteria. A PTB or SGA (as a proxy for FGR) infant increased the risk of subsequent stillbirth ((pooled OR 1.70; 95% confidence interval, 95% CI, 1.34-2.16) and (pooled OR 1.98; 95% CI 1.70-2.31), respectively). A combination of exposures, such as a preterm SGA (as a proxy for FGR) birth, doubled the risk of subsequent stillbirth (pooled OR 4.47; 95% CI 2.58-7.76). The risk of stillbirth also varied with prematurity, increasing three-fold following PTB <34 weeks of gestation (pooled OR 2.98; 95% CI 2.05-4.34) and six-fold following preterm SGA (as a proxy for FGR) <34 weeks of gestation (pooled OR 6.00; 95% CI 3.43-10.49). A previous stillbirth increased the risk of PTB (pooled OR 2.82; 95% CI 2.31-3.45), and subsequent SGA (as a proxy for FGR) (pooled OR 1.39; 95% CI 1.10-1.76). CONCLUSION: The risk of stillbirth, PTB, or SGA (as a proxy for FGR) was moderately elevated in women who previously experienced a single exposure, but increased between two- and three-fold when two prior adverse outcomes were combined. Clinical guidelines should consider the inter-relationship of stillbirth, PTB, and SGA, and that each condition is an independent risk factor for the other conditions. TWEETABLE ABSTRACT: Risk of adverse birth outcomes in next pregnancy increases with the combined number of previous adverse events. PLAIN LANGUAGE SUMMARY: Why and how was the study carried out? Each year, around 2.6 million babies are stillborn, 15 million are born preterm (<37 weeks of gestation), and 32 million are born small for gestational age (less than tenth percentile for weight, smaller than usually expected for the relevant pregnancy stage). Being born preterm or small for gestational age can increase the chance of long-term health problems. The effect of having a stillbirth, preterm birth, or small-for-gestational-age infant in a previous pregnancy on future pregnancy health has not been summarised. We identified 3399 studies of outcomes of previous pregnancies, and 17 were summarised by our study. What were the main findings? The outcome of the previous pregnancy influenced the risk of poor outcomes in the next pregnancy. Babies born to mothers who had a previous preterm birth or small-for-gestational-age birth were more likely to be stillborn. The smaller and the more preterm the previous baby, the higher the risk of stillbirth in the following pregnancy. The risk of stillbirth in the following pregnancy was doubled if the previous baby was born both preterm and small for gestational age. Babies born to mothers who had a previous stillbirth were more likely to be preterm or small for gestational age. What are the limitations of the work? We included a small number of studies, as there are not enough studies in this area (adverse birth outcomes followed by adverse cross outcomes in the next pregnancy). We found very few studies that compared the risk of small for gestational age after preterm birth or stillbirth. Definitions of stillbirth, preterm birth categories, and small for gestational age differed across studies. We did not know the cause of stillbirth for most studies. What are the implications for patients? Women who have a history of poor pregnancy outcomes are at greater risk of poor outcomes in following pregnancies. Health providers should be aware of this risk when treating patients with a history of poor pregnancy outcomes.

Comparison of DMSA scan 99 m and EC scan 99 m in diagnosis of cortical defect and differential renal function.

INTRODUCTION: Diagnosis of renal cortical lesions by radioisotopes in nuclear medicine is one of the most common techniques and procedures can be performed by different radiotracer. However, all these materials are accurate in determining kidney function, but there are differences between them in the field. The purpose of this study was to evaluate the effectiveness of EC scans compared with DMSA scan in the detection of cortical lesions and DRF. METHODS: 65 cases, which have been referred for various reasons, for DMSA scans were enrolled. Patients 1 week after DMSA scan with the previous consent of the EC being scanned. The results were compared in terms of convergence as well as sensitivity, specificity, positive and negative predictive value of EC with respect to the results of DMSA scan. RESULTS: PPV of EC was 100%, negative predictive value of EC was 68.75%, sensitivity of EC was 90.74% and specificity of EC was 100% in the detection of cortical lesions. DMSA scan and EC convergence rates result in cortical lesions in our study was high. DISCUSSION: We suggest EC scan as an alternative to reduce the cost of therapy and radiation, but considering the benefits of DMSA scan, it could remain the gold standard method of diagnosis. 

Developmental changes in diaphragm muscle function in the preterm and postnatal lamb.

RATIONALE: The preterm diaphragm is structurally and functionally immature, potentially contributing to an increased risk of respiratory distress and failure. We investigated developmental changes in contractile function and susceptibility to fatigue of the costal diaphragm in the fetal lamb to understand factors contributing to the risk of developing diaphragm dysfunction and respiratory disorders. We hypothesized that the functional capacity of the diaphragm will vary with maturational stage as will its susceptibility to fatigue. METHODS: Lambs were studied at 75, 100, 125, 145, 154, 168, and 200 days postconceptional age (term = 147 days). Lambs were euthanized (sodium pentobarbitone, 100 mg/kg) either at delivery or immediately prior to post-mortem for postnatal lambs. Contractile function was assessed on longitudinal strips of intact muscle fibers and the remaining tissue frozen in liquid nitrogen for analysis of myosin heavy chain (MHC) mRNA expression and protein content. RESULTS: Fetal development of diaphragm function was characterized by a significant increase in maximum specific force, increased susceptibility to fatigue, reduced twitch contraction times, and a progressive increase in MHCI and MHCII protein content. Postnatally, there was a progressive decrease in the susceptibility to fatigue that coincided with an increase in the MHC I:II protein ratio. CONCLUSION: These data indicate that the functional capacity of the diaphragm varies with maturational age and may be an important determinant of the susceptibility to preterm respiratory failure.

Effects of phytase supplementation in mature horses fed alfalfa hay and pelleted concentrate diets.

An experiment was conducted to study P digestibility in mature horses because of the growing environmental concerns regarding P runoff and previous equine research focused mostly on young and growing animals or used ponies as a model. Phytase supplementation of swine and poultry diets can result in greater phytate-P digestibility, leading to a decreased need for inorganic P supplementation and a decrease in P excreted to the environment; this, however, has not been demonstrated in the horse. Six mature Arabian geldings were fed 6 diets consisting of pelleted concentrate and alfalfa hay. The concentrates consisted mainly of soybean hulls, ground corn, wheat midds, broken rice, and beet pulp, and phytase was added to the concentrates accordingly before pelleting. There were 3 diet types: control (concentrate and hay), high P (greater P concentrate and hay), and forage only, and each diet type included 1 phytase-supplemented diet and 1 non-phytase-supplemented diet, resulting in 6 total diets. Phytase supplementation for the forage only diet was accomplished by feeding a nominal amount of concentrate formulated solely as a vehicle for the phytase. Horses had unrestricted access to water throughout the experiment. Using a Latin square design, all horses received all diets over a period of 12 wk. In each week, the new diet was fed for 11 d of diet acclimation, which was followed by a 3-d total collection of feces and urine for each horse. There was no effect (P < 0.05) of phytase supplementation on P output in the urine or feces, resulting in no differences in P apparent digestibility. Analysis of the feed and feces for phytate revealed a 93% average disappearance rate of phytate, indicating that horses are highly capable of degrading phytate and that phytase supplementation was not beneficial. Thus, the results indicate that mature horses are able to maintain a near 0 P balance, with adequate P provided in the diet even as phytate, and increased P intakes above requirement may result in increased potentially detrimental outputs to the environment.

Protein quality and utilization of timothy, oat-supplemented timothy, and alfalfa at differing harvest maturities in exercised Arabian horses.

To evaluate the protein quality and postgut N utilization of full-bloom timothy hay, oat-supplemented timothy-hay diets, and alfalfa hay harvested at different maturities, apparent whole tract N digestibility, urinary N excretion, and serum AA profiles were determined in light to moderately exercised Arabian horses. Six Arabian geldings (16.0 ± 0.3 yr; 467 ± 11 kg of BW) were randomly allocated to a 6 × 6 Latin square design. Diets included full-bloom timothy grass hay (G), G + 0.2% BW oat (G1), G + 0.4% BW oat (G2), mid-bloom alfalfa (A1), early-bloom alfalfa (A2), and early-bud alfalfa hay (A3). Forages were fed at 1.6% of the BW of the horse (as-fed). Each period consisted of an 11-d adaptation period followed by total collection of feces and urine for 3 d. Blood samples were taken on d 11 for analysis of serum AA concentrations. During the 3-d collection period, urine and feces were collected every 8 h and measured and weighed, respectively. Approximately 10% of the total urine volume and fecal weight per period was retained for N analyses. Fecal DM output was less (P < 0.05) in A1, A2, or A3 compared with G, G1, or G2. Apparent whole tract N digestibility was greater (P < 0.01) in A1, A2, and A3 compared with G, G1, or G2, and was greater (P < 0.05) in G1 and G2 compared with G. Nitrogen retention was not different from zero, and there were no differences (P > 0.05) in N retention among diets. Urinary N excretion and total N excretion were greater (P < 0.05) in A1, A2, and A3 compared with G, G1, or G2. Plasma concentrations for the majority of AA increased curvilinearly in response to feeding G, A1, A2, and A3 (quadratic, P < 0.05), with values appearing to maximize 2-h postfeeding. Although alfalfa N digestibility increased with decreasing harvest maturity, N retention did not differ and urinary volume and N excretion increased, indicating that postabsorptive N utilization decreased. In contrast, inclusion of oats at either 0.2 or 0.4% of the BW of the horse to timothy hay markedly enhanced N digestibility without increasing N excretion, indicating improvement in postgut N utilization. These findings indicate that feeding oat-supplemented timothy hay is more environmentally sustainable than feeding alfalfa to the horse at maintenance or under light to moderate exercise.

Blockade of TNF in vivo using cV1q antibody reduces contractile dysfunction of skeletal muscle in response to eccentric exercise in dystrophic mdx and normal mice.

This study evaluated the contribution of the pro-inflammatory cytokine, tumour necrosis factor (TNF) to the severity of exercise-induced muscle damage and subsequent myofibre necrosis in mdx mice. Adult mdx and non-dystrophic C57 mice were treated with the mouse-specific TNF antibody cV1q before undergoing a damaging eccentric contraction protocol performed in vivo on a custom built mouse dynamometer. Muscle damage was quantified by (i) contractile dysfunction (initial torque deficit) immediately after the protocol, (ii) subsequent myofibre necrosis 48 h later. Blockade of TNF using cV1q significantly reduced contractile dysfunction in mdx and C57 mice compared with mice injected with the negative control antibody (cVaM) and un-treated mice. Furthermore, cV1q treatment significantly reduced myofibre necrosis in mdx mice. This in vivo evidence that cV1q reduces the TNF-mediated adverse response to exercise-induced muscle damage supports the use of targeted anti-TNF treatments to reduce the severity of the functional deficit and dystropathology in DMD.

Screening of substance misuse during early pregnancy in Blyth: an anonymous unlinked study.

Substance abuse during pregnancy is rising and often remains undiagnosed. This harms both the mother and the baby. We conducted an anonymous unlinked study in Blyth valley, Northumberland, to determine the prevalence of substance abuse and alcohol use during pregnancy. Urine toxicology screening was performed on 150 women who attended antenatal clinic for booking. Seven commonly misused substances (amphetamine, benzodiazepine, barbiturates, cannabinoids, cocaine, methadone and opiates) and alcohol were tested in the urine. A total of 16 (10.7%) women were found positive and all 16 of them had denied use of any substance. Amphetamine was the most common among the substances misused. A total of 12 out of the 16 were from social classes 4 and 5. None of the positive women was nulliparous. Because of the difficulty in identifying substance misuse in pregnant women, consideration should be given to the implementation of routine substance screening at the booking antenatal visit in general population with a high incidence of substance misuse.

Topography of somatosensory processing: cerebral lateralization and focused attention.

Healthy dextrals underwent fMRI during a task of graphesthesia requiring detection of any number written consecutively from an otherwise random number sequence. Test conditions included (1) focus on unilateral right hand stimuli, (2) focus on unilateral left hand stimuli, (3) focus on right hand only during bilateral hand stimulation, (4) focus on left hand only during bilateral hand stimulation, and (5) rest. Attention to unilateral hand stimulation produced bihemispheric activation with minimal or no activation of ipsilateral primary sensorimotor region. Attention to unilateral left hand stimuli resulted in more activation than attention to unilateral right hand stimuli. Stimulation of the nonattended hand activated the contralateral somatosensory area, but to a lesser spatial extent than attended stimuli. Comparing focused attention to the left versus right side during identical sensory inputs (i.e., bilateral hand stimulation), focused attention to the right hand increased activation in the left somatosensory region, but focused attention to the left hand increased activation in both cerebral hemispheres. Thus, focused attention to unilateral somatosensory stimuli produces bilateral cerebral activation, but the increase in blood flow is greater in the contralateral hemisphere. Unattended stimuli activate the contralateral primary somatosensory area. Left/right asymmetries were demonstrated consistent with cerebral lateralization.

Now you see it, now you don't: statistical and methodological considerations in fMRI.

We illustrate the effects of statistical threshold, spatial clustering, voxel size, and two approaches to multiple comparison correction on fMRI results. We first analyzed fMRI images obtained from a single subject during a noun-verb matching task. Data were analyzed with Statistical Parametric Mapping (SPM) using two different voxel sizes, and results were displayed at three different levels of statistical significance. At each statistical threshold, results were first uncorrected for multiple comparisons and spatial extent and then presented using a spatial extent cluster of 20 voxels. We then statistically controlled the Type I error rate associated with multiple comparisons by using the false discovery rate and by the random field adjustment for false-positive rate used by SPM. We also examined group results from language and graphesthesia paradigms at three levels of statistical significance. In all circumstances, apparent random activations decreased as more conservative statistical approaches were employed, but activation in areas considered to be functionally significant was also reduced. These issues are important in the choice of analytic approach and interpretation of fMRI results, with clear implications for the surgical management of individual patients when fMRI results are used to delineate specific areas of eloquent cortex.

Specific prorenin/renin binding (ProBP). Identification and characterization of a novel membrane site.

Renin can be detected in cardiovascular and other tissues but it disappears after bilateral nephrectomy indicating that tissues can take up or bind renal renin from the circulation. If renin uptake is the result of specific binding, plasma prorenin may be a natural antagonist of tissue directed renin-angiotensin systems. To investigate if specific prorenin/renin uptake occurs in rat tissues, binding studies were performed, with rat microsomal membrane preparations using recombinant rat prorenin metabolically labeled with 35S-methionine as a probe. A high affinity binding site for both renin and prorenin was identified. Affinities for prorenin and renin were approximately 200 and 900 pmol/L, respectively. Binding was reversible, saturable, and pH and temperature dependent. The relative binding capacities of membranes from various rat tissues were as follows (fmol/mg): renal cortex (55), liver (54), testis (63), lung (31), brain (18), renal medulla (15), adrenal (17), aorta (7), heart (4), and skeletal muscle (1). Bound prorenin was displaced by rat and human renin or prorenin but not by the prosequence of rat prorenin, angiotensin I or II, rat or human angiotensinogen, the renin inhibitor SQ30697, atrial natriuretic factor, amylase, insulin, bovine serum albumin, hemoglobin, heparin, lysozyme, ovalbumin, cytochrome C, pepsin, pepsinogen, ribonuclease A, mannose-6-phosphate, alpha-methyl mannoside, gonadotropin releasing hormone, or an antibody to hog renin binding protein. these results demonstrate specific binding of prorenin to a site in rat tissues, herein named ProBP, that also binds renin. It is possible that differences in prorenin/renin binding capacity determine the activity of tissue-directed renin-angiotensin systems and that prorenin is a natural antagonist. Alternatively, a prorenin/renin receptor may have been identified that may function by transducing an intracellular signal.

Renin gene promoter activity in GC cells is regulated by cAMP and thyroid hormone through Pit-1-dependent mechanisms.

Transcriptional activity of human renin gene (hREN) 5'-flanking DNA sequences in pituitary cells is highly dependent on binding of the pituitary-specific transcription factor Pit-1. Pit-1 has been implicated in cAMP regulation of a number of pituitary genes and has also been shown to interact with thyroid hormone (T3) receptors in mediating T3 responsiveness of the rat growth hormone gene. In the present study we examine the effects of forskolin and T3 on the expression of luciferase hybrid genes containing hREN 5'-flanking DNAs (hREN.luc) transiently transfected into the pituitary cell line GC. Basal activities of all hREN.luc constructs transfected into cells grown in media containing serum stripped of hormones were low. Addition of forskolin stimulated expression up to 48-fold, depending on the hREN sequences present. The hREN sequence -148 to +18 was sufficient for both maximal expression and maximal stimulation by forskolin. Mutagenesis of the Pit-1 site between -82 and -58 reduced forskolin induction 4-5-fold. In addition to the Pit-1 site, the sequence between -148 and -98 was also required for maximal activity and forskolin induction. T3 on its own had no effect on hREN promoter activity in GC cells, but suppressed the effects of forskolin. Gel mobility shift and Western blot analyses indicated that forskolin treatment had no effect on Pit-1 DNA binding or Pit-1 levels. However, T3 reduced Pit-1 levels which was reflected in lower DNA binding under the conditions employed. Taken together, these findings emphasize the importance of cAMP-dependent mechanisms in directing renin gene expression.

Thyroid hormone receptor-induced bending of specific DNA sequences is modified by an accessory factor.

Transcriptional regulation by thyroid and steroid hormone receptors requires their recognition and binding of specific DNA sequences. However, little is known about the mechanisms whereby DNA bound receptors regulate transcription. In the present study, we examined the effects of thyroid hormone receptor (TR) binding on DNA conformation using various TR recognition sites contained within sets of circularly permuted flanking sequences. We show that under conditions where TR binds predominantly as monomer, the conformation of a number of binding sites is changed in a manner consistent with receptor induced bending. Despite similar affinities for receptor binding, not all binding sites tested showed evidence for receptor-induced bending. Notably, the conformation of a sequence from the frog vitellogenin 2 gene, which confers a positive transcriptional response when bound by estrogen receptor (ER), but a negative response when bound by TR, appeared to be unaffected by binding of either TR or ER. The observations suggest that the ability of the receptor to alter DNA architecture is strongly dependent on sequence characteristics other than those required for receptor binding. While both partly purified TR from rat liver and TR translated in vitro were able to induce DNA bending, the bend centers and bend angles produced by these different sources of receptor differed. However, addition of a receptor-depleted fraction from the rat liver TR preparation to in vitro translated receptor stimulated TR binding and appeared to form heterodimers with TR. This resulted in changes in both bend centers and bend angles to resemble more closely those produced by native receptor. Together, these results suggest that receptor-induced DNA bending may be specific to TRs and that the position and degree of bending is further modulated by the formation of heterodimers between TRs and accessory protein(s).

The rat growth hormone gene contains multiple thyroid response elements.

The thyroid hormone receptor exerts transcriptional control over a variety of genes. This report describes four sites that bind this receptor with high affinity within the 5'-flanking DNA of the rat growth hormone gene, approximately centered at -180, -160, -60 and -20 nucleotides from the transcription start site. These sites were defined by gel retardation of short synthetic oligonucleotides using native receptor purified several hundred-fold from rat liver. Binding sites were also defined by methylation interference and methidium-propyl-EDTA footprinting. Alignment of the four binding sites suggests that each contains two purine-rich regions, the more downstream of which, GGGATCGC, is highly conserved. Mutations made within each of the two upstream sites reduce receptor binding affinity. For one mutation, a partial loss of receptor binding strength correlated with a change in electrophoretic mobility, indicating that receptor binding may alter DNA conformation. Mutations at each of the four sites also reduce thyroid hormone responsiveness of the -237/+11 promoter linked to the chloramphenicol acetyltransferase gene coding sequences and transfected into cultured pituitary (GC) cells. These results suggest that several different receptor-binding elements interact to control thyroid hormone responsiveness of the rat growth hormone gene and reveal common sequences that may be important for receptor-DNA recognition.