Congenital Uterine Malformation by Experts (CUME): better criteria for distinguishing between normal/arcuate and septate uterus?

OBJECTIVES: To assess the level of agreement between experts in distinguishing between septate and normal/arcuate uterus using their subjective judgment when reviewing the coronal view of the uterus from three-dimensional ultrasound. Another aim was to determine the interobserver reliability and diagnostic test accuracy of three measurements suggested by recent guidelines, using as reference standard the decision made most often by experts (Congenital Uterine Malformation by Experts (CUME)). METHODS: Images of the coronal plane of the uterus from 100 women with suspected fundal internal indentation were anonymized and provided to 15 experts (five clinicians, five surgeons and five sonologists). They were instructed to indicate whether they believed the uterus to be normal/arcuate (defined as normal uterine morphology or not clinically relevant degree of distortion caused by internal indentation) or septate (clinically relevant degree of distortion caused by internal indentation). Two other observers independently measured indentation depth, indentation angle and indentation-to-wall-thickness (I:WT) ratio. The agreement between experts was assessed using kappa, the interobserver reliability was assessed using the concordance correlation coefficient (CCC), the diagnostic test accuracy was assessed using the area under the receiver-operating characteristics curve (AUC) and the best cut-off value was assessed using Youden's index, considering as the reference standard the choice made most often by the experts (CUME). RESULTS: There was good agreement between all experts (kappa, 0.62). There were 18 septate and 82 normal/arcuate uteri according to CUME; European Society of Human Reproduction and Embryology (ESHRE)-European Society for Gynaecological Endoscopy (ESGE) criteria (I:WT ratio > 50%) defined 80 septate and 20 normal/arcuate uteri, while American Society for Reproductive Medicine (ASRM) criteria defined five septate (depth > 15 mm and angle < 90°), 82 normal/arcuate (depth < 10 mm and angle > 90°) and 13 uteri that could not be classified (referred to as the gray-zone). The agreement between ESHRE-ESGE and CUME was 38% (kappa, 0.1); the agreement between ASRM criteria and CUME for septate was 87% (kappa, 0.39), and considering both septate and gray-zone as septate, the agreement was 98% (kappa, 0.93). Among the three measurements, the interobserver reproducibility of indentation depth (CCC, 0.99; 95% CI, 0.98-0.99) was better than both indentation angle (CCC, 0.96; 95% CI, 0.94-0.97) and I:WT ratio (CCC, 0.92; 95% CI, 0.90-0.94). The diagnostic test accuracy of these three measurements using CUME as reference standard was very good, with AUC between 0.96 and 1.00. The best cut-off values for these measurements to define septate uterus were: indentation depth ≥ 10 mm, indentation angle < 140° and I:WT ratio > 110% . CONCLUSIONS: The suggested ESHRE-ESGE cut-off value overestimates the prevalence of septate uterus while that of ASRM underestimates this prevalence, leaving in the gray-zone most of the uteri that experts considered as septate. We recommend considering indentation depth ≥ 10 mm as septate, since the measurement is simple and reliable and this criterion is in agreement with expert opinion. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Utilizing pretreatment and fungal incubation to enhance the nutritional value of canola meal.

AIMS: The objective of this study was to determine the optimal pretreatment and fungal strain to reduce glucosinolates (GLS), fibre and residual sugars while increasing the nutritional value of canola meal. METHODS AND RESULTS: Submerged incubation conditions were used to evaluate four pretreatment methods (extrusion, hot water cook, dilute acid and dilute alkali) and three fungal cultures (Aureobasidium pullulans Y-2311-1, Fusarium venenatum NRRL-26139 and Trichoderma reesei NRRL-3653) in hexane-extracted (HE) and cold-pressed (CP) canola meal. CONCLUSIONS: The combination of extrusion pretreatment followed by incubation with T. reesei resulted in the greatest overall improvement to HE canola meal, increasing protein to 51·5%, while reducing NDF, GLS and residual sugars to 18·6%, 17·2 µmol l-1  g-1 and 5% w/w, respectively. Extrusion pretreatment and incubation with F. venenatum performed the best with CP canola meal, resulting in 54·4% protein while reducing NDF, GLS and residual sugars to 11·6%, 6·7 µmol l-1  g-1 and 3·8% w/w respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The work is significant in that it provides a method of reducing GLS (up to 98%) and neutral detergent fibre (up to 65%) while increasing the protein content (up to 45%) of canola meal. This novel pretreatment and submerged incubation process could be used to produce a canola product with higher nutritional value for livestock consumption.

Growth performance of calves fed microbially enhanced soy protein in pelleted starters.

Our objective was to determine effects of feeding calves pelleted starters with microbially enhanced (fungi-treated) soy protein (MSP) in replacement of soybean meal (SBM) with different milk replacers (MR). Thirty-six Holstein calves (2 d old; 24 females, 12 males) in individual hutches were used in a 12-wk randomized complete block design study. Treatments were (1) MSP pellets with MR formulated for accelerated growth (28% crude protein, 18% fat; MSPA), (2) SBM pellets with MR formulated for accelerated growth (SBMA), and (3) MSP pellets with conventional MR (20% crude protein, 20% fat; MSPC). Pellets were similar except for 23% MSP or 23% SBM (dry matter basis). Pellets and water were fed ad libitum throughout the study. Feeding rates of MR on a dry matter basis were 0.37kg twice daily during wk 1, 0.45kg twice daily during wk 2 to 5, and 0.45kg once daily during wk 6. Intakes were recorded daily. Body weights, frame size measurements, and jugular blood samples were collected 2 d every 2 wk at 3 h after the morning feeding. Fecal grab samples were collected 5 times per d for 3 d during wk 12 and then composited by calf for analysis of apparent total-tract digestibility of nutrients using acid detergent insoluble ash as an internal marker. Total and starter pellet dry matter intake were greatest for calves fed SBMA and least for MSPC. Calves had similar average daily gain among treatments, but there was a treatment by week interaction and during the last few weeks of the study calves on MSPC had less body weight compared with MSPA or SBMA. Gain-to-feed ratio was similar among treatments; however, there was a treatment by week interaction. Serum glucose was similar among treatments. Plasma urea nitrogen was greatest for calves fed MSPA and least for MSPC. Plasma concentrations of IGF-1 were greatest for calves fed SBMA. Plasma concentrations of triglycerides were greatest for calves fed MSPC. Plasma concentrations of ß-hydroxybutyrate had a treatment by time interaction. Treatments had similar total-tract dry matter digestibility, but calves fed MSPC had greater crude protein digestibility than SBMA, with MSPA similar to both. Results demonstrated calves fed pelleted starters with MSP had maintained growth performance with less starter intake compared with SBM.

Association of ovarian response with picoAMH in women undergoing controlled ovarian hyperstimulation.

OBJECTIVE: Our study aims to evaluate the diagnostic performance of a high-sensitivity picoAnti-Müllerian Hormone (picoAMH) for predicting ovarian response in women undergoing controlled ovarian hyperstimulation and occurrence of ovarian hyperstimulation syndrome. METHODS: Retrospective cohort study at a single academic fertility center including all patients with picoAMH ELISA who underwent controlled ovarian hyperstimulation. The primary outcome was the number of oocytes retrieved, and secondary outcomes included cycle cancellation and ovarian hyperstimulation syndrome. Patients were grouped into poor, normal, and hyper-responders based on number of oocytes retrieved. RESULTS: The mean AMH and antral follicle count (AFC) were significantly different between normal response vs. hyper response group (p < 0.0001). Only serum AMH and not AFC was significantly increased in patients diagnosed with ovarian hyperstimulation syndrome (OHSS). For prediction of OHSS, receiver operating characteristic (ROC) analysis revealed that AMH (area under the ROC curve [AUC] = 0.85) was significantly better than the AFC (AUC = 0.64). The serum AMH cut-off at sensitivity of 80% for predicting OHSS among hyper responders from ROC curve was 3.67 ng/ml. Serum AMH measured by picoAMH ELISA showed superior correlation to number of oocytes retrieved when compared to AFC in the age group over 40 years old (r2 = 0.74 and r2 = 0.4, respectively) CONCLUSION: This study shows great utility of picoAMH ELISA for predicting ovarian response to controlled ovarian hyperstimulation (COH). Diagnostic performance of picoAMH for prediction of OHSS is superior to the AFC in our cohort.

Comparative analysis of the ileal bacterial composition of post-weaned pigs fed different high-quality protein sources.

To further understand the contribution of feedstuff ingredients to gut health in swine, gut histology and intestinal bacterial profiles associated with the use of two high-quality protein sources, microbially enhanced soybean meal (MSBM) and Menhaden fishmeal (FM) were assessed. Weaned pigs were fed one of three experimental diets: (1) basic diet containing corn and soybean meal (Negative Control (NEG)), (2) basic diet + fishmeal (FM; Positive Control (POS)) and (3) basic diet + MSBM (MSBM). Phase I POS and MSBM diets (d 0 to d 7 post-wean) included FM or MSBM at 7.5%, while Phase II POS and MSBM diets (d 8 to d 21) included FM or MSBM at 5.0%. Gastrointestinal tissue and ileal digesta were collected from euthanised pigs at d 21 (eight pigs/diet) to assess gut histology and intestinal bacterial profiles, respectively. Data were analysed using Proc Mixed in SAS, with pig as the experimental unit and pig (treatment) as the random effect. Histological and immunohistochemical analyses of stomach and small intestinal tissue using haematoxylin-eosin, Periodic Acid Schiff/Alcian blue and inflammatory cell staining did not reveal detectable differences in host response to dietary treatment. Ileal bacterial composition profiles were obtained from next-generation sequencing of PCR generated amplicons targeting the V1 to V3 regions of the 16S rRNA gene. Lactobacillus-affiliated sequences were found to be the most highly represented across treatments, with an average relative abundance of 64.0%, 59.9% and 41.80% in samples from pigs fed the NEG, POS and MSBM diets, respectively. Accordingly, the three most abundant Operational Taxonomic Units (OTUs) were affiliated to Lactobacillus, showing a distinct abundance pattern relative to dietary treatment. One OTU (SD_Ssd_00001), most closely related to Lactobacillus amylovorus, was found to be more abundant in NEG and POS samples compared to MSBM (23.5% and 35.0% v. 9.2%). Another OTU (SD_Ssd_00002), closely related to Lactobacillus johnsonii, was more highly represented in POS and MSBM samples compared to NEG (14.0% and 15.8% v. 0.1%). Finally, OTU Sd_Ssd-00011, highest sequence identity to Lactobacillus delbrueckii, was found in highest abundance in ileal samples from MSBM-fed pigs (1.9% and 3.3% v. 11.3, in POS, NEG and MSBM, respectively). There was no effect of protein source on bacterial taxa to the genus level or diversity based on principal component analysis. Dietary protein source may provide opportunity to enhance presence of specific members of Lactobacillus genus that are associated with immune-modulating properties without altering overall intestinal bacterial diversity.

Conversion of volatile fatty acids into polyhydroxyalkanoate by Ralstonia eutropha.

AIMS: The aims of this study were to optimize condensed corn solubles (CCS) as a medium for growth of Ralstonia eutropha and to determine the effects of individual volatile fatty acids (VFAs) on polyhydroxyalkanoate (PHA) production. METHODS AND RESULTS: A CCS medium of concentration 240 g l(-1) with a carbon : nitrogen ratio of 50 : 1 was developed as the optimal medium. Cultures were grown in 1-l aerated flasks at 250 rev min(-1) at 30 degrees C for 120 h. Comparable growth rates were observed in CCS vs a defined medium. At 48 h, VFAs were fed individually at different levels. Optimal levels of all the acids were determined to maximize PHA production. An overall comparison of the VFAs indicated that butyric and propionic acids provided the best results. CONCLUSION: An optimized CCS medium supported growth of R. eutropha. Butyric and propionic acids were the most efficient carbon sources to maximize PHA production when added at the 5 g l(-1) level. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that a byproduct of ethanol industry can be effectively used as a low cost medium for PHA production, thus partly reducing the cost of commercialization of biopolymers.

Profiling transient daytime peaks in urban air pollutants: city centre traffic hotspot versus urban background concentrations.

Averaged annual and daily limit values imposed for the protection of human health from urban air pollutants fail to address the potential influence of variations in personal exposure during the day, even though asthma attacks, myocardial ischaemia, and other effects have been linked to highly transient rises in atmospheric contamination. There is, therefore, a general need for the wider availability of information on hourly variations in urban pollutant levels, as well as on spatial differences between urban background and localised contamination hotspots. We compare data from urban background and traffic hotspot monitoring stations over a 12-month period to demonstrate the hourly flux of air pollutants, notably NO(x), SO(2) and PM(10), and illustrate the effects of controlling influences, such as traffic movements and exotic dust intrusions. Our methodology focuses on daytime transient peaks, which occur when much of the urban population is awake and active, and when striking increases in pollutant concentrations are especially common at traffic hotspots. Greater emphasis on the daily reality of transient peaks in urban atmospheric pollutants offers a higher degree of spatial and temporal data resolution for use in epidemiological studies.

Rhodospirillum rubrum: utilization of condensed corn solubles for poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) production.

AIMS: This study sought to develop a less expensive medium for growth of the polyhydroxyalkanoate-producing bacterium Rhodospirillum rubrum from the ethanol production coproduct, condensed corn solubles (CCS). METHODS AND RESULTS: Small-scale trials using R. rubrum were performed in aerated or anaerobic stoppered serum bottles filled with media. The CCS (240 g l(-1)) achieved a maximum cell density and growth rate comparable with the defined supplemented malate-ammonium medium (mSMN) or tryptic soy broth. Microaerophilic solubles medium cultures exhibited significantly higher maximum cell densities and growth rates than did strictly anaerobic cultures; while illumination, nickel or biotin addition had no effect. Growth of R. rubrum in a pH controlled bioreactor was significantly better in CCS (240 g l(-1)) than in mSMN medium and supported production of 0.36% (cell dry weight) poly-(3-hydroxybutyrate-Co-3-hydroxyvalerate) after 24 h. CONCLUSIONS: A CCS medium was devised that supported R. rubrum growth for biopolymer production as effective as the defined medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that a more economical medium can be developed for biopolymer production using a low value coproduct from ethanol production. The impact is that this inexpensive solubles medium may make it more economical to produce the biopolymer on a commercial scale.

Optimization of oestradiol assays to improve utility in an in vitro fertilization setting.

Background The measurement of oestradiol is an integral component for the management of ovarian stimulation for in vitro fertilization. Automated immunoassays offer fast assay times and high throughput, with less sensitivity and specificity. The aim of this study is to optimize the oestradiol assay in patients undergoing ovarian stimulation for in vitro fertilization via comparison of oestradiol values obtained using two immunoassays compared with mass spectrometry. Methods Patients undergoing ovarian stimulation were prospectively recruited. Serum samples were analysed with ADVIA Centaur® CP Immunoassay, Abbott Architect i1000® immunoassay and AB Sciex 5500 liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems. Per cent bias was determined for each system to report the average tendency of the values to be larger or smaller than the LC-MS/MS value. Linear regression of total follicular volume and oestradiol was computed. Results The ADVIA Centaur® CP assay had a positive bias of 20% compared with LC-MS/MS, while the Architect i1000® had a non-significant, negative bias of 0.3%. With regression fit, a clear, positive relationship was seen between follicular volume and oestradiol. The Architect i1000® assay had a greater correlation (R2 = 0.46) compared with Centaur® CP (R2 = 0.36), when oestradiol values were >1000 pg/mL (3670 pmol/L). Conclusions The Abbott Architect i1000® oestradiol assay exhibits greater agreement with LC-MS/MS and exhibited better correlation to follicular volume when oestradiol values are >1000 pg/mL (3670 pmol/L), prompting a change in the clinic's oestradiol platform. Attention to assay quality assurance via LC-MS/MS can improve the oestradiol accuracy and permit more informed clinical decisions for improved patient outcomes.

Evaluation of a bioprocessed soybean meal on nursery pig performance and immune status.

A total of 239 pigs (initial BW 6.56 ± 0.87 kg, 21 d of age) were used in a 35-d study to investigate the effects of fish meal (FM) and spray dried plasma (SDP) in combination with a bioprocessed soybean meal (SBM) on growth performance and immune responses in weaned pigs. Equal numbers of barrows and gilts were randomly allotted to 1 of 4 dietary treatments (10 pens/treatment) according to initial BW and sex: positive control (CON; corn/SBM diet) containing SDP and FM, the CON with bioprocessed SBM replacing FM (BPSBM), the CON with bioprocessed SBM replacing SDP (BPSBM), and the CON with bioprocessed SBM replacing both SDP and FM (BPSBM). Experimental diets were fed in Phase I (d 1-7 post-wean) and II (d 8-21) followed by a common Phase III diet (d 22-35); changes in BW and feed disappearance were determined accordingly. Pigs were sensitized against ovalbumin (OVA) and Candida albicans (CAA) on d 7 and 21. Assessment of immune response was based on lymphocyte proliferation in response to mitogens Concanavalin A and phytohemagglutinin (d 14 post-wean), dermal hypersensitivity to OVA and CAA (% increase in local swelling at 2, 6, 24, and 48 h post-injection) on d 28, and primary and secondary anti-OVA IgG at d 21 and 28, respectively. Pigs fed CON were heavier (P < 0.01) than pigs fed BPSBM and BPSBM, and not different from pigs fed BPSBM, at the end of Phase I and II (6.99, 6.80, 6.52, or 6.60 kg, pooled SEM 0.08, respectively in Phase I or 12.47, 12.18, 11.42, and 11.85 kg, pooled SEM 0.21, in Phase II, respectively). Hypersensitivity to OVA peaked at 2h in pigs fed CON, BPSBM, and BPSBM or peaked at 6h in pigs fed BPSBM (121.4, 165.6, 139.0, and 144.1%, pooled SEM 22.9, respectively, at 2 h and 86.7, 114.5, 95.0, and 156.8%, pooled SEM 29.4, respectively at 6 h). Peak response to CAA occurred at 2h in all groups (42.6, 55.2, 48.2, and 50.6%, pooled SEM 11.9, respectively, in the CON, BPSBM, BPSBM, and BPSBM, respectively). There was no difference in hypersensitivity due to experimental diet at any time point. Secondary anti-OVA IgG was 2-fold lower based on optical density values in pigs fed CON compared with BPSBM and BPSBM (0.78 vs. 1.56 and 1.55 optical density at 405 nm, pooled SEM 0.42, respectively). Dietary treatment did not impact lymphocyte proliferation. The bioprocessed SBM is a suitable alternative for FM and/or SDP in Phase I and II nursery diets based on pig growth. The prolonged hypersensitivity to OVA indicate that bioprocessed SBM may have a positive impact on pig immune function and the 2-fold increase in anti-OVA IgG warrants further investigation on the impact of bioprocessed SBM on pig immune function.

Evaluation of microbially enhanced soybean meal as an alternative to fishmeal in weaned pig diets.

An experimental, microbially enhanced soybean product (MEPRO) was evaluated as a replacement for fishmeal (FM). Assessment of feedstuffs should include estimation of digestibility as well as pig performance and in combination with dietary additives. Digestibility values determined in growing pigs may not apply to nursery pigs; thus, standardized ileal digestibility (SID) of amino acids (AA) in MEPRO and FM were determined using 30±1.6 kg BW ileal-cannulated barrows (n=6) and 9.8±1.2 kg BW barrows (n=37; serial slaughter). Experimental diets included MEPRO, FM and nitrogen free where FM and MEPRO were included as the sole protein source. The SID of AAs was 3% to 5% lower in MEPRO than FM when fed to 30 kg pigs. The SID of arginine and methionine was greater (P<0.05) in MEPRO than FM when fed to 10 kg pigs. The SID of AAs was 12% to 20% lower in FM when fed to 10 v. 30 kg pigs but only 3% to 9% lower in MEPRO. A total of 336 barrows and gilts were weaned at 21 days of age (initial BW=6.1±0.8 kg) and used in a performance trial. Pens of pigs were assigned to one of the six experimental diets (8 pens/diet in two blocks). Treatment diets were fed in Phase I (7 days) and Phase II (14 days) with all pigs fed a common Phase III diet (14 days). Experimental diets included (1) negative control (NEG) containing corn, soybean meal and whey, (2) NEG+acidifier, (3) NEG+FM (POS), (4) POS+acidifier (POS A+), (5) NEG+MEPRO (MEPRO) and (6) MEPRO+acidifier. The FM and MEPRO were included at 7.5% and 5.0% in Phase I and II diets, respectively. Diets were formulated to meet the standard nutrient requirements for weaned pigs. Pig BW and feed disappearance was measured weekly and fecal scores were measured daily for the first 14 days post-weaning as an indicator of post-weaning diarrhea syndrome (PWDS). Performance (BW, daily gain, feed intake and gain : feed) was not significantly different among treatments. Treatment for PWDS occurred on different days in each block. Analysis of fecal score was completed separately by block. Pigs fed the NEG diets had higher (P=0.02) fecal scores than pigs fed the POS diets on days 2 and 3 (block 1) and higher (P<0.05) than pigs fed MEPRO or POS diets and diets with dietary acidifier on days 6 and 3 (block 2). The MEPRO holds promise as an alternative to FM in nursery pig diets.

Tetrahymena thermophila: analysis of phospholipids and phosphonolipids by high-field 1H-NMR.

The phospholipids of control and lipid-modified Tetrahymena thermophila were identified and quantified, using 1-D and 2-D COSY proton NMR spectroscopy on intact lipids, before and after HPLC separation. The results are comparable to those obtained using classical lipid analytical techniques. The results indicate that the study of enzyme pathways and other metabolic processes involving phospholipids in Tetrahymena and related protozoa can be carried out using proton NMR spectroscopy as the investigating technique.

The role of molecular conformation in ion capture by carboxylic ionophores: a circular dichroism study of narasin A in single-phase solvents and liposomes.

Conformational and thermodynamic aspects of cation binding by the carboxylic ionophore narasin A were studied by circular dichroism (CD). In single-phase solvents, dramatic increases in the maximum differential absorption (delta epsilon) of the C-11 carbonyl were observed upon the binding of K+, Na+ and protons to the free anionic form. These changes were associated with major shifts in the conformation equilibrium between extended and pseudocyclic conformers of narasin. Similar CD changes observed upon the binding of K+ to narasin A in dimyristoylphosphatidylcholine vesicles provided evidence that in the membrane environment, comparable conformation changes were associated with ion binding. Variation of the polar and protic properties of single-phase solvents was also found to influence the delta epsilon of the cation bound species of narasin A, supporting previous evidence for polarity-mediated modulation of conformation. Comparison of cation binding affinities indicated that in both single-phase solvents and liposomes, narasin had a marked equilibrium selectivity for K+ over Na+.

1H-NMR lipid profiles of human blood platelets; links with coronary artery disease.

Blood platelets are closely involved in the early development of atherosclerosis and in the events that lead to thrombosis, both of which are dominating factors in coronary artery disease (CAD). The aim of the present study was to evaluate the platelet lipid profiles of patients suffering from CAD and explore the possibility of a link between platelet lipids and CAD, using high-resolution high-field proton nuclear magnetic resonance spectroscopy as the analytical tool. The total platelet lipid profiles of healthy volunteers were compared with those of patients presenting with chest pain requiring coronary angiography. Two lipid groups changed significantly: cholesterol increased by 16.5% and total diacylglycerophospholipids decreased by 15.7%. There was also a significant decrease of the ethanolamine-containing phospholipids, by 4.7%; the extent of unsaturation of the fatty acid chains, by 0.2, and increase of the linoleate content of the fatty acid chains, by 1.9%. Our results suggest that platelet lipid abnormalities occur in patients with CAD and these changes may predate the development of overt atherosclerosis.

Analytical subcellular fractionation of rat pituitary homogenates with special reference to the subcellular localization and properties of alkaline phosphatases.

Alkaline phosphatase activities of the virgin rat anterior pituitary were studied with a highly sensitive fluorometric assay. Tissue whole homogenates were fractionated on sucrose density gradients in a Beaufay automatic zonal rotor and the gradient fractions assayed for alkaline phosphatase, prolactin and various organelle marker enzymes. Alkaline phosphatase was distributed between two peaks on the gradient. The low-density (1.10-1.15 g . cm-3) alkaline phosphatase component co-sedimented with the plasma membrane marker, 5'-nucleotidase, had an apparent Km for 4-methylumbelliferyl phosphate of approx. 59 microM, and was inhibited by levamisole. The high-density (1.20-1.25 g . cm-3) peak was resistant to levamisole-inhibition, had an apparent Km of approx. 30 microM and its distribution was distinct from plasma membrane, Golgi, lysosome, endoplasmic reticulum, mitochondria and prolactin granule markers on the isopycnic gradients.

Properties of the calcium-activated chloride current in heart.

We used the whole cell patch clamp technique to study transient outward currents of single rabbit atrial cells. A large transient current, IA, was blocked by 4-aminopyridine (4AP) and/or by depolarized holding potentials. After block of IA, a smaller transient current remained. It was completely blocked by nisoldipine, cadmium, ryanodine, or caffeine, which indicates that all of the 4AP-resistant current is activated by the calcium transient that causes contraction. Neither calcium-activated potassium current nor calcium-activated nonspecific cation current appeared to contribute to the 4AP-resistant transient current. The transient current disappeared when ECl was made equal to the pulse potential; it was present in potassium-free internal and external solutions. It was blocked by the anion transport blockers SITS and DIDS, and the reversal potential of instantaneous current-voltage relations varied with extracellular chloride as predicted for a chloride-selective conductance. We concluded that the 4AP-resistant transient outward current of atrial cells is produced by a calcium-activated chloride current like the current ICl(Ca) of ventricular cells (1991. Circulation Research. 68:424-437). ICl(Ca) in atrial cells demonstrated outward rectification, even when intracellular chloride concentration was higher than extracellular. When ICa was inactivated or allowed to recover from inactivation, amplitudes of ICl(Ca) and ICa were closely correlated. The results were consistent with the view that ICl(Ca) does not undergo independent inactivation. Tentatively, we propose that ICl(Ca) is transient because it is activated by an intracellular calcium transient. Lowering extracellular sodium increased the peak outward transient current. The current was insensitive to the choice of sodium substitute. Because a recently identified time-independent, adrenergically activated chloride current in heart is reduced in low sodium, these data suggest that the two chloride currents are produced by different populations of channels.

High potassium and low sodium contractures in sheep cardiac muscle.

Contractures develop in sheep atrial trabeculae if Tyrode's solution is rapidly replaced by a solution containing elevated potassium, reduced sodium, or both. Two phases of the contracture can be identified on the basis of differences in physiological behavior: a rapid and transient phase that predominates during the first few seconds of the contracture, and a slowly developed phase that is responsible for the steady level of tension reached later in the contracture. The transient phase is particularly prominent if the muscle is stimulated rapidly before the contracture, and reduced or absent if the muscle is not stimulated or if calcium is not present before the contracture. Recovery of the transient phase after a contracture parallels the recovery of twitches. This transient phase appears to reflect the depolarization-induced release of activator (calcium) from an internal store, possibly the same store that is involved in the normal contraction. The slowly developed tension is dependent on the contracture solution used, and is decreased if the calcium concentration is reduced or if the sodium concentration is increased. It does not depend on conditions before the contracture and does not require time to recover. This phase of the contracture may be due to entry of calcium from the extracellular solution.

Relationships between voltage and tension in sheep cardiac Purkinje fibers.

The two-microelectrode technique of voltage clamping sheep cardiac Purkinje fibers was used to examine the changes in contraction which occur during trains of voltage clamps. (A "train" is defined as a series of voltage clamps delivered at a particular rate, beginning after a rest long enough that the effects of previous stimulation have died away.) Contractions showed striking staircases, or progressive changes in peak isometric tension, during trains. Short clamps, clamps to voltages more negative than --20 or --30 mV, or holding potentials less negative than the resting potential favored negative staircases, while long clamps, clamps to positive voltages, and holding potentials near the resting potential each favored positive staircases. The staircase behavior appeared to be due to changes in the initial rate of recovery of the ability to contract. The changes in staircase behavior as a function of clamp voltage suggested that the relationship between peak tension and clamp voltage should depend on the experimental design. When the steady-state contraction was plotted as a function of clamp voltage, voltage-tension relations like those recently reported for working ventricle were obtained, with a threshold between --30 and --40 mV and a steep relation between tension and voltage. When the first contraction after a rest was plotted, the threshold voltage was more negative, the curve was flatter, and the peak tensions at inside positive voltages were reduced.

Slow inward current and contraction of sheep cardiac Purkinje fibers.

A "slow" inward current (Is) has been identified in ventricular muscle and Purkinje fibers of several mammalian species. The two-microelectrode voltage clamp technique is used to examine some of the relationships between Is and contraction of the sheep cardiac Purkinje fiber. "Tails" of inward current occurring on repolarization and extrapolation of Is recovery each show that the Is system may not inactivate completely during prolonged depolarization. The rate of recovery of Is after a depolarization is slow, and when a train of 300-ms clamps (frequency 1 s-1) is begun after a rest, Is is larger for the first clamp than it is for succeedings clamps. For the first clamp after a rest, the thresholds for Is and tension are the same and there is a direct correlation between peak tension and peak Is for clamp voltages between threshold and minus 40 mV. After a clamp, however, the ability to contract recovers much more slowly than does Is. Therefore, since Is may occur under certain conditions without tension, the realtionship between Is and tension must be indirect. Calcium entering the cell via this current may replenish or augment an intracellular calcium pool.