Two levels of resting potential in cardiac purkinje fibers

In an appropriate ionic environment, the resting potential of canine cardiac purkinje fibers may have either of two value. By changing the external K concentration, [K](0), in small steps, it was shown that, in the low (1 mM) Cl, Na-containing solutions used in this study, the two levels of resting potential could be obtained only within a narrow range of [K](0) values; that range was usually found between 1 and 4 mM. Within the critical [K](0) range the resting potential could be shifted from either level to the other by the application of small current pulses. It was shown that under these conditions the steady-state current- voltage relationship was “N-shaped,” and that a region of both negative slope, and negative chord conductance lay between the two stable zero-current potentials. The negative chord conductance was largely due to inward sodium current, only part of which was sensitive to tetrodotoxin (TTX). Under appropriate conditions, the negative chord conductance could be abolished by several experimental interventions and the membrane potential thereby shifted from the lower to the higher resting level: those interventions which were effective by presumably diminishing the steady-state inward current included reducing the external sodium concentration, adding TTX, or adding lidocaine; those which presumably increased the steady-state outward current included small increases in [K](0), brief depolarizations to around -20 mV, or the addition of acetylcholine chloride.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Factors influencing heifer survival and fertility on commercial dairy farms

The average dairy cow survives only three lactations, reducing the availability of replacement heifers. Prenatal losses occur due to early embryonic mortality (about 40%), later embryo loss (up to 20% in high-yielding herds) or abortion (about 5%). A recent survey of 19 UK herds showed that 7.9% of calves were born dead and 3.4% died within 1 month. During the rearing phase, 6.7% of animals were lost before reaching first service at 15 months due to disease or accident and another 2.3% failed to conceive. Many potential replacements therefore never enter the milking herd. This severely limits opportunities for on-farm selection of breeding cows in addition to presenting a welfare issue and causing economic loss. The most profitable animals once lactation is reached combine good milk production with a regular calving pattern. Some aspects of performance are related to age at first calving (AFC), which in turn is influenced by heifer growth rates. Poorly growing animals required more services to conceive, calved later and subsequently performed badly. Optimum fertility and maximum yield in the first lactation were associated with an AFC of 24 to 25 months. However, heifers calving at 22 to 23 months performed best in terms of total milk yield and survival over the first 5 years, partly because good heifer fertility was associated with better fertility later. We have investigated some possible juvenile predictors of future performance. Low-birth-weight calves were more likely to come from either primiparous mothers or older dams (3+ lactations) with higher peak milk yields, suggesting that the uterine environment may limit prenatal calf growth due to competition for nutrients with maternal growth or milk production. Linear trait classification scores for frame size show genetic correlations with longevity. The skeletal measures of height and crown rump length in 1-month-old calves was correlated to subsequent stature, and frame size was correlated to weight at 15 months. It may thus be possible to predict performance from simple size measurements as juveniles. Neither endogenous nor stimulated growth hormone (GH) release in 6-month-old calves were related to milk yield in the first three lactations, but size of a stimulated GH peak was positively related to milk energy values in the first lactation. Cows with delayed ovulation (>45 days) in the first lactation had a higher GH pulse amplitude and lower IGF-I as a juvenile. Cows that partition excess energy into milk in their first lactation may suffer reduced longevity.     

Relationships between transition period diet,metabolic parameters and fertility in lactating dairy cows

Cows in severe negative energy balance after calving have reduced fertility, mediated by metabolic signals influencing the reproductive system. We hypothesised that transition diet could alter metabolic status after calving, and thus influence fertility. Multiparous dairy cows were assigned to four transition groups 6 weeks pre-calving and fed: (a) basal control diet (n = 10); (b) basal diet plus barley (STARCH, n = 10); (c) basal diet plus Soypass (high protein, HiPROT, n = 11); or (d) no transition management (NoTRANS, n = 9). All cows received the same lactational diet. Blood samples, body weights and condition scores (BCS) were collected weekly. Fertility parameters were monitored using milk progesterone profiles and were not affected by transition diet. Data from all cows were then combined and analysed according to the pattern of post-partum ovarian activity. Cows with low progesterone profiles had significantly lower insulin-like growth factor-I (IGF-I) and insulin concentrations accompanied by reduced dry matter intakes (DMIs), BCS and body weight. Cows with prolonged luteal activity (PLA) were older and tended to have lower IGF-I. Analysis based on the calving to conception interval revealed that cows which failed to conceive (9/40) also had reduced IGF-I, BCS and body weight. Fertility was, therefore, decreased in cows which were in poor metabolic status following calving. This was reflected in reduced circulating IGF-I concentrations and compromised both ovarian activity and conception. There was little effect of the transition diets on these parameters.     

Real-time PCR quantitation of glucocorticoid receptor alpha isoform

Background  

The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision.     

Do pigs find a familiar odourant attractive in novel surroundings?

Sixty-four DurocxLandrace pigs (Sus scrofa) of both sexes were reared from birth to 6 weeks of age in either fresh air or an atmosphere containing an artificial odourant (Ambi-Pur). Their behavioural responses to a novel environment (a modified open field) were then observed over 1h when the test arena contained either the familiar Ambi-Pur odourant or ammonia gas at 45ppm. Although members of other mammalian and avian species are attracted to familiar odours in otherwise novel surroundings, the presence of Ambi-Pur had no effect on the pigs' readiness to enter the novel arena from a sheltered area or on other fear-related behaviours following entry (p>0.05). Pigs kept in fresh air from weaning until test were generally less active and spent less time at the centre of the open field if the test was carried out in an ammoniated atmosphere rather than fresh air; this suggests that they found ammonia aversive. On the other hand, the presence of ammonia at test accelerated approach to the centre of the open field and increased the number of entries into that area and the percentage of time spent there as well as overall activity in pigs that had also been exposed chronically to this gas at 43ppm from weaning until testing at 6 weeks (p<0.05). Thus, after chronic exposure to either ammonia gas or an artificial odourant, ammonia was the only stimulus that appeared to act as an attractive agent for pigs placed in an otherwise novel environment.     

Uterine lymphocyte distribution and interleukin expression during early pregnancy in cows

Both the production of cytokines and the distribution of immune cells within the uterus change during early pregnancy. Evidence obtained mainly from mice indicates that these changes are important for implantation and in preventing a maternal immune response to the conceptus. The ruminant embryo also produces interferon tau at this time, the signal for the maternal recognition of pregnancy. The relationship between these events in cows was studied using uteri from three groups of animals on day 16 after observed oestrus: (i) cyclic controls, (ii) pregnant and (iii) inseminated but with no embryo present. Embryo size and the antiviral activity in uterine flushings (indicative of the interferon tau concentration) were measured. Sections of intact uterus were frozen for the localization and quantitation of CD4(+) (T lymphocytes), CD14(+) (macrophages) and CD21(+) (B lymphocytes) uterine cells by immunohistochemistry. The expression of interleukin (IL)-1alpha, IL-2, IL-6 and IL-10 mRNAs in uterine extracts was measured by RT-PCR. Neither embryo size, interferon tau concentration nor pregnancy status influenced the distribution of CD4(+), CD14(+) or CD21(+) cells in the day 16 uterus. Endometrial IL-1alpha mRNA was detected in most cows across the groups, whereas IL-2 mRNA was only present in the non-pregnant uterus. IL-6 and IL-10 mRNAs were not detectable in any uteri. In conclusion, IL-2 mRNA expression is detectable in the non-pregnant but not the pregnant uterus on day 16 and interferon t is unlikely to play a role in the redistribution of immune cells in the uterus during early bovine pregnancy.     

Influence of negative energy balance on cyclicity and fertility in the high producing dairy cow

The peripartum period is of critical importance to subsequent health and fertility. Most cows enter a state of negative energy balance (NEB) associated with many metabolic changes which have carry over effects on the resumption and normality of estrous cyclicity and the success of subsequent inseminations. A dataset on 500 lactations explored the relationships between metabolic traits measured before and after calving with fertility. Stepwise multiple regression analysis showed that longer calving to conception intervals were associated with altered profiles of IGF-I, urea and body condition score. These relationships between metabolic profiles and fertility differed between first lactation cows (which are still growing but produce less milk) and mature animals. Early postpartum the liver undergoes extensive biochemical and morphological modifications to adapt to NEB, the uterus is extensively remodeled and must clear bacterial infections, and the ovary must resume ovulatory cycles. RNA isolated from liver and uterine tissues harvested 2 weeks postpartum from cows in mild (MNEB) and severe (SNEB) energy balance was used to screen the Affymetrix 23K bovine microarray. In liver, SNEB resulted in differential expression of key genes involved in lipid catabolism, gluconeogenesis, and the synthesis and stability of IGF-I. This was accompanied by reduced systemic concentrations of IGF-I which is likely to impact on ovarian function and early embryo development. Within endometrium, cows in SNEB showed histological evidence for higher levels of inflammation and the microarray analysis identified groups of differentially expressed genes involved in tissue remodeling and immune response. This may delay uterine repair after calving, likely contributing to the observed reduction in fertility.     

Identification of relaxin in the placenta of the ewe

Sheep placentomes were collected at the abattoir and the stage of gestation was estimated from the crown-rump length and appearance of the fetus. Samples were extracted and either freeze dried (crude extracts) or fractionated on Sephadex G-50 and CM-cellulose. Relaxin immunoreactivity (RXN-IR) was detected in all samples by a pig relaxin RIA and diluted in parallel with the standard curve. Two patterns of RXN-IR were seen after Sephadex G50 purification: (a) a single main peak of RXN-IR eluting at a position similar to pig relaxin; or (b) a 3-peak pattern with additional higher (void volume) and lower (approximately 1000) molecular weight peaks. These peaks were all found with 4 different and specific antisera. The 6000 molecular weight peak eluted at a similar position to pig relaxin on CM cellulose and inhibited electrically stimulated rat uterine contractions in vitro. The amount of relaxin measured in crude extracts of placentomes from different ewes was very variable. Most samples were within the range 0.05-11.2 ng/g wet weight of tissue (3.0 +/- 0.45 (s.e.m.), n = 44) but a few contained much higher concentrations (25.5-61.4 ng/g, n = 3). There was no obvious variation in concentration with stage of pregnancy (20 days to term). Samples of intercotyledonary endometrium, allantochorion and whole ovaries from pregnant ewes were also extracted. All contained low concentrations of RXN-IR (0.6 +/- 0.13 ng/g, n = 4; 0.6 +/- 0.29 ng/g, n = 3; 1.0 +/- 0.66 ng/g, n = 7, respectively). We conclude that relaxin-like peptides are present in the pregnant ewe and that, as the placentomes are the largest component by weight, they represent the major source.     

Effect of oxytocin infusion on secretion of progesterone and luteinizing hormone and the concentration of uterine oxytocin receptors during the periovulatory period in cloprostenol-treated ewes.

Oxytocin infusions were initiated on day 10 of the oestrous cycle in ewes, and luteal regression was induced by injection of 100 micrograms cloprostenol on day 12. Blood samples were collected at frequent intervals via an indwelling jugular vein cannula to measure concentrations of progesterone and luteinizing hormone (LH) during the luteal and follicular phases in saline (n = 6) and oxytocin (n = 5) infused animals. The oxytocin infusion maintained peripheral plasma concentrations of 53 +/- 3.2 pg oxytocin ml-1 (mean +/- SEM) compared with values of about 1 pg ml-1 during oestrus in control ewes. Oxytocin infusion had no effect on luteal phase progesterone concentrations, the timing of luteolysis, basal luteinizing hormone (LH) secretion, LH pulse frequency, or the timing or height of the LH surge. Treated ewes came into oestrus significantly earlier than controls (P < 0.05) but ovulated normally. Uterine samples collected 96 h after cloprostenol injection (approximately day 2 of the cycle) showed that oxytocin receptor concentrations were significantly higher in the endometrium in ewes that had been given a 5 day oxytocin infusion than in control animals (556 and 262 fmol mg-1 protein, respectively: geometric means from ANOVA, P < 0.001), whereas myometrial receptor concentrations were not affected (113 and 162 fmol mg-1 protein, respectively). We conclude that the previously reported delay in luteal development caused by oxytocin infusion (Wathes et al., 1991) is not due to the inhibition or delay of ovulation, but must instead occur via a direct influence on the developing corpus luteum.(ABSTRACT TRUNCATED AT 250 WORDS)