Jun kinase delays caspase-9 activation by interaction with the apoptosome

Activation of c-Jun N-terminal kinase 1/2 (JNK) can delay oxidant-induced cell death, but the mechanism is unknown. We found that oxidant stress of cardiac myocytes activated both JNK and mitochondria-dependent apoptosis and that expression of JNK inhibitory mutants accelerated multiple steps in this pathway, including the cleavage and activation of caspases-3 and -9 and DNA internucleosomal cleavage, without affecting the rate of cytochrome c release; JNK inhibition also increased caspase-3 and -9 cleavage in a cell-free system. On activation by GSNO or H(2)O(2), JNK formed a stable association with oligomeric Apaf-1 in a approximately 1.4-2.0 mDa pre-apoptosome complex. Formation of this complex could be triggered by addition of cytochrome c and ATP to the cell-free cytosol. JNK inhibition abrogated JNK-Apaf-1 association and accelerated the association of procaspase-9 and Apaf-1 in both intact cells and cell-free extracts. We conclude that oxidant-activated JNK associates with Apaf-1 and cytochrome c in a catalytically inactive complex. We propose that this interaction delays formation of the active apoptosome, promoting cell survival during short bursts of oxidative stress.     

Endometrial thickness, Caucasian ethnicity, and age predict clinical pregnancy following fresh blastocyst embryo transfer: a retrospective cohort

Background  

In-vitro fertilization (IVF) with blastocyst as opposed to cleavage stage embryos has been advocated to improve success rates. Limited information exists on which to predict which patients undergoing blastocyst embryo transfer (BET) will achieve pregnancy. This study's objective was to evaluate the predictive value of patient and cycle characteristics for clinical pregnancy following fresh BET.     

Benefits to host sea anemones from ammonia contributions of resident anemonefish

Large ectosymbionts (especially fishes and crustaceans) may have major impacts on the physiology of host cnidarians (sea anemones and corals), but these effects have not been well quantified. Here we describe impacts on giant sea anemone hosts (Entacmaea quadricolor) and their endosymbiotic zooxanthellae (Symbiodinium spp.) from the excretion products of anemonefish guests (Amphiprion bicinctus) under laboratory conditions. Starved host anemones were maintained with anemonefish, ammonia supplements (= NH₃ gas and NH₄⁺ ion), or neither for 2 mo. In the presence of external ammonia supplements or resident anemonefish, the zooxanthellae within host anemones increased in abundance (173% and 139% respectively), and provided the hosts with energy that minimized host body size loss. In contrast, anemones cultured with neither ammonia nor anemonefish harbored significantly lower abundances of zooxanthellae (84% of initial abundance) and decreased > 60% in body size. Although they maintained higher zooxanthella abundances, anemones cultured with either ammonia supplements or resident anemonefish exhibited significantly lower ammonia uptake rates (0.065 ± 0.005 µmol g^- 1 h^- 1, and 0.052 ± 0.018 µmol g^- 1 h^- 1 respectively) than did control anemones (0.119 ± 0.009 µmol g^- 1 h^- 1), indicating that their zooxanthellae were more nitrogen sufficient. We conclude that, in this multi-level mutualism, ammonia supplements provide essentially the same level of physiological contribution to host anemones and zooxanthellae as do live resident fish. This nutrient supplement reduces the dependence of the zooxanthellae on host feeding, and allows them to provide abundant photosynthetically-produced energy to the host.     

GnRH antagonist enhance follicular growth in FSH-treated sheep but affect developmental competence of oocytes collected by ovum pick-up

This study evaluates the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from sheep treated with GnRH antagonists (GnRHa) and high doses of FSH. Eighteen Sarda ewes were treated with progestagen sponges (day 0). On day 7, 10 ewes received 3 mg of GnRHa s.c., while 8 served as control receiving saline. On day 10, all animals were treated with 96 IU of ovine FSH in four equal doses given i.m. every 12 h. We monitored follicular development by ultrasonography, twice daily from day 7 to 11, and found that GnRHa induced a significant increase in the number of total follicles in 72 h (11.7+/-0.9 to 21+/-2.4, r(2)=0.598, P<0.0001), while this number remained stable in control sheep. We found that FSH induced a significant rise in the number of follicles in both groups; but always higher (P<0.05) in GnRHa treated sheep, confirming that GnRHa enhances ovarian response to exogenous FSH stimulation. Twelve hours after the last FSH dose, oocytes were collected by OPU. Recovery percentage, morphological quality, ability to resume meiosis, fertilization and cleavage were similar in oocytes from treated and untreated sheep. However, the final blastocysts output was lower in GnRHa group (10.1% versus 27.4% in control group; P<0.05). In addition, re-expansion rates after vitrification, thawing and in vitro culture were lower in GnRHa treated ewes, although differences did not reach statistical significance (55.5% versus 74.1% in GnRHa treated and in control sheep, respectively).     

Detection of more than 50 different CFTR mutations in a large group of German cystic fibrosis patients

We have conducted a comprehensive study of the molecular basis of cystic fibrosis (CF) in 350 German CF patients. A screening approach based on single-strand conformation analysis and direct sequencing of genomic polymerase chain reaction products has allowed us to detect the molecular defects on 95.4% of the CF chromosomes within the coding region and splice sites of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The spectrum of sequence changes comprises 54 different mutations, including 17 missense mutations, 14 nonsense mutations, 11 frameshift mutations, 10 splice site variants and two amino acid deletions. Eleven of these mutations have not previously been described. Our results reflect the marked mutational heterogeneity of CF in a large sample of patients from a non-isolated population.     

Effects of LH administration at the end of an FSH superovulatory regimen on ovulation rate and embryo production in three breeds of sheep

In 3 experiments, 168 ewes of Manchega (n = 72), Churra (n = 62), and Merina (n = 34) breeds were used to test the hypothesis that administration of pure LH, coincident with progestogen removal during superovulation with FSH, causes an increase in the ovulation rate and number of embryos. This administration of LH can further interact with genotype, resulting in breed differential response. In each experiment, the animals were randomly assigned to 1 of 3 treatments. Estrus in all sheep was synchronized with intravaginal sponges of 30 mg of FGA for 12 d, then 270 microg of FSH were administered in 6 injections at 12-h intervals in decreasing doses, starting 48 h before sponge removal. The FSH/LH ratio of the original preparation was 3, and remained constant throughout the treatment in the control group (C). In Treatment 1, (T1) and Treatment 2, (T2), pure LH was administered coincident with progestogen removal-5th FSH injection, and with the 6th FSH injection, at 2 dose levels: 60 and 120 microg, (T1), and 120 and 240 microg (T2). Mating occurred 36 and 48 h after the progestogen removal, and the embryos were surgically collected and morphologically evaluated on Days 7 and 8 after sponge withdrawal. Overall, the results showed that LH administration at the end of the FSH treatment did not increase the ovulation rate and number of embryos in Merino (5.9 +/- 1.4 and 5.6 +/- 1.4, respectively, T1; 7.0 +/- 1.0 and 5.7 +/- 1.2, T 2; 4.9 +/- 1.1 and 2.6 +/- 0.7, C), Churra (6.8 +/-1.4 and 5.2 +/- 1.4, T1; 8.1 +/- 1.5 and 6.3 +/- 1.4, T2; 6.1 +/- 1.5 and 5.4 +/- 1.3, C) and Manchega (6.0 +/- 1.0 and 4.4 +/- 1.0, T1; 5.0 +/- 0.8 and 4.2 +/- 0.8, T2; 4.8 +/- 1.5 and 3.8 +/- 1.0, C). Administration of LH induced a significant (P < 0.05) increase in the frequency of multiple ovulations (72.3 +/- 4.3 %, T1; 74.1 +/- 11.5 %, T2; 55.6 +/- 5.9 %, C) paralleled to a decrease in the occurrence of ewes with no ovulations (8.7 +/- 2.6 %, T1; 7.6 +/- 4.6 % T2; 17.3 +/- 3.2 %, C) or 1 to 2 ovulations (18.7 +/- 4.6 %, T1; 18.1 +/- 7.5 %, T2; 26.8 +/- 5.8 % C), regardless of breed or dose of LH. No increase in the mean number of viable embryos was observed, probably due to both the high individual variability and the lower fertilization rates observed in sheep showing multiple ovulations.     

Effect of immunization on reproducvive performance, embryo quality and progesterone in Rasa Aragonesa ewes actively immunized against androstenedione or passively immunized against testosterone

A total of 217 Rasa Aragonesa ewes were used to test two immunization treatments: 1.Active immunization against androstenedione: ewes immunized in previous matings (androstenedione, reimmunized; AR groups, n=58) or not (first immunization; AF groups n=64) were boosted either 2 or 4 wk before mating. 2.Passive immunization against testosterone: antisera were injected either at sponge withdrawal (zero time; T0 group, n=21) or 1 wk previously (Tl group, n=22). We used 52 ewes as controls (C group). Half of each group was used either to record reproductive performance or to embryo viability assessment. Prolificacy was significantly increased in ewes which reached a moderate antibody level, independently of the treatment. Fertility was lower in AR ewes that attained a high antibody titre (P<0.01). The percentage of viable embryos recovered was lower in AF ewes (P<0.01), and in ewes whose testosterone antibody titre was high (P<0.05), compared to C group. It was proven that similar or lower antibody levels were more harmful for ewes from AF and Tl than for ewes from AR or T0 groups. The proportion of nonfertilized recovered ova was not significant. Progesterone levels were notably increased in AR ewes (P<0.001) independently of ovulation rate and were positively correlated to antibody titre at mating (P<0.01) but these events were not observed in T ewes. These findings indicate that after androgen immunoneutralization, only those ewes having antibody titres within a limited range at mating had improved reproductive performance. Further research is needed in order to understand the role that progesterone plays in immunized ewes.     

A termination mutation (2143delT) in the CFTR gene of German cystic fibrosis patients

German patients with cystic fibrosis (CF) were screened for molecular lesions in exon 13 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by single strand conformation polymorphism (SSCP) and chemical cleavage of mismatch analyses. Direct sequencing of four samples that displayed the same SSCP pattern and that were susceptible to cleavage of heteroduplexes by osmium tetroxide revealed, in all cases, a deletion of a single T residue at nucleotide position 2143 within codon 671 of the CFTR gene. As a result, leucine codon 671 is changed into a termination codon. In total, the 2143delT mutation was confirmed in 6 out of 271 German non-F508 CF chromosomes by artificial restriction fragment length polymorphism analysis, indicating that this frameshift mutation accounts for about 2% of German non-508 mutations. The 6 pancreas insufficient patients who are compound heterozygous for 2143-delT suffer from the typical features of pulmonary and gastrointestinal CF disease. The 2143delT mutation completes the panel of the more frequent CFTR mutations that reside on the F508 haplotype and that contribute to its overpresentation among German non-F508 alleles that are associated with severe forms of disease.