Recovery Rate and Quality of Embryos from Mares Inseminated at the First Post-Partum Oestrus

The pregnancy rate is lower in mares inseminated at the first post-partum (p.p.) oestrus (40-50%) compared with pregnancy rates in subsequent oestrous cycles (55-65%). The causes of the lowered pregnancy rate are not fully understood. The aim of the present study was to examine if embryonic defects could be one of the reasons for lowered pregnancy rate. A total of 23 p.p. and 14 non-lactating control mares were flushed 7 days after detection of ovulation. Embryo recovery rate was 48% and 71% in p.p. and control mares, respectively (p=0.16). Embryos were photographed, measured, graded and stained with fluorescein diacetate to assess their viability. Thereafter embryos were bisected and stained with Hoechst 33342 to count the cell nuclei. Embryos in both groups were equally viable and the cell numbers were not significantly different. According to morphological evaluation all embryos were classified as excellent or good. Embryos aged 7.3 to 7.6 days (± 0.25 days) were smaller in the p.p. group than in the control group (p<0.05). Forty-seven (9/19) and 8% (1/13) of the uterine swabs, taken before the first insemination, yielded bacteria and neutrophils in p.p. and control mares, respectively. The amount of neutrophils and/or bacteria had no statistically significant effect on embryo recovery rate (p>0.10). Recovery of embryos was not related to histological findings in uterine biopsies taken after embryo recovery. Embryo recovery rate in p.p. mares (48%) was similar to previously reported foal heat pregnancy rates (40- 50%). Hence, early embryonic death in utero would not be the most likely reason for lowered pregnancy rate in mares inseminated at the first p.p. oestrus. Sperm transport and oviductal conditions by the time of the first p.p. oestrus would need to be studied to clarify the role of fertilisation failure as the cause of lower pregnancy rate in mares inseminated at foal heat.     

The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

The "Goldilocks Effect" in Cystic Fibrosis: identification of a lung phenotype in the <Emphasis Type="Italic">cftr</Emphasis> knockout and heterozygous mouse

Background  

Cystic Fibrosis is a pleiotropic disease in humans with primary morbidity and mortality associated with a lung disease phenotype. However, knockout in the mouse of cftr, the gene whose mutant alleles are responsible for cystic fibrosis, has previously failed to produce a readily, quantifiable lung phenotype.     

Ryanodine receptors, a family of intracellular calcium ion channels, are expressed throughout early vertebrate development

Background

We developed a new version of the open source software package Peptrix that can yet compare large numbers of Orbitrap? LC-MS data. The peptide profiling results for Peptrix on MS1 spectra were compared with those obtained from a small selection of open source and commercial software packages: msInspect, Sieve? and Progenesis?. The properties compared in these packages were speed, total number of detected masses, redundancy of masses, reproducibility in numbers and CV of intensity, overlap of masses, and differences in peptide peak intensities. Reproducibility measurements were taken for the different MS1 software applications by measuring in triplicate a complex peptide mixture of immunoglobulin on the Orbitrap? mass spectrometer. Values of peptide masses detected from the high intensity peaks of the MS1 spectra by peptide profiling were verified with values of the MS2 fragmented and sequenced masses that resulted in protein identifications with a significant score.

Findings

Peptrix finds about the same number of peptide features as the other packages, but peptide masses are in some cases approximately 5 to 10 times less redundant present in the peptide profile matrix. The Peptrix profile matrix displays the largest overlap when comparing the number of masses in a pair between two software applications. The overlap of peptide masses between software packages of low intensity peaks in the spectra is remarkably low with about 50% of the detected masses in the individual packages. Peptrix does not differ from the other packages in detecting 96% of the masses that relate to highly abundant sequenced proteins. MS1 peak intensities vary between the applications in a non linear way as they are not processed using the same method.

Conclusions

Peptrix is capable of peptide profiling using Orbitrap? files and finding differential expressed peptides in body fluid and tissue samples. The number of peptide masses detected in Orbitrap? files can be increased by using more MS1 peptide profiling applications, including Peptrix, since it appears from the comparison of Peptrix with the other applications that all software packages have likely a high false negative rate of low intensity peptide peaks (missing peptides).     

Inflammatory responses to induced infectious endometritis in mares resistant or susceptible to persistent endometritis

Background

Canine hemangiosarcoma (HSA) is a malignant tumor with poor long-term prognosis due to development of metastasis despite aggressive treatment. The phosphatidyl-inositol-3 kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is involved in its endothelial pathologies; however, it remains unknown how this pathway plays a role in canine HSA. Here, we characterized new canine HSA cell lines derived from nude mice-xenografted canine HSAs and investigated the deregulation of the signaling pathways in these cell lines.

Results

Seven canine HSA cell lines were established from 3 xenograft canine HSAs and showed characteristics of endothelial cells (ECs), that is, uptake of acetylated low-density lipoprotein and expression of canine-specific CD31 mRNA. They showed varied morphologies and mRNA expression levels for VEGF-A, bFGF, HGF, IGF-I, EGF, PDGF-B, and their receptors. Cell proliferation was stimulated by these growth factors and fetal bovine serum (FBS) in 1 cell line and by FBS alone in 3 cell lines. However, cell proliferation was not stimulated by growth factors and FBS in the remaining 3 cell lines. Phosphorylated p44/42 Erk1/2 was increased by FBS stimulation in 4 cell lines. In contrast, phosphorylation of Akt at Ser⁴⁷³, mTOR complex 1 (mTORC1) at Ser²⁴⁴⁸, and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) at Ser⁶⁵ was high in serum-starved condition and not altered by FBS stimulation in 6 cell lines, despite increased phosphorylation of these residues in normal canine ECs. This suggested that the mTORC2/Akt/4E-BP1 pathway was constitutively activated in these 6 canine HSA cell lines. After cell inoculation into nude mice, canine HSA tumors were formed from 4 cell lines and showed Akt and 4E-BP1 phosphorylation identical to the parental cell lines.

Conclusions

Our findings suggest that the present cell lines may be useful tools for investigating the role of the mTORC2/Akt/4E-BP1 pathway in canine HSA formation both in vivo and in vitro.     

Ornithine Decarboxylase Activity in Brain Regulated by a Specific Macromolecul, the Antizyme

Mouse brain ornithine decarboxylase activity is about 70-fold higher at the time of birth compared with that of adult mice. Enzyme activity declines rapidly after birth and reaches the adult level by 3 weeks. Immunoreactive enzyme concentration parallels very closely the decrease of enzyme activity during the first postnatal week, remaining constant thereafter. The content of brain antizyme, the macromolecular inhibitor to ornithine decarboxylase, in turn is very low during the first 7 days and starts then to increase and at the age of 3 weeks it is about six times the level of that in newborn mice. This may explain the decrease in enzyme activity during brain maturation, and suggests the regulation of polyamine biosynthesis by an antizyme-mediated mechanism in adult brain.     

Automated genome mining for natural products

Background  

Discovery of new medicinal agents from natural sources has largely been an adventitious process based on screening of plant and microbial extracts combined with bioassay-guided identification and natural product structure elucidation. Increasingly rapid and more cost-effective genome sequencing technologies coupled with advanced computational power have converged to transform this trend toward a more rational and predictive pursuit.     

Increased activation of blood neutrophils after cigarette smoking in young individuals susceptible to COPD

Background

Cigarette smoking is the most important risk factor for Chronic Obstructive Pulmonary Disease (COPD). Only a subgroup of smokers develops COPD and it is unclear why these individuals are more susceptible to the detrimental effects of cigarette smoking. The risk to develop COPD is known to be higher in individuals with familial aggregation of COPD. This study aimed to investigate if acute systemic and local immune responses to cigarette smoke differentiate between individuals susceptible or non-susceptible to develop COPD, both at young (18-40 years) and old (40-75 years) age.

Methods

All participants smoked three cigarettes in one hour. Changes in inflammatory markers in peripheral blood (at 0 and 3 hours) and in bronchial biopsies (at 0 and 24 hours) were investigated. Acute effects of smoking were analyzed within and between susceptible and non-susceptible individuals, and by multiple regression analysis.

Results

Young susceptible individuals showed significantly higher increases in the expression of FcγRII (CD32) in its active forms (A17 and A27) on neutrophils after smoking (p = 0.016 and 0.028 respectively), independently of age, smoking status and expression of the respective markers at baseline. Smoking had no significant effect on mediators in blood or inflammatory cell counts in bronchial biopsies. In the old group, acute effects of smoking were comparable between healthy controls and COPD patients.

Conclusions

We show for the first time that COPD susceptibility at young age associates with an increased systemic innate immune response to cigarette smoking. This suggests a role of systemic inflammation in the early induction phase of COPD.

Trial registration

Clinicaltrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT00807469","term_id":"NCT00807469"}}NCT00807469

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0121-2) contains supplementary material, which is available to authorized users.     

Peroxiredoxin I and II in Human Eyes: Cellular Distribution and Association with Pterygium and DNA Damage

Peroxiredoxin I and II are both 2-Cys members of the peroxiredoxin family of antioxidant enzymes and inactivate hydrogen peroxide. On western blotting, both enzymes appeared as 22-kD proteins and were present in the sclera, retina and iris. Immunohistochemistry showed strong cytoplasmic labeling in the basal cells of the corneal epithelial layer and the corneoscleral limbus. The melanocytes within the stroma of the iris and the anterior epithelial cells of the lens also showed strong cytoplasmic labeling. The fibrous structure of the stroma and the posterior surface of the ciliary body were also labeled. There was also strong labeling for both enzymes in the photoreceptors and the inner and outer plexiform layers of the retina. There was increased labeling of peroxiredoxin I and II in pterygium. In normal conjunctiva and cornea, only the basal cell layer showed labeling for peroxiredoxin I and II, whereas, in pterygia, there was strong cytoplasmic labeling in most cells involving the full thickness of the epithelium. Co-localization of the DNA oxidation product 8-hydroxy-2’-deoxyguanosine antibody with the nuclear dye 4’,6’-diamidino-2-phenylindole dihydrochloride indicated that the majority of the oxidative damage was cytoplasmic; this suggested that the mitochondrial DNA was most affected by the UV radiation in this condition.     

Viability of bovine demi- and quarter-embryos after transfer

The viability of bovine demi- and quarter-embryos was investigated. Early compacting morulae were nonsurgically flushed from superovulated donor cows and were bisected by two microneedles. One of the halves was then split further into two quarters. Each demi- and quarter-embryo was placed in an evacuated zona pellucida. One demi- or two quarter-embryos were transferred non-surgically into cow or heifer recipients. Viability was measured by ultrasound scanning of the fetuses on Days 35, 48 and 60 of pregnancy. The pregnancy rates at Day 60 were 46.2% (6 13 ) for heifers and 33.3% (4 12 ) for cows after the transfer of a single demi-embryo. The transfer of two quarter-embryos resulted in a pregnancy rate of 61.5% (8 13 ) for heifers and 8.3% (1 12 ) for cows. Seven (53.8%) and four (33.3%) live fetuses were found on Day 60 following the transfer of demi-embryos into heifers and cows, respectively. The transfer of quarter-embryos resulted in 10 fetuses (38.5%) in the heifer recipients and only one fetus (4.2%) in the cow recipients. The results of this study suggest that heifers are more suitable than cows as recipients for quarter-embryos.