Homologous prostatic fluid added to frozen-thawed dog spermatozoa prior to intravaginal insemination of bitches resulted in better fertility than albumin-free TALP

The aim of this study was to determine whether canine prostatic fluid has intrinsic effects resulting in higher fertility than albumin-free Tyrode's albumin lactate pyruvate (afTALP) when added to thawed semen prior to intravaginal insemination. Twenty-four German shepherd bitches were inseminated intravaginally with frozen-thawed spermatozoa to which either homologous prostatic fluid (Group P; 12 bitches) or afTALP (Group T; 12 bitches) was added to give a final insemination volume of 7mL. Each bitch was inseminated daily starting when the vaginal folds first became angular and continuing until the day before a diestrus vaginal smear was first seen. Bitches were spayed about 3 weeks after the onset of diestrus and the number of corpora lutea and the number of conceptuses counted. Group P and Group T bitches were, respectively, inseminated 5.3+/-1.0 and 5.8+/-2.1 times with 48.9+/-8.6 and 50.4+/-8.3 million progressively motile spermatozoa per insemination. Eight Group P bitches and 10 Group T bitches conceived with totals of 76 and 45 conceptuses and 126 and 117 corpora lutea, respectively. Odds of conception were taken as the number of conceptuses divided by (the number of corpora lutea minus the number of conceptuses). After adjustment for the number of progressively motile spermatozoa per day and the random effect of bitch, the addition of prostatic fluid resulted in an increased odds of conception compared to afTALP. This effect decreased as the number of progressively motile spermatozoa per day increased.     

Characterization of follistatin-type domains and their contribution to myostatin and activin a antagonism

Follistatin (FST)-type proteins are important antagonists of some members of the large TGF-β family of cytokines. These include myostatin, an important negative regulator of muscle growth, and the closely related activin A, which is involved in many physiological functions, including maintenance of a normal reproductive axis. FST-type proteins, including FST and FST-like 3 (FSTL3), differentially inhibit various TGF-β family ligands by binding each ligand with two FST-type molecules. In this study, we sought to examine features that are important for ligand antagonism by FST-type proteins. Previous work has shown that a modified construct consisting of the FST N-terminal domain (ND) followed by two repeating follistatin domains (FSD), herein called FST ND-FSD1-FSD1, exhibits strong specificity for myostatin over activin A. Using cell-based assays, we show that FST ND-FSD1-FSD1 is unique in its specificity for myostatin as compared with similar constructs containing domains from FSTL3 and that the ND is critical to its activity. Furthermore, we demonstrate that FSD3 of FST provides affinity to ligand inhibition and confers resistance to perturbations in the ND and FSD2, likely through the interaction of FSD3 of one FST molecule with the ND of the other FST molecule. Additionally, our data suggest that this contact provides cooperativity to ligand antagonism. Cross-linking studies show that this interaction also potentiates formation of 1:2 ligand-FST complexes, whereas lack of FSD3 allows formation of 1:1 complexes. Altogether, these studies support that domain differences generate FST-type molecules that are each uniquely suited ligand antagonists.     

Effect of calf removal on serum luteinizing hormone and cortisol concentrations in postpartum beef cows

Serum luteinizing hormone (LH) and cortisol concentrations were measured in ten fall calving, Angus cows averaging 38 +/- 8 days postpartum. Calves from five cows were weaned at the beginning of the study. Blood samples were collected at 20 min. intervals for 48 h after weaning and for 8 h on day 4 and day 6 postweaning. Mean serum LH concentrations increased (P<0.01) in weaned cows (W) from 0.55 +/- 0.01 ng/ml at time of calf removal to 1.3 +/- 0.04 ng/ml 48 h afterwards. Comparable LH concentrations for suckled cows (S) were 0.65 +/- 0.08 ng/ml and 0.62 +/- 0.03 ng/ml respectively. Average serum LH concentrations at 48 h after weaning were greater (P<0.01) for W cows than S cows and a treatment by time interaction occurred (P<0.01) with serum LH concentrations increasing (P<0.01) from time of calf removal to 48 h after calf removal in W cows. Frequency of LH peaks increased (P<0.01) in W cows and by 48 h after weaning was greater (P<0.01) in W cows than in S cows. Magnitude of LH peaks did not differ between the two groups. Serum cortisol concentrations were not different between W and S cows except for a transient elevation (P<0.01) in W cows from 7.6 +/- 0.9 ng/ml to 11.9 +/- 1.0 ng/ml 9 to 12 h after calf removal. Since serum LH concentrations were increased in W cows but not in S cows at 48 h and serum cortisol concentrations increased transiently in W cows we suggest that circulating cortisol levels may not be a physiological inhibitor of LH secretion in the suckled postpartum beef cow.     

Exome Sequencing Identifies Rare Deleterious Mutations in DNA Repair Genes FANCC and BLM as Potential Breast Cancer Susceptibility Alleles

Despite intensive efforts using linkage and candidate gene approaches, the genetic etiology for the majority of families with a multi-generational breast cancer predisposition is unknown. In this study, we used whole-exome sequencing of thirty-three individuals from 15 breast cancer families to identify potential predisposing genes. Our analysis identified families with heterozygous, deleterious mutations in the DNA repair genes FANCC and BLM, which are responsible for the autosomal recessive disorders Fanconi Anemia and Bloom syndrome. In total, screening of all exons in these genes in 438 breast cancer families identified three with truncating mutations in FANCC and two with truncating mutations in BLM. Additional screening of FANCC mutation hotspot exons identified one pathogenic mutation among an additional 957 breast cancer families. Importantly, none of the deleterious mutations were identified among 464 healthy controls and are not reported in the 1,000 Genomes data. Given the rarity of Fanconi Anemia and Bloom syndrome disorders among Caucasian populations, the finding of multiple deleterious mutations in these critical DNA repair genes among high-risk breast cancer families is intriguing and suggestive of a predisposing role. Our data demonstrate the utility of intra-family exome-sequencing approaches to uncover cancer predisposition genes, but highlight the major challenge of definitively validating candidates where the incidence of sporadic disease is high, germline mutations are not fully penetrant, and individual predisposition genes may only account for a tiny proportion of breast cancer families.     

Evaluation of laboratory indexes of absorption of soil phosphorus by plants: II

Summary A comparison was made of several laboratory methods for estimating the yield of phosphorus in plants grown in greenhouse cultures on samples of 22 soils from different parts of the United States. The methods investigated and their rank in order of increasing precision of the estimates of the yield of phosphorus were as follows: extraction with lactic acid, calcium lactate buffer solution extraction with 2 per cent citric acid solution < extraction with 0.03N NH₄F, 0.025N HCl solution < percentage phosphorus saturation (found as follows: 100 × labile phosphorus by isotopic dilution/ phosphorus adsorption capacity according to Langmuir adsorption equation) labile phosphorus by isotopic dilution phosphorus extracted by water.Journal Paper No. J-3747 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project No. 1183. Contribution from the Department of Agronomy.     

Characteristics of a membrane-associated lipoxygenase in tomato fruit

Microsomal membranes isolated from the pericarp of maturegreen tomato (Lycopersicon esculentum) fruit rapidly metabolize exogenous radiolabeled linoleic acid into fatty acid oxidation products at 22°C. The reaction is strongly inhibited by n-propyl gallate, an inhibitor of lipoxygenase. The membranes also rapidly metabolize 16:0/18:2^* phosphatidylcholine into radiolabeled oxidation products that comigrate on TLC plates with those formed from free linoleic acid. At 30°C, the formation of fatty acid oxidation products from 16:0/18:2^* phosphatidylcholine is slower, and there is an initial accumulation of radiolabeled linoleic acid that is not evident at 22°C, which can be attributed to the action of lipolytic acyl hydrolase. Radiolabeled phosphatidic acid and diacylglycerol are also formed during metabolism of 16:0/18:2^* phosphatidylcholine by the microsomal membranes, and there is no breakdown of either linoleic acid or phosphatidylcholine by heat-denatured membranes. When Triton X-100 treated membranes were used, the same patterns of metabolite formation from radiolabeled linoleic acid and 16:0/18:2^* phosphatidylcholine were observed. Thus, the enzymes mediating the breakdown of these radiolabeled compounds appear to be tightly associated with the membranes. Collectively, the data indicate that there is a lipoxygenase associated with microsomal membranes from tomato fruit that utilizes free fatty acid substrate released from phospholipids. The microsomal lipoxygenase is strongly active over a pH range of 4.5 to 8.0, comprises approximately 38% of the total (microsomal plus soluble) lipoxygenase activity in the tissue, has an apparent K_m of 0.52 millimolar and an apparent V_max of 0.186 millimoles per minute per milligram of protein. The membranous enzyme also cross-reacts with polyclonal antibodies raised against soybean lipoxygenase-1 and has an apparent molecular mass of 100 kilodaltons.     

Preliminary X-ray crystallographic and NMR studies on the exonuclease domain of the epsilon subunit of Escherichia coli DNA polymerase III

The structured core of the N-terminal 3'-5' exonuclease domain of epsilon, the proofreading subunit of Escherichia coli DNA polymerase III, was defined by multidimensional NMR experiments with uniformly (15)N-labeled protein: it comprises residues between Ile-4 and Gln-181. A 185-residue fragment, termed epsilon(1-185), was crystallized by the hanging drop vapor diffusion method in the presence of thymidine-5'-monophosphate, a product inhibitor, and Mn(2+) at pH 5.8. The crystals are tetragonal, with typical dimensions 0.2 mm x 0.2 mm x 1.0 mm, grow over about 2 weeks at 4 degrees C, and diffract X-rays to 2.0 A. The space group was determined to be P4(n)2(1)2 (n = 0, 1, 2, 3), with unit cell dimensions a = 60.8 A, c = 111.4 A.