Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells

There are growing numbers of recombinant proteins that have been expressed in milk. Thus one can consider the placement of any gene of interest under the control of the regulatory elements of a milk protein gene in a dairy farm animal. Among the transgene introducing techniques, only nuclear transfer (NT) allows 100?% efficiency and bypasses the mosaicism associated with counterpart techniques. In this study, in an attempt to produce a transgenic goat carrying the human coagulation factor IX (hFIX) transgene, goat fetal fibroblasts were electroporated with a linearized marker-free construct in which the transgene was juxtaposed to ??-casein promoter designed to secret the recombinant protein in goat milk. Two different lines of transfected cells were used as donors for NT to enucleated oocytes. Two transgenic goats were liveborn. DNA sequencing of the corresponding transgene locus confirmed authenticity of the cloning procedure and the complementary experiments on the whey demonstrated expression of human factor IX in the milk of transgenic goats. In conclusion, our study has provided the groundwork for a prosperous and promising approach for large-scale production and therapeutic application of hFIX expressed in transgenic goats.     

Pregnancy-associated glycoprotein concentrations during pregnancy and the postpartum period in Azawak Zebu cattle

Specific RIA systems were developed and used to measure pregnancy-associated glycoprotein (PAG) concentrations during gestation and the postpartum period in Azawak Zebu cows. Twelve females were palpated per rectum and diagnosed as pregnant. Blood samples were taken at 5-10-day intervals from approximately Week 8 of gestation until Week 10 postpartum (pp). One Zebu cow (Z15) initially diagnosed as pregnant showed PAG concentrations lower than the assay sensitivity (<0.20 ng/ml) and did not calve. Another cow (ZSand) showed abnormally high PAG concentrations during gestation and was excluded from the general PAG profile. The 10 other Zebu cows exhibited a very similar PAG profile. In these animals, concentrations increased progressively from Week 8 to 35 of gestation (from 6.0+/-4.2 to 196.0+/-34.8 ng/ml), remaining relatively constant until Week 39 (210.8+/-74.8 ng/ml), when they increased sharply to reach their highest level (1095.6+/-607.2 ng/ml) at around parturition. After delivery, PAG concentrations declined significantly (P<0.05) until Week 2 postpartum (348.4+/-85.6 ng/ml) and slowly until Week 10 postpartum. Our results revealed that the PAG pattern in Zebu cattle was similar to those of taurine breeds during the first two trimesters of pregnancy, but differed in the peripartum period.     

Schistosoma mansoni: cell-specific expression and secretion of a serine protease during development of cercariae.

Eukaryotic serine proteases are an important family of enzymes whose functions include fertilization, tissue degradation by neutrophils, and host invasion by parasites. To avoid damaging the cells or organisms that produced them, serine proteases must be tightly regulated and sequestered. This study elucidates how the parasitic blood fluke Schistosoma mansoni synthesizes, stores, and releases a serine protease during differentiation of its invasive larvae. In situ hybridization with a cDNA probe localized the protease mRNA to acetabular cells, the first morphologically distinguishable parasite cells that differentiate from the embryonic cell masses present in the intermediate host snail. The acetabular cells contained vimentin but not cytokeratins, consistent with a mesenchymal, not epithelial, origin. Antiprotease antibodies, localized by immunoperoxidase, showed that the protease progressively accumulated in these cells and was packaged in vesicles of three morphologic types. Extension of cytoplasmic processes containing protease vesicles formed "ducts" which reached the anterior end of fully differentiated larvae. During invasion of human skin, groups of intact vesicles were released through the acetabular cytoplasmic processes and ruptured within the host tissue. Ruptured protease vesicles were noted adjacent to degraded epidermal cells and dermal-epidermal basement membrane, as well as along the surface of the penetrating larvae themselves. These observations are consistent with the proposed dual role for the enzyme in facilitating invasion of host skin by larvae and helping to release the larval surface glycocalyx during metamorphosis to the next stage of the parasite.     

Induction of multipotency in umbilical cord-derived mesenchymal stem cells cultivated under suspension conditions

Due to the limitations in the clinical application of embryonic stem cells (ESC) and induced pluripotent stem cells, mesenchymal stem cells (MSCs) are now much more interesting for cell-based therapy. Although MSCs have several advantages, they are not capable of differentiating to all three embryonic layers (three germ layers) without cultivation under specific induction media. Hence, improvement of MSCs for cell therapy purposes is under intensive study now. In this study, we isolated MSCs from umbilical cord tissue at the single-cell level, by treatment with trypsin, followed by cultivation under suspension conditions to form a colony. These colonies were trypsin resistant, capable of self-renewal differentiation to the three germ layers without any induction, and they were somewhat similar to ESC colonies. The cells were able to grow in both adherent and suspension culture conditions, expressed both the MSCs markers, especially CD105, and the multipotency markers, i.e., SSEA-3, and had a limited lifespan. The cells were expanded under simple culture conditions at the single-cell level and were homogenous. Further and complementary studies are required to understand how trypsin-tolerant mesenchymal stem cells are established. However, our study suggested non-embryonic resources for future cell-based therapy.     

Triploidy induction in the Caspian salmon, Salmo trutta caspius, by heat shock

Induction of triploidy was attempted in the Caspian salmon, Salmo trutta caspius, using heat shocks. The optimal temperature level (26, 28 and 30°C), initiation time [5, 10, 20 and 40 min post-fertilization (PF)] and duration of thermal shock (5, 10 and 20 min) required for effective induction of triploidy were investigated. Incidence of triploid fry was determined by surface and volume measurements of erythrocytes as well as from flow-cytometric analysis of some blood samples. Survival from fertilization to swim-up, triploid rates and triploid yields were in the range of 0–70%, 0–97% and 0–57%, respectively. The highest triploid yield was obtained with a shock treatment at 26°C for 10 min duration initiated 40 min PF.     

High-coordinated lanthanum(III) complexes with new mono- and bidentate phosphoryl donors; spectroscopic and structural aspects

Monodentate and bidentate ligands PhNHP(O)(NC₄H₈O)₂ (1) and PhC(O)NHP(O)(NH(tert-C₄H₉))₂ (2) were used to prepare new 7, 9 and 10-coordinated lanthanum(III) complexes; La(1)₂Cl₃(H₂O)₂ (3), La(1)₂(NO₃)₃H₂O.La(1)₂(NO₃)₃CH₃CN (4) and La(2)₂(NO₃)₃ (5), respectively. Crystallization of compound 2 in CH₃OH:CH₃CN leads to one conformer in contrast to the crystallization result from CHCl₃:n-C₇H₁₆ (two conformers). Compound 4 contains two independent nine-coordinated La(III) complexes that are different in the solvated molecules (H₂O and CH₃CN). Some structural and electronic perturbations in coordinated ligand were occurred upon complexation, that are confirmed by increase of ²J_PH, ³J_PH and ⁶J_PH coupling constants from the free ligand 1 to complexes 3 and 4. The steric repulsions in the first coordination sphere of La³⁺ ion, metal-ligand (M-L) binding strength and PO stretching frequency are very influenced by changing the counter ion from Cl⁻ to . Comparing the X-ray crystallography data of free ligand 2 with bis-chelated complex 5, it is found that the phosphoryl group is more reactive than carbonyl counterpart. A blue shift of the ν(N-H) vibration is observed in line with the weakening of the hydrogen bond from N-H···O_Phosphoryl in 1 to N-H···Cl in 3. Three dimensional butterfly-shape structures are seen in the unit cell of complex 3, which are produced by O_Water-H···O_Morpholine hydrogen bonds.     

Simulation of DNA bases in water: Comparison of the Monte Carlo algorithm with molecular mechanics force fields

The interaction between the nucleic acid bases and solvent molecules has an important effect in various biochemical processes. We have calculated total energy and free energy of the solvation of DNA bases in water by Monte Carlo simulation. Adenine, guanine, cytosine, and thymine were first optimized in the gas phase and then placed in a cubic box of water. We have used the TIP3 model for water and OPLS for the nucleic acid bases. The canonical (T, V, N) ensemble at 25 degrees C and Metropolis sampling technique have been used. Good agreement with other available computational data was obtained. Radial distribution functions of water around each site of adenine, guanine, cytosine, and thymine have been computed and the results have shown the ability of the sites for hydrogen bonding and other interactions. The computations have shown that guanine has the highest value of solvation free energy and N7 and N6 in adenine and guanine, N3 in cytosine, and N3 and O4 in thymine have the largest radial distribution function. Monte Carlo simulation has also been performed using the CHARMM program under the same conditions, and the results of two procedures are compared.     

Comparison of accuracy of transabdominal ultrasonography, progesterone and pregnancy-associated glycoproteins tests for discrimination between single and multiple pregnancy in sheep

The aim of the present study was to evaluate and, compare the accuracy of transabdominal ultrasonographic (US) and the progesterone (P4-RIA) and ovine pregnancy-associated glycoprotein (ovPAG-RIA) tests for the discrimination between single and multiple pregnancy in sheep. One hundred pregnant AwassixMerino ewes were scanned by transabdominal ultrasonography (3.5 MHz linear-array transducer) at Days 43-56 and 81 of these ewes were scanned at Days 76-87 of gestation. The ewes were scanned in dorsal recumbency at the bare area of the inguinal regions (without pre-scanning shaving of the ventral abdominal wall). After each scan, blood samples were withdrawn from the jugular vein to estimate the levels of P4 and ovPAG by radioimmunoassay. At lambing, 61 ewes gave birth to single lambs and 39 ewes gave birth to multiples. The sensitivity of the transabdominal US, the P4-RIA and the ovPAG-RIA tests for determining ewes carrying multiples was 54, 64.1 and 64.1% at Days 43-56. At Days 76-87 of gestation these accuracies were 60.0, 66.7 and 76.6% for the US, P4-RIA and PAG-RIA tests, respectively. The specificity of the transabdominal US, the P4-RIA and the ovPAG-RIA tests for determining ewes carrying singles, was 78.6, 60.7 and 62.3% at Days 43-56 and 78.4, 64.7 and 70.6% at Days 76-87 of gestation, respectively. It is concluded that the accuracy of transabdominal ultrasonographic (without pre-scanning shaving of the ventral abdominal wall), the P4- and the ovPAG-RIA tests for determination of the fetal numbers in AwassixMerino crossbred ewes is too low to be used in the field.     

Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mammalian cells

The mammalian mitochondrial inner membrane fusion protein OPA1 is controlled by complex patterns of alternative splicing and proteolysis. A subset of OPA1 isoforms is constitutively cleaved by YME1L. Other isoforms are not cleaved by YME1L, but they are cleaved when mitochondria lose membrane potential or adenosine triphosphate. In this study, we show that this inducible cleavage is mediated by a zinc metalloprotease called OMA1. We find that OMA1 small interfering RNA inhibits inducible cleavage, helps retain fusion competence, and slows the onset of apoptosis, showing that OMA1 controls OPA1 cleavage and function. We also find that OMA1 is normally cleaved from 60 to 40 kD by another as of yet unidentified protease. Loss of membrane potential causes 60-kD protein to accumulate, suggesting that OMA1 is attenuated by proteolytic degradation. We conclude that a proteolytic cascade controls OPA1. Inducible cleavage provides a mechanism for quality control because proteolytic inactivation of OPA1 promotes selective removal of defective mitochondrial fragments by preventing their fusion with the mitochondrial network.