Molecular characterization of the porcine TYK2 gene on SSC 2q1.3-->q2.1.

TYK2 is a member of the janus protein kinase family and plays an important role in the signal transduction of various cytokines including interferon alpha/beta. Cloning and characterization of the porcine TYK2 gene revealed a conserved organization with respect to other mammalian TYK2 orthologs. The porcine gene consists of 25 exons spanning approximately 26 kb and encoding a 5.3-kb mRNA. It is located in a GC-rich and gene-rich chromosome region and contains several CpG islands. The predicted 132-kDa TYK2 protein consists of 1,184 amino acids and shows 85% identity to the human TYK2 protein. The porcine TYK2 gene was localized by FISH and RH-mapping on SSC 2q1.3-->q2.1, which is in good agreement with established human-mouse-pig comparative maps.

Expression and characterization of functional recombinant bovine follicle-stimulating hormone (boFSHalpha/beta) produced in the milk of transgenic rabbits.

Bovine follicle-stimulating hormone (boFSH) is a heterodimeric glycoprotein that belongs to the pituitary gonadotropins. Bioactive FSH is composed of alpha and beta subunits which require extensive N-glycosylation and sialylation. The mammary gland of transgenic livestock is an attractive source for the synthesis of post-translationally modified proteins. Two mammary gland-specific gene constructs with the cDNA for the boFSH alpha (boFSHalpha) and beta (boFSHbeta) subunits controlled by bovine alpha-s1 casein regulatory sequences were co-microinjected into fertilized rabbit oocytes. Two FSHalpha/FSHbeta double transgenic rabbit lines were established. The transgene expression was strictly lactation and mammary gland specific. Protein analysis revealed the presence of the boFSH heterodimer in the milk of transgenic rabbits showing a molecular weight similar to that of purified pituitary gland derived boFSH (boFSH-P). Subunit specific antibodies detected both polypeptides with the expected molecular sizes. Biochemical characterization demonstrated the expected isoelectric points of the recombinant boFSH. The presence of the post-translationally added terminal sialic acid residues was indicated by wheat germ agglutinin (WGA) lectin Western blotting. The biological activity of the recombinant mammary gland produced boFSH was determined using a FSH-dependent reporter cell line. The bioactivity of the recombinant boFSH was comparable to that of purified boFSH-P.

Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts.

The efficiency of nuclear transfer (NT) using two primary cultures of fetal fibroblasts (FF1 and FF2) was compared vs. the same cultures transfected with an expression vector in which the bovine prochymosin coding sequence is placed under the control of the bovine alpha(S1)-casein promoter (TFF1 and TFF2). In addition, fibroblasts of a cloned transgenic fetus (TRFF1) derived from TFF1 and ear skin fibroblasts of a 1-month-old cloned transgenic calf (TRCF1) derived from TRFF1 were used as nuclear donors. Embryos reconstructed from FF1 (44%) and FF2 (52%) developed to the blastocyst stage at a significantly (P < 0.05) higher rate than those derived from TFF1 (24%) and TFF2 (27%). The proportions of cleaved embryos and blastocysts were significantly (P < 0.05) higher with TRFF1 than with TRCF1 used as nuclear donors (75 vs. 66% and 33 vs. 16%, respectively). Transfer of NT embryos derived from FF2 and TFF2 to recipients resulted in similar pregnancy rates on day 30 (52 and 48%, respectively). However, with TFF2 embryos, the majority of pregnancies (8/11; 73%) was lost in the first and second trimesters of gestation, whereas 4/11 (36%) pregnancies with FF2 embryos were lost during the full period of in vivo development. Of 11 FF2 and 6 TFF2 born calves (25 and 13% of transferred embryos, respectively), 6 and 3 survived including one oversized FF2 calf. After transfer of TRFF1 and TRCF1 NT embryos to recipients, initial pregnancy rate was as a tendency higher in the TRFF1 (49%) than in the TRCF1 group (30%). The majority (14/17) of TRFF1 pregnancies and all TRCF1 pregnancies were lost in the first and second trimester. A high proportion of TRFF1 calves (5/8) showed increased body weights, and only two calves which were also large survived. These findings demonstrate that (i) extended culture associated with transfection and selection procedures may induce changes of donor cells which markedly decrease the efficiency of nuclear transfer and (ii) these changes are not reversed by recloning.

Partial impairment of cytokine responses in Tyk2-deficient mice.

To assess the role of the Janus kinase (Jak) family member Tyk2, we have generated Tyk2-/- mice. In contrast to other Jaks, where inactivation leads to a complete loss of the respective cytokine receptor signal, Tyk2-/- mice display reduced responses to IFNalpha/beta and IL-12 and a selective deficiency in Stat3 activation in these pathways. Unexpectedly, IFNgamma signaling is also impaired in Tyk2-/- mice. Tyk2-/- macrophages fail to produce nitric oxide upon lipopolysaccharide induction. Tyk2-/- mice are unable to clear vaccinia virus and show a reduced T cell response after LCMV challenge. These data imply a selective contribution of Tyk2 to the signals triggered by various biological stimuli and cytokine receptors.

Chimeric pigs following blastocyst injection of transgenic porcine primordial germ cells.

Porcine primordial germ cell (PGC) derived cell lines of WAPhGH-transgenic pigs have been established that were able to contribute to chimeras. PGCs were isolated from day 25 to 28 genital ridges of more than 30 individual transgenic fetuses in order to have an easy to follow marker gene. To support undifferentiated growth, cell lines were derived and stable maintained on STO no. 8 feeder cells, a murine embryonic fibroblast cell line expressing recombinant, membrane-bound porcine stem cell factor (SCF). Fifteen lines proliferated in an undifferentiated state up to passage 13; two lines were maintained for more than 23 passages. Cell staining experiments for differentiation markers in several cell lines, indicated the presence of pluripotent cells in prolonged cultures. Further characterization using karyotyping revealed a normal, euploid set of chromosomes in cells of passages 15 and higher. Pluripotency of freshly isolated, short-term (up to 24 hr before injection) and long-term cultured, frozen/thawed cells was tested by injection into day 6 recipient blastocysts to give rise to chimeric piglets. The injected embryos (n = 209) were endoscopically transferred into the uterine horns of 11 recipient gilts. Tissue analysis from 49 fetuses and eighteen liveborn piglets for PGC contribution in chimeras was carried out using PCR analysis for the presence of the marker transgene. Thirty-two fetuses showed detectable chimerism in up to five out of 12 tissues analyzed. Skin samples from eight piglets were positive for the transgene, four of them displayed coat colour chimerism.

Stable production of human insulin-like growth factor 1 (IGF-1) in the milk of hemi- and homozygous transgenic rabbits over several generations.

One transgenic rabbit line was generated carrying a fusion gene consisting of the cDNA for human IGF-1 fused to a mammary gland specific expression cassette derived from bovine alpha-S1-casein sequences. Transgene expression was shown to be strictly tissue and lactation period specific. The transgenic rabbit line was bred for six generations. All transgenic animals showed stable production of biologically active IGF-1 over the generations and no apparent effect on the physiological or reproductive performance was observed. The absence of adverse effects on homozygous transgenic rabbits suggested the absence of insertional mutagenesis. Eight hemizygous transgenic offspring analysed produced on average 363 +/- 12 micrograms/ml (ranging from 223 +/- 61 to 484 +/- 39 micrograms/ml) mature human IGF-1 in their milk, whereas three homozygous animals produced on average 543 +/- 41 micrograms/ml (ranging from 360 +/- 15 to 678 +/- 80 micrograms/ml). Homozygous hulGF-1 females clearly showed a significantly increased production performance of the recombinant protein.

[The application of microwave energy in preparation of herbal drugs. 2]. / Der Einsatz von Mikrowellenenergie zur Aufbereitung von Arzneidrogen. 2. Mitteilung.

On the basis of various plant organs the relation between the used microwave energy and the drying processes, temperatures of the surface and the interior of the drying materials as well as the remaining moisture in the drugs was investigated. Leaf, flower and herb drugs showed a similar drying behaviour. Plant material of high humidity, e.g. meaty fruits or roots, should be dried by means of pulsed microwave application to avoid too high temperatures. In almost all investigated drugs microwave drying resulted in a lower remaining moisture than air drying. The effect of microwave irradiation on carbohydrates, fats and proteins in plant materials was preliminarily investigated.

[The application of microwave energy in preparing medicinal plant drugs. 1]. / Der Einsatz von Mikrowellenenergie zur Aufbereitung von Arzneidrogen. 1. Mitteilung.

Using a specially constructed microwave device, containing a steady-state-broadcasting magnetron that is infinitely adjustable within a range of 100 to 1200 W, the applicability of microwave energy for drying medicinal plants was investigated. When optimising the drying parameters (e.g. microwave power, time of irradiation, mass of plant material, thickness of layer) most of the obtained leave, flower, herb and root drugs showed a significant better appearance (colour) than the air dried drugs.

Phylogenetic evidence for the relationship between the genera Mobiluncus and Actinomyces.

Partial reverse transcriptase sequencing of 16S rRNA from Mobiluncus curtisii and M. mulieris clearly indicate that the genus Mobiluncus is not a member of the Bacteroidaceae but belongs to the order Actinomycetales. The highest degree of relationship is found with the genus Actinomyces which is supported by the presence of common physiological properties.