Production of ZFN-mediated GGTA1 knock-out pigs by microinjection of gene constructs into pronuclei of zygotes.

Animals as a source of organs and tissues for xenotransplantation could become a backup solution for the growing shortage of human donors. The presence of human xenoreactive anti- bodies directed against Galα1,3Gal antigens on the cell surface of a pig donor triggers the activa- tion of the complement leading to a hyperacute reaction. The development of genetic engineer- ing techniques has enabled the modification of genomes by knocking in and/or knocking out genes. In this paper, we report the generation of modified pigs with ZFN mediated disruption of the GGTA1 gene encoding the enzyme responsible for synthesis of Galα1,3Gal antigens. ZFN plasmids designed to target the exon 9 region of the pig GGTA1 gene encoding the catalytic domain were injected into the pronuclei of fertilized egg cells. Among 107 piglets of the F0 gene- ration analyzed, one female with 9-nt deletion in exon 9 of the GGTA1 gene was found. 13 of 33 piglets of the F1 generation represented the +/- GGTA1 genotype and 2 of 13 F2 piglets repre- sented the -/- GGTA1 genotype. No changes in the animals' behavior, phenotype or karyotype were observed. Analysis confirmed heredity of the trait in all animals. A complex functional analysis of the modified animals, including flow cytometry, human serum cytotoxicity test and immunohistochemical detection, was performed to estimate the phenotype effect of genetic modification and this indicated an efficient GGTA1 knock-out in modified pigs.

[Importance of cytogenetic analysis in patients with azoospermia or severe oligozoospermia undergoing in vitro fertilization]. / Znaczenie badan cytogenetycznych u pacjentów z azoospermia lub ciezka postacia oligozoospermii, korzystajacych z zaplodnienia in vitro.

The karyotypic analysis was performed to assess the importance of genetic factor in male infertility. For that purpose, chromosomal analysis in blood lymphocytes was performed in 28 males, candidates for ICSI with azoospermia or severe oligozoospermia and in their spouses. Although chromosomal aberrations were identified in as many as 11 couples, (in 6 couples aberrations were identified in male, in 4 other couples in female partner, whereas in 1 one couple they were detected in both partners) their risk for potential offspring is unequal. Balanced autosomal aberrations detected in two males (7%) constitute a high risk since they can cause not only infertility but also severe somatic abnormalities if transferred as the unbalanced ones to the next generation. The remaining 9 chromosomal aberrations identified in this study were present in mosaic additional cell lines with low representation. In 8 of them sex chromosomes and in 1 an autosom were involved. Although these mosaic chromosomal aberrations can lower efficiency of in vitro fertilisation, the probability that they can be transferred to the next generation causing somatic abnormalities is not high. This study indicates that in case of azoospermia or severe oligozoospermia, the karyotypic analysis should be performed in both partners prior to in vitro fertilisation.

Overexpression of Mxi1 inhibits the induction of the human ornithine decarboxylase gene by the Myc/Max protein complex.

We have previously shown that the Myc/Max protein complex plays a role in the growth-associated expression of the human ornithine decarboxylase gene. Mxi1 and Mad, novel Max-associated proteins have been identified and shown to form heterodimers with Max which bind efficiently to the Myc/Max consensus recognition sequence, CACGTG, in vitro. However, formation of Max/Mxi1 or Max/Mad heterodimers results in a reduction in Myc/Max dependent transcriptional activation of reporter plasmid constructs containing the consensus element. In light of the evidence that ODC is transcriptionally regulated in vitro and in vivo by the Myc/Max protein complex and the potential role of Mxi1 and Mad as antagonists of Myc transactivation activity, we set out to determine if one of these Max associated proteins, Mxi1, could affect the regulation of ODC expression by Myc/Max and if this regulation was correlated to growth status. Our results show that overexpression of Mxi1 does in fact inhibit ODC gene expression in a dose-dependent manner both in vivo and in vitro. In addition, evidence is presented which shows that levels of Mxi1 are up-regulated during long term quiescence and down-regulated following growth stimulation by serum. These results suggest that alterations in the levels of Max-associated proteins such as Mxi1 can modulate critical levels of functional Myc/Max protein complexes. This can alter transcriptional transactivation of Myc-regulated targets and as a consequence affect levels of genes essential for initiation and/or maintenance of growth.

Tyrosine protein kinase expression in long-term quiescent WI-38 cells following growth factor simulation.

We have used the WI-38 cell long-term quiescent model system to study the regulation of cell cycle progression at the molecular level. By modulating the length of time that WI-38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G-1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Stimulation of long- and short-term density arrested WI-38 cells with different growth factors or higher concentrations of individual growth factors does not alter the time required by long-term cells to enter S after stimulation. However, the time during the prereplicative period for which these growth factors are needed is different. Long-term quiescent WI-38 cells require EGF to traverse the G-0/G-1 border but do not need and apparently cannot respond to IGF-1 during the first 10 h after EGF stimulation, the length of the prolongation of the prereplicative phase. This suggests that EGF stimulation of long-term quiescent WI-38 cells initiates a series of molecular events which make these cells "competent" to respond to the "progression" growth factor, IGF-1. In light of the well-established role of protein tyrosine kinases in signal transduction, we set out to identify, clone, and analyze the expression of receptor and non-receptor tyrosine kinases which potentially could play a role during the prolongation of the prereplicative phase in making the long-term quiescent WI-38 cells competent to respond to IGF-1. We obtained 49 clones representing 11 different receptor and non-receptor type protein tyrosine kinases. Analysis of expression of these clones revealed a variety of different patterns of expression. However, the most striking pattern was exhibited by IGF-1 receptor. Our results suggest that induction of IGF-1 receptor mRNA by EGF may be an important event in the establishment of competence by EGF in long-term density arrested WI-38 cells.

[Changes of alpha-amylase activity in serum and urine of patients with various histological types of lung cancer]. / Zmiany aktywnosci alfa-amylazy w surowicy i moczu u chorych z róznymi typami histologicznymi raka pluca.

Serum and urinary activity of alpha-amylase was assessed in different types and clinical stages of lung cancer. Significantly increased activity was seen in adenocarcinoma and in stage III of the disease. Following surgical removal of the tumor alpha-amylase decreased on the 7th day by 19% and on the 21st day by 26.8%. The activity of urinary alpha-amylase also decreased. The authors have determined the thermostable fraction of the alpha-amylase which may imply the source of the enzyme in the serum.

Immunochemical properties of elongation factors 1 of plant origin.

Elongation factors 1 (EF-1) have been isolated from different plants: wheat, yellow lupine, blue lupine, Chinese cabbage and Norway maple. Antibodies for EF-1 from yellow lupine have been obtained in rabbits; antibodies for wheat EF-1 were elicited in mice. The immunological properties of EF-1 were assayed by the following methods: western blotting, double immunodiffusion and rocket immunoelectrophoresis. Our results suggest that one antigenic site is similar for all plant elongation binding factors tested. This epitope probably overlaps the centre of biological activity of EF-1, as was shown for wheat EF-1. The hypothesis concerning the potential presence of plant EF-1 as a subunit of turnip yellow mosaic virus RNA replicase (similar to prokaryotic EF-Tu in the Q beta RNA replicase system) has also been tested using immunotechniques as well as tests of biological activity, but has not been confirmed.

Effects of light deprivation on RNA synthesis, accumulation of guanosine 3'(2')-diphosphate 5'-diphosphate, and protein synthesis in heat-shocked Synechococcus sp. strain PCC 6301, a cyanobacterium.

The rate of total RNA synthesis, the extent of guanosine 3'(2')-diphosphate 5'-diphosphate (ppGpp) accumulation, and the pattern of protein synthesis were studied in light-deprived and heat-shocked Synechococcus sp. strain PCC 6301 cells. There was an inverse correlation between the rate of total RNA synthesis and the pool of ppGpp, except immediately after a temperature shift up, when a parallel increase in the rate of RNA synthesis and accumulation of ppGpp was observed. The inverse correlation between RNA synthesis and ppGpp accumulation was more pronounced when cells were grown in the dark. Heat shock treatment (47 degrees C) had an unexpected effect on ppGpp accumulation; there was a fairly stable level of ppGpp under heat shock conditions, which coincided with a stable steady-state rate of RNA synthesis even in the dark. We found that the pattern of dark-specific proteins was altered in response to heat shock. The transient synthesis of several dark-specific proteins was abolished by an elevated temperature (47 degrees C) in the dark; moreover, the main heat shock proteins were synthesized even in the dark. This phenomenon might be of aid in the study of cyanobacterial gene expression.

Effect of heat shock on protein synthesis in the cyanobacterium Synechococcus sp. strain PCC 6301.

The response to heat shock at 47 degrees C was examined in the cyanobacterium (blue-green alga) Synechococcus sp. strain PCC 6301. On heat shock, the growth of the cells decreased and they preferentially synthesized a limited number of polypeptides. The rate of synthesis of these proteins increased markedly in the early period of temperature shift up and gradually decreased afterwards. Among the proteins greatly affected by temperature shift up were those with apparent molecular weights of 91,000 (91K), 79K, 78K, 74K, 65K, 64K, 61K, 49K, 45K, 24K, 22K, 18K, 16K, 14K, 12K, and 11.4K, based on their mobilities in sodium dodecyl sulfate-polyacrylamide gels. From these initial studies on Synechococcus sp. strain PCC 6301 we conclude that in cyanobacteria a heat shock response similar to that known to occur in other eucaryotes and procaryotes might exist.