Expression of the human milk protein beta-casein in transgenic potato plants.

A 1177 bp cDNA fragment encoding the human milk protein beta-casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase (mas1',2') promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human beta-casein cDNA. The presence of human beta-casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human beta-casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human beta-casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human beta-casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as beta-casein. The above experiments demonstrate the expression of human milk beta-casein as part of an edible food plant. These findings open the way for reconstitution of human milk in edible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children.

Ethanol-induced signal transducing mechanism associated with a transient antiviral state in human amniotic cells.

Short-term effects of ethanol on human amnion cells were investigated by studying the cellular signaling processes and the replication of vesicular stomatitis virus. Treatment of human amniotic cells with ethanol transiently triggers the breakdown of inositol phospholipids, stimulates intracellular [Ca2+]i mobilization and activates the translocation of protein kinase C. Activation of this signal transduction mechanism is associated with the development of an antiviral state, as proven by studying 3H-uridine incorporation into the RNA of vesicular stomatitis virus. Induction of the antiviral state in human amniotic cells correlates with the solubility of the alcohols in the lipid membrane of the cells.

Oxytocin stimulates translocation of protein kinase C and induces antiviral state in human amnion cells.

Association of protein kinase C (PKC) activity to the membrane fraction was observed in oxytocin treated human amnion cells (UAC). In addition, oxytocin was shown to induce an antiviral state and to inhibit multiplication of vesicular stomatitis virus (VSV) in UAC. These observations together with earlier findings indicate that activation of inositol phospholipid breakdown with a consecutive activation of PKC is a common signal transduction pathway in interferon action and hormonal stimulation.

Protein phosphorylation and kinase activities in tumour cells after hyperthermia.

Phosphorylation of various proteins and the activities of specific kinases were studied in tumour cells after hyperthermia. P388 lymphoid tumour cells were treated at 40-45 degrees C for 1 h in vitro. Immediately after heat treatment, particulate and cytosol cell fractions were isolated, phosphorylated proteins separated and various kinase activities were measured. Hyperthermic treatment of the cells caused a significant decrease in protein kinase C activity while the activity of calcium-ion and phospholipid-independent protein kinases increased. Phosphorylation of cytosol proteins of 120, 80, 33, 25 and 14 kDa increased significantly after hyperthermia, and protein kinase C selectively phosphorylated the last three of these proteins. The phosphorylation of three heat shock proteins (44, 70 and 85 kDa) was not changed after hyperthermic treatment. Four tyrosine kinase activities were separated. The protein tyrosine kinase activity decreased to one-tenth of the control value after 45 degrees C for 1 h hyperthermia. The changes in kinase activities and protein phosphorylation induced by hyperthermia proved to be temperature- and time-dependent.

Ultrastructural changes of human amniotic cells induced by natural human interferon-alpha.

Human interferon-alpha (Hu-IFN alpha) and phorbol myristate acetate (PMA), a direct activator of protein kinase C (PK-C), induce the translocation of protein kinase C from the cytosol to the membrane fraction. By the use of transmission (TEM) and scanning (SEM) electron microscopy we have shown that treatment of human amniotic cells (UAC) with Hu-IFN alpha resulted in profound changes in the shape, volume and ultrastructure of the cells. Most treated cells had enlarged nuclei with marginal condensation of chromatin. Nucleolar segregation, disintegration and clumping of nucleolar components were also observed. The number of interdigitating cell processes decreased and the cell surface microvilli became shortened. Similar ultrastructural alterations were induced by PMA also. All these functional and morphological data strongly support the hypothesis that protein kinase C is a key factor in IFN-mediated cell reactions.

Phospholipase C and phospholipase A2 are involved in the antiviral activity of human interferon-alpha.

Treatment of human amniotic cells (UAC) with human interferon-alpha (Hu-IFN alpha) or phorbol myristate acetate (PMA) resulted in translocation of protein kinase C (PK-C) activity from the cytosol fraction to that of the membranes. Analysis of 32P incorporation into phospholipid fractions and studies of alterations in fatty acid content for the major phospholipids of IFN-treated cells suggest that phospholipases C and A2 are activated by Hu-IFN alpha. Addition of neomycin (an inhibitor of phospholipase C), as well as mepacrine (an inhibitor of phospholipase A2) to IFN-treated cells inhibited the antiviral activity of Hu-IFN alpha in the vesicular stomatitis virus (VSV)-UAC system used. These observations indicate that (i) activation of PK-C and (ii) diacylglycerol formation, arachidonic acid and/or lysophosphatidylcholine release are important steps in the mechanism of action of IFN.

Interferon (IFN)-like antiviral effect is induced by unspecific cross-linking of cell surface receptors.

Treatment of human amniotic cells (UAC) with Cytodex 1 (DEAE-dextran) results in the development of an antiviral state of the cells, as proven by studying (i) the cytopathic effect and (ii) [3H]uridine incorporation into the RNA of vesicular stomatitis virus (VSV) after VSV infection. The same treatment transiently triggers the breakdown of inositol phospholipids and activates the translocation of protein kinase C (PKC). On the basis of these data it can be suggested that cross-linking of cell surface receptors by a solid carrier bearing covalently bound positive charges may result in IFN-like effects.

Inverse correlation between growth and degrading enzyme activity of seedlings after gamma and neutron irradiation of pea seeds.

The effect of gamma-, 14 MeV neutron- and fission neutron irradiation was investigated on the growth rate and degrading enzyme activities of pea seedlings. Both dormant pea seeds and 4-day-old growing seedlings were used for the experiments. Depending on the gamma dose between 15 and 300 Gy the height of pea seedlings was found shorter, and parallel with this the endogenous RNase and peroxidase activities were higher than in the unirradiated controls. Seedlings proved to be more sensitive by about one order of magnitude than seeds. Irradiation of seeds between 5 and 10 Gy slightly enhanced the growth rate of seedlings (10 per cent) and parallel with this, the RNase activity measured was lower than that in the controls. On irradiation of seedlings with 14 MeV neutrons the growth inhibition and RNase activity enhancement was only 1.3 times more effective than in the case of irradiation of seeds. The following RBE values were obtained after irradiation of seeds, related to the biological effect of gamma rays: in growth inhibition, 6 for 14 MeV neutrons and 12 for fission neutrons, and the enhancement of two enzyme activities was 15-30 for 14 MeV neutrons and 45-58 for fission neutrons. In the case of seedling irradiation with 14 MeV neutrons the RBE was 1.0 for growth inhibition and between 3 and 6 for enhancement of enzyme activity. The isoenzyme pattern of RNase also changed: two isoenzymes became predominant after the gamma irradiation of seeds, characterized by molecular weights of 21,000 and 30,000, respectively. As a result of enhanced RNase activity, the degradation of longer polysomes to monomeric ribosomes occurred. Thus after ionizing irradiation of pea seeds and seedlings an inverse correlation was found between the growth rate of pea seedlings and the activities of degrading enzymes.

Inositol phospholipid turnover and protein kinase C translocation are stimulated by poly(I).poly(C) in human amnion cells (UAC).

Polyinosinic-polycytidylic acid, a potent inducer of inducer of interferon (IFN) production and activator of some IFN-induced enzymes, inhibits [3H]uridine incorporation into the RNA of vesicular stomatitis virus even in the absence of IFN synthesis, transiently triggers the breakdown of inositol phospholipids and activates the translocation of protein kinase C. Since IFNs also have similar activities these results suggest that IFN induction and IFN function are realised through common biochemical pathways.

Sequence composition of the template-active fraction of rat liver chromatin.

Rat liver chromatin has been separated into nuclease-sensitive and -resistant fractions after mild digestion with DNAase II. The nuclease-sensitive material is further fractionated into Mg2+ -soluble and -insoluble chromatin fractions. The kinetics of production of these chromatin fractions have been investigated. After a brief enzyme treatment (5 min at 10 enzyme units/A260 unit of chromatin at pH 6.6), 11% of the input chromatin DNA is found in the Mg2+ -soluble fraction. This DNA has a weight-average single-strand length of about 400 nucleotides and, as determined by renaturation kinetics, comprises a subset of nonrepetitive DNA sequences and a subset of families of middle repetitive sequences. This demonstrates the nonrandom distribution of repetitive and single copy sequences in the Mg2+ -soluble fraction of chromatin. Previous studies have shown that the Mg2+ -soluble fraction is enriched in nonrepeated sequences which are transcribed in vivo (Gottesfeld, J.M., Garrard, W.T., Bagi, G., Wilson, R.F., and Bonner, J. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 2193-2197). We now report that the Mg2+ -soluble fraction of liver chromatin contains a low proportion of sequences in common with the Mg2+ -soluble fraction of brain chromatin. Thus, fractionation does not depend on some general property of chromatin but is specific with regard to the template activity of the tissue from which the chromatin was obtained.

Partial purification of the template-active fraction of chromatin: a preliminary report.

A fraction of rat-liver chromatin that is transcriptionally active in vivo has been purified 6- to 7-fold over whole chromatin. This was accomplished by selectively shearing chromatin with DNase II followed by fractionating the released portion on the basis of its solubility properties in 2 mM MgCl(2). The resulting soluble material comprises 11% of the total chromatin DNA and is impoverished in histone and enriched in nonhistone protein. Compared with unsheared chromatin, this minor fraction exhibits marked differences in chromosomal protein species. DNA renaturation studies indicate that this fraction is composed of a specific subset of whole genomal DNA sequences. Furthermore, DNA.RNA hybridization experiments suggest that almost 60% of the nonrepetitious DNA sequences of this minor fraction could code for cellular RNA.