The secretion of tropoelastin by chick-embryo artery cells.

Chymotryptic fingerprint analyses of tropoelastin a and tropoelastin b demonstrated a very close relationship between these two polypeptides synthesized in a cell-free system under the direction of chick-embryo polyribosomal mRNA. A similar study on tropoelastin polypeptides extracted in their hydroxylated and under-hydroxylated forms from artery cells incubated with [3H]valine in the absence and presence of alpha alpha'-bipyridine or 3,4-dehydroproline confirmed this close relationship and suggested that tropoelastins a and b are likely to be the products of a single gene. Pulse-chase experiments in which the synthesis and secretion of tropoelastin by artery cells were monitored demonstrated that, after a pulse with [3H]proline, the polypeptides rapidly appeared in the medium and the half-time of tropoelastin secretion was approx. 30 min. Further pulse-chase studies, in which [3H]tropoelastin contents of subcellular fractions were determined, showed that rough and smooth microsomal fractions contained maximal amounts of tropoelastin at different times. The quantity of tropoelastin in the smooth-microsomal fraction was always only a small proportion of that in the rough-microsomal fraction, suggesting rapid translocation of the polypeptides to the plasma membrane. Incubation of the cells with 0.1 mM-colchicine did not markedly alter the rate of secretion or the distribution of tropoelastin between the subcellular fractions, whereas when 1 microM-monensin was included in the incubations the polypeptides were retained in the rough microsomal fraction. The results are consistent with the proposal that tropoelastin may follow a pathway of secretion from rough endoplasmic reticulum to the plasma membrane via secretory vesicles.     

Interferon production in human T lymphoblastoid cells

Human T lymphoblastoid cell (RPMI 8402 cell) produced interferon (IFN) through the induction by Sendai virus. The priming effect on the interferon production in the RPMI 8402 cell could be found by the pretreatment of human leukocyte IFN (Hu IFN-alpha), but not by that of the IFN produced in the RPMI 8402 cell (T-IFN). The superinduction by the irradiation of ultraviolet rays or the treatment of antimetabolites (actinomycin D and cycloheximide) or 5-bromodeoxyuridine was not found. The T-IFN was completely neutralized by the anti-Hu IFN-beta serum, but not by the anti-Hu IFN-alpha serum at all. In conclusion, it was confirmed that the IFN produced in the RPMI 8402 cell through the induction by Sendai virus was antigenically identical to Hu IFN-beta.     

Interferon production by mammalian cells grown in a serum-free medium.

Interferon, produced by rabbit heart cells grown in a serum-free medium, failed to protect rabbit heart serum-free cells, but protected rabbit heart serum-containing-medium cells against vaccinia and vesicular stomatitis virus. Interferon produced in serum-free cells had a greater species specificity than that produced in serum-containing media. The difference in activity was shown to be due to lack of adsorption by serum-free-medium cells.     

Metabolism of p-fluorophenylalanine in p-fluorophenylalanine sensitive and resistant tobacco cell cultures

The metabolism of D- and L-p-fluorophenylalanine (PFP) in DL-PFP resistant and sensitive tobacco cell cultures (Nicotiana tabacum), cell lines TX4 and TX1, respectively, has been compared. The amino acid analogue was taken up at a lower rate by the resistant cell line TX4. Incorporation of PFP into protein was also considerably reduced in TX4 cells, compared to TX1 cells. This, however, resulted mainly from a diminished availability of PFP due to a more rapid conversion of PFP by TX4 cells. TX1 cells and TX4 cells converted PFP qualitatively in the same way. The only detectable metabolite of D-PFP was N-malonyl-D-PFP, while all metabolites of L-PFP were identified as sequent products of the initial deamination of L-PFP by the enzyme phenylalanine ammonia-lyase (PAL). As TX4 cells were endowed with higher PAL-activity than TX1 cells, the resistant cells were able to metabolize L-PFP more rapidly to give, e.g., p-fluorocinnamoyl glucose ester and p-fluorocinnamoyl putrescine. In the presence of the specific PAL-inhibitor -aminooxy--phenylpropionic acid TX4 cells were slightly more sensitive to PFP. This suggests that the better detoxification contributes to the acquired resistance. The use of PFP as specific indicator for cell lines with increased PAL-activity, and hence increased levels of phenolic compounds, is discussed.Abbreviations AOPP -aminooxy--phenylpropionic acid - MCW methanol:chloroform:water - PAL phenylalanine ammonia-lyase - PFP p-fluorophenylalanine - Phe phenylalanine     

Labelling of prolyl hydroxylase tetrameric subunits in freshly isolated chick-embryo tendon cells and in certain chick-embryo tissues in vivo.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) may be formed in the back reaction of the amino acid-activation reaction [Zamecnik, Stephenson, Janeway & Randerath (1966) Biochem. Biophys. Res. Commun. 24, 91-98]. On the basis of a number of observations of the properties of Ap4A it has been suggested that it may have a signal function for the initiation of DNA replication in eukaryotic cells] Grummt (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 371-375]. In the present paper human platelets have been shown to contain relatively large amounts of Ap4A. The compound is apparently metabolic inactive in platelets, but it is almost quantitatively released when platelets are activated to aggregate by treatment with thrombin. The results are discussed in connection with the known growth-stimulating activity of platelets.     

Evidence that p-fluorophenylalanine has a direct effect on tubulin in Aspergillus nidulans.

Three temperature-sensitive alleles of benA (benA11, 17 and 21) confer resistance to growth inhibition by p-fluorophenylalanine (FPA). FPA resistance cosegregates with the benA gene. Two back-mutations in benA which cause loss of temperature sensitivity cause loss of FPA resistance, and two indirect suppressors of benA temperature sensitivity also cause FPA resistance to be lost. These results indicate that FPA resistance is an intrinsic property of the benA mutations. The intracellular phenylalanine concentrations of these strains are normal as is their ability to take up phenylalanine from the medium. We conclude that FPA must inhibit growth and cause non-disjunction by a direct effect on the polymerization of tubulin.     

Regulation of collagen post-translational modification in transformed human and chick-embryo cells.

Changes in the regulation of collagen post-translational modification in transformed cells were studied in three established human sarcoma cell lines and in chick-embryo fibroblasts freshly transformed by Rous sarcoma virus. The collagens synthesized by all but one of these and by all the control human and chick-embryo cell lines were almost exclusively of types I and/or III. The relative rate of collagen synthesis and the amounts of prolyl hydroxylase activity and immunoreactive protein were markedly low in all the transformed human cell lines. The other enzymes studied, lysyl hydroxylase, hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase, never showed as large a decrease in activity as did prolyl hydroxylase, suggesting a more efficient regulation of the last enzyme than of the three others. The chick-embryo fibroblasts freshly transformed by Rous sarcoma virus differed from the human sarcoma cells in that prolyl hydroxylase activity was distinctly increased, whereas the decreases in immunoreactive prolyl hydroxylase protein and the three other enzyme activities were very similar to those in the simian-virus-40-transformed human fibroblasts. It seems possible that this increased prolyl hydroxylase activity is only a temporary phenomenon occurring shortly after the transformation, and may be followed by a decrease in activity later. The newly synthesized collagens of all the transformed cells that produced almost exclusively collagen types I and/or III had high extents of lysyl hydroxylation, and there was also an increase in the ratio of glycosylated to non-glycosylated hydroxylysine. The data suggest that one critical factor affecting modification is the rate of collagen synthesis, which affects the ratio of enzyme to substrate in the cell.     

The hydrating effect of lathyrogenic compounds on chick-embryo cartilage in vivo

1. Injection of 0.16mug. of actinomycin D into pupae of the beetle Tenebrio molitor L. results in the development of modified adults in which the head and thorax are essentially adult while the abdomen and wings remain pupal-like. It is suggested that the messenger RNA for the development of head and thorax is present in the animal from the first day of pupation. 2. Injection of 0.16mug. of actinomycin D brings about 51-67% inhibition of labelled uridine incorporation into RNA. 3. When thymus DNA is mixed with actinomycin D before injection into pupae the latter develop into normal adults. This protection does not occur when DNA and actinomycin D are injected separately. 4. The inhibition of incorporation of labelled uridine into RNA by actinomycin is diminished to some extent when DNA and actinomycin D are injected separately and abolished if they are injected together. 5. Inhibition of RNA synthesis by actinomycin D in vitro is fully reversible. DNA or deoxyguanosine can reverse the effect of actinomycin D. 6. Incorporation of labelled glycine into protein is not affected by actinomycin D injection during the first 6 days of pupation. On the seventh day it becomes diminished in control pupae but this effect is prevented by actinomycin D. It is suggested that the template for protein synthesis is stable during the first 6 days of metamorphosis and that on the seventh day there is a qualitative change in the protein synthesized on the template.     

Interferon production by primary culture of human endothelial cells

Endothelial cells of human umbilical vein are capable of producing interferon upon induction with Newcastle disease virus, influenza virus, and poly I: poly C, but not staphylococcal enterotoxin A. All the interferons produced belonged to the alpha-type. After treatment with influenza virus the endothelial cells produce two subtypes of alpha-interferon: acid-labile and acid-stable.     

Interferon production by mammalian cells grown in a serum-free medium

Summary Interferon, produced by rabbit heart cells grown in a serum-free medium, failed to protect rabbit heart serum-free cells, but protected rabbit heart serum-containing-medium cells against vaccinia and vesicular stomatitis virus. Interferon produced in serum-free cells had a greater species specificity than that produced in serum-containing media. The difference in activity was shown to be due to lack of adsorption by serum-free-medium cells.