Metabolic Implications in the Biochemical Responses to Iron Deficiency in Cucumber (Cucumis sativus L.) Roots.

Strategy I plants respond to Fe deficiency by inducing morphological and biochemical modifications at the root level that are apt to make iron available for uptake. Cucumber (Cucumis sativus L.) grown in the absence of Fe has been shown to increase the capacity to acidify the rhizosphere and Fe3+ reduction activity. We have determined in these roots some metabolic activities that might be correlated with the increased proton extrusion. Proton efflux from roots may be followed by a mechanism regulating the cytosolic pH according to the pH-stat theory. Roots grown in the absence of Fe showed an increase in dark 14CO2 fixation and organic acid synthesis and a 6-fold increase in the extractable phosphoenolpyruvate carboxylase activity with respect to the control roots. Dehydrogenase activities producing cytosolic NAD(P)H were also increased under Fe deficiency. The presence of Fe2+, but not Fe3+, inhibited dark 14CO2 fixation in a range between 24 and 52% but did not show any effect on the in vitro phosphoenolpyruvate carboxylase activity.

Calcium Transport in Membrane Vesicles Isolated from Maize Coleoptiles (Effect of Indoleacetic Acid and Fusicoccin).

Maize (Zea mays L.) coleoptile segments loaded with 45Ca released about 50% of the ion after 1 h when treated with indoleacetic acid (IAA). In contrast, fusicoccin (FC) had no effect. The same relation was found when ATP-dependent Ca2+ transport, measured as 45Ca uptake, was determined in a plasmalemma-rich membrane vesicle fraction isolated from coleoptiles treated or untreated for 1 h with IAA or FC. In fact, IAA-treated membranes showed an increase in ATP-dependent 45Ca uptake by more than 30% with respect to the control and the FC treatment. Ca2+ uptake in IAA-treated membranes was only slightly affected (+27%) by supplying calmodulin (Cam) exogenously. However, Ca2+ uptake in membranes from the control and FC-treated coleoptiles were stimulated (+80%) by exogenous Cam. Calmidazolium, a Cam antagonist, inhibited Ca2+ uptake in the IAA treatment (-48%) to a greater extent with respect to the control and FC treatment (-33 and -29%, respectively). A possible relationship between the effect of IAA on the ATP-dependent Ca2+ transport activity, the involvement of Cam, and their effect on growth are discussed.

[Integration of carcinogenic viruses in the genome of in-vitro cultured human fibroblasts]. / Intégration de virus cancérigènes dans le génome de fibrolastes humains cultivés in vitro.

Avian sarcoma viruses are known for inducing no transformation of human diploid fibroblasts. Nevertheless, we show that the Rous sarcoma virus can infect and transform some human fibroblastic cell lines, replicate and express viral proteins, integrate into the host genome and prevent expression of MHC class I antigens on cell membranes. Cell transformation happens together with important and significant abnormalities of the cell karyotype and proviral integration is most often close to the c-src oncogene on chromosomes 1 and 20.

Inhibition of HLA class I antigen and mRNA expression induced by Rous sarcoma virus in transformed human fibroblasts.

Cells from various human nonlymphoreticular neoplasms show reduced HLA class I antigen expression. In this report, a system of human fibroblasts transformed by an avian retrovirus has been employed to investigate the mechanism of this phenomenon. Rous sarcoma virus has been used to transform in vitro human dermal fibroblasts, and clonal cell lines have been established from these cultures. In all the clones studied the integration of the provirus induced a reduction of cell-surface HLA-A, -B, -C framework antigen and beta 2-microglobulin expression when compared to levels for the respective parental fibroblasts. The reduction was correlated with a diminished intracellular synthesis of these molecules. Uninfected cells derived from an osteogenic sarcoma exhibited a reduced expression comparable to that of dermal diploid fibroblasts obtained from the same donor and transformed by Rous sarcoma virus. RNA gel blot analysis of total cellular RNA and of poly(A)+ cytoplasmic RNA showed a markedly decreased amount of HLA class I transcripts in the transformed cells. Southern blot study of genomic DNAs digested with several restriction endonucleases showed that the banding patterns of the HLA genes were not altered in the cells harboring the Rous sarcoma provirus. Our data are consistent with the hypothesis that the Rous sarcoma provirus that does not seem to be linked to the major histocompatibility complex class I gene superfamily may negatively control HLA gene expression.

Integration and expression of provirus in human cells transformed by avian sarcoma virus.

Previously, human diploid fibroblasts from some donors infected in vitro by avian sarcoma virus (ASV) were transformed and found, by electron microscopy, to produce small numbers of virus particles that were infectious by bioassay; also, a line of human osteosarcoma cells infected with ASV developed additional characteristics of transformation and released a small number of infectious virus particles. In this study the complete proviral sequence was shown to be integrated in the genome of these cells. The env-related proteins gp85 and gp37 and the gag-related proteins pr76, pr60, and p19 can be detected in cytoplasmic extracts of ASV-infected human cells. Comparable amounts of pp60v-src were found in human and avian cells infected with ASV. The associated kinase activity in infected human cells was dramatically increased as compared to that of uninfected controls; the enzyme had the same cation and substrate requirements as those from ASV-transformed avian cells. Replicating particles from infected human cells were purified and were significantly modified compared to those from avian hosts as shown by a) higher specific gravity, b) the presence of RSV gag-related but not env-related antigens, and c) the fact that the virus-associated reverse transcriptase preferred the divalent cations Mn2+ and Fe2+ over Mg2+.

Reduced expression of MHC class I antigens in chicken fibroblasts transformed by Rous sarcoma virus.

Monoclonal monomorphic as well as polyclonal antibodies against H-B class I (B-F) antigen were used to evaluate the expression of these molecules on normal and RSV-transformed chick embryo fibroblasts. The results indicate that chicken fibroblasts transformed in vitro by the Schmidt-Ruppin strain Rous sarcoma virus of subgroup B (SR-RSV-B) present reduced H-B class I antigen expression as compared to uninfected cells of the same inbred strain. This quantitative reduction was observed at the cell membrane and in whole cell lysates. Chick embryo fibroblasts infected with avian leukosis virus RAV-1 show that the levels of H-B class I antigens were indistinguishable from the levels measured on the control cells. Normal embryo fibroblasts derived from the inbred lines B4/B4 express higher levels of H-B antigens than normal cells carrying the B12/B12 genotype.

Binding of proteins, including pp60src, to activated CH-sepharose 4B.

Activated CH-Sepharose 4B and protein A Sepharose CL-4B can bind, selectively and non-specifically, polypeptides from chick embryo cells. The major polypeptides bound have apparent molecular masses of 57-60 kDa and 47-49 kDa and cannot be eluted by extensive washing with buffers containing detergents. One of the 57-60 kDa polypeptides was identified by immunoblotting as the transforming protein of Rous Sarcoma Virus (RSV), pp60src. This polypeptide could be removed from the solid phase immunoabsorbent with 60% dimethylsulfoxide, but not with 2% SDS, 5% beta-mercaptoethanol, 1 M NaCl or 0.1% Tween 20.

[Changes of the expression of histocompatibility (HLA) antigens in human fibroblasts transformed by the avian leukosis-sarcoma virus]. / Modification de l'expression des antigènes d'histocompatibilité (HLA) dans des fibroblastes humains transformés par le virus de la leucosarcomatose aviaire (ASV).

Monoclonal monomorphic antibodies anti-HLA class I and II antigens, were used to evaluate the expression of these molecules on normal and RSV-transformed human fibroblasts. The results indicate that the human diploid fibroblasts transformed in vitro by RSV present a reduced HLA-class I antigens expression as compared to the uninfected fibroblasts of the same donor. In parallel, it is demonstrated that the class II molecules absent on normal cells are expressed after transformation.

[Transformation of diploid human fibroblasts and viral production after infection by avian retroviruses]. / Transformation de fibroblastes diploïdes humains avec production virale après infection par des rétrovirus aviaires.

Among the cultures established from skin biopsies of 128 donors, two were transformed by RSV and produced virus. One out of 6 cellular lines developed from six human sarcomas was likewise transformed and also produced virus.

[Gliomas induced in mammals by Rous sarcoma virus (RSV) (author's transl)]. / Gliomes induits chez les mammifères par le virus du sarcome de Rous (RSV).

Studies concerning the induction of gliomas in a variety of animal species by an oncogenic avian virus (Rous sarcoma virus) were presented. Experimental conditions (age of inoculation, doses of virus) were reviewed. Preliminary attempts to localize viral antigens in the tumors in vivo using labelled antibodies were discussed.

[Morphology of ribosomal RNA in chicken fibroblasts]. / Morphologiè des ARN ribosomiques de fibroblastes de poulets.

It is shown with the electron microscope that the 28 S RNA component of the ribosomal RNA extracted from Chicken fibroblasts contains secondary structures which are not present in the 18S component.

Transformation in vitro of glial hamster cells by Rous sarcoma virus.

Cell lines from the brains of inbred CF hamster embryos were established in vitro. The morphology of the cells in the light and electron microscopes was that of glial cells, and the cells contained the nervous system-specific protein S-100. Infection with the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup B, resulted in foci of transformation. The transformed cells were virogenic and upon intracerebral and sc inoculations into young hamsters, they developed into histologically typical gliomas.

[Susceptibility to avian tumor viruses: determination of chick phenotypes]. / Susceptibilité aux virus oncogènes aviaires: détermination du phénotype de Poussins.

The genetic cellular susceptibility to avian sarcoma viruses (RSV) of subgroups A, B, C and E has been determined in one week old Chicks. Fibroblasts from pin feathers were grown in vitro and tested by focus formation after infection. This technique will allow, for the first time, the study of the influence of the host phenotypes (susceptibility or resistance to the different subgroups of RSV) upon the immune response to a given virus.

[Cellular contaminants and structural proteins of Rous sarcoma virus (RSV), studied by polyacrylamide gel electrophoresis]. / Contaminants cellulaires et protéines structurales dans le virus du Sarcome de Rous (RSV), étude par électrophorèse sur gel de polyacrylamide.

The number of polypeptides in highly purified preparations of RSV, of two different subgroups, produced in culture, has been compared to the polypeptides present in the supernatant of uninfected cultures and processed in identical manner. The analysis of PAGE-SDS shows that from 13 to 18 polypeptides present in viral preparations may be cellular contaminants. Fewer contaminating polypeptides are found in the myeloblastosis virus purified from plasma of Chicken.

A quantitative autoradiographic study of nucleolus-associated RNA and DNA synthesis during the eclipse phase in Rous sarcoma virus-infected chicken fibroblasts.

Functional and morphologic differences between the sensitivity of nucleoli of Rous sarcoma virus-transformed cells and that of newly infected cells to the action of actinomycin D (AD) have been demonstrated by quantitative light and electron microscope autoradiography and utilized to investigate the function of the nucleolus in the early stages of infection. After a pulse exposure to low doses of AD, increased RNA synthesis is induced within 80 minutes in the fibrillar portion of the nucleolus by infection. A concomitant increase in the retention of tritiated AD in the nucleolus and a quantitative redistribution of intranuclear and cytoplasmic DNA label are interpreted as evidence for a virus-induced amplification of the binding sites of AD in nucleolar chromatin.