Synthesis of sulfated proteoglycans throughout the cell cycle in smooth muscle cells from pig aorta

Cultured smooth muscle cells from pig aorta arrested in G0 phase by serum deprivation were stimulated to proliferate by replacing the medium with one containing 10% serum. Studies in DNA replication and proliferation of cells showed a relatively good synchrony: 90% of the cells were in G1 phase for 16 h after addition of serum; they entered S phase between 18 and 24 h, completed S phase and traversed G2 phase between 24 and 30–32 h; 75% of these cells multiplied after 30–32 h and the remainder were blocked at the end of G2 phase. The synthesis and secretion of sulfated proteoglycans were examined throughout a full cell cycle using metabolic labelling with [³⁵S]sulfate. Smooth muscle cells in G1 or G2 phase synthesized and secreted sulfated proteoglycans with a possible pause at the end of the G2 phase but at the beginning of the S phase and during mitosis the incorporation of [³⁵S]sulfate into these macromolecules stopped entirely. Structural characteristics of sulfated proteoglycans secreted into the medium during G1 phase and an entire cell cycle were investigated. The proportion of proteoglycan complexes and the relative hydrodynamic size of monomers and of constituent subunits of complexes were determined after chromatography on Sepharose CL-2B and CL-6B columns run under both associative and dissociative conditions. No significant differences were observed for the periods of the cell cycle that were studied:

1. 1. [³⁵S]Proteoglycan complexes represented at the end of G1 phase and of the cell cycle respectively 19 and 16% of the total [³⁵S]proteoglycans secreted into the medium.

2. 2. More than 90% of the subunits, obtained after dissociation of complexes, were characterized by a similar k_av after chromatography on Sepharose CL-2B columns eluted under dissociative conditions (k_av 0.68 at the end of G1 phase and 0.65 at the end of full cell cycle).

3. 3. About 95% of monomers synthesized at the two stages of the cell cycle were eluted at k_av 0.25 after chromatography on Sepharose CL-6B column run under associative conditions and were characterized by a similar glycosaminoglycan distribution. These results suggest that smooth muscle cells in culture liberate similar populations of proteoglycans into the medium during the G1 and G2 phases.

     

Albumin evolution in polyploid species of the genusXenopus

Electrophoresis of serum from 21 Xenopus species and subspecies reveals variable numbers of albumin bands. The diploid X. tropicalis has one albumin, while the tetraploid species (laevis, borealis, muelleri, clivii, fraseri, epitropicalis) have two. The octoploid species (amieti, boumbaensis, wittei, vestitus, andrei) have two to three bands, and the dodecaploid X. ruwenzoriensis has three. The molecular weight of the Xenopus albumins varies from 68 kd (in the tropicalis group) to 74 kd. The subspecies of X. laevis possess two albumins of different molecular weights (70 and 74 kd), whereas most species have only 70-kd albumins. Peptide maps have been obtained from albumin electromorphs by limited proteolysis in sodium dodecyl sulfate (SDS) gels, using S. aureus V8 protease. The peptide patterns produced by electromorphs from the same tetraploid Xenopus species generally differ from each other, suggesting that the two albumin genes contain a substantial amount of structural differences. In addition, the peptide maps are diagnostic for most tetraploid species and for some subspecies of X. laevis as well. Proteolysis of albumins from most octoploid and dodecaploid species results in patterns which are very similar to the ones produced by the electromorphs from X. fraseri. The albumins of X. vestitus differ from those of the other octoploid species. X. andrei possesses two fraseri-type and one vestitus-type albumin, which indicates that it probably originated by allopolyploidy.     

Enhancement of Oxygen-Evolution in Photosystem II-Phycobilisome Particles from Porphyridium cruentum

Oxygen-evolving photosystem II-phycobilisome particles froma red alga were inhibited 50–80% by aging, dilution, lowpH and salt-washing. Bovine serum albumin, and dithiothreitolwere found to stimulate activity in all but salt-washed particles.CaCl₂ and MnCl₂ partially restored activity lost after agingor dilution. ¹Current address: Waksman Institute of Microbiology, RutgersUniversity, Piscataway, New Jersey 08854-0759, U.S.A. (Received October 5, 1985; Accepted March 31, 1986)     

The periosteum in experimental avian hyperostosis induced by MAV 2-0 myeloblastosis virus. Differential diagnosis from osteopetrosis

Diaphyseal tibial bones of 11-14-17 th day-old hatched chicks infected with retrovirus myeloblastic MAV 2-0 were examined by conventional optical microscopy. The characteristic lesion is osteoblastic hyperplasia with the development of spongious bone. The new formed bone contains abnormal chondroid tissue. The relation between osseous and cartilaginous tissues in this experimental model is discussed. As connective tissue took part in the constitution of this hyperostosis, this osteopathy may be considered as an hyperplastic, monodermic mesenchymatous, pluritissular tumour. The numerous works concerning the "avian hyperostosis osteopetrosis" are commented.     

How do retroviral oncogenes induce transformation in avian erythroid cells?

The v-erb B oncogene, as well as other oncogenes of the src-gene family transform immature erythroid cells from chick bone marrow in vivo and in vitro. The erb B-transformed erythroid cells differ from normal late erythroid precursors (CFU-E) in that they have acquired the capacity to undergo self-renewal as well as to differentiate terminally. They also do not require the normal erythroid differentiation hormone, erythropoietin, for either process. Cooperation of v-erb B with a second oncogene, v-erb A, results in a differentiation arrest of the transformed cells, which now only use the self-renewal pathway. Studies with conditional and non-conditional mutants in both v-erb B and v-erb A will be presented to elucidate further how the transforming proteins encoded by these oncogenes, gp74erb B and gp75gag-erb A, affect the differentiation programme of the infected erythroid precursor with the outcome of hormone-independent leukaemic cells arrested at an early stage of erythroid differentiation.     

Synthesis and phosphorylation of cytoskeleton components in foetal,regenerating and adult normal rat hepatocytes during culture

Summary Detergent insoluble material (DIM) was prepared by gentle treatment with detergent from foetal, regenerating and adult normal rat hepatocytes cultured for various times. It retained to some degree the morphology of the cells. After incubation of intact cells with ³⁵S-methionine, most of the labelled DIM proteins were found to be components of the cytoskeleton. They included several cytokeratins, vimentin and actin. The synthesis rate varied with the age of animals and culture conditions. The high synthetic rate of vimentin in foetal and regenerating hepatocytes could be associated with cell proliferation. No correlation was found between cytokeratin synthesis and hepatocyte growth. Most of the cytoskeleton proteins could be phosphorylated in intact cells and in DIM from cultured hepatocytes. However the degree of phosphorylation of these proteins was not related to their synthetic rate. The decreased phosphorylation level in cultured adult rat hepatocytes could be related to the rapid loss of specific functions.     

Use of tritium labeled amino acid conjugates of prostaglandins and thromboxanes as labeled ligands in prostanoid radioimmunoassay

Conjugates of prostaglandins and thromboxanes with tritium labeled amino acids were prepared and employed as labeled ligands in porstaglandin and thromboxane radioimmunoassays. Assays for PGF_2α, 15-keto-13, 14-dihydro-PGF_2α, TXB₂ and 15-keto-13, 14-dihydro-TXB₂ were evaluated in comparative studies using either these heterologous ligands or the corresponding homologus tritiated eicosanoid as tracers. Binding properties for the respective antibodies were found to be similar using either tracer.Three biological studies were also conducted, viz. study of the release of TXB₂ during collagen induced platelet aggregation, of 15-keto-13, 14-dihydro-TXB₂ during guinea pig pulmonary anaphylaxis, and of PGF_2α (measured as 15-keto-13, 14-dihydro-PGF_2α in peripheral plasma) during bovine luteolysis. The analyses gave comparable results using either the heterologous or the homologous assay.Thus, this type of labeled prostanoid conjugates may serve as a convenient alternative to homologous tracers in radioimmunoassay. Heterologous tracers may even in certain cases provide the only simple solution to the problem of preparing a labeled ligand of high specific activity.     

Microlocating lithium in the mouse embryo by use of a (n, α) nuclear reaction

Summary The quantitative imaging of lithium distribution, in histological sections of 15-days old mouse embryos (whose mother had been submitted to Li-treatment), was performed using⁶Li isotope as tracer,⁶Li(n,)³H nuclear reaction for detection, and dielectric track detectors. Despite the particular difficulties of cryosectioning the embryos without disturbing the lithium distribution, the Li regionalization appeared to be very clear-cut. The ectomesodermic tissues were significantly more loaded with lithium than the endodermic ones. This is probably related to the ectomesodermic tissues being also those most sensitive to the teratogenic effect of lithium. The Li-distribution in the embryo brain was almost homogeneous, instead of being heterogeneous as in adult brain. The mean Li-concentration in the embryo brain was not much below the Li concentration in the grey matter of the mother brain, but it was significantly larger than that in the white matter of the mother brain. Our results are discussed in the context of teratogenic effects observed in situ during mammalian development.     

DNA-polymorphic patterns linked to the β-globin genes in German families affected with hemoglobinopathies and thalassemias: a comparison to other ethnic groups

Summary DNA haplotype constellations of the β-globin gene cluster have been analyzed in German families with hemoglobinopathies (Hb Freiburg, Hb K?ln, Hb Presbyterian) and β-thalassemias. The polymorphis patterns obtained were compared to those found in families from Greece, Italy, and Turkey affected by β-thalassemia syndromes. With the combined analysis of seven restriction site polymorphisms a DNA-diagnostic prediction for additional offspring could be made with an overall frequency of 75% in the four ethnic groups.     

Mapping of cysteine genes on the chromosome of Pseudomonas aeruginosa PAO

Summary Three loci coding for different steps in the pathway of cysteine biosynthesis have been mapped by R68.45-mediated coconjugation analysis. The cysteine auxotrophic mutants could be subdivided into sulfite and sulfide-requiring mutants. Sulfide-requiring mutants (cysIV group) were localized at a single position between pyrF and pur-67, while sulfite-requiring mutants (cysI and cysII) mapped at two different regions. The cysI group was also localized between pyrF and pur-67, although more distal to pyrF than the cysIV group. This group included the cys-54 marker, which has been mapped previously. The second group of sulfite-requiring mutants, designated as cysII, was cotransducible with hisI and localized at the end of the PAO chromosomal map. This location was also confirmed for the marker cys-59.The marker cys-59 (which was cotransducible with his1) was cotransferred by R68.45-mediated conjugations with both the late marker pur-67 and the early marker ilv-226. As the late marker hisI was positioned at about 60–65 min (Herrmann and Günther, in press) the length of the PAO chromosome was estimated to be about 70 min.