Differentiation under the control of insulin of rat preadipocytes in primary culture: Isolation of homogeneous cellular fractions by gradient centrifugation

Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats. In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10^?9 M). During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid:CoA ligase. When VLDL and heparin were added with insulin to the medium, this effect was not potentiated. On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells. With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture. The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions. It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development.     

Interactions of Escherichia coli membrane lipoproteins with the murein sacculus.

Bifunctional cross-linking reagents were used to identify cell envelope proteins that interacted with the murein sacculus. This revealed that a number of [3H]leucine-labeled proteins and [3H]palmitate-labeled lipoproteins were reproducibly cross-linked to the sacculus in plasmolyzed cells. The results suggested that most of the cell envelope lipoproteins, and not only the murein lipoprotein, mediate interactions between the murein sacculus and the inner and/or outer membrane of the cell.     

Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage.

Human furin is a calcium-dependent serine endoprotease that can efficiently cleave many precursor proteins on the carboxyl side of the consensus cleavage sequence, -Arg-X-Lys/Arg-Arg-, both in vivo and in vitro. Analysis of furin proteins in extracts of cells infected with a vaccinia recombinant expressing human furin show that the enzyme is present as two prominent forms of 90 and 96 kDa. Because the structurally related bacterial subtilisins require endoproteolytic removal of the NH2-terminal pro-region by an autocatalytic intramolecular cleavage, we speculated that the size heterogeneity in the furin doublet similarly may result from a proteolytic removal of an NH2-terminal pro-region. Here we report identification of the 90-kDa furin NH2 terminus and, based on the reported sequence of the furin cDNA, demonstrate that this furin protein is derived from a larger precursor by an endoproteolytic cleavage on the COOH-terminal side of a consensus furin cleavage site, -Arg-Thr-Lys-Arg107-. Expression of mutant furin molecules containing an altered cleavage site (Arg104----Ala or Arg107----Gly) resulted in the production of only the 96-kDa furin protein. Assays of furin-dependent cleavage of a protein substrate in vitro showed that proteolytic activity was associated with the 90-kDa and not the 96-kDa furin protein, demonstrating that removal of the NH2-terminal pro-region is required for furin activity. Expression of a third furin construct containing a mutation of the active site aspartate (Asp153----Asn) similarly resulted in the expression of only the 96-kDa protein, suggesting that furin activation occurs by an autoproteolytic cleavage. Finally, the production of 90-kDa furin from either site-directed furin mutant could not be potentiated by overexpressing active furin, suggesting that the autoproteolytic activation was an intramolecular event.     

Fructose-bisphosphatase-deficient mutants of the yeast Schizosaccharomyces pombe

We showed that in the yeast Schizosaccharomyces pombe, fructose-bisphosphatase is not subject to catabolite inactivation as it was observed in Saccharomyces cerevisiae. However, this enzyme activity is sensitive to catabolite repression in both yeasts. Two mutants lacking completely fructose-bisphosphatase activity were found. They were unable to grow on glycerol medium. They were still respiratory competent and exhibited the ability to derepress partially malate dehydrogenase activity. In glucose exponential phase culture, the parental strain lacks completely the fructosebisphosphatase activity due to catabolite repression. In these conditions, the growth is slowed down only in the mutants eventhough both mutants and their parental strain lack this enzyme activity. Normal sporulation and poor spore germination were observed for one mutant whereas, only in the presence of glucose, normal sporulation and normal spore germination were observed for the second mutant. Mendelian segregation of glycerol growth was found for the well germinating mutant. It is of nuclear heredity. The two mutations appeared to be closely linked.Abbreviations FBPase Fructose-1,6-bisphosphatase - fbp ^- genetic symbol for FBPase deficiency - glr ^- symbol for inability to grow on glycerol A. M. Colson is Research Associate au Fonds National de la Recherche Scientifique     

The cell cycle of lymphocytes in Fanconi anemia

Summary BrdU-incorporation techniques were used to study the cell cycle in 18 cases of Fanconi's anemia (FA).By comparison with controls, a significant slowing of the cell cycle of lymphocytes in vitro was observed in all FA patients, and possibly in FA heterozygotes, although to a lesser degree. It is probable that the demonstration of the slowing is dependent on the culture conditions. No slowing was observed in other patients affected by at least one of the symptoms of FA. The slow cell cycle of FA cells is mostly due to a very long G₂-phase. A relationship between slow cell cycle and chromatid anomalies exists, the slower cells being significantly more frequently carriers of radial figures than the faster cells, in the same patient.     

Localization of Phloem Oxidases

Among oxidases, cytochrome oxidase has been localized in mitochondria of all phloem cells, catalase has been visualized in parenchyma peroxisomes and peroxidase has been localized in cell walls and in several cell organelles. In angiosperms, peroxidase is present in all phloem cell walls; it is sensitive to cyanide inhibition excepted in sieve areas and around plasmodesmata between sieve tubes and companion cells. In some species, this cyanide resistant oxidasic activity can be localized without exogenous H₂O₂. Peroxidase is localized on ribosomes, inside vacuoles, on the tonoplast and often on the plasmalemma in companion cells and differentiating sieve elements. In young sieve cells some dictyosomes can exhibit a strong peroxidasic activity. In mature parenchyma cells peroxidase can be associated with ER cisternae but not with vacuoles.     

Distribution of mRNAs of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of α-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5–6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2–3-fold). There were no α-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.     

Autolysis of Escherichia coli

Autolysis of unwashed exponential-phase Escherichia coli cells was efficiently promoted by first submitting them to a quick downshock with distilled water before an upshock with 0.5 M sodium acetate, pH 6.5. The association of these two osmotic shocks had a remarkable synergistic effect and led to significant decreases in turbidity and viability. Different factors influencing the rate of cell lysis were examined. A close correlation was established between autolysis and the degradation of peptidoglycan. Both phenomena were induced by the same shock treatment, followed similar kinetics, and were efficiently blocked by addition of divalent cations. Cell lysis was also inducible by a shock treatment with 10(-3) M ethylenediaminetetraacetic acid or ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid and blocked by the addition of divalent cations.     

The structure of cyanobacterial phycobilisomes: a model

Phycobilisomes, supramolecular complexes of water-soluble accessory pigments, serve as the major light-harvesting antennae in cyanobacteria and red algae. Regular arrays of these organelles are found on the surface of the thylakoid membranes of these organisms. In the present study, the hemi-discoidal phycobilisomes of several species of cyanobacteria were examined in thin sections of cells and by negative staining after isolation and fixation. Their fundamental structures were found to be the same. Isolated phycobilisomes possessed a triangular core assembled from three stacks of disc-shaped subunits. Each stack contained two discs which were 12 nm in diameter and 6–7 nm thick. Each of these discs was probably subdivided into halves 3–3.5 nm thick. Radiating from each of two sides of the triangular core were three rods 12 nm in diameter. Each rod consisted of stacks of 2 to 6 disc-shaped subunits 6 nm thick. These discs were subdivided into halves 3 nm thick.The average number of discs of 6 nm thickness forming the peripheral rods varied among the strains studied. For certain chromatically adapting strains, the average rod length was dependent upon the wavelength of light to which cells were exposed during growth. Analyses of phycobilisomes by spectroscopic techniques, polyacrylamide gel electrophoresis, and electron microscopy were compared. These analyses suggested that the triangular core was composed of allophycocyanin and that the peripheral rods contained phycocyanin and phycoerythrin (when present). A detailed model of the hemi-discoidal phycobilisome is proposed. This model can account for many aspects of phycobiliprotein assembly and energy transfer.Abbreviations PBS phycobilisome(s) - PBP phycobiliprotein(s) - AP allophycocyanin - PC phycocyanin - PE phycoerythrin - PEC phycoerythrocyanin - AP-B allophycocyanin B - C- cyanobacterial - R- rhodophytan - B- Bangiophycean - SDS sodium dodecyl sulfate - LPP Lyngbya-Plectonema-Phormidium group - Na-KPO₄ buffers NaH₂PO₄ titrated with a solution of KH₂PO₄ of equivalent molarity to a given pH     

L'intensité lumineuse,son influence sur la teneur en phosphore des carapaces d'Ostracodes

Actual and fossil shells of ostracodes have beenanalyzed with an electronic microprobe: all of them contain phosphorus. X—pictures teach us that the repartition of this chemical element is homogeneous in all the points of the shell. There is no relationship between phosphorus contents in the shells of ostracodes and salinity. In return, there is a relationship between phosphorus contents and temperature and, chiefly, organic phosphorus contents in the environment. However, it is interesting to observe that the analyzed shells of fresh-water ostracodes contain less phosphorus that the generality of the shells of marine ostracodes. The contents of organic phosphorus, in the biotope, depends on the growth of phytoplancton in the upper layers of water which needs a minimum light intensity.