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A Rattner

Publications and source records attributed to A Rattner.

35 records · Page 2Linked to original sources
PubMed1995

Collagen bioassay by the contraction of fibroblast-populated collagen lattices.

The ability of fibroblasts to induce contraction of a collagen gel was studied with respect to the quantity and the quality of type I acid-soluble collagen. The speed of contraction and the appearance of the fibre bundles obtained after contraction depend not only on the ratio of the amount of collagen to fibroblasts but also on the process of the collagen purification. When collagen lattices made with a pepsinized collagen were compared to lattices made with a non-pepsinized collagen of the same amount, the fibres from pepsinized collagen seemed fewer (only 13% of the observed surface against 51% in the case of non-pepsinized collagen) and the lattices appeared by electron microscopy to be almost empty as if the lattices were comprised of less collagen. The importance of the non-helical domain of the collagen molecule for the identification and organization of collagen by fibroblasts is discussed. Collagen retained by fibroblasts was maximum (80-99%) when collagen was prepared in the presence of protease inhibitors and decreased when proteolysis was not avoided, for example when collagen was prepared in the presence of pepsin. A test using an estimation of the percentage of collagen retained by fibroblasts in a contracted collagen lattice is proposed to check the biological quality of collagen samples.

Acetates↗
PubMed1993

NF-kappa B activates the HIV promoter in neurons.

Human immunodeficiency virus (HIV) infection of the brain leads to massive neuronal damage, resulting in the AIDS (acquired immunodeficiency syndrome) dementia complex (ADC). A recent study using transgenic mice indicates that neurons possess transcription factors capable of activating the HIV promoter. To identify these, we transfected two types of primary cultures of rat neurons with HIV promoter-reporter gene constructs. The two kappa B regulatory sites in the HIV long terminal repeat (LTR) are shown to be essential for strong promoter activity. Two proteins present in neurons, BETA and an NF-kappa B-like protein, can bind the kappa B sites. These proteins are shown to belong to distinct families of transcription factors. Mutation analysis and transfection of a dominant negative NF-kappa B mutant, indicate that the neuronal NF-kappa B-like activity mediates HIV promoter activation. cDNA cloning, biochemical and immunological analyses indicate that neuronal NF-kappa B is similar to NF-kappa B of other tissues. Transfections of primary neuron cultures with an HIV promoter-beta-galactosidase construct show that within these cultures, neurons are indeed the cells that highly activate the HIV promoter. Thus, analogous to the situation in T-lymphocytes and macrophages, NF-kappa B is an activator of HIV transcription in neurons.

AIDS Dementia Complex↗
PubMed1992

[Synthesis and supramolecular organization of components of the extracellular matrix by fibroblasts cultured in a collagen lattice].

Fibroblasts cultured in a collagen gel contract and organize the gel into a three-dimensional matrix of collagen fibers. Within this matrix, the fibroblast cell cycle is blocked at the G1 phase but also at the G2 phase. The fibroblasts produce the main extracellular matrix components (collagen, noncollagen proteins, glycosaminoglycans), although in small amounts. Studies using this in vitro model with radiolabeled precursor substances (14C proline, 3H glucosamine) demonstrated production of supermolecular complexes which resisted to proteolysis by pepsin and collagenase and could not be isolated by saline precipitation. Polyclonal antibodies identified type I collagen, type VI collagen and fibronectin in this coherent supermolecular structure. The presence of glycosaminoglycans was also demonstrated by alcian blue precipitation.

Cells, Cultured↗
PubMed1991

Nuclear factor kappa B activates proenkephalin transcription in T lymphocytes.

Upon activation, T lymphocytes accumulate high levels of the neuropeptide enkephalin which correlate with high levels of proenkephalin mRNA in the cells. Here we investigated the transcriptional basis for these changes. The proenkephalin promoter contains a sequence GGGGACGTCCCC, named B2, which is similar to the kappa B sequence GGGGACTTTCC, the binding site of the transcription factor nuclear factor (NF)-kappa B. Activation of T lymphocytes induces an NF-kappa B-like binding activity to the B2 site, concomitant with activation of the proenkephalin promoter. Mutations at the B2 site abolish this transcriptional activation. The purified homodimer (two p50s) of the DNA-binding subunit of NF-kappa B binds the B2 site of proenkephalin relatively better than does the heterotetramer (two p65s plus two p50s) form of the factor. Thus, it appears that the T-cell-specific activation of the proenkephalin promoter is mediated by NF-kappa B. However, as NF-kappa B is ubiquitous and the transcriptional activation through the B2 site is T cell specific, yet another T-cell-specific factor which synergizes with NF-kappa B should be considered.

Animals↗
PubMed1989

A brain-specific transcription activator.

We have identified a DNA binding protein, named BETA, that interacts with the same (B) transcriptional regulatory sequence as the known transcription factor NF-kappa B. BETA is found only in gray matter throughout the brain, and not in a variety of other rat tissues. Two binding sites for BETA are present adjacent to the promoter of the rat proenkephalin gene. Transfection of primary brain cultures that express BETA, with a reporter gene driven by the SV40 promoter linked to BETA DNA binding sites, results in transcriptional activation. We infer that BETA is a brain-specific transcription activator.

Animals↗
PubMed1989

Modification of enzyme activities concerned with collagen metabolism in the heart of spontaneously hypertensive and aortic-constricted rats.

The activities of three enzymes concerned with collagen metabolism 4-prolyl hydroxylase, UDP-glucose: collagen glucosyltransferase and glucosyl-galactosyl-hydroxylysine glucohydrolase and 4-hydroxyproline content have been studied in the cardiac ventricles of spontaneously hypertensive rats (SHR) during prehypertensive, hypertensive and sustained hypertensive stages (respectively 4.5, 12 and 19 weeks of age). They were compared with values observed in age-matched normotensive Wistar Kyoto rats (WKY). The same studies have been performed in parallel on aortic-constricted rats (ACR) 8 days after suprarenal constriction of the abdominal aorta. The most striking finding was a significant increase in cardiac prolyl hydroxylase specific activity in the ACR but not in the SHR. No variation in 4-hydroxyproline concentration was found in the hearts of ACR. In contrast, a decrease in 4-hydroxyproline concentration was found in the hearts of SHR at 19 weeks. Cardiac glucosyltransferase specific activity was significantly elevated only in the SHR at 12 weeks. No variation in glucohydrolase specific activity was detected in the hearts of either SHR or ACR. The cardiac enzyme activities all decreased with age. These data show that the alterations in cardiac collagen metabolism are different in SHR and ACR. The patterns of the alterations found in the heart mirror those observed in the aorta in both models under the same experimental conditions.

Age Factors↗
PubMed1987

Chronic liver iron overload in the baboon by ferric nitrilotriacetate. Morphologic and functional changes with special reference to collagen synthesis enzymes.

Two baboons receiving intramuscular injections of ferric nitrilotriacetate over a two-year period were compared with two control baboons. The results indicate that in iron-overloaded animals: liver iron excess was major (maximal liver iron concentration values of 42 mumol/100 mg dry weight for both animals vs 1.3 +/- 0.2 (mean +/- SD) in controls) and chronic (for 15 months liver iron concentrations were higher than 15); iron deposition, although less abundant than in sinusoidal cells, was pronounced within parenchymal cells; serum transaminase activities were markedly increased; rare foci of perisinusoidal fibrosis were observed in areas of massive iron overload; and a dramatic decrease in hepatic 4-prolyl-hydroxylase activity was found, in contrast with unchanged glucosyltransferase and galactosyltransferase activities. In conclusion these findings suggest that, in our model, chronic liver iron overload: exerts a marked biochemical cytolytic effect; and does not produce significant hepatic fibrosis, possibly related to an inhibiting effect of ferric nitrilotriacetate complex on 4-prolyl-hydroxylase activity.

Animals↗
PubMed1986

In vitro production of collagen by synovial fibroblasts from D-penicillamine-treated arthritic rabbits.

In order to get further insight into the mechanism of D-penicillamine action on synovial tissue collagen synthesis, fibroblasts derived from drug-treated arthritic rabbits were cultured and labelled with radioactive proline. No evident correlation was found between the amount of newly synthesized collagen and the previous treatment of animals. In contrast, the prolyl-hydroxylase activity was reduced in cells from rabbits receiving D-penicillamine. This finding suggests that culture conditions may influence the collagen-synthesizing potentiality of the synovial fibroblasts without changing the level of enzyme activity. Therefore, the prolyl-hydroxylase activity could be considered here as a more reliable reflection of the in vivo situation. The ratio of type III to type I procollagens, as estimated by DEAE-cellulose chromatography, showed a rise in cultures from D-penicillamine-treated rabbits as compared to controls. This result indicates that long-term administration of the drug may alter the collagen composition of synovial tissue matrix in rheumatoid arthritis. The question remains, however, whether this alteration contributes to the beneficial effect of the drug.

Animals↗
PubMed1986

D-penicillamine inhibition of interleukin-1 production: a possible mechanism for its effect on synovial collagen synthesis?

Collagen production was investigated in cultured rabbit synovial fibroblasts exposed in vitro to D-penicillamine (D-Pen). The results show that these cells are rather insensitive to the drug since only a slight increase of the collagen amount secreted was observed for 48-h exposure to concentrations of 200-400 micrograms/ml. However, fibroblasts derived from the synovium of arthritic rabbits proved to be more susceptible to D-Pen, responding by a marked increase of collagen secretion even for concentrations of 50 micrograms/ml. This finding suggests that synovial fibroblasts of arthritic patients, probably stimulated by the inflammation process, could be target cells for the D-Pen action. The activities of 4-prolyl-hydroxylase (4-PH) and galactosylglucosyl-transferase (GGT) were assayed in the same cultures. A correlation has been found between the 4-PH activity and the collagen amount produced. In contrast, no alteration in the level of GGT on exposure to D-Pen was detected. Finally, D-Pen was shown to reduce in vitro the production of collagen-inhibiting factors by phytohaemagglutinin-stimulated mononuclear cells. This effect was associated with an inhibition of the release of monocyte cell factor (MCF/interleukin-1), suggesting that D-Pen could indirectly affect synovial collagen synthesis by interfering with interleukin-1 secretion.

Animals↗
PubMed1985

Mononuclear cell-mediated modulation of synovial cell metabolism. I. Collagen synthesis.

Human PHA-stimulated mononuclear cells produce a factor which inhibits synovial cell collagen and non-collagen protein synthesis, whereas it enhances hyaluronic acid (HA) production. Indomethacin (10(-4)-10(-6) M), a cyclo-oxygenase inhibitor, suppresses this effect, suggesting that the mechanism is prostaglandin-mediated. The active material, of apparent molecular weight 12 000-20 000, also displays the properties of the mononuclear cell factor (MCF) previously described by others, since its stimulates collagenase and PGE2 release by the cultured synovial cells. Furthermore, it co-purifies with interleukin 1 (IL 1) as shown by lymphocyte-activating factor activity. This strongly suggests that IL 1 could be responsible for some (or all) the effects observed on MCF-exposed synovial cells. From these data, we deduce the possibility that mononuclear cells may participate in limiting synovial collagen deposition in rheumatoid arthritis.

Cells, Cultured↗
PubMed1985

Aorta collagen metabolism in spontaneously hypertensive and aortic-constricted rats: variations in enzyme activities concerned with disaccharide unit synthesis and degradation according to blood pressure and age.

Two enzyme activities concerned with collagen disaccharide unit metabolism (UDP-glucose: collagen glucosyltransferase and glucosyl-galactosyl-hydroxylysine glucohydrolase) have been studied in the thoracic aortic wall together with 4-prolyl hydroxylase activity and 4-hydroxyproline content in spontaneously hypertensive rats (SHR) at the prehypertensive, hypertensive and sustained hypertensive stages (respectively 32 days, 12 weeks and 19 weeks of age). They were compared with values observed in age-matched normotensive Wistar Kyoto rats (WKY). The same studies have been performed in parallel on aortic-constricted rats (ACR) 8 days after suprarenal constriction of the abdominal aorta. Negative regressions of all three specific activities as function of age were observed. The most striking difference observed between the SHR and the WKY was the increase of glucosyltransferase specific activity, already found at the prehypertensive stage and continuing thereafter; the glucohydrolase specific activity was increased only during the establishment of hypertension whereas no modification was found with prolyl hydroxylase at any stage. However, a diminution of hydroxyproline concentration was seen at all ages while total hydroxyproline mass remained unaffected. The alterations of the aortic collagen metabolism observed in the ACR recall those seen in the SHR at the prehypertensive stage: the only significant modification was that of glucosyltransferase activity. Correlation was found between glucosyltransferase activity and blood pressure level in the two animal models.

Age Factors↗
PubMed1984

A method for the measurement and characterization of protease activities responsible for extracellular or intracellular degradation of collagen precursors.

The first method for the qualitative and quantitative evaluation of extracellular and intracellular protease activities responsible for degradation of newly synthesized collagen is described. In a double incubation method, underhydroxylated collagen chains (protocollagen) serve as substrate for protease extract and then for the indicator enzyme, 4 prolyl hydroxylase. It was possible to characterize at least four types of protocollagen sites sensible to these proteases. The microsomal fraction of chick embryo liver contained a protease active on protocollagen and whose activity was similar to that of purified human synovial collagenase.

Animals↗
PubMed1980

Employment of gas-liquid chromatography for the analysis of collagen amino acids in biopsy tissue.

In this paper, gas-liquid chromatography, adapted for the determination of collagen amino acids, is described. This technique was attractive for its sensitivity in that only a small amount of protein such as in 0.5 mg of tissue, especially as obtained from biopsy tissue, was needed for the separation and determination of proline (Pro), 4-hydroxyproline (4-Hyp), 3-hydroxyproline (3-Hyp), lysine (Lys), hydroxylysine (Hyl) and epsilon-hydroxy-norleucine (epsilon-PH-Norleu), the characteristic amino acids of collagen. Thus, without purification of collagen, by determining the ratio Hyl/4-Hyp and 4-Hyp/Pro it was possible to determine some anomalies in the collagen content of biopsy tissue (skin or liver). The ratio Hyl/4-Hyp allows an estimation of the lack of hydroxylation of polypeptidic lysine as in the Ehlers-Danlos syndrome type VI; and the ratio 4-Hyp/Pro allows measurement of variations in collagen content in relation to protein, especially in the liver, as in alcoholic cirrhosis.

Adult↗
PubMed1979

[Determination of free intracellular proline pool in fibroblast cultures].

By using a direct, rapid, sensitive colorimetric method, free intracellular proline pool from human adult skin fibroblasts in monolayer was determined. Thus under identical experimental conditions, free intracellular proline pool was about 20 pmol proline/micrograms cellular proteins, whatever the cell lines from human adult skin fibroblasts, the passage and the time of subculture. Besides, there was a poor correlation between collagen biosynthesis in fibroblast cultures and free intracellular proline pool.

Adult↗
PubMed1976

[The action of calcium on the step of collagen metabolism, catalyzed by procollagen-proline hydroxylase].

Calcium effects on procollagen-proline-hydroxylase activity of chick embryo tibias was studied. Serial preincubation of the natural substrate, the enzymatic preparation or the mixture of both with and without calcium showed that calcium decreased procollagen-prolin-hydroxylase affinity for procollagen, decreased the maximum velocity of the reaction by binding to the substrate and by increasing the thermosensitivity of the enzyme -- substrate complex. Calcium could also increase the maximum velocity by dissociation of an enzyme -- inhibitor ligand complex. The resulting decrease of the overall reaction rate has been discussed in the context of the relationship between collagen metabolism and calcium fixation during calcification.

Animals↗
PubMedDate not supplied

Characterization of human osteoblastic cells: influence of the culture conditions.

Human osteoblastic cells were isolated enzymatically from adult human spongy bone and grown in MEM-Ham F12 1:1 medium supplemented with 2% Ultroser (USM). They were subcultured and examined for osteoblast features by morphological, histological, and biochemical approaches. The cells had a characteristic polyhedral morphology and produced a high level of alkaline phosphatase (ALKP). Confluent cultures were uniformly stained for ALKP and flow cytometry analysis with fluorescein diphosphate gave a single peak signal, reflecting a highly positive population, distinct from cultures of fibroblasts. The ALKP activity was stimulated by 1,25 (OH)2 vitamin D3. CD 44 was strongly expressed in these cultures, although osteoblasts are negative in vivo and osteocytes are positive. The main collagen synthesized was type I collagen and osteocalcin was produced after stimulation by vitamin D3. 10 mM betaGP induced mineralization and microprobe analysis of the crystals showed a composition close to hydroxyapatite. Changing the culture conditions to MEM-10% calf serum acted on cell behavior: it reduced the production of these biochemical markers of osteoblasts and the morphology became fibroblastlike with more rapid cell multiplication. The parameter most affected by the change in culture medium was ALKP, which was selected as the determinant criterion for defining an osteoblast culture. ALKP activity was then used to characterize a culture of cells seeded in a collagen gel.

Adult↗