Glutathione content of cultured human fibroblasts during in vitro ageing

The glutathione level of cultured human fibroblasts was determined with a micro-modification of a spectrophotometric glutathione cycling method. There was a slight increase in reduced glutathione (GSH) content during in vitro ageing of normal human fibroblasts. Fibroblasts from patients with Werner's syndrome of ceroid lipofuscinosis (Spielmeyer-Vogt syndrome) and healthy individuals exhibited similar patterns of GSH levels during in vitro ageing.The GSH content of non-proliferating confluent cultures of normal fibroblasts and of proliferating normal fibroblasts was identical. Moreover, autofluorescent “aged” cells isolated by cell sorting did not differ in GSH content from the non-autofluorescent cells in the same culture. It was concluded that the GSH content does not play a role in in vitro ageing, nor in the accumulation of autofluorescent material in human skin fibroblasts.     

Treatment of burns with cultured fibroblasts

Laboratory of Tissue Culture, Department of Pathological Anatomy, A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 4, pp. 400–402, April, 1990.     

Fidelity of histone synthesis in cultured human fibroblasts

The results in the literature to support Orgel's general error hypothesis of ageing only provide indirect evidence that errors in protein synthesis increase during senescence. This study attempts to provide direct evidence of errors in protein synthesis by measuring the misincorporation of 35S-methionine into histone H1 obtained from young and old fibroblasts (MRC-5). The conclusions that can be drawn from this study are: (a) the error level for the misincorporation of methionine into histone H1 is less than 7 methionines/10(5) amino acids and 2-3 methionines/10(4) amino acids in young and old cells respectively; (b) a methionine containing fraction associated with H1 is obtained after the final purification. The amount of this fraction increases with the age of the cell culture as does the number of methionine residues; (c) there is a variation in the complexity of H1 polypeptide chains, the complexity increasing with the age of cultured cells.     

Antigenicity of proteins from cultured synovial fibroblasts

We were able to characterize about 25 antigenic proteins of cultured synovial fibroblasts with apparent molecular weights of 15-230 kd by SDS-electrophoresis and immunoblotting using guinea pig antiserum. Some of these proteins were bovine-serum-derived, adsorbed onto fibroblasts from culture medium. Both rheumatoid and normal sera contained natural antibodies reacting with synovial fibroblast as well as bovine serum antigens when studied with immunoblotting. The amounts of antibodies were quantitated with enzyme immunoassay, measuring IgG and IgA class antibodies against plasma membrane preparations of rheumatoid and normal synovial fibroblasts. No rheumatoid arthritis-related changes were detected in synovial fibroblast protein antigens or antibodies against them in rheumatoid patient specimens.     

RhoA-induced changes in fibroblasts cultured on organic monolayers

Substantial previous work indicates that adherent cell morphology in culture is modulated by surface chemistry. Activation of the intracellular small molecular weight GTPase, RhoA, has recently been shown to play an essential role in controlling initiation of key integrin-mediated events in surface adhesion and proliferation. RhoA is interconvertible between an active, membrane-bound form and an inactive, cytosolic RhoGDI-bound form in response to integrin stimulation. This study reports the use of self-assembled functionalized organic alkylthiol monolayers (SAMs) as well-defined cell culture substrates to investigate the relationships between surface chemistry, RhoA activation and subsequent cell morphological and molecular level signal transduction responses in cells attaching to derivatized SAMs. Well-controlled alkylthiol surface chemistries were used to monitor and modulate the activation state of RhoA in attaching cells. Activation states were determined indirectly by fractionating cell lysates into membrane and cytosolic fractions by ultracentrifugation. Western blots were then performed, showing RhoA localization to be surface chemistry-dependent. RhoGDI levels and its intracellular localization were also shown to be surface-chemistry dependent. Cells cultured on -CH3 terminated SAMs, which normally exhibit a low-growth phenotype, were transfected with a constitutively active mutant form of RhoA. Subsequent cell morphological changes were observed on SAM surfaces by fluorescence microscopy. Results support surface chemistry influences on the activation state of RhoA mediated by adsorbed proteins and distinct changes in adherent cell morphology resulting from modulation of this activation state.     

新生大鼠心脏成纤维细胞体外分裂方式

目的观察体外培养状态下心脏成纤维细胞(CFs)的形态特征和分裂增殖方式。方法原代培养新生大鼠CFs,免疫荧光双标法分别检测波形蛋白和磷酸化组蛋白H3(H3P)共表达,波形蛋白和微管蛋白共表达。利用活细胞工作站和荧光显微镜观察处于不同分裂期的细胞时相。结果 CFs 0.5~1h贴壁,培养2~3 d细胞增殖旺盛,大量细胞进入有丝分裂期,其中多数CFs先隆起变圆,而少数则仍维持扁平状进行分裂。偶见无丝分裂现象。圆隆型有丝分裂时长为20~30min,扁平型分裂时长为40~50min。分裂前期胞核形状规则,H3P开始表达;前中期核膜破裂,纺锤体形成;中期染色体排列在赤道面,H3P高表达,纺锤体呈典型纺锤样;后期染色单体分开并移向两极,H3P在染色单体上持续表达;末期两个子核形成,微管聚集在两子核之间;胞质分裂期,两个子细胞形成,H3P不表达。结论体外培养的CFs存在圆隆型和扁平型两种有丝分裂形态,在分裂方式上存在有丝分裂和无丝分裂两种形式。     

The dynamics of fibronectin synthesis in cultured human fibroblasts

Two qualitatively different stages in fibronectin production by confluent cultures of human fibroblasts are demonstrated by immunomorphological and immunobiochemical methods. In the first stage fibronectin is accumulated exclusively in the cytoplasm, while excretory synthesis predominates in the second stage. The maximum production of fibronectin is observed on day 3 in culture. It is concluded that 3-day-old cultures of human fibroblasts are preferable for wound treatment. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, No. 5, pp. 575–577, May, 1996     

Regulation of interleukin 1 generation in immune-activated fibroblasts

In the present study we have demonstrated that fibroblasts can generate the inflammatory cytokine interleukin 1 (IL 1) under conditions similar to those abundant in cellular immune responses. Thus, induction of IL 1 requires a sequential two-step protocol which consists of preactivation of mouse embryo fibroblasts (MEF) with crude preparations of T cell or macrophage-derived conditioned media (CM; 72 h), followed by a challenge with lipopolysaccharide (LPS; 24 h). Unstimulated fibroblasts or such cells activated by either CM or LPS produced only low levels of IL 1, while a synergism between both signals was observed for obtaining maximal IL 1-like activity in MEF. Each of a series of individual recombinant lymphokines and cytokines (IL 2, granulocyte/macrophage-colony-stimulating factor, tumor necrosis factor, IL 1 beta and interferons-alpha, beta and gamma) was shown to serve as an efficient priming signal for the induction of IL 1. IL 1-like activity in fibroblasts was detected in cell lysates or associated with the producing-cell membrane but not in culture fluids. Immune-stimulated fibroblasts, activated under such experimental conditions, were shown to actively transcribe mRNA of both IL 1 genes (alpha and beta). For the expression of IL 1-specific mRNA in fibroblasts a single stimulus, provided by either LPS or a lymphokine/cytokine, was sufficient; however, a more intense signal was observed when both stimuli were applied. The IL 1-like biological activity of fibroblast origin was significantly reduced by anti-IL 1 alpha antibodies. Thus, fibroblasts, when activated by immune and bacterial products, generate IL 1 which in turn possibly amplifies cellular immune responses or inflammatory processes in connective tissues.     

Ascorbic acid stimulates production of glycosaminoglycans in cultured fibroblasts

The effect of ascorbic acid on collagen synthesis is well characterized. Proteoglycans and their attached glycosaminoglycans are components of the extracellular matrix closely associated with collagen fibers. We examined whether ascorbic acid also plays a role in glycosaminoglycan production. Synthesis and deposition of glycosaminoglycans into the extracellular matrix and secretion into the media were followed in human skin fibroblasts cultured in the presence and absence of ascorbic acid. Specific glycosaminoglycans were identified and quantitated by differential enzyme digestion, ion-exchange column chromatography, and cellulose-acetate electrophoresis. No major qualitative changes in glycosaminoglycans were observed. However, quantitatively, synthesis of glycosaminoglycans increased 30 to 90%, and deposition into the extracellular matrix increased 80% in the presence of ascorbic acid. This effect was only in part secondary to decreased levels of collagen, and the diminished capacity of underhydroxylated collagen to bind proteoglycans. The effect of ascorbic acid on extracellular macromolecules is thus more pervasive than previously assumed.     

Immunoperoxidase em localisation of cytoplasmic actin in cultured fibroblasts

Human smooth muscle autoantibody (SMA) of defined anti-actin specificity was tested by the indirect immunoperoxidase staining method on frozen sections of tissues and on cultured rat lung fibroblasts. The serum stained tissue sections and cultured fibroblasts in a pattern identical with that obtained by indirect immunofluorescence.Ultrastructural studies carried out on the immunoperoxidase stained cells showed that the long parallel filaments spanning the long axis of cultured fibroblasts seen by light microscopy correspond with the thick bundles of microfilaments.     

Vocal cord augmentation with cultured autologous fibroblasts

Bovine collagen is an acceptable agent for vocal cord medialization; however, it produces only a temporary effect. As a foreign protein bovine collagen is susceptible to host collagenase and can induce immune response. Autologous collagen has become recently available, but it is less effective as a medialization agent. The study examines human skin fibroblasts growing in culture. Human skin bioptates were taken from the retroauricular area. Fibroblasts in culture were tested for scar contractility and ability to produce type I collagen (by flow cytometry with labeled antibodies). After five passages in culture the cells produced normal type I collagen, exhibited normal contractility, and did not induce no tumors in nude mice. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 130, No. 8, pp. 207–209, August, 2000     

Trisomy 8 restricted to cultured fibroblasts.

In the course of re-examing cultured fibroblasts stored in liquid nitrogen from a patient with developmental retardation, solitary left kidney, and Wilms tumour, a cell line trisomic for chromosome 8 was found. Trisomy 8 was restricted to fibroblasts in the first 22 subcultures and was absent in later passages as well as in lymphocytes. A familial pericentric inversion of chromosome 2 was observed in three generations including the propositus but was though to be unrelated to the clinical problem. Multiple spontaneous chromosomal rearrangements were seen in several late subcultures.     

Vocal cord augmentation with cultured autologous fibroblasts

Bovine collagen is an acceptable agent for vocal cord medialization; however, it produces only a temporary effect. As a foreign protein bovine collagen is susceptible to host collage-nase and can induce immune response. Autologous collagen has become recently available, but it is less effective as a medialization agent. The study examines human skin fibroblasts growing in culture. Human skin bioptates were taken from the retroauricular area. Fibroblasts in culture were tested for scar contractility and ability to produce type I collagen (by flow cytometry with labeled antibodies). After five passages in culture the cells produced normal type I collagen, exhibited normal contractility, and did not induce no tumors in nude mice. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 8, pp. 207–209, August, 2000     

Biosynthesis of embryonic prealbumin by cultured human fibroblasts

Embryonic prealbumin (EPA) was found in human fibroblasts by immunodiffusion and immunofluorescence analysis. No quantitative or qualitative differences in the content and localization of this antigen were found between embryonic and adult human fibroblasts. It is concluded that human fibroblasts synthesize EPA, which can be used as a marker for cells of this type.Department of Biochemistry and Problem Laboratory for Immunochemistry of Malignant and Embryonic Tissues, N. I. Pirogov Second Moscow Medical Institute. Laboratory of General Cytogenetics, Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. P. Bochkov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 2, pp. 176–179, February, 1980.     

Thiol depletion induces apoptosis in cultured lung fibroblasts.

Thiol antioxidants are implicated in the protection of cells from oxidative injury. We studied the role of thiols in the regulation of apoptosis in cultured lung fibroblasts. Thiol depletion by culturing fibroblasts in cystine-free medium or with thiol-depleting agents induced oxidant accumulation and cell death by apoptosis. The cell death was prevented by the antioxidants ascorbic acid (AA) and catalase. Thiol depletion also induced leukotriene (LT) C4, LTD4, and LTE4 production and selective phosphorylation of p38-mitogen-activated protein kinase (MAPK) and its nuclear substrate ATF2. LT production and p38-MAPK phosphorylation were required for induction of apoptosis because thiol depletion-induced apoptosis was completely blocked by the 5-lipoxygenase inhibitor AA861, the LT antagonists FPL55712 and ONO1078, and the p38-MAPK inhibitor SB203580. LT production was inhibited by AA and p38-MAPK phosphorylation was inhibited by AA, AA861, and FPL55712. In an in vitro scratch wound model, repopulating fibroblasts at the wound margin, but not quiescent cells at the intact site, selectively underwent thiol depletion- induced apoptosis that was completely blocked by AA861, FPL55712, and SB203580. Thus, thiol depletion induces apoptosis through an ordered pathway involving oxidant accumulation, LT production, and p38-MAPK activation. Apoptosis of wound fibroblasts may be responsible for impaired wound healing in various organs, including the lung.     

Maternal inheritance of mitochondrial DNA polymorphisms in cultured human fibroblasts

We have isolated the total cellular DNA from the cultured diploid fibroblasts of a six-member, three-generation human family. Using a specific radioactive probe for mitochondrial (mt) sequences we have identified new polymorphic variants in this family for the Hhal restriction endonuclease cleavage pattern of the mtDNA. The inheritance of these cleavage patterns verifies the maternal inheritance of mtDNA through all three generations.     

Regulation of antral gastrin content

Five groups of rats were fasted for 3 days and injected with either NaCl or 5, 10, 20, or 40 micrograms/kg bombesin every 8 h. The animals were killed, and their serum and antral gastrin levels were compared with those of normally fed rats. Fasting reduced serum gastrin to 14% of control; antral gastrin was reduced to 21% of control. All doses of bombesin significantly increased serum gastrin in fasted rats, and 20 and 40 micrograms/kg significantly increased antral gastrin. A group of normally fed rats was also compared with one fed a liquid diet for 7 days. Half of each of these was injected with 20 micrograms/kg bombesin (3 times/day) and the other half with NaCl. Bombesin significantly increased serum and antral gastrin in the rats fed solid food. The liquid diet lowered serum and antral gastrin to 17 and 59% of control values, respectively. Bombesin injection totally prevented these decreases. These data indicate that food in the gastrointestinal tract is not required for either gastrin release or synthesis. Furthermore, the data suggest that gastrin synthesis is regulated primarily by gastrin release or by direct stimulation by bombesin rather than by specific food products.