Carnosine stimulates vimentin expression in cultured rat fibroblasts.

Two-dimensional electrophoretic gel profiles were compared between rat 3Y1 fibroblasts cultured in the presence and absence of 30 mM L-carnosine (beta-alanyl-L-histidine) for one week without any replenishment of medium. While a number of cellular proteins changed their expression levels by the addition of carnosine, we identified one of the most prominently varied proteins as vimentin. Immunoblot analysis with anti-vimentin antibody demonstrated that the vimentin levels increased about 2-fold after one-week culture in the presence of carnosine. We also confirmed that the increase of vimentin expression was dependent on the concentration of carnosine added to the medium. Moreover, when cultured cells were stained with anti-vimentin antibody and observed by light microscopy, most cells grown in the presence of carnosine were found to have markedly developed vimentin filaments. The increase of vimentin expression was also observed by adding with carnosine related dipeptides, N-acetylcarnosine and anserine.     

Hydrocortisone stimulates fibronectin synthesis in cultured fibroblasts

The effect of hydrocortisone on fibronectin synthesis was investigated in cultured skin fibroblasis. Confluent cells were treated with hydrocortisone (10^?7 M to 10^?5 M) for 2 days and labeled with [³H]proline for 24 h. Fibronectin levels in both the culture medium and the cell layer were studied by gelatin-Sepharose affinity chromatography and SDS-polyacrylamide gel electrophoresis. In control cultures of human fetal skin fibroblasts, fibronectin constituted 8% of the total labeled proteins in the medium. The proportion of fibronectin increased to 13.1% at 10^?7 M hydrocortisone, 15.5% at 10^?6 M and to 19.4% at 10^?5 M. The proportion of fibronectin associated with the cell layer remained at 2-3% of total [³H]prolne-labeled proteins and did not increase with hydrocortisone exposure. The stimulating effect of hydrocortisone on medium fibronectin was also demonstrated in cultured human newborn foreskin fibroblasts and in rabbit skin fibroblasts.     

Ascorbic acid stimulates collagen production without altering intracellular degradation in cultured human skin fibroblasts

The influence of ascorbic acid on intracellular degradation of collagen synthesized by cultured human-skin fibroblasts was examined. In confluent cells maintained in 0.5% serum-supplemented medium, ascorbic acid had no significant effect on collagen degradation measured with hydroxyproline as the marker. Similar results were obtained when collagen degradation was measured with the marker hydroxylysine, the cellular synthesis of which is independent of ascorbic acid. The stimulatory effects of ascorbic acid on collagen production therefore cannot be explained by a change in the rate of degradation. Ascorbic acid acts at some as yet undetermined level to increase the rate of collagen synthesis.     

Modulation of fibroblast proliferation by oxygen free radicals.

The major unexplained phenomenon in fibrotic conditions is an increase in replicating fibroblasts. In this report we present evidence that oxygen free radicals can both stimulate and inhibit proliferation of cultured human fibroblasts, and that fibroblasts themselves release superoxide (O2.-) free radicals. Fibroblasts released O2.- in concentrations which stimulated proliferation, a finding confirmed by a dose-dependent inhibition of proliferation by free radical scavengers. Oxygen free radicals released by a host of agents may thus provide a very fast, specific and sensitive trigger for fibroblast proliferation. Prolonged stimulation may result in fibrosis, and agents which inhibit free radical release may have a role in the prevention of fibrosis.     

The aging brain, metals and oxygen free radicals.

In this overview we bring together certa in facts and concepts that support the theory that the aging of "disease-free" brain is a consequence of the accumulated cellular-molecular modifications caused by oxygen free radicals. The relevance of transition metals, especially iron ions, in the production of oxygen free radicals, initiation of oxidative chain-reactions and in site-specific molecular modifications is documented. Mitochondria are identified as the major source of oxygen free radicals, and mitochondrial DNA is a likely target. Special attention is given to iron-sulfur clusters as sources of reactive iron and sites of modifications. Potential mechanisms by which oxygen free radicals can alter membrane receptors and intracellular signaling are cited. Although the evidence is still correlative, the oxygen free radical theory has strong experimental support and has promise for facilitating a better understanding of the "disease-free", aging brain.     

Sulfur oxidation of free methionine by oxygen free radicals

The oxidation of free methionine to methionine sulfoxide by chemically or enzymatically generated oxygen free radicals is presented. The physiological significance of this process in living cells is suggested.     

Altered glycosaminoglycan production in cultured osteogenesis-imperfecta skin fibroblasts.

Collagen and glycosaminoglycan syntheses were studied in skin fibroblasts cultured from patients with osteogenesis imperfecta (OI) and from age-matched controls. Collagen synthesis (measured as protein-bound [3H]hydroxyproline) was decreased in all four OI cell lines studied in the present experiments, comprising 16-24% of total protein synthesis (40% in normal cells). Hyaluronic acid production in OI skin fibroblasts per cell was higher than in age-matched controls, but the production of sulphated glycosaminoglycans was at the normal level. Thus the ratio of the hyaluronic acid and sulphated-glycosaminoglycan radioactivities was markedly higher in OI cultures than in control cultures, especially at the exponential phase of growth where the synthesis of hyaluronic acid was highest. Hyaluronic acid in OI had a normal molecular weight when determined by gel filtration on Sepharose 2B. The removal of high-molecular-weight hyaluronic acid from the medium by hyaluronidase had no effect on the rate of collagen secretion in OI cell line 1 (A.T.C.C. 1262), in which the rate of collagen secretion was lowest.     

Multinucleation in mouse fibroblasts cultured in methocel medium

3T3-4E cells formed multinucleate cells with high frequency when incubated in methocel medium. The experiment with hydroxyurea and the cytological observation of mitoses showed that multinucleate cells were produced by nuclear division in the absence of cytokinesis. When transferred onto a solid substratum, most of the multinucleate cells divided within seven hours into mononucleate cells through the process of cytoplasmic division, indicating that cell spreading induced cytokinesis. Other mouse fibroblast lines examined so far showed only the low frequency of multinucleation. These findings indicate that in 3T3-4E cells cultivated in methocel, nuclear division occurred independently of cytokinesis, and that cytokinesis was also anchorage-dependent. This system will be available for studying cytoplasmic division of mammalian cells.     

Alterations in cardiac contractile proteins due to oxygen free radicals.

In view of the potential role of free radicals in the genesis of cardiac abnormalities under different pathophysiological conditions and the importance of contractile proteins in determining heart function, this study was undertaken to examine the effects of oxygen free radicals on the rat heart myofibrils. Xanthine plus xanthine oxidase (X + XO) which is known to generate superoxide anions (O2-) and hydrogen peroxide (H2O2), an activated species of oxygen, was found to decrease Ca(2+)-stimulated ATPase activity, increase Mg(2+)-ATPase activity and reduce sulfhydryl (SH) group contents in myofibrils; these effects were completely prevented by superoxide dismutase (SOD) plus catalase (CAT). Both H2O2 and hypochlorous acid (HOCl), an oxidant, produced actions on cardiac myofibrils similar to those observed by X + XO. The effects of H2O2 and HOCl were prevented by CAT and L-methionine, respectively. N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), inhibitors of SH groups, also produced effects similar to those seen with X + XO. Dithiothreitol (DTT), a well known sulfhydryl-reducing agent, prevented the actions of X + XO, H2O2, HOCl, NEM and DTNB. These results suggest that marked changes in myofibrillar ATPase activities by different species of oxygen free radicals may be mediated by the oxidation of SH groups.     

Role of oxygen free radicals in carcinogenesis and brain ischemia

Even though oxygen is necessary for aerobic life, it can also participate in potentially toxic reactions involving oxygen free radicals and transition metals such as Fe that damage membranes, proteins, and nucleic acids. Oxygen free radical reactions and oxidative damage are in most cases held in check by antioxidant defense mechanisms, but where an excessive amount of oxygen free radicals are produced or defense mechanisms are impaired, oxidative damage may occur and this appears to be important in contributing to several pathological conditions including aging, carcinogenesis, and stroke. Several newer methods, such as in vivo spin-trapping, have become available to monitor oxygen free radical flux and quantitate oxidative damage. Using a combination of these newer methods collectively focused on one model, recent results show that oxidative damage plays a key role in brain injury that occurs in stroke. Subtle changes, such as oxidative damage-induced loss of glutamine synthetase activity, may be a key event in stroke-induced brain injury. Oxygen free radicals may play a key role in carcinogenesis by mediating formation of base adducts, such as 8-hydroxyguanine, which can now be quantitated to very low levels. Evidence is presented that a new class of free radical blocking agents, nitrone spin-traps, may help not only to clarify if free radical events are involved, but may help prevent the development of injury in certain pathological conditions.     

Reactive oxygen in skeletal muscle. II. Extracellular release of free radicals.

We have tested the hypothesis that diaphragm muscle fibers release superoxide anion radicals (O2-.) into the extracellular space. Fiber bundles were isolated from rat diaphragm and incubated in Krebs-Ringer solution containing cytochrome c (10(-5) M), a standard assay for O2-.. Bundles were either passive or active, i.e., directly stimulated to contract rhythmically. After 1 h, absorbance of reduced cytochrome c in the incubation medium was measured at 550 nm. Absorbance was greater in medium exposed to passive muscle than in medium without muscle (P < 0.01), indicating O2-. release by passive muscle. Absorbance was greater in medium exposed to active muscle than in that exposed to passive muscle (P < 0.01), an increase inhibited by superoxide dismutase (10(3) U/ml). Active bundles fatigued; bundles developing the lowest final stresses produced the greatest absorbance increases (P < 0.001), suggesting that the magnitude of fatigue was inversely related to O2-. release. We conclude that O2-. is released by diaphragm myocytes into the interstitium and surrounding medium, a process accelerated by fatiguing muscular contractions.     

Mitochondria, oxygen free radicals, disease and ageing

Superoxide is generated by the mitochondrial respiratory chain. The transformation of this superoxide into hydrogen peroxide and, under certain conditions, then into hydroxyl radicals is important in diseases where respiratory chain function is abnormal or where superoxide dismutase function is altered, as in amyotrophic lateral sclerosis. In addition, these reactive oxygen species can influence the ageing process through mechanisms involving mutagenesis of mtDNA or increased rates of shortening of telomeric DNA.     

Lys-bradykinin stimulates Na+ influx and DNA synthesis in cultured human fibroblasts

The effect of Lys-bradykinin on net Na+ influx in serum-deprived cultured human fibroblasts (HSWP cells) was measured. It was found that Lys-bradykinin stimulates net Na+ influx with a K1/2 of 1 nM. When Lys-bradykinin was combined with epidermal growth factor, vasopressin and insulin, the net Na+ influx stimulated was comparable with that measured in response to 10% serum. The above combination of growth factors also was found to stimulate DNA synthesis to 92% of the serum-stimulated levels in HSWP cells and to support cell growth over a period of 6 days. In addition, it was observed that the Na+ influx stimulated by Lys-bradykinin or by the combination of four growth factors was completely inhibited by the amiloride analog benzamil. Thus Lys-bradykinin presumably stimulates the same Na+ transport system as is stimulated by serum. Finally, the present results suggest that an increase in Na+ influx always accompanies DNA synthesis in HSWP cells. On the basis of these observations, it can be hypothesized that Na+ influx is a necessary event to stimulate DNA synthesis.     

In vitro production of free oxygen radicals induced by pulsed ultrasounds in whole blood exposed to diagnostical frequencies and intensities.

DNA alkalinization experiments on lymphocytes from sonicated whole blood and on in vitro cultured lymphocytes in presence of free radical scavengers (superoxide dismutase, catalase and mannitol) showed that lesions inflicted upon DNA by pulsed ultrasounds could be ascribed to production of free radicals (O2-, OH.) and H2O2, which could mediate the production of still unidentified organic radicals, likely to be responsible for DNA damage.     

Phytohemagglutinin isolectin stimulation of human lympocytes cultured in serum free medium.

The ability of the red kidney bean (Phaseolus vulgaris) Phytohemagglutinin (PHA) isolectins L4 and E4 to transform human lymphocytes cultured in serum free or serum supplemented medium was studied. Previous similar studies done in fetal bovine serum (FBS) supplemented medium have shown L4 to be 30–60 times more potent a mitogen than E4. In serum free conditions, this difference was much less, L4 being only 3–9 times more potent than E4. In serum free medium, optimal mitogenic concentrations of L4 and E4 were 1.1.–3.3 ug/culture and 3.3–10 ug/culture respectively as compared to 3.3–10 ug/culture and 90–270 ug/culture for L4 and E4, respectively, in FBS suplemented medium. L4 stimulated lymphocytes in serum containing medium transform more rapidly than do L4 treated cells cultured in serum free conditions. Fetuin added to serum free cultures of lymphocytes more effectively inhibited transformation induced by E4 than by L4. Although the binding of 1251 E4 and L4 to lymphocytes was greatly reduced by the addition of FBS to the medium, the reduction in E4 binding was much greater than that in L4 binding. Neither L4 or E4 caused the death of lymphocytes cultured in serum free or FBS supplemented medium. These results confirm the previously described difference in mitogenic potential between L4 and E4. However, the results of earlier studies done in FBS supplemented medium include several artifacts related to the differential interaction of PHA isolections with fetuin and other serum glycoproteins.     

Protective effects of ceruloplasmin against electrolysis-induced oxygen free radicals in rat heart.

The potentially injurious effects of oxygen-derived free radicals (OFR) on the myocardium can be prevented in part by pretreatment with OFR scavengers or antioxidants. Since ceruloplasmin (CP) has been shown to possess potent antioxidant activity and scavenge a variety of OFR in vitro, we have undertaken to study its protective effects against myocardial injury induced by OFR. CP was freshly purified by a fast method that minimized proteolytic enzyme degradation. Free radicals were generated by the electrolysis (10 mA DC current for 1 min) of a Krebs-Henseleit solution perfusing an isolated rat heart preparation under constant pressure conditions. CP (0.25 microM) afforded 80 and 63% protection (n = 8, p less than 0.05), respectively, against the deleterious effects of electrolysis-induced OFR on left ventricular pressure and coronary flow. The increase in left ventricular end diastolic pressure used here as an index of heart failure did not occur in the presence of 0.25 microM CP. Moreover, CP significantly reduced the increase of norepinephrine washout in the effluent perfusate after electrolysis suggesting a protection against free radical-induced injury to sympathetic nerve endings.     

Human breast milk stimulates prostaglandin synthesis in cultured human skin fibroblasts

Effect of human breast milk or its fractions on prostaglandin synthesis was investigated in cultured human skin fibroblasts. Prostaglandins released into the media were measured by radioimmunoassay. Incorporation of breast milk (2% level) into 10% fetal calf serum media (for 48 hours) stimulated the synthesis of 6-keto-PGF1 alpha (stable product of prostacyclin) by 800%. This stimulating effect of milk persisted after cold acetone extraction to remove phospholipids and potentiated further after dialysis. Stimulation by one of the commercial formulas (Similac) was less than 50% of the milk effect. Milk also stimulated PGE2 synthesis, although to a much lesser degree. These studies show for the first time that a) human breast milk contains potent factor(s) capable of influencing prostaglandin synthesis and suggest that b) these factors might have a role in the development of lipid synthetic pathways during early life.