Fibroblasts, vitamin

Factor VII. This is a vitamin K-dependent serine protease that functions in the extrinsic coagulation pathway and catalyzes the activation of Factors IX and X. Factor VII is present constitutively in the surface membrane of pericytes and fibroblasts in the adventitia of blood vessels, vascular endothehum, and monocytes. It is a single-chain glycoprotein of approximately 50,000 daltons. 

In vitro and ex vivo studies have shown that FATPs transport LCFAs and very long-chain fatty acids (VLCFAs) but no medium-chain fatty acids, fatty acid esters, or lipid-soluble vitamins [4]. LCFA transport is inhibited by prior protease treatment. Synthetic substrates for FATPs include 14C-labeled fatty acids and the fluorescently labeled fatty acid analogue C1 -BODEP Y-Cl 2. Using the latter substrate, differences in fatty acid uptake kinetics between FATP expressing 3T3 LI adipocytes and 3T3 LI fibroblasts, which are devoid of FATPs, can be readily appreciated (Fig. 2). 

Vitamins and lipids are often required for animal cells to grow in serum-free medium. Phosphoethanolamine and ethanolamine are key additives that facilitate the growth of the mammary tumor cell line 64024 (Kano-Sueoka and Errick, 1981). In addition, ethanolamine promotes the growth of human lymphocytes and mouse hybridoma cells. Short-term cultures of human diploid lung and foreskin fibroblasts grow in medium that includes among its supplements soybean lecithin, cholesterol, sphingomyelin, and vitamin E. 

Offord, EA, JC Gautier, O Avanti et al. 2002. Photoprotective potential of lycopene, beta-carotene, vitamin E, vitamin C and camosic acid in UVA-irradiated human skin fibroblasts. Free Radio Biol Med 32(12) 1293-1303. 

In contrast to transition metals iron and copper, which are well-known initiators of in vitro and in vivo lipid peroxidation (numerous examples of their prooxidant activities are cited throughout this book), the ability of nontransition metals to catalyze free radical-mediated processes seems to be impossible. Nonetheless, such a possibility is suggested by some authors. For example, it has been suggested that aluminum toxicity in human skin fibroblasts is a consequence of the enhancement of lipid peroxidation [74], In that work MDA formation was inhibited by SOD, catalase, and vitamins E and C. It is possible that in this case aluminum is an indirect prooxidant affecting some stages of free radical formation. 

Homocystinuria can be treated in some cases by the administration of pyridoxine (vitamin Bs), which is a cofactor for the cystathionine synthase reaction. Some patients respond to the administration of pharmacological doses of pyridoxine (25-100 mg daily) with a reduction of plasma homocysteine and methionine. Pyridoxine responsiveness appears to be hereditary, with sibs tending to show a concordant pattern and a milder clinical syndrome. Pyridoxine sensitivity can be documented by enzyme assay in skin fibroblasts. The precise biochemical mechanism of the pyridoxine effect is not well understood but it may not reflect a mutation resulting in diminished affinity of the enzyme for cofactor, because even high concentrations of pyridoxal phosphate do not restore mutant enzyme activity to a control level. 

The fibroblasts do not convert cyanocobalamin or hydroxocobalamin to methylcobalamin or adenosyl-cobalamin, resulting in diminished activity of both N5-methyltetrahydrofolate homocysteine methyltransferase and methylmalonyl-CoA mutase. Supplementation with hydroxocobalamin rectifies the aberrant biochemistry. The precise nature of the underlying defect remains obscure. Diagnosis should be suspected in a child with homocystinuria, methylmalonic aciduria, megaloblastic anemia, hypomethioninemia and normal blood levels of folate and vitamin B12. A definitive diagnosis requires demonstration of these abnormalities in fibroblasts. Prenatal diagnosis is possible. 

The receptor for RANKL is RANK, also known as ODAR (Anderson et al. 1997 Hsu et al. 1999). RANK is expressed in osteoclast precursors, mature osteoclasts, condrocytes, fibroblasts, and immune system cells (Anderson et al. 1997 Hsu et al. 1999). The binding of RANKL with RANK on preosteoclasts initiates the OCS and the activation of osteoclasts (Anderson et al. 1997 Hsu et al. 1999 Nakagawa et al. 1998). RANK-deficient mice display a phenotype characterized by osteopetrosis and several defects in the immune system similar to that observed in RANKL-deficient mice (Dougall et al. 1999). Consistent with this hypothesis, RANK-deficient mice are resistant to bone resorption induced by TNF-a, IL-l/J, or vitamin D3 (Li et al. 2000). In agreement with this, mice deficient in molecules implied in the transduction pathway from RANK like TRAF-6 or NF-/c Bl/NK-/c B2 also show an osteopetrotic phenotype,... 

Three hormones serve as the principal regulators of calcium and phosphate homeostasis parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and the steroid vitamin D (Figure 42-2). Vitamin D is a prohormone rather than a true hormone, because it must be further metabolized to gain biologic activity. PTH stimulates the production of the active metabolite of vitamin D, l,25(OH)2D. l,25(OH)2D, on the other hand, suppresses the production of PTH. l,25(OH)2D stimulates the intestinal absorption of calcium and phosphate. l,25(OH)2D and PTH promote both bone formation and resorption in part by stimulating the proliferation and differentiation of osteoblasts and osteoclasts. Both... 

Conversion of 7-dehydrocholesterol to vitamin D3 and metabolism of D3 to l,25(OH)2D3 and 24,25(OH)2D3. Control of the latter step is exerted primarily at the level of the kidney, where low serum phosphorus, low serum calcium, and high parathyroid hormone favor the production of l,25(OH)2D3, whereas fibroblast growth factor 23 inhibits its production. The inset shows the... 

In this hereditary disease up to 1 - 2 g of methylmalonic acid per day (compared to a normal of <5 mg/day) is excreted in the urine, and a high level of the compound is present in blood. Two causes of the rare disease are known/ One is the lack of functional vitamin B12-containing coenzyme. This can be a result of a mutation in any one of several different genes involved in the synthesis and transport of the cobalamin coenzyme.6 Cultured fibroblasts from patients with this form of the disease contain a very low level of the vitamin B12 coenzyme (Chapter 16), and addition of excess vitamin B12 to the diet may restore coenzyme synthesis to normal. Among elderly patients a smaller increase in methylmalonic acid excretion is a good indicator of vitamin B12 deficiency. A second form of the disease, which does not respond to vitamin B12, arises from a defect in the methylmalonyl mutase protein. Methylmalonic aciduria is often a very severe disease, frequently resulting in death in infancy. Surprisingly, some children with the condition are healthy and develop normally.3 1... 

For some time, the effects of and responses to vitamin E have been interpreted in terms of an antioxidant mechanism of action. However, several observations have raised the question as to whether other mechanisms could be involved. For example, the effects of selenium and vitamin E on growth and polyunsaturated fatty acid synthesis in cultured mouse fibroblasts could not be reproduced by artificial antioxidants [198, 199]. The specific requirement of (+ )-a-toco-pherol for the phenotypic differentiation of the rotifer [200] may not be through an antioxidant mechanism. The effects of vitamin E on differentiation of neuroblastoma cells [201] and metamorphosis of various species [202] are likely to be due to a growth-factor-like action. A study on the interaction... 

The vitamin can be carried by the high-affinity receptor for low-density lipoprotein in fibroblasts [251]. It has been suggested to act in vivo as a specific enzyme inhibitor (of lipoxygenase). A vitamin-E-lipoxygenase complex in vivo could terminate the initiation of free radicals and other oxidized products. The binding of the vitamin to the enzyme is probably through the hydrophobic chain and involves one peptide [135]. 

Traditionally, the production of mAbs uses complex culture media containing glucose and amino acids as the main sources of carbon for cell metabolism, as well as vitamins, micronutrients and sometimes animal serum, usually fetal bovine serum. Chapter 5 provides a discussion on composition of culture media and recent trends in the search for formulas that do not require the use of animal serum, or of proteins of animal origin. These serum-free formulations use substitutes such as peptones, epithelial and fibroblast growth factors, hydrolysates, yeast extract, choline, and inositol. For the production of mAbs, various serum-free formulas are available, some of these developed specifically for a given cell line (Chu and Robinson, 2001). The development of those media is easier for non-anchorage-dependent cells, such as those used for mAb production. Thus, approximately 50% of the antibodies for therapeutic use are already produced using serum-free media. In some circumstances, the elimination of serum should be accompanied by the addition of other substances with the same shear stress protective effect of serum proteins,... 

The structure of ascorbic acid resembles an alpha-hydroxy acid, which is generally not appreciated. Ascorbic acid is present in most fruits, and may underlie some of the effects attributed to fruit extracts. Vitamin C has pronounced HA-stimulating effects in the fibroblast assay. But its antioxidant activity confounds the effects it may induce. The deposition of HA is stimulated when Vitamin C is added to cultured fibroblasts. The most profound changes occur in the compartmentalization of HA. The preponderance of the enhanced HA becomes cell-layer instead of being secreted into the medium.240,261 The chemical reactions catalyzed by ascorbic acid that bind HA to cell or matrix components are not known. 

Metabolic studies failed to detect any methylmalonyl-CoA mutase in skin fibroblasts from the first patient, while in the second patient the level was found to be 10% of normal. Two forms of vitamin B12 were tried on each of the patients, cyanocobalamin and hydroxocobalamin, both without apparent effect. 

Homocystinuria may result from one or several abnormalities in the mechanism whereby homocysteine is methylated to form methionine. About half of the patients respond to treatment with pyridoxine and it is thought that the vitamin overcomes a block at the homocysteine/cystathionine level by mass action (C23). However, Schuh et al. (S22) have recently described a patient who responded to vitamin B12. The infant presented with severe developmental delay, homocystinuria, and a megaloblastic anemia. Treatment with cyanocobalamin was without effect but treatment with hydroxocobalamin resulted in a rapid clinical improvement, and the homocystinuria disappeared. Methionine synthetase activity in cell extracts was normal, while cultured fibroblasts showed an absolute growth requirement for methionine. The defect appeared to be limited to methyleobalamin accumulation and an inability to transfer the methyl group from 5-methyltetrahydrofolate to homocysteine. 

The striking increases in the formation of tritiated water and tritiated hydroxyproline on in vitro addition of ascorbate are consistent with a function of this vitamin in hydroxylation—probably at step 3. The present results do not support a systemic ascorbic acid-mediated effect, the belief that ascorbic acid functions in the maintenance of collagen, or acts by stimulating maturation of the fibroblasts in the system under study here. The present data do not support the possibility that intermediates containing hydroxyproline accumulate in scurvy. The proposal that ascorbic acid is involved in the hydroxylation reaction itself is consistent with studies on the nonenzymatic hydroxylation of proline (4) and on enzymatic hydroxylation of other compounds (5, 20, 21, 44, 51). 

Cibacron blue F3GA Human plasma binding protein for vitamin D, human fibroblast interferon, glutamine synthetase, T4 DNA ligase, aspartate carbamoyltransferase... 

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