Biosynthesis cell culture models

The vast majority of research focused on selenium in biology (primarily in the fields of molecular biology, cell biology, and biochemistry) over the past 20 years has centered on identification and characterization of specific selenoproteins, or proteins that contain selenium in the form of selenocysteine. In addition, studies to determine the unique machinery necessary for incorporation of a nonstandard amino acid (L-selenocysteine) during translation also have been central to our understanding of how cells can utilize this metalloid. This process has been studied in bacterial models (primarily Escherichia colt) and more recently in mammals in vitro cell culture and animal models). In this work, we will review the biosynthesis of selenoproteins in bacterial systems, and only briefly review what is currently known about parallel pathways in mammals, since a comprehensive review in this area has been recently published. Moreover, we summarize the global picture of the nonspecific and specific use of selenium from a broader perspective, one that includes lesser known pathways for selenium utilization into modified nucleosides in tRNA and a labile selenium cofactor. We also review recent research on newly identified mammalian selenoproteins and discuss their role in mammalian cell biology. 

Nevertheless, there are many questions still open because of problems to detect enzyme activities corresponding to each step of the pathway. The model of biosynthesis pathway was put together by studying the metabolism of exogenously applied intermediates in cell cultures of various origins and combining these results with data of native brassinosteroid patterns. It is more or less accepted that there are three pathways in parallel, the early and the late C6 oxidation pathway, as well as the 24/ -epimers follow ing the same route. Some observations in the analysis of native brassinosteroid patterns suggest a possible connection between the pathways. It was shown that seeds of Arabidopsis contain castasterone and 24-epi-brassinolide [34]. Also members of both 24-epimers, brassinolide and 24-epi-brassinolide were detected in tomato seeds [Winter, unpublished]. 

Figure 7.6 Stimulus-response coupling models for the induction of alkaloid biosynthesis in plant cell cultures. A. Terpenoid indole alkaloids. B. Benzylisoquinoline alkaloids.

With respect to fatty acid biosynthesis, plant cell suspension cultures resemble cells in green leaves of intact plants. In leaves of Flacourtiaceae, straight-chain and cyclic fatty acid biosynthesis is located in chloroplasts. The locus of this biosynthetic process in seeds, the main cyclopentenyl fatty acid-producing organ, is not known the results obtained with cultured cells may indicate localization in the cytosol. However, the model function of cell cultures should be considered with caution. 

Some biochemical functions defined by the Arabidopsis dwarf mutants were later confirmed by heterologous expression of genes and by in vivo conversion of postulated substrates [17-20]. As part of these physiological and biochemical studies, tomato cell suspension cultures have also been established to investigate intermediates and enzymes of brassinosteroid biosynthesis and metabolism [21-23]. Enzyme activities from partially purified protein extracts were first detected in this model system [24]. 

Burstein and Hunter (1995) observed that THC stimulated the biosynthesis of anandamide in neuroblastoma cells employing either ethanolamine or arachidonic acid as the label. Anandamide bios5mthesis has also been shown to occur in primary cultures of rat brain neurons labelled with [H]-ethanolamine when stimulated with ionomycin, a Ca ionophore (Di Marzo et al. 1994). These authors proposed an alternate model for the biosynthesis of anandamide in which N-arachidonoyl phosphatidyl ethanolamine is cleaved by a phospholipase D activity to yield phosphatidic acid and ararchidonoylethanolamide. This model is based upon extensive studies undertaken by Schmid and collaborators (1990), who have shown that fatty acid ethanolamide formation results from the N-acylation of phosphatidyl ethanolamine by a transacylase to form N-acyl phosphatidylethanolamine. Possibly resulting from postmortem changes, this compound is subsequently hydrolyzed to the fatty acid ethanolamide and the corresponding phosphatide by a phosphodiesterase, phospholipase D. 

GPI-deficient mammalian cells are viable in tissue culture and many GPI-deficient mutant cell lines have been established. However, GPI deficiency has major consequences at the level of tissues and the whole body. This was revealed in transgenic mouse models in which the PIG-A gene (required for the first step of GPI biosynthesis) was knocked out in specific tissues or in the whole animal. For example, keratinocyte-specific disruption of PIG-A caused abnormal development of skin leading to death of the mutant mice a few days after birth (M. Tarutani, 1997), and disruption of PIG-A in the whole animal resulted in embryos that did not develop beyond day 9 of gestation (M. Nozaki, 1999). A somatic mutation of PIG-A in multipotent hematopoietic human stem cells causes paroxysmal nocturnal hemoglobinuria, an acquired hemolytic disease in humans characterized by abnormal activation of complement on erythrocytes due to a deficiency of GPI-anchored complement regulatory proteins such as decay accelerating factor (N. Inoue, 2003). This disease is characterized by intravascular hemolysis and anemia. 

In several different models, treatment of cultured cells with anandamide as well as other cannab-inoids results iu increased arachidonic acid release and eicosanoid biosynthesis. Wartmaim et al. (1995) observed such an effect and gave evidence that MAP kinase contributes to this response. Exposure of Wl-38 hbroblasts to anandamide causes increased MAP kinase activity and increases the phosphorylation of cytoplasmic phospholipase A2 (cPLA2) resulting in its activation and the... 

Synchronized cultures of Chlorella ellipsoidea were used as a model system to identify biochemical modes of action of two herbicides/ butamiphos (0-ethyl--0-(3-methyl-6-nitropheny1)-N-sec-buty1-phosphorothioamidate, formerly coded S-2846), and chlorpropham (isopropyl inch lorocarban il ate). Cell cycle studies showed that butamiphos inhibited cell division, whereas its effects on respiration and general biosynthesis were slight. Confirmatory experiments with onion root apices showed that mitosis was blocked at the metaphase and that the spindle apparatus was disrupted. 

Several strategies to increase the production of electron shuttles have been developed to improve the MFC performance in the model exoeleetrogens. For Shewanella species, flavins (riboflavin and flavin mononucleotide) are the most well-known self-secreted electron shuttles. Using deletion mutants lacking various Mtr-associated proteins, the significance of the Mtr extracellular respiratory pathway for the reduction of flavins has been demonstrated. The decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of Mtr-associated proteins activity. Weakly acidic pH resulted in poor performance of the MFC and low riboflavin concentrations in the bacterial cultures, while enhanced electrochemical activity of riboflavin was reported at alkaline pH. The increase of riboflavin biosynthesis by Shewanella at the alkaline condition underlies the improvement in the electricity output in MFCs. ... 

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