Biologic Drug Substance and Drug Product Manufacture
Anthony J. Hickey, Sandro R.P. da Rocha in Pharmaceutical Inhalation Aerosol Technology, 2019
The nutritional requirements of the auxotrophs form the basis of selection of cells post-transfection for those expressing exogenous proteins and has been utilized to also increase the transgene copy number and expression levels. Commonly used auxotrophs of CHO cells are the DG44 and DUKXB-11 host cell lines that are deficient in the dihydrofolate reductase (DHFR) enzyme. This enzyme reduces dihydrofolic acid to tetrahydrofolic acid, an essential cellular biochemical product for purine and thymidylate synthesis. Cells lacking the DHFR enzyme require glycine, hypoxanthine, and thymidine to grow (and are thus called triple auxotrophs). This property is utilized for the expression of a heterologous gene by co-transfection with a functional copy of the DHFR gene, such that the transfected cells do not require exogenously supplied glycine, hypoxanthine, and thymidine in the growth medium. Hence, cell culture in a deficient growth medium allows the selection of transfected cells. Another recombinant DNA expression strategy is the glutamine synthetase (GS) system utilized in GS deficient CHO cells. GS catalyzes the production of glutamine, an essential amino acid required for cellular metabolism, from glutamate and ammonia. Upon co-transfection of the recombinant gene and GS into host cells, the cells are cultivated in glutamine-free media to select for producing clones.
Vitamins
Frank A. Barile in Barile’s Clinical Toxicology, 2019
Pteroylglutamic acid (folate) is found in leafy vegetables, organ meats, and yeast. Physiologically, folic acid is required for nucleoprotein synthesis and the maintenance of hematopoiesis. It is converted intracellularly to tetrahydrofolic acid, which acts as a cofactor in the biosynthesis of purines and thymidylates of nucleic acids. Consequently, deficiency of folic acid (along with vitamin B12) is often seen during pregnancy and in malabsorption conditions and is responsible for defective DNA synthesis. The condition is manifested by the production of enlarged immature red cells characteristic of megaloblastic anemia. Although folic acid is relatively nontoxic, some allergic, central nervous system (CNS), and GI reactions have been noted with large doses (Table 22.4).
Recent Studies on the Neoplasia and Abnormal Cellular Differentiation in Methyl Insufficiency
Maryce M. Jacobs in Vitamins and Minerals in the Prevention and Treatment of Cancer, 2018
These findings led us to reconsider other situations in which humans may be particularly sensitive to folate deficiency. Folic acid deficiency is fairly common in humans.27 Indeed, among four species investigated the hepatic levels of tetrahydrofolic acid (THFA) were lowest in humans; further, the hepatic content of dihydrofolate reductase (DHFR) was also much less in humans than in the monkey or the rat (Table 3).28 One might thus expect humans to be particularly sensitive to toxic agents which stress the folate pool. This is indeed the case with methanol, whose toxic metabolic, formic acid is detoxified by metabolism in the C1-folate pool. The relative toxicities of methanol to humans, monkeys, rats, and mice is inversely proportional to the capacity of the species to metabolize formic acid via the reduced folate pool.28 Thus, the limited experimental and clinical evidence available indicates that humans appear to be more sensitive to dietary folate deficiency than are rodents.
Pneumocystis jirovecii: a review with a focus on prevention and treatment
Published in Expert Opinion on Pharmacotherapy, 2021
R. Benson Weyant, Dima Kabbani, Karen Doucette, Cecilia Lau, Carlos Cervera
TMX-SMX is a combination of two antimicrobials that work together to inhibit the folic acid metabolic pathway. The two drugs work on different steps in the pathway, giving them synergistic activity (Figure 1). Sulfamethoxazole (SMX) is a sulfonamide that is structurally similar to para-aminobenzoic acid (PABA) and competes with PABA for the enzyme dihydropteroate synthetase (DHPS). DHPS converts PABA to dihydropteroic acid, an intermediate of tetrahydrofolic acid. Trimethoprim (TMP) is a competitive inhibitor of an enzyme further down the pathway, dihydrofolate reductase (DHFR). DHFR converts dihydrofolic acid to tetrahydrofolic acid. Tetrahydrofolic acid is essential for synthesis of purines, a component of DNA, and therefore essential for cellular reproduction. Humans can obtain and incorporate folate from food, but bacteria (and PJ) must create it endogenously.
The association between homocysteine and systemic sclerosis: A review of the literature and meta-analysis
Published in Modern Rheumatology, 2018
Yan-Jie Zhang, Li Zhang, Xiao-Lei Huang, Yu Duan, Li-Juan Yang, Jing Wang
Homocysteine (Hcy) is a nonessential sulfur-containing amino acid mainly derived from the diet of methionine, which occurs naturally in the blood plasma. Its chemical properties showed a similarity to cysteine, hence the name homocysteine. In the human body, about 1/2 of Hcy and methyltetrahydrofolate turn into methionine and tetrahydrofolic acid under the action of methylenetetrahydrofolate reductase (MTHFR), and this process needs the help of folic acid and vitamin B12 [4]. The rest of the Hcy turn into cysteine under the catalysis of cystathionine β-synthase (CBS), which require the participation of vitamin B6 (Figure 1). When any factors cause the impair of Hcy metabolism or defects in crucial co-factors that participate in its recycling, it will develop into hyperhomocysteinemia (HHey). There have been a lot of findings shown that HHey was recognized as a modifiable independent risk factor for coronary, cerebral and peripheral vascular diseases [5–8], and experimental studies have also suggested that Hcy induces vascular endothelium injury and leads to vascular damage [3]. These findings suggest that Hcy might be involved in the pathogeneses of both central and peripheral vascular disorders. Recently, many studies have also revealed that multiple rheumatic diseases may be associated with HHey [9–11], and the possible role of HHey in the complex damage mechanisms of systemic rheumatic diseases has also the reason for concern.
The relationship between gene polymorphism of MTRR A66G and lower extremity deep venous thrombosis
Published in Hematology, 2018
Tetrahydrofolate in the body can be combined with a carbon unit at the 5-N and 10-N positions into tetrahydrofolic acid. Methyltetrahydrofolic acid was synthesized by MTHFR [30]. Under the catalysis of MS, homocysteine reacts with 5-methyltetrahydrofolic acid, which is demethylated into tetrahydrofolate, and homocysteine is methylated into methionine, to complete a cycle of tetrahydrofolate. Under the action of cystathionine-β-synthase (CBS), with vitamin B6 as a cofactor, homocysteine and silk amino acids combined to form cystathionine, further forming cysteine [31]. MTRR is a cofactor for MS and catalyzes the regeneration of methylcobalamin. MTRR maintains the MS in a reduced state while maintaining its methylation status by reducing vitamin B6. Studies have shown that the polymorphism of 66 A-G loci in MTRR caused methionine to be replaced by isoleucine [32]. The decrease in enzyme activity leads to hyperhomocysteinemia due to the abnormal metabolism of folic acid, which can cause vascular endothelial damage and dysfunction and stimulate the proliferation of vascular smooth muscle cell, leading to the imbalance between vascular relaxing factor and vascular contraction factor eventually, thus DVT occurs.
Related Knowledge Centers
- Cofactor
- Dihydrofolate Reductase
- Sulfonamide
- Amino Acid
- Folate
- Dihydrofolic Acid
- Methotrexate
- 5,10-Methylenetetrahydrofolate
- Dihydropteroate
- 4-Aminobenzoic Acid