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Background: Pulmonary hypertension (PH) is a progressive disorder characterized by remodeling of the pulmonary vasculature and elevated mean pulmonary arterial pressure, resulting in right heart failure., Methods: Here, we show that direct targeting of the endothelium to uncouple eNOS (endothelial nitric oxide synthase) with DAHP (2,4-diamino 6-hydroxypyrimidine; an inhibitor of GTP cyclohydrolase 1, the rate-limiting synthetic enzyme for the critical eNOS cofactor tetrahydrobiopterin) induces human-like, time-dependent progression of PH phenotypes in mice., Results: Critical phenotypic features include progressive elevation in mean pulmonary arterial pressure, right ventricular systolic blood pressure, and right ventricle (RV)/left ventricle plus septum (LV+S) weight ratio; extensive vascular remodeling of pulmonary arterioles with increased medial thickness/perivascular collagen deposition and increased expression of PCNA (proliferative cell nuclear antigen) and alpha-actin; markedly increased total and mitochondrial superoxide production, substantially reduced tetrahydrobiopterin and nitric oxide bioavailabilities; and formation of an array of human-like vascular lesions. Intriguingly, novel in-house generated endothelial-specific dihydrofolate reductase (DHFR) transgenic mice (tg-EC-DHFR) were completely protected from the pathophysiological and molecular features of PH upon DAHP treatment or hypoxia exposure. Furthermore, DHFR overexpression with a pCMV-DHFR plasmid transfection in mice after initiation of DAHP treatment completely reversed PH phenotypes. DHFR knockout mice spontaneously developed PH at baseline and had no additional deterioration in response to hypoxia, indicating an intrinsic role of DHFR deficiency in causing PH. RNA-sequencing experiments indicated great similarity in gene regulation profiles between the DAHP model and human patients with PH., Conclusions: Taken together, these results establish a novel human-like murine model of PH that has long been lacking in the field, which can be broadly used for future mechanistic and translational studies. These data also indicate that targeting endothelial DHFR deficiency represents a novel and robust therapeutic strategy for the treatment of PH., Competing Interests: Disclosures None.

The mechanisms of adaptation to inactivation of essential genes remain unknown. Here we inactivate E. coli dihydrofolate reductase (DHFR) by introducing D27G,N,F chromosomal mutations in a key catalytic residue with subsequent adaptation by an automated serial transfer protocol. The partial reversal G27- > C occurred in three evolutionary trajectories. Conversely, in one trajectory for D27G and in all trajectories for D27F,N strains adapted to grow at very low metabolic supplement (folAmix) concentrations but did not escape entirely from supplement auxotrophy. Major global shifts in metabolome and proteome occurred upon DHFR inactivation, which were partially reversed in adapted strains. Loss-of-function mutations in two genes, thyA and deoB , ensured adaptation to low folAmix by rerouting the 2-Deoxy-D-ribose-phosphate metabolism from glycolysis towards synthesis of dTMP. Multiple evolutionary pathways of adaptation converged to a suboptimal solution due to the high accessibility to loss-of-function mutations that block the path to the highest, yet least accessible, fitness peak., Competing Interests: JR, ES No competing interests declared, (© 2019, Rodrigues and Shakhnovich.)

Since the use of protein therapeutics is effective for treating intractable human diseases, the production of biologic therapeutic agents has dramatically increased over the past three decades. The Chinese hamster ovary (CHO) cell lines are the most commonly used host cell expression system for recombinant protein production. High productive and stable clonal cell lines for recombinant protein production have been established from the DHFR-deficient CHO cell using the dihydrofolate reductase/methotrexate (DHFR/MTX) selection methods. Human embryonic kidney 293 (HEK293) cells are alternative host cells widely used for protein production. In most case, however, the cells are used for the transient expression, and there is no gene amplification system in HEK293 cells. In this study, we established a DHFR-deficient HEK293 cell line for the high yield of recombinant proteins. We doubly knocked out DHFR and DHFR2 in the MAN1A1/A2/B1/C1-quadruple knockout HEK293 (QD-KO) cells, using the CRISPR/Cas9 system. The DHFR-deficient QD-KO cells were used to overexpress two proteins, lysosomal acid lipase and the constant fragment of human immunoglobulin G 1 by the DHFR/MTX gene-amplification method. This method resulted in a dramatic increase in the two protein expressions in the DHFR-deficient QD-KO cells by increasing MTX concentration. Our system could be adopted in the production of several recombinant proteins including therapeutic proteins., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Hypertension and abdominal aortic aneurysm (AAA) are severe cardiovascular diseases with incompletely defined molecular mechanisms. In the current study we generated dihydrofolate reductase (DHFR) knockout mice for the first time to examine its potential contribution to the development of hypertension and AAA, as well as the underlying molecular mechanisms. Whereas the homozygote knockout mice were embryonically lethal, the heterozygote knockout mice had global reduction in DHFR protein expression and activity. Angiotensin II infusion into these animals resulted in substantially exaggerated elevation in blood pressure and development of AAA, which was accompanied by excessive eNOS uncoupling activity (featured by significantly impaired tetrahydrobiopterin and nitric oxide bioavailability), vascular remodeling (MMP2 activation, medial elastin breakdown and adventitial fibrosis) and inflammation (macrophage infiltration). Importantly, scavenging of mitochondrial reactive oxygen species with Mito-Tempo in vivo completely abrogated development of hypertension and AAA in DHFR knockout mice, indicating a novel role of mitochondria in mediating hypertension and AAA downstream of DHFR deficiency-dependent eNOS uncoupling. These data for the first time demonstrate that targeting DHFR-deficiency driven mitochondrial dysfunction may represent an innovative therapeutic option for the treatment of AAA and hypertension., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Oxidative stress plays an important role in the formation of abdominal aortic aneurysm (AAA), and we have recently established a causal role of uncoupled eNOS in this severe human disease. We have also shown that activation of NADPH oxidase (NOX) lies upstream of uncoupled eNOS. Therefore, identification of the specific NOX isoforms that are required for eNOS uncoupling and AAA formation would ultimately lead to novel therapies for AAA. In the present study, we used the Ang II infused hph-1 mice to examine the roles of NOX isoforms in the development of AAA. We generated double mutants of hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4. After two weeks of Ang II infusion, the incidence rate of AAA substantially dropped from 76.5% in Ang II infused hph-1 mice (n=34) to 11.1%, 15.0%, 9.5% and 0% in hph-1-NOX1 (n=27), hph-1-NOX2 (n=40), hph-1-p47phox (n=21), and hph-1-NOX4 (n=33) double mutant mice, respectively. The size of abdominal aortas of the four double mutant mice, determined by ultrasound analyses, was significantly smaller than the hph-1 mice. Aortic nitric oxide and H 4 B bioavailabilities were markedly improved in the double mutants, while superoxide production and eNOS uncoupling activity were substantially diminished. These effects seemed attributed to an endothelial specific restoration of dihydrofolate reductase expression and activity, deficiency of which has been shown to induce eNOS uncoupling and AAA formation in both Ang II-infused hph-1 and apoE null animals. In addition, over-expression of human NOX4 N129S or T555S mutant newly identified in aneurysm patients increased hydrogen peroxide production, further implicating a relationship between NOX and human aneurysm. Taken together, these data indicate that NOX isoforms 1, 2 or 4 lies upstream of dihydrofolate reductase deficiency and eNOS uncoupling to induce AAA formation. These findings may promote development of novel therapeutics for the treatment of the disease by inhibiting NOX signaling., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Adaptation of dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) DG44 cells to chemically defined suspension culture conditions is a time-consuming and labor-intensive process because nonadapted DHFR-deficient CHO DG44 cells normally show poor growth in chemically defined medium (CDM). We examined the effects of folate derivatives, ribonucleotides, and nucleobases on the growth of suspension-adapted DHFR-deficient CHO DG44 cells in CDM. Among the tested additives, tetrahydrofolate (THF) was identified as an effective component for increasing cell growth. THF supplementation in the range of 0.2-359 μM enhanced cell growth in in-house CDM. Addition of 3.6 μM THF to in-house CDM resulted in a more than 2.5-fold increase in maximum viable cell density. Moreover, supplementation of six different commercial CDMs with 3.6 μM THF yielded up to 2.9-fold enhancement of maximum viable cell density. An anchorage- and serum-dependent DHFR-deficient CHO DG44 cell line was adapted within two consecutive passages to suspension growth in in-house CDM supplemented with 3.6 μM THF. These data indicate that supplementation of chemically defined cell culture media with greater than 0.2 μM THF can help achieve a high density of suspension-adapted DHFR-deficient CHO DG44 cells and may facilitate rapid adaptation of nonadapted DHFR-deficient CHO DG44 cells to suspension culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1539-1546, 2016., (© 2016 American Institute of Chemical Engineers.)

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR-deficient DG44, and DUXB11-based DHFR deficient CHO. Current Genentech commercial full-length antibody products have all been produced in the DUXB11-derived DHFR-deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11-derived DHFR-deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11-based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14-day fed batch cultures in shake flasks. In contrast, the DUXB11-based host produced ∼0.1 g/l for both antibodies in the same 14-day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ∼2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass., (© 2013 American Institute of Chemical Engineers.)

In Chinese hamster ovary (CHO) cells, rapid glucose metabolism normally leads to inefficient use of glucose, most of which is converted to lactate during cell cultures. Since lactate accumulation during the culture often exerts a negative effect on cell growth and valuable product formation, several genetic engineering approaches have been developed to suppress lactate dehydrogenase-A (LDH-A), the enzyme converting pyruvate into lactate. However, despite the reduced lactate accumulation, such cell cultures are eventually terminated in the late period of the culture, mainly due to apoptosis. Therefore, we developed an apoptosis-resistant, less lactate-producing dhfr(-) CHO cell line (CHO-Bcl2-LDHAsi) by overexpressing Bcl-2, one of the most well-known anti-apoptotic proteins, and by downregulating LDH-A in a dhfr(-) CHO cell line. When the dhfr(-) CHO-Bcl2-LDHAsi cell line was used as a host cell line for the development of recombinant CHO (rCHO) cells producing an Fc-fusion protein, the culture longevity of the rCHO cells was extended without any detrimental effect of genetic engineering on specific protein productivity. Simultaneously, the specific lactate production rate and apparent yield of lactate from glucose were reduced to 21-65% and 37-78% of the control cells, respectively. Taken together, these results show that the use of an apoptosis-resistant, less lactate-producing dhfr(-) CHO cell line as a host cell line saves the time and the effort of establishing an apoptosis-resistant, less lactate-producing rCHO cells for producing therapeutic proteins., (© Springer-Verlag 2011)

DHFR-deficient CHO cells are the most commonly used host cells in the biopharmaceutical industry and over the years, individual substrains have evolved, some have been engineered with improved properties and platform technologies have been designed around them. Unexpectedly, we have observed that different DHFR-deficient CHO cells show only poor growth in fed-batch cultures even in HT supplemented medium, whereas antibody producer cells derived from these hosts achieved least 2-3 fold higher peak cell densities. Using a set of different expression vectors, we were able to show that this impaired growth performance was not due to the selection procedure possibly favouring fast growing clones, but a direct consequence of DHFR deficiency. Re-introduction of the DHFR gene reproducibly restored the growth phenotype to the level of wild-type CHO cells or even beyond which seemed to be dose-dependent. The requirement for a functional DHFR gene to achieve optimal growth under production conditions has direct implications for cell line generation since it suggests that changing to a selection system other than DHFR would require another CHO host which - especially for transgenic CHO strains and tailor-suited process platforms - this could mean significant investments and potential changes in product quality. In these cases, DHFR engineering of the current CHO-DG44 or DuxB11-based host could be an attractive alternative., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism and an important target of antineoplastic, antimicrobial, and antiinflammatory drugs. We describe three individuals from two families with a recessive inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency due to a germline missense mutation in DHFR, resulting in profound enzyme deficiency. We show that cerebral folate levels, anemia, and pancytopenia of DHFR deficiency can be corrected by treatment with folinic acid. The characterization of this disorder provides evidence for the link between DHFR and metabolism of cerebral tetrahydrobiopterin, which is required for the formation of dopamine, serotonin, and norepinephrine and for the hydroxylation of aromatic amino acids. Moreover, this relationship provides insight into the role of folates in neurological conditions, including depression, Alzheimer disease, and Parkinson disease., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The importance of intracellular folate metabolism is illustrated by the severity of symptoms and complications caused by inborn disorders of folate metabolism or by folate deficiency. We examined three children of healthy, distantly related parents presenting with megaloblastic anemia and cerebral folate deficiency causing neurologic disease with atypical childhood absence epilepsy. Genome-wide homozygosity mapping revealed a candidate region on chromosome 5 including the dihydrofolate reductase (DHFR) locus. DHFR sequencing revealed a homozygous DHFR mutation, c.458A>T (p.Asp153Val), in all siblings. The patients' folate profile in red blood cells (RBC), plasma, and cerebrospinal fluid (CSF), analyzed by liquid chromatography tandem mass spectrometry, was compatible with DHFR deficiency. DHFR activity and fluorescein-labeled methotrexate (FMTX) binding were severely reduced in EBV-immortalized lymphoblastoid cells of all patients. Heterozygous cells displayed intermediate DHFR activity and FMTX binding. RT-PCR of DHFR mRNA revealed no differences between wild-type and DHFR mutation-carrying cells, whereas protein expression was reduced in cells with the DHFR mutation. Treatment with folinic acid resulted in the resolution of hematological abnormalities, normalization of CSF folate levels, and improvement of neurological symptoms. In conclusion, the homozygous DHFR mutation p.Asp153Val causes DHFR deficiency and leads to a complex hematological and neurological disease that can be successfully treated with folinic acid. DHFR is necessary for maintaining sufficient CSF and RBC folate levels, even in the presence of adequate nutritional folate supply and normal plasma folate., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Chinese hamster ovary (CHO) cells are the major mammalian host for producing various therapeutic proteins. Among CHO cells, the dihydrofolate reductase-deficient CHO DG44 cell line has been used as a popular mammalian host because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the proteome level. Here, the first detailed proteome analysis of the CHO DG44 cell line is described. A protein reference map of the CHO DG44 cell line was established by analyzing whole cellular proteins using 2-DE with various immobilized pH gradients (pHs 3-10, 5-8, and 3-6) in the first dimension and a 12% acrylamide gel in the second dimension. The map is composed of over 1400 silver-stained protein spots. Among them, 179 protein spots, which represent proteins associated with various biological processes and cellular compartments, were identified based on MALDI-TOF-MS and MS/MS. This proteome database should be valuable for better understanding of CHO cell physiology and protein expression patterns which may lead to efficient therapeutic protein production.

Aim: To obtain high level expression of recombinant human truncated osteoprotegerin (TOPG) with higher bioactivity in CHO-DHFR(-) cells., Methods: The recombinant vector pcDNA3.1/DHFR-TOPG was constructed and transfected into CHO-DHFR(-) cells by the directions of LipofectAMINE 2000 for stable expression. The stable expression cell strains were screened by selective medium IMDM with 50 mL/L FCS, then serially passed in methotraxate (MTX) for gene amplification. The expression were analyzed by ELISA and RT-PCR. At last, the bioactivity analysis was performed in vitro., Results: The expression level of recombinant truncated human OPG was up to 6 mg/L x 72 h, and it had significant suppression effect on the formation of OLC (P<0.05)., Conclusion: Recombinant truncated human OPG has high expression and bioactivity. The results make it possible for further studying and clinical implying of OPG.

DHFR-deficient Chinese hamster ovary (CHO DHFR(-)) cells are the most popular mammalian expression system for inducible amplification of transgene. In order to obtain more stable transfected CHO DHFR(-) cell clones, transfection efficiency of electroporation under different conditions were systemically investigated using plasmid pSV-beta-Gal as reporter gene. Transfection efficiency was proportionally increased with pulse duration and number of pulse applied. In addition, higher transfection efficiency was found in high salt extracellular solution (Berg's and Hank's buffers) than in intracellular solution (cytomix buffer) under the same electroporation condition. The highest transfection efficiency in examined conditions was about 1 in 350 cells (or 0.289%) when cells were electroporated with twice pulses at 400V, 375microF. The present study offers an optimized guideline for introducing exogenous DNA into CHO DHFR(-) cells by electroporation.

Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural-but imperfect-DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR(-/-) cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2-3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR(-/-) cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production.

This review examines the history of folate, its metabolism and its relationship to drugs and diseases. The scientific interest in folate has been increasing in recent years because of new findings related to its important role in many diseases like macrocytic anemia, congenital malformations, vascular thrombosis, atherosclerotic disease and cancer. The fascinating puzzle of folate is analyzed and the most recent news about folate treatment in patients with chronic renal failure is reported.

Introduction: Homocysteine lies at an important metabolic branch point; it may be either converted to cystathionine through the transsulfuration pathway, or methylated to form methionine. Hyperhomocysteinemia may result from hereditary defects affecting one of these reactions., State of Art: Cystathionine beta synthase or 5,10-methylenetetrahydrofolate deficiency can both result in homocystinuria. Current knowledge about biochemical mechanisms leading to hyperhomocysteinemia, clinical and radiological features, pathogenesis and treatment are reviewed, focusing on late onset forms of these diseases which can be diagnosed in adulthood. CBS deficiency is characterized by lens dislocation, skeletal abnormalities, neurologic disturbances and thromboembolism. MTHFR deficiency leads to various neurological symptoms, ranging from developmental delay to encephalopathy, including motor and gait abnormalities, seizures, psychiatric manifestations and rarely strokes. The treatment of CBS deficiency depends on vitamin B6, whereas MTHFR deficiency can be efficiently treated by vitamin B12, folic acid, and betaine., Perspectives: Homocysteinemia should be measured in patients with unexplained neurological manifestations or thromboembolism.

Chinese hamster ovary (CHO) cells and dihydrofolate reductase (dhfr)/methotrexate (MTX) gene amplification system are routinely used to generate stable producer CHO cell clones in biopharmaceutical industries. The present study proposes a novel method by the co-amplification of the silencing vector targeted to dhfr gene for improvements of selecting high-producing clones in dhfr-deficient and wild-type CHO cells. Using the silencing vector also resulted in improving the stability of the recombinant protein expression in the absence of MTX in the CHO/dhFr(-) and wild-type CHO cells. This new method is proposed to generate highly expressed stable cell clones of both dhfr-deficient and wild-type CHO cells for recombinant antigen production. Utilization of the silencing vector designed in this study can improve antigen expression through dhfr-directed gene amplification in other dhfr-competent cell lines for vaccine development.

Background: Plasmodium falciparum is the predominant human malaria species in Mozambique and a lead cause of mortality among children and pregnant women nationwide. Sulphadoxine/pyrimethamine (S/P) is used as first line antimalarial treatment as a partner drug in combination with artesunate., Methods: A total of 92 P. falciparum-infected blood samples, from children with uncomplicated malaria attending the Centro de Saude de Bagamoyo in the Province of Maputo-Mozambique, were screened for S/P resistance-conferring mutations in the pfdhfr and pfdhps genes using a nested mutation-specific polymerase chain reaction and restriction digestion (PCR-RFLP). The panel of genetic polymorphisms analysed included the pfdhfr 164L mutation, previously reported to be absent or rare in Africa., Results: The frequency of the S/P resistance-associated pfdhfr triple mutants (51I/59R/108N) and of pfdhfr/pfdhps quintuple mutants (51I/59R/108N + 437G/540E) was 93% and 47%, respectively. However, no pfdhfr 164L mutants were detected., Conclusion: The observation that a considerably high percentage of P. falciparum parasites contained S/P resistance-associated mutations raises concerns about the validity of this drug as first-choice treatment in Mozambique. On the other hand, no pfdhfr 164L mutant was disclosed, corroborating the view that that this allele is still rare in Africa.

Homocystinuria, a rare autosomal recessive genetic disease, is generally classified into 3 types (types I-III). Extremely rare type III is from a defect of the enzyme tetrahydrofolate reductase which converts homocysteine to methionine. The anesthetic problem of type III homocystinuria is the elevation of serum homocysteine and the deficiency of essential amino acid methionine, which result in the perioperative thromboembolic events and nitrous oxide-induced neurological impairment. We provided successful anesthetic management for a patient with type III homocystinuria, during which we were careful to take measures against perioperative thromboembolism and did not use nitrous oxide.

Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 microg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 microM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future.

We examined the pharmacological profile of (3,4-dihydro-2H-pyrano[2,3]b quinolin-7-yl) (cis-4-methoxycyclohexyl) methanone (JNJ16259685). At recombinant rat and human metabotropic glutamate (mGlu) 1a receptors, JNJ16259685 non-competitively inhibited glutamate-induced Ca2+ mobilization with IC50 values of 3.24+/-1.00 and 1.21+/-0.53 nM, respectively, while showing a much lower potency at the rat and human mGlu5a receptor. JNJ16259685 inhibited [3H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone ([3H]R214127) binding to membranes prepared from cells expressing rat mGlu1a receptors with a Ki of 0.34+/-0.20 nM. JNJ16259685 showed no agonist, antagonist or positive allosteric activity toward rat mGlu2, -3, -4 or -6 receptors at concentrations up to 10 microM and did not bind to AMPA or NMDA receptors, or to a battery of other neurotransmitter receptors, ion channels and transporters. In primary cerebellar cultures, JNJ16259685 inhibited glutamate-mediated inositol phosphate production with an IC50 of 1.73+/-0.40 nM. Subcutaneously administered JNJ16259685 exhibited high potencies in occupying central mGlu1 receptors in the rat cerebellum and thalamus ( ED50=0.040 and 0.014 mg/kg, respectively). These data show that JNJ16259685 is a selective mGlu1 receptor antagonist with excellent potencies in inhibiting mGlu1 receptor function and binding and in occupying the mGlu1 receptor after systemic administration.

The effect of alteration of the glycolytic pathway on cell damage induced by oxidative stress was investigated with dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cells that either overexpress cytosolic glycerol-3-phosphate dehydrogenase (CHO/cGPDH cells) or are depleted of the A subunit of lactate dehydrogenase as a result of anti-sense RNA expression (CHO/anti-LDH cells). The extent of oxidative phosphorylation in CHO/anti-LDH and CHO/cGPDH cells was increased and decreased, respectively, relative to that in parental CHO cells, as revealed by measurement of the intracellular content of ATP, the rate of cellular O(2) consumption, the mitochondrial membrane potential (DeltaPsi(m)), and the generation of reactive oxygen species. The sensitivity of these cell lines to cell death induced by the exogenous oxidant tert-butyl hydroperoxide decreased according to the rank order CHO/anti-LDH>CHO>CHO/cGPDH. Exogenous pyruvate markedly increased the sensitivity of CHO/cGPDH cells to oxidant-induced death. The differences among the three cell lines in susceptibility to oxidant-induced death were reflected in the proportion of oxidant-treated cells with a subdiploid DNA content, with a collapsed DeltaPsi(m), and with cytochrome c in the cytosol, indicating that death was mediated by apoptosis. These results demonstrate that the influx of respiratory substrate into mitochondria is an important determinant of cell sensitivity to oxidant-induced apoptosis.

Nephrotic patients are at risk of developing venous and arterial thrombotic complications. Pulmonary embolism due to affected deep leg veins is by far the most common event. Renal or cerebral vein thromboses have been described. Thrombosis of arterial vessels is less frequent. Mesenteric infarction is a rare but severe complication in patients with nephrotic syndrome (NS). We report a 7-year-old boy with a steroid-dependent (SD) NS and a homozygous mutation of methylenetetrahydrofolate reductase, increasing the risk of thromboembolic events. He developed a thrombosis of his superior mesenteric artery during his ninth relapse, which was responsible for a necrosis of 240 cm of his small bowel, necessitating resection of necrotic parts and double external ostomy diversion. Remission was achieved with pulse prednisolone therapy. Corticoids were reduced over 4 months progressively. Oral cyclosporin A (CyA) was initiated for long-term treatment. Due to a short bowel syndrome with severe malabsorption, even oral administration of 22.5 mg/kg per day CyA did not lead to sufficient plasma levels. Intravenous cyclophosphamide pulse therapy over 6 months led to a complete remission. No relapse occurred over a period of more than 5 months after the last cyclophosphamide pulse. Anticoagulation and screening for increased susceptibility for thrombotic events are necessary in every nephrotic patient. Intravenous cyclophosphamide pulse therapy is a useful alternative in SDNS with impaired intestinal absorption of applied immunosuppressive drugs.

Apoptosis-resistant dihydrofolate reductase-deficient CHO cell line (dhfr(-) CHO-bcl2) was developed by introduction of the bcl-2 gene into the dhfr(-) CHO cell line (DUKX-B11, ATCC CRL-9096) and subsequent selection of clones stably overexpressing Bcl-2 in the absence of selection pressure. When the dhfr(-) CHO-bcl2 cell line was used as a host cell line for development of a recombinant CHO (rCHO) cell line expressing a humanized antibody, it displayed stable expression of the bcl-2 gene during rCHO cell line development and no detrimental effect of Bcl-2 overexpression on specific antibody productivity. Taken together, the results obtained demonstrate that the use of an apoptosis-resistant dhfr(-) CHO cell line as the host cell line saves the effort of establishing an apoptosis-resistant rCHO cell line and expedites the development process of apoptosis-resistant rCHO cells producing therapeutic proteins., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 872-876, 2003.)

An inducible reporter gene system for Chinese Hamster Ovary (CHO-DHFR(-)) cells has been developed and characterized with respect to its dynamic properties. The reporter gene system consists of the human c-fos promoter and variants of the green fluorescence protein (GFP), either EGFP with enhanced fluorescence or its destabilized form d2EGFP. The expression of wild-type EGFP or its destabilized form was studied in CHO-DHFR(-) cells in response to serum addition or deprivation. It was shown that serum-induced c-fos promoter mediated EGFP expression was considerably higher than expression from the human CMV promoter, a strong, constitutive promoter preferentially used for high-level expression in CHO cells. However, EGFP was less suitable for studying expression dynamics than d2EGFP due to the protein's long half-life in mammalian cells. The use of d2EGFP resulted in a significant improvement in the dynamic characteristics of the biomarker, particularly when the recombinant cells were selected for high-level GFP expression by subcloning or fluorescence activated cell/sorting (FACS). GFP expression in different subclones and cell populations sorted by FACS was characterized with respect to its dynamic responses in the presence or absence of serum in the culture medium. Significant differences in the GFP expression dynamics were observed for the isolated cell populations. The experimental results indicate that cells with high-level GFP expression also have a faster dynamic response and are thus, desirable for practical application of the reporter gene system e.g. in toxicity monitoring.

Inherited genetic deficiency of lysosomal acid alpha glucosidase or acid maltase (GAA) results in the autosomal recessive glycogen storage disease type II (GSD II). To investigate whether we could generate a functional recombinant human GAA (rhGAA) for enzyme replacement therapy, we subcloned the cDNAs for human GAA and mouse dihydrofolate reductase (DHFR) into DHFR(neg) Chinese hamster ovary cells and established a stable cotransformant that expressed rhGAA. We cultured the recombinant cells in media with progressively increasing concentrations of methotrexate and found that human GAA enzyme activity increased to over 2,000 IU per gram protein. Importantly, the human GAA enzyme activity correlated to equivalent amounts of human GAA protein by rocketimmunoelectrophoresis. We confirmed that the human GAA enzyme activity corresponded to an amplification in human GAA mRNA by Northern analysis and human GAA cDNA copy number by Southern analysis. Exposing the rhGAA to human GSDII fibroblast cells or patient's lymphocytes or monocytes resulted in uptake of the rhGAA and reversal of the enzymatic defect. Mannose-6-phosphate in the media blocked uptake. GAA -/- mice were treated with the rhGAA at 1 mg/kg, which resulted in heterozygous levels of GAA in tissues, most notably skeletal muscle, heart and diaphragm after two infusions. More importantly, after multiple infusions, hind, and fore-limb muscle weakness was reversed. This rhGAA would be ideal for enzyme replacement therapy in GSD II., (Copyright 2000 Academic Press.)

In this study we have compared the immune response of normal human cells cultured in vitro to two virulent strains of Leishmania major (CC1 and LV39), and to an avirulent vaccine strain (dhfr-ts-) made by targeted deletion of the essential gene DHFR-TS. We utilized an in vitro system in which naive T cells from normal human donors were primed with autologous Leishmania-infected macrophages. All three parasites infected macrophages and transformed into amastigotes within the cells. However, whereas LV39 and CC1 replicated in macrophages, dhfr-ts- did not. When peripheral blood lymphocytes (PBL) were stimulated with autologous macrophages infected with any of the three parasites, the lymphocytes produced a type-1-biased cytokine response. Finally, addition of IL-12 during the first stimulation period increased the production of interferon-gamma but decreased IL-5 secretion. On the other hand, anti-IL-12 resulted in the opposite effect.

To develop serum-free (SF) medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44), a statistical optimization approach based on a Plackett-Burman design was adopted. DG44 cells which were normally maintained in 10 serum medium were gradually weaned to 0.5% serum medium to increase the probability of successful growth in SF medium. A basal medium was prepared by supplementing Dulbecco's modified Eagle's medium and Ham's nutrient mixture F12 with hypoxanthine (10 mg/l) and thymidine (10 mg/l). Twenty-eight different supplements were selected as variables on the basis of their growth-promoting abilities. From statistical analysis, leucine, tryptophan, lysine, proline, histidine, hydrocortisone, ethanolamine, and phosphatidylcholine were identified as important components showing positive effects on cell growth. A new SF medium (SF-DG44) was formulated by supplementing the basal medium with these components. When the weaned cells were inoculated at 1.0 x 10(5) cells/ml, a maximum viable cell concentration of 6.4 x 10(3)) cells/ ml was achieved in SF-DG44 medium. In contrast, when the unweaned cells were used, a concentration of only 4.1 x 10(5) cells/ml was reached under the same culture conditions, indicating that weaning of cells improves cell growth in SF medium. In summary, we found that development of a novel SF medium for DG44 cells was facilitated using a Plackett-Burman design technique and weaning of cells.

In Escherichia coli, dihydrofolate reductase is required for both the de novo synthesis of tetrahydrofolate and the recycling of dihydrofolate produced during the synthesis of thymidylate. The coding region of the dihydrofolate reductase gene, folA, was replaced with a kanamycin resistance determinant. Unlike earlier deletions, this mutation did not disrupt flanking genes. When the mutation was transferred into a wild-type strain and a thymidine-(thy) requiring strain, the resulting strains were viable but slow growing on rich medium. Both synthesized less folate than their parents, as judged by the incorporation of radioactive para-aminobenzoic acid. The derivative of the wild-type strain did not grow on any defined minimal media tested. In contrast, the derivative of the thy-requiring strain grew slowly on minimal medium with thy but exhibited auxotrophies on some combinations of supplements. These results suggest that when folates are limited, they can be distributed appropriately to folate-dependent biosynthetic reactions only under some conditions.

The morphology of t-PA mutant engineered cell line FSGGI48 is similar to that of CHO-dhfr- cell in polygon. When aminopterin (MTX) concentration is 5 x 10(-6) mol/L, a part of cells become slim, but they are still in polygon. Therefore, the morphology of FSGGI48 cell line is normal. The expression level is 4000 IU/10(6) cells/24 h in serum-free medium. The cell line is bequeathed for three months in vitro and revived after freezing three months, the greater part of cells are stable. The expression level is 3000-4000 IU/10(6) cells/24 h. The assay of tumorigenesis shows that the cells, cellular DNA and purified products don't form tumors in nude mice. The cell line hasn't been contaminated by mycoplasma. The results of chromosomal analysis show that the chromosomal number of CHO-dhfr- cell is 20 and the abnormal rate is 6%. FSGGI48 cell line is same as CHO-dhfr- cell in chromosomal number and the abnormal rate is 15%-18%, it is normal in scope. Therefore, the engineered cell line FSGGI48 is excellent.

Eighteen mutants deficient in dihydrofolate reductase (DHFR) activity were induced with 0.5 microM N-hydroxy-aminofluorene in four separate experiments. This carcinogen dose killed approximately 80% of the treated cells and resulted in a mutational frequency approximately 3 x 10(-6). The nature of the induced changes in each of the mutants was determined by direct sequencing following polymerase chain reaction amplification, or in one instance, by Southern blot analysis. Nearly all (15/17) of the mutations were single base changes. Consistent with the binding specificity of this chemical, all mutations were targeted to guanine bases. The predominant change was G:C-->T:A transversion which was evident in 11/15 mutants. A single dG-AF mutational hotspot was noted at a site in the DHFR coding sequence of exon 4; one-third of the induced point mutations arose at this position. These results are compared with our previous analyses of mutants induced with the related aromatic amine, N-2-acetoxy-2-acetyl-aminofluorene.

Transposon Tn5tac1 can generate conditional mutations by virtue of an outward-facing tac promoter, which is regulated by the lac repressor and isopropyl-beta-D-thiogalactopyranoside (IPTG). We report here on a Tn5tac1 insertion in Escherichia coli that results in a conditional (IPTG-elicited) folA mutant phenotype: During aerobic growth, IPTG caused decreased synthesis of dihydrofolate reductase (DHFR; encoded by the folA gene) and hypersensitivity to trimethoprim (a DHFR inhibitor); during anaerobic growth, IPTG elicited auxotrophy that was satisfied by thymine or glycine or threonine. The Tn5tac1 insertion was downstream from folA, with the tac promoter pointing into the gene (antisense direction). Complementation tests indicated that the conditional folA deficiency was a cis effect of transcription from the tac promoter, perhaps due to head-to-head collision between converging RNA polymerases.

We have transformed Chinese hamster ovary cells with a plasmid containing mouse dihydrofolate reductase (DHFR) cDNA and the Escherichia coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene under the control of the mouse mammary tumor virus and SV40 early promoters, respectively. Selection for the expression of XGPRT using the dominant selection scheme described by Mulligan and Berg yields clones that simultaneously express DHFR. Growth of these cells in progressively increasing concentrations of methotrexate, results in selection of cells that overproduce DHFR and its messenger RNA 250-500 fold. ANalyses of the plasmid DNA sequences in these cells reveal that the increased production of DHFR is due in part to gene amplification (approximately 50-fold) and in part to a selective overproduction of DHFR RNA. Last, the methotrexate-resistant cells contain 50 times more XGPRT RNA and DNA than the initial transformant; this demonstrates the potential for using gene amplification as a means for overproducing the products of genes linked to DHFR cDNA in plasmid vectors.

A strain of Escherichia coli was isolated in which dihydrofolate reductase was not detected by an enzyme assay or by competition for antibody. This strain requires methionine, glycine, a purine, and thymidine for growth in addition to the auxotrophic requirements of the parent strain. It was found to be useful as a recipient of plasmids harboring dihydrofolate reductase genes.

Dihydrofolate reductase (DHFR), the main target for methotrexate and other antifolate compounds was found to be present in 100-200 times higher concentration in human cell lines grown in vitro than in human tumors or cells obtained in situ. The DHFR content of human cell lines in vitro however were equivalent to rodent tumor lines also measured in vitro. The enzyme was quantitated by [3H]methotrexate binding, [3H]dihydrofolate reduction to [3H]tetrahydrofolate, and immunoprecipitation with a monospecific anti-serum to DHFR. Additional studies revealed only a liver sample to contain significant amounts of an inhibitor of DHFR activity. It is postulated either that low levels of DHFR in fresh human tissue reflect low cell turnover or conversely that high levels in vitro and in animal tissues reflect high levels of enzyme due to selection because of high levels of folic acid in culture medium and prepared feeds.

Mutants of Chinese hamster ovary cells lacking dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate:NADP+ oxidoreductase; EC 1.5.1.3) activity were isolated after mutagenesis and exposure to high-specific-activity [3H]deoxyuridine as a selective agent. Fully deficient mutants could not be isolated starting with wild-type cells, but could readily be selected from a putative heterozygote that contains half of the wild-type level of dihydrofolate reductase activity. The heterozygote itself was selected from wild-type cells by using [3H]deoxyuridine together with methotrexate to reduce intracellular dihydrofolate reductase activity. Fully deficient mutants require glycine, a purine, and thymidine for growth; this phenotype is recessive to wild type in cell hybrids. Revertants have been isolated, one of which produces a heat-labile dihydrofolate reductase activity. These mutants may be useful for metabolic studies relating to cancer chemotherapy and for fine-structure genetic mapping of mutations by using available molecular probes for this gene.

To determine the cause of severe megaloblastosis detected at birth and at four weeks in two unrelated infants their bone marrow and liver cells were studied. Both patients had abnormal deoxyuridine suppression tests, corrected to normal by 5-formyl tetrahydrofolic acid. Liver-cell homogenate from one patient had a previously undetectable level of dihydrofolate reductase restored to normal by high cation concentration in the assay. Activity of the liver-cell homogenate from the other patient, which was one quarter of the normal level, was restored to only half normal activity by high cation concentration. Dihydrofolic acid reductase deficiency prevents this conversion of folic acid to tetrahydrofolic acid; the enzyme activity appears to differ in each patient. A satisfactory clinical response in both patients followed parenteral therapy with 5-formyl tetrahydrofolic acid. One sibling in each family died of a similar illness. Autosomal recessive inheritance is probable.

The translation of polycistronic mRNAs in mammalian cells was studied. Transcription units, constructed to contain one, two or three open reading frames (ORFs), were introduced stably into Chinese hamster ovary cells and transiently into COS monkey cells. The analysis of mRNA levels and protein synthesis in these cells demonstrated that the mRNAs transcribed were translated to generate multiple proteins. The efficiency of translation was reduced approximately 40- to 300-fold by the insertion of an upstream ORF. The results support a modified 'scanning' model for translation initiation which allows for translation initiation at internal AUG codons. High-level expression of human granulocyte-macrophage colony stimulating factor was achieved utilizing a vector that contains a polycistronic transcription unit encoding an amplifiable dihydrofolate reductase marker gene in its 3' end. Thus, polycistronic expression vectors can be exploited to obtain high-level expression of foreign genes in mammalian cells.

The deoxyuridine suppression (dU) test, first described in 1964, has gained an important place both in the diagnosis of megaloblastic anemias and in the study of vitamin B12 folate interrelationships in the pathogenesis of megaloblastic anemia. The test measures the integrity of the de novo synthetic pathway of DNA synthesis, in which vitamin B12 and folate play an essential role. The exact mechanism of the test is still largely unknown. However, it is probably the most sensitive and specific functional test for the establishment of vitamin B12 and/or folate deficiency. As such, it is an important diagnostic tool in the investigation of patients suspected of suffering from deficiency of either or both of these vitamins. The test may also have applications towards the study of other factors required for the de novo synthesis of DNA.

To study the influence of the level of dihydrofolate reductase (DHFR) on methotrexate (MTX) metabolism, the formation of methotrexate polyglutamates (MTXPGs) and the retention of the drug were examined in Chinese hamster ovary cells (DUKXB11) lacking DHFR and in control cells (CHO-UTC). Both cells accumulated MTXPGs poorly. After a 24-hr incubation with 1.0 microM [3H]MTX, the level of total MTX in DUKXB11 cells was 40% of that in CHO-UTC cells, reflecting the lack of DHFR-bound MTX and MTXPGs in the mutant cells. MTXPGs accounted for a higher proportion of the intracellular MTX in DUKXB11 than in CHO-UTC cells (25 vs 18%). Following exposure to 3.0 microM MTX for 24 hr, total drug levels were similar in both cell lines, and MTXPGs constituted even more of the intracellular drug in DUKXB11 cells compared to CHO-UTC cells (34 vs 23%). DUKXB11 cells accumulated longer MTXPGs (MTXG1u3,4) compared to CHO-UTC cells (MTXG1u2,3), following exposure to both 1.0 and 3.0 microM MTX. The longer MTXPGs in the mutant cells may have resulted from the lack of DHFR in them. Binding of MTXPGs to DHFR in CHO-UTC may interfere with their further polyglutamylation. When cells were resuspended in drug-free buffer for 1 hr following a 24-hr incubation with MTX, the retention of drug was less in DUKXB11 cells (46%) than in CHO-UTC cells (78%), due mainly to a greater loss of unmetabolized MTX in the mutant cells (89%) than in control cells (26%). Nevertheless, the amount of non-exchangeable unmetabolized MTX retained in DUKXB11 cells following exposure to 3.0 microM MTX exceeded the MTX-binding capacity. These studies demonstrate that DHFR-deficient cells accumulated more and longer MTXPGs than control cells. In addition, they suggest that some unmetabolized MTX was retained in cells not bound to DHFR.

We have used the technique of DNA-mediated gene transfer to introduce dihydrofolate reductase genes into dihydrofolate reductase-deficient Chinese hamster ovary cells. The transferred sequences include: dihydrofolate reductase minigenes, dihydrofolate reductase cDNA clones, and genomic DNA from mouse cells with highly amplified dihydrofolate reductase genes. The hamster cells were capable of utilizing the murine transcription initiation sites, splice junctions, and polyadenylation sites in complete murine dihydrofolate reductase genes. Only a short region of 5'-flanking sequence (160 base pairs (bp] was sufficient for proper initiation of dihydrofolate reductase transcription. In contrast only those clones with all of the dihydrofolate reductase introns and extensive (greater than 6.5 kilobase pairs) 3'-flanking regions utilized the dihydrofolate reductase polyadenylylation sites. Four of nine clones tested regulate the transfected genes normally, synthesizing dihydrofolate reductase preferentially at the onset of S phase. Regulated genes include one which entirely lacks intervening sequences, two with fewer than 420 bp of 5'-flanking sequence, and two in which polyadenylylation occurs predominantly in the adjacent hamster sequences. Two of the five cell lines which do not exhibit normal regulation contain genes which appear to be transcribed primarily from promoters present in the flanking hamster DNA. The other genes which fail to regulate do not appear to differ significantly in the structure of their transcripts or protein products from genes which regulate normally. We conclude that only the coding sequences, a region of less than 340 bp of sequence 5' of the translation initiation codon, and a short region of 3'-flanking sequence are required for regulated production of dihydrofolate reductase. The regulation can be abolished, however, by unknown properties of the site of insertion.

We assessed the role of acentric chromosome fragments in gene amplification by using cell fusion techniques to introduce the fragmented chromosomes of a donor Chinese hamster ovary (CHO) cell line that contained the dihydrofolate reductase (dhfr) gene(s) into a CHO cell line deficient for dhfr. Chromosome fragments were successfully integrated into cells at a frequency of approximately 3%. Methotrexate-resistant variants arose much more frequently in two cell lines derived from these successful cell fusions than in wild-type CHO cells. The hybrid cell lines also amplified their dhfr genes more readily than did the CHO cell line used as dhfr donor.