Enhancement of folates in plants through metabolic engineering

Humans depend on plants as a major source of dietary folates. Inadequate dietary levels of the vitamin folate can lead to megaloblastic anemia, birth defects, impaired cognitive development, and increased risk of cardiovascular disease and cancer. The biofortification of folate levels in food crops is a target for metabolic engineering. Folates are synthesized de novo from pterins and para-amino benzoic acid, which are subsequently combined to form dihydropteroate, the direct precursor to dihydrofolate. We postulated that GTP cyclohydrolase-1, which catalyzes the first committed step in pterin biosynthesis, was a rate-limiting step in pterin synthesis in plants and, therefore, in folate synthesis. On this basis, we proposed that the expression of an unregulated bacterial GTP cyclohydrolase-1 in plants would increase pterin biosynthesis with a concomitant enhancement of folate levels. The folE gene encoding GTP cyclohydrolase-1 was cloned from Escherichia coli and introduced into Arabidopsis thaliana through plant transformation. The expression of bacterial GTP cyclohydrolase-1 in transgenic Arabidopsis resulted in a 1,250-fold and 2- to 4-fold enhancement of pterins and folates, respectively. These results helped to identify other potential factors regulating folate synthesis, suggesting ways to further enhance folate levels in food crops.     

Demonstration of the inhibitory effect of human alpha-fetoprotein on in vitro transformation of human lymphocytes.

We have studied the effects of human alpha-fetoprotein (HAFP), isolated from the serum and ascitic fluid of a hepatoma-bearing patient, on the in vitro transformation of human peripheral blood lymphocytes by a variety of mitogenic stimuli. At a concentration of 2.5 mg/ml, HAFP inhibited the lymphocyte response to phytohemagglutinin, concanavalin A, and rabbit anti-human thymocyte serum, but failed to inhibit the response to pokeweed mitogen. HAFP was able to inhibit the one-way mixed lymphocyte culture at concentrations of 250-500 mug/ml, but failed to inhibit at 100 mug/ml. Exposure of lymphocytes to 2.2 mg/ml of HAFP for 18 hr did not result in significant lymphocytotoxicity, and such cells washed free of HAFP were fully capable of participating in the mixed lymphocyte culture. HAFP did not inhibit lymphocyte E-rosette formation. Fetal HAFP was more effective in inhibiting human lymphocyte responses than hepatoma HAFP. These experiments support the suggestion that HAFP plays an important immunoregulatory role during fetal development, possibly through the suppression of thymus-derived lymphocyte responses to antigenic stimuli; they also suggest that there are important differences in the biological properties of hepatoma and fetal HAFP.     

In vitro differentiation of a human erythroid cell line (KMOE) induced by some metabolic inhibitors

Summary KMOE-2/05, a continuous human erythroid cell line derived from a patient with acute erythremia was capable of differentiating into benzidine positive cells following exposure to cytosine arabinoside (CA), mitomycin C, or daunorubicin. Among the three substances, CA was the most effective inducer. Other compounds, reported as effective inducers on human and murine erythroid cells, were also tested but they were ineffective.Benzidine positive cells were counted to be approximately 500/1×10⁵ cells after 10 days incubation with CA at its optimal concentration of 1×10^–5 M. Under the same conditions, the hemoglobin (Hb) concentration quantitated by radioimmunoassay (RIA) was more than 500 ng/l×10⁶ cells. Quantitative kinetics of synthesized Hb and of benzidine-positive cell counts, after exposure to CA were closely correlated.This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Public Health and Welfare of Japan and also by a Research Grant from the Cancer Prevention Association in Fukuoka     

Chemotactic factor-induced generation of superoxide radicals by human neutrophils: effect of metabolic inhibitors and antiinflammatory drugs.

Human peripheral neutrophils generated superoxide radicals as assessed by ferricytochrome C reduction in response to activation by the synthetic chemotactic factor, N-formyl-methionyl-leucyl-phenylalanine. Superoxide generation was inhibited by 2-deoxy-D-glucose (ID50 4 X 10(-5)M), 2-iodoacetate (ID50 5 X 10(-5)M), and N-ethyl-maleimide (ID50 5 X 10(-6)M), suggesting a dependence on anaerobic glycolysis and sulfhydryl groups. Ouabain, microtubule-disrupting agents, inhibitors of respiration, oxidative phosphorylation, and protein and nucleic acid synthesis were without appreciable effects. Indomethacin (ID50 1 X 10(-4)M), ibuprofen (ID50 9 X 10(-4)M, and phenylbutazone (ID50 1 X 10(-5)M) all caused dose-dependent inhibition of superoxide generation at concentrations approximating those plasma and tissue levels obtained in human beings at therapeutic doses. Acetylsalicylic acid (125-500) microgram/ml) and aurothioglucose (10(-3)-10(-6)M) were without appreciable effects. Superoxide generation was inhibited only by relatively high concentrations of hydrocortisone (ID50 greater than 10(-3)M). Because superoxide radicals have been implicated in the pathogenesis of tissue injury in several forms of inflammation and arthritis in vivo, these studies suggest that the production of a potential cytotoxic factor may be subject to pharmacologic manipulation and that at least some of the antiphlogistic effects of the nonsteroidal antiinflammatory agents may be mediated through effects on superoxide production.     

The effect of certain glycolytic inhibitors on aerobic lactic acid production by human leukocytes in vitro

Oxamic acid depressed aerobic lactic acid formation by normal, human leukocytes by only 20-25 per cent. Glyceraldehyde increased lactic acid production.Sorbose-1-phosphate was without effect. Iodacetate inhibited markedly, butfluoride depressed only in high concentrations. The significance of these findingsregarding the mechanism of aerobic lactic acid formation by mature leukocytesis discussed.

Submitted on August 23, 1955 Accepted on October 28, 1955     

Regulation of the induction of colonies in vitro by normal human lymphocytes.

Lymphocytes isolated from normal human peripheral blood can be induced to form colonies in vitro by incubation with the appropriate inducer. Phytohemagglutinin can induce colonies with T (thymus-derived) lymphocytes. Optimun colony formation with about two colonies per 10(2) peripheral blood lymphocytes was obtained by adding, in addition to phytohemagglutinin, autologous plasma, autoologous red blood cells, and fresh L-glutamine or L-cystine. In the absence of these fresh amino acids, no colonies were formed at seeding levels below 10(5) cells per 35 mm petri dish. The addition of either of these amino acids gave a 10-fold decrease in the minimum number of cells that had to be seeded for colony formation. Lipopolysaccharide did not induce the formation of colonies, but enhanced the formation of T cell colonies by phytohemagglutinin. The mixing of lymphocytes from persons with and deficient in glucose-6-phosphate-dehydrogenase (EC 1-1-1-49) has shown that phytohemagglutinin-induced colonies can be derived from single cells and are, therefore, clones. No colonies were formed by lethally irradiated cells. Incubation with pokeweed mitogen also induced the formation of colonies. With autologous plasma and autologous red blood cells, pokeweek mitogen induced about one colony per 5 X 10(3) cells seeded and no colonies at seeding levels below 10(5) cells per petri dish. The minimum number of cells needed for colony formation by pokeweed mitogen was not decreased by fresh L-glutamine or L-cystine. The results indicate that normal human lymphocytes can be cloned in vitro and that induction of these lymphocyte colonies can be regulated by lectins and other specific factors.     

Influence of metformin on metabolic effect of insulin in human adipose tissue in vitro

To study the mechanism(s) of action of metformin, fragments of human subcutaneous adipose tissue were incubated with therapeutic blood concentrations of metformin. In the absence of insulin no effect of metformin was seen on either lipolysis or glucose metabolism. When insulin was present, however, metformin stimulated glucose conversion into both triglycerides and CO2. In marked contrast, no effect of metformin was observed on the antilipolytic effect of insulin. In agreement with this selective effect no change in insulin binding was found. In conclusion, metformin seems to exert its effect on glucose metabolism by potentiating the action of insulin at a post-receptor level, possibly on the rate of glucose transport.     

Ex vivo and in vitro effect of human immunodeficiency virus protease inhibitors on neutrophil apoptosis

Polymorphonuclear leukocytes (PMNL) from human immunodeficiency virus (HIV)-infected patients exhibit accelerated apoptosis and impaired functional activity. HIV protease inhibitor-based therapy produces improvements in both acquired and innate immune responses. Ex vivo and in vitro effects of HIV protease inhibitors on apoptosis and chemotaxis of PMNL were evaluated. After therapy, there was a rapid and significant decrease of PMNL apoptosis, which correlated with increased chemotactic function. These findings were found both in patients with immunological and virological response and in control subjects who showed an increase in CD4(+) T cell counts but no concomitant decline in HIV load. After in vitro treatment with ritonavir or indinavir, apoptosis of both HIV-infected and -uninfected PMNL markedly decreased and correlated with significant enhancement of chemotaxis. These results suggest that HIV protease inhibitors may improve the PMNL function by reducing the apoptosis rate and that this effect may, at least in part, be independent of their antiviral activity.     

Synthesis of prolactin by human decidua in vitro.

To determine whether human decidua and/or chorion synthesizes and secretes prolactin, explants of decidua obtained at Caesarian section and explants of chorion from the membranes separating dizygotic twins were cultured for periods of up to 6 days. The decidual explants released 366 +/- 37 ng prolactin/100 mg tissue (mean +/- S.D.) during each day in culture and incorporated 3H-labelled amino acids into immunoprecipitable prolactin. In the radioimmunoassay for prolactin, serial dilutions of incubation medium displaced 125I-labelled prolactin parallel to the displacement by pituitary prolactin and the prolactin in the medium eluted from Sephadex G-150 in a position indentical to that of pituitary prolactin. Chorionic explants released prolactin into the incubation medium during day 1 of culture only and did not incorporate 3H-labelled amino acids into prolactin. These results demonstrate that prolactin is synthesized by the decidua and not by the chorion and suggest that the decidua is the source of prolactin in amniotic fluid.     

Uptake and retention of moulting hormones by the integument of crayfishes in vitro. II. Influence of metabolic inhibitors and sulphydryl group inhibitors

Antimycin A and 2,4-dinitrophenol reduced both the initial uptake and the total uptake of ecdysone into crayfish hypodermis in vitro. Antimycin A did not reduce the retention of moulting hormone, in contrast to 2,4-dinitrophenol. The inhibitory effect of antimycin A could be completely overcome. The membrane-mediated ecdysteroid uptake was sensitive to the sulphydryl group inhibitors N-ethylmaleinimide (NEM) and p-chloromercuriphenylsulphonate (PCMPS). Inhibition of steroid uptake by PCMPS was completely reversed by dithiothreitol.     

In vitro effect of duodenal juice on R binders cobalamin complexes in subjects with pancreatic insufficiency: correlation with cobalamin absorption.

Absorption of cobalamin free or bound to chicken serum was assessed in nine patients with pancreatic insufficiency. Simultaneously the in vitro effect of duodenal juice collected from six patients and seven controls was tested on labelled cobalamin complexed to chicken serum or to R salivary binder. Malabsorption of free cobalamin was observed in one of nine patients and in four of nine patients when cobalamin was administered bound to chicken serum. The in vitro effect of duodenal juice on cobalamin complexed to chicken serum or to R salivary binder was studied: the percentage of free cobalamin released was significantly decreased in pancreatic insufficiency compared with controls whatever the binder used; the degradation of R salivary binder was different in pancreatic insufficiency and in controls. Despite the in vitro abnormalities observed in pancreatic insufficiency, these did not correlate with the in vivo absorption of cobalamin which was often normal in our patients.     

Synthesis and release of glycosylated prolactin by human decidua in vitro

To determine whether human decidual tissue synthesizes and secretes a glycosylated form of PRL (G-hPRL), explants of decidua obtained after normal term delivery were cultured for up to 5 days. During each day of incubation, the decidual explants incorporated [3H]mannose and [3H]glucosamine into immunoprecipitable G-hPRL. The explants released 33-50% less G-hPRL than hPRL. The release of decidual G-hPRL was unaffected by treatment with TRH (10(-4)-10(-8) M) or dopamine (10(-5)-10(-9) M) during a 4-h incubation period. G-hPRL also was sought in samples of amniotic fluid. Glycosylated hPRL was detected in all amniotic fluid samples from normal pregnant women at 32-40 weeks of gestation. These results indicate that human decidual explants in vitro may be used as a model system to study the secretion of G-hPRL and its regulation during pregnancy.     

Transformation of human lymphocytes in vitro by autologous and allogeneic rheumatoid synovial fluids.

To assess the possible participation of cellular immune mechanisms in the pathogenesis of rheumatoid arthritis (RA) in vitro studies of the blastogenicity of rheumatoid and non-rheumatoid synovial fluids for human peripheral blood lymphocytes were conducted. In autologous cultures it was found that 13 of 19 rheumatoid fluids induced significant lymphocyte blastogenesis, whereas only 1 of 13 nonrheumatoid fluids induced such a response. In allogeneic cultures rheumatoid fluids induce significant blastogenesis of RA lymphocytes in 18 of 23 experiments, and of non-RA lymphocytes in 8 of 18 experiments. By contrast, nonrheumatoid fluids induced significant blastogenesis of RA lymphocytes in 2 of 13 experiments, and of non-RA lymphocytes in 1 of 14 experiments. The blastogenicity of fluids was found to correlate significantly with the presence therein of immunofluorescent intracellular inclusions of immunoglobulin and complement. These studies support the concept that the presence of immune complexes in the majority of rheumatoid synovial fluids might render the latter blastogenic for human lymphocytes in vivo, thereby perpetuating rheumatoid synovitis.     

In vitro synthesis of the nucleotide loop of cobalamin by Salmonella typhimurium enzymes

In Salmonella typhimurium, the CobU, CobS, CobT, and CobC proteins have been proposed to catalyze the late steps in adenosylcobalamin biosynthesis, which define the nucleotide loop assembly pathway. This paper reports the in vitro assembly of the nucleotide loop of adenosylcobalamin from its precursors adenosylcobinamide, 5, 6-dimethylbenzimidazole, nicotinate mononucleotide, and GTP. Incubation of these precursors with the CobU, CobS, and CobT proteins resulted in the synthesis of adenosylcobalamin-5'-phosphate. This cobamide was isolated by HPLC, identified by UV-visible spectroscopy and mass spectrometry, and shown to support growth of a cobalamin auxotroph. Adenosylcobalamin-5'-phosphate was also isolated from reaction mixtures containing adenosylcobinamide-GDP (the product of the CobU reaction) and alpha-ribazole-5'-phosphate (the product of the CobT reaction) as substrates and CobS. These results allowed us to conclude that CobS is the cobalamin(-5'-phosphate) synthase enzyme in S. typhimurium. The CobC enzyme, previously shown to dephosphorylate alpha-ribazole-5'-phosphate to alpha-ribazole, was shown to dephosphorylate adenosylcobalamin-5'-phosphate to adenosylcobalamin. Adenosylcobinamide was converted to adenosylcobalamin in reactions where all four enzymes were present in the reaction mixture. This in vitro system offers a unique opportunity for the rapid synthesis and isolation of cobamides with structurally different lower-ligand bases that can be used to investigate the contributions of the lower-ligand base to cobalamin-dependent reactions.     

In vitro immunoglobulin synthesis by human intestinal lamina propria lymphocytes.

The concentration of IgG and IgA was measured in the supernatants of peripheral blood mononuclear cells and of cells harvested from the intestinal lamina propria, which were cultured in vitro in the presence or absence of mitogens. The lamina propria mononuclear cells were harvested by collagenase digestion of macroscopically normal mucosa from 10 fresh surgical resections for carcinoma. Secretion of IgA in cultures of unstimulated lamina propria mononuclear cells greatly exceeded that of IgG. The addition of pokeweed mitogen increased Ig secretion by cultures of peripheral blood mononuclear cells but decreased Ig secretion by lamina propria mononuclear cells. The addition of concanavalin A suppressed Ig synthesis by pokeweed mitogen stimulated cells more in cultures of peripheral blood mononuclear cells than in lamina propria mononuclear cells. Cycloheximide inhibited Ig secretion by more than 90% in cultures of peripheral blood mononuclear cells, but there was less inhibition in cultures of lamina propria mononuclear cells. In the four unstimulated cultures of lamina propria mononuclear cells examined, over 75% of the Ig was secreted in the first three to four days of culture. The results indicate that lamina propria mononuclear cells are refractory to the inductive and suppressive signals of mitogens, and represent an activated cell population which is committed to Ig secretion before being cultured.