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PubMed2004

Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterization.

Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue-specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B-domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte-produced FVIII.

Bioreactors↗
PubMed1991

Regulation of the chlA locus of Escherichia coli K12: involvement of molybdenum cofactor.

The chlA locus encodes functions required for the biosynthesis of the molybdopterin part of the molybdenum cofactor. Mutants, carrying gene fusions at the chlA locus, which place beta-galactosidase expression under the control of the chlA promoter, have been isolated employing lambda placMu1 as the mutagen. The mutants exhibited beta-galactosidase expression which was greatly enhanced when grown anaerobically. Secondary mutations at the chlB, D, E or G loci did not affect the high level of expression. The fnr gene product was not required for the anaerobic expression. Bacteriophage lambda transducing phages were isolated which carried the phi(chlA-lac) mutations and were used to construct chlA+/phi(clA-lac) merodiploids. The merodiploids exhibited a much lower level of expression but showed the same characteristics as strains carrying lac fusions to the single chromosomal chlA locus. Genetic evidence is presented which strongly suggests that the molybdenum cofactor is a repressor of chlA expression. The anaerobic enhancement of chlA expression is mediated via a mechanism that is distinct from the molybdenum cofactor effect.

Anaerobiosis↗
PubMed2006

Estimation of cytoplasmic nitrate and its electrochemical potential in barley roots using 13NO3 and compartmental analysis.

13NO3 was used to determine the intracellular compartmentation of NO3 in barley roots (Hordeum vulgare cv. Klondike), followed by a thermodynamic analysis of nitrate transport. Plants were grown in one-tenth Johnson's medium with 1 mol m3 NO3 (NO3-grown plants) or 1 mol m3 NH4NO3 (NH4NO3-grown plants). The cytoplasmic concentrations of NO3 in roots were only approx. 3-6 mol m3 (half-time for exchange approx. 21 s) in both NO3 and NH4NO3 plants. These pool sizes are consistent with published nitrate microelectrode data, but not with previous compartmental analyses. The electrochemical potential gradient for nitrate across the plasmalemma was +26 +/-1 kJ mol1 in both NO3- and NH4NO3-grown plants, indicating active uptake of nitrate. At an external pH of 6, the plasmalemma electrochemical potential for protons would be approx. -29 +/- 4 kJ mol1. If the cytoplasmic pH was 7.3 +/- 0.2, then 2H+/1NO3 cotransport, or a primary ATP-driven pump (2NO3/1ATP), are both thermodynamically possible. NO3 is also actively transported across the tonoplast (approx. +6 to 7 kJ mol1).

Biological Transport↗
PubMed1974

Mechanism of anion permeation through the muscle fibre membrane of an elasmobranch fish, Taeniura lymma.

1. Properties of anion permeation through the membrane of skeletal muscle fibres of the stingray, Taeniura lymma, were studied with intracellular recording and polarization techniques.2. The Cl conductance of the resting membrane in the normal stingray saline at pH 7.7 is 8-10 times greater than the K conductance.3. The Cl conductance decreases with decreasing external pH, with an apparent pK of 5.3, whereas the K conductance is independent of pH between 4 and 9.4. The Q(10) of the Cl conductance is about 2.0, compared with a value of 1.2-1.4 for the K conductance.5. The Cl conductance is proportional to the external Cl concentration when observed after the fibre is equilibrated in the test solution.6. The permeability sequence obtained by potential measurement is SCN > NO(3) > Cl = Br > I > ClO(3) and the permeability ratio is independent of the mole fraction of anions.7. The conductance sequence determined by total replacement of the external Cl with other anion species differs from the permeability sequence and the conductance observed for partial replacement deviates significantly from that expected from the independence principle.8. Possible mechanisms of anion permeation are discussed.

Animals↗
PubMed1986

Passive transport and binding of lead by human red blood cells.

The uptake of Pb into human red blood cells has been studied using Pb buffers. Passive Pb movements can be studied conveniently when the cells are depleted of adenosine 5'-triphosphate (ATP), to eliminate active transport, and of inorganic phosphate, to prevent precipitation of lead phosphate. Pb can cross the membrane passively in either direction. Influx and efflux show similar properties. Passive Pb transport is strongly stimulated by HCO3-, and is reduced by replacing Cl- with ClO4-. It is inhibited by low concentrations of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanostilbene-2.2'-disulphonic acid (DIDS), characteristic inhibitors of anion transport. Pb uptake is unaffected by varying the external concentrations of Na+, K+ and Ca2+. When Pb enters the cell, it binds mainly to haemoglobin. The ratio of bound Pb:free Pb2+ in the cytosol is estimated to be 6000:1. Pb binding to haemoglobin is unaffected by oxygenation. Binding to albumin is quantitatively similar to binding to haemoglobin. The implications of these results for the transport and binding of Pb in the blood are discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation.

Basic fibroblast growth factor (bFGF) binds to heparan sulfate proteoglycans at the cell surface and to receptors with tyrosine kinase activity. Prevention of binding between cell surface heparan sulfate and bFGF (i) substantially reduces binding of fibroblast growth factor to its cell-surface receptors, (ii) blocks the ability of bFGF to support the growth of Swiss 3T3 fibroblasts, and (iii) induces terminal differentiation of MM14 skeletal muscle cells, which is normally repressed by fibroblast growth factor. These results indicate that cell surface heparan sulfate is directly involved in bFGF cell signaling.

Animals↗
PubMed1998

TNF-alpha induction of CD44-mediated leukocyte adhesion by sulfation.

Regulation of cell adhesion is important for immune system function. CD44 is a tightly regulated cell adhesion molecule present on leukocytes and implicated in their attachment to endothelium during an inflammatory immune response. The proinflammatory cytokine tumor necrosis factor-alpha, but not interferon-gamma, was found to convert CD44 from its inactive, nonbinding form to its active form by inducing the sulfation of CD44. This posttranslational modification was required for CD44-mediated binding to the extracellular matrix component hyaluronan and to vascular endothelial cells. Sulfation is thus a potential means of regulating CD44-mediated leukocyte adhesion at inflammatory sites.

Cell Adhesion↗
PubMed1971

Phenotypic restoration by molybdate of nitrate reductase activity in chlD mutants of Escherichia coli.

ChlD mutants of Escherichia coli are pleiotropic, lacking formate-nitrate reductase activity as well as formate-hydrogenlyase activity. Whole-chain formate-nitrate reductase activity, assayed with formate as the electron donor and measuring the amount of nitrite produced, was restored to wild-type levels in the mutants by addition of 10(-4)m molybdate to the growth medium. Under these conditions, the activity of each of the components of the membrane-bound nitrate reductase chain increased after molybdate supplementation. In the absence of nitrate, the activities of the formate-hydrogenlyase system were also restored by molybdate. Strains deleted for the chlD gene responded in a similar way to molybdate supplementation. The concentration of molybdenum in the chlD mutant cells did not differ significantly from that in the wild-type cells at either low or high concentrations of molybdate in the medium. However, the distribution of molybdenum between the soluble protein and membrane fractions differed significantly from wild type. We conclude that the chlD gene product cannot be a structural component of the formate-hydrogenlyase pathway or the formate-nitrate reductase pathway, but that it must have an indirect role in processing molybdate to a form necessary for both electron transport systems.

Anaerobiosis↗
PubMed1972

Deletion and complementation analysis of biotin gene cluster of Escherichia coli.

Genetic deletions that terminate within the cluster of genes needed for biotin biosynthesis in Escherichia coli have been isolated and mapped by transduction with phages lambda and P1. These deletions order the point mutations in each of the five genes. Mutations causing biotin dependence were incorporated into lambdapbio transducing phages. New bio(-) mutations were induced by exposure of lambdapbio particles to ultraviolet light. Tests of complementation between such bio(-)pbio particles and bio(-) mutant cells divide the bio(-) mutations into five cistrons: bioA, bioB, bioF, bioC, and bioD. Certain bioA and bioF mutations exhibit intragenic complementation, suggesting that these genes determine enzymes composed of identical subunits.

Biotin↗
PubMed1975

Positive selection of mutants with deletions of the gal-chl region of the Salmonella chromosome as a screening procedure for mutagens that cause deletions.

We have developed a convenient and specific positive selection for long deletions through the gal region of the chromosomes of Salmonella typhimurium and Escherichia coli. Through simultaneous selection for mutations in the two closely linked genes, gal and chlA, a variety of deletions of varying length, some extending through as much as 1 min of the chromosome, could be readily obtained. Many of these deletions resulted in the loss of a gene, which we named dhb, concerned with the ability of the bacterium to synthesize the iron chelating agent enterobactin. The selection was adapted for the screening of mutagens for their ability to generate long deletions in the bacterial deoxyribonucleic acid. Forty agents were screened for this capability. Nitrous acid, previously reported to be an efficient mutagen for this purpose, increased the frequency of deletion mutations 50-fold in our system. Three others, nitrogen mustard, mitomycin C, and fast neutrons, were shown to increase the frequency of long deletions between five- and eightfold. The remainder were found to be incapable of generating these deletions.

Benzoates↗
PubMed1983

Membrane cytochromes of Escherichia coli chl mutants.

The cytochromes present in the membranes of Escherichia coli cells having defects in the formate dehydrogenase-nitrate reductase system have been analyzed by spectroscopic, redox titration, and enzyme fractionation techniques. Four phenotypic classes differing in cytochrome composition were recognized. Class I is represented by strains with defects in the synthesis or insertion of molybdenum cofactor. Cytochromes of the formate dehydrogenase-nitrate reductase pathway are present. Class II strains map in the chlC-chlI region. The cytochrome associated with nitrate reductase (cytochrome bnr) is absent in these strains, whereas that associated with formate dehydrogenase (cytochrome bfdh) is the major cytochrome in the membranes. Class III strains lack both cytochromes bfdh and bnr but overproduce cytochrome d of the aerobic pathway even under anaerobic conditions in the presence of nitrate. Class III strains have defects in the regulation of cytochrome synthesis. An fdhA mutant produced cytochrome bnr but lacked cytochrome bfdh. These results support the view that chlI (narI) is the structural gene for cytochrome bnr and that chlC (narG) and chlI(narI) are in the same operon, and they provide evidence of the complexity of the regulation of cytochrome synthesis.

Aldehyde Oxidoreductases↗
PubMed1987

Molybdenum effector of fumarate reductase repression and nitrate reductase induction in Escherichia coli.

In Escherichia coli the presence of nitrate prevents the utilization of fumarate as an anaerobic electron acceptor. The induction of the narC operon encoding the nitrate reductase is coupled to the repression of the frd operon encoding the fumarate reductase. This coupling is mediated by nitrate as an effector and the narL product as the regulatory protein (S. Iuchi and E. C. C. Lin, Proc. Natl. Acad. Sci. USA 84:3901-3905, 1987). The protein-ligand complex appears to control narC positively but frd negatively. In the present study we found that a molybdenum coeffector acted synergistically with nitrate in the regulation of frd and narC. In chlD mutants believed to be impaired in molybdate transport (or processing), full repression of phi(frd-lac) and full induction of phi(narC-lac) by nitrate did not occur unless the growth medium was directly supplemented with molybdate (1 microM). This requirement was not clearly manifested in wild-type cells, apparently because it was met by the trace quantities of molybdate present as a contaminant in the mineral medium. In chlB mutants, which are known to accumulate the Mo cofactor because of its failure to be inserted as a prosthetic group into proteins such as nitrate reductase, nitrate repression of frd and induction of narC were also intensified by molybdate supplementation. In this case a deficiency of the molybdenum coeffector might have resulted from enhanced feedback inhibition of molybdate transport (or processing) by the elevated level of the unutilized Mo cofactor. In addition, mutations in chlE, which are known to block the synthesis of the organic moiety of the Mo cofactor, lowered the threshold concentration of nitrate (< 1 micromole) necessary for frd repression and narC induction. These changes could be explained simply by the higher intracellular nitrate attainable in cells lacking the ability to destroy the effector.

Chlorates↗
PubMed1989

Genetic regulation of nitrate assimilation in Klebsiella pneumoniae M5al.

We isolated Mu dI1734 insertion mutants of Klebsiella pneumoniae that were unable to assimilate nitrate or nitrite as the sole nitrogen source during aerobic growth (Nas- phenotype). The mutants were not altered in respiratory (anaerobic) nitrate and nitrite reduction or in general nitrogen control. The mutations were linked and thus defined a single locus (nas) containing genes required for nitrate assimilation. beta-Galactosidase synthesis in nas+/phi(nas-lacZ) merodiploid strains was induced by nitrate or nitrite and was inhibited by exogenous ammonia or by anaerobiosis. beta-Galactosidase synthesis in phi(nas-lacZ) haploid (Nas-) strains was nearly constitutive during nitrogen-limited aerobic growth and uninducible during anaerobic growth. A general nitrogen control regulatory mutation (ntrB4) allowed nitrate induction of phi(nas-lacZ) expression during anaerobic growth. This and other results suggest that the apparent anaerobic inhibition of phi(nas-lacZ) expression was due to general nitrogen control, exerted in response to ammonia generated by anaerobic (respiratory) nitrate reduction.

Anaerobiosis↗