Chenopods synthesize betaine by a two-step oxidation of choline: choline -. betaine aldehyde -. betaine. Both oxidation reactions are carried out by isolated spinach (Spinacia oleracea L.) chloroplasts in darkness and are promoted by light. The mechanism of betaine aldehyde oxidation was investigated with subcellular fractions from spinach leaf protoplasts. The chloroplast stromal fraction contained a specific pyridine nucleotide-dependent betaine aldehyde dehydrogenase (about 150 to 250 nanomoles per milligram chlorophyll per hour) which migrated as one isozyme on native polyacrylamide gels stained for enzyme activity. The cytosol fraction contained a minor isozyme of betaine aldehyde dehydrogenase. Leaves of pea (Pisum sativum L.), a species that lacks betaine, had no betaine aldehyde dehydrogenase isozymes. The specific activity of betaine aldehyde dehydrogenase rose three-fold in spinach plants grown at 300 millimolar NaCl; both isozymes contributed to the increase. Stimulation of betaine aldehyde oxidation in illuminated spinach chloroplasts was due to a thylakoid activity which was sensitive to catalase; this activity occurred in pea as well as spinach, and so appears to be artifactual. We conclude that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H202-mediated reaction cannot be ruled out.Betaine (glycinebetaine) accumulates in response to salinization or to water deficit in chenopods, grasses, and in other angiosperm families (8) as well as in a number of prokaryotes (11,14). For higher plants, Wyn Jones et al. (30) have proposed that betaine is localized mainly in the cytoplasm, where it acts as a nontoxic osmoticum, allowing osmotic adjustment to occur without perturbing metabolic functions. Much evidence (7) now supports this proposal, which accords betaine synthesis a major role in adaptation to osmotic stress.In-vivo radiotracer studies (8) show that betaine is synthesized in leaves from a two-step oxidation of choline:Little is known about the nature of these reactions in plants or about the enzymes involved. However, the enzymology of choline oxidation is quite well known for mammalian liver, in which both steps are mitochondrial (10, 29), and for certain microorganisms (15-17). In these nonplant systems the choline -+ betaine aldehyde step is catalyzed by a flavoprotein dehydrogenase or oxidase, the betaine aldehyde --betaine step by a specific 'Funded We recently showed that both steps in choline oxidation are chloroplastic in spinach, that they are light-promoted, and that the effect of light is sensitive to DCMU (9). On the other hand, Pan et al. (19) reported that the cytosolic fraction from spinach leaves contained NAD-dehydrogenase activity specific for betaine aldehyde, and that the chloroplast fraction lacked this activity. Therefore, in this work we examined (a) the mechanisms by which spinach chloroplasts oxidize betaine aldehyde in darkness and light, and (b) the subcell distributio...

Chenopods synthesize betaine in the chloroplast via a twostep oxidation of choline: choline betaine aldehyde betaine.Our previous experiments with intact chloroplasts, and in vivo 1802 labeling studies, led us to propose that the first step is mediated by a monooxygenase which uses photosynthetically generated reducing power (C Lerma, AD Hanson, D Rhodes [1988] insensitive to carbon monoxide. The specific activity was increased threefold in plants growing in 200 millimolar NaCI. Gel filtration experiments gave a molecular weight of 98 kilodaltons for the choline-oxidizing enzyme, and provided no evidence for other electron carriers which might mediate the reduction of the 98-kilodalton enzyme by ferredoxin.supports Wyn Jones's hypothesis (28) that betaine accumulation is a biochemical adaptation to salinity, with betaine acting as a major cytoplasmic osmolyte in salt-stressed plants (1 1, 17, 26). In stressed or unstressed plants, betaine is synthesized by a two-step oxidation of choline; both steps occur in the chloroplast in spinach and other chenopods (3):-2H -2H Choline -. betaine aldehyde -* betaine In spinach, the second step is catalyzed by a stromal BADH,4 which is encoded by a single nuclear gene (24,25). The enzyme for the first step is not yet known, although two lines of indirect evidence indicate that it is a monooxygenase (which could produce the hydrate form of betaine aldehyde by introducing an OH group at Cl of choline). First, in vivo labeling studies demonstrate that spinach leaf disks incorporate 180 from 1802 into betaine, and that this occurs at the choline -. betaine aldehyde step (9). Second, experiments with intact chloroplasts show that the choline -+ betaine aldehyde reaction requires photosynthetically generated reducing power as well as 02(23). The choline-oxidizing enzyme from spinach chloroplasts would therefore appear to be unlike the choline dehydrogenases (8,12,21)

The argJ gene coding for N 2 -acetyl-l-ornithine: l-glutamate N-acetyltransferase, the key enzyme involved in the acetyl cycle of l-arginine biosynthesis, has been cloned from thermophilic procaryotes: the archaeon Methanoccocus jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearothermophilus. Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the linear biosynthetic pathway), whereas bacterial genes additionally complement an argA mutant (deficient in N-acetylglutamate synthetase, the first enzyme of the pathway). In keeping with these in vivo data the purified His-tagged ArgJ enzyme of M. jannaschii only catalyzes N 2 -acetylornithine conversion to ornithine, whereas T. neapolitana and B. stearothermophilus ArgJ also catalyze the conversion of glutamate to N-acetylglutamate using acetylCoA as the acetyl donor. M. jannaschii ArgJ is therefore a monofunctional enzyme, whereas T. neapolitana and B. stearothermophilus encoded ArgJ are bifunctional. Kinetic data demonstrate that in all three thermophilic organisms ArgJ-mediated catalysis follows ping-pong bi±bi kinetic mechanism. Acetylated ArgJ intermediates were detected in semireactions using [ 14 C]acetylCoA or [ 14 C]N 2 -acetyl-l-glutamate as acetyl donors. In this catalysis l-ornithine acts as an inhibitor; this amino acid therefore appears to be a key regulatory molecule in the acetyl cycle of l-arginine synthesis. Thermophilic ArgJ are synthesized as protein precursors undergoing internal cleavage to generate a and b subunits which appear to assemble to a2b2 heterotetramers in E. coli. The cleavage occurs between alanine and threonine residues within the highly conserved PXM-ATML motif detected in all available ArgJ sequences.

International audienceSubsets of mature B cell neoplasms are linked to infection with intracellular pathogens such as Epstein-Barr virus (EBV), hepatitis C virus (HCV), or Helicobacter pylori. However, the association between infection and the immunoglobulin-secreting (Ig-secreting) B proliferative disorders remains largely unresolved. We investigated whether the monoclonal IgG (mc IgG) produced by patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM) targets infectious pathogens. Antigen specificity of purified mc IgG from a large patient cohort (n = 244) was determined using a multiplex infectious-antigen array (MIAA), which screens for reactivity to purified antigens or lysates from 9 pathogens. Purified mc IgG from 23.4% of patients (57 of 244) specifically recognized 1 pathogen in the MIAA. EBV was the most frequent target (15.6%), with 36 of 38 mc IgGs recognizing EBV nuclear antigen-1 (EBNA-1). MM patients with EBNA-1-specific mc IgG (14.0%) showed substantially greater bone marrow plasma cell infiltration and higher β2-microglobulin and inflammation/infection-linked cytokine levels compared with other smoldering myeloma/MM patients. Five other pathogens were the targets of mc IgG: herpes virus simplex-1 (2.9%), varicella zoster virus (1.6%), cytomegalovirus (0.8%), hepatitis C virus (1.2%), and H. pylori (1.2%). We conclude that a dysregulated immune response to infection may underlie disease onset and/or progression of MGUS and MM for subsets of patients

Human Rad51 is a key element of recombinational DNA repair and is related to the resistance of cancer cells to chemo‐ and radiotherapies. The protein is thus a potential target of anti‐cancer treatment. The crystallographic analysis shows that the BRC‐motif of the BRCA2 tumor suppressor is in contact with the subunit–subunit interface of Rad51 and could thus prevent filament formation of Rad51. However, biochemical analysis indicates that a BRC‐motif peptide of 69 amino acids preferentially binds to the N‐terminal part of Rad51. We show experimentally that a short peptide of 28 amino acids derived from the BRC4 motif binds to the subunit–subunit interface and dissociates its filament, both in the presence and absence of DNA, certainly by binding to dissociated monomers. The inhibition is efficient and specific for Rad51: the peptide does not even interact with Rad51 homologs or prevent their interaction with DNA. Neither the N‐terminal nor the C‐terminal half of the peptide interacts with human Rad51, indicating that both parts are involved in the interaction, as expected from the crystal structure. These results suggest the possibility of developing inhibitors of human Rad51 based on this peptide.