Lipid peroxidation in plumbagin administered rats

Plumbagin was administered to rats at a concentration of 1,2,4,8 and 16 mg per kg body weight. After 24 h lipid peroxide levels were found to decrease in subcellular fractions of liver. Plumbagin inhibited ascorbate and nicotinafde adenine dinucleotide phosphate (reduced) dependent lipid peroxidation but was without any effect on cumene hydroperoxide dependent lipid peroxidation. Injection of 16 mg of plumbagin per kg body weight was found to decrease liver total reduced glutathione and also fcrosomal glucose-6-phosphatase. The results are discussed with reference to the anti- and prooxidant properties of plumbagin.     

Synthesis of ribulose 1,5-bisphosphate carboxylase by isolatedSorghum mesophyll chloroplasts

Chloroplasts isolated fromSorghum vulgare are active in light-dependent, organelle protein synthesis. Intact chloroplasts can use light as an energy source; photosynthetically inactive chloroplasts require the addition of ATP for this protein synthesis. Preincubation of chloroplasts in light at 25°C for 1 h depleted the endogenous templates completely; such preincubated chloroplasts translated exogenously added heterologous templates efficiently. When total cellular RNA fromChlorella protothecoides, a C₃ plant, was used as template for translation in a cell-free light-dependent system of isolated mesophyll chloroplasts fromSorghum vulgare, a C₄ type plant, polypeptides of 55 kDa (large subunit) and 15 kDa (small subunit) were detectable in the fluorographic profile of the newly synthesized proteins; these polypeptides were absent in the products obtained with endogenous RNA. Evidence for the fidelity of the system was obtained by immunological analysis of ribulose 1, 5-bisphosphate carboxylase obtained by the translation ofChlorella cellular RNAs.     

Self-binding antibodies (autobodies) form specific complexes in solution

In this report we have shown that members of the murine self-binding antibody family, S107, form soluble complexes and precipitate under conditions in which non-self-binding antibodies remain in solution. Two approaches were used to demonstrate the self-association of autobodies: size-exclusion column chromatography and polyethylene glycol (PEG)-mediated precipitation assay. The anti-phosphorylcholine antibody T15 and two somatic variants, U4, which binds DNA, and U10, which has no identified specificity, produced larger precipitates in 10% PEG than other non-self-binding antibodies. The selectivity of PEG-mediated precipitation of self-binding antibodies is demonstrated by reduction of precipitation with specific haptens known to inhibit self-binding in solid-phase assays. Phosphorylcholine and nucleotides reduced precipitation of T15 and U4, respectively, but not U10. To rule out Fc-Fc mediated self-association in solution, we have also demonstrated self-complexing of F(ab')2 fragments of T15 using PEG. The self-binding locus was further dissected using peptides derived from V regions. A 24-residue peptide derived from the second hypervariable region of the VH of S107 specifically enhanced precipitation of T15, U4, and U10, but not other antibodies. These results provide evidence of a dormant potential of self-binding antibodies to precipitate under conditions that reduce the solubility of proteins. The implication of this potential is discussed with respect to pathological complex formation.     

Tumor idiotype vaccines. VII. Analysis and correlation of structural, idiotypic, and biologic properties of protective and nonprotective Ab2

We have previously generated and used anti-Id mAb (Ab2) to induce protective immunity against the L1210 DBA/2 tumor and for immunotherapy of established tumors. Among various anti-Id that were typed serologically as internal image Ab2 of the mouse mammary tumor virus tumor-associated Ag gp52, only one induced protective immunity and was effective in immunotherapy. In this study we compared the structural, idiotypic, and network properties of the protective and nonprotective antiidiotypic antibodies. The DNA sequence of the variable regions of six anti-Id was determined. The VH sequence of four Ab2, including the protective Ab2, are highly homologous, whereas the VL sequences differ and were assigned to different Vk families. In addition, the DH sequence region of the same four Ab2 are identical, whereas one is highly homologous and another one without homology. Search for amino acid sequence homologies between the Ab2 and gp52 showed the strongest similarities in the CDR2 of the L chain from the protective Ab2. In addition, the CDR2 region also had homology with a T cell epitope on gp52. The biologic basis of effective idiotypic mimicry was studied at the level of Ab3 induced by the Ab2. Id inhibition analysis using Ab3 induced by either protective or nonprotective Ab2, revealed differences. Thus, there is evidence for differences among the Ab1-Ab2-Ab3 cascade induced by protective and nonprotective anti-Id.     

The evolutionary origin of the 35 kb circular DNA of Plasmodium falciparum: new evidence supports a possible rhodophyte ancestry

In common with other Apicomplexan parasites, Plasmodium falciparum carries two extrachromosomal DNAs, one of which, the 6 kb element, is undoubtedly mitochondrial. The second, generally referred to as the 35 kb circle, is of unknown provenance, but the nature and organization of its genetic content makes a mitochondrial association unlikely and the molecule has features reminiscent of plastid genomes. We now report the occurrence on the circle of an open reading frame specifying a predicted 470 amino acid protein that shares more than 50% identity with a gene currently known only on the plastome of red algae. This high degree of conservation confirms the 35 kb circle's plastid ancestry, and we speculate that it may have originated from the rhodoplast of an ancient red algal endosymbiont in the progenitor of the Apicomplexa.     

Structure and mechanism of action of a novel phosphoglycerate mutase from Bacillus stearothermophilus

Bacillus stearothermophilus phosphoglycerate mutase (PGM), which interconverts 2- and 3-phosphoglyceric acid (PGA), does not require 2,3-diphosphoglyceric acid for activity. However, this enzyme does have an absolute and specific requirement for Mn(2+) ions for catalysis. Here we report the crystal structure of this enzyme complexed with 3PGA and manganese ions to 1.9 A resolution; this is the first crystal structure of a diphosphoglycerate-independent PGM to be determined. This information, plus the location of the two bound Mn(2+) ions and the 3PGA have allowed formulation of a possible catalytic mechanism for this PGM. In this mechanism Mn(2+) ions facilitate the transfer of the substrate's phosphate group to Ser62 to form a phosphoserine intermediate. In the subsequent phosphotransferase part of the reaction, the phosphate group is transferred from Ser62 to the O2 or O3 positions of the reoriented glycerate to yield the PGA product. Site-directed mutagenesis studies were used to confirm our mechanism and the involvement of specific enzyme residues in Mn(2+) binding and catalysis.     

Differential incision of bulky carcinogen-DNA adducts by the UvrABC nuclease: comparison of incision rates and the interactions of Uvr subunits with lesions of different structures

The UvrABC nuclease system from Escherichia coli removes DNA damages induced by a wide range of chemical carcinogens with variable efficiencies. The interactions with UvrABC proteins of the following three lesions site-specifically positioned in DNA, and of known conformations, were investigated: (i) adducts derived from the binding of the (-)-(7S,8R,9R,10S) enantiomer of 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] by cis-covalent addition to N(2)-2'-deoxyguanosine [(-)-cis-anti-BP-N(2)-dG], (ii) an adduct derived from the binding of the (+)-(1R,2S,3S,4R) enantiomer of 1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-5-methylchrysene [(+)-anti-5-MeCDE] by trans addition to N(2)-2'-deoxyguanosine [(+)-trans-anti-MC-N(2)-dG], and (iii) a C8-2'-deoxyguanosine adduct (C8-AP-dG) formed by reductively activated 1-nitropyrene (1-NP). The influence of these three different adducts on UvrA binding affinities, formation of UvrB-DNA complexes by quantitative gel mobility shift analyses, and the rates of UvrABC incision were investigated. The binding affinities of UvrA varied among the three adducts. UvrA bound to the DNA adduct (+)-trans-anti-MC-N(2)-dG with the highest affinity (K(d) = 17 +/- 2 nM) and to the DNA containing C8-AP-dG with the least affinity (K(d) = 28 +/- 1 nM). The extent of complex formation with UvrB was also the lowest with the C8-AP-dG adduct. 5' Incisions occurred at the eighth phosphate from the modified guanine. The major 3' incision site corresponded to the fifth phosphodiester bond for all three adducts. However, additional 3' incisions were observed at the fourth and sixth phosphates in the case of the C8-AP-dG adduct, whereas in the case of the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG lesions additional 3' cleavage occurred at the sixth and seventh phosphodiester bonds. Both the initial rate and the extent of 5' and 3' incisions revealed that C8-AP-dG was repaired less efficiently in comparison to the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG containing DNA adducts. Our study showed that UvrA recognizes conformational changes induced by structurally different lesions and that in certain cases the binding affinities of UvrA and UvrB can be correlated with the incision rates. The size of the bubble formed around the damaged site with mismatched bases also appears to influence the incision rates. A particularly noteworthy finding in this study is that UvrABC repair of a substrate with no base opposite C8-AP-dG was quite inefficient as compared to the same adduct with a C opposite it. These findings are discussed in terms of the available NMR solution structures.     

Is dipalmitoylphosphatidylcholine a substrate for convertase?

Convertase has homology with carboxylesterases, but its substrate(s) is not known. Accordingly, we determined whether dipalmitoylphosphatidylcholine (DPPC), the major phospholipid in surfactant, was a substrate for convertase. We measured [(3)H]choline release during cycling of the heavy subtype containing [(3)H]choline-labeled DPPC with convertase, phospholipases A(2), B, C, and D, liver esterase, and elastase. Cycling with liver esterase or peanut or cabbage phospholipase D produced the characteristic profile of heavy and light peaks observed on cycling with convertase. In contrast, phospholipases A(2), B, and C and yeast phospholipase D produced a broad band of radioactivity across the gradient without distinct peaks. [(3)H]choline was released when natural surfactant containing [(3)H]choline-labeled DPPC was cycled with yeast phospholipase D but not with convertase or peanut and cabbage phospholipases D. Similarly, yeast phospholipase D hydrolyzed [(3)H]choline from [(3)H]choline-labeled DPPC after incubation in vitro, whereas convertase, liver esterase, or peanut and cabbage phospholipases D did not. Thus convertase, liver esterase, and plant phospholipases D did not hydrolyze choline from DPPC either on cycling or during incubation with enzyme in vitro. In conclusion, conversion of heavy to light subtype of surfactant by convertase may require a phospholipase D type hydrolysis of phospholipids, but the substrate in this reaction is not DPPC.