IgG anti-IgE autoantibodies in visceral leishmaniasis

Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 +/- 0.43) than sera from healthy individuals (OD = 1.35 +/- 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 +/- 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.     

Molecular phylogenetic relationships among Lemnaceae and Araceae using the chloroplast trnL-trnF intergenic spacer

We test competing hypotheses of relationships among Aroids (Araceae) and duckweeds (Lemnaceae) using sequences of the trnL-trnF spacer region of the chloroplast genome. Included in the analysis were 22 aroid genera including Pistia and five genera of Lemnaceae including the recently segregated genus Landoltia. Aponogeton was used as an outgroup to root the tree. A data set of 522 aligned nucleotides yielded maximum parsimony and maximum likelihood trees similar to those previously derived from restriction site data. Pistia and the Lemnaceae are placed in two separate and well-supported clades, suggesting at least two independent origins of the floating aquatic growth form within the aroid clade. Within the Lemnaceae there is only partial support for the paradigm of sequential morphological reduction, given that Wolffia is sister to Wolffiella+Lemna. As in the results of the restriction site analysis, pantropical Pistia is placed with Colocasia and Typhonium of southeastern Asia, indicative of Old World affinities. Branch lengths leading to duckweed terminal taxa are much longer relative to other ingroup taxa (including Pistia), evidently as a result of higher rates of nucleotide substitutions and insertion/deletion events. Morphological reduction within the duckweeds roughly correlates with accelerated chloroplast genome evolution.     

Hydrocarbon utilization by nodule bacteria and plant growth-promoting rhizobacteria

Standard and locally isolated nodule bacteria and plant growth-promoting rhizobacteria (PGPR) were grown on crude oil and individual pure hydrocarbons as sole sources of carbon and energy. The nodule bacteria included two standard Rhizobium leguminosarum strains, two standard Bradyrhizobium japonicum strains, and one unknown nodule bacterial strain that was locally isolated from Vicia faba nodules. The PGPR included one standard Serratia liquefaciens strain and two locally isolated strains of Pseudomonas aeruginosa and Flavobacterium sp. The pure hydrocarbons tested included n-alkanes with chain lengths from C9 to C40 and the aromatic hydrocarbons benzene, biphenyle, naphthalene, phenanthrene, and toluene. Quantitative gas liquid chromatographic analyses confirmed that pure cultures of representative nodule bacteria and PGPR could attenuate n-octadecane and phenanthrene in the surrounding nutrient medium. Further, intact nodules of V. faba containing bacteria immobilized on and within those nodules reduced hydrocarbon levels in a medium in which those nodules were shaken. It was concluded that legume crops are suitable phytoremediation tools for oily soil, since they enrich such soils not only with fixed nitrogen, but also with hydrocarbon-utilizing microorganisms. Further, legume nodules may have biotechnological value as materials for cleaning oily liquid wastes.     

Field study of the fate of labelled fertilizer ammonium-N applied to sesame and sunflower in a sandy soil

The recovery in crop and soil of labelled fertilizer ammonium-N applied to sesame and sunflower growing on sandy soil was measured. The sesame and sunflower received respectively 238 and 143 kg Nha⁻¹ as (NH₄)₂SO₄ enriched with 4.63 At. %¹⁵N excess. In the plants, the Ndff was 31.19% and 31.96% in sesame and sunflower, respectively. The fertilizer recovery by sunflower was 22.3%, by sesame only 12.3%. The amount of fertilizer N remaining in the soil at harvest was 13.04% for the sesame and 5.95% for the sunflower plot. The loss of fertilizer N under sesame was 74.66% and 71.75% under sunflower. The average of seed yield of the plants inside the¹⁵N plot was compared with the seed yield of the same amount of plants from outside the¹⁵N plot. They did not differ significantly, indicating that the results obtained from the¹⁵N plot can be extrapolated to the rest of the field.     

Post-translational Modification of Ribosomal Proteins: STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF RimO FROM THERMOTOGA MARITIMA, A RADICAL S-ADENOSYLMETHIONINE METHYLTHIOTRANSFERASE

Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826–1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 Å crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family.     

Synthesis and characterization of a series of N3O-ligated mononuclear Mn(II) halide complexes

Treatment of a N₃O-donor chelate ligand (mpppa = N-methyl-N-((6-pivaloylamido-2-pyridyl)methyl)-N-(2-pyridylethyl)amine; bpppa = N-benzyl-N-((6-pivaloylamido-2-pyridyl)methyl)-N-(2-pyridylmethyl)amine) with equimolar amounts of Mn(ClO₄)₂ · 6H₂O and Me₄NX (X = Cl, Br, I) in methanol resulted in the production of a series of mononuclear Mn(II) halide complexes of the formula [(L)Mn-X(CH₃OH)]ClO₄ (L = mpppa or bpppa). X-ray crystallographic studies of [(mpppa)Mn-Cl(CH₃OH)]ClO₄ · CH₃OH (2 · CH₃OH), [(mpppa)Mn-Br(CH₃OH)]ClO₄ · CH₃OH (4 · CH₃OH), [(mpppa)Mn-I(CH₃OH)]ClO₄ · CH₃OH (6 · CH₃OH), and [(bpppa)Mn-I(CH₃OH)]ClO₄ · O₂(CH₂CH₃)₂ (7 · O(CH₂CH₃)₂) revealed for each a mononuclear Mn(II) center having tetradentate coordination of the chelate ligand, one coordinated halide anion, and one molecule of coordinated methanol. An increase in the Mn-X distance through the halide series (Cl, Br, I) correlates linearly with the increase in the radius of the anion. The magnetic moment of each halide complex, measured via Evans method in methanol, is consistent with the presence of a high-spin distorted octahedral Mn(II) center. The EPR features of the halide complexes in methanol do not change as a function of the nature of the halide coordinated to the Mn(II) center.     

Two novel mutations in exon 2 of bone morphogenetic protein (BMP) 15 gene in Pakistani infertile females

ObjectiveTo determine the proportion of fertility in Pakistani infertile females and discover if there are considerable connection among BMP15 gene polymorphism, follicle maturation and hormonal regulation in Pakistani infertile females.MethodsAll selected participants were initially examined through follicle-stimulating hormones (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), Prolactin, and Trans-vaginal scan (TVS). BMP15 gene polymorphism among infertile and fertile females was done by extracted Genomic DNA from whole blood. Sanger sequencing was performed for the identification of mutation in exons-intron boundaries of the BMP15 gene. Bioinformatics tools were used to assess the protein structure.ResultsThe total five mutations including two novel missense variants of BMP15 in exon 2, whereas three previously reported i.e. two cosmic mutations (c.615delC), (c.584InsG) and one frame shift mutations (c.635delA) were also observed. The first novel mutation was found at (c.1038InsGG) (p.346Gln < Gly) in which the insertion of GG at DNA position 1038 of exon 2 resulting in a substitution of glutamine into glycine at 346th amino acid of BMP15 protein. The second novel variant (c.1049delT) (p. Ser334Pro) was also observed in exon 2 of the BMP15 gene, which substituted serine into proline at 334th amino acid of the BMP15 protein.ConclusionIt is concluded that there are various missense mutations present in exon 2 of the BMP15 gene of Pakistani infertile females, consequently expected function of protein changes due to change in codons of amino acids. Provean and SIFT suggest the two novel variants as potentially deleterious. Although three other variants were also found in Pakistani infertile females which were previously reported. These mutations may result in early blockage of folliculogenesis and ovaries become streaky. Further research is required to resolve the actual allusion of these variations in the BMP15 gene.     

Guiding protein aggregation with macromolecular crowding

Macromolecular crowding is expected to have a significant effect on protein aggregation. In the present study we analyzed the effect of macromolecular crowding on fibrillation of four proteins, bovine S-carboxymethyl-alpha-lactalbumin (a disordered form of the protein with reduced three out of four disulfide bridges), human insulin, bovine core histones, and human alpha-synuclein. These proteins are structurally different, varying from natively unfolded (alpha-synuclein and core histones) to folded proteins with rigid tertiary and quaternary structures (monomeric and hexameric forms of insulin). All these proteins are known to fibrillate in diluted solutions, however their aggregation mechanisms are very divers and some of them are able to form different aggregates in addition to fibrils. We studied how macromolecular crowding guides protein between different aggregation pathways by analyzing the effect of crowding agents on the aggregation patterns under the variety of conditions favoring different aggregated end products in diluted solutions.     

X-ray structure of the [FeFe]-hydrogenase maturase HydE from Thermotoga maritima

Maturation of the [FeFe]-hydrogenase active site depends on at least the expression of three gene products called HydE, HydF, and HydG. We have solved the high resolution structure of recombinant, reconstituted S-adenosine-L-methionine-dependent HydE from Thermotoga maritima. Besides the conserved [Fe(4)S(4)] cluster involved in the radical-based reaction, this HydE was reported to have a second [Fe(4)S(4)] cluster coordinated by three Cys residues. However, in our crystals, depending on the reconstitution and soaking conditions, this second cluster is either a [Fe(2)S(2)] center, with water occupying the fourth ligand site or is absent. We have carried out site-directed mutagenesis studies on the related HydE from Clostridium acetobutylicum, along with in silico docking and crystal soaking experiments, to define the active site region and three anion-binding sites inside a large, positive cavity, one of which binds SCN(-) with high affinity. Although the overall triose-phosphate isomerase-barrel structure of HydE is very similar to that of biotin synthase, the residues that line the internal cavity are significantly different in the two enzymes.