Adsorption of ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase on calcium oxalate

We used ethylenediaminetetraacetic acid dianhydride (EDTAD) to modify oxalate decarboxylase (OXDC) to improve its adsorption on calcium oxalate stones. The modified sites were identified by Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and the adsorption mechanism of the EDTAD-modified OXDC on calcium oxalate (CaOx) was investigated. We investigated adsorption time, initial enzyme concentration, temperature and solution pH on the adsorption process. Data were analyzed using kinetics, thermodynamics and isotherm adsorption models. UPLC-MS showed that EDTAD was attached to OXDC covalently and suggested that the chemical modification occurred at both the free amino of the side chain and the α-NH₂ of the peptide. The adsorption capacity of the EDTAD-OXDC on calcium oxalate was 53.37% greater than that of OXDC at the initial enzyme concentration of 5 mg/ml, pH = 7.0, at 37° C. The modified enzyme (EDTAD-OXDC) demonstrated improved oxalate degradation activity at pH 4.5?6.0. Kinetic data fitting analysis suggested a pseudo second order kinetic model. Estimates of the thermodynamic parameters including ΔG⁰, ΔH⁰ and ΔS⁰ of the adsorption process showed it to be feasible, spontaneous and endothermic. Isotherm data fitting analysis indicated that the adsorption process is reduced to monolayer adsorption at a low enzyme concentration and to multilayer adsorption at a high enzyme concentration. It may be possible to apply OXDC to degradation of calcium oxalate stones.     

Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy

Renal fibrosis is the common histological feature of advanced glomerular and tubulointerstitial disease leading to end-stage renal disease (ESRD). However, specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persistent inflammation is a key event in the development of fibrosis, we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that, in the unilateral ureteral obstruction model of renal fibrosis, the B1R is overexpressed and that delayed treatment with an orally active nonpeptide B1R antagonist blocks macrophage infiltration, leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.     

Identification of novel DNA repair proteins via primary sequence, secondary structure, and homology

Background  

DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM).     

Dimeric Dnm1-G385D interacts with Mdv1 on mitochondria and can be stimulated to assemble into fission complexes containing Mdv1 and Fis1

Interactions between yeast Dnm1p, Mdv1p, and Fis1p are required to form fission complexes that catalyze division of the mitochondrial compartment. During the formation of mitochondrial fission complexes, the Dnm1p GTPase self-assembles into large multimeric complexes on the outer mitochondrial membrane that are visualized as punctate structures by fluorescent labeling. Although it is clear that Fis1p.Mdv1p complexes on mitochondria are required for the initial recruitment of Dnm1p, it is not clear whether Dnm1p puncta assemble before or after this recruitment step. Here we show that the minimum oligomeric form of cytoplasmic Dnm1p is a dimer. The middle domain mutant protein Dnm1G385Dp forms dimers in vivo but fails to assemble into punctate structures. However, this dimeric mutant stably interacts with Mdv1p on the outer mitochondrial membrane, demonstrating that assembly of stable Dnm1p multimers is not required for Dnm1p-Mdv1p association or for mitochondrial recruitment of Dnm1p. Dnm1G385Dp is reported to be a terminal dimer in vitro. We describe conditions that allow assembly of Dnm1G385Dp into functional fission complexes on mitochondria in vivo. Using these conditions, we demonstrate that multimerization of Dnm1p is required to promote reorganization of Mdv1p from a uniform mitochondrial localization into punctate fission complexes. Our studies also reveal that Fis1p is present in these assembled fission complexes. Based on our results, we propose that Dnm1p dimers are initially recruited to the membrane via interaction with Mdv1p.Fis1p complexes. These dimers then assemble into multimers that subsequently promote the reorganization of Mdv1p into punctate fission complexes.     

Chemical composition and cytotoxicity of oils and eremophilanes derived from various parts of Eremophila mitchellii Benth. (Myoporaceae)

A detailed investigation of the wood, leaf, branch and root oil of Eremophila mitchellii (Benth.) was carried out by a combination of GC-FID, GC-MS and NMR. The wood oil was composed predominantly of eremophilanes, a rare class of biologically active, bicyclic sesquiterpenoids. The root oil was also found to contain the eremophilanes together with the zizaene sesquiterpene, sesquithuriferone. 9-Hydroxy-1,7(11),9-eremophilatrien-8-one and the known 1(10),11-eremophiladien-9-one (eremophilone), 9-hydroxy-7(11),9-eremophiladien-8-one (2-hydroxyeremophilone), 8-hydroxy-11-eremophilen-9-one (santalcamphor), 8-hydroxy-10,11-eremophiladien-9-one, sesquithuriferone and 8-hydroxy-1,11-eremophiladien-9-one were purified and elucidated by NMR. Three approaches to the purification of the major eremophilanes from the wood oil are described. (+) Spathulenol, α-pinene, globulol, viridiflorene were the major constituents of the leaf oil. All of the essential oils and the eremophilanes exhibited cytotoxicity against P388D(1) mouse lymphoblast cells in-vitro.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Austrocolorins A1 and B1: atropisomeric 10,10'-linked dihydroanthracenones from an Australian Dermocybe sp

The atropisomeric austrocolorins A(1) (7) and B(1) (8), new members of the rare tricolorin class of 10,10'-coupled dihydroanthracenones, are isolated from an indigenous Australian toadstool belonging to the subgenus Dermocybe of Cortinarius, and their structure and absolute central and axial configuration is deduced from the spectroscopic data, and confirmed by chemical degradation and chiral HPLC analysis.     

Substitution bias, rapid saturation, and the use of mtDNA for nematode systematics

Only relatively recently have researchers turned to molecular methods for nematode phylogeny reconstruction. Thus, we lack the extensive literature on evolutionary patterns and phylogenetic usefulness of different DNA regions for nematodes that exists for other taxa. Here, we examine the usefulness of mtDNA for nematode phylogeny reconstruction and provide data that can be used for a priori character weighting or for parameter specification in models of sequence evolution. We estimated the substitution pattern for the mitochondrial ND4 gene from intraspecific comparisons in four species of parasitic nematodes from the family Trichostrongylidae (38-50 sequences per species). The resulting pattern suggests a strong mutational bias toward A and T, and a lower transition/transversion ratio than is typically observed in other taxa. We also present information on the relative rates of substitution at first, second, and third codon positions and on relative rates of saturation of different types of substitutions in comparisons ranging from intraspecific to interordinal. Silent sites saturate extremely quickly, presumably owing to the substitution bias and, perhaps, to an accelerated mutation rate. Results emphasize the importance of using only the most closely related sequences in order to infer patterns of substitution accurately for nematodes or for other taxa having strongly composition-biased DNA. ND4 also shows high amino acid polymorphism at both the intra- and interspecific levels, and in higher level comparisons, there is evidence of saturation at variable amino acid sites. In general, we recommend using mtDNA coding genes only for phylogenetics of relatively closely related nematode species and, even then, using only nonsynonymous substitutions and the more conserved mitochondrial genes (e.g., cytochrome oxidases). On the other hand, the high substitution rate in genes such as ND4 should make them excellent for population genetics studies, identifying cryptic species, and resolving relationships among closely related congeners when other markers show insufficient variation.      

Phylogeny of the Drosophila saltans species group based on combined analysis of nuclear and mitochondrial DNA sequences

Nucleotide sequences from two nuclear loci, alcohol dehydrogenase and internal transcribed spacer-1 of the nuclear ribosomal DNA repeats, and two mitochondrial genes, cytochrome oxidase I and cytochrome oxidase II, were determined from nine species in the Drosophila saltans species group. The partition homogeneity test and partitioned Bremer support were used to measure incongruence between phylogenetic hypotheses generated from individual partitions. Individual loci were generally congruent with each other and consistent with the previously proposed morphological hypothesis, although they differed in level of resolution. Since extreme conflict between partitions did not exist, the data were combined and analyzed simultaneously. The total evidence method gave a more resolved and highly supported phylogeny, as indicated by bootstrap proportions and decay indices, than did any of the individual analyses. The cordata and elliptica subgroups, considered to have diverged early in the history of the D. saltans group, were sister taxa to the remainder of the saltans group. The sturtevanti subgroup, represented by D. milleri and D. sturtevanti, occupies an intermediate position in this phylogeny. The saltans and parasaltans subgroups are sister clades and occupy the most recently derived portion of the phylogeny. As with previous morphological studies, phylogenetic relationships within the saltans subgroup were not satisfactorily resolved by the molecular data.      

Phylogenetic analysis supports horizontal transfer of P transposable elements

Nucleotide sequence comparisons were used to investigate the evolution of P transposable elements and the possibility that horizontal transfer has played a role in their occurrence in natural populations of Drosophila and other Diptera. The phylogeny of P elements was examined using published sequences from eight dipteran taxa and a new, partial sequence from Scaptomyza elmoi. The results from a number of different analyses are highly consistent and reveal a P-element phylogeny that contradicts the phylogeny of the species. At least three instances of horizontal transfer are necessary to explain this incongruence, but other explanations cannot be ruled out at this time.