Some dicopper complexes of benzimidazole-containing ligands.

Dicopper complexes of the following benzimidazole-containing ligands have been studied as possible models for the active site of hemocyanin: EDTB (N,N,N',N'-tetrakis-(2-benzimidazolylmethyl)-1,2-ethanediamine), EGTB (1,1,10,10-tetrakis-(2-benzimidazolylmethyl)-1,10-diaza-4,7- dioxadecane), and MEGTB (1,1,10,10-tetrakis-(1-methylbenzimidazol-2-y lmethyl)-1,10-diaza-4,7-dioxadecane). The initial oxygenation product of Cu2(EDTB)(ClO4)2 in Me2SO gives optical absorption maxima at 315 nm (epsilon = 3750 M-1 cm-1) and 690 nm (epsilon = 100 M-1 cm-1). The fluorescence emission intensities of Cu2(EDTB)(ClO4)2 at 400 and 700 nm (excitation at 350 nm) decreases rapidly on exposure to air. This suggests oxidation of Cu2(I) to Cu2(II). The x-ray absorption edge spectra suggest that both coppers in the oxygenation product, analyzed as Cu2(EDTB)(ClO4)2(O).3H2O, are Cu(II). From spectrophotometric titration of Cu2(MEGTB)Cl4 with azide, formation constant of the Cu2(MEGTB)N3Cl3 complex has been obtained. Data from cyclic voltammetry experiments suggest that in the presence of azide, Cu(II)(N3)Cu(II) species is present.     

Identification of a second pyridoxine (pyridoxamine) 5′-phosphate oxidase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Pyridoxine (pyridoxamine) 5′-phosphate oxidase (PPOX) is involved in the biosynthetic pathway of vitamin B₆, converting pyridoxine 5′-phosphate (PNP) or pyridoxamine 5′-phosphate (PMP) into pyridoxal 5′-phosphate (PLP). PLP is a well-known cofactor of numerous enzymes including transamination and decarboxylation reactions. We have previously identified a PPOX (AtPPOX-1) protein encoded by At5g49970 in Arabidopsis thaliana. Here, we report a second PPOX in Arabidopsis, which was named as AtPPOX-2 encoded by At2g46580. The RT-PCR amplified cDNA of AtPPOX-2 was cloned into an Escherichia coli expression vector and a yeast shuttle vector. Both PPOX enzyme assay and complementation of the oxidative stress sensitivity phenotype of a yeast PDX3 deletion mutant demonstrated that At2g46580 encodes a PPOX protein (AtPPOX-2). The catalytic efficiency of AtPPOX-1 is approximately 300-fold higher than that of AtPPOX-2 for PNP. Based on bioinformatic analysis, AtPPOX-2 has a putative mitochondrial transit peptide at the N-terminus. The truncated AtPPOX-2 without 18 amino acids at the N-terminal end lost PPOX activity, suggesting that the N-terminal 18 amino acids are necessary for the enzyme activity of AtPPOX-2. Phylogenetic analysis of AtPPOX-2 homologs from all domains of life suggests that AtPPOX-2 homologs in plants are the product of lateral gene transfer from the cyanobacterial endosymbionts from which plastids are derived.     

Effects of Phosphate,HEDTA, and Light Sources on Cr(VI) Retention by Goethite

Iron hydrous hydro(oxide) has been regarded as an important sorbent for Cr(VI) in soil systems due to its wide distribution. However, many factors, such as phosphate (P), organic ligands, and light sources, could influence Cr(VI) retention by the soil components. The existence of inorganic or organic ligands not only competes with solution Cr(VI) for surface sites, but also results in releasing sorbed Cr(VI). Although organic matter can reduce Cr(VI) to less toxic Cr(III), the reduction rate is extremely slow. The objective of this study was to evaluate the influence of P on Cr(VI) sorption by goethite. The reduction of Cr(VI) by N-hydroxyethyl-ethylenediamine-triacetic acid (HEDTA) and goethite under different intensity of light was also investigated. Competitive sorption experiment indicated that P had lower inhibition of Cr(VI) sorption when the initial Cr(VI) concentration was higher than P. Goethite suspensions could catalyze Cr(VI) reduction under growth chamber light. Goethite accompanied with light could also accelerate Cr(VI) reduction by HEDTA. This phenomenon could be evidenced by the formation of Cr(III) and decreasing desorption of retained Cr(VI) by P.     

Modeling helix-turn-helix protein-induced DNA bending with knowledge-based distance restraints.

A crucial element of many gene functions is protein-induced DNA bending. Computer-generated models of such bending have generally been derived by using a presumed bending angle for DNA. Here we describe a knowledge-based docking strategy for modeling the structure of bent DNA recognized by a major groove-inserting alpha-helix of proteins with a helix-turn-helix (HTH) motif. The method encompasses a series of molecular mechanics and dynamics simulations and incorporates two experimentally derived distance restraints: one between the recognition helix and DNA, the other between respective sites of protein and DNA involved in chemical modification-enabled nuclease scissions. During simulation, a DNA initially placed at a distance was "steered" by these restraints to dock with the binding protein and bends. Three prototype systems of dimerized HTH DNA binding were examined: the catabolite gene activator protein (CAP), the phage 434 repressor (Rep), and the factor for inversion stimulation (Fis). For CAP-DNA and Rep-DNA, the root mean square differences between model and x-ray structures in nonhydrogen atoms of the DNA core domain were 2.5 A and 1.6 A, respectively. An experimental structure of Fis-DNA is not yet available, but the predicted asymmetrical bending and the bending angle agree with results from a recent biochemical analysis.     

An Examination of the Association between FOXA1 Staining Level and Biochemical Recurrence following Salvage Radiation Therapy for Recurrent Prostate Cancer

Background

Standardly collected clinical and pathological patient information has demonstrated only moderate ability to predict risk of biochemical recurrence (BCR) of prostate cancer in men undergoing salvage radiation therapy (SRT) for a rising PSA after radical prostatectomy (RP). Although elevated FOXA1 staining has been associated with poor patient outcomes following RP, it has not been studied in the specific setting of SRT after RP. The aim of this study was to evaluate the association between FOXA1 staining level and BCR after SRT for recurrent prostate cancer.

Methods

A total of 141 men who underwent SRT at our institution were included. FOXA1 staining levels in primary tumor samples were detected using immunohistochemistry. FOXA1 staining percentage and intensity were measured and multiplied together to obtain a FOXA1 H-score (range 0–12) which was our primary staining measure. P-values ≤ 0.0056 were considered as statistically significant after applying a Bonferroni correction for multiple comparisons.

Results

There was not a significant association between FOXA1 H-score and risk of BCR when considering H-score as an ordinal variable or as a categorical variable (all P≥0.090). Similarly, no significant associations with BCR were observed for FOXA1 staining percentage or staining intensity (all P≥0.14).

Conclusions

FOXA1 staining level does not appear to have a major impact on risk of BCR after SRT.     

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice¹. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin², and those interested in myasthenia gravis inject them with the acetylcholine receptor³. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified⁴, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.Download video file.^{(63M, mov)}     

Directional shape complementarity at the protein-DNA interface

Nature utilizes various styles of architecture for DNA-binding proteins to recognize diverse DNA sequences, a process facilitated by a complementary surface between protein and DNA. However, the extent and ways this 'shape complementarity' occurs at the protein-DNA interface have yet to be characterized. Here, by analyzing a set of diverse protein-DNA complexes of known three-dimensional structures, we investigated whether the normal vectors of a protein surface at the interface exhibited any relationship with DNA conformation. Generally, the normal vectors of a DNA-contacting protein surface distinctly preferred certain angles, enabling them to align with certain axes characterizing the conformation of DNA. Thus, a new geometric property of DNA-binding protein is demonstrated, i.e. the "shape complementarity" of protein-DNA recognition clearly bears the property of "directionality".