A new role for heme, facilitating release of iron from the bacterioferritin iron biomineral

Bacterioferritin (BFR) from Escherichia coli is a member of the ferritin family of iron storage proteins and has the capacity to store very large amounts of iron as an Fe(3+) mineral inside its central cavity. The ability of organisms to tap into their cellular stores in times of iron deprivation requires that iron must be released from ferritin mineral stores. Currently, relatively little is known about the mechanisms by which this occurs, particularly in prokaryotic ferritins. Here we show that the bis-Met-coordinated heme groups of E. coli BFR, which are not found in other members of the ferritin family, play an important role in iron release from the BFR iron biomineral: kinetic iron release experiments revealed that the transfer of electrons into the internal cavity is the rate-limiting step of the release reaction and that the rate and extent of iron release were significantly increased in the presence of heme. Despite previous reports that a high affinity Fe(2+) chelator is required for iron release, we show that a large proportion of BFR core iron is released in the absence of such a chelator and further that chelators are not passive participants in iron release reactions. Finally, we show that the catalytic ferroxidase center, which is central to the mechanism of mineralization, is not involved in iron release; thus, core mineralization and release processes utilize distinct pathways.     

以肠道外症状为首发表现的结肠癌47例临床分析

目的:分析以肠道外症状为首发表现的结肠癌临床特征,旨在提高结肠癌诊治水平。方法:收集我院1994年1月至2008年1月间以肠道外症状为首发表现的结肠癌47例,对其临床特点进行回顾性分析。结果:47例以肠道外症状为首发表现的结肠癌中位发病年龄为65岁;贫血症状多见,占78.7%(37/47),长期低热3例,肝转移瘤4例,肺转移瘤2例,皮肌炎1例;肿瘤多发生于右半结肠,占74.5%(35/47);病理类型:乳头状腺癌为主,占61.7%(29/47);Dukes分期:B期占44.7%(21/47),C期占42.6%(20/47);结肠镜是重要诊断方法,结肠镜与钡灌肠有互补优势;手术是主要治疗手段,综合治疗可延长患者生存期。结论:以肠道外症状为首发表现的结肠癌以贫血多见,病变多位于右半结肠,结肠镜是主要确诊方法,以手术为主的综合治疗可改善患者预后。     

A Novel 1,4-Dihydropyridine Derivative Improves Spatial Learning and Memory and Modifies Brain Protein Expression in Wild Type and Transgenic APPSweDI Mice

Ca²⁺ blockers, particularly those capable of crossing the blood-brain barrier (BBB), have been suggested as a possible treatment or disease modifying agents for neurodegenerative disorders, e.g., Alzheimer’s disease. The present study investigated the effects of a novel 4-(N-dodecyl) pyridinium group-containing 1,4-dihydropyridine derivative (AP-12) on cognition and synaptic protein expression in the brain. Treatment of AP-12 was investigated in wild type C57BL/6J mice and transgenic Alzheimer’s disease model mice (Tg APP_SweDI) using behavioral tests and immunohistochemistry, as well as mass spectrometry to assess the blood-brain barrier (BBB) penetration. The data demonstrated the ability of AP-12 to cross the BBB, improve spatial learning and memory in both mice strains, induce anxiolytic action in transgenic mice, and increase expression of hippocampal and cortical proteins (GAD67, Homer-1) related to synaptic plasticity. The compound AP-12 can be seen as a prototype molecule for use in the design of novel drugs useful to halt progression of clinical symptoms (more specifically, anxiety and decline in memory) of neurodegenerative diseases, particularly Alzheimer’s disease.     

STRUCTURE AND DEVELOPMENT OF VIRUSES OBSERVED IN THE ELECTRON MICROSCOPE : IV. VIRUSES OF THE RI-APC GROUP

Representative viruses of the RI-APC group were observed with the electron microscope in thin sections of infected HeLa cells. The viral particles varied in density, were approximately 60 mµ in diameter and had a center to center spacing when close packed of about 65 mµ. Many of the less dense particles exhibited an internal body averaging 24 mµ in diameter. It was suggested that within the nucleus the virus differentiated from dense granular and reticular material and formed crystals. Disintegration of the crystals and disruption of the nuclear membrane with release of virus into the cytoplasm appeared to occur at any stage. No evidence to suggest development of the virus in the cytoplasm was obtained. It was possible to deduce the structure of the viral crystal from the electron micrographs. The viral particles are packed in a cubic body—centered lattice. Correlative histochemical observations in the light microscope which are now in progress revealed that the crystals and non-crystalline aggregates of virus were strongly Feulgen-positive.     

The mechanism of inhibition of Ran-dependent nuclear transport by cellular ATP depletion

Rran-dependent nuclear transport requires a nuclear pool of RanGTP both for the assembly of export complexes and the disassembly of import complexes. Accordingly, in order for these processes to proceed, Ran-dependent nuclear import and export assays in vitro require the addition of GTP to produce RanGTP. Notably, no ATP requirement can be detected for these transport processes in vitro. But in vivo, when cells are depleted of ATP by the addition of sodium azide and 2-deoxyglucose to block ATP production by oxidative phosphorylation and glycolysis, respectively, Ran-dependent nuclear import and export are rapidly inhibited. This raised the question of whether there is an ATP requirement for these nuclear transport pathways in an intact cell that has remained undetected in vitro. Here we report that the free (but not total) GTP concentration rapidly drops to an undetectable level upon ATP depletion as does the availability of RanGTP. Our conclusion is that the inhibition of Ran-dependent nuclear transport observed upon ATP depletion in vivo results from a shortage of RanGTP rather than the inhibition of some ATP-dependent process.     

The Z-DNA motif d(TG)30 promotes reception of information during gene conversion events while stimulating homologous recombination in human cells in culture.

Tracts of the alternating dinucleotide polydeoxythymidylic-guanylic [d(TG)].polydeoxyadenylic-cytidylic acid [d(AC)], present throughout the human genome, are capable of readily forming left-handed Z-DNA in vitro. We have analyzed the effects of the Z-DNA motif d(TG)30 upon homologous recombination between two nonreplicating plasmid substrates cotransfected into human cells in culture. In this study, the sequence d(TG)30 is shown to stimulate homologous recombination up to 20-fold. Enhancement is specific to the Z-DNA motif; a control DNA fragment of similar size does not alter the recombination frequency. The stimulation of recombination is observed at a distance (237 to 1,269 base pairs away from the Z-DNA motif) and involves both gene conversion and reciprocal exchange events. Maximum stimulation is observed when the sequence is present in both substrates, but it is capable of stimulating when present in only one substrate. Analysis of recombination products indicates that the Z-DNA motif increases the frequency and alters the distribution of multiple, unselected recombination events. Specifically designed crosses indicate that the substrate containing the Z-DNA motif preferentially acts as the recipient of genetic information during gene conversion events. Models describing how left-handed Z-DNA sequences might promote the initiation of homologous recombination are presented.     

Multifactor dimensionality reduction software for detecting gene-gene and gene-environment interactions

MOTIVATION: Polymorphisms in human genes are being described in remarkable numbers. Determining which polymorphisms and which environmental factors are associated with common, complex diseases has become a daunting task. This is partly because the effect of any single genetic variation will likely be dependent on other genetic variations (gene-gene interaction or epistasis) and environmental factors (gene-environment interaction). Detecting and characterizing interactions among multiple factors is both a statistical and a computational challenge. To address this problem, we have developed a multifactor dimensionality reduction (MDR) method for collapsing high-dimensional genetic data into a single dimension thus permitting interactions to be detected in relatively small sample sizes. In this paper, we describe the MDR approach and an MDR software package. RESULTS: We developed a program that integrates MDR with a cross-validation strategy for estimating the classification and prediction error of multifactor models. The software can be used to analyze interactions among 2-15 genetic and/or environmental factors. The dataset may contain up to 500 total variables and a maximum of 4000 study subjects. AVAILABILITY: Information on obtaining the executable code, example data, example analysis, and documentation is available upon request. SUPPLEMENTARY INFORMATION: All supplementary information can be found at http://phg.mc.vanderbilt.edu/Software/MDR.     

The Aspergillus fumigatus sialidase is a 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid hydrolase (KDNase): structural and mechanistic insights

Aspergillus fumigatus is a filamentous fungus that can cause severe respiratory disease in immunocompromised individuals. A putative sialidase from A. fumigatus was recently cloned and shown to be relatively poor in cleaving N-acetylneuraminic acid (Neu5Ac) in comparison with bacterial sialidases. Here we present the first crystal structure of a fungal sialidase. When the apo structure was compared with bacterial sialidase structures, the active site of the Aspergillus enzyme suggested that Neu5Ac would be a poor substrate because of a smaller pocket that normally accommodates the acetamido group of Neu5Ac in sialidases. A sialic acid with a hydroxyl in place of an acetamido group is 2-keto-3-deoxynononic acid (KDN). We show that KDN is the preferred substrate for the A. fumigatus sialidase and that A. fumigatus can utilize KDN as a sole carbon source. A 1.45-Å resolution crystal structure of the enzyme in complex with KDN reveals KDN in the active site in a boat conformation and nearby a second binding site occupied by KDN in a chair conformation, suggesting that polyKDN may be a natural substrate. The enzyme is not inhibited by the sialidase transition state analog 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) but is inhibited by the related 2,3-didehydro-2,3-dideoxy-d-glycero-d-galacto-nonulosonic acid that we show bound to the enzyme in a 1.84-Å resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5-Å resolution structure of a covalently bound catalytic intermediate. The A. fumigatus sialidase is therefore a KDNase with a similar catalytic mechanism to Neu5Ac exosialidases, and this study represents the first structure of a KDNase.     

Construction, expression, and biologic activity of murine/human chimeric antibodies with specificity for the human alpha/beta T cell receptor.

Murine/human chimeric antibodies with specificity for the human TCR-alpha/beta have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 or gamma 4 C region exons. The chimeric genes were transfected into murine Sp2/O hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1 to 7 pg/cell/24 h. The chimeric antibodies bound specifically to T cells and competed effectively with the parental murine mAb for binding to these sites. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced in the chimeric antibodies as compared with murine BMA 031. C-dependent cytolysis, however, was not detectable with any of the antibodies. Chimeric BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft-vs-host disease, autoimmune diseases and other T cell-related disorders.