Module M1 of Zebrafish Neuroglobin Acts as a Structural and Functional Protein Building Block for a Cell-Membrane-Penetrating Activity

Neuroglobin (Ngb) is a recently discovered vertebrate globin that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection during oxidative stress that occurs, for example, during ischemia and reperfusion. Recently, we found that zebrafish, but not human, Ngb can translocate into cells. Moreover, we demonstrated that a chimeric ZHHH Ngb protein, in which the module M1 of human Ngb is replaced by the corresponding region of zebrafish Ngb, can penetrate cell membranes and protect cells against oxidative stress-induced cell death, suggesting that module M1 of zebrafish Ngb is important for protein transduction. Furthermore, we recently showed that Lys7, Lys9, Lys21, and Lys23 in module M1 of zebrafish Ngb are crucial for protein transduction activity. In the present study, we have investigated whether module M1 of zebrafish Ngb can be used as a building block to create novel cell-membrane-penetrating folded proteins. First, we engineered a chimeric myoglobin (Mb), in which module M1 of zebrafish Ngb was fused to the N-terminus of full-length human Mb, and investigated its functional and structural properties. Our results showed that this chimeric Mb protein is stable and forms almost the same heme environment and α-helical structure as human wild-type Mb. In addition, we demonstrated that chimeric Mb has a cell-membrane-penetrating activity similar to zebrafish Ngb. Moreover, we found that glycosaminoglycan is crucial for the cell-membrane-penetrating activity of chimeric Mb as well as that of zebrafish Ngb. These results enable us to conclude that such module substitutions will facilitate the design and production of novel functional proteins

Human XPC-hHR23B interacts with XPA-RPA in the recognition of triplex-directed psoralen DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair (NER), XPA-RPA, recognizes DNA ICLs. We now report the use of triplex technology to direct a site-specific psoralen ICL to a target DNA substrate to determine whether the human global genome NER damage recognition complex, XPC-hHR23B, recognizes this lesion. Our results demonstrate that XPC-hHR23B recognizes psoralen ICLs, which have a structure fundamentally different from other lesions that XPC-hHR23B is known to bind, with high affinity and specificity. XPC-hHR23B and XPA-RPA protein complexes were also observed to bind psoralen ICLs simultaneously, demonstrating not only that psoralen ICLs are recognized by XPC-hHR23B alone, but also that XPA-RPA may interact cooperatively with XPC-hHR23B on damaged DNA, forming a multimeric complex. Since XPC-hHR23B and XPA-RPA participate in the recognition and verification of DNA damage, these results support the hypothesis that interplay between components of the global genome repair sub-pathway of NER is critical for the recognition of psoralen DNA ICLs in the mammalian genome

Adenomatoid tumour of the liver

An unusual primary adenomatoid tumour arising in the normal liver is described. Hepatectomy was performed, and the patient is alive and free of disease 1 year postsurgery. Grossly, the tumour showed a haemorrhagic cut surface with numerous microcystic structures. Histological examination revealed cystic or angiomatoid spaces of various sizes lined by cuboidal, low-columnar, or flattened epithelioid cells with vacuolated cytoplasm and round to oval nuclei. The epithelioid cells were entirely supported by proliferated capillaries and arteries together with collagenous stroma. Immunohistochemical studies showed that the epithelioid cells were strongly positive for a broad spectrum of cytokeratins (AE1/AE3, CAM5.2, epithelial membrane antigen and cytokeratin 7) and mesothelial markers (calretinin, Wilms’ tumour 1 and D2-40). These cells were negative for Hep par-1, carcinoembryonic antigen, neural cell adhesion molecule, CD34, CD31 and HMB45. Atypically, abundant capillaries were observed; however, the cystic proliferation of epithelioid cells with vacuoles and immunohistochemical profile of the epithelioid element were consistent with hepatic adenomatoid tumour

Characterization of Reaction Intermediates of Human Excision Repair Nuclease

Nucleotide excision repair in humans is a complex reaction involving 14 polypeptides in six repair factors for dual incisions on either sides of a DNA lesion. To identify the reaction intermediates that form by the human excision repair nuclease, we adopted three approaches: purification of functional DNA.protein complexes, permanganate footprinting, and the employment as substrate of presumptive DNA reaction intermediates containing unwound sequences 5' to, 3' to, or encompassing the DNA lesion. The first detectable reaction intermediate was formed by substrate binding of XPA, RPA, XPC.HHR23B plus TFIIH (preincision complex 1, PIC1). In this complex the DNA was unwound on either side of the lesion by no more than 10 bases. Independent of the XPG nuclease function, the XPG protein stabilized this complex, forming a long lived preincision complex 2 (PIC2). The XPF.ERCC1 complex bound to PIC2, forming PIC3, which led to dual incisions and the release of the excised oligomer. With partially unwound DNAs, thymine cyclobutane dimer was excised at a fast rate independent of XPC.HHR23B, indicating that a major function of this protein is to stabilize the unwound DNA or to aid lesion unwinding in preincision complexes

Nuclear Physics Experiments with Ion Storage Rings

In the last two decades a number of nuclear structure and astrophysics experiments were performed at heavy-ion storage rings employing unique experimental conditions offered by such machines. Furthermore, building on the experience gained at the two facilities presently in operation, several new storage ring projects were launched worldwide. This contribution is intended to provide a brief review of the fast growing field of nuclear structure and astrophysics research at storage rings.Comment: XVIth International Conference on Electro-Magnetic Isotope Separators and Techniques Related to their Applications, December 2--7, 2012 at Matsue, Japa

Comparison of Technetium-99m-MIBI imaging with MRI for detection of spine involvement in patients with multiple myeloma

BACKGROUND: Recently, radiopharmaceutical scanning with Tc-99m-MIBI was reported to depict areas with active bone disease in multiple myeloma (MM) with both high sensitivity and specificity. This observation was explained by the uptake of Tc-99m-MIBI by neoplastic cells. The present investigation evaluates whether Tc-99m-MIBI imaging and magnetic resonance imaging (MRI) perform equally well in detecting myelomatous bone marrow lesions. METHODS: In 21 patients with MM, MRIs of the vertebral region TH12 to S1 and whole body scans with Tc-99m-MIBI were done. RESULTS: Tc-99m-MIBI scanning missed bone marrow infiltration in 43 of 87 vertebrae (50.5%) in which MRI showed neoplastic bone marrow involvement. In patients with disease stage I+II, Tc-99m-MIBI scanning was negative in all of 24 vertebrae infiltrated according to MRI. In patients with disease stage III, Tc-99m-MIBI scanning detected 44 of 63 (70%) vertebrae involved by neoplastic disease. CONCLUSION: Tc-99m-MIBI scanning underestimated the extent of myelomatous bone marrow infiltration in the spine, especially in patients with low disease stage

Association between Ngb polymorphisms and ischemic stroke in the Southern Chinese Han population

<p>Abstract</p> <p>Background</p> <p><it>Neuroglobin </it>(<it>Ngb</it>), one of novel members of the globin superfamily, is expressed predominantly in brain neurons, and appears to modulate hypoxic-ischemic insults. The mechanisms underlying <it>Ngb</it>-mediated neuronal protection are still unclear. For it is one of the candidate protective factors for ischemic stroke, we conducted a case-control study to clarify the association of <it>Ngb </it>polymorphisms with ischemic stroke in the Southern Chinese Han population.</p> <p>Methods</p> <p>355 cases and 158 controls were recruited. With brain imaging, cases were subdivided into large-artery atherosclerosis (LVD) and small-vessel occlusion (SVD) stroke. PCR amplified all the four exons of <it>Ngb </it>and flanking intron sequence for each exon. Genotyping for <it>Ngb </it>was achieved by direct sequencing and mismatched PCR-RFLP. Polymorphisms were studied both individually and as haplotypes in each group and subgroup which subdivided according to gender or age.</p> <p>Results</p> <p>Two intronic polymorphisms 89+104 c>t and 322-110 (6a)>5a were identified. The allele frequency of 89+104 t was decreased in stroke cases. The protective effect seems to be more pronounced in subgroups of female patients and age > 60 years. Also, we have confirmed decreased LDL-C level and reduced hypertension and hypercholesterolemia in 89+104 t allele carriers. In contrast, the 322-110 (6a)>5a genotype distribution was similar between cases and controls. However, the haplotype 89+104 c>t/322-110 (6a)>5a was related with LVD and SVD stroke. The haplotype c-5a was more frequent in both LVD and SVD groups while t-6a was more frequent in controls.</p> <p>Conclusion</p> <p>Ngb polymorphism 89+104 t had protective effects on LVD and SVD in the Southern Chinese Han population. A "hitchhiking" effect was observed for the 89+104 t/322-110 (6a) genotype combination especially for LVD.</p

Entamoeba lysyl-tRNA Synthetase Contains a Cytokine-Like Domain with Chemokine Activity towards Human Endothelial Cells

Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II). This Entamoeba EMAPII-like polypeptide (EELP) is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS) that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity

The Chloroplast Genome Sequence of Mungbean (Vigna radiata) Determined by High-throughput Pyrosequencing: Structural Organization and Phylogenetic Relationships

Mungbean is an economically important crop which is grown principally for its protein-rich dry seeds. However, genomic research of mungbean has lagged behind other species in the Fabaceae family. Here, we reported the complete chloroplast (cp) genome sequence of mungbean obtained by the 454 pyrosequencing technology. The mungbean cp genome is 151 271 bp in length which includes a pair of inverted repeats (IRs) of 26 474 bp separated by a small single-copy region of 17 427 bp and a large single-copy region of 80 896 bp. The genome contains 108 unique genes and 19 of these genes are duplicated in the IR. Of these, 75 are predicted protein-coding genes, 4 ribosomal RNA genes and 29 tRNA genes. Relative to other plant cp genomes, we observed two distinct rearrangements: a 50-kb inversion between accD/rps16 and rbcL/trnK-UUU, and a 78-kb rearrangement between trnH/rpl14 and rps19/rps8. We detected sequence length polymorphism in the cp homopolymeric regions at the intra- and inter-specific levels in the Vigna species. Phylogenetic analysis demonstrated a close relationship between Vigna and Phaseolus in the phaseolinae subtribe and provided a strong support for a monophyletic group of the eurosid I