Parallel evolution in eight-barbel loaches of the genus Lefua (Balitoridae, Cypriniformes) revealed by mitochondrial and nuclear DNA phylogenies

The evolutionary history of eight-barbel loaches of the genus Lefua contains important phylogenetic information that will aid in resolution of the faunal formations and evolutionary histories of Japanese and East Asian freshwater fishes. Our sequencing of the mitochondrial D-loop region in a large number of samples allowed construction of the most comprehensive phylogeny of these loaches to date; we demonstrated monophyly of five Lefua species and identified populations of Lufua. sp. and Lefua echigonia. Loaches inhabiting the Tokai region in Japan were morphologically and ecologically indistinguishable from Lefua sp. However, they were included in the L. echigonia lineage. We determined a novel phylogeny by sequencing the nuclear ribosomal S7 subunit and showed that nuclear DNA phylogeny essentially matched the mitochondrial DNA phylogeny. Loaches from the Tokai region were part of the L. echigonia lineage, indicating parallel evolution between Tokai loaches and Lefua sp. in western Japan. We presented the most robust phylogeny to date using concatenated mitochondrial and nuclear sequences. The wealth of molecular information allowed us to speculate on evolutionary processes in the genus Lefua.     

Sialic Acid-focused Quantitative Mouse Serum Glycoproteomics by Multiple Reaction Monitoring Assay

Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation. However, in-depth knowledge of protein glycosylation to uncover functions and their clinical applications requires quantitative glycoproteomics eliciting both peptide and glycan sequences concurrently. Here we describe a novel strategy for the multiplexed quantitative mouse serum glycoproteomics based on a specific chemical ligation, namely, reverse glycoblotting technique, focusing sialic acids and multiple reaction monitoring (MRM). LC-MS/MS analysis of de-glycosylated peptides identified 270 mouse serum peptides (95 glycoproteins) as sialylated glycopeptides, of which 67 glycopeptides were fully characterized by MS/MS analyses in a straightforward manner. We revealed the importance of a fragment ion containing innermost N-acetylglucosamine (GlcNAc) residue as MRM transitions regardless the sequence of the peptides. Versatility of the reverse glycoblotting-assisted MRM assays was demonstrated by quantitative comparison of 25 targeted glycopeptides from 16 proteins between mice with homo and hetero types of diabetes disease model.Clinical proteomics focusing on the identification and validation of biomarkers and the discovery of proteins as therapeutic targets is an emerging and highly important area of proteomics. Biomarkers are measurable indicators of a specific biological state (particularly one relevant to the risk of contraction) and the presence or the stage of disease, and are thus expected to be useful for the prediction, detection, and diagnosis of disease as well as to follow the efficacy, toxicology, and side effects of drug treatment, and to provide new functional insights into biological processes.At present, proteomics methods based on mass spectrometry (MS) have emerged as the preferred strategy for discovery of diagnostic, prognostic, and therapeutic protein biomarkers. Most biomarker discovery studies use unbiased, “identified-based” approaches that rely on high performance mass spectrometers and extensive sample processing. Semiquantitative comparisons of protein relative abundance between disease and control patient samples are used to identify proteins that are differentially expressed and, thus, to populate lists of potential biomarkers. De novo proteomics discovery experiments often result in tens to hundreds of candidate biomarkers that must be subsequently verified in serum. However, despite the large numbers of putative biomarkers, only a small number of them are passed through the development and validation process into clinical practice, and their rate of introduction is declining. The first non-standard abbreviation (MS above is standard) must be footnoted the same as the abbreviation footnote, and MRM must be the first abbreviation in the list because it is the one footnoted. After that the order does not matter.Targeted proteomics using multiple reaction monitoring (MRM)1 is emerging as a technology that complements the discovery capabilities of shotgun strategies as well as an alternative powerful novel MS-based approach to measure a series of candidate biomarkers (1–7). Therefore, MRM is expected to provide a powerful high throughput platform for biomarker validation, although clinical validation of novel biomarkers has been traditionally relying on immunoassays (8, 9). MRM exploits the unique capabilities of triple quadrupoles (QQQ) MS for quantitative analysis. In MRM, the first and the third quadrupoles act as filters to specifically select predefined m/z values corresponding to the peptide precursor ion and specific fragment ion of the peptide, whereas the second quadrupole serves as collision cell. Several such transitions (precursor/fragment ion pairs) are monitored over time, yielding a set of chromatographic traces with retention time and signal intensity for a specific transition as coordinates. These measurements have been multiplexed to provide 30 or more specific assays in one run. Such methods are slowly gaining acceptance in the clinical laboratory for the routine measurement of endogenous metabolites (10) (e.g. in screening newborns for a panel of inborn errors of metabolism) some drugs (11) (e.g. immunosuppressants), and the component analysis of sugars (12).One of the profound challenges in clinical proteomics is the need to handle highly complex biological mixtures. This complexity presents unique analytical challenges that are further magnified with the use of clinical serum/plasma samples to search for novel biomarkers of human disease. The serum proteome is composed of tens of thousands of unique proteins, of which concentrations may exceed 10 orders of magnitude. Protein glycosylation, one of the most common post-translational modifications, generates tremendous diversity, complexity, and heterogeneity of gene products. It changes the biological and physical properties of proteins, which include functions as signals or ligands to control their distribution, antigenicity, metabolic fate, stability, and solubility. Protein glycosylation, in particular by N-linked glycans, is prevalent in proteins destined for extracellular environments. These include proteins on the extracellular side of the plasma membrane, secreted proteins, and proteins contained in body fluids (such as blood serum, cerebrospinal fluid, urine, breast milk, saliva, lung lavage fluid, or pancreatic juice). Considering that such body fluids are most easily accessible for diagnostic and therapeutic purposes, it is not surprising that many clinical biomarkers and therapeutic targets are glycoproteins. These include, for example, cancer antigen 125 (CA125) in ovarian cancer, human epidermal growth factor receptor 2 (Her2/neu) in breast cancer, and prostate-specific antigen (PSA) in prostate cancer. In addition, changes in the extent of glycosylation and the structure of N-glycans or O-glycans attached to proteins on the cell surface and in body fluids have been shown to correlate with cancer and other disease states, highlighting the clinical importance of this modification as an indicator or effector of pathologic mechanisms (13–16). Thus, clinical proteomic platforms should have capability to provide protein glycosylation information as well as sufficient analytical depth to reliably detect and quantify specific proteins with sufficient accuracy and throughput.To improve the detection limits to the required sensitivities, one needs to dramatically reduce the complexity of the sera samples. For focused glycoproteomics, several techniques using lectins or antibodies enabling the large-scale identification of glycoproteins have recently been developed (17–19). Notably, Zhang et al. reported a method for the selective isolation of peptides based on chemical oxidation of the carbohydrate moiety and subsequent conjugation to a solid support using hydrazide chemistry (20–26). However, it is not possible to provide any structural information about N-glycans because the MS analysis is performed on peptides of which N-glycans are removed preferentially by treating with peptide N-glycanase (PNGase). In 2007, we developed a method for rapid enrichment analysis of peptides bearing sialylated N-glycans on the MALDI-TOF-MS platform (27). The method involves highly selective oxidation of sialic acid residues of glycopeptides to elaborate terminal aldehyde group and subsequent enrichment by chemical ligation with a polymer reagent, namely, reverse glycoblotting technique inspired from an original concept of glycoblotting method (28). This method, in principle, is capable identifying both glycan and peptide sequences concurrently. Recently, Nilsson et al. reported that glycopeptides from human cerebrospinal fluid can be enriched on the basis of the same principle as the reverse glycoblotting protocol, and captured glycopeptides were analyzed with ESI FT-ICR MS (29). Because it is well known that sialic acids play important roles in various biological processes including cell differentiation, immune response, and oncogenesis (30–34), our attention has been directed toward feasibility of the reverse glycoblotting technique in quantitative analysis of the specific glycopeptides carrying sialic acid(s) by combining with multiplexed MRM-based MS.     

Hydrodynamics-based delivery of plasmid DNA encoding CTLA4-Ig prolonged cardiac allograft survival in rats

BACKGROUND: Although gene therapy using plasmid vectors is thought to be safer compared with viral vectors, poor efficacy of gene transfer is the obstacle preventing wide application of plasmid vectors. However, high levels of foreign gene expression have been achieved by rapid tail vein injection of a large volume of a plasmid DNA solution into rats. Using this technique, we examined the effect of rat CTLA4-Ig gene transfer on prevention of cardiac allograft rejection in this animal model. METHODS: Recipient Lewis rats were injected with either plasmid pCAGGS-CTLA4-Ig-Glu-tag as a treatment vector or plasmid pCAGGS-signal peptide (SP)-Ig as a control vector by hydrodynamics-based delivery technique on the day before heart transplantation. Hearts from Brown Norway donors were transplanted into the neck of Lewis recipients and graft survival was assessed. RESULTS: The plasma level of CTLA4-Ig reached a peak of nearly 5 microg/mL 1 day after injection, and then slowly decreased but still remained above 0.9 microg/mL until 100 days after injection. The recipient rats treated with the control vector and untreated rats rejected cardiac allografts within 7 days. On the other hand, the median survival time of the grafts treated with pCAGGS-CTLA4Ig-Glu-tag was more than 100 days. Histological examination revealed that long-term survival allografts contained fewer infiltrating lymphocytes. The serum from recipients with long-term survival allograft suppressed allogenic mixed lymphocyte reaction. CONCLUSIONS: CTLA4-Ig gene transfer by means of tail vein injection of plasmid DNA into a recipient rat resulted in remarkable prolongation of cardiac allograft survival with persistent plasma level of CTLA4-Ig protein.     

Bid truncation mediated by caspases-3 and -9 in vinorelbine-induced apoptosis

Vinorelbine is a chemotherapeutic vinca alkaloid clinically prescribed for non-small cell lung cancer and breast cancer. Here we studied the mechanism for vinorelbine-induced apoptosis in a human T-cell lymphoma. Although vinorelbine induces DNA fragmentation that is inhibited by specific peptide inhibitors for caspases-9 and -3 in Jurkat cells, caspase-8 deficiency retards vinorelbine-induced apoptosis. Activation of caspase-8 is also observed in vinorelbine-treated cells, and the activity is diminished when the caspase-3 activity is blocked by a specific peptide inhibitor, Ac-DNLC-CHO. Blocking of the Fas receptor with an antagonistic anti-Fas antibody does not affect vinorelbine-induced DNA fragmentation. These results suggest that vinorelbine-induced apoptosis is enhanced by the activation of caspase-8 via caspase-9-mediated activation of caspase-3, but not through a Fas-triggered signal. Western blotting suggests that vinorelbine cleaves caspase-3, -9 and -8 and reduces the amount of mitochondrial cytochrome c. Caspase-8 deficiency suppresses all of these events. A downstream substrate for caspase-8, Bid, is also cleaved in vinorelbine-treated cells, but the Bid truncation is also observed in caspase-8-deficient Jurkat cells. Importantly, recombinant caspases-3 and -9, as well as caspase-8, directly cleaves recombinant Bid in vitro. These results suggest that caspases-3 and -9 participate in Bid truncation, indicating a new mechanism for vinorelbine-induces apoptosis.     

The lmx1b gene is pivotal in glomus development in Xenopus laevis

We have previously shown that lmx1b, a LIM homeodomain protein, is expressed in the pronephric glomus. We now show temporal and spatial expression patterns of lmx1b and its potential binding partners in both dissected pronephric anlagen and in individual dissected components of stage 42 pronephroi. Morpholino oligonucleotide knock-down of lmx1b establishes a role for lmx1b in the development of the pronephric components. Depletion of lmx1b results in the formation of a glomus with reduced size. Pronephric tubules were also shown to be reduced in structure and/or coiling whereas more distal tubule structure was unaffected. Over-expression of lmx1b mRNA resulted in no significant phenotype. Given that lmx1b protein is known to function as a heterodimer, we have over-expressed lmx1b mRNA alone or in combination with potential interacting molecules and analysed the effects on kidney structures. Phenotypes observed by over-expression of lim1 and ldb1 are partially rescued by co-injection with lmx1b mRNA. Animal cap experiments confirm that co-injection of lmx1b with potential binding partners can up-regulate pronephric molecular markers suggesting that lmx1b lies upstream of wt1 in the gene network controlling glomus differentiation. This places lmx1b in a genetic hierarchy involved in pronephros development and suggests that it is the balance in levels of binding partners together with restricted expression domains of lmx1b and lim1 which influences differentiation into glomus or tubule derivatives in vivo.     

BlotGlycoABCTM, an integrated glycoblotting technique for rapid and large scale clinical glycomics

Recent progress in mass spectrometry has led to new challenges in glycomics, including the development of rapid glycan enrichment techniques. A facile technique for exploration of a carbohydrate-related biomarker is important because proteomics research targets glycosylation, a posttranslational modification. Here we report an "all-in-one" protocol for high throughput clinical glycomics. This new technique integrates glycoblotting-based glycan enrichment onto the BlotGlycoABC bead, on-bead stabilization of sialic acids, and fluorescent labeling of oligosaccharides in a single workflow on a multiwell filter plate. The advantage of this protocol and MALDI-TOF MS was demonstrated through differentiation of serum N-glycan profiles of subjects with congenital disorders of glycosylation and hepatocellular carcinoma and healthy donors. The method also permitted total cellular glycomics analysis of human prostate cancer cells and normal human prostate epithelial cells. These results demonstrate the potentials of glycan enrichment/processing for biomarker discovery.     

Brain Atrophy in a Murine Model of Chronic Fatigue Syndrome and Beneficial Effect of Hochu-ekki-to (TJ-41)

Brain-derived neurotrophic factor (BDNF) is associated with the main symptoms of chronic fatigue sydrome (CFS) and neuron apoptosis. Nevertheless, no study has been performed directly to explore the relationship between CFS, BDNF and neuron apoptosis. We induced a CFS model by six injections of killed Brucella abortus antigen in BALB/c mice and treated them with Hochu-ekki-to (TJ-41). Daily running activity, body weight (BW), ratio of cerebral weight to BW (CW/BW) and expression levels of BDNF and Bcl-2 mRNA in the hippocampus were determined. The daily activity and CW/BW decreased significantly in the CFS model. BDNF and Bcl-2 mRNA expression levels in the hippocampus were suppressed in the CFS model and TJ-41 treated mice, while no significant difference was found between them. We improved a murine model to investigate the relationship between CFS and brain dysfunction. In this model, reduced daily activity might have been associated with decreased hippocampal BDNF mRNA expression, hippocampal apoptosis and brain atrophy. TJ-41 increased the daily running activity of the model, which was independent of brain recovery.