Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration

Rho family GTPases, particularly Rac1 and Cdc42, are key regulators of cell polarization and directional migration. Adenomatous polyposis coli (APC) is also thought to play a pivotal role in polarized cell migration. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts directly with APC. IQGAP1 and APC localize interdependently to the leading edge in migrating Vero cells, and activated Rac1/Cdc42 form a ternary complex with IQGAP1 and APC. Depletion of either IQGAP1 or APC inhibits actin meshwork formation and polarized migration. Depletion of IQGAP1 or APC also disrupts localization of CLIP-170, a microtubule-stabilizing protein that interacts with IQGAP1. Taken together, these results suggest a model in which activation of Rac1 and Cdc42 in response to migration signals leads to recruitment of IQGAP1 and APC which, together with CLIP-170, form a complex that links the actin cytoskeleton and microtubule dynamics during cell polarization and directional migration.     

苹果一个锌指蛋白基因的cDNA克隆及其表达特性分析

采集了处于营养生长向生殖行长转化期(6~8月)的红玉苹果(Malus domestica Borkh.)茎顶,构建了其cDNA文库,并从中分离得到了一个具有锌指结构的EST序列,又通过5`RACE的方法,从cDNA文库中找到了其上游779bp的cDNA片段.最后用PCR的方法获得了苹果锌指蛋白的全长cDNA,并命为MdZF1.该cDNA序列已在GenBank登录,登录号为AB116545.MdZF1的锌指结构域与玉米的开花转换基因ID1有高度同源性.通过对苹果不同组织、器官的Northem和RT-PCR分析表明MdZF1在根、茎、叶、顶芽以及花器官(萼片、花瓣、雄蕊、雌蕊)中都有表达.Southern分析表明MdZF1的基因组中是以单拷贝存在的.     

Decreased expression of Apaf‐1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease‐activating factor‐1 (Apaf‐1) is a cell death effector that acts with cytochrome c and caspase‐9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf‐1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf‐1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf‐1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf‐1 expression was observed when comparing nevi and melanomas (chi‐square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf‐1 (chi‐square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf‐1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf‐1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf‐1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.     

Environmental DNA enables detection of terrestrial mammals from forest pond water

Terrestrial animals must have frequent contact with water to survive, implying that environmental DNA (eDNA) originating from those animals should be detectable from places containing water in terrestrial ecosystems. Aiming to detect the presence of terrestrial mammals using forest water samples, we applied a set of universal PCR primers (MiMammal, a modified version of fish universal primers) for metabarcoding mammalian eDNA. The versatility of MiMammal primers was tested in silico and by amplifying DNAs extracted from tissues. The results suggested that MiMammal primers are capable of amplifying and distinguishing a diverse group of mammalian species. In addition, analyses of water samples from zoo cages of mammals with known species composition suggested that MiMammal primers could successfully detect mammalian species from water samples in the field. Then, we performed an experiment to detect mammals from natural ecosystems by collecting five 500‐ml water samples from ponds in two cool‐temperate forests in Hokkaido, northern Japan. MiMammal amplicon libraries were constructed using eDNA extracted from water samples, and sequences generated by Illumina MiSeq were subjected to data processing and taxonomic assignment. We thereby detected multiple species of mammals common to the sampling areas, including deer (Cervus nippon), mouse (Mus musculus), vole (Myodes rufocanus), raccoon (Procyon lotor), rat (Rattus norvegicus) and shrew (Sorex unguiculatus). Many previous applications of the eDNA metabarcoding approach have been limited to aquatic/semiaquatic systems, but the results presented here show that the approach is also promising even for forest mammal biodiversity surveys.     

Effects of early experience with low-quality roughage on liver metabolome in lambs

Introduction

Early experience with low-quality roughage has the potential to improve its utilization in ruminants.

Objectives

This study determined the effects of early experience with low-quality roughage on dry matter intake (DMI), body weight (BW) and the hepatic metabolism of lambs.

Methods

Ten lambs (3 month old) were randomly allocated to two treatments: the low-quality roughage group (LR) or the control group (C). The study lasted 7 months. In the first 4 months, LR was fed low-quality roughage, whereas C was fed normal roughage. In the last 3 months, both groups were fed low-quality roughage. Dry matter intake (DMI) was determined at the 1st, 3rd, 5th and 7th months (P1, P2, P3 and P4). Analysis of liver tissue metabolomics were carried out in P2 and P3.

Results

DMI was greater in LR than C in P1 (p?<?0.05), but did not differ in P2. In P3 and P4, DMI was greater in LR than in C (p?<?0.01). The concentration of five glycolysis/gluconeogenesis intermediates, some lipid metabolism-related metabolites, six amino acids and several amino acid metabolism-related metabolites were different between treatments in P2. From P2 to P3, concentrations of these metabolites changed in LR. However, no difference was found between treatments in P3.

Conclusion

Feeding low-quality roughage to lambs early in life influenced hepatic glycolysis/gluconeogenesis, fatty acid oxidation and amino acid metabolism. However, for lambs who had no early experience with low-quality roughage, similar alterations were induced in the liver after 1 month of eating low-quality roughage.

     

Crystallization and Preliminary X-Ray Analysis of Neuropsin, a Serine Protease Expressed in the Limbic System of Mouse Brain

Neuropsin (M_r25 032) is a serine protease expressed in the limbic system of mouse brain. It has been implicated in various neurological processes including formation of memory and may be important as a drug target in the treatment of epilepsy. The recombinant protein was produced using a baculovirus expression system and was purified. Two crystal forms were obtained by a hanging-drop vapor-diffusion method with polyethylene glycol. Preliminary X-ray crystallographic analysis revealed that crystal form I belongs to triclinic space groupP1 with unit cell dimensionsa= 97.16 Å,b= 97.12 Å,c= 46.75 Å and α = 99.17°, β = 99.77°, γ = 117.35°. Self-rotation function analysis of these data of form I indicates the position of a noncrystallographic threefold axis. There are six molecules in the crystallographic asymmetric unit. Crystal form II also belongs to triclinic space groupP1 but has unit cell dimensions ofa= 38.40 Å,b= 55.16 Å,c= 65.37 Å and α = 95.38°, β = 89.98°, γ = 110.46° with two molecules in the crystallographic asymmetric unit. Form II has a noncrystallographic twofold axis. Intensity data to 3.1 Å resolution for form I and to 2.2 Å resolution for form II have been collected.     

Isolation and characterization of the mouse ortholog of the Fukuyama-type congenital muscular dystrophy gene

Fukuyama-type congenital muscular dystrophy (FCMD) is a severe autosomal-recessive muscular dystrophy accompanied by brain malformation. Previously, we identified the gene responsible for FCMD through positional cloning. Here we report the isolation of its murine ortholog, Fcmd. The predicted amino acid sequence of murine fukutin protein encoded by Fcmd is 90% identical to that of its human counterpart. Radiation hybrid mapping localized the gene to 2.02 cR telomeric to D4Mit272 on chromosome 4. Northern blot analysis revealed ubiquitous expression of Fcmd in adult mouse tissues. Through in situ hybridization, we observed a wide distribution of Fcmd expression throughout embryonic development, most predominantly in the central and peripheral nervous systems. We also detected high Fcmd expression in the ventricular zone of proliferating neurons at 13.5 days post-coitum. Brain malformation in FCMD patients is thought to result from defective neuronal migration. Our data suggest that neuronally expressed Fcmd is likely to be important in the development of normal brain structure.     

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.     

Viral replication rate regulates clinical outcome and CD8 T cell responses during highly pathogenic H5N1 influenza virus infection in mice

Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-β or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans.