Degradation of p0071 and p120-catenin during adherens junction disassembly by Leptospira interrogans.

Leptospira interrogans disseminates hematogenously to reach the target organs by disrupting epithelial adherens junctions (AJs), thus causing leptospirosis, which is a globally neglected zoonotic disease. L. interrogans induces E-cadherin (E-cad) endocytosis and cytoskeletal rearrangement during AJ disassembly, but the detailed mechanism remains unknown. Elucidation of AJ disassembly mechanisms will guide new approaches to developing vaccines and diagnostic methods. In this study, we combine proteomic and imaging analysis with chemical inhibition studies to demonstrate that disrupting the AJs of renal proximal tubule epithelial cells involves the degradation of two armadillo repeat-containing proteins, p0071 and p120-catenin, that stabilize E-cad at the plasma membrane. Combining proteasomal and lysosomal inhibitors substantially prevented p120-catenin degradation, and monolayer integrity destruction without preventing p0071 proteolysis. In contrast, the pan-caspase inhibitor Z-VAD-FMK inhibited p0071 proteolysis and displacement of both armadillo repeat-containing proteins from the cell-cell junctions. Our results show that L. interrogans induces p120-catenin and p0071 degradation, which mutually regulates E-cad stability by co-opting multiple cellular degradation pathways. This strategy may allow L. interrogans to disassemble AJs and disseminate through the body efficiently.

Basic eluent for rapid and comprehensive analysis of fatty acid isomers using reversed-phase high performance liquid chromatography/Fourier transform mass spectrometry.

Comprehensive separation of fatty acids, including various isomers, is very important for chemical analyses associated with detailed physiological function investigations. We found that a basic eluent is effective for realizing clear separation of double-bond regioisomers by reversed-phase high-performance liquid chromatography. We carried out a comprehensive analysis of fatty acids, including double-bond positional isomers, trans-fatty acids, and iso-fatty acids using reversed-phase high-performance liquid chromatography coupled with Fourier transform mass spectrometry (LC/FTMS). Clear separation of these fatty acids was achieved using a C18 column and a basic eluent. A three-pump gradient system using acetone provided rapid elution within 22 min. In a single run, 184 fatty acid molecular species contained in a dietary fish oil supplement were detected by the FTMS full scan. A previously unreported peak was also detected, which was assigned as tetratriacontadecaenoic acid by comparison with the MS2 profiles of fatty acids with known chemical structures. This result demonstrates that the developed method is useful for clarifying the composition of fatty acid molecular species in target samples, providing a promising approach to discover unreported fatty acids.

New approach to predict photoallergic potentials of chemicals based on murine local lymph node assay.

Photoallergic dermatitis, caused by pharmaceuticals and other consumer products, is a very important issue in human health. However, S10 guidelines of the International Conference on Harmonization do not recommend the existing prediction methods for photoallergy because of their low predictability in human cases. We applied local lymph node assay (LLNA), a reliable, quantitative skin sensitization prediction test, to develop a new photoallergy prediction method. This method involves a three-step approach: (1) ultraviolet (UV) absorption analysis; (2) determination of no observed adverse effect level for skin phototoxicity based on LLNA; and (3) photoallergy evaluation based on LLNA. Photoallergic potential of chemicals was evaluated by comparing lymph node cell proliferation among groups treated with chemicals with minimal effect levels of skin sensitization and skin phototoxicity under UV irradiation (UV+) or non-UV irradiation (UV-). A case showing significant difference (P < .05) in lymph node cell proliferation rates between UV- and UV+ groups was considered positive for photoallergic reaction. After testing 13 chemicals, seven human photoallergens tested positive and the other six, with no evidence of causing photoallergic dermatitis or UV absorption, tested negative. Among these chemicals, both doxycycline hydrochloride and minocycline hydrochloride were tetracycline antibiotics with different photoallergic properties, and the new method clearly distinguished between the photoallergic properties of these chemicals. These findings suggested high predictability of our method; therefore, it is promising and effective in predicting human photoallergens.

Applicability of an Integrated Testing Strategy consisting of in silico, in chemico and in vitro assays for evaluating the skin sensitization potencies of isocyanates.

The skin sensitization potential of chemicals has been traditionally assessed using regulatory accepted in vivo methods, such as guinea pig maximization test or mouse local lymph node assays (LLNAs). A huge effort to reduce and replace the use of animals for safety assessments of chemicals because of regulatory requirements and ethical issues is presently underway, and alternative non-animal methods have been greatly developed. So far, a few studies have investigated the sensitization potencies of isocyanates which is a group of highly reactive chemicals that are known to be occupational allergens. The present study evaluated nine commonly used isocyanates using an in vivo LLNA and assessed the applicability of an Integrated Testing Strategy (ITS) consisting of an in silico Derek Nexus prediction, an in chemico direct peptide reactivity assay (DPRA), and an in vitro human Cell Line Activation Test (h-CLAT) to isocyanates. All nine isocyanates were evaluated as positive using the LLNA, Derek Nexus and DPRA, whereas seven chemicals tested positive using the h-CLAT: hexamethylene diisocyanate tested negative, and 1,5-diisocyanatonaphthalene could not be examined because of a solubility issue. When assessed using the ITS, the positive/negative evaluations of skin sensitization hazard were consistent with those assessed using the LLNA for all nine chemicals. However, the potency prediction results of the ITS tended to be underestimated, compared with those of the LLNA. The data presented in this work provide insights into the performance of non-animal testing approaches for evaluating the skin sensitization potencies of isocyanates.

Identification of sheep red blood cell (SRBC) surface immune-responsive peptides detected by antisera from SRBC-immunized rats.

To identify the sheep red blood cell (SRBC) surface immune-responsive peptides, immuno-reactive fraction of SRBC was detected by SDS-PAGE and western blot analysis with antisera from SRBC-immunized rats. Then the most intense immuno-reactive band on SDS-PAGE was subjected to nanoLC-ESI-MS/MS analysis, and 17 proteins were identified including membrane proteins of erythrocytes such as band 3 anion transport protein isoform 1 (Anion exchange protein 1; AE-1, CD233), Ammonium transporter Rh type A (Rh type A glycoprotein, CD241) and Ankyrin-1 (ANK-1), Spectrin beta chain. Among them, plasma protein AE-1 (CD233) and Rh type A glycoprotein (CD241) have transmembrane domain and correspond to extracellular region in their sequences. These extracellular regions of the plasma membrane proteins are supposed to be major immune-responsive peptides of SRBC in rats. These peptides are promising for the construction of an ELISA system which does not require the processing of SRBC membrane ghosts.

Establishment of combined analytical method to extract the genes of interest from transcriptome data.

Techniques for analyzing genome-wide expression profiles, such as the microarray technique and next-generation sequencers, have been developed. While these techniques can provide a lot of information about gene expression, selection of genes of interest is complicated because of excessive gene expression data. Thus, many researchers use statistical methods or fold change as screening tools for finding gene sets whose expression is altered between groups, which may result in the loss of important information. In the present study, we aimed to establish a combined method for selecting genes of interest with a small magnitude of alteration in gene expression by coupling with proteome analysis. We used hypercholesterolemic rats to examine the effects of a crude herbal drug on gene expression and proteome profiles. We could not select genes of interest by using standard methods. However, by coupling with proteome analysis, we found several effects of the crude herbal drug on gene expression. Our results suggest that this method would be useful in selecting gene sets with expressions that do not show a large magnitude of alteration.

Methacarn as a whole brain fixative for gene and protein expression analyses of specific brain regions in rats.

For molecular analysis in anatomically-specific brain regions for rodent studies, it is necessary to establish a fast and accurate procedure for tissue sampling to achieve high integrity and expression fidelity of extracted molecules. The present study was performed to examine suitability of whole brain fixation with methacarn and subsequent tissue sampling using punch-biopsy devices for gene expression analysis in rats. After fixation, each specific region, i.e., hippocampal dentate gyrus, corpus callosum, cingulate cortex or cerebellar vermis was collected, and the integrity and variability of expression data of extracted total RNAs and polypeptides were examined. Methacarn fixation, acetone fixation, and unfixed tissues were compared. Methacarn fixation resulted in high integrity of total RNAs sufficient for conducting global expression analysis and superior in terms of uniformity in the integrity among brain regions to that of acetone fixation. Extracted polypeptide after methacarn fixation revealed similar integrity to that without fixation or with acetone fixation. Methacarn fixation resulted in lower mRNA expression variability between samples than acetone fixation in microarray analysis. The fidelity of polypeptide expression was mostly equivalent between methacarn and acetone fixation in 2-dimensional differential in-gel electrophoresis, although the expression levels of a small number of polypeptides from acetone-fixed tissues were affected. These results suggest that whole brain fixation with methacarn retains advantages for global analyses of mRNAs and polypeptides in rodent studies.

Estimation of tracheal pressure and imposed expiratory work of breathing by the endotracheal tube, heat and moisture exchanger, and ventilator during mechanical ventilation.

BACKGROUND: The resistance of the endotracheal tube (ETT), the heat and moisture exchanger (HME), and the ventilator may affect the patient's respiratory status. Although previous studies examined the inspiratory work of breathing (WOB), investigation of WOB in the expiratory phase is rare. We estimated tracheal pressure at the tip of the ETT (Ptrach) and calculated expiratory WOB imposed by the ETT, the HME, and the expiratory valve. We examined imposed expiratory WOB in patients under a continuous mandatory ventilation (CMV) mode and during spontaneous breathing trials (SBTs). We hypothesized that imposed expiratory WOB would increase with heightened ventilatory demand. METHODS: We measured airway pressure (Paw) and respiratory flow (V). We estimated Ptrach using the equation Ptrach = Paw - K1 × V(K2) - 2.70 × V(L/s)(1.42). K1 and K2 were determined by the inner diameter (ID) of the ETT. Imposed expiratory WOB was calculated from the area of Ptrach above PEEP versus lung volume. We examined imposed expiratory WOB and imposed expiratory resistance in relation to mean expiratory flow. RESULTS: We examined 28 patients under CMV mode, and 29 during SBT. During both CMV and SBT, as mean expiratory flow increased, imposed expiratory WOB increased. The regression curves between mean expiratory flow (x) (L/s) and imposed expiratory WOB (y) (J/L) were y = 1.35x(0.83) (R(2) = 0.79) for 7 mm ID ETT under CMV, y = 1.12x(0.82) (R(2) = 0.73) for 8 mm ID ETT under CMV, y = 1.07x(1.04) (R(2) = 0.85) for 7 mm ID ETT during SBT, and y = 0.84x(0.93) (R(2) = 0.75) for 8 mm ID ETT during SBT. Levels of imposed expiratory WOB were affected by ETT diameter and ventilator mode. The reason for increasing imposed expiratory WOB was an increase in expiratory resistance imposed by the ETT and HME. CONCLUSIONS: Under mechanical ventilation, imposed expiratory WOB should be considered in patients with higher minute ventilation.

Tudor domain containing 7 (Tdrd7) is essential for dynamic ribonucleoprotein (RNP) remodeling of chromatoid bodies during spermatogenesis.

In the male germline in mammals, chromatoid bodies, a specialized assembly of cytoplasmic ribonucleoprotein (RNP), are structurally evident during meiosis and haploidgenesis, but their developmental origin and regulation remain elusive. The tudor domain containing proteins constitute a conserved class of chromatoid body components. We show that tudor domain containing 7 (Tdrd7), the deficiency of which causes male sterility and age-related cataract (as well as glaucoma), is essential for haploid spermatid development and defines, in concert with Tdrd6, key biogenesis processes of chromatoid bodies. Single and double knockouts of Tdrd7 and Tdrd6 demonstrated that these spermiogenic tudor genes orchestrate developmental programs for ordered remodeling of chromatoid bodies, including the initial establishment, subsequent RNP fusion with ubiquitous processing bodies/GW bodies and later structural maintenance. Tdrd7 suppresses LINE1 retrotransposons independently of piwi-interacting RNA (piRNA) biogenesis wherein Tdrd1 and Tdrd9 operate, indicating that distinct Tdrd pathways act against retrotransposons in the male germline. Tdrd6, in contrast, does not affect retrotransposons but functions at a later stage of spermiogenesis when chromatoid bodies exhibit aggresome-like properties. Our results delineate that chromatoid bodies assemble as an integrated compartment incorporating both germline and ubiquitous features as spermatogenesis proceeds and that the conserved tudor family genes act as master regulators of this unique RNP remodeling, which is genetically linked to the male germline integrity in mammals.

[Epidural blood patch for postdural puncture headache in a five-year-old child].

A case was presented of a 5-year-old girl who suffered an accidental dural puncture during placement of an epidural catheter under general anesthesia for orthopedic surgery. She complained of headache 4 days after the operation, which was relieved on supine position but became worse on sitting position. Her symptoms failed to respond to conservative management. An epidural blood patch was performed under general anesthesia and completely resolved her symptoms. The reported incidence of epidural blood patch for post dural puncture headache following accidental dural puncture in children is low. We outline this case and the consideration for management for epidural blood patch in pediatric patients.

[Two cases of transfusion-related acute lung injury following massive transfusion during the perioperative period].

Transfusion-related acute lung injury (TRALI) is a serious complication of transfusion of blood and blood components. TRALI is reported to be the most common cause of transfusion-associated death. TRALI has increasingly become known in the medical community. However, TRALI has been overlooked frequently because of poor knowledge of medical staffs. We report two cases of TRALI after massive transfusion due to massive bleeding during cardiovascular surgery. In the perioperative period, the diagnosis of TRALI is difficult to make because of coexistence of various factors leading to hypoxia. Thus, in this report, we discuss the management of these two cases focusing upon the differential diagnosis of TRALI.

High-accuracy prediction of carcinogenicity by global quantitative analysis of post-translational modifications in a 28-day in vivo rat study.

A global quantitative analysis of post-translational modifications (PTMs) of distinct proteins was executed at the proteomic level using two-dimensional fluorescence differential gel electrophoresis. We evaluated the effects of 66 chemical compounds, including 15 genotoxic carcinogens, 28 non-genotoxic carcinogens, and 23 non-carcinogens, in the male F344 rat liver in a 28-day repeated dose study. In the master gel of rat liver protein, we identified 728 spots by hybrid quadrupole time-of-flight mass spectrometry. They collapsed into 356 distinct proteins. Of these, 126 were represented by two or more spots in the 2-D gel. We calculated the logarithmic ratio of volume changes of all 1028 combinations generated from 126 proteins and investigated the relevance to carcinogenicity. This quantitative proteomic study revealed the existence of several PTMs characteristic of carcinogens that may play an important role in early stage of carcinogenicity. Prediction of carcinogenicity from PTM data gave a higher concordance (92.4%) than prediction from protein expression data (74.2%). This novel approach holds great promise as a way of revealing the roles of charge modifications and molecular weight variations of proteins in biological processes.

Quantitative proteomic analysis of rat liver for carcinogenicity prediction in a 28-day repeated dose study.

The potential of quantitative proteomic analysis to predict carcinogenicity of chemical compounds was investigated. Using 2D-DIGE, we analyzed the effects of 63 chemical compounds on protein expression in the rat liver after 28 daily doses. Types of carcinogens were categorized depending on the species and organ specificity. The carcinogen characteristic proteins for each classification were identified by Welch's t value. For evaluation of the predictive concordance we used support vector machines. The rat hepatic carcinogen-specific classification gave higher concordance than the other classification. The generalization performance was measured by leave-one-out cross-validation. For genotoxic and non-genotoxic compounds, a concordance of 79.3 and 76.5%, respectively, was obtained by the top 30 ranked proteins with Welch's t value. Furthermore, we found that the increase of the expression level of the stress response proteins as the common feature of poorly predicted chemical compounds in the leave-20%-out cross-validation. Quantitative proteomics could be promising technique for developing biomarker panels that can be used for carcinogenicity prediction. The list of proteins identified in this study and the zoomed gel images of the top ranked proteins in statistic analysis are provided in Supplementary Data.

Comprehensive identification of cytochrome p450 isoforms from solubility-based fractionated rat liver microsomes.

Cytochrome P450 isoforms from male rat liver microsomes were comprehensively identified using nano liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). The enrichment of P450, an endomembrane-anchored heme protein, was achieved by solubility-based protein fractionation, and greatly improved the total number of identified P450 isoforms. LC-MS/MS analysis of fractions resulted in the identification of total 36 P450 isoforms. The combination of proteomic analysis and the solubility-based fractionation would provide powerful tool for the expression analysis of the superfamily proteins having great similarities between the amino acids sequences.

Comparison of hippocampal synaptosome proteins in young-adult and aged rats.

The hippocampus is important in learning and memory functions but its ability to aid in these functions declines during aging. In this study, we examined hippocampal proteins whose expressions changed in the aging process. A comparison of synaptosome proteins of hippocampus prepared from young-adult (9-week-old) rats with those from aged (30-month-old) rats by two-dimensional fluorescence difference gel electrophoresis revealed 24 spots that were expressed differently among about 1000 spots detected in both young-adult and aged rat samples. Nineteen of these 24 spots were identified by peptide mass fingerprinting. These proteins included chaperone proteins and proteins related to the cytoskeleton, neurotransmission, signal transduction and energy supply. The cytoskeleton-related proteins included actin and T-complex 1, which is thought to play a role in actin folding. Actin was up-regulated but T-complex 1 was down-regulated in aged rat synapses. These results suggest that age-dependent changes of actin filament formation are related to neuronal dysfunction associated with aging.

Detection of oligosaccharides labeled with cyanine dyes using matrix-assisted laser desorption/ionization mass spectrometry.

The sensitivity of oligosaccharides in mass spectrometry lags far behind that of peptides. This is a critical factor in realizing the high-throughput analysis of posttranslational modifications in proteomics. We here described that hydrazide derivatives of cyanine dyes (Cy3, Cy5) with a positive charge made excellent labeling reagents for the detection of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. Cy3-labeled standard N-glycan could be detected at 200 amol on the MALDI target plate in reflectron mode without any purification procedures after the labeling reaction, which may meet the level of sensitivity required in proteome research. Despite the general recognition that the production of signals of oligosaccharides under MALDI conditions would be highly dependent on the matrix, most of the known N-glycans from chicken ovalbumin could be detected upon Cye derivatization nearly independent of the kind of matrix tested (e.g., nor-harman, 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid) without spoiling the signal strength. Postsource decay afforded simple spectra mainly consisting of Y-type fragment ions, thus simplifying the sequence analysis. In-source decay afforded a similar fragmentation pattern only when acidic matrixes were used. In addition, this derivatization technique was successfully applied to the profiling of N-glycans of gel-separated glycoproteins.

Quantitative chemical proteomics for identifying candidate drug targets.

We have developed a systematic strategy for drug target identification. This consists of the following sequential steps: (1) enrichment of total binding proteins using two differential affinity matrixes upon which are immobilized positive and negative chemical structures for drug activity, respectively; (2) covalent labeling of the proteins with a new cleavable isotope-coded affinity tag (ICAT) reagent, followed by proteolysis of the combined proteins; (3) isolation, identification, and relative quantification of the tagged peptides by liquid chromatography-mass spectrometry; (4) array-based transcription profiling to select candidate proteins; and (5) confirmation of direct interaction between the activity-associated structure and the selected proteins by using surface plasmon resonance. We present a typical application to identify the primary binding protein of a novel class of anticancer agents exemplified by E7070. Our results suggest that this approach provides a new aspect of quantitative proteomics to find specific binding proteins from protein mixture and should be applicable to a wide variety of biologically active small molecules with unidentified target proteins.

Affinity chromatography with collapsibly tethered ligands.

We introduce a novel affinity chromatography mode in which affinity ligands are secured to the media surface via collapsible tethers. In traditional affinity chromatography, the immobilized ligands act passively, and their local concentration is static. In collapsibly tethered affinity chromatography, the ligand can move dynamically in response to external stimuli, a design that enables marked changes in both the local concentration of the ligand and its surrounding environment without exchange of solvent. Using the thermoresponsive polymer poly(N-isopropylacrylamide) (PIPAAm) as a scaffold for ligand and hapten attachment, we were able to achieve controlled mobility and microenvironment alteration of the affinity ligand Ricinus communis agglutinin (RCA120). The glycoprotein target, asialotransferrin, was loaded onto a column in which PIPAAm was partially substituted with both RCA120 and lactose. At 5 degrees C, the column retained the glycoprotein, but released most (95%) of the asialotransferrin upon warming to 30 degrees C. This temperature-induced elution was much greater than can be explained by temperature dependency of sugar recognition by RCA120. The simplest explanation is that upon thermally induced dehydration and collapse of the PIPAAm chains, coimmobilized RCA120 ligand and lactose hapten are brought into closer proximity to each other, enabling immobilized lactose to displace affinity-bound asislotransferrin from the immobilized RCA120 lectin.

Regulation of protein binding toward a ligand on chromatographic matrixes by masking and forced-releasing effects using thermoresponsive polymer.

A novel concept of affinity regulation based on masking and forced-releasing effects using a thermoresponsive polymer was elucidated. Affinity chromatographic matrixes were prepared using either poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) or poly(glycidyl methacrylate-co-triethyleneglycol dimethacrylate) beads immobilized with ligand molecule, Cibacron Blue F3G-A (CB), together with poly(N-isopropylacrylamide) (PIPAAm), a polymer with a cloud point of 32 degrees C. Two different lengths of spacer molecules were used for the immobilization of CB while maintaining the PIPAAm size constant. Chromatographic analyses using bovine serum albumin as a model protein showed a clear correlation between spacer length and binding capacity at temperatures lower than the lower critical solution temperature (LCST) of PIPAAm. The binding capacity under the LCST was significantly reduced only when the calculated spacer length was shorter than the mean size of the extended PIPAAm. Furthermore, the adsorbed protein could be desorbed (released) from the matrix surface by lowering the temperature to below the LCST while maintaining other factors such as pH and ion strength. Selective recovery of human albumin from human sera was demonstrated using this newly developed thermoresponsive affinity column.

The high specificities of Phaseolus vulgaris erythro- and leukoagglutinating lectins for bisecting GlcNAc or beta 1-6-linked branch structures, respectively, are attributable to loop B.

Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities. While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgaris erythroagglutinating and leukoagglutinating lectins (E(4)- and L(4)-PHA) are unique in recognizing larger structures. E(4)- and L(4)-PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a beta1,6-linked branch, respectively. However, the detailed mechanisms of molecular recognition are poorly understood. In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E(4)- and L(4)-PHA. We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433-445). The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance. These comparisons indicate that the high specificities of E(4)- and L(4)-PHA toward bisecting GlcNAc and beta1,6-linked branch structures are almost solely attributable to loop B. The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible. Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus. Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E(4)- and L(4)-PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a beta1,6-linked branch.