Engineering the Propeptide of Microbial Transglutaminase Zymogen: Enabling Substrate-Dependent Activation for Bioconjugation Applications.

Microbial transglutaminase (MTG) from Streptomyces mobaraensis is a powerful biocatalytic glue for site-specific cross-linking of a range of biomolecules and synthetic molecules that have an MTG-reactive moiety. The preparation of active recombinant MTG requires post-translational proteolytic digestion of a propeptide that functions as an intramolecular chaperone to assist the correct folding of the MTG zymogen (MTGz) in the biosynthesis. Herein, we report engineered active zymogen of MTG (EzMTG) that is expressed in soluble form in the host Escherichia coli cytosol and exhibits cross-linking activity without limited proteolysis of the propeptide. We found that the saturation mutagenesis of residues K10 or Y12 in the propeptide domain generated several active MTGz mutants. In particular, the K10D/Y12G mutant exhibited catalytic activity comparable to that of mature MTG. However, the expression level was low, possibly because of decreased chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. The K10R/Y12A mutant exhibited specific substrate-dependent reactivity toward peptidyl substrates. Quantitative analysis of the binding affinity of the mutated propeptides to the active site of MTG suggested an inverse relationship between the binding affinity and the catalytic activity of EzMTG. Our proof-of-concept study provides insights into the design of a new biocatalyst using the MTGz as a scaffold and a potential route to high-throughput screening of EzMTG mutants for bioconjugation applications.

Possible roles of human mast cells in the formation of xanthelasma palpebrarum.

Cutaneous xanthoma consist of foam cells that originate from monocytes or macrophages and accumulate in perivascular areas of the skin. The main component of these cells is oxidized low-density lipoprotein (oxLDL). In this study, we show that mast cells surround the accumulated foam cells, suggesting their involvement in xanthoma formation. Coculture of THP-1 or U937 monocytes with the human mast cell line LUVA upregulated their uptake of oxLDL. Positive staining for intracellular cell adhesion molecule-1 (ICAM-1) at the borders between mast cells and foam cells was seen in pathological specimens of the most common cutaneous xanthoma, xanthelasma palpebrarum, and in cocultures. In the latter, ICAM1 messenger RNA levels were upregulated. The administration of anti-ICAM-1 blocking antibody inhibited the increase in oxLDL uptake by THP-1 or U937 monocytes cocultured with LUVA. Taken together, these results suggest a role for mast cells in the formation of xanthelasma palpebrarum and the involvement of ICAM-1 in this process.

A peroxisome deficiency-induced reductive cytosol state up-regulates the brain-derived neurotrophic factor pathway.

The peroxisome is a subcellular organelle that functions in essential metabolic pathways, including biosynthesis of plasmalogens, fatty acid ß-oxidation of very-long-chain fatty acids, and degradation of hydrogen peroxide. Peroxisome biogenesis disorders (PBDs) manifest as severe dysfunction in multiple organs, including the central nervous system (CNS), but the pathogenic mechanisms in PBDs are largely unknown. Because CNS integrity is coordinately established and maintained by neural cell interactions, we here investigated whether cell-cell communication is impaired and responsible for the neurological defects associated with PBDs. Results from a noncontact co-culture system consisting of primary hippocampal neurons with glial cells revealed that a peroxisome-deficient astrocytic cell line secretes increased levels of brain-derived neurotrophic factor (BDNF), resulting in axonal branching of the neurons. Of note, the BDNF expression in astrocytes was not affected by defects in plasmalogen biosynthesis and peroxisomal fatty acid ß-oxidation in the astrocytes. Instead, we found that cytosolic reductive states caused by a mislocalized catalase in the peroxisome-deficient cells induce the elevation in BDNF secretion. Our results suggest that peroxisome deficiency dysregulates neuronal axogenesis by causing a cytosolic reductive state in astrocytes. We conclude that astrocytic peroxisomes regulate BDNF expression and thereby support neuronal integrity and function.

In vivo uterine local gene delivery system using TAT-displaying bionanocapsules.

BACKGROUND: The uterus is an organ that is directly accessible via the transvaginal route, whereas the drug delivery system and the gene delivery system (GDS) for the uterus are very limited, even in animal models. In the present study, we optimized a bionanocapsule (BNC) comprising a hepatitis B virus envelope L-protein particle, for which a structurally similar particle has been used as an immunogen of a conventional HB vaccine worldwide for more than 30 years, as a local uterine GDS using a mouse model. METHODS: To display various antibodies for re-targeting to different cells other than hepatic cells, the pre-S1 region of BNC was replaced with a tandem form of the protein A-derived immunoglobulin G Fc-interacting region (Z domain, ZZ-BNC). To induce strong cell adhesion after local administration into the uterine cavity, ZZ-BNC was modified with a transactivator of transcription (TAT) peptide. RESULTS: Gene transfer using TAT-modified ZZ-BNC is approximately 5000- or 18-fold more efficient than the introduction of the same dose of naked DNAs or the use of the cationic liposomes, respectively. TAT-modified ZZ-BNC was rapidly eliminated from the uterus and had no effect on the pregnancy rate, litter size or fetal growth. CONCLUSIONS: TAT-modified ZZ-BNC could be a useful GDS for uterine endometrial therapy via local uterine injection.

Wnt activator CHIR99021-stimulated human dermal papilla spheroids contribute to hair follicle formation and production of reconstituted follicle-enriched human skin.

The aim of the present study was to accomplish de novo generation of reconstituted human skin with enriched hair follicles. Dermal papillae (DP) are known to play a crucial organizing role in hair follicle induction. However, generation of enriched human hair follicles using cultured DP cells has not been accomplished because DP cells easily lose their hair-inducing ability with culturing. To enhance the hair-inducing ability of DP cells, Wnt signaling pathway activation or three-dimensional (3D) spheroid culture methods were employed in previous studies. Herein, we assessed effects of the canonical Wnt/ß-catenin signaling activator CHIR99021 and found that it enhanced the expression of DP signature genes associated with hair-inducing ability. Further comparison of three different 3D culture methods revealed the highest expression of DP signature genes in spheroids generated by a floating drop method compared with other methods. CHIR99021 synergistically increased expression of DP signature genes in combination with floating drop culture. "Reconstituted skin assay" prepared using the most promising CHIR99021-stimulated 3D spheroids showed enrichment for human hair follicles. Labeled DP spheroids and derived cells were primarily found to be DP and dermal sheath cup (DSC) cells, implying organization of hair formation by DP spheroids. Finally, to evaluate the functional features of generated human skin and hair follicles, we injected human DSC cells, which reportedly show DP precursor behavior, and exhibit hair-inducing ability through incorporation into hair follicles, into mice. Histological studies revealed injected DSC cells in dermal sheath of hair follicles, consistent with a previous report, thus verifying the functionality of generated skin and hair follicles. Collectively, our findings demonstrate that DP spheroids synergistically stimulated by CHIR99021 and 3D culture contributed to hair follicle formation, thus making it possible to generate reconstituted hair follicle-enriched human skin with functional features.

Chemical Endoplasmic Reticulum Chaperone Alleviates Doxorubicin-Induced Cardiac Dysfunction.

RATIONALE: Doxorubicin is an effective chemotherapeutic agent for cancer, but its use is often limited by cardiotoxicity. Doxorubicin causes endoplasmic reticulum (ER) dilation in cardiomyocytes, and we have demonstrated that ER stress plays important roles in the pathophysiology of heart failure. OBJECTIVE: We evaluated the role of ER stress in doxorubicin-induced cardiotoxicity and examined whether the chemical ER chaperone could prevent doxorubicin-induced cardiac dysfunction. METHODS AND RESULTS: We confirmed that doxorubicin caused ER dilation in mouse hearts, indicating that doxorubicin may affect ER function. Doxorubicin activated an ER transmembrane stress sensor, activating transcription factor 6, in cultured cardiomyocytes and mouse hearts. However, doxorubicin suppressed the expression of genes downstream of activating transcription factor 6, including X-box binding protein 1. The decreased levels of X-box binding protein 1 resulted in a failure to induce the expression of the ER chaperone glucose-regulated protein 78 which plays a major role in adaptive responses to ER stress. In addition, doxorubicin activated caspase-12, an ER membrane-resident apoptotic molecule, which can lead to cardiomyocyte apoptosis and cardiac dysfunction. Cardiac-specific overexpression of glucose-regulated protein 78 by adeno-associated virus 9 or the administration of the chemical ER chaperone 4-phenylbutyrate attenuated caspase-12 cleavage, and alleviated cardiac apoptosis and dysfunction induced by doxorubicin. CONCLUSIONS: Doxorubicin activated the ER stress-initiated apoptotic response without inducing the ER chaperone glucose-regulated protein 78, further augmenting ER stress in mouse hearts. Cardiac-specific overexpression of glucose-regulated protein 78 or the administration of the chemical ER chaperone alleviated the cardiac dysfunction induced by doxorubicin and may facilitate the safe use of doxorubicin for cancer treatment.

Anti-HB-EGF Antibody-Mediated Delivery of siRNA to Atherosclerotic Lesions in Mice.

For atherosclerotic cardiovascular diseases (ACD), gene therapy may be a potential therapeutic strategy; however, lack of effective and safe methods for gene delivery to atherosclerotic plaques have limited its potential therapeutic applications. To overcome this limitation, we developed a novel antibody-based gene delivery system (anti-HB-EGF/NA vector) by chemically crosslinking antibodies against human heparin-binding epidermal growth factor-like growth factor (HB-EGF). It has been shown to be excessively expressed in human atherosclerotic plaques and NeutrAvidin (NA) for conjugating biotinylated siRNA. Immunofluorescence staining and quantitative flow cytometry analysis using human HB-EGF-expressing cells showed both antibody-mediated selective cellular targeting and efficient intracellular delivery of conjugated biotin-fluorescence. Moreover, we demonstrated antibody-mediated significant and selective gene knockdown via conjugation with anti-HB-EGF/NA vector and biotinylated siRNA (anti-HB-EGF/NA/b-siRNA) in vitro. Furthermore, using high fat-fed human HB-EGF knock-in and apolipoprotein E-knockout (Hbegf hz/hz; Apoe-/-) mice, we demonstrated that the anti-HB-EGF/NA vector, conjugating biotin-fluorescence, increasingly accumulated within the atherosclerotic plaques of the ascending aorta in which human HB-EGF expression levels were highly elevated. Moreover, in response to a single intravenous injection of anti-HB-EGF/NA/b-siRNA in a dose-dependent manner, qPCR analysis of laser-dissected atherosclerotic plaques of the ascending aorta showed significant knockdown of the reporter gene expression. These results suggest that the anti-HB-EGF antibody-mediated siRNA delivery could be a promising delivery system for gene therapy of ACD.

Development of a novel one-step production system for injectable liposomes under GMP.

There are few methods available for injectable liposome production under good manufacturing practices (GMP). Injectable liposome production processes under GMP generally consist of liposome formation, size homogenization, organic solvent removal, liposome concentration control and sterilization. However, these complicated and separate processes make it difficult to maintain scalability, reproducibility and sterility. To overcome these limitations, we developed a novel one-step in-line closed liposome production system that integrated all production processes by combining the in-line thermal mixing device with modified counterflow dialysis. To validate the system, we produced liposomal cyclosporine A (Lipo-CsA) and lyophilized the liposomes. The three independent pilot batches were highly reproducible and passed the quality specifications for injectable drugs, demonstrating that this system could be used under GMP. The accelerated stability test suggested that the liposomes would be stable in long-term storage. This one-step system facilitates a fully automated and unattended production of injectable liposomes under GMP.

Evaluation of intramitochondrial ATP levels identifies G0/G1 switch gene 2 as a positive regulator of oxidative phosphorylation.

The oxidative phosphorylation (OXPHOS) system generates most of the ATP in respiring cells. ATP-depleting conditions, such as hypoxia, trigger responses that promote ATP production. However, how OXPHOS is regulated during hypoxia has yet to be elucidated. In this study, selective measurement of intramitochondrial ATP levels identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS. A mitochondria-targeted, FRET-based ATP biosensor enabled us to assess OXPHOS activity in living cells. Mitochondria-targeted, FRET-based ATP biosensor and ATP production assay in a semiintact cell system revealed that G0s2 increases mitochondrial ATP production. The expression of G0s2 was rapidly and transiently induced by hypoxic stimuli, and G0s2 interacts with OXPHOS complex V (FoF1-ATP synthase). Furthermore, physiological enhancement of G0s2 expression prevented cells from ATP depletion and induced a cellular tolerance for hypoxic stress. These results show that G0s2 positively regulates OXPHOS activity by interacting with FoF1-ATP synthase, which causes an increase in ATP production in response to hypoxic stress and protects cells from a critical energy crisis. These findings contribute to the understanding of a unique stress response to energy depletion. Additionally, this study shows the importance of assessing intramitochondrial ATP levels to evaluate OXPHOS activity in living cells.

[Anaphylactoid Reactions Suspected to Be Caused by Neostigmine in Pediatric Patients under General Anesthesia].

Anaphylactoid reaction is a rapid systemic allergic reaction to many kinds of allergen. The peak age of onset is in the forties and there are not many reports on anaphylactoid reactions in pediatric patients. We report two cases of pediatric patients who underwent surgical treatment on retinoblastoma and developed anaphylactoid reaction probably caused by neostigmine. General anesthesia was induced with fentanyl, sevoflurane, dinitrogen monoxide, and rocronium. The procedure was uneventfully completed. Just after the administration of neostigmine to reverse rocronium, the patients showed red flare on the face and chest, and wheezes were heard, but the vital signs were relatively stable. The rapid onset from the administration of neostigmine to the allergic reaction accompanied by skin and respiratory manifestations strongly suggested the anaphylactoid reaction to neostigmine.

Noninvasive and quantitative live imaging reveals a potential stress-responsive enhancer in the failing heart.

Recent advances in genome analysis have enabled the identification of numerous distal enhancers that regulate gene expression in various conditions. However, the enhancers involved in pathological conditions are largely unknown because of the lack of in vivo quantitative assessment of enhancer activity in live animals. Here, we established a noninvasive and quantitative live imaging system for monitoring transcriptional activity and identified a novel stress-responsive enhancer of Nppa and Nppb, the most common markers of heart failure. The enhancer is a 650-bp fragment within 50 kb of the Nppa and Nppb loci. A chromosome conformation capture (3C) assay revealed that this distal enhancer directly interacts with the 5'-flanking regions of Nppa and Nppb. To monitor the enhancer activity in a live heart, we established an imaging system using the firefly luciferase reporter. Using this imaging system, we observed that the novel enhancer activated the reporter gene in pressure overload-induced failing hearts (failing hearts: 5.7±1.3-fold; sham-surgery hearts: 1.0±0.2-fold; P<0.001, repeated-measures ANOVA). This method will be particularly useful for identifying enhancers that function only during pathological conditions.

Serum bta-miRNA-375 as a potential biomarker for the early diagnosis of enzootic bovine leukosis.

To identify a biomarker for the early diagnosis of enzootic bovine leukosis (EBL) caused by bovine leukemia virus (BLV), we investigated the expression of a microRNA, bta-miR-375, in cattle serum. Using quantitative reverse-transcriptase PCR analysis, we measured bta-miR-375 levels in 27 samples from cattle with EBL (EBL cattle), 45 samples from animals infected with BLV but showing no clinical signs (NS cattle), and 30 samples from cattle uninfected with BLV (BLV negative cattle). In this study, we also compared the kinetics of bta-miR-375 with those of the conventional biomarkers of proviral load (PVL), lactate dehydrogenase (LDH), and thymidine kinase (TK) from the no-clinical-sign phase until EBL onset in three BLV-infected Japanese black (JB) cattle. Bta-miR-375 expression was higher in NS cattle than in BLV negative cattle (P < 0.05) and greater in EBL cattle than in BLV negative and NS cattle (P < 0.0001 for both comparisons). Receiver operating characteristic curves demonstrated that bta-miR-375 levels distinguished EBL cattle from NS cattle with high sensitivity and specificity. In NS cattle, bta-miR-375 expression was increased as early as at 2 months before EBL onset-earlier than the expression of PVL, TK, or LDH isoenzymes 2 and 3. These results suggest that serum miR-375 is a promising biomarker for the early diagnosis of EBL.

Recruiting mechanism of the AAA peroxins, Pex1p and Pex6p, to Pex26p on the peroxisomal membrane.

A peroxisomal C-tail-anchored type-II membrane protein, Pex26p, recruits AAA ATPase Pex1p-Pex6p complexes to peroxisomes. We herein attempted to gain mechanistic insight into Pex26p function. Pex26pΔ33-40 truncated in amino-acid residues at 33-40 abolishes the recruiting of Pex1p-Pex6p complex to peroxisomes and fails to complement the impaired phenotype of pex26 CHO cell mutant ZP167, thereby suggesting that peroxisomal localization of Pex1p and Pex6p is indispensable for the transport of matrix proteins. In in vitro transport assay using semipermeabilized CHO cells, Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. This finding is confirmed by the assay using Walker-motif mutants. Transport of Pex1p and Pex6p is temperature-dependent. In vitro binding assays with glutathione-S-transferase-fused Pex26p, Pex1p and Pex6p bind to Pex26p in a manner dependent on ATP binding but not ATP hydrolysis. These results suggest that ATP hydrolysis is required for stable localization of Pex1p to peroxisomes, but not for binding to Pex26p. Moreover, Pex1p and Pex6p are altered to a more compact conformation upon binding to ATP, as verified by limited proteolysis. Taken together, Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by the ATPase cycle.

Peroxisome biogenesis disorders: molecular basis for impaired peroxisomal membrane assembly: in metabolic functions and biogenesis of peroxisomes in health and disease.

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS). Gene defects of peroxins required for both membrane assembly and matrix protein import are identified: ten mammalian pathogenic peroxins for ten complementation groups of PBDs, are required for matrix protein import; three, Pex3p, Pex16p and Pex19p, are shown to be essential for peroxisome membrane assembly and responsible for the most severe ZS in PBDs of three complementation groups 12, 9, and 14, respectively. Patients with severe ZS with defects of PEX3, PEX16, and PEX19 tend to carry severe mutation such as nonsense mutations, frameshifts and deletions. With respect to the function of these three peroxins in membrane biogenesis, two distinct pathways have been proposed for the import of peroxisomal membrane proteins in mammalian cells: a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex16p-dependent class II pathway. In class II pathway, Pex19p also forms a soluble complex with newly synthesized Pex3p as the chaperone for Pex3p in the cytosol and directly translocates it to peroxisomes. Pex16p functions as the peroxisomal membrane receptor that is specific to the Pex3p-Pex19p complexes. A model for the import of peroxisomal membrane proteins is suggested, providing new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p. Another model suggests that in Saccharomyces cerevisiae peroxisomes likely emerge from the endoplasmic reticulum.

Liposomal amiodarone augments anti-arrhythmic effects and reduces hemodynamic adverse effects in an ischemia/reperfusion rat model.

PURPOSE: Although amiodarone is recognized as the most effective anti-arrhythmic drug available, it has negative hemodynamic effects. Nano-sized liposomes can accumulate in and selectively deliver drugs to ischemic/reperfused (I/R) myocardium, which may augment drug effects and reduce side effects. We investigated the effects of liposomal amiodarone on lethal arrhythmias and hemodynamic parameters in an ischemia/reperfusion rat model. METHODS AND RESULTS: We prepared liposomal amiodarone (mean diameter: 113 ± 8 nm) by a thin-film method. The left coronary artery of experimental rats was occluded for 5 min followed by reperfusion. Ex vivo fluorescent imaging revealed that intravenously administered fluorescent-labeled nano-sized beads accumulated in the I/R myocardium. Amiodarone was measurable in samples from the I/R myocardium when liposomal amiodarone, but not amiodarone, was administered. Although the intravenous administration of amiodarone (3 mg/kg) or liposomal amiodarone (3 mg/kg) reduced heart rate and systolic blood pressure compared with saline, the decrease in heart rate or systolic blood pressure caused by liposomal amiodarone was smaller compared with a corresponding dose of free amiodarone. The intravenous administration of liposomal amiodarone (3 mg/kg), but not free amiodarone (3 mg/kg), 5 min before ischemia showed a significantly reduced duration of lethal arrhythmias (18 ± 9 s) and mortality (0 %) during the reperfusion period compared with saline (195 ± 42 s, 71 %, respectively). CONCLUSIONS: Targeting the delivery of liposomal amiodarone to ischemic/reperfused myocardium reduces the mortality due to lethal arrhythmia and the negative hemodynamic changes caused by amiodarone. Nano-size liposomes may be a promising drug delivery system for targeting I/R myocardium with cardioprotective agents.

Dermal Papilla Cell-Derived Extracellular Vesicles Increase Hair Inductive Gene Expression in Adipose Stem Cells via ß-Catenin Activation.

Recently, extracellular vesicle (EV)-mediated cell differentiation has gained attention in developmental biology due to genetic exchange between donor cells and recipient cells via transfer of mRNA and miRNA. EVs, also known as exosomes, play a role in maintaining paracrine cell communication and can induce cell proliferation and differentiation. However, it remains unclear whether adipose-derived stem cells (ASCs) can adopt dermal papilla (DP)-like properties with dermal papilla cell-derived extracellular vesicles (DPC-EVs). To understand the effect of DPC-EVs on cell differentiation, DPC-EVs were characterized and incubated with ASCs, of monolayer and spheroid cell cultures, in combination with the CAO1/2FP medium specialized for dermal papilla cells (DPCs). DPC-like properties in ASCs were initially evaluated by comparing several genes and proteins with those of DPCs via real-time PCR analysis and immunostaining, respectively. We also evaluated the presence of hair growth-related microRNAs (miRNAs), specifically mir-214-5P, mir-218-5p, and mir-195-5P. Here, we found that miRNA expression patterns varied in DPC-EVs from passage 4 (P4) or P5. In addition, DPC-EVs in combination with CAP1/2FP accelerated ASC proliferation at low concentrations and propagated hair inductive gene expression for versican (vcan), alpha-smooth muscle actin (α-sma), osteopontin (opn), and N-Cam (ncam). Comparison between the expression of hair inductive genes (vcan, α-sma, ctnb, and others), the protein VCAN, α-SMA and ß-Catenin (CTNB), and hair inductive miRNAs (mir-214-5P, mir-218-5p, and mir-195-5p) of DPC-EVs revealed similarities between P4 DPC-EVs-treated ASCs and DPCs. We concluded that early passage DPC-EVs, in combination with CAP1/2FP, enabled ASCs to transdifferentiate into DPC-like cells.

Chimerism through the activation of invariant natural killer T cells prolongs graft survival after transplantation of induced pluripotent stem cell-derived allogeneic cardiomyocytes.

The loss of functional cells through immunological rejection after transplantation reduces the efficacy of regenerative therapies for cardiac failure that use allogeneic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Recently, mixed-chimera mice with donor-specific immunotolerance have been established using the RGI-2001 (liposomal formulation of α-galactosyl ceramide) ligand, which activates invariant natural killer T (iNKT) cells. The present study aimed to investigate whether mixed chimerism, established using RGI-2001, prolongs graft survival in allogeneic iPSC-CM transplantation. Mixed-chimera mice were established via combinatorial treatment with RGI-2001 and anti-CD154 antibodies in an irradiated murine bone marrow transplant model. Luciferase-expressing allogeneic iPSC-CMs were transplanted into mixed-chimera and untreated mice, followed by in vivo imaging. RGI-2001 enhanced iNKT cell activation in mice, and mixed chimerism was successfully established. In vivo imaging revealed that while the allografts were completely obliterated within 2 weeks when transplanted to untreated mice, their survivals were not affected in the mixed-chimera mice. Furthermore, numerous CD3+ cells infiltrated allografts in untreated mice, but fewer CD3+ cells were present in mixed-chimera mice. We conclude that mixed-chimera mice established using RGI-2001 showed prolonged graft survival after allogeneic iPSC-CM transplantation. This donor-specific immunotolerance might increase the efficacy of regenerative therapies for heart failure with allogeneic iPSC-CMs.

Tropomyosin micelles are the major components contributing to the white colour of boiled shellfish soups.

Basket clam soup, a popular Asian dish, is prepared by boiling clams in hot water. The soup is generally cloudy, and it is considered that increased cloudiness enhances taste. However, the composition of the whitening ingredients and their association with taste enhancement remains unclear. In this study, we aimed to identify the components contributing to the white colour of the boiled soup. The white component upon precipitation with trichloroacetic acid reacted positively with ninhydrin, indicating the presence of proteins. The separation of proteins using sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed an intense band of size 33 kDa. Peptide mass fingerprinting of the identified protein using matrix-assisted laser desorption/ionisation-time-of-flight tandem mass spectrometry revealed the protein as tropomyosin. To validate the involvement of tropomyosin in the turbidity of the soup, tropomyosin was expressed and extracted from Escherichia coli. As expected, the purified protein suspended in water resulted in turbid appearance. To determine whether lipids have any association with the observed cloudiness of the soup, the amounts of fatty acids were measured. The proportion of estimated fatty acids was very low compared to that of proteins. Overall, we identified the major component contributing to soup cloudiness as tropomyosin forming micelles.

A novel glycosylation signal regulates transforming growth factor beta receptors as evidenced by endo-beta-galactosidase C expression in rodent cells.

The αGal (Galα1-3Gal) epitope is a xenoantigen that is responsible for hyperacute rejection in xenotransplantation. This epitope is expressed on the cell surface in the cells of all mammals except humans and Old World monkeys. It can be digested by the enzyme endo-ß-galactosidase C (EndoGalC), which is derived from Clostridium perfringens. Previously, we produced EndoGalC transgenic mice to identify the phenotypes that would be induced following EndoGalC overexpression. The mice lacked the αGal epitope in all tissues and exhibited abnormal phenotypes such as postnatal death, growth retardation, skin lesion and abnormal behavior. Interestingly, skin lesions caused by increased proliferation of keratinocytes suggest the role of a glycan structure [in which the αGal epitope has been removed or the N-acetylglucosamine (GlcNAc) residue is newly exposed] as a regulator of signal transduction. To verify this hypothesis, we introduced an EndoGalC expression vector into cultured mouse NIH3T3 cells and obtained several EndoGalC-expressing transfectants. These cells lacked αGal epitope expression and exhibited 1.8-fold higher proliferation than untransfected parental cells. We then used several cytokine receptor inhibitors to assess the signal transduction cascades that were affected. Only SB431542 and LY364947, both of which are transforming growth factor ß (TGFß) receptor type-I (TßR-I) inhibitors, were found to successfully reverse the enhanced cell proliferation rate of EndoGalC transfectants, indicating that the glycan structure is a regulator of TßRs. Biochemical analysis demonstrated that the glycan altered association between TßR-I and TßR-II in the absence of ligands.

Exposure to external environment of low ion concentrations is the trigger for rapid wound closure in Xenopus laevis embryos.

Wounds in Xenopus laevis embryos close rapidly, as previously described. In this study, we examined the dependence on extracellular Na(+) and/or Cl(-) ion concentrations of the closure of wounds in Xenopus embryos inflicted by thermal injury. Wound closure did not occur in normal amphibian medium (100% NAM), while wound areas remarkably decreased either in 10-50% NAM or in 100% NAM lacking Na(+) or Cl(-). Similarly, wound areas did not change in a set of Na(+) and Cl(-) ion concentrations equivalent to those of the humoral fluids of intact Xenopus embryos, but rapid wound closure was induced by decreasing the concentration of either of the two ions. A tangential accumulation of actin cytoskeleton along the wound edge was associated with wound closure. However, a similar actin alignment formed even under the 100% NAM condition, in which wounds did not close, as stated above. The epidermis around the wound edge exhibited ellipse-shaped hypertrophy, and the marginal cells centripetally elongated during wound closure. On the other hand, no distinct morphological changes occurred in 100% NAM, although the epidermis was somewhat thickened. Thus, the morphological changes in the epidermis specific to the low ionic environment most likely play active roles in the wound closure of Xenopus laevis embryos, whereas the tangential actin alignment alone may be insufficient. Taken together, we propose that the wound closure in Xenopus embryos is triggered by a decline in either the extracellular Na(+) or Cl(-) ion concentration, and that this process is required for the abovementioned changes in the shape of the marginal cells.