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BACKGROUND Fetal alcohol spectrum disorder (FASD) is caused by prenatal alcohol exposure (PAE), the intake of ethanol (C2 H5 OH) during pregnancy. Features of FASD cover a range of structural and functional defects including congenital heart defects (CHDs). Folic acid and choline, contributors of methyl groups to one-carbon metabolism (OCM), prevent CHDs in humans. Using our avian model of FASD, we have previously reported that betaine, another methyl donor downstream of choline, prevents CHDs. The CHD preventions are substantial but incomplete. Ethanol causes oxidative stress as well as depleting methyl groups for OCM to support DNA methylation and other epigenetic alterations. To identify more compounds that can safely and effectively prevent CHDs and other effects of PAE, we tested glutathione (GSH), a compound that regulates OCM and is known as a "master antioxidant." METHODS/RESULTS Quail embryos injected with a single dose of ethanol at gastrulation exhibited congenital defects including CHDs similar to those identified in FASD individuals. GSH injected simultaneously with ethanol not only prevented CHDs, but also improved survival and prevented other PAE-induced defects. Assays of hearts at 8 days (HH stage 34) of quail development, when the heart normally has developed 4-chambers, showed that this single dose of PAE reduced global DNA methylation. GSH supplementation concurrent with PAE normalized global DNA methylation levels. The same assays performed on quail hearts at 3 days (HH stage 19-20) of development, showed no difference in global DNA methylation between controls, ethanol-treated, GSH alone, and GSH plus ethanol-treated cohorts. CONCLUSIONS GSH supplementation shows promise to inhibit effects of PAE by improving survival, reducing the incidence of morphological defects including CHDs, and preventing global hypomethylation of DNA in heart tissues.

While major coronary artery development and pathologies affecting them have been extensively studied, understanding the development and organization of the coronary microvasculature beyond the earliest developmental stages requires new tools. Without techniques to image the coronary microvasculature over the whole heart, it is likely we are underestimating the microvasculature’s impact on normal development and diseases. We present a new imaging and analysis toolset to visualize the coronary microvasculature in intact embryonic hearts and quantify vessel organization. The fluorescent dyes DiI and DAPI were used to stain the coronary vasculature and cardiomyocyte nuclei in quail embryo hearts during rapid growth and morphogenesis of the left ventricular wall. Vessel and cardiomyocytes orientation were automatically extracted and quantified, and vessel density was calculated. The coronary microvasculature was found to follow the known helical organization of cardiomyocytes in the ventricular wall. Vessel density in the left ventricle did not change during and after compaction. This quantitative and automated approach will enable future cohort studies to understand the microvasculature’s role in diseases such as hypertrophic cardiomyopathy where misalignment of cardiomyocytes has been observed in utero.

Background Despite decades of public education about dire consequences of prenatal alcohol exposure, drinking alcohol during pregnancy remains prevalent. As high as 40% of live-born infants exposed to alcohol during gestation and diagnosed with Fetal Alcohol Syndrome have congenital heart defects that can be life-threatening. In animal models, the methyl donor betaine, found in foods such as wheat bran, quinoa, beets and spinach, ameliorated neurobehavioral deficits associated with prenatal alcohol exposure (PAE) but effects on heart development are unknown. Methods Previously we modeled a binge drinking episode during the first trimester in avian embryos. Here we investigated whether betaine could prevent adverse effects of alcohol on heart development. Embryos exposed to ethanol with and without an optimal dose of betaine (5 μM) were analyzed at late developmental stages. Cardiac morphology parameters were rapidly analyzed and quantified using optical coherence tomography. DNA methylation at early stages was detected by immunofluorescent staining for 5-methylcytosine in sections of embryos treated with ethanol or co-treated with betaine. Results Compared to ethanol-exposed embryos, betaine-supplemented embryos had higher late-stage survival rates and fewer gross head and body defects than seen after alcohol exposure alone. Betaine also reduced the incidence of late-stage cardiac defects such as absent vessels, abnormal atrio-ventricular (AV) valves, and hypertrophic ventricles. Furthermore, betaine co-treatment brought measurements of great vessel diameters, interventricular septum (IVS) thickness, and AV leaflet volumes in betaine-supplemented embryos close to control values. Early-stage 5-methycytosine staining revealed that DNA methylation levels were reduced by ethanol exposure and normalized by co-administration with betaine. Conclusions This is the first study demonstrating efficacy of the methyl donor betaine in alleviating cardiac defects associated with PAE. These findings highlight the therapeutic potential of low-concentration betaine doses in mitigating PAE induced birth defects and has implications for prenatal nutrition policies, especially for women who may not be responsive to folate supplementation. This article is protected by copyright. All rights reserved.

Repeated-dose toxicity (RDT) studies are one of the critical studies to assess chemical safety. There have been some studies attempting to predict RDT endpoints based on chemical substructures, but it remains very difficult to establish such a method, and a more detailed characterization of chemical compounds seems necessary. Cytochrome P450s (P450s) comprise multiple forms with different substrate specificities and play important roles in both the detoxification and metabolic activation of xenobiotics. In this study, we investigated possible use of P450 reactivity of chemical compounds to classify the compounds. A total of 148 compounds with available rat RDT test data were used as test compounds and subjected to inhibition assays against 18 human and rat P450s. Among the tested compounds, 82 compounds inhibited at least one P450 form. Hierarchical clustering analyses using the P450 inhibitory profiles divided the 82 compounds into nine groups, some of which showed characteristic chemical and biological properties. Principal component analyses of the P450 inhibition data in combination with the calculated chemical descriptors demonstrated that P450 inhibition data were plotted differently than most chemical descriptors in the loading plots. Finally, association analyses between P450 inhibition and RDT endpoints showed that some endpoints related to the liver, kidney and hematology were significantly associated with the inhibition of some P450s. Our present results suggest that the P450 reactivity profiles can be used as novel descriptors for characterizing chemical compounds for the investigation of the toxicity mechanism and/or the establishment of a toxicity prediction model.

Epicardium-derived cells (EPDCs) are an important pool of multipotent cardiovascular progenitor cells. Through epithelial-to-mesenchymal-transition (EMT), EPDCs invade the subepicardium and myocardium and further differentiate into several cell types required for coronary vessel formation. We previously showed that epicardial hypoxia inducible factor (HIF) signaling mediates the invasion of vascular precursor cells critical for patterning the coronary vasculature. Here, we examine the regulatory role of hypoxia (1% oxygen) on EPDC differentiation into vascular smooth muscle cells (VSMCs). Results: Hypoxia stimulates EMT and enhances expression of several VSMC markers in mouse epicardial cell cultures. This stimulation is specifically blocked by inhibiting transforming growth factor-beta (TGFβ) receptor I. Further analyses indicated that hypoxia increases the expression level of TGFβ-1 ligand and phosphorylation of TGFβ receptor II, suggesting an indispensable role of the TGFβ pathway in hypoxia-stimulated VSMC differentiation. We further demonstrate that the non-canonical RhoA/Rho kinase (ROCK) pathway acts as the main downstream effector of TGFβ to modulate hypoxia’s effect on VSMC differentiation. Conclusion: Our results reveal a novel role of epicardial HIF in mediating coronary vasculogenesis by promoting their differentiation into VSMCs through noncanonical TGFβ signaling. These data elucidate that patterning of the coronary vasculature is influenced by epicardial hypoxic signals.

Over 500,000 women per year in the United States drink during pregnancy, and 1 in 5 of this population also binge drink. Up to 40% of live-born children with prenatal alcohol exposure (PAE) present with congenital heart defects (CHDs) including life-threatening outflow and valvuloseptal anomalies. Previously we established a PAE model in the avian embryo and used optical coherence tomography (OCT) imaging to assay looping-stage (early) cardiac function/structure and septation-stage (late) cardiac defects. Early-stage ethanol-exposed embryos had smaller cardiac cushions (valve precursors) and increased retrograde flow, while late-stage embryos presented with gross head/body defects, and exhibited smaller atrio-ventricular (AV) valves, interventricular septae, and aortic vessels. However, supplementation with the methyl donor betaine reduced gross defects, prevented cardiac defects such as ventricular septal defects and abnormal AV valves, and normalized cardiac parameters. Immunofluorescent staining for 5-methylcytosine in transverse embryo sections also revealed that DNA methylation levels were reduced by ethanol but normalized by co-administration of betaine. Furthermore, supplementation with folate, another methyl donor, in the PAE model appeared to normalize retrograde flow levels which are typically elevated by ethanol exposure. Studies are underway to correlate retrograde flow numbers for folate with associated cushion volumes. Finally, preliminary findings have revealed that glutathione, a key endogenous antioxidant which also regulates methyl group donation, is particularly effective in improving alcohol-impacted survival and gross defect rates. Current investigations will determine whether glutathione has any positive effect on PAE-related CHDs. Our studies could have significant implications for public health, especially related to prenatal nutrition recommendations.

Cholangiocarcinoma is a disease with a poor prognosis. A human cholangiocarcinoma cell line, TK, was previously established to enable further understanding of the disease. We conducted this investigation to determine whether or not the TK line is useful for pharmacokinetic study of the chemotherapeutic agent gemcitabine (GEM). Along with the BXPC3 human pancreatic adenocarcinoma cell line, the sensitivity to and effects on the TK cell line of GEM were compared. The influence of deoxycytidine kinase (dCK) transduction was also comparatively investigated. The effects of GEM in terms of drug sensitivity of the TK cell line, cell cycle and levels of transcripts of key enzymes were comparable to the BXPC3 cell line. Responses to the drug were similar in both cell lines. In contrast to pancreatic carcinoma, cell lines for research on cholangiocarcinoma have been limited. This study suggests the application of the TK cell line to the pharmacokinetic study of the chemosensitization of therapeutic drugs, such as GEM.

Aims The transcription factor hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) regulates myocardial vascularization and growth during cardiogenesis. Our aim was to determine whether HEXIM1 also has a beneficial role in modulating vascularization, myocardial growth, and function within the adult heart. Methods and results To achieve our objective, we created and investigated a mouse line wherein HEXIM1 was re-expressed in adult cardiomyocytes to levels found in the foetal heart. Our findings support a beneficial role for HEXIM1 through increased vascularization, myocardial growth, and increased ejection fraction within the adult heart. HEXIM1 re-expression induces angiogenesis, that is, essential for physiological hypertrophy and maintenance of cardiac function. The ability of HEXIM1 to co-ordinate processes associated with physiological hypertrophy may be attributed to HEXIM1 regulation of other transcription factors (HIF-1-α, c-Myc, GATA4, and PPAR-α) that, in turn, control many genes involved in myocardial vascularization, growth, and metabolism. Moreover, the mechanism for HEXIM1-induced physiological hypertrophy appears to be distinct from that involving the PI3K/AKT pathway. Conclusion HEXIM1 re-expression results in the induction of angiogenesis that allows for the co-ordination of tissue growth and angiogenesis during physiological hypertrophy.

Some foods from plant sources, particularly of Moringeceae origin, contained compounds that enhanced bacterial ice-nucleation activity. A hot water extract from mustard seeds was so potent in its enhancing activity that the compounds responsible could be isolated from the seeds as yellow crystals. By instrumental analyses, this enhancer was identified as 4-hydroxy-3-nitrophenylacetic acid. The addition of a purified preparation of this compound at 0.1-1 ppm to the cultivation medium for Xanthomonas campestris was effective for enhancing its ice-nucleation activity.

Over 500,000 women per year in the United States drink during pregnancy, and 1 in 5 of this population also binge drink. As high as 20-50% of live-born children with prenatal alcohol exposure (PAE) present with congenital heart defects including outflow and valvuloseptal anomalies that can be life-threatening. Previously we established a model of PAE (modeling a single binge drinking episode) in the avian embryo and used optical coherence tomography (OCT) imaging to assay early-stage cardiac function/structure and late-stage cardiac defects. At early stages, alcohol/ethanol-exposed embryos had smaller cardiac cushions and increased retrograde flow. At late stages, they presented with gross morphological defects in the head and chest wall, and also exhibited smaller or abnormal atrio-ventricular (AV) valves, thinner interventricular septae (IVS), and smaller vessel diameters for the aortic trunk branches. In other animal models, the methyl donor betaine (found naturally in many foods such as wheat bran, quinoa, beets and spinach) ameliorates neurobehavioral deficits associated with PAE but the effects on heart structure are unknown. In our model of PAE, betaine supplementation led to a reduction in gross structural defects and appeared to protect against certain types of cardiac defects such as ventricular septal defects and abnormal AV valvular morphology. Furthermore, vessel diameters, IVS thicknesses and mural AV leaflet volumes were normalized while the septal AV leaflet volume was increased. These findings highlight the importance of betaine and potentially methylation levels in the prevention of PAE-related birth defects which could have significant implications for public health.

We developed multi-scale, live-time interactive visualization of color image data, including microscopic whole-mouse cryo-images serving many biomedical applications. Using true-color volume rendering, we interactively, selectively enhanced anatomy using feature detection. For example, to enhance red organs (vessels, liver, etc.) and internal surfaces, we computed a red feature from R /( R + G + B ) and surface features from color/gray-scale gradients, respectively. For >70GB cryo-image volumes, we developed multi-resolution visualization, which provided low-resolution rendering of an entire mouse and zooming to organs, tissues, and cells. Fusions of fluorescence and color cryo-volumes uniquely showed biodistribution of metastatic and stem cells within an anatomical context.

Objective Fabry disease is characterized by genetic alpha-galactosidase A deficiency, resulting in accumulation of glycolipids (GL-3) and tissue damage. Hearing loss is also common and attributed to GL-3 accumulation in the inner ear. The only reported histological studies dealt with murine and human specimens. Accordingly, histopathological studies of the cochlea were performed on an alpha-galactosidase A deficient murine model of Fabry disease, using C57BL6/J mice as the controls. Methods The hearing ability was evaluated using the ABR threshold, while cochlear specimens were observed light microscopically and ultrathin temporal bone sections by TEM. Results HE staining showed no accumulation of GL-3 or abnormal cochlear morphology in the alpha-galactosidase A deficient mice, but toluidine blue staining and TEM revealed GL-3 accumulation in the stria vascularis and kidney. No GL-3 accumulation was detected in the C57BL6/J controls by either HE staining or TEM. The alpha-galactosidase A deficient mice and the controls showed no clear differences in the ABR threshold (hearing acuity), but for older animals the threshold was higher in the C57BL6/J controls. Conclusion In summary, although the alpha-galactosidase A deficient mice showed no clear hearing loss, GL-3 accumulation was demonstrated in the cochlea.

Recently, we found that mutation of the C-terminus of transcription factor hexamethylene bisacetamide-inducible protein 1 (HEXIM1) in mice leads to abnormalities in cardiovascular development because of aberrant vascular endothelial growth factor (VEGF) expression. HEXIM1 regulation of some genes has also been shown to be positive transcription elongation factor b (P-TEFb) dependent. However, it is not known whether HEXIM1 regulates VEGF in the mammary gland. We demonstrate that HEXIM1 regulates estrogen-induced VEGF transcription through inhibition of estrogen receptor-alpha recruitment to the VEGF promoter in a P-TEFb-independent manner in MCF-7 cells. Under hypoxic conditions, HEXIM1 inhibits estrogen-induced hypoxia-inducible factor-1 alpha (HIF-1alpha) protein expression and recruitment of HIF-1alpha to the hypoxia-response element in the VEGF promoter. In the mouse mammary gland, increased HEXIM1 expression decreased estrogen-driven VEGF and HIF-1alpha expression. Conversely, a mutation in the C-terminus of HEXIM1 (HEXIM1(1-312)) led to increased VEGF and HIF-1alpha expression and vascularization in mammary glands of heterozygous HEXIM1(1-312) mice when compared with their wild-type littermates. In addition, HEXIM1(1-312) mice have a higher incidence of carcinogen-induced mammary tumors with increased vascularization, suggesting an inhibitory role for HEXIM1 during angiogenesis. Taken together, our data provide evidence to suggest a novel role for HEXIM1 in cancer progression.

Our previous studies and those of others indicated that the transcription factor Hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) is a tumor suppressor and cyclin-dependent kinase inhibitor, and that these HEXIM1 functions are mainly dependent on its C-terminal region. We provide evidence here that the HEXIM1 C-terminal region is critical for cardiovascular development. HEXIM1 protein was detected in the heart during critical time periods in cardiac growth and chamber maturation. We created mice carrying an insertional mutation in the HEXIM1 gene that disrupted its C-terminal region and found that this resulted in prenatal lethality. Heart defects in HEXIM1 1 to 312 mice included abnormal coronary patterning and thin ventricular walls. The thin myocardium can be partly attributed to increased apoptosis. Platelet endothelial cell adhesion molecular precursor-1 staining of HEXIM1 1 to 312 heart sections revealed decreased vascularization of the myocardium despite the presence of coronary vasculature in the epicardium. The expression of vascular endothelial growth factor (VEGF), known to affect angioblast invasion and myocardial proliferation and survival, was decreased in HEXIM1 1 to 312 mice compared with control littermates. We also observed decreased fibroblast growth factor 9 (FGF9) expression, suggesting that effects of HEXIM1 in the myocardium are partly mediated through epicardial FGF9 signaling. Together our results suggest that HEXIM1 plays critical roles in coronary vessel development and myocardial growth. The basis for this role of HEXIM1 is that VEGF is a direct transcriptional target of HEXIM1, and involves attenuation a repressive effects of C/EBPα on VEGF gene transcription.

Cited2 is a cAMP-responsive element-binding protein (CBP)/p300 interacting transcriptional modulator and a proposed negative regulator for hypoxia-inducible factor (HIF)-1α through its competitive binding with HIF-1α to CBP/p300. Disruption of the gene encoding Cited2 is embryonic lethal because of defects in the development of heart and neural tube. Morphological and Doppler echocardiographic analyses of Cited2 −/− embryos reveal severe cardiovascular abnormalities, including pulmonic arterial stenosis and ventricular septal defects accompanied by high peak outflow velocities, features of the human congenital cardiac defect termed tetralogy of Fallot. The mRNA levels of several HIF-1α-responsive genes, such as vascular endothelial growth factor (VEGF), Glut1, and phosphoglycerate kinase 1, increased in the Cited2 −/− hearts. The increase of VEGF levels is significant, because defects in the Cited2 −/− embryos closely resemble the major defects observed in the VEGF transgenic embryos. Finally, compared with wild-type, cultured fibroblasts from Cited2 −/− embryos demonstrate an enhanced expression of HIF-1α-responsive genes under hypoxic conditions. These observations suggest that functional loss of Cited2 is responsible for defects in heart and neural tube development, in part because of the modulation of HIF-1 transcriptional activities in the absence of Cited2. These findings demonstrate that Cited2 is an indispensable regulatory gene during prenatal development.

Hypoallergenic wheat flour produced by modification with cellulase and actinase showed inhibitory activity against ovalbumin permeation in an in vitro model by using the Caco-2 cell monolayer. The activity was found in the cellulase preparation used for producing the flour. An active compound was isolated by HPLC and identified as Trp-Ser-Asn-Ser-Gly-Asn-Phe-Val-Gly-Gly-Lys by 1H-NMR data and Edman degradation. The undecapeptide, some oligopeptides with the N-terminal sequences and Trp ethyl ester showed activity at 10(-7) M, acetyl Trp being active at 10(-2) M. These data suggest that the Trp residue without a free carboxyl group would be required for the inhibitory activity of ovalbumin absorption through the intestinal tract.

Brown Norway rats were immunized with gluten, and then fed a diet containing hypoallergenic flour or an amino acid mixture. The rats were then made to inhale a solubilized gluten to induce gluten-specific bronchial asthma. The antibody levels in the serum of rats were measured by ELISA, and cell counts were done on cytospin preparations of bronchoalveolar lavage fluid. Body weight was decreased after allergen challenge in rats fed the amino acid mixture but not in rats fed the hypoallergenic flour. Antibody levels in the serum were significantly lower in rats fed hypoallergenic flour than in those fed the amino acid mixture. Differential cell counts in the bronchoalveolar lavage fluid showed that the numbers of eosinophils, lymphocytes, and neutrophils were significantly lower in rats fed the hypoallergenic flour than in those fed the amino acid mixture. These results suggest that hypoallergenic flour actively suppresses the allergic reactions, probably by inducing oral tolerance.

One class of the nuclear receptor AF-2 coactivator complexes contains the SRC-1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identified a subfamily of RNA-binding DEAD-box proteins (p72/p68) as a human estrogen receptor alpha (hER alpha) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC-1/TIF2 family protein and the hER alpha A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC-1) and SRA were co-immunoprecipitated with estrogen-bound hER alpha in MCF7 cells and in partially purified complexes associated with hER alpha from HeLa nuclear extracts. Estrogen induced co-localization of p72 with hER alpha and TIF2 in the nucleus. The presence of p72/p68 potentiated the estrogen-induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen-induced transactivation of hER alpha. These findings indicate that p72/p68 acts as an ER subtype-selective coactivator through ER alpha AF-1 by associating with the coactivator complex to bind its AF-2 through direct binding with SRA and the SRC-1/TIF2 family proteins.

A new polysaccharide with a molecular weight of 5.0 x 10(4) was isolated as a possible wheat allergen from a water-soluble fraction of flour by affinity chromatography and gel filtration. The isolated polysaccharide was found to be a possible wheat allergen, as it bound specifically to IgE antibodies in the sera of patients allergic to the water-soluble fraction of flour. Chemically, the sugar moiety of the polysaccharide consisted of D-glucose and D-mannose with beta-1,4-linkages in a molar ratio of 4.4:1. Since this mannoglucan is thought to be stable in our body, it would act as a remaining allergen to cause a long-lasting allergic reaction to wheat flour.

グルテンボールを凍結乾燥後粉砕し, ふるいにかけたところ, 多孔質のグルテンが得られた。この基質にアクチナーゼをpH7, 40℃で1時間作用させたところ, 基質の約90%が可溶化した。反応物を Sephadex G-15カラムクロマトに供し, Glx含量が50.6%の高グルタミンペプチドを得た。このペプチドを濾過除菌することにり, 安定なグルタミン供給源として臨床応用されることが期待された。

An allergenic peptide responsible for atopic dermatitis was isolated from the chymotryptic hydrolysate of gluten. The primary structure of the peptide was determined as (Ser-Gln-Gln-Gln-(Gln-)Pro-Pro-Phe)4. The epitope in the allergenic peptide was Gln-Gln-Gln-Pro-Pro. The N-terminal glutamine and the two proline residues were essential for epitopic function. Based on this epitope structure, a practical method is proposed to produce hypoallergenic flour using food-processing enzyme. Soft flour was mixed with water dissolving actinase and then incubated at 37°C for 1 h. The product was determined to be negative allergenicity by ELISA (enzyme-linked immunosorbent assay). Hypoallergenic wheat products in the form of bread and pasta-like noodles were fabricated.

NMR analysis of a model pentapeptide, acetyl (Ac)–Gln–Gln–Gln–Pro–Pro, as an epitope of a wheat allergen was performed. The problem of severe signal overlapping in the 1H NMR spectrum was overcome by elaborate two-dimensional methods using 13C information. HMQC-TOCSY allowed the assignments of the 1H and 13C NMR signals except the γ-glutamylamide parts. E-HSQC-ROESY, which was constructed by modification of the E-HSQC, could serve for discriminating ROEs of protons whose δH values were close to each other. Although the sensitivity of HSQC-ROESY itself was low, selecting only CH carbons by adjusting the proper delay and flip angle of a pulse allowed a narrow F1 spectral width and hence the collection of numerous transients. As a result, the configurations of the amide bonds of the backbone were determined as all-trans. ©1998 John Wiley & Sons, Ltd.

In our series of studies aimed at improving food proteins by enzymatic hydrolysis and resynthesis (plastein reaction), we first isolated and identified a series of bitter peptides occurring in a peptic hydrolysate of soybean protein. We then applied the plastein reaction to the bitter hydrolysate and succeeded in debittering it. We also found that the papain-catalyzed resynthesis reaction permitted covalent incorporation of added amino acid esters. This led to improving the functional and nutritional properties of food proteins, giving rise to peptide surfactants with considerable oil-emulsifying capacity and modified peptides with balanced amino acid compositions. The products we then developed are exemplified by a low-phenylalanine, high-tyrosine peptide for patients with phenylketonuria. The products aiso include peptides with maximized branched-chain amino acids and minimized aromatic amino acids as well as those with an enriched glutamine content. Recently, we extended our studies to analysis of cereal allergens and the design of hypoallergenic foods. Our first success was the design of rice grains with removed allergenic prateins. The process for producing this kind of functional food was subsequently industrialized, and the product “Fine Rice” was approved as the first “food far specified health use” by the Japan Ministry of Health and Welfare. This was followed by the current study, aimed at producing a hypoallergenic wheat flour preparation by controlled enzymatic hydrolysis. In this study, we have determined a major epitope unit (Gln-Gln-Gln-Pro-Pro) of glutenin origin and also developed a haptenic peptide that may be usable as an antiallergenic factor for patients with wheat-associated atopic dermatitis. An important aspect of these studies is that we took special care not to jeopardize acceptability or palatability, which is one of the most important considerations when dealing with food.

For application to the treatment of wheat-sensitive allergy, we developed a practical method for producing a haptenic peptide mixture, and evaluated its haptenic properties. Wheat flour was hydrolyzed with chymotrypsin to obtain a hydrolysate, and the diffusible fraction from the hydrolysate was subjected to gel-filtration. The resulting oligopeptide fraction could bind to wheat-specific IgE antibodies in the serum of patients allergic to wheat. Since this peptide mixture did not induce histamine release from basophils in these patients, it was concluded that the peptide mixture was composed of monovalent haptens. Histamine release from antigenstimulated basophils was almost completely inhibited when the basophils were preincubated with the haptenic peptide mixture. These results suggest that this haptenic peptide mixture can regulate the allergenic reaction in an epitope-specific manner.

The minimum primary structure of the IgE-binding epitope in wheat gluten was determined as Gln-Gln-Gln-Pro-Pro. The N-terminal glutamine and the two proline residues were essential for epitopic function. The occurrence of the second glutamine residue and acetylation of the N-terminal amino group were found to exert some auxiliary functions, whereas only a lesser contribution was expected for the third glutamine residue. It has also been confirmed that acetyl-Gln-Gln-Gln-Pro-Pro bound to wheat-specific IgE antibodies in the sera of patients allergic to wheat, although it did not induce histamine release from the basophils of these patients. Taken together, we concluded that the Gln-Gln-Gln-Pro-Pro motif constituted an IgE-binding rather than immunogenic epitope and also that acetyl-Gln-Gln-Gln-Pro-Pro might act as a hapten capable of binding to specific IgE molecules. The possibility exists that this peptide will have practical application to therapy for or prevention of wheat-sensitive allergy.

We propose a novel method to produce hypoallergenic wheat flour suitable for patients allergic to wheat by using enzymatic fragmentation with cellulase and actinase. The hypoallergenic flour displayed potent inhibitory activity against allergen absorption and actively suppressed allergic reactions, probably inducing oral tolerance. The results suggest that hypoallergenic wheat flour has allergy-suppressive effects without inducing side effects.

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (KA = 1.9 x 10(5)-1.4 x 10(8) M-1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (KA = 2.0 x 10(5)-1.8 x 10(7) M-1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.

固形分濃度が22.5%以上の凍結濃縮生乳を高圧処理すると,ゲルが生成した.尿素とEDTAはゲル化を阻害した.適度の薦糖の存在はゲル化を助けた.高圧処理によって,カゼインミセルとサブミセルの中間的な大きさの粒子が生成し,この粒子の凝集による網目構造の形成がゲル化に寄与した.ゲル強度は空隙構造に依存した.

Light-microscopic and immunoblot immunochemical procedures were used to study the distribution and biochemical characteristics of neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) in the suprachiasmatic nuclei (SCN) of the adult Siberian hamster. In the adult brain PSA-NCAM is located in regions capable of undergoing morphological rearrangements and thus is generally considered to be an indicator of neural plasticity. Immunostaining for PSA in the Siberian hamster SCN (using a monoclonal antibody against the alpha 2,8-linked PSA of NCAM) was evident throughout the rostrocaudal axis of the SCN, with the most intense reaction in the ventrolateral region. Immunoreactivity was present in the neuropil, which delineated groups of cells with unstained cytoplasm. Many of the SCN cells were aggregated into cords or clusters. The optic chiasm was free from label, except for short processes apparently extending from the densely stained neuropil in the ventrolateral SCN. Immunoreactivity was abolished by preincubating sections in an endoneuraminidase (endo-N) or by preincubation of the primary antibody with PSA-NCAM. Immunostaining of the nonsialylated NCAM polypeptide was also limited to the neuropil, but this was more diffuse and less regionally specific than PSA staining. The ventral SCN exhibited the most intense labeling for NCAM. Immunoblot analyses revealed the immunoreactive PSA-NCAM as a broad band migrating between apparent molecular weights in the range of 150-300 kD, which was ablated by treatment of SCN tissue extract with endo-N. This electrophoretic migration pattern of PSA-NCAM from the SCN region was similar to that seen in several other brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Among various techniques for the removal of water from foods, freeze concentration is unique in that solutes in aqueous media are concentrated with the growth of ice crystals during freezing. In the presence of added ice-nucleation-active bacterial cells as ice nuclei, the bulk water in foods freezes at a subzero temperature near the melting point of ice. Since in general the bacterial ice-nucleation activity is so stable below 25°C, the seeding can be done at room temperature. For applications, we thus concentrated raw egg white and found the characteristic that the product formed a hard gel when heated and also gave a fine foam when whipped. The freeze-concentrated product from fresh milk was characterized by forming a gel when pressurized. A similar technique was applied to fresh lemon juice to obtain a concentrated product still retaining its original flavor. As another application, strawberry paste was separated into juice and pulp fractions, and the juice fraction alone was freeze-concentrated. The pulp fraction was then put back together with sugar, pectin and citric acid. We thus succeeded in making a non-heated jam which, compared to conventional jam, was almost equal in texture and superior in fresh flavor and colour.

Hypoallergenic wheat products in the form of pasta-like noodles and bread were fabricated. Wheat flour was added with a 0.6-fold weight of water dissolving collagenase to obtain hypoallergenic flour batter. Methods for producing hypoallergenic pasta-like noodles and bread from the batter were designed. In the noodle making, the batter was mixed with an oligosaccharide with a mild sweetness, a surfactant, and salt, and the mixture was extruded under heating. Both retorting and refrigerating processes were applied to the noodles. In the bread making a mixture of the batter, glucose, citric acid, sodium hydrogen carbonate and salt was baked at 180°C. The addition of glucose contributed to generation of faborable flavor and color.

Commercially available egg white was freeze-concentrated using bacterial ice nuclei, which improved its foaming properties. Baked meringue, angel cake, snow-jelly, and marshmallow were prepared using freeze-concentrated egg white foam and their physical properties were evaluated. We concluded that freeze-concentrated egg white can be used to make some foam foods.

The application of both pressurization and enzymatic treatment to aged rice grains was investigated to improve their cooking properties. Pressurization at 100 MPa was favorable in improving the properties. For treatment of the grains with enzymes, actinase was more effective than cellulase and pectolyase. Actinase-treated grains, when cooked, gave a favorable result in terms of the stickiness/hardness ratio and brightness . Stickiness and brightness of cooked rice were developed by removing albumins and globulins from grains . In newly-harvested grains, the proteins were easily removed by extraction with water which contained rice-originating minerals. In aged grains, however, the proteins were already denatured and extracted with difficulty. Proteases hydrolyzed the denatured proteins to be extractable . As a result, starch granules existing on the surface of grains were liberated. Some proteases also hydrolyzed a starch granule-associated protein, and, consequently, made the starch highly gelatinized by heating. The protease treatment of aged grains was effective for two main reasons ; liberation of starch granules and removal of the granule-associated protein . During cooking of rice the liberated starch without the associated proteins gelatinized to give rise to stickiness . The gelatinized starch is considered to form a more isotropic film on the surface of every cooked rice grain to give brightness as well.

The ice nucleation-active protein of Erwinia ananas IN-10 (inaA protein) was over-expressed as inclusion bodies in Escherichia coli in a yield of 15.3 mg of inaA protein from 60 mg of bacterial cells on a dry-matter basis. The inaA protein was purified from the inclusion bodies by solubilization with detergents to obtain a protein preparation free from sugar and lipid. This preparation had a distinct ice nucleation activity, indicating that the inaA protein per se is able to act as a nucleus.

This study aims to improve the stickiness and brightness of cooked rice from aged rice grains by removing its albumins and globulins with proteases. In newly–harvested grains, the proteins were easily removed by extraction with water which contained minerals from rice. In aged grains, however, the proteins were already denatured and extracted with difficulty. Proteases hydrolyzed even the denatured proteins to make them extractable. As a result, starch granules on the surface of grains were liberated. Some proteases also hydrolyzed a starch granule-associated protein, and consequently, made the starch highly gelatinizable by heating. The protease treatment of aged grains was effective for two main reasons: liberation of starch granules and removal of the granule-associated protein. During cooking of rice the liberated starch without the associated proteins gelatinized to give rise to stickiness. The gelatinized starch is considered to form a more isotropic film on the surface of every cooked rice grain to...

We applied the Case cryo-imaging system to detect signals of developmental toxicity in transgenic mouse fetuses resulting from maternal exposure to a developmental environmental toxicant (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD). We utilized a fluorescent transgenic mouse model that expresses Green Fluorescent Protein (GFP) exclusively in smooth muscles under the control of the smooth muscle gamma actin (SMGA) promoter (SMGA/EGFP mice kindly provided by J. Lessard, U. Cincinnati). Analysis of cryo-image data volumes, comprising of very high-resolution anatomical brightfield and molecular fluorescence block face images, revealed qualitative and quantitative morphological differences in control versus exposed fetuses. Fetuses randomly chosen from pregnant females euthanized on gestation day (GD) 18 were either manually examined or cryo-imaged. For cryo-imaging, fetuses were embedded, frozen and cryo-sectioned at 20 μm thickness and brightfield color and fluorescent block-face images were acquired with an in-plane resolution of ≈15 μm. Automated 3D volume visualization schemes segmented out the black embedding medium and blended fluorescence and brightfield data to produce 3D reconstructions of all fetuses. Comparison of Treatment groups TCDD GD13, TCDD GD14 and control through automated analysis tools highlighted differences not observable by prosectors performing traditional fresh dissection. For example, severe hydronephrosis, suggestive of irreversible kidney damage, was detected by cryoimaging in fetuses exposed to TCDD. Automated quantification of total fluorescence in smooth muscles revealed suppressed fluorescence in TCDD-exposed fetuses. This application demonstrated that cryo-imaging can be utilized as a routine high-throughput screening tool to assess the effects of potential toxins on the developmental biology of small animals.

In order to utilize peptide-bound rather than free glutamine for enteral nutrition, a method was developed for producing a high-glutamine oligopeptide mixture from gluten by treatment with protease. Commercially available gluten was hydrolyzed first with molsin and then with actinase to produce a hydrolysate. Sephadex G-15 adsorption chromatography applied to the hydrolysate afforded a nearly 50% glutamine containing oligopeptide fraction with a yield of 33% on a nitrogen basis. We evaluated this oligopeptide fraction by feeding trials with fasted and methotrexate-administrated rats. The oligopeptide fraction, when compared to a simulated free amino acid mixture, was significantly more effective in enhancing the mucosal protein content in fasted rats. Also, methotrexate-administrated rats fed the oligopeptide fraction were more resistant to methotrexate-induced enterocolitis than those fed a simulated free amino acid mixture.

A novel method for producing a low ovomucoid egg white preparation is proposed. Egg white powder (0.5 g) was dissolved in a 10-fold weight of distilled water and adjusted to pH 5, and ethanol was added to the solution at a final concentration of 20% (v/v). The mixture was vigorously stirred and centrifuged. The precipitate was washed three times with 20% ethanol (6.25 ml each), with about 65% of egg white proteins occurring in the precipitate. The use of ELISA demonstrated that 70% of ovomucoid was recovered from the supernatant fraction. However, functionally important proteins such as ovalbumin, ovotransferrin, and lysozyme still remained in the precipitate. These results may be due primarily to the much higher solubility of ovomucoid in this aqueous ethanol. Food quality evaluation showed that high whippability and foam stability were retained in the low ovomucoid preparation as in its material egg white. This product would thus be applicable as a new processed food for ovomucoid-sensitive allergic patients.

Quantification of microvascular remodeling as a meaningful discovery tool requires mapping and measurement of site-specific changes within vascular trees and networks. Vessel density and other critical vascular parameters are often modulated by molecular regulators as determined by local vascular architecture. For example, enlargement of vessel diameter by vascular endothelial growth factor (VEGF) is restricted to specific generations of vessel branching (Parsons-Wingerter et al., Microvascular Research72: 91, 2006). The averaging of vessel diameter over many successively smaller generations is therefore not particularly useful. The newly automated, user-interactive software VESsel GENeration Analysis (VESGEN) quantifies major vessel parameters within two-dimensional (2D) vascular trees, networks, and tree-network composites. This report reviews application of VESGEN 2D to angiogenic and lymphangiogenic tissues that includes the human and murine retina, embryonic coronary vessels, and avian chorioallantoic membrane. Software output includes colorized image maps with quantification of local vessel diameter, fractal dimension, tortuosity, and avascular spacing. The density of parameters such as vessel area, length, number, and branch point are quantified according to site-specific generational branching within vascular trees. The sole user input requirement is a binary (black/white) vascular image. Future applications of VESGEN will include analysis of 3D vascular architecture and bioinformatic dimensions such as blood flow and receptor localization. Branching analysis by VESGEN has demonstrated that numerous regulators including VEGF(165), basic fibroblast growth factor, transforming growth factor beta-1, angiostatin and the clinical steroid triamcinolone acetonide induce 'fingerprint' or 'signature' changes in vascular patterning that provide unique readouts of dominant molecular signaling.

We have proposed a method to produce an oligopeptide mixture from zein by enzymatic modification to improve the Fischer ratio. In this study we produced the oligopeptide mixture on an enlarged scale and evaluated it in comparison with a corresponding amino acid mixture by feeding tests with normal and liver-injured rats. Both of the oligopeptide mixture and the amino acid mixture enhanced the Fischer ratio in plasma and brain at a similar degree. In rats suffering from severe hepatic injury, undesirable aromatic amino acids were absorbed from the oligopeptide mixture to a less extent than from the amino acid mixture. Peptide feeding, as well as amino acid feeding, resulted in decreasing concentrations of tryptophan and serotonin and in keeping the concentrations of dopamine and norepinephrine in cerebrum. These results suggest that the oligopeptide mixture is useful for patients with severe heptic disease.

The application of both pressurization and enzymatic treatment to aged rice grains was investigated to improve their cooking properties. Pressurization at 100 MPa was favorable in improving the properties. For treatment of the grains with enzymes, actinase was more effective than cellulase and pectolyase. Neither lipase nor transglutaminase showed any imporving effect. Actinase-treated grains, when cooked, gave the most farvorable result in terms of the stickiness/hardness ratio, brightness, flavor, and texture.

A method was proposed to produce a peptide fraction from zein by enzymatic hydrolysis to improve the Fischer ratio, i.e., the molar ratio of the branched chain amino acids, leucine, isoleucine and valine, vs. the aromatic amino acids, phenylalanine and tyrosine. Zein was hydrolyzed first with alkalophilic proteinase and then with actinase to liberate aromatic amino acids. The hydrolysate, however, tasted bitter and it was modified with transglutaminase to decrease the bitterness. Sephadex G-15 adsorption chromatography was used on the debittered product to remove the aromatic amino acids liberated. A series of these enzymatic modifications and the chromatography afforded a final product with a Fischer ratio of 20.0 at a yield of 56% on a nitrogen basis. Such a very high Fischer ratio and a reasonable yield would warrant economic feasibility for producing this formula on an industrial scale.

The proper sequence of electrical activation of the mature four-chambered heart requires specialized conduction pathways including the His-Purkinje system and a nearly complete separation of the atrial and ventricular myocardium. We tracked the emergence of the structure of the mature His-Purkinje system in the developing chicken embryo with anti-polysialylated neural cell adhesion molecule (PSA-NCAM) and the HNK1 antibody against a sulfated carbohydrate epitope. The function of the His-Purkinje system was assayed using extracellular electrodes and high-resolution voltage-sensitive two-dimensional optical mapping. The appearance of the mature form of the His-Purkinje system delineated by the markers coincided with the onset of the mature electrophysiological pattern of ventricular activation. These data suggest that, at the completion of ventricular septation, the His-Purkinje system undergoes critical structural and functional transitions that impact on the global pattern of conduction and contraction of the developing four-chambered heart.

This work proposes a new method for improving the functional properties of food proteins by intentional modification. Hen’s egg white was used as an example of the food proteins to be improved. Commercially available egg white was freeze-concentrated using bacterial ice nuclei. The resulting increase in its solid content led to formation of a hard, but fragile gel by heat treatment at 90–120°C for 20 min. When modified with arginine, it was changed to a viscoelastic product. Scanning electron microscopic and pulsed NMR observations suggested that tight aggregation of constituent protein molecules occurred in the hard gel network by the heat treatment and that the aggregation partly became loose by the modification with arginine.