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We investigated the IgE reactivity to crude and purified milk antigens in the sera of 112 dogs with cutaneous adverse food reactions (CAFRs). Of the 112 dogs, 33 (29%) had specific IgE for crude milk antigens. In the dogs with milk-specific IgE, IgE reactivity to casein, bovine serum albumin (BSA), α-lactalbumin, β-lactoglobulin, and bovine IgG were 81%, 85%, 39%, 27%, and 35%, respectively. Casein and BSA may be important allergens in dogs with CAFRs. Some canine vaccines contain casein hydrolysate as a stabilizer and the pooled serum with anti-casein IgE showed IgE reactivity to the vaccines containing it. Information about IgE reactivity to casein in dogs with CAFRs could be useful for predicting adverse reactions to the vaccines including casein hydrolysate.

Interleukin-4 (IL4) and interleukin-13 (IL13) are involved in the initial response of T helper 2 lymphocytes through the activation of the IL4 receptor alpha (IL4RA), which is a common receptor chain for these cytokines. In humans, several single-nucleotide polymorphisms (SNPs) identified in the IL4R and in interleukin coding genes were associated with atopic disorders. However, the association between canine IL4R polymorphisms and atopic disorders has not been investigated yet. This study aimed to determine the associations between four non-synonymous SNPs and canine atopic dermatitis (CAD) in shiba inu and miniature dachshund populations. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis were used to genotype four polymorphisms of canine IL4R and IL13 in 34 shiba inu and 19 miniature dachshund patients with CAD, as well as 29 shiba inu and 39 miniature dachshund patients without the condition. Results from miniature dachshunds revealed a potential association between the presence of minor A allele rs24378020 and CAD (odds ratio, 0.10; 95% confidence interval, 0.01-0.85; Poriginal=0.0062). This CAD resistance allele led to an amino acid substitution (Arg688Cys) that could impair IL4 and IL13 signaling. In shiba inu patients, rs24378020 was fixed by homozygosity of the major G allele. No association was found between the remaining three evaluated SNPs and CAD. Nevertheless, the study suggests that the IL4R Cys688 variant reduces the risk of CAD in miniature dachshunds.

Persistent papillomatosis on footpads related to canine papillomavirus type 2 (CPV-2) infection has been described in dogs with immunocompromised condition. A 9-year-old, male French bulldog was presented with cauliflower-like nodules on the footpads of his left front leg. Histopathological examination revealed multiple finger-like projections of squamous epithelium with intranuclear inclusion bodies. Immunohistochemistry using an anti-bovine papillomavirus antibody demonstrated immunostaining in the keratinocytes. Partial genome DNA of CPV-2 was amplified from the lesion. Full genome sequence of CPV-2 in the subject showed 99.95% nucleotide identity with that of CPV-2 from the reference data. Two weeks after a biopsy, the skin lesion spontaneously regressed without any specific treatment. In non-immunocompromised dogs, CPV-2-related footpad papillomatosis could spontaneously resolve after a biopsy.

Papillomavirus (PV) is a well-known pathogen associated with epithelial and mucosal neoplastic diseases. In contrast to human PVs, characterization of animal PVs from the aspect of anogenital neoplasm is still on a learning curve. In the present study, two vulval and one anal warts, histologically diagnosed as fibropapillomas, excised from dairy cattle were analyzed. PCR and sequencing revealed that bovine papillomavirus type 1 (BPV-1) and BPV-2 were detected from anal and vulval fibropapillomas, respectively. Immunohistochemistry detected PV antigen in a few differentiated keratinocytes of one vulval case. Reverse-transcriptase PCR detected the early region, but not the late region of BPV mRNA in all three cases. The present study will provide new insight into the relationship between BPV and anogenital papilloma in cattle.

Background IgE reactivity to fish allergens in atopic dogs, which are used as models for food allergy, has not been elucidated to date. We investigated IgE reactivity to crude extracts and purified allergens derived from the Pacific cod (Gadus macrocephalus) in atopic dogs to identify the allergenic proteins of cod. Results The levels of specific IgE to crude cod extracts were measured in the sera of 179 atopic dogs, including 27 dogs with cod allergy, using enzyme-linked immunosorbent assay (ELISA). Specific IgE to crude cod extracts were present in 36 (20%) of the 179 atopic dogs and in 12 (44%) of the 27 dogs with cod allergy. The allergens in crude cod extracts were analyzed by ELISA, immunoblotting, and liquid chromatography-tandem mass spectrometry. In allergen component analysis, IgE reactivity to tropomyosin and enolase was observed in the sera of dogs with cod allergy. IgE reactivity to parvalbumin, collagen, and tropomyosin was evaluated using the sera of atopic dogs that tested positive for specific IgE to crude cod extracts. Among the 36 dogs with IgE reactivity to crude cod extracts, 9 (25%), 14 (39%), and 18 (50%) dogs tested positive for specific IgE to parvalbumin, collagen, and tropomyosin, respectively. Conclusions The IgE reactivity to cod allergens observed in dogs was similar to that in humans, and this finding further supports the use of atopic dogs with fish allergy as a model for fish allergy in humans.

Severe papillomatosis occasionally causes astasia leading to euthanizing cattle. There are currently a limited number of reports on virologic approach in severe bovine papillomatosis. Here we report a full genome characterization of bovine papillomavirus type 1 (BPV-1) from the case of severe papillomatosis. A calf developed numerous papillomas on the skin and some nodules in the upper gastrointestinal tract at seven months old. The skin lesion was diagnosed as the epithelial papilloma with BPV antigen expression, while the gastrointestinal lesions were diagnosed as the fibropapilloma without BPV antigen. Full genome analysis revealed that BPV-1s detected in all the lesions were exactly the same. Compared with the reference BPV-1 sequence, there was a single nucleotide insertion in the upstream regulatory region.

Association of felis catus papillomaviruses (FcaPVs) with feline squamous cell carcinoma (SCC) has been reported worldwide, while there is limited information about FcaPVs in Asia. In this study, 21 feline SCC biopsy samples from cats in Japan were analyzed by PCR with PV consensus primers and type-specific primers for FcaPV type 2 (FcaPV-2), FcaPV-3 and FcaPV-4 designed in this study. Sequence analysis revealed that one sample was FcaPV-3, and two were FcaPV-4. In both FcaPV-4 positive samples, 334th tryptophan in L1 ORF was deleted compared with the reference sequence. Moreover, immunohistochemistry showed that p16 protein was positive in both FcaPV-4 detected samples. This study would contribute to the molecular epidemiological and pathological understanding of FcaPV in Japan.

In this study, dogs with atopic dermatitis were separated into non-food-induced atopic dermatitis (NFIAD) group (n = 15) and food-induced atopic dermatitis (FIAD) group (n = 37) based on an elimination diet test. IgE reactivity for crude Malassezia pachydermatis (M. pachydermatis) and house dust mites (HDM) allergen extracts was investigated in the two groups using fluorometric enzyme-linked immunosorbent assay (ELISA) and intradermal skin test (IDST). Nine (60%) of the 15 dogs in NFIAD group and 6 (16%) of the 37 dogs in FIAD group showed specific IgE for M. pachydermatis (Mann-Whitney U-test, P 0.01). By immunoblotting analysis, the pooled serum samples from dogs with IgE for M. pachydermatis showed IgE reactivity for 50 kDa protein of M. pachydermatis. Twelve (80%) of the 15 dogs in NFIAD group and 8 (22%) of the 37 dogs in FIAD group showed specific IgE for HDM (Mann-Whitney U-test, P 0.01). In addition, the dogs in NFIAD group significantly show a positive IDST to M. pachydermatis and HDM extracts compared with the dogs in FIAD group. The results suggest that dogs with NFIAD are at increased risk of becoming sensitized to the normal commensal organism M. pachydermatis compared with dogs with FIAD, perhaps co-sensitization occurred due to an HDM protease antigen's, Der f 1 and/or Der p 1, proteolytic activity related epidermal skin barrier defects. Treatment to limit skin colonization may thus be especially important in NFIAD.

Diarrhea is a major cause of death in calves and this is linked directly to economic loss in the cattle industry. Fermented milk replacer (FMR) has been used widely in clinical settings for calf feeding to improve its health and growth. However, the protective efficacy of FMR on calf diarrhea remains unclear. In this study, we verified the preventive effects of FMR feeding on calf diarrhea using an experimental infection model of bovine rotavirus (BRV) in newborn calves and a field study in dairy farms with calf diarrhea. In addition, we evaluated the protective efficacy of lactic acid bacteria-supplemented milk replacer (LAB-MR) in an experimental infection model. In the experimental infection, calves fed FMR or high-concentrated LAB-MR had diarrhea, but the water content of feces was lower and more stable than that of calves fed normal milk replacer. The amount of milk intake also decreased temporarily, but recovered immediately in the FMR- and LAB-MR-fed calves. As compared with the control calves, FMR- or LAB-MR-fed calves showed less severe or reduced histopathological lesions of enteritis in the intestinal mucosa. In a field study using dairy calves, FMR feeding significantly reduced the incidence of enteritis, mortality from enteritis, duration of a series of treatment for enteritis, number of consultations, and cost of medical care for the disease. These results suggest that feeding milk replacer-based probiotics to calves reduces the severity of diarrhea and tissue damage to the intestinal tract caused by BRV infection and provides significant clinical benefits to the prevention and treatment of calf diarrhea.

Dogs with cutaneous adverse food reactions (CAFR) often have specific IgE to food allergens. Egg white, which is majorly composed of ovomucoid, ovalbumin, ovotransferrin, and lysozyme, is a food allergen in dogs. Information of the IgE reactivity to purified egg white allergens supports accurate diagnosis and efficiency treatment in humans. However, to the best of our knowledge, there have been no studies on the IgE reactivity to purified egg white allergens in dogs. Here, we investigated the IgE reactivity to crude and purified allergens of hen egg white in dogs with CAFR. First, when we examined serum samples from 82 dogs with CAFR for specific IgE to crude egg white by ELISA, 9.8% (8/82) of the dogs with CAFR showed the IgE reactivity to crude egg white. We then used sera from the eight dogs with positive IgE reactivity to crude egg white to examine the IgE reactivity to four purified allergens, ovomucoid, ovalbumin, ovotransferrin, and lysozyme, by ELISA. We found that 75% (6/8) of the dogs showed IgE reactivity to both ovomucoid and ovalbumin, and that 37.5% (3/8) of the dogs showed IgE reactivity to ovotransferrin. None (0/8) showed IgE reactivity to lysozyme. Moreover, validating these results, the immunoblot analyses were performed using the sera of the three dogs showing the highest IgE reactivity to crude egg white. Both anti-ovomucoid and anti-ovalbumin IgE were detected in the sera of these dogs, while anti-ovotransferrin IgE was not detected. Considering these, ovomucoid and ovalbumin appears to be the major egg white allergens in dogs with CAFR.

Bacteriophages (phages) belonging to the family Podoviridae genus N4-like viruses have been used as therapeutic agent in phage therapy against Pseudomonas aeruginosa infections. P. aeruginosa phage KPP21 was isolated in Japan, and phylogenetically investigated the phages belonging to this viral genus. Morphological and genetic analyses confirmed that phage KPP21 belongs to the family Podoviridae genus N4-like viruses. Moreover, phylogenetic analyses based on putative DNA polymerase and major virion protein showed that P. aeruginosa phages belonging to the genus N4-like viruses are separated into two lineages and that phage KPP21 is in the same clade as phage LUZ7.

Regulatory T cells (Tregs) regulate immune responses and maintain host immune homeostasis. Tregs contribute to the disease progression of several chronic infections by oversuppressing immune responses via the secretion of immunosuppressive cytokines, such as transforming growth factor (TGF)-β and interleukin-10. In the present study, we examined the association of Tregs with Mycoplasma bovis infection, in which immunosuppression is frequently observed. Compared with uninfected cattle, the percentage of Tregs, CD4+CD25highFoxp3+ T cells, was increased in M. bovis-infected cattle. Additionally, the plasma of M. bovis-infected cattle contained the high concentrations of TGF-β1, and M. bovis infection induced TGF-β1 production from bovine immune cells in in vitro cultures. Finally, we analyzed the immunosuppressive effects of TGF-β1 on bovine immune cells. Treatment with TGF-β1 significantly decreased the expression of CD69, an activation marker, in T cells, and Th1 cytokine production in vitro. These results suggest that the increase in Tregs and TGF-β1 secretion could be one of the immunosuppressive mechanisms and that lead to increased susceptibility to other infections in terms of exacerbation of disease during M. bovis infection.

Purpose Umbilical cord blood-derived platelet-rich plasma (UCB-PRP) containing growth factors has attracted attention as a biomaterial useful for regenerative medicine. The osteoblastic differentiation of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) can be induced by UCB-PRP. Materials and methods Nine samples of UC and UCB were used to conduct an in vitro study that determined the contents of three growth factors (i.e., platelet-derived growth factor, transforming growth factor β-1, and vascular endothelial growth factor) and that examined, by staining with Alizarin red, their ability to induce the osteoblastic differentiation of UC-MSCs at the baseline, 3 months, and 3 years of cryopreservation. Results The contents of growth factors in cryopreserved UCB-PRP were markedly elevated compared to those found in UCB at baseline. The samples of UCB that were added with cryopreserved UCB-PRP and those with bone morphogenetic protein-2 were stained granularly with Alizarin red, thus indicating the presence of calcium. The samples of UCB that were not added with UCB-PRP were not stained with Alizarin red. The above-mentioned contents and ability were maintained at 3 years of cryopreservation. Cryopreserved UCB-PRP possibly and advantageously induced the osteoblastic differentiation of UC-MSCs. Conclusion The potential clinical application of cryopreserved UCB-PRP to regenerative medicine was suggested.

The nematode Anisakis simplex is a representative parasite infecting marine animals. When third stage larvae of A. simplex infecting fish and squids are ingested by humans, individuals previously sensitized by this parasite may experience IgE-mediated allergic reactions. So far, as many as 13 kinds of proteins (Ani s 1-13) have been identified as A. simplex allergens but several more unknown allergens are suggested to exist. In this study, therefore, chemiluminescent immunoscreening of an expression cDNA library constructed from the third stage larvae was conducted to identify a new allergen. As a result, an IgE-positive clone coding for a 23.5 kDa protein (named Ani s 14) composed of 217 amino acid residues was isolated. The regions 4-147 and 34-123 of Ani s 14 share 31% identity with the region 796-940 of Ani s 7 and 32% identity with the region 2-91 of Ani s 12, respectively. Recombinant Ani s 14 was successfully expressed in Escherichia coli as a His-tagged protein and shown to be IgE reactive to 14 (54%) of 26 sera from Anisakis-allergic patients. In conclusion, Ani s 14 is a new major allergen of A. simplex that is specific to Anisakis-allergic patients.

Survival analysis is applied when the time until the occurrence of an event is of interest. Such data are routinely collected in plant diseases, although applications of the method are uncommon. The objective of this study was to use two studies on post-harvest diseases of peaches, considering two harvests together and the existence of random effect shared by fruits of a same tree, in order to describe the main techniques in survival analysis. The nonparametric Kaplan-Meier method, the log-rank test and the semi-parametric Cox's proportional hazards model were used to estimate the effect of cultivars and the number of days after full bloom on the survival to the brown rot symptom and the instantaneous risk of expressing it in two consecutive harvests. The joint analysis with baseline effect, varying between harvests, and the confirmation of the tree effect as a grouping factor with random effect were appropriate to interpret the phenomenon (disease) evaluated and can be important tools to replace or complement the conventional analysis, respecting the nature of the variable and the phenomenon. A análise de sobrevivência é aplicada quando o tempo até a ocorrência de um evento for o objeto de interesse. Em doenças de plantas, dados dessa natureza são rotineiramente coletados, embora aplicações do método sejam pouco comuns. O objetivo deste trabalho foi utilizar dois estudos de doenças em pós-colheita de pêssegos, considerando-se safras conjuntamente e a existência de efeito aleatório, compartilhado por frutos de uma mesma árvore, para descrever as principais técnicas em análise de sobrevivência. Aplicaram-se a técnica não paramétrica de Kaplan-Meier e a estatística log-rank, além do modelo semiparamétrico de riscos proporcionais, de Cox, para estimar o efeito de cultivares e do número de dias após a floração plena sobre a sobrevivência ao sintoma de podridão parda e sobre o risco instantâneo de expressá-lo, em duas safras consecutivas. A análise conjunta com efeito basal, variando entre safras, e a verificação do efeito de árvore como fator de agrupamento com efeito aleatório, mostraram-se adequadas para interpretar o fenômeno avaliado (doença) e podem ser ferramentas importantes para substituir ou complementar as análises convencionais, respeitando-se as naturezas da variável e do fenômeno.

Background The identification of low-level antibodies by single-antigen bead methodology has brought advancements to risk evaluation of kidney transplant recipients. However, the use of mean fluorescence intensity (MFI) to quantify antibodies and to guide therapy is not enough. Notably, immunoglobulin G (IgG) subclass switching is hypothesized to follow a programmed sequence after an emergency signal from the germinal center. In transplantation this process is not clear yet. In the present study, we sequentially evaluate anti-HLA donor specific antibody (DSA) subclasses, their profile changes, and C1q-binding ability and the influence of those characteristics on antibody mediated rejection (AMR) occurrence and allograft function. Methods A total of 30 DSA-positive patients were tested for IgG subclass content and C1q-binding in sequential serum samples. Results Twenty-one patients were DSA-positive before transplant; patients sensitized only by transfusion or pregnancies had IgG1 and/or IgG3, and patients sensitized by both transfusion and pregnancies or previous transplant showed a broader range of IgG subclasses. C1q binding was detected in high MFI made up of IgG1 or multiple IgG subclasses. Only 4 patients were positive for C1q posttransplantation and 3 of these showed an increase in MFI, changes in subclasses patterns, AMR, and allograft dysfunction. Conclusions Posttransplant evaluation of DSA subclasses and the ability to bind C1q may be informative for both AMR occurrence and allograft dysfunction. Monitoring these events may help to better define risk and interventional time points.

The group of phages belonging to the family Podoviridae, genus P68virus, including Staphylococcus viruses S13′ and S24-1, are important because of their benefits in phage therapy against Staphylococcus aureus infections. The O-glycosidic linkage patterns of wall teichoic acids (WTAs) in S. aureus cell walls seem to be important for adsorption of this phage group. In this study, the adsorption of Staphylococcus viruses S13′ and S24-1 to S. aureus was examined using strains with modified WTA glycosidic linkage patterns. We found that the β-O-N-acetylglucosamine of WTAs was essential for S13′ adsorption, while N-acetylglucosamine, regardless of the α- and β-O-glycosidic linkages of the WTAs, was essential for S24-1 adsorption. Next, examining the binding activities of their receptor-binding proteins (RBPs) to cell walls with different WTA glycosidic patterns, the β-O-N-acetylglucosamine of the WTAs was essential for S13′ RBP binding, while N-acetylglucosamine, regardless of the α- and β-O-glycosidic linkages of the WTAs, was essential for S24-1 RBP binding. Therefore, the results of the RBP binding assays were consistent with those of the phage adsorption assays. Bioinformatic analysis suggested that the RBPs of Staphylococcus viruses S13′ and S24-1 were structurally similar to the RBPs of phage phi11 of thefamily Siphoviridae. Phylogenetic analysis of the RBPs indicated that two phylogenetic subclusters in the family Podoviridae were related to the glycosidic linkage patterns required for phage adsorption, possibly mediated by RBPs. We hope that this study will encourage the future development of therapeutic phages.

Japanese cedar (Cryptomeria japonica; CJ) pollinosis is a type I allergy induced by CJ pollen, and Cry j 2 is one of the major allergens in this pollen. In a previous study, we analyzed IgE epitopes on Cry j 2 in humans by using synthetic peptides. The main purpose of this study was to identify B-cell epitopes on Cry j 2 in patients with CJ pollinosis by using monoclonal antibodies (mAbs) for Cry j 2. We used ELISA with mAbs for the epitope analysis. Sera samples were collected from 80 patients with CJ pollinosis, and allergenic epitopes for mAbs and human IgE were identified using ELISA with synthetic peptides. The importance of the epitopes for human IgE was analyzed using an inhibition ELISA. Four independent epitopes (epitope #1, #2, #3, and #4) were identified on Cry j 2 with the use of mAbs. Epitope #3 and #4, corresponding to peptides No. 25 and No. 33, respectively, were newly determined as epitopes for mAbs and human IgE. Inhibition ELISA showed that not only epitope #2 (sequential) but epitope #1 (conformational) may play an important role in the CJ pollinosis. Our results revealed 4 epitopes, including two new ones, on Cry j 2. We also found that inhibition ELISA with appropriate mAbs could be a viable method of evaluating the importance of the conformational and sequential epitopes for human IgE. These results are beneficial for the development of safer and more efficient therapeutic strategies for treating CJ pollinosis.

Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060 , orf065 , and orf086 . The information obtained in this study should be useful for further development of safe and efficient phage therapy. IMPORTANCE Pseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed.

Objective and methods Dysferlin encoded by DYSF deficiency leads to two main phenotypes, limb girdle muscular dystrophy (LGMD) 2B and Miyoshi myopathy. To reveal in detail the mutational and clinical features of LGMD2B in Japan, we observed 40 Japanese patients in 36 families with LGMD2B in whom dysferlin mutations were confirmed. Results and conclusions Three mutations (c.1566C>G, c.2997G>T and c.4497delT) were relatively more prevalent. The c.2997G>T mutation was associated with late onset, proximal dominant forms of dysferlinopathy, a high probability that muscle weakness started in an upper limb and lower serum creatine kinase (CK) levels. The clinical features of LGMD2B are as follows: (1) onset in the late teens or early adulthood, except patients homozygous for the c.2997G>T mutation; (2) lower limb weakness at onset; (3) distal change of lower limbs on muscle CT at an early stage; (4) impairment of lumbar erector spinal muscles on muscle CT at an early stage; (5) predominant involvement of proximal upper limbs; (6) preservation of function of the hands at late stage; (7) preservation of strength in neck muscles at late stage; (8) lack of facial weakness or dysphagia; (9) avoidance of scoliosis; (10) hyper-Ckaemia; (11) preservation of cardiac function; and (12) a tendency for respiratory function to decline with disease duration. It is important that the late onset phenotype is found with prevalent mutations.

The present study investigated IgE reactivity to a new Cryptomeria japonica pollen allergen (Cry j 3) in dogs with atopic dermatitis by using a fluorometric ELISA. Serum samples from 15 dogs that showed IgE sensitivity to crude C. japonica pollen allergen by ELISA were tested for specific IgE to each allergen, individually. All 15 dogs had anti-Cry j 1 IgE, 6 (40%) had anti-Cry j 2 IgE, and 11 (73%) had anti-Cry j 3 IgE. Further, we found that these anti-Cry j 3 IgE reacted to Cry j 3 with immunoblotting analysis. These findings indicate that Cry j 3 may be a major allergen in dogs.

Anisakis simplex is a representative nematode parasitizing marine organisms, such as fish and squids, and causes not only anisakiasis but also IgE-mediated allergy. Although 10 kinds of proteins have so far been identified as A. simplex allergens, many unknown allergens are considered to still exist. In this study, a chemiluminescent immunoscreening method with higher sensitivity than the conventional method was developed and used to isolate IgE-positive clones from an expression cDNA library of A. simplex. As a result, three kinds of proteins, Ani s 11 (307 amino acid residues), Ani s 11-like protein (160 residues) and Ani s 12 (295 residues), together with three known allergens (Ani s 5, 6 and 9), were found to be IgE reactive. Furthermore, ELISA data showed that both recombinant Ani s 11 and 12 expressed in Escherichia coli are recognized by about half of Anisakis-allergic patients. Ani s 11 and Ani s 11-like protein are characterized by having six and five types of short repetitive sequences (5-16 amino acid residues), respectively. Both proteins share as high as 78% sequence identity with each other and also about 45% identity with Ani s 10, which includes two types of short repetitive sequences. On the other hand, Ani s 12 is also structurally unique in that it has five tandem repeats of a CX(13-25)CX(9)CX(7,8)CX(6) sequence, similar to Ani s 7 having 19 repeats of a CX(17-25)CX(9-22)CX(8)CX(6) sequence. The repetitive structures are assumed to be involved in the IgE-binding of the three new allergens.

Intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits food intake in goldfish, unlike the orexigenic action in rodents, via the melanocortin system with suppression of neuropeptide Y (NPY) mRNA expression. We therefore investigated the neuronal relationship between MCH- and NPY-containing neurons in the goldfish brain, using a double-immunofluorescence method and confocal laser scanning microscopy. MCH- and NPY-like immunoreactivities were distributed throughout the brain. In particular, MCH-containing nerve fibers or endings lay in close apposition to NPY-containing neurons in a specific region of the hypothalamus, the nucleus posterioris periventricularis (NPPv). These observations suggest that MCH-containing neurons provide direct input to NPY-containing neurons in the NPPv of goldfish, and that MCH plays a crucial role in the regulation of feeding behavior as an anorexigenic neuropeptide, inhibiting the orexigenic activity of NPY.

Intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits food intake in goldfish, unlike in rodents, suggesting that its anorexigenic action is mediated by alpha-melanocyte-stimulating hormone (alpha-MSH) but not corticotropin-releasing hormone. This led us to investigate whether MCH-containing neurons in the goldfish brain have direct inputs to alpha-MSH-containing neurons, using a confocal laser scanning microscope, and to examine whether the anorexigenic action of MCH is also mediated by other anorexigenic neuropeptides, such as cholecystokinin (CCK) and pituitary adenylate cyclase-activating polypeptide (PACAP), using their receptor antagonists. MCH- and alpha-MSH-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. MCH-containing nerve fibers or endings lay in close apposition to alpha-MSH-containing neurons in the hypothalamus in the posterior part of the nucleus lateralis tuberis (NLTp). The inhibitory effect of ICV-injected MCH on food intake was not affected by treatment with a CCK A/CCK B receptor antagonist, proglumide, or a PACAP receptor (PAC(1) receptor) antagonist, PACAP((6-38)). ICV administration of MCH at a dose sufficient to inhibit food consumption also did not influence expression of the mRNAs encoding CCK and PACAP. These results strongly suggest that MCH-containing neurons provide direct input to alpha-MSH-containing neurons in the NLTp of goldfish, and that MCH plays a crucial role in the regulation of feeding behavior as an anorexigenic neuropeptide via the alpha-MSH (melanocortin 4 receptor)-signaling pathway. (c) 2008 Elsevier Inc. All rights reserved.

In this study, we investigated the hepatic uptake clearance (CL uptake ) of tetrodotoxin (TTX) in the marine puffer fish Takifugu rubripes by integration plot analysis after a single bolus injection of 0.25 mg TTX/kg body weight into the hepatic vein at 20 °C. The blood concentration of TTX decreased over time after the injection, from 1451 ± 45 ng/mL at 10 min to 364 ± 59 ng/mL at 60 min. TTX concentrations in the spleen and kidney decreased in parallel with the blood concentrations, whereas those in the muscle and skin remained almost the same throughout the experiment. In contrast, the TTX concentration in the liver gradually increased, reaching 1240 ± 90 ng/g liver at 60 min after injection. The amount of TTX that had accumulated in the liver 60 min after injection accounted for 63 ± 5% of the administered dose. Integration plot analysis indicated a CL uptake of 3.1 mL/min/kg body weight in the liver for TTX, a rate far below that of the hepatic portal vein blood flow rate (at most, 9%). This finding is consistent with negligible extraction of TTX by the liver. The results demonstrated conclusively that the liver-specific distribution of TTX in T. rubripes is achieved by removal from the systemic circulation, but not by the hepatic first-pass effect.

The intestinal H+/peptide cotransporter 1 (PEPT1) plays important roles as a nutrient and drug transporter. Previously, we reported that rat intestinal PEPT1 showed a diurnal rhythm and that this rhythm is closely related to the feeding schedule. Furthermore, we also demonstrated that transcription factors, Sp1, Cdx2, and peroxisome proliferator-activated receptor-α (PPAR-α) contribute to the basal, intestine-specific, and fasting-induced expression of PEPT1, respectively. In this study, to clarify the molecular mechanism governing the diurnal rhythm of PEPT1 expression, we compared expression profiles of these transcription factors under two kinds of feeding schedules. The intestinal Sp1 and Cdx2 did not show a circadian accumulation of mRNA or response to the daytime feeding regimen. Plasma free fatty acids, endogenous PPAR-α ligands, exhibited a robust circadian fluctuation in phase with that of PEPT1. However, subsequent experiments using PPAR-α-null mice revealed the absence of any association between the circadian rhythm of PEPT1 and PPAR-α. We then focused on the clock genes (Clock, Bmal1, Per1–2, and Cry1) and clock-controlled gene, albumin D site-binding protein (DBP). A robust and coordinated circadian expression of the clock genes was observed, and daytime feeding entirely inverted the phase except for Clock. The expression of DBP was in phase with that of PEPT1 in both groups. Electrophoretic mobility shift assays and reporter assays revealed that DBP has the ability to bind the DBP binding site located in the distal promoter region of the rat PEPT1 gene and induce the transcriptional activity. These findings indicate that DBP plays pivotal roles in the circadian oscillation of PEPT1.

Gonadotropin-releasing hormone (GnRH) is an evolutionarily conserved neuropeptide with 10 amino acid residues, which possesses some structural variants. A molecular form known as chicken GnRH II ([His(5) Trp(7) Tyr(8)] GnRH, cGnRH II) is widely distributed in vertebrates, and has recently been implicated in the regulation of sexual behavior and food intake in an insectivore, the musk shrew. However, the influence of cGnRH II on feeding behavior has not yet been studied in model animals such as rodents and teleost fish. In this study, therefore, we investigated the role of cGnRH II in the regulation of feeding behavior in the goldfish, and examined its involvement in food intake after intracerebroventricular (ICV) administration. ICV-injected cGnRH II at graded doses, from 0.1 to 10 pmol/g body weight (BW), induced a decrease of food consumption in a dose-dependent manner during 60 min after treatment. Cumulative food intake was significantly decreased by ICV injection of cGnRH II at doses of 1 and 10 pmol/g BW during the 60-min post-treatment observation period. ICV injection of salmon GnRH ([Trp(7) Leu(8)] GnRH, sGnRH) at doses of 0.1-10 pmol/g BW did not affect food intake. The anorexigenic action of cGnRH II was completely blocked by treatment with the GnRH type I receptor antagonist, Antide. However, the anorexigenic action of cGnRH II was not inhibited by treatment with the corticotropin-releasing hormone (CRH) 1/2 receptor antagonist, *-helical CRH((9-41)), and the melanocortin 4 receptor antagonist, HS024. These results suggest that, in the goldfish, cGnRH II, but not sGnRH, acts as an anorexigenic factor, as is the case in the musk shrew, and that the anorexigenic action of cGnRH II is independent of CRH- and melanocortin-signaling pathways.

Marine puffer fish accumulates tetrodotoxin (TTX) in the liver and ovary. In this study, we examined the pharmacokinetics of TTX in Takifugu rubripes by a single administration under general anesthesia at 20 degrees C for 300 min. The blood concentration-time profile showed multiple distinct phases after injection into hepatic portal vein. The area under the blood concentration-time curve (AUC) increased linearly at the dosage of 0.25-0.75 mg TTX/kg body weight, and the total body clearance was 2.06+/-0.17 mL/min/kg body weight. The AUCs following administration into the hepatic portal vein and hepatic vein were closely similar (147+/-33 versus 141+/-1 ng.min/microL), indicating negligible hepatic first-pass effect. Comparison of the AUCs following an administration to the hepatic vein and gastrointestinal tract (0.25 mg TTX/kg body weight) elucidated the bioavailability of TTX to be 62%. There was no significant increase in the AUCs following direct injection into the gastrointestinal tract (0.50 versus 1.0 mg TTX/kg body weight). At the dosage of 0.25 mg TTX/kg body weight into the hepatic vein, hepatic portal vein or gastrointestinal tract, TTX amount in the liver accounted for 84+/-6%, 70+/-9% or 49+/-17% of the total TTX amount applied, respectively. These results demonstrate that TTX is absorbed into the systemic circulation from the gastrointestinal tract by saturable mechanism and finally accumulated in the liver within 300 min.

L-amino acid oxidase (LAO) shows broadly antibacterial activity against Gram-positive and Gram-negative bacteria by H(2)O(2) generated in the oxidative process of L-amino acids. However, LAO (termed SSAP) isolated from the rockfish Sebastes schlegelii skin mucus acted selectively on Gram-negative bacteria. Therefore, this study was undertaken to clarify the antibacterial action of SSAP as compared with H(2)O(2). SSAP inhibited potently the growth of Aeromonas salmonicida, Photobacterium damselae subsp. piscicida and Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 0.078, 0.16 and 0.63 microg/mL, respectively. H(2)O(2) inhibited the growth of both Gram-positive and Gram-negative bacteria with an MIC ranging from 0.31 to 2.5 mM. When SSAP was incubated with P. damselae subsp. piscicida and Escherichia coli, SSAP was demonstrated to bind to P. damselae subsp. piscicida but not to E. coli by Western blotting and LAO activity measurement. These results show that the bacteria binding activity may be involved in the bacterial cell selectivity of SSAP. Electron microscopic observation of A. salmonicida, P. damselae subsp. piscicida and V. parahaemolyticus revealed that the treatments with SSAP and H(2)O(2) induced cell surface damage to A. salmonicida, remarkable elongation of P. damselae subsp. piscicida bodies and pores into V. parahaemolyticus cells.

Background: Tropomyosin and arginine kinase have been identified as crustacean allergens. During purification of arginine kinase from black tiger shrimp Penaeus monodon, we found a new allergen of 20-kDa. Methods: A 20-kDa allergen was purified from the abdominal muscle of black tiger shrimp by salting-out, anion-exchange HPLC and reverse-phase HPLC. Following digestion of the 20-kDa allergen with lysyl endopeptidase, peptide fragments were isolated by reverse-phase HPLC, and 2 of them were sequenced. The 20-kDa allergen, together with tropomyosin and arginine kinase purified from black tiger shrimp, was evaluated for IgE reactivity by ELISA. Five species of crustaceans (kuruma shrimp, American lobster, pink shrimp, king crab and snow crab) were surveyed for the 20-kDa allergen by immunoblotting. Results: The 20-kDa allergen was purified from black tiger shrimp and identified as a sarcoplasmic calcium-binding protein (SCP) based on the determined amino acid sequences of 2 enzymatic fragments. Of 16 sera from crustacean-allergic patients, 8 and 13 reacted to SCP and tropomyosin, respectively; the reactivity to arginine kinase was weakly recognized with 10 sera. In immunoblotting, an IgE-reactive 20-kDa protein was also detected in kuruma shrimp, American lobster and pink shrimp but not in 2 species of crab. Preadsorption of the sera with black tiger shrimp SCP abolished the IgE reactivity of the 20-kDa protein, suggesting the 20-kDa protein to be an SCP. Conclusions: SCP is a new crustacean allergen, and distribution of IgE-reactive SCP is probably limited to shrimp and crayfish.

The nematode Anisakis simplex is a representative parasite for marine animals and occasionally causes not only anisakiasis but also allergic reactions in sensitized subjects. Besides the known allergens, a number of unidentified allergens have been suggested to still exist in A. simplex. In this study, a new heat-stable allergen of 15kDa (named Ani s 8) was purified from the third stage larvae of A. simplex by gel filtration on Sephacryl S-300, anion-exchange HPLC on Mono Q and reverse-phase HPLC on TSKgel Phenyl-5PW RP. Analysis by fluorescence ELISA showed that 7 of 28 Anisakis-allergic patients had elevated serum levels of IgE to Ani s 8. On the basis of the determined partial amino acid sequence, the complete sequence of Ani s 8 (composed of 150 amino acid residues) was elucidated by cDNA cloning, in which as many as 32 homologs of the cDNA encoding 10 isoforms of Ani s 8 were detected. Ani s 8 shares amino acid sequence homology (up to 36%) with several members of the SXP/RAL-2 protein family, including Ani s 5 (15kDa) previously identified as an A. simplex allergen. Inhibition ELISA data demonstrated the IgE cross-reactivity between Ani s 8 and Ani s 5.

Intracerebroventricular (ICV) injection of melanin-concentrating hormone (MCH) influences feeding behavior in the goldfish and exerts an anorexigenic action in goldfish brain, unlike its orexigenic action in mammals. Despite a growing body of knowledge concerning MCH function in mammals, the role of MCH in appetite has not yet been well studied in fish. The aim of the present study was to investigate the involvement of endogenous MCH in the feeding behavior of the goldfish. We examined the distribution of MCH-like immunoreactivity (MCH-LI) in the goldfish brain and the effect of feeding status upon this distribution. Neuronal cell bodies containing MCH-LI were localized specifically to four areas of the hypothalamus. Nerve fibers with MCH-LI were found mainly in the neurohypophysis, with a few in the telencephalon, mesencephalon, and diencephalon. The number of neuronal cell bodies containing MCH-LI in the dorsal area adjoining the lateral recess of the third ventricle in the posterior and inferior lobes of the hypothalamus showed a significant decrease in fasted fish compared with that in normally fed fish, although other areas showed no evident differences. We also administered an antiserum against fish MCH (anti-MCH serum) by ICV injection and examined its immuno-neutralizing effect on food intake by using an automatic monitoring system. Cumulative food intake was significantly increased by ICV injection of the anti-MCH serum. These results indicate that MCH potentially functions as an anorexigenic neuropeptide in the goldfish brain, and that the further study of the evolutionary background of the MCH system and its role in appetite is warranted.

Background Cry j 2 and Cha o 2 are major allergens in Japanese cedar ( Cryptomeria japonica ; CJ) and Japanese cypress ( Chamaecyparis obtusa ; CO) pollen, respectively. Here, we assessed the epitopes related to the cross-reactivity between Cry j 2 and Cha o 2 using in vitro analyses. Methods Peptides were synthesized based on Cry j 2 sequential epitopes and relevant Cha o 2 amino acid sequences. Four representative monoclonal antibodies (mAbs) against Cry j 2 were used according to their epitope recognitions. Serum samples were collected from 31 patients with CJ pollinosis. To investigate cross-reactivity between Cry j 2 and Cha o 2, ELISA and inhibition ELISA were performed with mAbs and sera from patients with CJ pollinosis. Results Two of four mAbs had reactivity to both Cry j 2 and Cha o 2. Of these two mAbs, one mAb (T27) recognized the amino acid sequence 169 KVVNGRTV 176 on Cha o 2. This is related to the core epitope 169 KWVNGREI 176 on Cry j 2, which is an important IgE epitope. In addition, we found that these correlative sequences and purified allergens showed cross-reactivity between Cry j 2 and Cha o 2 in IgE of CJ patients. Conclusions We demonstrated the importance of 169 KVVNGRTV 176 in Cha o 2 for cross-reactivity with the Cry j 2 epitope 169 KWVNGREI 176 , which plays an important role in allergenicity in CJ pollinosis. Our results are useful for the development of safer and more efficient therapeutic strategies for the treatment of CJ and CO pollen allergies.

Fish is an important causative material of food allergy. Although the allergenicity of fish is considered to correlate with the content of parvalbumin, the major fish allergen, available information about the parvalbumin content in fish is limited. In this study, a simple and reliable quantification method for fish parvalbumin by SDS-PAGE was first established. Application of the SDS-PAGE method to 22 species of fish revealed a marked variation in parvalbumin content among fish. Furthermore, the parvalbumin content was found to be higher in dorsal white muscle than in ventral white muscle, in rostral part of white muscle than in caudal part of white muscle and in white muscle than in dark muscle. IgE reactivity of fish was roughly proportional to parvalbumin content. Interestingly, large-sized migratory fish, such as salmon, swordfish and tuna, were commonly very low in both parvalbumin content and IgE reactivity.

The nematode Anisakis simplex is a marine parasite that causes allergy as well as anisakiasis. Although five Anisakis allergens have already been identified, immunoblotting studies suggested that unidentified allergens still exist. In this study, an expression cDNA library constructed from A. simplex was subjected to immunoscreening using an Anisakis-allergic patient serum, and two positive clones coding for allergens (named Ani s 5 and 6) were obtained. Ani s 5 (152 amino acid residues) is homologous with nematode proteins belonging to the SXP/RAL-2 protein family and Ani s 6 (84 amino acid residues) with serine protease inhibitors from various animals. Of the 28 patient sera examined, seven and five reacted to recombinant Ani s 5 and 6 expressed in Escherichia coli, respectively. By inhibition immunoblotting experiments using the recombinant allergens as inhibitors, natural Ani s 5 could be identified as a 15-kDa protein in the crude extract of A. simplex but natural Ani s 6 could not be identified probably due to its low expression. In conclusion, Ani s 5 and 6 are new allergens of A. simplex that are specific to some Anisakis-allergic patients.

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3′ RACE, 5′ RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H2O2-generation assay and substrate specificity for only l-Lys with a Km of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H2O2 is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.

We previously demonstrated that starvation markedly increased the amount of mRNA and protein levels of the intestinal H+/peptide cotransporter (PEPT1) in rats, leading to altered pharmacokinetics of the PEPT1 substrates. In the present study, the mechanism underlying this augmentation was investigated. We focused on peroxisome proliferator-activated receptor alpha (PPARalpha), which plays a pivotal role in the adaptive response to fasting in the liver and other tissues. In 48-h fasted rats, the expression level of PPARalpha mRNA in the small intestine markedly increased, accompanied by the elevation of serum free fatty acids, which are endogenous PPARalpha ligands. Oral administration of the synthetic PPARalpha ligand WY-14643 to fed rats increased the mRNA level of intestinal PEPT1. Furthermore, treatment of the human intestinal model, Caco-2 cells, with WY-14643 resulted in enhanced PEPT1 mRNA expression and uptake activity of glycylsarcosine. In the small intestine of PPARalpha-null mice, augmentation of PEPT1 mRNA during fasting was completely abolished. In the kidney, fasting did not induce PEPT1 expression in either PPARalpha-null or wild-type mice. Together, these results indicate that PPARalpha plays critical roles in fasting-induced intestinal PEPT1 expression. In addition to the well-established roles of PPARalpha, we propose a novel function of PPARalpha in the small intestine, that is, the regulation of nitrogen absorption through PEPT1 during fasting.

H(+)/peptide cotransporter 1 (PEPT1, SLC15A1) localized at the brush-border membranes of intestinal epithelial cells plays important roles in the intestinal absorption of small peptides and a variety of peptidemimetic drugs. We previously demonstrated that transcription factor Sp1 functions as a basal transcriptional regulator of human PEPT1. However, the factor responsible for the intestine-specific expression of PEPT1 remains unknown. In the present study, we investigated the effect of the intestinal transcription factors on the transcription of the PEPT1 gene and found that only Cdx2 markedly trans-activated the PEPT1 promoter. However, the promoter region responsible for this effect lacked a typical Cdx2-binding sequence, but instead, possessed some Sp1-binding sites. In vitro experiments using Caco-2 cells showed that (1) mutation of the Sp1-binding site diminished the effect of Cdx2, (2) co-expression of Cdx2 and Sp1 synergistically trans-activated the PEPT1 promoter and (3) Sp1 protein was immunoprecipitated with Cdx2 protein. These results raise the possibility that Cdx2 modulates the PEPT1 promoter by interaction with Sp1. The significance of Cdx2 in vivo for PEPT1 regulation was shown by the determination of mRNA levels of Cdx2 and PEPT1 in human tissue. In gastric samples, some with intestinal metaplasia, the levels of PEPT1 and Cdx2 mRNA were highly correlated. Taken together, the present study suggests that Cdx2 plays a key role in the transcriptional regulation of the intestine-specific expression of PEPT1, possibly through interaction with Sp1.

In vitro accumulation of tetrodotoxin (TTX) and paralytic shellfish toxin (PST) in tiger puffer fish Takifugu rubripes was investigated using liver tissue slices. When T. rubripes liver slices were incubated with Leibovitz’s L-15 medium containing 0.13 mM TTX at 20 °C in air with saturated humidity, they accumulated 21.5 ± 7.3 μg TTX g−1 liver after the incubation for 12 h and increased to 55.3 ± 8.2 μg TTX g−1 liver at 48 h. In the incubation of T. rubripes liver slices with 0.13 mM PST-containing medium, PST was detected 6.3 ± 0.9 μg g−1 liver at 12 h and reached a plateau thereafter. These results reveal the difference between TTX and PST in accumulation in T. rubripes liver tissue slices. To examine the variation in PST accumulation among fish species, the liver tissue slices from tiger puffer fish T. rubripes, parrot-bass Oplegnathus fasciatus and green ling Hexagrammos otakii were incubated at a concentration of 0.027 mM PST. The toxin contents of 3.0 μg g−1 liver were observed at 8 h regardless of fish species but were not increased subsequently, showing no variety among these three species as to accumulation patterns of PST. It is noted that the tiger puffer fish T. rubripes liver specifically accumulate TTX in preference to PST.

H+-coupled peptide transporter 1 (PEPT1, SLC15A1) localized at the brush-border membranes of intestinal epithelial cells plays an important role in the intestinal absorption of small peptides and a variety of peptidemimetic drugs. PEPT1 is regulated by various factors, including hormones, dietary conditions, some pharmaceutics, and diurnal rhythm. But there is little information about the transcriptional regulation of PEPT1. In the present study, therefore, we cloned the human (h)PEPT1 promoter region and examined its promoter activity using a human intestinal cell line, Caco-2. Deletion analysis of the hPEPT1 promoter suggested that the region spanning −172 to −35 bp was essential for basal transcriptional activity. This region lacked a TATA-box but contained some GC-rich sites that supposedly bind with the transcription factor Sp1. Mutational analysis revealed that three of these putative Sp1 sites contributed to the transcriptional activity. EMSA showed that Sp1 bound to two GC-rich sites. Furthermore, inhibition of Sp1 binding by mithramycin A treatment significantly reduced the transcriptional activity. Finally, overexpression of Sp1 increased the transcriptional activity in a dose-dependent manner. This study reports the first characterization of the hPEPT1 promoter and shows the significant role of Sp1 in the basal transcriptional regulation of hPEPT1.

This study was undertaken to evaluate and reduce the allergenicity of crustacean extractives that are added to a variety of processed foods as seasonings. Tropomyosin, the major crustacean allergen, was detected in only one of the six kinds of crustacean extractives by SDS–PAGE and immunoblotting. However, all the extractives were shown to be considerably allergenic by inhibition ELISA using sera from crustacean-allergic patients. Analyses by gel filtration HPLC suggested that tropomyosin is mostly degraded to peptide fragments during manufacturing of the extractives but at least some of the fragments are still IgE-reactive. On digestion with proteases, the allergenicity of the extractives was almost completely lost. The effectiveness of protease digestion to reduce the allergenicity of tropomyosin was also confirmed in model experiments using the heated extracts from four species of crustaceans.

The effect of cattle breed on stearoyl-CoA desaturase (SCD) gene expression was investigated in this study. Detailed comparisons of SCD mRNA level were made among three steers each of Japanese Black, Holstein and their crossbreed which were age-matched had been fed the same diet and were sampled by biopsy of the longissimus dorsi (LD) muscle and subcutaneous fat. The levels of SCD mRNA were measured in samples of muscle and subcutaneous fat. The levels of SCD mRNA demonstrated a breed effect in each tissue, though the relative expression was higher (P

An antibacterial protein in the skin secretion of rockfish (Sebastes schlegeli) was purified by lectin affinity chromatography on Con A-Sepharose and gel filtration on TSKgel G3000SW. The antibacterial protein featured the high molecular mass and selective action against Gram-negative bacteria. The molecular mass of the protein was estimated to be approximately 150 kDa in gel filtration and approximately 75 kDa by SDS-PAGE, suggesting that it is dimeric. The antibacterial principle was an acidic glycoprotein with pI 4.5, 3.4% reducing sugar and 2.8% amino sugar. Its sugar chains had N-type (high mannose-type) oligosaccharide and sialic acid components. It inhibited strongly the growth of Aeromonas salmonicida, Photobacterium damselae and Shewanella putrefaciens with a minimum inhibitory concentration (MIC) of approximately 3 microg/ml, and moderately the growth of Vibrio parahaemolyticus and A. hydrophila with a MIC of 12.5 microg/ml and 25 microg/ml, respectively. The values of the minimum bactericidal concentration were almost equivalent to those of MIC. The potent sensitivity against virulent pathogens such as A. hydrophila, A. salmonicida and P. damselae may contribute considerably to the innate host defense mechanism to combat microbes on the mucosal surfaces of the rockfish.

In this article we combine ideas from spatial statistics with lifetime data analysis techniques to investigate possible spatial variation in survival of adult acute myeloid leukemia patients in northwest England. Exploratory analysis suggests both clinically and statistically significant variation in survival rates across the region. A multivariate gamma frailty model incorporating spatial dependence is proposed and applied, with results confirming the dependence of hazard on location.

The ability of CD34+ leukemic cells to differentiate to dendritic cells (DCs) was investigated in 18 acute myeloid leukemia (AML) and 4 lymphoid leukemia (ALL) patients. The generation of DCs was determined by the expression of DC-associated CD1a or CD83 (more than 30%) with costimulatory molecules, by CD80 antigens (>20%), and by the exhibition of allostimulatory activity. In the AML patients, allostimulatory mature DCs were generated from 3 of 9 M0 or M1, 2 of 5 M2,2 of 4 M4 or M5, and 3 of 4 ALL (L2) cases. In total, DCs were more efficiently induced from cases expressing over 75% of CD34+ among whole bone marrow mononuclear cells (8 of 12), compared with those under 75% (2 of 10; P < .05). B-cell (CD19), natural killer (NK)-cell (CD56), or T-cell (CD7) lineage markers, which were aberrantly expressed on the blasts, were rarely found on leukemic DCs at the end of the culture period, and myeloid (CD13, CD33), not lymphoid (CD10), markers were shown on ALL-derived DCs. In Philadelphia chromosome-positive ALL or AML patients with t (8;21), DCs were confirmed to be of leukemic origin by fluorescence in situ hybridization analysis.

Background & Aims : Differentiation-inducing factor-1 (DIF-1) is a small lipophilic substance that induces stalk cell differentiation in the cellular mold Dictyostelium discoideum. It has been shown that DIF-1 has an anti-tumor activity in mammalian cells. In the present study, we examined the effects of DIF-1 on the growth of some bacteria, fungi, and an influenza virus.Method : We used in vitro systems for the assay for growth of microorganisms.Results : DIF-1 did not show marked effect on the bacteria and fungi tested. Interestingly, DIF-1 inhibited viral plaque formation in its host Madin-Darby canine kidney (MDCK) cells in a dose dependent manner and IC50 was 18μM. DIF-1 also inhibited the growth of MDCK cells and IC50 was 30μM.Conclusions : These results indicate that DIF-1 or its analogs may be utilized as a tool for the analysis of virus functions.