Evaluation of a receptor gene responsible for maternal blood IgY transfer into egg yolks using bursectomized IgY-depleted chickens.

In avian species, maternal immunoglobulin Y (IgY) is transferred from the blood to the yolks of maturing oocytes; however, the mechanism underlying this transfer is unknown. To gain insight into the mechanisms of maternal IgY transfer into egg yolks, IgY-depleted chickens were generated by removing the bursa of Fabricius (bursectomy) during egg incubation, and their egg production and IgY transport ability into egg yolks were determined. After hatching, blood IgY concentrations of the bursectomized chickens decreased gradually until sexual maturity, whereas those of IgA remained low from an early stage of growth (from at least 2 wk of age). Chickens identified as depleted in IgY through screening of blood IgY and IgA concentrations were raised to sexual maturity. At 20 wk of age, both blood and egg yolk IgY concentrations in the IgY-depleted group were 600-fold lower than those of the control group, whereas egg production did not differ between the groups. Intravenously injected, digoxigenin-labeled IgY uptake into the egg yolk was approximately 2-fold higher in the IgY-depleted chickens than in the controls, suggesting that IgY depletion may enhance IgY uptake in maturing oocytes. DNA microarray analysis of the germinal disc, including the oocyte nucleus, revealed that the expression levels of 73 genes were upregulated more than 1.5-fold in the IgY-depleted group, although we could not identify a convincing candidate gene for the IgY receptor. In conclusion, we successfully raised IgY-depleted chickens presenting a marked reduction in egg yolk IgY. The enhanced uptake of injected IgY into the egg yolks of the IgY-depleted chickens supports the existence of a selective IgY transport mechanism in maturing oocytes and ovarian follicles in avian species.

Ingestion of paddy rice increases intestinal mucin secretion and goblet cell number and prevents dextran sodium sulfate-induced intestinal barrier defect in chickens.

Paddy rice is a potential feed grain for chickens, whose strong gizzards can crush the hull. Here, we investigated whether paddy rice rich in hull-derived water-insoluble dietary fiber stimulates intestinal mucin secretion and production, as well as the possible involvement of paddy rice in intestinal barrier function. Layer male chicks at 7 d of age were divided into four groups according to the diet: corn, polished rice, brown rice, or paddy rice (650 g/kg diet), which they ate for 14 consecutive days. At 21 d of age, the birds were refed their experimental diets, and small intestinal mucin fractions were collected to determine intestinal mucin content. Small intestinal mucin secretion was induced most strongly in the paddy rice group (Experiment 1). The rank order of diet-induced mucin secretion was paddy rice > corn = brown rice > polished rice. Ileal MUC2 gene expression and ileal number of goblet cells were highest in the paddy rice group (Experiment 1). A study of bromodeoxy-U uptake into ileal epithelial cells indicated the increase in goblet cells in the paddy rice group was related to accelerate epithelial cell migration (Experiment 2). A single supplementation of isolated rice hulls without kernels increased MUC2 gene expression and goblet cell numbers (Experiment 3), suggesting the importance of the hull's bulk-forming capacity on mucin production. Finally, chicks fed corn or paddy rice were orally administered dextran sodium sulfate (DSS) to disrupt intestinal barrier function. In the DSS-treated birds, the intestinal permeability of fluorescein isothiocyanate dextran in the everted gut sacs was much lower in the paddy rice group than in the corn group (Experiment 4), showing that paddy rice protects against mucosal disruption. In conclusion, ingestion of paddy rice increases intestinal mucin secretion and production through enhanced MUC2 gene expression and epithelial turnover and prevents DSS-induced intestinal barrier defects in chickens.

Immunohistochemical analysis of the Akt/mTOR/4E-BP1 signalling pathway in canine haemangiomas and haemangiosarcomas.

The specific signalling pathways that are deregulated in canine endothelial tumours have not yet fully elucidated. Therefore, the aim of the present study was to examine activation of the Akt/mammalian target of rapamycin (mTOR)/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) signalling pathway in spontaneously arising canine haemangiomas (HAs) and haemangiosarcomas (HSAs) in order to identify novel molecular targets for treatment. Surgically-resected samples of HA (n = 27), HSA (n = 37), granulation tissue (n = 4) and normal skin (n = 4) were investigated by immunohistochemistry. Approximately 80% of the HSA samples had moderate to intense expression of phosphorylated Akt at Ser473 (p-Akt Ser473), p-Akt Thr308, p-4E-BP1 Thr37/46 and eukaryotic initiation factor 4E, which was significantly higher than in the HAs and was similar to the expression in activated endothelial cells (ECs). Although p-mTOR complex1 (p-mTORC1) Ser2448 was expressed by most of the activated ECs, only 35% of the HSA samples had weak to moderate expression. Because mTORC2 and phosphorylates Akt Ser473 was activated in HSA samples, the present findings suggest that the mTORC2/Akt/4E-BP1 pathway, regulated independently of mTORC1, may be important for targeting therapy in canine HSAs.

Pathological features of Mycobacterium kansasii infection in black bearded sakis (Chiropotes satanas).

Two black bearded sakis (Chiropotes satanas), kept in the same cage in a zoological park, developed multifocal subcutaneous nodular lesions and were diagnosed as having mycobacterial infection by microscopical examination of tissues and 16S rRNA polymerase chain reaction (PCR) and subsequent sequencing of amplicons. One animal died despite both being treated with prolonged antimicrobial therapy. This animal had disseminated disease with lesions in the liver, spleen, lymph nodes and brain. The lesions were granulomatous in nature, but organisms were not identified by acid-fast staining other than on an impression smear of one of the skin nodules. The granulomatous lesions lacked epithelioid macrophages, multinucleated giant cells and fibrous encapsulation. Mycobacterium kansasii was identified by PCR in the lymph nodes of the animal with disseminated disease. Mycobacterial speciation was not as readily achieved in the animal with cutaneous nodules only.

Expression of platelet-derived growth factor and its receptors in spontaneous canine hemangiosarcoma and cutaneous hemangioma.

Hemangiosarcoma (HSA) is a malignant neoplasia of vascular endothelial cells (ECs). Our previous report on the expression of vascular endothelial growth factor, basic fibroblast growth factor, and their receptors in canine HSA suggested an autocrine/ paracrine mechanism of tumor growth. However, the influence of other angiogenic growth factors in canine HSA was not elucidated; therefore, the expression of platelet-derived growth factor (PDGF) and its receptors was investigated by immunohistochemical analysis. Forty-six canine HSAs and 21 canine cutaneous hemangiomas (HAs) were analyzed. For immunohistochemistry, anti-PDGF-BB, anti-PDGFR-α, and anti-PDGFR-ß antibodies were utilized as primary antibodies. Immunoreactivities were scored as strongly positive (>25% positive neoplastic cells), weakly positive (1-25% positive neoplastic cells), and negative if not staining at all. In cutaneous HA, 33.3% and 57.1% of cases were strongly and weakly positive, respectively, and 43.5% and 13.0% of HSAs were strongly and weakly positive for PDGF-BB, respectively. Moreover, 38.1% and 28.6% of cutaneous HAs cases were strongly and weakly positive, respectively, and 23.9% and 4.3% of HSAs cases were strongly and weakly positive, respectively, for PDGFR-α. Thirty-five HSAs cases (76.1%) were strongly positive, and the remaining 11 (23.9%) were weakly positive for PDGFR-ß. In contrast, 18 (72.0%) cutaneous HAs were negative, and only 3 cases (12.0%) were weakly positive, for PDGFR-ß. The proportion of strongly positive cases of HSAs was significantly higher than that of cutaneous HA for PDGFR-ß (P<0.01), while PDGFR-α was highly expressed in cutaneous HA and may be related to pathogenesis of cutaneous HA. Therefore, PDGFR-ß may be associated with the malignant nature of canine HSA.

Transcatheter arterial embolisation for paediatric inflammatory pseudotumour of the maxillary sinus.

BACKGROUND: Inflammatory pseudotumours are mostly seen in the lung, and occasionally in the head and neck region including the sinonasal area. Reported treatment modalities comprise corticosteroid treatment, surgical excision and radiotherapy. The latter option is required because wide surgical resection may be difficult for head and neck lesions, especially in children. However, clinicians should be aware of the risk of late-onset side effects of radiotherapy in children. CASE REPORT: We present a two-year-old girl with a massive inflammatory pseudotumour of the maxillary sinus. Transcatheter arterial embolisation was performed, and the lesion was successfully managed without additional therapy. There was no evidence of recurrence over the next five years. CONCLUSION: This is the first report presenting the utility of arterial embolisation for inflammatory pseudotumour.

Higher incorporation of heterologous chicken immunoglobulin Y compared with homologous quail immunoglobulin Y into egg yolks of Japanese quail (Coturnix japonica).

In avian species, blood IgY is selectively incorporated into the yolks of maturing oocytes, although the precise mechanism is poorly understood. Our previous study showed that 22% of i.v.-injected heterologous chicken IgY (cIgY) was incorporated into egg yolks of Japanese quail (Coturnix japonica). However, it is not known whether homologous quail IgY (qIgY) can be more efficiently incorporated into quail egg yolks than cIgY. Therefore, we compared the uptakes of qIgY and cIgY i.v. administered into quail egg yolks and further characterized the uptakes of these 2 antibodies into quail ovarian follicles. Quail IgY and cIgY purified from the blood of the respective bird were labeled with digoxigenin, and their uptakes into quail egg yolks were determined by ELISA. Unexpectedly, total incorporation of the injected qIgY was only one-third of that of cIgY, although much more qIgY was left in blood compared with cIgY, suggesting that qIgY is the less preferable antibody as a transport ligand into quail egg yolks. On the other hand, deposition of the qIgY into heart, lung, liver, spleen, kidney, and ovarian follicular membrane was markedly higher than that of cIgY. Amino acid sequence analysis of 3 peptides derived from the trypsin-digested qIgY heavy chain revealed low homology between qIgY and cIgY. In conclusion, our results show that heterologous cIgY is more efficiently incorporated into quail egg yolks than homologous qIgY, possibly due to a distinctive antibody transport system existing in oocytes. The present results also may provide a new strategy for delivering useful proteinaceous substances into egg yolks in an attempt to produce designer eggs.

Immunohistochemical demonstration of angiogenesis-associated homeobox proteins in canine vascular tumours.

Angiogenic homeobox genes regulate the behaviour of endothelial cells (ECs) during angiogenesis, so the aim of this study was to determine whether expression of these genes may be a determinant of malignancy in canine haemangiosarcoma (HSA). Homeobox proteins were evaluated immunohistochemically in tissue samples from canine HSAs (n=78), haemangiomas (HAs; n=30) and samples of granulation tissue (n=8). Active ECs in granulation tissue were positively labelled by antisera specific for HoxA9, HoxB3, HoxD3, HoxB7, Pbx1 and Meis1. Quiescent ECs in granulation tissue did not express HoxD3 and Pbx1. There were significantly more neoplastic cells positively labelled for HoxA9, HoxB3, HoxD3 and Pbx1 in HSA compared with HA. Almost all tumours were positive for HoxB7 and Meis1. HoxB3, HoxD3, Pbx1 and Meis1 proteins were detected in 80-90% of the HSAs, but in <20% of the HAs. Overall, homeobox protein expression in HSA appears to have a phenotype similar to that of active ECs in angiogenesis. The expression of homeobox genes associated with angiogenesis might be associated with the malignant growth of HSA.

Teratoma of the umbilical cord in a giraffe (Giraffa camelopardalis reticulata).

A 12-year-old pregnant female giraffe (Giraffa camelopardalis reticulata) died approximately 2 months prior to her anticipated parturition date. At necropsy, a mass measuring approximately 20 x 36 x 20 cm was observed, attached to the umbilical cord, the latter being otherwise normal in appearance. Histologically, the mass contained 3 germinal tissue components with areas of squamous epithelium, respiratory epithelium, primitive neural tissues, glial tissue, peripheral nerve, adipose tissue, cartilage, and smooth muscle. Based on these findings, the tumor was diagnosed as a teratoma originating from the umbilical cord. This is possibly the second reported case of umbilical cord teratoma in animals.

An Aneurinibacillus sp. strain AM-1 produces a proline-specific aminopeptidase useful for collagen degradation.

AIMS: We have been for a species of thermophilic bacteria that can effectively decompose collagen and collagen peptides that tend to be hard-to-degrade proteins because of their high content of proline residues. This study focused upon the enzymatic degradation of prolyl peptides by thermophilic bacteria. METHODS AND RESULTS: A strain, AM-1, producing a proline-specific aminopeptidase was isolated using a medium containing gelatin that was taken from soil samples collected at Arima Hot Spring located near Kobe, Japan. The strain showed the strongest level of hydrolysing activity toward prolyl-p-nitroanilide, and the activity proved to be thermostable. Phylogenetic analysis based on 16S rDNA sequences revealed that the isolated strain AM-1 was closest to Aneurinibacillus thermoaerophilus DSM10154T in its characteristics. Analysis of the purified proline-specific aminopeptidase suggested that the enzyme is an aminopeptidase containing metal that includes important disulphide bond(s). The strain AM-1 aminopeptidase has more similarities with leucyl aminopeptidases, but its activity level differs greatly with prolyl peptides. CONCLUSIONS: The proline-specific aminopeptidase from strain AM-1 is the first from the genus Aneurinibacillus and may be a new type of aminopeptidase for hydrolysing prolyl peptide. This enzyme also contributed to the degradation of collagen when used in combination with another collagenolytic protease. SIGNIFICANCE AND IMPACT OF THE STUDY: The proline-specific aminopeptidase obtained from strain AM-1 may be used in the treatment of wastewater containing collagen that is encountered in the meat industries, and for decreasing bitter peptides in milk products.

[Coronary artery bypass grafting in the patients with renal dysfunction].

We studied coronary artery bypass grafting (CABG) in patients with renal dysfunction. From April 1994 to October 1999, 59 patients with renal dysfunction underwent CABG. The patients were divided into 2 groups: group A: 18 patients with end stage renal disease who were receiving hemodialysis, group B; 41 patients with creatinine levels higher than 1.5 mg/dl who were not supported by dialysis. We compared and analyzed the findings of there 2 groups. Regarding preoperative factors, the incidence of old myocardial infarctions, diabetes mellitus and old cerebral infarctions did not differ significantly between the 2 groups. Regarding perioperative factors, the incidence of the number of vessel diseases, emergency operations, operation times and blood transfusions did not differ significantly between the 2 groups. Regarding the post-operative course, the hospital mortality rates demonstrated low levels in 2 groups. The graft patency of group A was 95%, while it was 99% in group B. The post-operative in-hospital days was 24.8 days in the group A, while it was 30.1 days in the group B. No significant difference was observed between the 2 groups. As a results, post-operative hemodialysis was needed in 8 of the patients who underwent on-pump CABG from group A. The actual survival rates were 75.3% in group A and 84.3% in group B at 4 years. The cardiac event free rate for group A was 93.3%, while it was 97.5% in group B at 4 years. In conclusion, CABG may improve the post-operative outcome in renal dysfunction patients. In addition, the use of off-pump CABG is also considered to achieve a better renal function than on-pump CABG.

Improvement of growth and nutritive value in chicks with non-genetically modified phytase product from Aspergillus niger.

1. Non-genetically modified (non-GM) phytase product derived from Aspergillus niger possesses various side active enzymes including alpha-amylase, protease, cellulase and hemicellulase. In contrast, the product of genetically modified (GM) phytase product has much less side active enzyme since the capacity of phytase production is reinforced by gene modification. In the present study we have tried to determine whether the difference of side enzyme activity of phytase product affects growth performances and nutritive value in chicks; in addition we tried to characterise the physiological change induced by the difference of side active enzymes. 2. Single Comb White Leghorn male chicks at 7 d of age were fed on experimental barley-based diets for 10 d. The feeding trial was of a factorial design (3 x 2 x 2), having three types of dietary phytase products (control, non-GM or GM phytase products derived from A. niger at 1000 U/kg diet), two levels of dietary available P supplement (0 or 6 g/kg diet) and two levels of dietary protein (CP 180 or 120 g/kg). 3. The non-GM phytase product caused a 6% increase in final body weight and feed efficiency compared with the control and the GM phytase product without interacting with dietary protein and available P level. However, in birds given available P-free diet, both non-GM and GM phytase products induced a 20% increase in plasma P concentration, suggesting no difference in phytase activity between the non-GM and GM phytase products. 4. The balance study showed that the metabolisable energy of the non-GM phytase product (15.6 +/- 0.05 kJ/g diet) was significantly higher among the treatments (control, 15.1 +/- 0.05; GM phytase product 15.3 +/- 0.07). The non-GM phytase product also increased the rate of food passage through the crop, and caused a drastic reduction in intestinal weight, perhaps as a consequence of digestion of non-starch polysaccharides. 5. We conclude that the side active enzymes in non-GM phytase product improve growth performance and nutritive value of the diet in chicks. However, the efficacy of phytase activity should not be different between non-GM and GM phytase products.

Insulin administration suppresses an increase in insulin-like growth factor binding protein-2 gene expression stimulated by fasting in the chicken.

1. We examined the changes in plasma IGF-I concentration and tissue IGFBP-2 gene expression of young fasted chickens refed a commercial diet or administered bovine insulin intravenously. 2. Plasma IGF-I concentration was decreased by fasting for 2 d. Although plasma IGF-I concentration was increased by refeeding, it didn't recover to the level of chickens fed a commercial diet ad libitum. 3. Insulin administration lowered plasma IGF-I concentration compared to other groups. 4. Hepatic IGFBP-2 mRNA was increased by fasting for 2 d and decreased by refeeding for 6 h. Insulin administration also decreased hepatic IGFBP-2 gene expression stimulated by fasting to the level of refed chickens. 5. IGFBP-2 mRNA in the gizzard was increased by fasting for 2 d and tended to decrease after refeeding for 6 h. Insulin administration decreased gizzard IGFBP-2 gene expression to less than that in refed chickens. 6. There was no between-treatment difference in IGFBP-2 mRNA in the brain and kidney. 7. These results suggest that the changes in IGFBP-2 gene expression in the liver and gizzard by fasting and refeeding might be partly regulated by the alteration in plasma insulin concentration.

The vaso-contractile action of zooxanthellatoxin-B from a marine dinoflagellate is mediated via Ca2+ influx in the rabbit aorta.

We previously showed that zooxanthellatoxin-B, isolated from dinoflagellate, caused a sustained contraction of the aorta in an external Ca2+-dependent manner. To clarify the role of Ca2+ in this action, we examined the effects of zooxanthellatoxin-B as well as a depolarizing stimulus (60 mM KCl), using the simultaneous recording for cytosolic Ca2+ level (fura-2) and developed tension in the rabbit aorta. KCl (60 mM) elicited a rapid cytosolic Ca2+ elevation followed by a pronounced contraction, and time required for half-maximum contraction was 2 min. Zooxanthellatoxin-B caused an increase in cytosolic Ca2+ followed by a gradual contraction, with a time for half-maximum contraction of 5-10 min in a concentration-dependent manner. We found a strong correlation between Ca2+ elevation and the contraction in zooxanthellatoxin-B action. In a Ca2+-free solution, zooxanthellatoxin-B caused neither the contraction nor the increase in cytosolic Ca2+. Furthermore, both pre- and post-treatment with verapamil, a voltage-operated Ca2+-channel blocker, partially suppressed both an increase in cytosolic Ca2+ and the contraction by zooxanthellatoxin-B. Zooxanthellatoxin-B-induced contraction was also inhibited by other voltage-operated Ca2+-channel blockers: nifedipine or diltiazem. These results suggest that zooxanthellatoxin-B-elicited contraction is caused by a Ca2+ influx into the smooth muscle cells, partially via voltage-operated Ca2+ channels.

Off-pump CABG for a patient with a brain tumor.

Coronary artery bypass grafting (CABG) without cardiopulmonary bypass (off-pump CABG) was performed on a patient with a brain tumor. A patient with effort angina of the left anterior descending artery (LAD), and diagonal branch stenosis was referred to us for CABG. He had a mass lesion in the brain that was diagnosed as meningioma involving the internal carotid artery and a middle cerebral artery. To avoid brain complications, we performed off-pump CABG using the internal thoracic and radial arteries to the LAD and a diagonal branch. Off-pump CABG was an effective method to avoid brain complications for patients with a brain tumor involving cerebral vessels.

Factors regulating amylase secretion from chicken pancreatic acini in vitro.

In mammals, cholecystokinin regulates pancreatic exocrine secretion under physiological conditions. We have shown, however, that cholecystokinin at physiological concentrations does not induce pancreatic amylase secretion in birds. Therefore, we investigated the effects of various neurotransmitters and gut hormones on the pancreatic amylase secretory response in isolated chicken pancreatic acini. Acetylcholine (half-maximal stimulation at 800 nM) and vasoactive intestinal polypeptide (half-maximal stimulation at 40 pM) produced a concentration-dependent increase in amylase secretion at physiological concentrations. The combination of acetylcholine and vasoactive intestinal polypeptide produced an additive response in amylase secretion. Sodium nitroprusside, a spontaneous nitric oxide releaser, and bombesin, induced amylase secretion at concentrations greater than 10 nM and 100 nM, respectively. Gastrin and secretin increased amylase secretion at pharmacological concentrations (10 to 100 nM). Our findings suggest that neural regulation is important for pancreatic enzyme secretion in birds and the contribution of gut hormones seems to be physiologically unimportant.

Control of c-fos expression in STC-1 cells by peptidomimetic stimuli.

Enteroendocrine cells respond to nutrient and non-nutrient stimuli in the gut lumen. The intestinal hormone cholecystokinin (CCK) is secreted in response to luminal fatty acids, amino acids, peptides and proteins. The peptidomimetic cephalosporins have been reported to provide model, stable, compounds with similar secretagogue activity to peptide. Putative luminal stimuli also influence transcriptional activity in enteroendocrine cells, but the mechanisms are uncertain. In the present study we have investigated the control of c-fos expression in STC-1 cells (an enteroendocrine cell line). Peptidomimetics stimulated calcium-dependent release of CCK, and increased intracellular calcium, phosphorylation of p42/44 mitogen-activated protein kinase (MAP kinase) and c-fos mRNA abundance. Hypotonic stress also increased p42/44 MAP kinase phosphorylation and c-fos mRNA, but not CCK release. The increase in c-fos mRNA was strikingly potentiated by peptidomimetics in hypotonic medium. Increased c-fos expression, but not CCK release, was suppressed by the MAP kinase (MEK) inhibitor PD98059, and by the tyrosine kinase inhibitor genistein. We conclude that in STC-1 cells, peptidomimetics act through the p42/44 MAP kinase pathway to increase c-fos expression but not exocytosis. Moreover, a putative non-nutritive stimulus, hypotonic stress, may interact with this pathway to enhance c-fos expression, independently of hormone release.