The posthatch physiology of the turkey poult--II. Hematology

Packed cell volume, total leukocytes, total erythrocytes, hemoglobin, mean cell volume and mean corpuscular hemoglobin concentration of the poult were analyzed daily for the first 10 days posthatch. Sexually dimorphic effects on hematological parameters were not apparent, but there was a sex X day interaction for all parameters except hemoglobin indicating that males were slower to develop/respond to critical stimuli. The study revealed a latency in production of erythrocytes and leukocytes in posthatch males and females which coincided temporally with early poult mortality.     

Serum chemistries of Coturnix coturnix japonica given dietary manganese oxide (Mn3O4)

1. Plasma creatinine and inorganic phosphorus were increased in manganese oxide (Mn3O4)-treated adult male Coturnix quail, but BUN, BUN/creatinine ratio, uric acid, and total calcium were decreased. 2. Serum enzymes (alkaline phosphatase, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and lactic dehydrogenase) were elevated in Mn3O4-treated adult male Coturnix quail, but creatine phosphokinase was not affected. 3. Dietary Mn3O4 at 5000 ppm did not produce overt signs of toxicosis.     

Tissue monoamine and plasma corticosterone concentrations following sympathetic alteration during acute endotoxicosis in chickens.

1. Splenic and heart ventricular NE levels and plasma corticosterone concentrations were increased following E. coli endotoxin administration in three-week-old chicks. 2. Chicks receiving either reserpine or propranolol injection before endotoxin exhibited increased NE in both tissues while only splenic 5-HT increased in birds receiving propranolol and endotoxin. 3. The effects of acute bacterial infection in chickens involves the peripheral nervous system neurotransmitters, NE and 5-HT, in the regulation of immune response.     

Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells

Introduction

Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro.

Methods

Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.

Results

We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.

Conclusion

We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.     

Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease

Celiac disease is a T cell-driven intolerance to wheat gluten. The gluten-derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine, the site where gluten induces inflammatory T cell responses that lead to celiac disease. We have now determined the usefulness of a newly identified prolyl endoprotease from Aspergillus niger for this purpose. Gluten and its peptic/tryptic digest were treated with prolyl endoprotease, and the destruction of the T cell epitopes was tested using mass spectrometry, T cell proliferation assays, ELISA, reverse-phase HPLC, SDS-PAGE, and Western blotting. We observed that the A. niger prolyl endoprotease works optimally at 4-5 pH, remains stable at 2 pH, and is completely resistant to digestion with pepsin. Moreover, the A. niger-derived enzyme efficiently degraded all tested T cell stimulatory peptides as well as intact gluten molecules. On average, the endoprotease from A. niger degraded gluten peptides 60 times faster than a prolyl oligopeptidase. Together these results indicate that the enzyme from A. niger efficiently degrades gluten proteins. Future studies are required to determine if the prolyl endoprotease can be used as an oral supplement to reduce gluten intake in patients.     

Non-Smad transforming growth factor-β signaling regulated by focal adhesion kinase binding the p85 subunit of phosphatidylinositol 3-kinase

TGF-β modulates numerous diverse cellular phenotypes including growth arrest in epithelial cells and proliferation in fibroblasts. Although the Smad pathway is fundamental for the majority of these responses, recent evidence indicates that non-Smad pathways may also have a critical role. Here we report a novel mechanism whereby the nonreceptor tyrosine focal adhesion kinase (FAK) functions as an adaptor necessary for cell type-specific responses to TGF-β. We show that in contrast to Smad actions, non-Smad pathways, including c-Abl, PAK2, and Akt, display an obligate requirement for FAK. Interestingly, this occurs in Src null SYF cells and is independent of FAK tyrosine phosphorylation, kinase activity, and/or proline-rich sequences in the C-terminal FAT domain. FAK binds the phosphatidylinositol 3-kinase (PI3K) p85 regulatory subunit following TGF-β treatment in a subset of fibroblasts but not epithelial cells and has an obligate role in TGF-β-stimulated anchorage-independent growth and migration. Together, these results uncover a new scaffolding role for FAK as the most upstream component regulating the profibrogenic action of TGF-β and suggest that inhibiting this interaction may be useful in treating a number of fibrotic diseases.     

Effect of ascorbic acid and acute heat exposure on heat shock protein 70 expression by young white Leghorn chickens

Dietary ascorbic acid (AA) and heat stress (HS) affect heat shock protein 70 (hsp70) and body temperature (BT) of strain cross white Leghorn chickens. At five weeks of age, chicks fed either no supplemental AA (N-AA) or 200 mg/kg AA (AA) were subjected to either HS (42 degrees C) or maintained in control (CN, 23 degrees C) ambient temperature (T(a)) for 1 h. Body temperature (BT) was recorded for each bird before collection of heart and liver for hsp70 assay. In the CN AA-fed groups, neither the lower constitutive hsc70 nor the decreased hsp70 response to HS in the heart and liver were sex-dependent. The BT was increased by HS, but neither AA nor sex of the bird affected BT response. A diet X T(a) interaction revealed that BT of CN AA-fed chickens was lower than in N-AA-fed chickens, but BT of HS AA-fed chicks was greater than BT in HS N-AA-fed chickens. The BT and hsp70 responses were positively correlated. A lower expression of hsp70 indicated less of a stress response in the AA-fed chickens.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.