Retinal neural progenitors express topographic map markers

Transplantation of neural stem cells for replacing neurons after neurodegeneration requires that the transplanted stem cells accurately reestablish the lost neural circuits in order to restore function. Retinal ganglion cell axons project to visual centers of the brain forming circuits in precise topographic order. In chick, dorsal retinal neurons project to ventral optic tectum, ventral neurons to dorsal tectum, anterior neurons to posterior tectum and posterior neurons to anterior tectum; forming a continuous point-to-point map of retinal cell position in the tectal projection. We found that when stem cells derived from ventral retina were implanted in dorsal host retina, the stem cells that became ganglion cells projected to dorsal tectum, appropriate for their site of origin in retina but not appropriate for their site of implant in retina. This led us to ask if retinal progenitors exhibit topographic markers of cell position in retina. Indeed, retinal neural progenitors express topographic markers: dorsal stem cells expressed more Ephrin B2 than ventral stem cells and, conversely, ventral stem cells expressed more Pax-2 and Ventroptin than dorsal stem cells. The fact that neural progenitors express topographic markers has pertinent implications in using neural stem cells in cell replacement therapy for replacing projecting neurons that express topographic order, e.g., analogous neurons of the visual, auditory, somatosensory and motor systems.     

Roles of Acidic Phospholipids and Nucleotides in Regulating Membrane Binding and Activity of a Calcium-independent Phospholipase A2 Isoform

Phospholipase A₂ activity plays key roles in generating lipid second messengers and regulates membrane topology through the generation of asymmetric lysophospholipids. In particular, the Group VIA phospholipase A₂ (GVIA-iPLA₂) subfamily of enzymes functions independently of calcium within the cytoplasm of cells and has been implicated in numerous cellular processes, including proliferation, apoptosis, and membrane transport steps. However, mechanisms underlying the spatial and temporal regulation of these enzymes have remained mostly unexplored. Here, we examine the subset of Caenorhabditis elegans lipases that harbor a consensus motif common to members of the GVIA-iPLA₂ subfamily. Based on sequence homology, we identify IPLA-1 as the closest C. elegans homolog of human GVIA-iPLA₂ enzymes and use a combination of liposome interaction studies to demonstrate a role for acidic phospholipids in regulating GVIA-iPLA₂ function. Our studies indicate that IPLA-1 binds directly to multiple acidic phospholipids, including phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidic acid, and phosphorylated derivatives of phosphatidylinositol. Moreover, the presence of these acidic lipids dramatically elevates the specific activity of IPLA-1 in vitro. We also found that the addition of ATP and ADP promote oligomerization of IPLA-1, which probably underlies the stimulatory effect of nucleotides on its activity. We propose that membrane composition and the presence of nucleotides play key roles in recruiting and modulating GVIA-iPLA₂ activity in cells.     

The plasmamembrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I

The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646-8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152-14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59-61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693-18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.     

Oestrone Sulphate Measurements for the Prediction of Small or Large Litters in Pigs

Serum from 88 pregnant sows and gilts was sampled 24 and 28 days after their first insemination or mating day. The oestrone sulphate (E1S) concentration in the samples was assessed with a commercially available radioimmunoassay kit modified for use with swine serum. The first aim was to test whether it was possible to predict litters of total number <10 piglets at term. The second aim was to compare the use of day 24 or day 28 samples, or of both, in this prediction.     

Mutation of proline-1003 to glycine in the epidermal growth factor (EGF) receptor enhances responsiveness to EGF.

We have shown previously that the epidermal growth factor (EGF) receptor is phosphorylated at Ser-1002 and that this phosphorylation is associated with desensitization of the EGF receptor. Ser-1002 is followed immediately by Pro-1003, a residue that may promote the adoption of a specific conformation at this site or severe as a recognition element for the interaction of the EGF receptor with other proteins. To examine these possibilities, we have mutated Pro-1003 of the EGF receptor to a Gly residue and have analyzed the effect of this mutation on EGF-stimulated signaling. Cells expressing the P1003G EGF receptors exhibited higher EGF-stimulated autophosphorylation and synthetic peptide phosphorylation compared to cells expressing wild-type EGF receptors. In addition, the ability of EGF to stimulate PI 3-kinase activity and mitogen-activated protein kinase activity was enhanced in cells expressing the P1003G EGF receptor. Cells expressing P1003G receptors also demonstrated an increased ability to form colonies in soft agar in response to EGF. These results indicate that mutation of Pro-1003 leads to a potentiation of the biological effects of EGF. The findings are consistent with the hypothesis that Pro-1003 plays a role in a form of regulation that normally suppresses EGF receptor function.     

Novel technique for biliary reconstruction using an isolated gastric tube with a vascularized pedicle: a live animal experimental study and the first clinical case

Background

Biliary tract reconstruction continues to be a challenging surgical problem. Multiple experimental attempts have been reported to reconstruct biliary defects with different materials and variable outcome. Our aim was to evaluate a new method for biliary reconstruction using an isolated pedicled gastric tube in a live animal trial and also to present the first clinical case.

Methods

Seven mongrel dogs underwent biliary reconstruction using gastric tube harvested, completely separated from the greater curvature, and based on a vascularized pedicle with the right gastroepiploic vessels. The tube was interposed between the common bile duct (CBD) and the duodenum. Postoperative mortality, morbidity, liver functions, gross and microscopic histological picture were assessed. The first clinical case was also presented where, in a patient with post-cholecystectomy biliary injury, an isolated pedicled gastric tube was interposed between the proximal and distal ends of the CBD.

Results

One dog did not recover from anesthesia and another one died postoperatively from septic peritonitis. Five dogs survived the procedure and showed uneventful course and no cholestasis. The mean anastomotic circumference was 4.8 mm (range 4-6) for CBD anastomosis and 6.2 mm (range 5-7) for duodenal anastomosis. Histologically, anastomotic sites showed good evidence of healing. In the first clinical case, the patient showed clinical and biochemical improvement. Endoscopic retrograde cholangiography was feasible and assured patent biliary anastomoses.

Conclusion

In mongrel dogs, biliary reconstruction using pedicled gastric tube interposition between CBD and duodenum is feasible with satisfactory clinical results, anastomotic circumference and histological evidence of healing. The technique is also feasible in human and seems to be promising.     

Studies of the transmissibility of the agent of bovine spongiform encephalopathy to the domestic chicken

Background

Escherichia coli O157:H7 is the most common serovar of enterohemorrhagic E. coli associated with serious human disease outbreaks. Cattle are the main reservoir with E. coli O157:H7 inducing hemorrhagic enteritis in persistent shedding beef cattle, however little is known about how this pathogen affects cattle health. Jejunal Hemorrhage Syndrome (JHS) has unclear etiology but the pathology is similar to that described for E. coli O157:H7 challenged beef cattle suggestive that E. coli O157:H7 could be involved. There are no effective treatments for JHS however new approaches to managing pathogen issues in livestock using prebiotics and probiotics are gaining support. The first objective of the current study was to characterize pathogen colonization in hemorrhaged jejunum of dairy cattle during natural JHS outbreaks. The second objective was to confirm the association of mycotoxigenic fungi in feeds with the development of JHS and also to identify the presence of potential mycotoxins. The third objective was to determine the impact of a prebiotic, Celmanax?, or probiotic, Dairyman's Choice? paste, on the cytotoxicity associated with feed extracts in vitro. The fourth objective was to determine the impact of a prebiotic or a probiotic on E. coli O157:H7 colonization of mucosal explants and a bovine colonic cell line in vitro. The final objective was to determine if prebiotic and probiotic feed additives could modify the symptoms that preceded JHS losses and the development of new JHS cases.

Findings

Dairy cattle developed JHS after consuming feed containing several types of mycotoxigenic fungi including Fusarium culmorum, F. poae, F. verticillioides, F. sporotrichioides, Aspergillus flavus, Penicillium roqueforti, P. crustosum, P. paneum and P. citrinum. Mixtures of Shiga toxin - producing Escherichia coli (STEC) colonized the mucosa in the hemorrhaged tissues of the cattle and no other pathogen was identified. The STECs expressed Stx1 and Stx2, but more significantly, Stxs were also present in the blood clot blocking the jejunum. Mycotoxin analysis of the corn crop confirmed the presence of fumonisin, NIV, ZEAR, DON, 15-ADON, 3-ADON, NEO, DAS, HT-2 and T-2. Feed extracts were toxic to enterocytes and 0.1% Celmanax? removed the cytotoxicity in vitro. There was no effect of Dairyman's Choice? paste on feed-extract activity in vitro. Fumonisin, T-2, ZEAR and DON were toxic to bovine cells and 0.1% Celmanax? removed the cytotoxicity in vitro. Celmanax? also directly decreased E. coli O157:H7 colonization of mucosal explants and a colonic cell line in a dose-dependent manner. There was no effect of Dairyman's Choice? paste on E. coli O157:H7 colonization in vitro. The inclusion of the prebiotic and probiotic in the feed was associated with a decline in disease.

Conclusion

The current study confirmed an association between mycotoxigenic fungi in the feed and the development of JHS in cattle. This association was further expanded to include mycotoxins in the feed and mixtures of STECs colonizing the severely hemorrhaged tissues. Future studies should examine the extent of involvement of the different STEC in the infection process. The prebiotic, Celmanax?, acted as an anti-adhesive for STEC colonization and a mycotoxin binder in vitro. Future studies should determine the extent of involvement of the prebiotic in altering disease.     

Recording the Motor Activity of Aquatic Animals by Ultrasound

An ultrasonic technique based on the Doppler effect is presented to allow the registration of aquatic animal movements. The method was tested on crucians (Carassius carassius) in an aquarium as part of chronobiological investigations. Comparison with light barrier measurements shows that the Doppler technique is suitable for detecting typical circadian and seasonal changes in the motor activity of the fishes. The features of the ultrasonic method are discussed.     

IL-13 fusion cytotoxin ameliorates chronic fungal-induced allergic airway disease in mice.

IL-13 has emerged as a major contributor to allergic and asthmatic responses, and as such it represents an attractive target in these diseases. In this study, IL-13-responsive cells in the lung were targeted via the intranasal administration of IL-13-PE38QQR (IL-13-PE), comprised of human IL-13 and a derivative of Pseudomonas exotoxin, to Aspergillus fumigatus-sensitized mice challenged with A. fumigatus spores, or conidia. Mice received 50, 100, or 200 ng of IL-13-PE or diluent alone (i.e., control group) on alternate days from day 14 to day 28 after the conidia challenge. The control group of mice exhibited significant airway hyperreactivity, goblet cell hyperplasia, and peribronchial fibrosis at day 28 after conidia. Although the two lower doses of IL-13-PE had limited therapeutic effects in mice with fungal-induced allergic airway disease, the highest dose of IL-13-PE tested significantly reduced all features of airway disease compared with the control group. Whole lung mRNA expression of IL-4Ralpha and IL-13Ralpha1 was markedly reduced, whereas bronchoalveolar lavage and whole lung levels of IFN-gamma were significantly elevated in mice treated with 200 ng of IL-13-PE compared with the control group. This study demonstrates that a therapy designed to target IL-13-responsive cells in the lung ameliorates established fungal-induced allergic airway disease in mice.