Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

BACKGROUND: There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. METHODOLOGY/PRINCIPAL FINDINGS: We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. CONCLUSIONS/SIGNIFICANCE: Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale

The Receptor Tyrosine Kinase Alk Controls Neurofibromin Functions in Drosophila Growth and Learning

Anaplastic Lymphoma Kinase (Alk) is a Receptor Tyrosine Kinase (RTK) activated in several cancers, but with largely unknown physiological functions. We report two unexpected roles for the Drosophila ortholog dAlk, in body size determination and associative learning. Remarkably, reducing neuronal dAlk activity increased body size and enhanced associative learning, suggesting that its activation is inhibitory in both processes. Consistently, dAlk activation reduced body size and caused learning deficits resembling phenotypes of null mutations in dNf1, the Ras GTPase Activating Protein-encoding conserved ortholog of the Neurofibromatosis type 1 (NF1) disease gene. We show that dAlk and dNf1 co-localize extensively and interact functionally in the nervous system. Importantly, genetic or pharmacological inhibition of dAlk rescued the reduced body size, adult learning deficits, and Extracellular-Regulated-Kinase (ERK) overactivation dNf1 mutant phenotypes. These results identify dAlk as an upstream activator of dNf1-regulated Ras signaling responsible for several dNf1 defects, and they implicate human Alk as a potential therapeutic target in NF1

Dynamic Switch of Negative Feedback Regulation in Drosophila Akt–TOR Signaling

Akt represents a nodal point between the Insulin receptor and TOR signaling, and its activation by phosphorylation controls cell proliferation, cell size, and metabolism. The activity of Akt must be carefully balanced, as increased Akt signaling is frequently associated with cancer and as insufficient Akt signaling is linked to metabolic disease and diabetes mellitus. Using a genome-wide RNAi screen in Drosophila cells in culture, and in vivo analyses in the third instar wing imaginal disc, we studied the regulatory circuitries that define dAkt activation. We provide evidence that negative feedback regulation of dAkt occurs during normal Drosophila development in vivo. Whereas in cell culture dAkt is regulated by S6 Kinase (S6K)–dependent negative feedback, this feedback inhibition only plays a minor role in vivo. In contrast, dAkt activation under wild-type conditions is defined by feedback inhibition that depends on TOR Complex 1 (TORC1), but is S6K–independent. This feedback inhibition is switched from TORC1 to S6K only in the context of enhanced TORC1 activity, as triggered by mutations in tsc2. These results illustrate how the Akt–TOR pathway dynamically adapts the routing of negative feedback in response to the activity load of its signaling circuit in vivo

The restorative role of annexin A1 at the blood–brain barrier

Annexin A1 is a potent anti-inflammatory molecule that has been extensively studied in the peripheral immune system, but has not as yet been exploited as a therapeutic target/agent. In the last decade, we have undertaken the study of this molecule in the central nervous system (CNS), focusing particularly on the primary interface between the peripheral body and CNS: the blood–brain barrier. In this review, we provide an overview of the role of this molecule in the brain, with a particular emphasis on its functions in the endothelium of the blood–brain barrier, and the protective actions the molecule may exert in neuroinflammatory, neurovascular and metabolic disease. We focus on the possible new therapeutic avenues opened up by an increased understanding of the role of annexin A1 in the CNS vasculature, and its potential for repairing blood–brain barrier damage in disease and aging

Loss of cholinergic innervation differentially affects eNOS-mediated blood flow, drainage of Aβ and cerebral amyloid angiopathy in the cortex and hippocampus of adult mice

Vascular dysregulation and cholinergic basal forebrain degeneration are both early pathological events in the development of Alzheimer’s disease (AD). Acetylcholine contributes to localised arterial dilatation and increased cerebral blood flow (CBF) during neurovascular coupling via activation of endothelial nitric oxide synthase (eNOS). Decreased vascular reactivity is suggested to contribute to impaired clearance of β-amyloid (Aβ) along intramural periarterial drainage (IPAD) pathways of the brain, leading to the development of cerebral amyloid angiopathy (CAA). However, the possible relationship between loss of cholinergic innervation, impaired vasoreactivity and reduced clearance of Aβ from the brain has not been previously investigated. In the present study, intracerebroventricular administration of mu-saporin resulted in significant death of cholinergic neurons and fibres in the medial septum, cortex and hippocampus of C57BL/6 mice. Arterial spin labelling MRI revealed a loss of CBF response to stimulation of eNOS by the Rho-kinase inhibitor fasudil hydrochloride in the cortex of denervated mice. By contrast, the hippocampus remained responsive to drug treatment, in association with altered eNOS expression. Fasudil hydrochloride significantly increased IPAD in the hippocampus of both control and saporin-treated mice, while increased clearance from the cortex was only observed in control animals. Administration of mu-saporin in the TetOAPPSweInd mouse model of AD was associated with a significant and selective increase in Aβ40-positive CAA. These findings support the importance of the interrelationship between cholinergic innervation and vascular function in the aetiology and/or progression of CAA and suggest that combined eNOS/cholinergic therapies may improve the efficiency of Aβ removal from the brain and reduce its deposition as CAA

dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1.

Genetic studies in Drosophila melanogaster underscore the importance of the insulin-signalling pathway in controlling cell, organ and animal size. Effectors of this pathway include Chico (the insulin receptor substrate homologue), dPI(3)K, dPKB, dPTEN, and dS6K. Mutations in any of these components have a striking effect on cell size and number, with the exception of dS6K. Mutants in dS6K affect cell size but not cell number, seemingly consistent with arguments that dS6K is a distal effector in the signalling pathway, directly controlled by dTOR, a downstream effector of dPI(3)K and dPKB. Unexpectedly, recent studies showed that dS6K activity is unimpaired in chico-deficient larvae, suggesting that dS6K activation may be mediated through the dPI(3)K docking sites of the Drosophila insulin receptor. Here, we show genetically, pharmacologically and biochemically that dS6K resides on an insulin signalling pathway distinct from that of dPKB, and surprisingly also from that of dPI(3)K. More striking, despite dPKB-dPI(3)K-independence, dS6K activity is dependent on the Drosophila homologue of the phosphoinositide-dependent protein kinase 1, dPDK1, demonstrating that both dPDK1, as well as dTOR, mediated dS6K activation is phosphatidylinositide-3,4,5-trisphosphate (PIP3)-independent

Long-term exposure to ambient ultrafine particles and respiratory disease incidence in in Toronto, Canada: a cohort study

Abstract Background Little is known about the long-term health effects of ambient ultrafine particles (<0.1 μm) (UFPs) including their association with respiratory disease incidence. In this study, we examined the relationship between long-term exposure to ambient UFPs and the incidence of lung cancer, adult-onset asthma, and chronic obstructive pulmonary disease (COPD). Methods Our study cohort included approximately 1.1 million adults who resided in Toronto, Canada and who were followed for disease incidence between 1996 and 2012. UFP exposures were assigned to residential locations using a land use regression model. Random-effect Cox proportional hazard models were used to estimate hazard ratios (HRs) describing the association between ambient UFPs and respiratory disease incidence adjusting for ambient fine particulate air pollution (PM2.5), NO2, and other individual/neighbourhood-level covariates. Results In total, 74,543 incident cases of COPD, 87,141 cases of asthma, and 12,908 cases of lung cancer were observed during follow-up period. In single pollutant models, each interquartile increase in ambient UFPs was associated with incident COPD (HR = 1.06, 95% CI: 1.05, 1.09) but not asthma (HR = 1.00, 95% CI: 1.00, 1.01) or lung cancer (HR = 1.00, 95% CI: 0.97, 1.03). Additional adjustment for NO2 attenuated the association between UFPs and COPD and the HR was no longer elevated (HR = 1.01, 95% CI: 0.98, 1.03). PM2.5 and NO2 were each associated with increased incidence of all three outcomes but risk estimates for lung cancer were sensitive to indirect adjustment for smoking and body mass index. Conclusions In general, we did not observe clear evidence of positive associations between long-term exposure to ambient UFPs and respiratory disease incidence independent of other air pollutants. Further replication is required as few studies have evaluated these relationships