Impaired functional activity of alveolar macrophages from GM-CSF-deficient mice

We hypothesized that pulmonary granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically involved in determining the functional capabilities of alveolar macrophages (AM) for host defense. To test this hypothesis, cells were collected by lung lavage from GM-CSF mutant mice [GM(-/-)] and C57BL/6 wild-type mice. GM(-/-) mice yielded almost 4-fold more AM than wild-type mice. The percentage of cells positive for the beta(2)-integrins CD11a and CD11c was reduced significantly in GM(-/-) AM compared with wild-type cells, whereas expression of CD11b was similar in the two groups. The phagocytic activity of GM(-/-) AM for FITC-labeled microspheres was impaired significantly compared with that of wild-type AM both in vitro and in vivo (after intratracheal inoculation with FITC-labeled beads). Stimulated secretion of tumor necrosis factor-alpha (TNF-alpha) and leukotrienes by AM from the GM(-/-) mice was greatly reduced compared with wild-type AM, whereas secretion of monocyte chemoattractant protein-1 was increased. Transgenic expression of GM-CSF exclusively in the lungs of GM(-/-) mice resulted in AM with normal or supranormal expression of CD11a and CD11c, phagocytic activity, and TNF-alpha secretion. Thus, in the absence of GM-CSF, AM functional capabilities for host defense were significantly impaired but were restored by lung-specific expression of GM-CSF.     

Use of lacticin 481 to facilitate delivery of the bacteriophage resistance plasmid, pCBG104 to cheese starters

AIMS: Use of lacticin 481 to facilitate the conjugal transfer of the bacteriophage resistance plasmid pCBG104 to various starter cultures. METHODS AND RESULTS: A raw milk isolate of Lactococcus was found to harbour determinants for lacticin 481 production and immunity and phage resistance on a plasmid designated pCBG104. The lacticin 481 was successfully used to mobilize the phage resistance determinant to a variety of cheese starters enabling the formation of highly phage resistant starters. In addition, it facilitated the stacking of a number of phage resistance genes, namely a type I restriction modification system, a phage abortive infection system and a phage adsorption blocking system in a single Lactococcus strain without the use of recombinant techniques. The transconjugants were all shown to produce lacticin 481 and to contain the entire 481 operon. Subsequently one transconjugant was selected and successfully used for large-scale cheddar cheese manufacture. CONCLUSIONS: Lacticin 481 could be used as a food-grade selectable marker to facilitate the introduction of advantageous traits to starter cultures for industrial food fermentations. SIGNIFICANCE AND IMAPCT OF THE STUDY: Food-grade selectable markers greatly facilitate the introduction of various advantageous traits to starter cultures for industrial food fermentation. Indeed self-cloning which is becoming increasingly important for strain improvement has a requirement for the identification and demonstration of the utility of tools such as lacticin 481.     

Effect of hepatic denervation on the counterregulatory response to insulin-induced hypoglycemia in the dog

Our aim was to determine whether complete hepatic denervation would affect the hormonal response to insulin-induced hypoglycemia in dogs. Two weeks before study, dogs underwent either hepatic denervation (DN) or sham denervation (CONT). In addition, all dogs had hollow steel coils placed around their vagus nerves. The CONT dogs were used for a single study in which their coils were perfused with 37 degrees C ethanol. The DN dogs were used for two studies in a random manner, one in which their coils were perfused with -20 degrees C ethanol (DN + COOL) and one in which they were perfused with 37 degrees C ethanol (DN). Insulin was infused to create hypoglycemia (51 +/- 3 mg/dl). In response to hypoglycemia in CONT, glucagon, cortisol, epinephrine, norepinephrine, pancreatic polypeptide, glycerol, and hepatic glucose production increased significantly. DN alone had no inhibitory effect on any hormonal or metabolic counterregulatory response to hypoglycemia. Likewise, DN in combination with vagal cooling also had no inhibitory effect on any counterregulatory response except to reduce the arterial plasma pancreatic polypeptide response. These data suggest that afferent signaling from the liver is not required for the normal counterregulatory response to insulin-induced hypoglycemia.     

Prolonged exposure to lipopolysaccharide inhibits macrophage 5-lipoxygenase metabolism via induction of nitric oxide synthesis

LPS from bacteria can result in the development of sepsis syndrome and acute lung injury. Although acute exposure to endotoxin primes leukocytes for enhanced synthesis of leukotrienes (LT), little is known about the effect of chronic exposure. Therefore, we determined the effect of prolonged LPS treatment on 5-lipoxygenase (5-LO) metabolism of arachidonic acid in alveolar macrophages (AM) and in peripheral blood monocytes. Pretreatment of AM with LPS caused time- and dose-dependent suppression of LT synthetic capacity. LPS pretreatment failed to inhibit arachidonic acid (AA) release. The fact that LPS inhibited LT synthesis from endogenous AA more than from exogenous AA suggested an effect on 5-LO-activating protein (FLAP). In addition, an inhibitory effect of LPS treatment on AM 5-LO activity was suggested by cell-free 5-LO enzyme assay. No effect on the expression of either 5-LO or FLAP proteins was observed. New protein synthesis was necessary for LPS-induced reduction of 5-LO metabolism in AM, and immunoblotting demonstrated marked induction of NO synthase (NOS). Inhibition by LPS was reproduced by an NO donor and was abrogated by inhibitors of constitutive and inducible NOS. Compared with AM, peripheral blood monocytes exhibited no suppression by LPS of 5-LO metabolism and no induction of inducible NOS. We conclude that prolonged exposure to LPS impairs AM 5-LO metabolism by NO-mediated suppression of both 5-LO and FLAP function. Because LT contribute to antimicrobial defense, this down-regulation of 5-LO metabolism may contribute to the increased susceptibility to pneumonia in patients following sepsis.     

Characterization of glucokinase regulatory protein-deficient mice

The glucokinase regulatory protein (GKRP) inhibits glucokinase competitively with respect to glucose by forming a protein-protein complex with this enzyme. The physiological role of GKRP in controlling hepatic glucokinase activity was addressed using gene targeting to disrupt GKRP gene expression. Heterozygote and homozygote knockout mice have a substantial decrease in hepatic glucokinase expression and enzymatic activity as measured at saturating glucose concentrations when compared with wild-type mice, with no change in basal blood glucose levels. Interestingly, when assayed under conditions to promote the association between glucokinase and GKRP, liver glucokinase activity in wild-type and null mice displayed comparable glucose phosphorylation capacities at physiological glucose concentrations (5 mM). Thus, despite reduced hepatic glucokinase expression levels in the null mice, glucokinase activity in the liver homogenates was maintained at nearly normal levels due to the absence of the inhibitory effects of GKRP. However, following a glucose tolerance test, the homozygote knockout mice show impaired glucose clearance, indicating that they cannot recruit sufficient glucokinase due to the absence of a nuclear reserve. These data suggest both a regulatory and a stabilizing role for GKRP in maintaining adequate glucokinase in the liver. Furthermore, this study provides evidence for the important role GKRP plays in acutely regulating of hepatic glucose metabolism.     

Identification of rCop-1, a New Member of the CCN Protein Family,as a Negative Regulator for Cell Transformation

By using a model system for cell transformation mediated by the cooperation of the activated H-ras oncogene and the inactivated p53 tumor suppressor gene, rCop-1 was identified by mRNA differential display as a gene whose expression became lost after cell transformation. Homology analysis indicates that rCop-1 belongs to an emerging cysteine-rich growth regulator family called CCN, which includes connective-tissue growth factor, CYR61, CEF10 (v-src inducible), and the product of the nov proto-oncogene. Unlike the other members of the CCN gene family, rCop-1 is not an immediate-early gene, it lacks the conserved C-terminal domain which was shown to confer both growth-stimulating and heparin-binding activities, and its expression is lost in cells transformed by a variety of mechanisms. Ectopic expression of rCop-1 by retroviral gene transfers led to cell death in a transformation-specific manner. These results suggest that rCop-1 represents a new class of CCN family proteins that have functions opposing those of the previously identified members.Oncogenic conversion of a normal cell into a tumor cell requires multiple genetic alterations (12). Of particular interest is the fact that mutations in both ras oncogenes (3) and the p53 tumor suppressor gene cooperate in transformation of mammalian cells (11). Mutations in both ras and the p53 gene were also found at high frequencies in a variety of human cancers, including those of the colon, lung, and pancreas (2, 18). It has been proposed that both p53 and Ras function, whether directly or through other signaling molecules, to control expression of genes that are important for cell growth and differentiation (13, 17, 37). To this end, several ras target genes (10) and p53 target genes, including those encoding p21/CIP1/WAF1, an inhibitor of G₁ cyclin-dependent kinase (9); Mdm-2, a negative regulator of p53 (1); GADD45, a protein involved in DNA repair (36); and Bax, which promotes apoptosis (28), have been identified. Most of these genes, except p21/CIP1/WAF1, which was cloned by subtractive hybridization, were identified by the candidate gene hypothesis. Recently, more p53 target genes have been isolated by the differential display technique, including those coding for cyclin G (31); MAP4, a microtubule-associated protein negatively regulated by p53 (29); and PAG608, a novel nuclear zinc finger protein whose overexpression promotes apoptosis (14). Functional characterizations of these genes have shed light on the role of p53 in cell cycle control and apoptosis. However, genes that mediate tumor suppression activity by p53 remain elusive.The fact that neither the inactivation of p53 nor the activation of Ras alone is able to transform primary mammalian cells (34), whereas both mutations together can do so, suggests that genes regulated by p53 and Ras cooperate in upsetting normal cell growth control cells (11). Using differential display (22), we set out to identify genes whose expression is altered by both mutant ras and p53 by comparing the mRNA expression profiles of normal rat embryo fibroblasts (REFs) and their derivatives transformed by either a constitutively inactivated or a temperature-sensitive mutant p53 in cooperation with the activated H-ras oncogene (11, 27). In this report we describe the identification and give a functional characterization of rCop-1, a gene whose expression is abolished by cell transformation. By sequence homology, rCop-1 was found to belong to an emerging cysteine-rich growth regulator family called CCN (which stands for connective-tissue growth factor [CTGF], CEF10/Cyr61, and Nov) (4). Here we show that rCop-1 may represent a novel class of CCN family proteins based on its unique cell cycle expression pattern, its lack of the C-terminal (CT) domain conserved in all CCN proteins, its loss of expression in all transformed cells analyzed, and its ability to confer cytotoxicity to the transformed cells.     

Pathogen Sources Estimation and Scenario Analysis Using the Soil and Water Assessment Tool (SWAT)

Concerns over water quality in Ireland have increased in recent years, in part due to the more frequent contamination of drinking water by pathogens such as Escherichia coli and Cryptosporidium. The objective of this study was to assess the use of SWAT for pathogen source estimation and to analyze the effects of various source scenarios on pathogen outputs in Irish catchments. Two agricultural catchments in Ireland susceptible to pathogen contamination of source water were the center of the SWAT model development with the primary focus on levels of E. coli in surface water. Model simulations used site and source specific information which was analyzed considering the total E. coli count for the simulation period (Fergus: January 2005–October 2006; Kilshanvey: January 2006–July 2007). Pathogen source estimation identified point sources as the most significant contributors to E. coli output with direct deposition the primary contributor (95%) in Kilshanvey and wastewater treatment plant outflow (89%) the main contributor in the Fergus catchment. A scenario analysis evaluated possible situations that may occur in study locations. The analysis indicated that restriction of livestock access to water sources and improved wastewater treatment would represent effective methods of improving water quality in both catchments.     

Orally active and brain permeable proline amides as highly selective 5HT2c agonists for the treatment of obesity

Brain-penetrable proline amides were developed as 5HT2c agonists with more than 1000-fold binding selectivity against 5HT2b receptor. After medicinal chemistry optimization and SAR studies, orally active proline amides with robust efficacy in a rodent food intake inhibition model were uncovered.     

Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes

Introduction  

Nucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells, and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells.