Mechanisms for the inhibition of amiloride-sensitive Na+ absorption by extracellular nucleotides in mouse trachea

Purinergic stimulation of airway epithelial cells induces Cl- secretion and modulates Na+ absorption by an unknown mechanism. To gain insight into this mechanism, we used a perfused micro-Ussing chamber to assess transepithelial voltage (V-te) and amiloride-sensitive short-circuit current (Isc-Amil) in mouse trachea. Exposure to apical ATP or UTP (each 100 mumol/l) caused a large initial increase in lumen negative V-te and I-sc corresponding to a transient Cl- secretion, while basolateral application of ATP/UTP induced only a small secretory response. Luminal, but not basolateral, application of nucleotides was followed by a sustained and reversible inhibition of Isc-Amil that was independent of extracellular Ca2+ or activation of protein kinase C and was not induced by carbachol (100 mumol/l) or the Ca2+ ionophore ionomycin (1 mumol/l). Removal of extracellular Cl- or exposure to 200 muM DIDS reduced UTP-mediated inhibition of Isc-Amil Substantially. The phospholipase inhibitor U73122 (10 mumol/l) and pertussis toxin (PTX 200 ng/ml) both attenuated UTP-induced Cl- secretion and inhibition of Isc-Amil. Taken together, these data imply a contribution of Cl- conductance and PTX-sensitive G proteins to nucleotide-dependent inhibition of the amiloride-sensitive Na+ current in the mouse trachea.

Mast cells and oral inflammation

Mast cells are mobile granule-containing secretory cells that are distributed preferentially about the microvascular endothelium in oral mucosa and dental pulp. The enzyme profile of mast cells in oral tissues resembles that of skin, with most mast cells expressing the serine proteases tryptase and chymase. Mast cells in oral tissues contain the pro-inflammatory cytokine tumour necrosis factor-alpha in their granules, and release of this promotes leukocyte infiltration during evolving inflammation in several conditions, including lichen planus, gingivitis, pulpitis, and periapical inflammation, through induction of endothelial-leukocyte adhesion molecules. Mast cell synthesis and release of other mediators exerts potent immunoregulatory effects on other cell types, while several T-lymphocyte-derived cytokines influence mast cell migration and mediator release. Mast cell proteases may contribute to alterations in basement membranes in inflammation in the oral cavity, such as the disruptions that allow cytotoxic lymphocytes to enter the epithelium in oral lichen planus. A close relationship exists among mast cells, neural elements, and laminin, and this explains the preferential distribution of mast cells in tissues. Mast cells are responsive to neuropeptides and, through their interaction with neural elements, form a neural immune network with Langerhans cells in mucosal tissues. This facilitates mast cell degranulation in response to a range of immunological and non-immunological stimuli. Because mast cells play a pivotal role in inflammation, therapies that target mast cell functions could have value in the treatment of chronic inflammatory disorders in the oral cavity.

Immunoglobulins in saliva of preterm and full-term infants. A longitudinal study from 0-18 months of age

The aim of this longitudinal study was to determine salivary levels of total IgA, IgG and IgM in 84 preterm and 214 full-term infants, from birth to 18 months of age. Unstimulated whole saliva was collected from each infant at birth, and subsequently at 3-monthly intervals. Immunoglobulin levels were estimated using an ELISA technique. At birth, IgA was detected in 147/214 (69%) full-term infants but only 47/84 (56%) preterm infants (P

Structure of the gut contents of Trichogramma australicum Girault (Hymenoptera : Trichogrammatidae) larvae fixed in situ in eggs of its host Helicoverpa armigera (Hubner) (Lepidoptera : Noctuidae)

Trichogramma australicum larvae develop most rapidly in younger eggs of its host, the pest lepidopteran Helicoverpa armigera . To establish how the developmental stage of the host affects the diet of T. australicum , larvae were fixed in situ in eggs of H. armigera of different ages and the structure of the egg contents and parasitoid gut contents examined histologically. Larvae feeding on newly laid host eggs contain primarily yolk particles in their gut, while larvae feeding on older hosts contain necrotic cells and yolk particles. The gut of T. australicum larvae does not contain organised tissue remnants, indicating that larvae feed primarily by sucking food into their pharynx and feed best on a mixture of particulate semisolids in a liquid matrix. Secretory structures of T. australicum larvae that could be involved in modifying the host environment were examined. The hindgut is modified to form an anal vesicle with a number of attributes suggesting that it may be a specialised secretory structure. The paired salivary glands open to the exterior via a common duct.

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, intergrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations. (C) 2003 Elsevier Science Ltd. All rights reserved.

Mouse proteome analysis

A general overview of the protein sequence set for the mouse transcriptome produced during the FANTOM2 sequencing project is presented here. We applied different algorithms to characterize protein sequences derived from a nonredundant representative protein set (RPS) and a variant protein set (VPS) of the mouse transcriptome. The functional characterization and assignment of Gene Ontology terms was done by analysis of the proteome using InterPro. The Superfamily database analyses gave a detailed structural classification according to SCOP and provide additional evidence for the functional characterization of the proteome data. The MDS database analysis revealed new domains which are not presented in existing protein domain databases. Thus the transcriptome gives us a unique source of data for the detection of new functional groups. The data obtained for the RPS and VPS sets facilitated the comparison of different patterns of protein expression. A comparison of other existing mouse and human protein sequence sets (e.g., the International Protein Index) demonstrates the common patterns in mammalian proteornes. The analysis of the membrane organization within the transcriptome of multiple eukaryotes provides valuable statistics about the distribution of secretory and transmembrane proteins

The mouse secretome: functional classification of the proteins secreted into the extracellular environment

We have developed a computational strategy to identify the set of soluble proteins secreted into the extracellular environment of a cell. Within the protein sequences predominantly derived from the RIKEN representative transcript and protein set, we identified 2033 unique soluble proteins that are potentially secreted from the cell. These proteins contain a signal peptide required for entry into the secretory pathway and lack any transmembrane domains or intracellular localization signals. This class of proteins, which we have termed the mouse secretome, included >500 novel proteins and 92 proteins

Computational differentiation of N-terminal signal peptides and transmembrane helices

Signal peptides and transmembrane helices both contain a stretch of hydrophobic amino acids. This common feature makes it difficult for signal peptide and transmembrane helix predictors to correctly assign identity to stretches of hydrophobic residues near the N-terminal methionine of a protein sequence. The inability to reliably distinguish between N-terminal transmembrane helix and signal peptide is an error with serious consequences for the prediction of protein secretory status or transmembrane topology. In this study, we report a new method for differentiating protein N-terminal signal peptides and transmembrane helices. Based on the sequence features extracted from hydrophobic regions (amino acid frequency, hydrophobicity, and the start position), we set up discriminant functions and examined them on non-redundant datasets with jackknife tests. This method can incorporate other signal peptide prediction methods and achieve higher prediction accuracy. For Gram-negative bacterial proteins, 95.7% of N-terminal signal peptides and transmembrane helices can be correctly predicted (coefficient 0.90). Given a sensitivity of 90%, transmembrane helices can be identified from signal peptides with a precision of 99% (coefficient 0.92). For eukaryotic proteins, 94.2% of N-terminal signal peptides and transmembrane helices can be correctly predicted with coefficient 0.83. Given a sensitivity of 90%, transmembrane helices can be identified from signal peptides with a precision of 87% (coefficient 0.85). The method can be used to complement current transmembrane protein prediction and signal peptide prediction methods to improve their prediction accuracies. (C) 2003 Elsevier Inc. All rights reserved.

Will chymase inhibitors be the next major development for the treatment of cardiovascular disorders?

Chymase is contained in the secretory granules of mast cells. In addition to the synthesis of angiotensin II, chymase is involved in transforming growth factor-beta activation and cleaves Type I procollagen to produce collagen. NK301 and BCEAB are orally-active inhibitors of chymase. NK301 was tested in a dog model of vascular intimal hyperplasia after balloon injury and shown to reduce the increased chymase activity in the injured arteries and prevent intimal thickening. In a hamster model of cardiac fibrosis associated with cardiomyopathy, BCEAB reduced the increased cardiac chymase activity in cardiomyopathy and reduced fibrosis. Chymase inhibitors may be an important development for the treatment of cardiovascular injury associated with mast cell degranulation.

GLUT4 recycles via a trans-Golgi network (TGN) subdomain enriched in Syntaxins 6 and 16 but not TGN38: Involvement of an acidic targeting motif

Insulin stimulates glucose transport in fat and muscle cells by triggering exocytosis of the glucose transporter GLUT4. To define the intracellular trafficking of GLUT4, we have studied the internalization of an epitope-tagged version of GLUT4 from the cell surface. GLUT4 rapidly traversed the endosomal system en route to a perinuclear location. This perinuclear GLUT4 compartment did not colocalize with endosomal markers (endosomal antigen I protein, transferrin) or TGN38, but showed significant overlap with the TGN target (t)-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Syntaxins 6 and 16. These results were confirmed by vesicle immunoisolation. Consistent with a role for Syntaxins 6 and 16 in GLUT4 trafficking we found that their expression was up-regulated significantly during adipocyte differentiation and insulin stimulated their movement to the cell surface. GLUT4 trafficking between endosomes and trans-Golgi network was regulated via an acidic targeting motif in the carboxy terminus of GLUT4, because a mutant lacking this motif was retained in endosomes. We conclude that GLUT4 is rapidly transported from the cell surface to a subdomain of the trans-Golgi network that is enriched in the t-SNAREs Syntaxins 6 and 16 and that an acidic targeting motif in the C-terminal tail of GLUT4 plays an important role in this process.

The effects of peripheral and central warming on body temperature during canine laparotomy

Actively warming patients during surgery is considered the best method of preventing inadvertent hypothermia due to multiple causes: anaesthetic depression of the hypothalamic thermoregulatory centre, cutaneous vasodilatation, reduction of heat production by skeletal muscles, cold intravenous fluid administration and heat loss from opened body cavities. To compare the effects of active peripheral skin warming and trunk warming on body temperature during surgery, 15 dogs undergoing ovariohysterectomy were studied using a prospective randomised trial design. Dogs were randomised into two groups: one group was warmed by compress leg pads (n=7) on limbs and the other group by a circulating warm water mattress (n=8), applied to the trunk. The rectal, oesophageal and room temperatures and relative humidity were measured. The results showed that the compress leg pads (active peripheral warming) were significantly (P

The ultrastructure and function of the silk-producing basitarsus in the Hilarini (Diptera : Ernpididae)

The tribe Hilarini (Diptera: Empididae), commonly known as dance flies, can be recognised by their swollen silk-producing prothoracic basitarsus, a male secondary sexual characteristic. The ultrastructure and function of the silk-producing basitarsus from one undescribed morphospecies of Hilarini, 'Hilarempis 20', is presented. Male H. 20 collect small parcels of diatomaceous algae from the surface of freshwater creeks that they bind with silk produced by the gland in the basitarsus. The gift is then presented to females in a nearby swarm, composed predominately of females. The basitarsus houses approximately 12 pairs of class III dermal glandular units that congregate on the ventral side of the cavity. Each gland cell has a large extracellular lumen where secretion accumulates. The lumen drains to the outside via a conducting canal encompassed by a canal cell and a duct extending through the shaft of a specialised secretory spine. The secretory spines lie in pairs in a ventral groove that runs the length of the basitarsus. A comparison of the basitarsal secretory spines with sensilla on the basitarsi of non gland-bearing legs of males, and with non gland-bearing prothoracic. basitarsi of females, suggests that the glandular units are derived from contact chemosensory sensilla. (C) 2003 Elsevier Ltd. All rights reserved.

The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion

Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response [1, 2]. TNFalpha plays a key role in inflammatory disease [3]; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types [4, 5] was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.

GAIP participates in budding of membrane carriers at the trans-Golgi network

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans -Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.

Prevalence of periodontal disease in dogs and owners' level of awareness - a prospective clinical trial

Periodontal disease (PD) is widely known among veterinarians for its high prevalence and serious consequences to the dogs. The objective of this study was to assess the occurrence of PD in dogs that live in the micro-region of Viçosa, treated at the Veterinary Hospital of the Federal University of Viçosa (HVT - Hospital Veterinário da Universidade Federal de Viçosa), as well as to assess how aware of this disease dog owners are. In order to do so, all dogs treated at the HVT from March 10th, 2009 to November 30th, 2009, on alternate days, had their oral cavities examined. Medical history data, such as age, type of food, main complaint and owner consent, halitosis, presence of dental calculus, inflammation and gingival recession and tooth loss, were collected. A prevalence of 88.67% was found for PD in dogs referred to the HVT, and 2.67% were referred due to this disease. Of all the owners who participated in the study, 43.83% knew about periodontal disease and of these 17.46% made use of some type of prevention or treatment. Therefore, periodontal disease is highly prevalent and the owners are not aware of the disease. Thus, a dog owner clarification program on periodontal disease is needed in the area where HVT-UFV operates.