The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase.

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.

Neutering increases the risk of obesity in male dogs but not in bitches - A cross-sectional study of dog- and owner-related risk factors for obesity in Danish companion dogs.

Knowledge of risk factors for canine obesity is an important pre-requisite of effective preventative strategies. This study aimed to investigate risk factors for canine obesity in adult companion dogs across Zealand, Denmark. Client-owned dogs (>2 years of age and without chronic illness) were recruited and examined at eight companion animal veterinary practices in areas with varying socio-economic characteristics. The body condition score (BCS) of the dogs was examined by two investigators based on a 9-point scoring scheme. Dog owners answered a questionnaire that had prompts regarding: 1) dog characteristics, including neuter status, 2) owner characteristics, 3) feeding and exercise practices and 4) the owners' attachment to the dog. The effect of these factors on BCS and the risk of being heavy/obese (BCS scores 7-9) were analysed in two separate analyses. A total of 268 dogs were included in the analysis, of which 20.5% were found to be heavy/obese. The average BCS was 5.46. In terms of dog characteristics, neutering dramatically increased both BCS and the risk of being heavy/obese in male dogs but not in bitches. BCS and the risk of being heavy/obese increased in senior bitches and decreased in senior male dogs. The risk of being heavy/obese was higher in dogs with overweight and obese owners. Regarding feeding and exercise practices, providing only one meal per day increased BCS and risk of being heavy/obese. Treats during relaxation increased the risk of dogs being heavy/obese. It also increased the dogs' BCS, but only if the owners were overweight or obese. An increased duration of daily walking increased the risk of the dog being heavy/obese, but only if the owner was overweight or obese. Allowing the dog to run free in the garden/property decreased the risk of the dog being heavy/obese. The owners' attachment to the dog was not associated with the dogs' BCS or dogs' being heavy/obese. An important and novel finding was that neutering increased the risk of being overweight or obese for male dogs while bitches were at risk irrespective of neuter status. Furthermore, a complex interaction between owners' weight status, feeding practices and the risk of dogs being overweight or obese was found, which stresses the need to consider companion animal obesity from a One Health perspective in future prospective studies. Finally, this study was unable to confirm that canine obesity is a product of owners being too attached to their dogs.

High-resolution native and complex structures of thermostable beta-mannanase from Thermomonospora fusca - substrate specificity in glycosyl hydrolase family 5.

BACKGROUND: . beta-Mannanases hydrolyse the O-glycosidic bonds in mannan, a hemicellulose constituent of plants. These enzymes have potential use in pulp and paper production and are of significant biotechnological interest. Thermostable beta-mannanases would be particularly useful due to their high temperature optimum and broad pH tolerance. The thermophilic actinomycete Thermomonospora fusca secretes at least one beta-mannanase (molecular mass 38 kDa) with a temperature optimum of 80 degreesC. No three-dimensional structure of a mannan-degrading enzyme has been reported until now. RESULTS: . The crystal structure of the thermostable beta-mannanase from T. fusca has been determined by the multiple isomorphous replacement method and refined to 1.5 A resolution. In addition to the native enzyme, the structures of the mannotriose- and mannohexaose-bound forms of the enzyme have been determined to resolutions of 1.9 A and 1.6 A, respectively. CONCLUSIONS: . Analysis of the -1 subsite of T. fusca mannanase reveals neither a favourable interaction towards the axial HO-C(2) nor a discrimination against the equatorial hydroxyl group of gluco-configurated substrates. We propose that selectivity arises from two possible mechanisms: a hydrophobic interaction of the substrate with Val263, conserved in family 5 bacterial mannanases, which discriminates between the different conformations of the hydroxymethyl group in native mannan and cellulose; and/or a specific interaction between Asp259 and the axial hydroxyl group at the C(2) of the substrate in the -2 subsite. Compared with the catalytic clefts of family 5 cellulases, the groove of T. fusca mannanase has a strongly reduced number of aromatic residues providing platforms for stacking with the substrate. This deletion of every second platform is in good agreement with the orientation of the axial hydroxyl groups in mannan.

Species differences in the sites of cleavage of pro-lactase to lactase supports lack of selective pressure.

The pro-sequences in pro-lactase-phlorizin hydrolase (LPH) are needed for lactase to proceed past the ER, but are irrelevant as to the enzymatic activities. Hence, in all species removal of the pro- sequences (or most of them) must take place after the ER. Contrary to this, the details of the removal of these pro-sequences are to be expected to differ in the various species, since they are not subjected to selective pressure. Using site-directed mutagenesis we investigated processing in rabbit. The first cleavage occurs by furin (or furin-like PCs) and takes place at R-A-A-R(349) in the pro-sequence, generating the known 180 kDa intermediate. Replacing R(349) by Q results in a mutant which is not cleaved but nevertheless transported to the cell surface as demonstrated by immunofluorescence. Further processing of either the 180 kDa intermediate or the mutant is not directly mediated by furin-like PCs, but involves (also) other proteases. These results demonstrate that formation of the 180 kDa intermediate, consistently found only in rabbits, but not in man, is not essential for lactase transport: in all likelihood lack of selective pressure has led to species-specific processing of pro-LPH.

A vector for the synthesis of cRNAs encoding Myc epitope-tagged proteins in Xenopus laevis oocytes.

We describe a plasmid, pNKS2-myc, designed for convenient in-frame fusion of an antibody-specific epitope sequence to the N terminus of a desired cDNA and subsequent synthesis of transcripts that direct the synthesis of the tagged polypeptide in Xenopus laevis (Xl) oocytes. pNKS2-myc contains an SP6 promoter, followed by the translation initiation sequence of the Na,K-pump beta 3 subunit of Xl and the sequence encoding an epitope derived from the human c-myc proto-oncogene product. Appropriate restriction sites allow one to insert virtually any desired cDNA fragment directly behind the epitope-specific sequence and before a long poly(A) tail. After linearization with EcoRI or NotI, polyadenylated cRNA can be synthesized that is efficiently translated in Xl oocytes. The utility of pNKS2-myc is demonstrated by cloning cDNAs coding for Na,K-pump subunits into this vector and injecting the corresponding cRNAs into oocytes. The tagged mouse beta 1 and beta 2 subunit isoforms could be purified from detergent extracts of these cells by immunoprecipitation with a generally available monoclonal antibody (mAb) to the tag, 9E10, as well as with specific mAb that recognize individual beta subunit isoforms. Under native conditions, endogenous and coexpressed exogenous alpha 1 subunits (the catalytic subunit of the Na,K-pump) were co-precipitated, indicating that the N-terminal addition of the decapeptide epitope has no adverse effect on the folding of beta subunits nor on their assembly with alpha subunits. Furthermore, the Myc-specific mAb likewise precipitated a Myc-tagged Na,K-pump alpha 1 subunit together with any of the co-synthesized beta subunits.

Production and secretion in CHO cells of the extracellular domain of AMOG/beta 2, a type-II membrane protein.

A hybrid gene consisting of the sequences coding for the signal peptide and N terminus of a type-I membrane protein, the neural cell adhesion molecule (N-CAM), and the extracellular domain of the adhesion molecule on glia (AMOG/beta 2), a type-II membrane protein, was constructed. The sequence was inserted into a eukaryotic expression vector containing the human cytomegalovirus promoter and the glutamine synthetase selection marker, and used to transfect Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of soluble recombinant AMOG/beta 2 (reAMOG/beta 2), which was secreted into the culture medium as a heavily glycosylated 40-55-kDa protein. N-terminal sequence analysis revealed that the protein is not cleaved at the natural signal peptide cleavage site of N-CAM, but two amino acids (aa) further downstream. Treatment of reAMOG/beta 2 with N-glycosidase F (GlycoF) reduced the molecular mass to 27 kDa, corresponding to the calculated mass of the unglycosylated form. In contrast to AMOG/beta 2 isolated from mouse brain, which is sensitive to endoglycosidase H, the immunoaffinity-purified re-protein is more resistant to this treatment, indicating that the sugars attached to reAMOG/beta 2 are mainly of the complex type. Our results demonstrate the feasibility of secreting the extracellular domain of a type-II membrane protein, which is usually inserted into the membrane with the C terminus facing the extracellular side.

Relevance of Na,K-ATPase to local extracellular potassium homeostasis and modulation of synaptic transmission.

The ion gradients generated by the Na,K-ATPase are essential for Na+-coupled transport systems, osmoregulation and restoration of ion concentrations in excitable tissues. Indirectly, the sodium pump controls intracellular Ca2+ concentration through the Na/Ca exchanger. In the nervous system various neurotransmitters can modulate Na,K-ATPase activity. The great diversity of Na,K-ATPase subunit isoforms, their complex spatial and temporal regulation of expression and their cellular localisation imply a functional role of the sodium pump in different regulatory pathways. Among these, potassium homeostasis and modulation of synaptic transmission are discussed here.

Inhibition of Na,K-ATPase activity by cGMP is isoform-specific in brain endothelial cells.

cGMP has been shown to either activate or inhibit Na,K-ATPase activity. Using mouse brain endothelial cells which express both ouabain-resistant alpha1 and ouabain-sensitive alpha2 and alpha3 isoforms, we show that cGMP reduces total Na,KATPase activity to about 58%. The inhibition is prevented by the protein kinase G (PKG)-specific inhibitor KT5823, indicating that cGMP-mediated activation of PKG leads to inhibition of the pump. A similar extent of inhibition is obtained with nitric oxide. cGMP-induced inhibition acts mainly on alpha1 isoforms but hardly affects alpha2/alpha3 isoforms. These data suggest that inhibition of Na,K-ATPase activity by cGMP occurs in an isoform-selective manner in brain endothelial cells.

Intestinal lactase-phlorizin hydrolase (LPH): the two catalytic sites; the role of the pancreas in pro-LPH maturation.

Brush border lactase-phlorizin hydrolase carries two catalytic sites. In the human enzyme lactase comprises Glu-1749, phlorizin hydrolase Glu-1273. The proteolytic processing of pro-lactase-phlorizin hydrolase by (rat) enterocytes stops two amino acid residues short of the N-terminus of 'mature' final, brush border lactase-phlorizin hydrolase. Only these two amino acid residues are removed by luminal pancreatic protease(s), probably trypsin.

Co-culture blood-brain barrier models and their use for pharmatoxicological screening.

The availability of an in vitro blood-brain barrier model would represent a powerful alternative to experimental animals in pharmacological and toxicological research. This overview collects the various current approaches to build an in vitro model of the blood-brain barrier for these purposes. Purified bovine, porcine and human brain microcapillary endothelial cells as well as several immortalized cell lines have been used to model the blood-brain barrier in vitro, partly in co-culture with astrocytes of various species, or various cell lines such as C6 glioma or N2a neuroblastoma cells. The collected data indicate that functional parameters often can be induced by soluble and membrane-bound factors in such cell systems. Relevant barrier-specific parameters are reviewed: electrical resistance, and structure and function of the multidrug resistance P-glycoprotein and the y-glutamyl transpeptidase. Both P-glycoprotein and gamma-glutamyl transpeptidase have great influence on the pharmacodynamics, toxicology and metabolic capacity of the blood-brain barrier (drug efflux, oxidative damage, detoxification of endotoxins, etc.). Several available in vitro models appear to be suited for pharmacotoxicological screening, if the functional parameters gamma-glutamyl transpeptidase, P-glycoprotein as well as transendothelial resistance are monitored.

The extracellular domain of the sodium pump beta isoforms determines complex stability with alpha 1.

Both subunits of the Na,K-ATPase are encoded by several genes giving rise to at least six isozymes. To examine whether beta isoforms assemble with alpha 1 in a selective manner, we have overexpressed wild-type and chimeric beta subunits in L929 cells and examined assembly as a function of resistance towards detergent-mediated dissociation. In the presence of digitonin all beta chimeras coimmunoprecipitate the endogenous alpha 1 subunit. Only beta proteins with the ectodomain of beta 1 coimmunoprecipitate alpha 1 in the presence of Triton X-100. All beta chimeras stimulate Na,K-ATPase activity in L929 cells. These data indicate that the beta subunit ectodomains mediate interactions with alpha 1 and influence the stability of this complex.

Quantitative aspects of membrane behavior in rat parathyroid cells after depression or elevation of serum calcium.

The behavior of membranes concerned with parathyroid hormone secretion was studied by electron microscopic morphometry in parathyroid cells of rats with temporarily reduced serum calcium concentration resulting from phosphate ion application and in rats with elevated serum calcium concentration following vitamin D3 administration. The phosphate ion application resulted in an increase of the cell surface area and a concomitant decrease of the surface area of the Golgi complex and secretory granules within 3 hours. After 3 hours, the cell surface area decreased, whereas the surface area of the Golgi complex and the secretory granules increased, and after 12 hours, the surface area of the rough endoplasmic reticulum also increased. In the vitamin D3-treated rats the surface area of the secretory granules increased, but the cell surface area had decreased by 24, 48, and 72 hours after application. These data suggest that parathyroid cells respond to a transient depression of the serum calcium concentration by an initial centrifugal membrane shift indicating enhanced exocytosis, followed by a centripedal membrane shift indicating enhanced endocytic retrieval of the plasma membrane. Later, membrane synthesis led to an increase of the membrane compartments concerned with parathyroid hormone secretion. Elevation of the serum calcium concentration following vitamin D3 treatment resulted in reduced release of parathyroid hormone by exocytosis and enhanced retrieval of plasma membranes by endocytosis. The fate of the retrieval plasma membrane remains unclear.

[The adverse effects of local anesthetics on the eye in the development of ocular irritation test]. / De l'agressivité des anesthésiques locaux envers l'oeil au développement d'un test d'irritation oculaire.

Referring to the ocular damage produced by local anaesthetics applied to the eye, the authors suggest the use of a test, to reveal the danger that some ophthalmic preparations may represent for patients. They review the studies devoted to the evaluation of ocular damage, particularly those which resulted into tests based on the methodology of Draize. A test scaled down to the mouse eye is described, in which the subjective appreciation is completed by an objective measurement. The test is based on the increased permeability of injured cornea for fluorescein. The fluorescence emitted by this tracer depends on the extent of the micro-lesions and is measured with an optical electronic device. The possibilities and limitations of this test are determined by the relationship between dose and extent of ocular damage. Such a relationship has been established for oxybuprocain. An appropriate concentration of this local anaesthetic allows to investigate the influence of adjuvants, preservatives and viscosifiers on the effect of oxybuprocain on the cornea. The authors conclude that it is possible to apply this test to the development of well-tolerated ophthalmic preparations.

Functional characterization of beta isoforms of murine Na,K-ATPase. The adhesion molecule on glia (AMOG/beta 2), but not beta 1, promotes neurite outgrowth.

We have previously provided evidence for a dual function of the adhesion molecule on glia (AMOG/beta 2), the beta 2 subunit of the murine Na,K-ATPase, both as neural recognition molecule mediating neuron-glia interactions and as functional beta subunit of the sodium pump. To analyze the functional role of AMOG/beta 2 in neurite outgrowth, AMOG/beta 2-expressing L-cells were generated by transfection and used as substrates for neurite outgrowth of cerebellar and hippocampal neurons. AMOG/beta 2-transfected L-cells led to an increase in neurite length after 6 h, which was specifically inhibited by antibodies to AMOG/beta 2 and a neuronal membrane fraction. Moreover, the extracellular domain of AMOG/beta 2 generated as a soluble recombinant protein in Chinese hamster ovary cells partially inhibited the increase in neurite outgrowth on AMOG/beta 2-transfected L-cells. L-cells transfected with the mouse beta 1 subunit had no effect on neurite extension. Our observations show for the first time differences in functional properties for different beta isoforms of the Na,K-ATPase and suggest that AMOG/beta 2 but not beta 1 is able to interact with an unknown neuronal receptor leading to increased neurite outgrowth, most likely via signal transduction.

The adhesion molecule on glia (AMOG/beta 2) and alpha 1 subunits assemble to functional sodium pumps in Xenopus oocytes.

The adhesion molecule on glia, AMOG, an integral cell surface glycoprotein highly expressed by cerebellar astrocytes and involved in neuron to astrocyte adhesion and granule neuron migration (Antonicek, H., Persohn, E., and Schachner, M. (1987) J. Cell Biol. 104, 1587-1595) has been identified as a beta 2 subunit isoform of the mouse sodium pump (Gloor, S., Antonicek, H., Sweadner, K.J., Pagliusi, S., Frank, R., Moos, M., and Schachner, M. (1990) J. Cell Biol. 110, 165-174). Here we demonstrate that AMOG/beta 2 expressed by cRNA injection in Xenopus oocytes is capable of combining with endogenous Xenopus alpha 1 subunits or coexpressed Torpedo alpha 1 subunits to yield a functional alpha 1/AMOG sodium pump isozyme. Determinations of the number of ouabain binding sites and ouabain-sensitive 86Rb+ uptake suggest that the alpha 1/AMOG isozyme has slightly lower maximum transport rate and apparent affinity for external K+ than the alpha 1/beta 1 isozyme. Immunoprecipitation of alpha 1/AMOG complexes from digitonin extracts of [35S]methionine-labeled oocytes with a monoclonal anti-AMOG antibody provides direct evidence for a stable association between AMOG and the alpha 1 subunits of Xenopus and Torpedo.

Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates.

The functional unit of the Na,K-ATPase consists of a catalytic alpha subunit noncovalently linked with a glycoprotein subunit, beta. Using ouabain binding assays and immunoprecipitation of rodent alpha/beta complexes, we show here that all six possible isozymes between three alpha and two beta isoforms can be formed in Xenopus oocytes. Two isoform-specific differences in alpha/beta interactions are observed: (i) alpha1/beta1 and alpha2/beta2 complexes, in contrast to alpha1/beta2 complexes, are stable against Triton X-100-mediated dissociation, and (ii) beta2 subunits must carry N-glycans to combine with alpha1 but not with alpha2. The interacting surfaces are mainly exposed to the extracellular side because coexpression of a truncated beta1 subunit comprising the ectodomain results in assembly with alpha1 and alpha2, but not with alpha3; the beta2 ectodomain combines with alpha2 only. A chimera consisting of 81% and 19% of the alpha1 N terminus and alpha2 C terminus, respectively, behaves like alpha2 and coprecipitates with the beta2 ectodomain. In contrast, the reciprocal chimera does not coprecipitate with the beta2 ectodomain. These results provide evidence for a selective interaction of Na,K-ATPase alpha and beta subunits.

Tenascin-R is an intrinsic autocrine factor for oligodendrocyte differentiation and promotes cell adhesion by a sulfatide-mediated mechanism.

O4(+) oligodendrocyte (OL) progenitors in the mammalian CNS are committed fully to terminal differentiation into myelin-forming cells. In the absence of other cell types in vitro, OL differentiation reproduces the in vivo development with a correct timing, suggesting the existence of an intrinsic regulatory mechanism that presently is unknown. We have examined the effect of two isoforms of the extracellular matrix (ECM) molecule tenascin-R (TN-R), which is expressed by OLs during the process of myelination, on the adhesion and maturation of OLs in vitro. Here we show that the substrate-bound molecules supported the adhesion of O4(+) OLs independently of the CNS region or age from which they were derived. At the molecular level this process was mediated by protein binding to membrane surface sulfatides (Sulf), as indicated by the interference of O4 antibody and Sulf with the attachment of OLs or other Sulf+ cells, erythrocytes, to TN-R substrates and by direct protein-glycolipid binding studies. In the absence of platelet-derived growth factor (PDGF), exogenous TN-R induced myelin gene expression and the upregulation of its own synthesis by cultured cells, resulting in a rapid terminal differentiation of O4(+) progenitors. Our findings strongly suggest that TN-R represents an intrinsic regulatory molecule that controls the timed OL differentiation by an autocrine mechanism and imply the relevance of TN-R for CNS myelination and remyelination.

Interleukin-1 modulates protein tyrosine phosphatase activity and permeability of brain endothelial cells.

Interleukin-1 alpha (IL-1 alpha) and interleukin-6 (IL-6), both known to be able to open the blood-brain barrier (BBB), downregulated plasma membrane-associated tyrosine phosphatase activity in primary porcine brain endothelial cells (PBEC). In contrast, transforming growth factor beta (TGF-beta) upregulated PTP activity and tumor necrosis factor alpha (TNF-alpha) had no effect. Plasma membrane-associated PTP activity of PBEC was upregulated at contact inhibited growth arrest. Tightly confluent cells reduced 3H-inulin permeability by 34% compared with just confluent cells indicating the formation of barrier properties. The decrease in permeability temporally correlated with the elevated PTP activity of the cells at growth arrest and was reversed to control by IL-1 alpha. Vanadate, a broad-specificity PTP inhibitor, also enhanced 3H-inulin permeability. These data suggest that IL-1 alpha-induced endothelial permeability could be controlled through lowering PTP activity.

Processing of human intestinal prolactase to an intermediate form by furin or by a furin-like proprotein convertase.

Human lactase-phlorizin hydrolase (human-LPH) is synthesized as a large precursor (prepro-LPH), then cleaved to a pro-LPH of 220 kDa which is further cut to a "mature-like LPH" of a size close to that of mature LPH, i.e. about 150 kDa (in the processing of rabbit pro-LPH the intermediate has a mass of approximately 180 kDa). By coexpression of human prepro-LPH with furin in COS-7 cells we show that furin generates a mature-like LPH. Radioactive amino acid sequence analysis reveals that furin recognizes the motif R-T-P-R832, a protein convertase consensus, to generate a NH2 terminus located 36 amino acids upstream of the NH2 terminal found in vivo at Ala869. This intermediate is ultimately cleaved to the mature LPH form by other proteases including the pancreatic ones. These data demonstrate that human pro-LPH, like the rabbit enzyme, is processed to the mature enzyme by furin or furin-like enzymes through at least an intermediate form that has, however, an apparent mass close to that of the mature enzyme.