Endocytosis of the non-catalytic ADAM23: Recycling and long half-life properties.

A Disintegrin And Metalloprotease 23 (ADAM23) is a member of the ADAMs family of transmembrane proteins, mostly expressed in nervous system, and involved in traffic and stabilization of Kv1-potassium channels, synaptic transmission, neurite outgrowth, neuronal morphology and cell adhesion. Also, ADAM23 has been linked to human pathological conditions, such as epilepsy, cancer metastasis and cardiomyopathy. ADAM23 functionality depends on the molecule presence at the cell surface and along the secretory pathway, as expected for a cell surface receptor. Because endocytosis is an important functional regulatory mechanism of plasma membrane receptors and no information is available about the traffic or turnover of non-catalytic ADAMs, we investigated ADAM23 internalization, recycling and half-life properties. Here, we show that ADAM23 undergoes constitutive internalization from the plasma membrane, a process that depends on lipid raft integrity, and is redistributed to intracellular vesicles, especially early and recycling endosomes. Furthermore, we observed that ADAM23 is recycled from intracellular compartments back to the plasma membrane and thus has longer half-life and higher cell surface stability compared with other ADAMs. Our findings suggest that regulation of ADAM23 endocytosis/stability could be exploited therapeutically in diseases in which ADAM23 is directly involved, such as epilepsy, cancer progression and cardiac hypertrophy.

Active fractions of mannoproteins derived from yeast cell wall stimulate innate and acquired immunity of adult and elderly dogs.

Nutritional intervention in older dogs aims to increase lifespan and improve life quality as well as delay the development of diseases related to ageing. It is believed that active fractions of mannoproteins (AFMs) obtained through extraction and fractionation of yeast cell walls (Saccharomyces cerevisiae) may beneficially modulate the immune system. However, studies that have evaluated this component and the effects of ageing on the immune system of dogs are scarce. This study aimed to evaluate the immunological effects of AFMs in adult and elderly dogs. Three extruded iso-nutrient experimental diets were formulated: without addition of AFM (T0); with AFM at 400 mg/kg (T400); and with AFM at 800 mg/kg (T800). Thirty-six beagle dogs were used, and six experimental treatments, resulting in combinations of age (adult and elderly) and diet (T0, T400, and T800), were evaluated. On days zero, 14, and 28, blood samples were obtained for leucocyte phenotyping and phagocytosis assays. On days zero and 28, a lymphoproliferation test, quantification of reactive oxygen (H2O2) and nitrogen (NO) intermediate production, evaluation of faecal immunoglobulin A (IgA) content, and a delayed cutaneous hypersensitivity test (DCHT) were performed. Statistical analyses were performed with SAS software. Repeated measure variance analyses were performed, and means were compared by the Tukey test. Values of P ≤ 0.05 were considered significant, and values of P ≤ 0.10 were considered tendencies. Dogs fed T400 tended to have higher neutrophilic phagocytic activity than dogs fed T800 (P = 0.073). Regarding reactive oxygen intermediates, bacterial lipopolysaccharide (LPS)-stimulated neutrophils from animals that were fed T400 had a tendency to produce more H2O2 than those from animals fed the control diet (P = 0.093). Elderly dogs, when compared to adult dogs, had lower absolute T and B lymphocyte counts, lower auxiliary T lymphocyte counts, and higher cytotoxic T lymphocyte counts (P < 0.05). A significant effect of diet, age, and time with saline inoculation was noted for the DCHT. There was no effect of diet or age on faecal IgA content in dogs. This study suggests beneficial effects of mannoproteins on the specific and nonspecific immune responses in adult and elderly dogs.

Monoclonal Antibody DL11C8 Identifies ADAM23 as a Component of Lipid Raft Microdomains.

A disintegrin and metalloprotease protein 23 (ADAM23) is a transmembrane type I glycoprotein involved with the development and maintenance of the nervous system, including neurite outgrowth, neuronal adhesion and differentiation and regulation of synaptic transmission. In addition, ADAM23 seems to participate in immune response and tumor establishment through interaction with different members of integrin receptors. Here, we describe a novel monoclonal antibody (DL11C8) that specifically recognizes the cysteine-rich domain of both pre-protein (100 kDa) and mature (70 kDa) forms of ADAM23 from different species, including human, rodents and avian orthologs. Using this antibody, we detected both forms of ADAM23 on the cell surface of three neuronal cell lineages (Neuro-2a, SH-SY5Y and CHLA-20), with a higher relative content of ADAM23100 kDa. Furthermore, we demonstrate for the first time that a catalytically inactive member of the ADAM family is present in the membrane signaling platforms, namely lipid rafts. Indeed, the mature ADAM2370 kDa partitions between raft and non-raft membrane domains, while the pro-protein ADAM23100 kDa is mainly expressed in non-raft domains. These membranous distributions were observed in both different brain regions homogenates and primary cultured neurons lysates from mouse cortex and cerebellum. Taken together, these findings point out ADAM23 as a lipid raft molecular component.

Enzyme effects on extruded diets for dogs with soybean meal as a substitute for poultry by-product meal.

The effects of exogenous enzymes supplementation on kibble diets for dogs formulated with soybean meal (SBM) as a substitute for poultry by-product meal (PM) was investigated on nutrient digestibility, fermentation products formation, post-prandial urea response and selected faecal bacteria counts. Two kibble diets with similar compositions were used in two trials: PM-based diet (28.9% of PM; soybean hulls as a fibre source) and SBM-based diet (29.9% of SBM). In experiment 1, the SBM diet was divided into three diets: SBM-0, without enzyme addition; SBM-1, covered after extrusion with 7500 U protease/kg and 45 U cellulase/kg; and SBM-2, covered with 15,000 U protease/kg and 90 U cellulase/kg. In experiment 2, the SBM diet was divided into three diets: SBM-0; SBM-1, covered with 140 U protease/kg; 8 U cellulase/kg, 800 U pectinase/kg, 60 U phytase/kg, 40 U betaglucanase/kg and 20 U xylanase/kg; and SMB-2, covered with 700 U protease/kg, 40 U cellulase/kg, 4000 U pectinase/kg, 300 U phytase/kg, 200 U betaglucanase/kg and 100 U xylanase/kg. Each experiment followed a block design with six dogs per diet. Data were submitted to analysis of variance and means compared by orthogonal and polynomial contrasts (p < 0.05). In both experiments, nutrients and energy digestibility did not differ between diets (p > 0.05). SBM consumption resulted in increased faecal moisture and production (p < 0.05), without effect on faecal score. Higher concentration of propionate, acetate and lactate, and lower ammonia and pH were found in the faeces of dogs fed SBM (p < 0.05). Higher post-prandial urea was verified in dogs fed SBM (p < 0.05). In experiment 2, the addition of enzymes increased faecal concentration of propionate, acetate and total short-chain fatty acid (p < 0.05) and tended to reduce post-prandial urea concentration (p = 0.06). Although with similar digestibility, SBM shows a worse utilization of absorbed amino acids than the PM. Soybean oligosaccharides can beneficially change gut fermentation product formation. Enzymes can increase the gut fermentation activity and improve the SBM proteic value.