Incidence of Vitis fruit-induced clinical signs and acute kidney injury in dogs and cats.

OBJECTIVES: To determine the incidence of clinical signs and Vitis fruit-induced acute kidney injury in dogs and cats with a Vitis fruit ingestion reported to the Dutch Poisons Information Center, and a description of the therapies instituted by the veterinarians. MATERIALS AND METHODS: All cases of Vitis fruit ingestions in dogs and cats reported to the center between January 1, 2018 and December 31, 2018 were included in this study. Veterinarians and pet owners were contacted by phone or email to obtain follow-up information. Information was collected using a standardised data collection sheet. RESULTS: Ninety-five dogs and 13 cats with proven Vitis fruit ingestion were included. Fourteen dogs and two cats developed clinical signs: emesis (11/16, 68.8%), lethargy (5/16, 31.3%), diarrhoea (3/16, 18.8%), anorexia (3/16, 18.8%), tremor (2/16, 12.5%) and restlessness (1/16, 6.3%). The overall incidence for developing clinical signs was 14.7% in dogs and 15.4% in cats. One (1/95, 1%) dog developed acute kidney injury after ingestion of Vitis fruit. No cats developed acute kidney injury. Induction of emesis and/or administration of activated charcoal was instituted in 72 of 82 (88%) and eight of 11 (73%) of asymptomatic dogs and cats and six of 14 (43%) and two of two (100%) of symptomatic dogs and cats, respectively. Overall, emesis was induced in 72 of 95 (76%) dogs (100% success rate) and removed Vitis fruits in the majority of cases (98% when induced <4 hours after ingestion and 83% when induced 4 to 12 hours after ingestion). Emesis was induced in seven of 13 (54%) cats (86% success rate) and removed Vitis fruits in 83% of the cases. CLINICAL SIGNIFICANCE: In this study, a significant proportion (around 15%) of dogs and cats developed clinical signs after ingestion of Vitis fruits, which were predominantly related to the gastrointestinal tract. Symptomatic acute kidney injury was rare. Our findings suggest the use of decontamination measures, i.e. induction of emesis, may be warranted up to 12 hours after ingestion.

Structural determinants of nucleobase modification recognition in the AlkB family of dioxygenases.

Iron-dependent dioxygenases of the AlkB protein family found in most organisms throughout the tree of life play a major role in oxidative dealkylation processes. Many of these enzymes have attracted the attention of researchers across different fields and have been subjected to thorough biochemical characterization because of their link to human health and disease. For example, several mammalian AlkB homologues are involved in the direct reversal of alkylation damage in DNA, while others have been shown to play a regulatory role in epigenetic or epitranscriptomic nucleic acid methylation or in post-translational modifications such as acetylation of actin filaments. These studies show that that divergence in amino acid sequence and structure leads to different characteristics and substrate specificities. In this review, we aim to summarize current insights in the structural features involved in the substrate selection of AlkB homologues, with focus on nucleic acid interactions.

Polyoxometalates as artificial nucleases: hydrolytic cleavage of DNA promoted by a highly negatively charged ZrIV-substituted Keggin polyanion.

A highly negatively charged binuclear ZrIV-substituted Keggin polyoxometalate [{α-PW11O39Zr(µ-OH)(H2O)}2]8- (ZrK 2 : 2) has been shown to promote the hydrolytic cleavage of phosphoester bonds in the supercoiled plasmid pUC19 DNA under physiological pH and temperature, giving relaxed and linear forms of pUC19 as hydrolysis products. The interaction between ZrK 2 : 2 and DNA was experimentally proven by circular dichroism (CD) spectroscopy and 31P diffusion ordered NMR spectroscopy.

Functional variation in the arginine vasopressin 2 receptor as a modifier of human plasma von Willebrand factor levels.

SUMMARY OBJECTIVES: Stimulation of arginine vasopressin 2 receptor (V2R) with arginine vasopressin (AVP) results in a rise in von Willebrand factor (VWF) and factor VIII plasma levels. We hypothesized that gain-of-function variations in the V2R gene (AVPR2) would lead to higher plasma levels of VWF and FVIII. METHODS AND RESULTS: We genotyped the control populations of two population-based studies for four AVPR2 variations: a-245c, G12E, L309L, and S331S. Rare alleles of a-245c, G12E, and S331S, which were in linkage disequilibrium, were associated with higher VWF propeptide, VWF and FVIII levels. The functionality of the G12E variant was studied in stably transfected MDCKII cells, expressing constructs of either 12G-V2R or 12E-V2R. Both V2R variants were fully glycosylated and expressed on the basolateral membrane. The binding affinity of V2R for AVP was increased three-fold in 12E-V2R-green fluorescent protein (GFP) cells, which is in accordance with increased levels of VWF propeptide associated with the 12E variant. The dissociation constant (K(D)) was 4.5 nm [95% confidence interval (CI) 3.6-5.4] for 12E-V2R-GFP and 16.5 nm (95% CI 10.1-22.9) for 12G-V2R-GFP. AVP-induced cAMP generation was enhanced in 12E-V2R-GFP cells. CONCLUSIONS: The 12E-V2R variant has increased binding affinity for AVP, resulting in increased signal transduction, and is associated with increased levels of VWF propeptide, VWF, and FVIII.

Potential of nonpeptide (ant)agonists to rescue vasopressin V2 receptor mutants for the treatment of X-linked nephrogenic diabetes insipidus.

According to the body's need, water is reabsorbed from the pro-urine that is formed by ultrafiltration in the kidney. This process is regulated by the antidiuretic hormone arginine-vasopressin (AVP), which binds to its type 2 receptor (V2R) in the kidney. Mutations in the gene encoding the V2R often lead to the X-linked inheritable form of nephrogenic diabetes insipidus (NDI), a disorder in which patients are unable to concentrate their urine despite the presence of AVP. Many of these mutations are missense mutations that do not interfere with the intrinsic functionality of V2R, but cause its retention in the endoplasmic reticulum (ER), making it unavailable for AVP binding. Because the current treatments for NDI relieve its symptoms to some extent, but do not cure the disorder, cell-permeable antagonists (pharmacological chaperones) have been successfully used to stabilise the mutant receptors and restore their plasma membrane localisation. Recently, cell-permeable agonists also were shown to rescue ER-retained V2R mutants, leading to increased cAMP levels and translocation of aquaporin-2 to the apical membrane. This makes V2R-specific cell-permeable agonists very promising therapeutics for NDI as a result of misfolded V2R receptors.

Phage proteomics: applications of mass spectrometry.

Current techniques in mass spectrometry (MS) allow sensitive and accurate identification of proteins thanks to the in silico availability of these protein sequences within databases. This chapter provides a short overview of MS techniques used in the identification of phage structural proteins and focuses on an electron spray peptide ionization (ESI-MS/MS) approach to identify the phage structural proteome in a comprehensive and systematic ways. Such analyses provide an experimental examination of structural proteins and confirm genome-based gene predictions.

Response of cats to familiar and unfamiliar human contact using continuous direct arterial blood pressure measurement.

Continuous direct measurement of feline arterial blood pressure (ABP) was carried out via a modified method with percutaneous, ultrasound guided catheterization of the common carotid artery. In 21 healthy, conscious cats the ABP was measured during rest, alertness and activity. Furthermore, the ABP response to being petted by familiar and unfamiliar persons was assessed. Linear mixed modelling revealed that the mean blood pressure (MBP) in resting cats (114.6mmHg) was lower (P<0.001) than in alert cats (122.7mmHg), which was lower (P<0.001) than that of active cats (136.8mmHg). The MBP during petting by a familiar person (144.7mmHg) tended to be higher (P=0.065) than that during petting by an unfamiliar person (139.4mmHg). The MBP of active cats was lower (P=0.003) than MBP of cats petted by a familiar person, but not different from MBP of cats petted by an unfamiliar person. The MBP returned to resting values between 16 and 20min after the familiar person had left, whereas resting values were reached between 11 and 15min after the unfamiliar person had left. The complications of the described method were limited considering the potential risks of continuous direct ABP measurement. In conclusion, the described technique enables accurate measurement of feline ABP, which is influenced by the cat's activity level and the familiarity of persons.

Alpha-smooth muscle actin (alpha-SMA) and nestin expression in reactive astrocytes in multiple sclerosis lesions: potential regulatory role of transforming growth factor-beta 1 (TGF-beta1).

AIMS: Rapid and extensive activation of astrocytes occurs subsequent to many forms of central nervous system (CNS) injury. Recent studies have revealed that the expression profile of reactive astrocytes comprises antigens present during astrocyte development. Elevated levels of the injury-related cytokine transforming growth factor-beta 1 (TGF-beta1) secreted by microglial cells and invading macrophages have been correlated with the reactive astrocyte phenotype and glial scar formation. METHODS: In the present study, the expression profile of alpha-smooth muscle actin (alpha-SMA) and nestin, two cytoskeletal proteins expressed during astrocyte development, was studied in multiple sclerosis (MS) lesions. In addition, alpha-SMA and nestin organization and expression were analysed in rat primary astrocyte cultures in response to TGF-beta1. RESULTS: In active lesions and in the hypercellular margin of chronic active MS lesions, immunostaining for alpha-SMA revealed a subpopulation of reactive astrocytes, whereas the majority of reactive astrocytes expressed nestin. alpha-SMA and nestin expressing reactive astrocytes were in close relationship with TGF-beta1 expressing macrophages or microglia. In addition, TGF-beta1 expression within alpha-SMA or nestin expressing astrocytes was also detected. Our in vitro experiments showed that TGF-beta1 regulated the organization and expression of alpha-SMA and nestin in astrocytes. CONCLUSIONS: Reactive astrocytes in active MS lesions re-express alpha-SMA and nestin. We suggest that the in vivo re-expression might be under regulation of TGF-beta1. These results further clarify the regulation of astrocyte activity after CNS injury, which is important for the astroglial adaptation to pathological situations.

Water in health and disease: new aspects of disturbances in water metabolism.

Vasopressin is a critical regulator of water homeostasis. There are two major receptors for vasopressin: V1 and V2 receptors. Disturbances in water balance are commonly encountered in clinical practice and can be divided into disorders of urinary dilution and concentration. The major representatives of such disorders are diabetes insipidus and the syndrome of inappropriate secretion of antidiuretic hormone (SI ADH). Recent studies show that genetic forms of nephrogenic diabetes insipidus are due to mutations in the genes coding for the vasopressin V2 receptor (V2R) or aquaporin-2 (AQP2). Identification of the genes involved and analysis of the cellular fate of the V2R and AQP2 mutants are relevant for understanding the functioning of the V2R and AQP2 protein. These developments also have implications for future therapeutic options. The development of nonpeptide vasopressin receptor antagonists (VRAs) offers prospects for the treatment of euvolaemic (SI ADH) or hypervolaemic hyponatraemia (congestive heart failure or cirrhosis). Several nonpeptide VRAs are now in various stages of clinical trials. At present, only conivaptan is registered by the FD A for intravenous treatment of euvolaemic and hypervolaemic hyponatremia. A recent long-term study comparing tolvaptan with placebo in patients with chronic heart failure showed no reduction in risk of death and hospitalisation.

Insulin sensitivity and beta-cell function in healthy cats: assessment with the use of the hyperglycemic glucose clamp.

A hyperglycemic clamp (HGC) was developed for use in conscious cats. In 21 healthy, normal glucose tolerant cats glucose disposal rate (M), insulin sensitivity (ISI (HGC)), and beta-cell response (I) at arterial plasma glucose of 9 mmol.l (-1) were measured. The HGC was tolerated well and steady state glucose infusion was achieved. Compared to values reported for humans, M values for the cats were low, which appeared to relate to both a low ISI (HGC) and a low I. HGC measures correlated with fasting plasma glucose and insulin concentrations as well as with their HOMA (homeostasis model assessment) and QUICKI (quantitative insulin sensitivity check index) counterparts. Also, I and ISI (HGC) correlated with their counterparts derived from intravenous glucose tolerance tests. In conclusion, this is the first report of hyperglycemic glucose clamping in cats. The procedure (HGC) allows for measurements of glucose disposal, beta-cell response and insulin sensitivity. Compared to human data, both insulin sensitivity and insulin secretion appeared to be low in cats. This is compatible with the carnivorous nature of this species, for which insulin resistance would be advantageous during periods of restricted food availability.

Functional rescue of vasopressin V2 receptor mutants in MDCK cells by pharmacochaperones: relevance to therapy of nephrogenic diabetes insipidus.

Intracellular retention of a functional vasopressin V2 receptor (V2R) is a major cause of congenital nephrogenic diabetes insipidus (NDI) and rescue of V2R mutants by nonpeptide antagonists may restore their basolateral membrane (BM) localization and function. However, the criteria for efficient functional rescue of G protein-coupled receptor (GPCR) mutants at clinically feasible antagonist concentrations are unknown. We found that the four nonpeptide antagonists SR49059, OPC31260, OPC41061, and SR121463B induced maturation and rescued the BM expression of eight of nine different V2R mutants, stably expressed in physiologically relevant polarized cells. The extent of maturation and rescued BM expression correlated with the antagonists' concentration and affinity for the V2R. Displacement of the antagonists by AVP and subsequent cAMP generation inversely correlated with the antagonists' affinities for the V2R but is partially influenced by antagonist-specific aspects. Despite limited increases in maturation and cell-surface expression of V2R mutants, the low-affinity SR49059 optimally induced functional rescue at high concentrations, due to its easy displacement by vasopressin. At clinically feasible antagonist concentrations, however, only the high-affinity antagonists OPC31260 and OPC41061 induced functional rescue, as at these concentrations the extent of BM expression became limited. In conclusion, functional rescue of mutant V2Rs at clinically feasible concentrations is most effective with high-affinity antagonists. As OPC31260 and OPC41061 are clinically safe, they are promising candidates to relieve NDI. Moreover, as numerous other diseases are caused by endoplasmic reticulum-retained GPCRs for which cell-permeable antagonists become available, our finding that high-affinity antagonists are superior is anticipated to be important for pharmacotherapy development of these diseases.

Effect of octreotide on plasma concentrations of glucose, insulin, glucagon, growth hormone, and cortisol in healthy dogs and dogs with insulinoma.

The inhibitory effect of the somatostatin analogue octreotide on the secretion of insulin could be used in the treatment of insulinoma. However, current information on the effectiveness of octreotide in dogs is conflicting. Therefore, the endocrine effects of a single subcutaneous dose of 50 microg octreotide were studied in healthy dogs in the fasting state (n=7) and in dogs with insulinoma (n=12). Octreotide did not cause any adverse effects. In healthy dogs in the fasting state, both plasma insulin and glucagon concentrations declined significantly. Basal (non-pulse related) GH and ACTH concentrations were not affected. A slight but significant decrease in the plasma glucose concentrations occurred. Dogs with insulinoma had significantly higher baseline insulin concentrations and lower baseline glucose concentrations than healthy dogs in the fasting state. Plasma glucagon, GH, ACTH, and cortisol concentrations did not differ from those in healthy dogs. Baseline plasma insulin concentrations decreased significantly in dogs with insulinoma after octreotide administration, whereas plasma concentrations of glucagon, GH, ACTH, and cortisol did not change. In contrast to the effects in the healthy dogs, in the dogs with insulinoma plasma glucose concentrations increased. Thus, the consistent suppression of plasma insulin concentrations in dogs with insulinoma, in the absence of an suppressive effect on counter-regulatory hormones, suggests that further studies on the effectiveness of slow-release preparations in the long-term medical treatment of dogs with insulinoma are warranted.

Rescue of vasopressin V2 receptor mutants by chemical chaperones: specificity and mechanism.

Because missense mutations in genetic diseases of membrane proteins often result in endoplasmic reticulum (ER) retention of functional proteins, drug-induced rescue of their cell surface expression and understanding the underlying mechanism are of clinical value. To study this, we tested chemical chaperones and sarco(endo)plasmic reticulum Ca2+ ATPase pump inhibitors on Madin-Darby canine kidney cells expressing nine ER-retained vasopressin type-2 receptor (V2R) mutants involved in nephrogenic diabetes insipidus. Of these nine, only V2R-V206D showed improved maturation and plasma membrane rescue with glycerol, dimethyl sulfoxide (DMSO), thapsigargin/curcumin, and ionomycin but not with other osmolytes or growth at 27 degrees C. This revealed that rescue is mutant specific and that this mutant is prone to rescue by multiple compounds. Rescue did not involve changed expression of molecular chaperones calnexin, heat-shock protein (HSP) 70, or HSP90. V2R antagonist SR121463B treatment revealed that V2R-V206D and V2R-S167T were rescued and matured to a greater extent, suggesting that the rescuing activity of a pharmacological versus chemical chaperone is broader and stronger. Calcium measurements showed that rescue of V2R-V206D by thapsigargin, curcumin, and ionomycin was because of increased cytosolic calcium level, rather than decreased endoplasmic reticulum calcium level. The molecular mechanism underlying rescue by DMSO, glycerol, and SR121463B is different, because with these compounds intracellular calcium levels were unaffected.

Characterization of vasopressin V2 receptor mutants in nephrogenic diabetes insipidus in a polarized cell model.

X-linked nephrogenic diabetes insipidus (NDI) is caused by mutations in the gene encoding the vasopressin V2 receptor (V2R). For the development of a tailored therapy for NDI, knowledge of the cellular fate of V2R mutants is needed. It would be useful when this fate could be predicted from the location and type of mutation. To identify similarities and differences in localization, maturation, stability, and degradation of COOH-terminal GFP-tagged V2R mutants, we stably expressed nine mutants in polarized Madin-Darby canine kidney cells. The mutants V2R-L44P, -Delta62-64, -I130F, -S167T, -S167L, and -V206D were mainly expressed in the endoplasmic reticulum (ER) as immature proteins. These mutants had relatively short half-lives due to proteasomal degradation, except for V2R-Delta62-64. In contrast, V2R-R113W, -G201D, and -T204N were expressed in the ER and in the basolateral membrane as immature, high-mannose glycosylated, and mature complex-glycosylated proteins. The immature forms of V2R-R113W and -T204N, but not V2R-G201D, were rapidly degraded. The mature forms varied extensively in their stability and were degraded by only lysosomes (V2R-T204N and wild-type V2R) or lysosomes and proteasomes (V2R-G201D, -R113W). These data reveal that most missense V2R mutations lead to retention in the ER and suggest that mutations that likely distort a transmembrane domain or introduce a charged amino acid close to it make a V2R mutant more prone to ER retention. Because six of the mutants tested showed significant increases in intracellular cAMP levels on transient expression in COS cells, activation of these six receptors following rescue of cell-surface expression might provide a cure for NDI patients.

Protein profiling of 3T3-L1 adipocyte differentiation and (tumor necrosis factor alpha-mediated) starvation.

The increased incidence of obesity and related disorders in Western societies requires a thorough understanding of the adipogenic process. Data at the protein level of this process are scarce. Therefore we performed a proteome analysis of differentiating and starving 3T3-L1 cells using two-dimensional gel electrophoresis combined with mass spectrometry. Effects of different starvation conditions were examined by subjecting 3T3-L1 adipocytes to caloric restriction, either in the absence or the presence of the lipolysis inducer tumor necrosis factor-alpha. Ninety-three differentially expressed proteins were found during differentiation and starvation of 3T3-L1 cells, 50 of which were identified. GenMAPP/MAPP-finder software revealed a non-reciprocal regulation of the glycolytic pathway during 3T3-L1 differentiation followed by starvation. Furthermore, proteins involved in growth regulation, cytoskeletal rearrangements and protein modification, 16 of which have not been described before in 3T3-L1 cells, were identified. In conclusion, our data provide valuable information for further understanding of the adipogenic process.

Functional display of family 11 endoxylanases on the surface of phage M13.

Two family 11 endoxylanases (EC 3.2.1.8) were functionally displayed on the surface of bacteriophage M13. The genes encoding endo-1,4-xylanase I from Aspergillus niger (ExlA) and endo-1,4-xylanase A from Bacillus subtilis (XynA) were fused to the gene encoding the minor coat protein g3p in phagemid vector pHOS31. Phage rescue resulted in functional monovalent display of the enzymes as was demonstrated by three independent tests. Firstly, purified recombinant phage particles showed a clear hydrolytic activity in an activity assay based on insoluble, chromagenic arabinoxylan substrate. Secondly, specific binding of endoxylanase displaying phages to immobilized endoxylanase inhibitors was demonstrated by interaction ELISA. Finally, two rounds of selection and amplification in a biopanning procedure against immobilized endoxylanase inhibitor were performed. Phages displaying endoxylanases were strongly enriched from background phages displaying unrelated proteins. These results open perspectives to use phage display for analysing protein-protein interactions at the interface between endoxylanases and their inhibitors. In addition, this technology should enable engineering of endoxylanases into novel variants with altered binding properties towards endoxylanase inhibitors.

Identification and characterization of a highly thermostable bacteriophage lysozyme.

Pseudomonas aeruginosa bacteriophage phiKMV is a T7-like lytic phage. Liquid chromatography-mass spectrometry of the structural proteins revealed gene product 36 (gp36) as part of the phiKMV phage particle. The presence of a lysozyme domain in the C terminal of this protein (gp36C) was verified by turbidimetric assays on chloroform-treated P. aeruginosa PAO1 and Escherichia coli WK6 cells. The molecular mass (20,884 Da) and pI (6.4) of recombinant gp36C were determined, as were the optimal enzymatic conditions (pH 6.0 in 16.7 mM phosphate buffer) and activity (4800 U/mg). Recombinant gp36C is a highly thermostable lysozyme, retaining 26% of its activity after 2 h at 100 degrees C and 21% after autoclaving. This thermostability could prove an interesting characteristic for food conservation technology.

Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells.

Binding of arginine-vasopressin (AVP) to its V2 receptor (V2R) in the basolateral membrane of principal cells induces Aquaporin-2-mediated water reabsorption in the kidney. To study the regulation of the V2R by dDAVP in a proper model, a polarized renal cell line stably-expressing V2R-GFP was generated. Labeled AVP-binding studies revealed an equal basolateral vs. apical membrane distribution for V2R-GFP and endogenous V2R. In these cells, GFP-V2R was expressed in its mature form and localized for 75% in the basolateral membrane and for 25% to late endosomes/lysosomes. dDAVP caused a dose- and time-dependent internalization of V2R-GFP, which was completed within 1 h with 100 nM dDAVP, was prevented by coincubation with a V2R antagonist, and which reduced its half-life from 11.5 to 2.8 h. Semiquantification of the V2R-GFP colocalization with E-cadherin (basolateral membrane), early endosomal antigen-1 (EEA-1) and lysosome-associated membrane protein-2 (LAMP-2) in time revealed that most dDAVP-bound V2R was internalized via early endosomes to late endosomes/lysosomes, where it was degraded. The dDAVP-internalized V2R did not recycle to the basolateral membrane. In conclusion, we established the itinerary of the V2R in a polarized cell model that likely resembles the in vivo V2R localization and regulation by AVP to a great extent.

Profiling of the secreted proteins during 3T3-L1 adipocyte differentiation leads to the identification of novel adipokines.

Adipose tissue is an endocrine organ capable of secreting a number of adipokines with a role in the regulation of adipose tissue and whole-body metabolism. We used two-dimensional gel electrophoresis combined with mass spectrometry to profile the secreted proteins from (pre)adipocytes. The culture medium of 3T3-L1 cells during adipocyte differentiation was screened, and 41 proteins that responded to blocking of secretion by 20 degrees C treatment and/or brefeldin A treatment were identified. Prohibitin, stress-70 protein, and adhesion-regulating molecule 1 are reported for the first time as secreted proteins. In addition, procollagen C-proteinase enhancer protein, galectin-1, cyclophilin A and C, and SF20/IL-25 are newly identified as adipocyte secreted factors. Secretion profiles indicated a dynamic environment including an actively remodeling extracellular matrix and several factors involved in growth regulation.

Approach for defining endogenous reference genes in gene expression experiments.

The quantification of gene expression by real-time polymerase chain reaction (PCR) has revolutionized the field of gene expression analysis. Due to its sensitivity and flexibility it is becoming the method of choice for many investigators. However, good normalization protocols still have to be implemented to facilitate data exchange and comparison. We have designed primers for 10 unrelated genes and developed a simple protocol to detect genes with stable expression that are suitable for use as endogenous reference genes for further use in the normalization of gene expression data obtained by real-time PCR. Using this protocol, we were able to identify human proteosome subunit Y as a reliable endogenous reference gene for human umbilical vein endothelial cells treated for up to 18 h with TNFalpha, IL-4, or IFNgamma and for B cells isolated from healthy controls and patients suffering from IgA nephropathy. Other optional endogenous reference genes that can be considered are phosphomannomutase (PPMM) and actin for endothelial cells and glyceraldehyde-3-phosphate dehydrogenase and PPMM for B cells.