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.

Antiglomerular basement membrane nephritis in beige mice. Deficiency of leukocytic neutral proteinases prevents the induction of albuminuria in the heterologous phase.

Antiglomerular basement membrane (GBM) nephritis with massive albuminuria can be induced in mice by injection of heterologous antibodies against mouse GBM. The albuminuria and the glomerular lesions in this model are not mediated by complement, but are dependent on the presence of polymorphonuclear granulocytes (PMN) in the glomeruli. Neutral serine proteinases and reactive oxygen metabolites produced by activated PMN have been implicated as agents contributing to tissue damage. We examined the role of leukocytic neutral proteinases by comparing the glomerular damage and albuminuria after injection of rabbit anti-mouse GBM antibodies in normal control mice (C57BL/6J, +/+) and in beige mice (C57BL/6J,bg/bg) in which PMN are deficient of the neutral proteinases elastase and cathepsin G. The dose-dependent albuminuria that occurred in control mice after injection of 1.4-22 mg of anti-GBM antibodies was not observed in beige mice, despite a comparable influx of PMNs in the glomeruli. By electron microscopy both strains showed a similar attachment of PMN to the denuded GBM together with swelling and necrosis of endothelial cells. Elastase activity of extracts from PMN of beige mice was only 10-15% of the activity of control mice. In vitro, GBM degradation by PMN extracts of beige mice was 70% lower than that seen in control experiments. PMNs of beige and control mice showed no differences in superoxide production. In addition, administration of scavengers of reactive oxygen metabolites, such as catalase and desferrioxamine, did not prevent the albuminuria in this model. These findings support the important contribution of leukocytic neutral proteinases to the induction of albuminuria in the acute phase of anti-GBM nephritis in the mouse.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Identification of local carboxy-terminal hydrophobic interactions essential for folding or stability of chloramphenicol acetyltransferase.

The role of the carboxy terminal in folding and stabilization of type I chloramphenicol acetyltransferase (CAT1) has been studied by mutagenesis and Fourier transform infrared analysis. We have shown that a CAT mutant truncated by seven amino acid residues folds into active protein. In this study, the last three residues of this truncated CAT mutant were randomized to detect structural information required for achieving a native enzyme conformation. Statistical analysis of sequencing data from randomly chosen mutants revealed that the amino-terminal CAT fragment of 212 amino acid residues is the shortest deletion mutant able to adopt a soluble, enzymatically active structure. This minimal length corresponds to a protein with full-length alpha5-helix in the three-dimensional crystal structure of CAT type III. The amino acid preferences at the carboxy terminal in the randomization experiments suggest that this helix also forms completely in the shortened CAT mutants. In addition correct folding and/or stabilization requires the formation of a hydrophobic + microdomain at the end of the alpha5-helix. The role of this hydrophobic interaction in CAT folding and structure stabilization is discussed.

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.

An Escherichia coli plasmid vector system for high-level production and purification of heterologous peptides fused to active chloramphenicol acetyltransferase.

A very small plasmid vector system is described for construction and high-level production of C-terminal chloramphenicol acetyltransferase (CAT) fusion proteins in Escherichia coli. The only functional elements of the plasmid are a minimal region of the ColE1 origin of DNA replication and the Tn9 cat gene, both under control of a tac promoter. Since C-terminal fusion to CAT does not interfere with chloramphenicol (Cm) resistance, plasmids are maintained under Cm selection. Because of its small size (1392 bp), the system is especially convenient for building and expression of synthetic genes and gene fragments. This concept was utilized to generate a fusion with a synthetic gene encoding the multiple-epitope fragment from the rubella virus E1 membrane protein. Affinity-purified fusion proteins were obtained in mg amounts from 100-ml batches of culture fluid, and incorporated as a specific antigen in a rubella immunoglobulin G enzyme-linked immunosorbent assay.

AgarCyto: a novel cell-processing method for multiple molecular diagnostic analyses of the uterine cervix.

In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer.

In vitro and in vivo detection of functional somatostatin receptors in canine insulinomas.

UNLABELLED: Ten dogs with hypoglycemia due to insulinomas were studied to assess the expression of somatostatin receptors (SSTRs) in canine insulinomas and its potential diagnostic value. METHODS: The response of circulating glucose and insulin concentrations to the subcutaneous administration of a somatostatin analog, octreotide, was measured. SSTRs were visualized in vitro by autoradiography. [Iodine-125-Tyr3]-octreotide and [125I-Tyr11]-somatostatin-14 (SRIF-14) were used as radioligands. SPECT was performed 6 hr after the injection of [111In-DTPA-D-Phe1]-octreotide. RESULTS: After subcutaneous injection of 50 micrograms octreotide, plasma glucose concentration rose from 2.3 +/- 0.2 mmol/liter to 3.2 +/- 0.3 mmol/liter at 3.5 hr (p < 0.05) and plasma insulin concentration decreased from 451 +/- 135 pmol/liter to a nadir of 249 +/- 115 pmol/liter at 30 min (p < 0.05). In vitro autoradiography revealed that all primary insulinomas and their metastases had specific SSTRs for both [125I-Tyr3]-octreotide and [126I-Tyr11]-SRIF-14. Scatchard analysis of SSTR binding in the tumor tissue of one dog revealed high-affinity binding sites for [125I-Tyr3]-octreotide (dissociation constant (Kd) 1.7 nM, maximum binding capacity (Bmax) 499 fmol/mg membrane protein). The primary tumor and/or metastases in five of six dogs could be visualized and localized by SPECT with [111In-DTPA-D-Phe1]-octreotide. In the remaining dog, multiple metastases (< 3 mm) were found in the liver at necropsy, apparently too small to be visualized by SPECT. CONCLUSION: The in vitro autoradiography and ligand binding studies indicate that canine insulinomas express one type of SSTR. This is in contrast with findings in humans where, on the basis of ligand binding studies, different subtypes of SSTRs have been identified. The uniformity of SSTRs, their high frequency of expression and the high incidence of metastatic disease make canine insulinomas very suitable for investigation of the value of SRIF analogs in the diagnosis and treatment of metastasized endocrine pancreatic tumors.

Locally produced growth hormone in canine insulinomas.

The production and release of GH has been demonstrated in a variety of extra-pituitary tissues. In this respect insulin-producing pancreatic tumours are also of interest because it has been observed that GH may promote islet cell proliferation. However, these effects have only been related to GH of pituitary origin and the possibility of local production of GH with autocrine-/paracrine effects has not been considered. In this study, a reverse transcriptase polymerase chain reaction (RT-PCR) was used to demonstrate the presence of GH mRNA in pancreatic tissue of five healthy dogs and insulinomas of 14 dogs. After Southern blotting of the RT-PCR products, blots were hybridized using a canine-specific GH-probe and quantified using phosphor imaging. GH gene expression was further demonstrated by in situ hybridization using a canine digoxigenin-labelled GH-specific cDNA probe. In addition, GH immunohistochemistry was performed. In five samples of normal pancreatic tissue a weak hybridization signal was found. This signal was significantly higher in nine of 12 primary tumours. In ten of 11 metastases there was a positive hybridization signal, and this signal was also significantly higher than in the primary tumours. In situ hybridization in one sample demonstrated that GH mRNA was only produced in the tumour cells. The local production of GH was confirmed by positive staining of tumour tissue with anti-GH antibodies in ten of 12 samples. It is concluded that canine insulinomas express the gene encoding GH mRNA. The locally produced GH may have an autocrine/paracrine effect on tumour progression. The relatively high expression levels in metastases of these tumours may be related to the low inhibitory influence of somatostatin outside the pancreas.

Two Streptomyces lividans 66 transfer RNA genes with anticodons corresponding to serine (AGC) and arginine (CGU) codons.

From the nucleotide sequence of a 2110-bp Streptomyces lividans 66 DNA fragment two transfer RNA genes were identified: tRNA(Ser) (AGC) and tRNA(Arg) (CGU). These tRNA genes are transcribed from the same DNA strand and both are preceded by a putative promoter structure. They are separated by an intergenic region of 191 bp. Like most Streptomyces tRNA genes described so far, they do not encode the 3' terminal CCA of mature tRNAs. Both genes are followed by extensive inverted repeats, which could serve as transcriptional terminator signals. Remarkably, these hairpin structures share an identical 9 base pair stretch (5'-GAAGCCCCG-3'). Furthermore, the tRNA(Arg) region is followed by two potential open reading frames, which are encoded on complementary strands and have 3' overlapping ends. The gene for tRNA(Arg) (CGU) is the first Streptomyces tRNA gene described so far which encodes the translation of a codon with uridine at the third (wobble) position.

Progress in Arabidopsis genome sequencing and functional genomics.

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.

Pyothorax in nine dogs.

The results of treatment of pyothorax using systemic antibiotics, drainage, and lavage of the pleural space, are reported for 9 dogs. All 9 dogs recovered completely. In 8 of the 9 dogs the follow-up period was at least 6 months and in none was there a relapse. The results obtained with this treatment are excellent in comparison with the results that have been reported for treatment with systemic antibiotics and drainage of the pleural space but without lavage. Apart from the addition of pleural lavage to the treatment protocol, the better result might be because migrating plant related foreign bodies did not seem to play an important role in the pathogenesis of pyothorax in this group of dogs.