Purification and partial characterization of feline pepsinogen

OBJECTIVE: To purify and partially characterize feline pepsinogen (fPG) from the gastric mucosa and compare fPG with PGs of other species. SAMPLE POPULATION: Stomachs of 6 cats. PROCEDURE: A crude protein extract was prepared from the gastric mucosa of feline stomachs. Feline PG A was purified by ammonium sulfate precipitation, weak-anion-exchange chromatography, size-exclusion chromatography, and strong-anion exchange chromatography. Partial characterization consisted of estimation of molecular weights (MWs) and isoelectric points, N-terminal amino acid sequencing, and investigation of susceptibility to pepstatin inhibition. RESULTS: Several fPG A-group isoforms were identified. The MWs of the isoforms ranged from 37,000 to 44,820. Isoelectric points were all < pH 3.0. The proteolytic activity of the activated PGs was inhibited completely by pepstatin in a range of equimolar to 10-fold molar excess. The specific absorbance of fPG A was 1.29. The N-terminal amino acid sequence of the first 25 residues of the predominant fPG A7 had 75%, 72%, 64%, and 56% homology with PG A of dogs, rabbits, cattle, and humans, respectively. Sequences of 4 other fPG A-group isoforms were similar to fPG A7. All isoforms were immunologically cross-reactive with sheep anti-fPG A7 antiserum. CONCLUSIONS AND CLINICAL RELEVANCE: PG A is the only identified type of PG in cats and, similar to pg in other species, comprises multiple isoforms. The availability of fPG A may be used to facilitate the development of an immunoassay to quantify serum fPG A as a potential marker for gastric disorders in cats.     

The effect of ticlopidine on canine platelets, fibrinogen, and antithrombin III activity

The effect of ticlopidine on platelet function, platelet number, mean platelet volume, antithrombin III activity, and fibrinogen was evaluated in 10 laboratory beagles. Ticlopidine (62 mg/kg) significantly inhibited ADP- and collagen-induced platelet aggregation within 2 days of the beginning of oral administration. Collagen-induced platelet 14C-serotonin release was not inhibited by day 9 of medication but was inhibited by day 20 in two of three beagles given medication for 32 days. Significant increases in mean platelet number were observed on days 2 and 5. The trend toward increased platelet number continued until day 16, at which time platelet number began to decrease toward baseline in three of three dogs treated for 32 days. Mean platelet volume (MPV) was significantly decreased compared to baseline on days 5 and 9. In three dogs treated for 32 days, the lowest MPV was observed on day 9 in two dogs and on day 12 in one dog. Significant changes were not observed in antithrombin III activity or fibrinogen with ticlopidine treatment.     

Altered plasma concentrations of sex hormones in cats infected by feline immunodeficiency virus or feline leukemia virus

Gender differences may affect human immunodeficiency virus (HIV) infection in humans and may be related to fluctuations in sex hormone concentration. The different percentage of male and female cats observed to be infected by feline leukemia virus (FeLV) or feline immunodeficiency virus (FIV) has been traditionally explained through the transmission mechanisms of both viruses. However, sexual hormones may also play a role in this different distribution. To study this possibility, 17β-estradiol, progesterone, testosterone, and dehydroepiandrosterone (DHEA) concentrations were analyzed using a competitive enzyme immunoassay in the plasma of 258 cats naturally infected by FIV (FIV(+)), FeLV (FeLV(+)), or FeLV and FIV (F(-)F(+)) or negative for both viruses, including both sick and clinically healthy animals. Results indicated that the concentrations of 17β-estradiol and testosterone were significantly higher in animals infected with FIV or FeLV (P < 0.05) than in negative cats. Plasma concentrations of DHEA in cats infected by either retrovirus were lower than in negative animals (P < 0.05), and F(-)F(+) cats had significantly lower plasma values than monoinfected cats (P < 0.05). No significant differences were detected in the plasma concentration of progesterone of the four groups. No relevant differences were detected in the hormone concentrations between animal genders, except that FIV(+) females had higher DHEA concentrations than the corresponding males (P < 0.05). In addition, no differences were observed in the hormone concentrations between retrovirus-infected and noninfected animals with and without clinical signs. These results suggest that FIV and FeLV infections are associated with an important deregulation of steroids, possibly from early in the infection process, which might have decisive consequences for disease progression.     

Purification and characterization of a feline hepatic insulin receptor

OBJECTIVE: To elucidate the functional characteristics of a highly purified soluble liver insulin receptor in cats. SAMPLE POPULATION: Frozen livers from domestic cats were obtained commercially. PROCEDURES: The feline hepatic insulin receptor was purified from Triton X-100 solubilized plasma membranes by the use of several chromatography matrices, including affinity chromatography on an insulin-Sepharose matrix. RESULTS: The receptor, although not homogeneous, was purified 3,000-fold. Two silver-stained protein bands were identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weight of 134,000 and 97,000, which are similar to insulin receptors isolated from other animals. This isolated receptor had steady-state insulin binding by 40 minutes at 24 C. Optimal insulin binding occurred at pH 7.8 and with 150 mM NaCl. Under these conditions, a curvilinear Scatchard plot was obtained with the isolated receptor. Using a 2 binding-site model, the feline insulin receptor had a high-affinity low-capacity site with a dissociation constant (KD; nM) of 3 and a low-affinity high-capacity site with a K(D) of 1,180. The receptor also had tyrosine kinase activity toward an exogenous substrate that was stimulated by insulin and protamine. CONCLUSIONS AND CLINICAL RELEVANCE: Many of the reported characteristics of the liver insulin receptor in cats are similar to those for the receptor isolated from other animals and tissues, although some differences exist. These similarities suggest that characterization of the feline insulin receptor is important to understanding insulin resistance in cats with diabetes as well as in humans with diabetes.     

Measurement of plasma antithrombin III activity in healthy horses

A fluorometric assay was used to determine plasma antithrombin III (AT III) activities in 15 healthy adult horses. Nearly all plasma samples had an initial value of greater than 100% thrombin inhibited, so a 1:1 dilution of the prepared samples was performed. Following dilution, the mean value of the animals was 59.17 +/- 7.4% thrombin inhibited. Mares had significantly greater AT III activity than did geldings (P less than 0.01). The results of this study indicate the horse has more AT III activity than did other domestic species in which AT III activity has been reported.     

Anaesthesia in the dyspneic canine or feline patient

The different aspects of the anaesthetic approach of dyspneic patients are outlined, and the various possibilities for sedation, anaesthetic induction and maintenance, and facilitating recovery are discussed. More specific anaesthetic approaches are presented for each of the different types of stridoric dyspnea i.e. dyspnea associated with nasal, pharyngeal, laryngeal, or tracheal stridor.     

Anaesthesia in the dyspneic canine or feline patient

Summary

The different aspects of the anaesthetic approach of dyspneic patients are outlined, and the various possibilities for sedation, anaesthetic induction and maintenance, and facilitating recovery are discussed. More specific anaesthetic approaches are presented for each of the different types of stridoric dyspnea i.e. dyspnea associated with nasal, pharyngeal, laryngeal, or tracheal stridor.     

Purification and characterization of feline ghrelin and its possible role

Ghrelin, a novel 28-amino acid peptide with an n-octanoyl modification at Ser3, has been isolated from rat and human stomach as an endogenous ligand for the growth hormone secretagogue receptor. Here, we purified feline ghrelin and examined its possible physiological role in cats. The major active form of feline ghrelin is a 28-amino acid peptide octanoylated (C8:0) at Ser3; except for one amino acid residue replacement, this structure is identical to those of rat and human ghrelins. However, much structural divergence in peptide length and fatty acid modification was observed in feline ghrelin: peptides consisting of 27 or 26 amino acids lacking Gln14 and/or Arg28 were found, and the third serine residue was modified by octanoic acid (C8:0), decanoic acid (10:0), or unsaturated fatty acids (C8:1, C10:1 and C10:2). In agreement with the structural divergence, two kinds of cDNA with different lengths were isolated. Administration of synthetic rat ghrelin increased plasma growth hormone levels in cats, with a potency similar to that in rat or human. Plasma levels of ghrelin in cats increased approximately 2.5-fold after fasting. The present study indicates the existence of structural divergence in feline ghrelin and suggests that, as in other animals, ghrelin may play important roles in GH release and feeding in cats.     

Total selenium concentrations in canine and feline foods commercially available in New Zealand

AIMS: To determine the total selenium concentrations in petfoods commercially available in New Zealand and to establish whether these meet the current minimum recommended requirements of selenium in foods for cats and dogs. METHODS: Samples (n=89) from petfoods commercially available in New Zealand were analysed for total selenium concentration using a fluorometric method. Data, expressed on a dry matter (DM) basis, were analysed according to petfood type (dog or cat, and wet or dry), predominant flavour (chicken, seafood, chicken and seafood, beef, meat mix, other), manufacturer and country of manufacture. RESULTS: Fifty percent of petfoods purchased for this study were manufactured in Australia, and the remainder were produced in the United States of America (USA), New Zealand or Thailand. Mean total selenium concentrations were similar (0.61-0.80 mg/kg DM) in petfoods produced in Australia, New Zealand and the USA, but higher (mean 3.77 mg/kg DM; p<0.05) in petfoods produced in Thailand. Petfoods produced in Australia, New Zealand and the USA contained a variety of predominant flavours, whereas petfoods from Thailand contained only seafood flavour. Seafood-based flavours had the highest selenium concentrations in both cat and dog foods. Wet and dry dog foods had similar concentrations of selenium to dry cat foods, but wet cat foods had higher and more variable concentrations of selenium than these others (p<0.05). The mean selenium concentrations in cat and dog foods were 1.14 and 0.40 mg/kg DM, respectively, and there were no significant differences between manufacturers. CONCLUSIONS: Selenium concentrations in commercial petfoods sold in New Zealand appeared to meet recommended dietary requirements, although the range of concentrations was highly variable. Whether these recommendations are adequate for the maintenance of optimal health in cats and dogs has yet to be determined. CLINICAL RELEVANCE: Overt selenium deficiency disorders are unlikely in dogs and cats in New Zealand fed commercial petfoods unless the bioavailability of selenium in particular petfoods is low.     

Purification and partial characterization of canine pepsinogen A and B

OBJECTIVE: To purify and partially characterize various isoforms of canine pepsinogen (PG) from gastric mucosa. SAMPLE POPULATION: Stomachs obtained from 6 euthanatized dogs. PROCEDURE: Mucosa was scraped from canine stomachs, and a crude mucosal extract was prepared and further purified by use of weak anion-exchange chromatography, hydroxyapatite chromatography, size-exclusion chromatography, and strong anion-exchange chromatography. Pepsinogens were characterized by estimation of molecular weights, estimation of their isoelectric points (IEPs), and N-terminal amino acid sequencing. RESULTS: Two different groups of canine PG were identified after the final strong anion-exchange chromatography: PG A and PG B. Pepsinogens differed in their molecular weights and IER Pepsinogen B appeared to be a dimer with a molecular weight of approximately 34,100 and an IEP of 4.9. Pepsinogen A separated into several isoforms. Molecular weights for the various isoforms of PG A ranged from 34,200 to 42,100, and their IEPs ranged from 4.0 to < 3.0.The N-terminal amino acid sequence for the first 25 amino acid residues for PG A and B had good homology with the amino acid sequences for these proteins in other species. CONCLUSIONS AND CLINICAL RELEVANCE: Canine PG B and several isoforms of canine PG A have been purified. Availability of these PGs will facilitate development of immunoassays to measure PG in canine serum as a potential diagnostic marker for gastric disorders in dogs.     

Canine haemobartonellosis, canine hepatozoonosis, and feline cytauxzoonosis

Although Lyme borreliosis (Lyme disease), ehrlichiosis, Rocky Mountain spotted fever, and babesiosis occur more frequently in dogs or cats, from a clinical standpoint, other tick-borne diseases such as canine haemobartonellosis, canine hepatozoonosis, and feline cytauxzoonosis are just as important to recognize. Information concerning these less common tick-borne diseases are discussed, including their causative agents and their relationship to disease pathogenesis, diagnosis, treatment, and prevention.     

Localization of canine, feline, and mouse renal membrane proteins

Immunohistochemistry allows the localization of proteins to specific regions of the nephron. This article reports the identification and localization of proteins in situ within normal canine, feline, and mouse kidney by immunohistochemistry; maps their distribution; and compares results to previously reported findings in other species. The proteins investigated are aquaporin 1, aquaporin 2, calbindin D-28k, glutathione S-transferase-α, and Tamm-Horsfall protein. Aquaporins are integral membrane proteins involved in water transport across cell membranes. Calbindin D-28k is involved in renal calcium metabolism. Glutathione S-transferase-α is a protein that aids in detoxification and drug metabolism. The role of Tamm-Horsfall protein is not fully understood. Proposed functions include inhibition of calcium crystallization and reduction of bacterial urinary tract infection. The authors' findings in the dog are similar to those in other species: Specifically, the authors localize aquaporin 1 to the proximal convoluted tubule epithelium, vasa recta endothelium, and descending thin limbs; aquaporin 2 to collecting duct epithelium; and calbindin D-28k within distal convoluted tubule epithelium. Glutathione S-transferase-α has variable expression and is found in only the renal transitional epithelium in some individuals, in only the proximal straight tubules in others, or in both locations in others. Tamm-Horsfall protein localizes to thick ascending limb epithelium. These findings are similar in the cat, with the exception that aquaporin 1 is located in glomerular podocytes, in addition to proximal convoluted tubule epithelium, and glutathione S-transferase-α is found solely within the proximal convoluted tubule within all kidney samples examined. The mouse kidney is almost identical to the dog but expresses glutathione S-transferase-α in the glomeruli only.     

Effects of storage temperature and time on canine plasma ammonia concentrations

Stability of ammonia in canine plasma was determined, with regard to temperature and time of storage. Heparinized venous blood samples were collected from 8 healthy dogs, immediately placed in an ice water bath, and centrifuged at 5 C. Plasma was harvested from the blood samples, and the initial analysis of each sample for plasma ammonia was performed within 30 minutes after collection. Separate aliquots of the plasma from each dog were stored at 21 C, 4 C, -15 C, or -40 C. Ammonia concentrations of the aliquots stored at the various temperatures were determined at 24, 48, and 96 hours after collection. Statistical analysis of the data from each dog did not indicate a significant relationship between the initial concentration of plasma ammonia and subsequent determinations. Correlation or significance was not found among samples stored at similar temperatures and evaluated at similar times.     

Pathophysiology of antithrombin III deficiency

This article focuses on the pathophysiology of thrombosis in patients with acquired antithrombin III deficiency. Antithrombin III is an important natural inhibitor of the hemostatic mechanism, and a hypercoagulable state is often induced in diseases causing antithrombin III deficiency. Laboratory determination of antithrombin III activity is particularly useful in the clinical evaluation and therapeutic management of patients with disseminated intravascular coagulation, nephrotic syndrome, and severe hepatopathy.     

Ultrastructure of canine, feline, and bovine mast cell neoplasms

Biopsy specimens were collected from skin or spleen from 8 dogs, 7 cats, and 1 cow with mast cell neoplasms. Following histopathologic grading, the neoplasms were examined by transmission electron microscopy with the intention of reviewing their fine structure and recording new findings. For 2 feline specimens, short-term cell cultures were established, and adherent cells were fixed in situ and examined with the electron microscope. In addition to the usual array of mast cell organelles, including Golgi apparatus, secretory granules, mitochondria, vesicles, tubules, microfilaments, and ribosomes, important findings included coarse interdigitation of cytoplasmic projections between adjacent mast cells; intracytoplasmic parallel stacks of 10 nm diameter filaments as well as parallel arrays of coarse, 120 to 150 nm diameter tubules; the appearance of coated vesicles and caveolae on cell surfaces; the appearance of endocytosed erythrocytes; and the formation of giant secretory granules.     

Measurement of aldosterone in feline, canine and human urine

BACKGROUND: Systemic hypertension is an important problem in older cats associated with kidney disease and hypokalaemia, suggesting that excessive activity of the renin-angiotensin-aldosterone system might contribute to the hypertensive state. Fluctuations in plasma renin activity and plasma aldosterone concentrations complicate the interpretation of these assays. OBJECTIVES: The aim of this study was to determine whether measurement of urinary aldosterone excretion in cats aided the investigation of hypertension. METHODS: Urine concentrations of free (ethyl acetate extract) and 18-glucuronidated aldosterone (acid hydrolysis before extraction) were measured by radioimmunoassay in normal, normotensive and hypertensive azotaemic cats (n=11 per group). Urine samples from 11 healthy human volunteers and eight normal dogs were also analysed for comparison. Urinary aldosterone concentration was corrected for the urinary creatinine concentration. RESULTS: Cats excreted 7.3 times less free aldosterone than human beings, and no free aldosterone was detected in dog urine. Acid hydrolysis led to large increases in aldosterone recovery from both human beings and dog but not feline urine. No significant effect of hypertension or azotaemia on feline urinary aldosterone concentration was found. CLINICAL SIGNIFICANCE: Measurement of aldosterone in feline urine using the available methodology has limited or no utility in investigating feline hypertension.     

Preservative effect of aprotinin on canine plasma immunoreactive adrenocorticotropin concentrations

The susceptibility of adrenocorticotropin (ACTH) in canine blood and plasma to enzymatic degradation has limited the availability of endogenous ACTH assay for veterinary use. This study examined if a proteinase (enzyme) inhibitor, aprotinin, mixed with blood at the time of collection, would limit the loss of immunoreactive (IR) ACTH from canine plasma stored at various temperatures. Blood was collected from laboratory-maintained dogs or dogs with hyperadrenocorticism and placed into EDTA-containing tubes in the presence or absence of aprotinin. Plasma obtained was stored for 4 d at temperatures ranging from −86° C to room temperature (22° C). Results showed that addition of aprotinin preserved IR-ACTH concentrations in plasma stored for 4 d at temperatures ≤ 4° C, or in unfrozen plasma stored inside insulated shipping containers containing frozen refrigerant packs. Plasma collected with aprotinin and stored at 22° C showed a slight (17–23%) but significant (P < 0.05) decline in IR-ACTH. Unfrozen plasma collected without aprotinin showed significant (P < 0.05) loss of IR-ACTH during storage under identical conditions. These data indicate that aprotinin has a profound preservative effect upon canine plasma IR-ACTH and that it may be possible to submit unfrozen samples collected with this inhibitor to appropriate reference laboratories for analysis of IR-ACTH.     

Basics in canine and feline rhinoscopy

Patients suffering from upper respiratory disease such as chronic nasal congestion, sneezing, nasal discharge, and epistaxis invite complete evaluation of their paired nasal cavities. Thorough assessment of these cavities employs sundry diagnostic procedures that enable the investigating clinician to characterize the internal structures of the nasal cavities. After the conscious patient undergoes a complete physical examination, a gross assessment of its external nasal structures is established and areas of physical asymmetry are noted. A working anatomic knowledge of these asymmetric foci helps to guide the next diagnostic steps. The patient is then placed under general anesthesia, during which, in list order, imaging studies, rhinoscopy, and nasal biopsy or foreign body retrieval, are performed.