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.     

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.     

A role of ghrelin in cancerogenesis

Ghrelin is a 28 amino-acid multi-functional peptide hormone, which was identified as a natural ligand of the growth hormone secretagogue receptor (GHS-R). Pituitary growth hormone-releasing activity in both animals and humans has been well documented. It has various biological functions, including regulation of appetite and body weight, control of energy homeostasis, modulation of cardiovascular and gastrointestinal system and anti-inflammatory effect. However, both ghrelin and its receptor (GHS-R) are widely distributed in various tumors, which strongly implies their role in neoplastic cell growth trough autocrine/paracrine mechanism. Multiple studies have demonstrated the role of ghrelin in cancer cells proliferation, differentiation, invasiveness and apoptosis inhibition. The ghrelin axis is more complex than it was originally thought and consist of several compounds that might interact with each other and affect ghrelin activities. Here, we provide an overview of the ghrelin and its receptor role in tumor progression.     

Basic biological characterization of feline morbillivirus

Feline morbillivirus (FmoPV) is an emerging virus that was recently discovered in domestic cats with chronic nephritis. Despite the potential role of FmoPV in chronic nephritis, little is known about its biological characteristics. In this study, we established a quantitative assay of FmoPV by using an indirect immunofluorescence technique. Viral titers of FmoPV were determined in one week. Treatment with polybrene^® or trypsin which was previously used in virus isolation did not augment the virus titers. FmoPV was notably stable at 4°C, retaining high titers for at least 12 days. Heat-treatment at 60°C and 70°C effectively inactivated FmoPV in 10 and 2 min, respectively. The biological characteristics of FmoPV reported here will be beneficial for establishing an efficient virus isolation method and will provide important information to take a measure to reduce the risk of FmoPV infection.     

Cytochemical characterization of feline leukemic cells

Hematopoietic cells in blood and/or bone marrow from 23 leukemic cats and ten control cats were characterized using a battery of cytochemical enzyme stains. The results of cytochemical staining led to modification of diagnosis based on clinical, hematologic and histopathologic findings in four (17%) of the leukemias. Sudan black-B and chloroacetate esterase served as granulocytic markers in both the control and leukemic groups. Peroxidase activity was seen in the granulocytes and monocytes of control animals but not in the blasts of leukemic cats. Diffuse alpha naphthyl butyrate esterase staining marked monocytes in both control and leukemic animals. Cytochemical staining was found to be a valuable aid in the classification of leukemias in the cat.     

相思子毒素-a的纯化及鉴定

采用硫酸铵分级分离法和凝胶层析法,从相思子种仁中分离纯化出一种毒蛋白P1′。凝胶薄层扫描测得纯度≥97%;SDS-PAGE电泳测定其相对分子质量为64300;经β-巯基乙醇处理,P1′分离为A、B 2条链,相对分子质量分别为29100和35400;经腹腔注射,小鼠LD50为2.11μg/kg;P1′质量浓度≥3.91 mg/L时,可凝集兔红细胞;P1′的B链N末端8个氨基酸序列为：I-V-E-K-S-I/L-I-N,与相思子毒素-a（abrin-a）和相思子毒素-b（abrin-b）同源性较高,为75%;肽质量指纹谱（PMF）分析表明,P1′与abrin-a匹配强度最高,为86%,与abrin-b、相思子毒素-c（abrin-c）和相思子毒素-d（abrin-d）匹配强度均为10%,表明毒蛋白P1′为abrin-a。abrin-a经1%甲醛减毒处理制备类毒素,家兔5次免疫后获得了效价高、特异性较强的抗血清。     

Identification and characterization of feline UBE1L gene

Interferon-stimulated gene 15 (ISG15) is one of the type I interferon-inducible proteins. Addition of ISG15 known as ISGylation is an ubiquitin-like posttranslational modification. Coexpression of ISG15 and ubiquitin-activating enzyme E1-like protein (UBE1L) is required to induce ISGylation. Previously, we identified feline ISG15 gene and found that the capsid protein of feline immunodeficiency virus was ISGylated in vitro by treatment with feline interferon-ω. In this study, we cloned feline UBE1L (FeUBE1L) gene to further study the mechanism of the antiviral activities induced by ISGylation. Sequencing analysis revealed that active sites of FeUBE1L were highly conserved. These data suggest that FeUBE1L has an enzymatic activity. Further, expression of FeUBE1L was induced in feline cell lines by treatment with feline interferon-ω and ovine interferon-τ.     

Total sialic acid: An acute phase reactant in cats with a possible role in feline coronavirus infection

The aims of this study were to validate a colorimetric method to measure total sialic acid (TSA) in feline serum and to investigate the serum concentration of TSA in clinically healthy cats seronegative (n = 9) and seropositive (n = 48) for feline coronavirus (FCoV), and in cats affected by feline infectious peritonitis (FIP, n = 28), tumors (n = 20), or inflammation (n = 16). The correlation between TSA and α₁-acid glycoprotein (AGP) was also investigated. The method employed in this study is precise and accurate at TSA levels (in mg/L) commonly encountered in feline serum. No significant differences between seropositive (385.6 ± 192.2 mg/L) and seronegative (433.5 ± 179.0 mg/L) cats were detectable, suggesting that the simple infection by FCoVs does not influence TSA levels. Compared with seropositive controls, the concentration of TSA was higher in cats with FIP (556.7 ± 268.3 mg/L, P = 0.003), tumors (522.5 ± 294.4 mg/L, P = 0.028), and inflammation (546.8 ± 208.3 mg/L, P = 0.018). The discriminating power of TSA for FIP is moderate (area under the ROC curve = 0.65) and the likelihood ratio is higher than 3.0 only at high TSA levels. Consequently, TSA could support a diagnosis of FIP only at extremely high serum concentration (> 800 mg/L) or when the pre-test probability of FIP is high. No correlations were found between the TSA and AGP concentrations in cats with FIP, suggesting that sialylated proteins other than AGP are present. Both the antibody titre and the degree of AGP sialylation were negatively correlated with TSA levels, suggesting that increased TSA may contribute to reduce the burden of FCoVs.     

Ghrelin in domestic animals: distribution in stomach and its possible role

Ghrelin, a novel growth-hormone-releasing acylated peptide, was recently isolated from rat and human stomachs. In rat, peripheral or central administration of ghrelin stimulates the secretion of growth hormone (GH) from the pituitary gland. Recent work suggests that ghrelin plays an important role in energy homeostasis, body weight, and food intake. We examined the distribution of cells immunoreactive to ghrelin in the stomachs of domestic animals and rats, using a polyclonal antibody for the N-terminal fragment of rat ghrelin [1-11]. We measured the plasma levels of ghrelin before and after feeding in cows, and GH levels after central administration of ghrelin in Shiba goats, to elucidate the possible role of ghrelin. Immunostained cells were widely distributed from the neck to the base of the oxyntic gland in all animals. The plasma ghrelin concentration in cows decreased significantly 1 h after feeding, and then recovered to pre-feeding levels. Administration of ghrelin into the third ventricle in Shiba goats dramatically increased the plasma GH concentration dose-dependently. These results suggest that ghrelin plays an important role in GH secretion and feeding regulation in domestic animals.     

Parameters of production and partial characterization of feline interleukin 2

The conditions for the production of feline interleukin 2 (IL-2) from peripheral blood leukocytes (PBL) and splenocytes by concanavalin A (Con A) stimulation are described. Feline IL-2 was quantitated by measuring DNA synthesis in the murine IL-2-dependent cell line, CTLL-20. In addition, feline IL-2 was generated for the maintenance of long-term cultures of Con A-stimulated feline PBL and for biochemical characterization. Finally, IL-2 production was evaluated from the PBL of feline leukemia virus (FeLV)-infected cats. Con A at 9.6 micrograms/ml produced a plateau of peak IL-2 activity from 24 to 48 h following stimulation. The tumor promoter, phorbol myristic acetate, stimulated feline IL-2 production and enhanced Con A-stimulated feline IL-2 production. Fetal calf serum (FCS) was not required for IL-2 production; however, FCS at 5% (v/v) allowed for maximal Con A-stimulated IL-2 production. Feline IL-2 generated from Con A-stimulated splenocytes migrated with an apparent molecular size of 13.7 to 23 kD by gel filtration chromatography and supported the proliferation of Con A-activated feline PBL at a final concentration of 0.3 to 0.9 units/ml.     

Molecular characterization of feline Hepatozoon species from Brazil

Feline Hepatozoon species from Brazil was molecular identified and characterized for the first time in S?o Paulo state, Brazil. Partial sequences of the 18S rRNA gene from the Hepatozoon from three naturally infected cats were analyzed. Sequences revealed that feline Hepatozoon was closely related to the canine Hepatozoon canis from Brazil.     

Assembly of feline calicivirus-like particle and its immunogenicity

Virus-like particles (VLPs) were produced in insect cells infected with a recombinant baculovirus containing the capsid gene of feline calicivirus strain F9 (FCV-F9). The FCV VLPs were morphologically and antigenically similar to the native virus and contained a single capsid protein with a molecular weight of approximately 60 kDa that reacted with FCV antiserum. Moreover, following immunization of rabbits, VLPs were able to elicit neutralizing antibodies against several FCV strains isolated from clinical samples. Our preliminary results showed that FCV-VLP could be considered a candidate vaccine against FCV infections.     

Establishment and characterization of eight feline mammary adenocarcinoma cell lines

Eight new feline mammary adenocarcinoma cell lines derived from either primary or metastatic lesions were established. The morphology of all the cell lines was epithelioid and round to spindle in shape, with cell growth occurring in a monolayer fashion. On immunohistochemistry, these cells reacted with anti-keratin and anti-vimentin antisera. The doubling time of these cells was between 19 and 54 hr. Tumor masses were developed in nude mice by subcutaneous inoculation of the cells that were histologically identical to their original mammary tumor lesions. Telomerase activities measured using the telomeric repeat amplification protocol assay revealed high telemetric activity in all of the cells.     

Desmin as a possible immunohistochemical marker for feline hypertrophic cardiomyopathy

Desmin has been suggested as a possible histopathological marker for hypertrophic cardiomyopathy (HCM) in humans. To test whether a similar pattern of desmin staining applies to HCM in cats, we conducted an immunohistochemical study on myocardial samples from 13 cats (HCM 4, other cardiomyopathies (OCM) 4, and control 5). The pattern of staining for desmin in HCM cats was not the same as that reported in humans, but was weaker than in OCM cats and controls. This suggested that desmin may be a possible histochemical marker for feline HCM, but our data was insufficient to clearly confirm this.     

Some ultrastructural findings on feline mammary carcinomas and their possible immunological significance

Feline mammary carcinoma is an aggressive highly metastatic malignant neoplasm occurring spontaneously in an outbred population. Tumour tissues from 20 clinical cases of simpletype adenocarcinomas (primary and metastatic tumours, cell suspensions, and cultured cells), were examined ultrastructurally by transmission and scanning electron microscopy.No evidence of an aetiological viral agent was found. Tumour cell morphology suggested that well-developed microvilli and cell surface structure may reflect the relative low immunogenicity and high metastasising potential of these carcinomas in the cat.