Plasma leptin concentration in dogs: effects of body condition score, age, gender and breeds.

Leptin is a cytokine produced by adipocytes, and plays a key role in the regulation of energy balance. In the present study, we measured plasma leptin concentrations of 166 normal and obese dogs visiting veterinary practices, and clarified the influence of age, gender and breed on plasma leptin levels in dogs. Leptin levels were higher in the dogs with higher body condition scores. There was no noticeable influence of age, gender and breed, but those in optimal puppies and obese Miniature Dachshund tended to be lower than those in corresponding groups. We conclude that plasma leptin is a reliable marker of adiposity in dogs regardless of age, gender and breed variations, and thereby useful as a blood biochemistry test for health examinations and treatment of obesity.

Effect of ovariectomy and ad libitum feeding on body composition, thyroid status, ghrelin and leptin plasma concentrations in female dogs.

The objective of this study was to evaluate the effects of ovariectomy (i) and ad libitum feeding (ii) on energy intake, body weight (BW), body composition, thyroid status, leptin and ghrelin plasma concentrations. Four young adult female Beagle dogs were fed a maintenance diet for 6 weeks prior to ovariectomy, then 6 months after. Food allowance was adjusted in order to maintain optimal BW. Then, a diet slightly higher in energy concentration was fed ad libitum for 4 months. The maintenance diet was then fed ad libitum for one additional month. The maintenance of optimal BW after ovariectomy required a significant decrease in energy allowance. No increase in fat mass was observed. Ghrelin concentration remained unchanged. During the first month of ad libitum feeding, plasma ghrelin concentration and energy intake increased, then they decreased. Mean BW, plasma leptin, thyrotropin (TSH), total triiodothyronine (TT3) and total thyroxine (TT4) concentrations significantly increased over the study. The BW increase was exclusively due to an increase in body fat. In conclusion, energy allowance should be strictly controlled in spayed female dogs. The results suggest that in dogs, thyroid hormones, leptin and ghrelin concentrations change in response to a positive energy balance in an attempt to limit weight gain. However, the significant weight gain shows that this goal was not achieved.

Canine adiponectin: cDNA structure, mRNA expression in adipose tissues and reduced plasma levels in obesity.

Adiponectin is a protein synthesized and secreted by adipocytes. Decreased adiponectin is responsible for insulin resistance and atherosclerosis associated with human obesity. We obtained a cDNA clone corresponding to canine adiponectin, whose nucleotide and deduced amino acid sequences were highly identical to those of other species. Adiponectin mRNA was detected in adipose tissues, but not in other tissues, of dogs. When 22 adult beagles were given a high-energy diet for 14 weeks, they became obese, showing heavier body weights, higher plasma leptin concentrations, but lower plasma adiponectin concentrations. The adiponectin concentrations of plasma samples collected from 71 dogs visiting veterinary practices were negatively correlated to plasma leptin concentrations, being lower in obese than non-obese dogs. These results are compatible with those reported in other species, and suggest that adiponectin is an index of adiposity and a target molecule for studies on diseases associated with obesity in dogs.

Diurnal variations of serum leptin in dogs: effects of fasting and re-feeding.

Leptin is a protein synthesized and secreted primarily by adipocytes, and plays a key role in the regulation of energy balance. We have reported that serum leptin is elevated in obese dogs. In the present study, we examined diurnal variations of serum leptin in the dog, with special references to feeding and fasting cycles. Four male beagles were accustomed to feed once a day at 10:00 h, and blood samples were taken every 3 h for 24-36 h. Serum leptin concentration showed clear diurnal variations, being lowest before food intake (2.3+/-0.5 ng/mL) at 09:00 h, and highest (10.5+/-2.4 ng/mL) at 18:00 h. Such diurnal variations disappeared when the dogs were fasted. Serum insulin also showed diurnal variation with higher levels at 12:00-15:00 h. When insulin or glucose was injected in the fasted dogs to mimic the post-prandial insulin rise, serum leptin concentration was significantly increased in 4-8 h, but in both cases to a lesser extents than those after food intake. The results indicate that serum leptin concentrations change diurnally in association with feeding-fasting cycles in the dog, partially due to changes in insulin secretion.

CDNA cloning of feline leptin and its mRNA expression in adipose tissue.

Leptin, the product of the obese (ob) gene, is an adipocyte-derived hormone involved in regulating food intake and energy expenditure in humans and rodents. To determine the primary structure of feline leptin, we cloned the feline leptin cDNA using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of complementary DNA (cDNA) ends (RACE) methods. The full-length feline leptin cDNA was 2935 bp with a 501 bp open reading frame encoding the precursor peptide of 167 amino acids including 21 residues of signal peptide. The sequence of a 146-amino acid mature leptin was 81.5-91.8% homologous to those of other species. RT-PCR analysis revealed that the leptin mRNA was expressed in adipose tissues and not detected in liver, heart, kidney, lung, pancreas. brain and skeletal muscle. These data show that feline leptin is highly homologous to leptins of other species, and expressed in adipose tissues in cats.

Influence of the presence of OB-Re on leptin radioimmunoassay.

Leptin, a hormone derived from adipose tissue, regulates energy homeostasis and body weight. In the mouse, serum leptin levels, when measured by radioimmunoassay (RIA), increase by a factor of more than 50 times during pregnancy, compared with those in the non-pregnant state. It is well known that mouse placenta produces the secretory isoform of the leptin receptor, OB-Re. In order to investigate the issue of whether serum leptin levels are actually increased during pregnancy or whether the increased OB-Re concentration plays a role in this phenomenon, serum leptin levels were determined by the immunoprecipitation of leptin using anti-leptin antibody, and were found to be increased only by about ten times during pregnancy. To investigate the influence of OB-Re on leptin measurement by the RIA procedure, serum leptin levels were measured by the RIA after the addition of OB-Re to the serum. The apparent values of leptin levels increased in parallel with the amount of OB-Re added to the serum. Leptin levels, as determined by the RIA, might therefore provide artificially high values when serum levels of the secretory form of OB-R are high, in cases, for example, such as the last period of pregnancy in mice.

Sandwich enzyme-linked immunosorbent assay of canine leptin.

Leptin is the ob gene product secreted from adipocytes in mammals, and thereby its plasma level reflects body fat content. To establish an assay method for leptin in the dog, rabbit anti-canine leptin antibody was obtained using canine recombinant leptin as an antigen. This antibody reacted to canine leptin much stronger than mouse, rat and human leptins. Sandwich enzyme-linked immunosorbent assay (ELISA) using this antibody was developed. The serum leptin levels of 13 healthy dogs were in a range from 1.4 to 5.6 ng/ml with the mean +/- SEM of 3.0 +/- 0.3 ng/ml.

Comparison of venoarterial versus venovenous access in the cerebral circulation of newborns undergoing extracorporeal membrane oxygenation.

This study was designed to compare venoarterial (VA) with venovenous (VV) access in the cerebral circulation of newborn infants during extracorporeal membrane oxygenation (ECMO). Among 14 infants with VA ECMO, 7 had no intracranial complications (group 1), while the others (group 2) developed intracranial hemorrhage (ICH). In contrast, among 19 infants with VV ECMO, only 1 developed ICH. Serial echocardiograms were performed before and after 1, 6, 12, and 24 h and 2 and 3 days of ECMO. The mean cerebral blood flow (CBF) velocities were measured in the anterior cerebral artery (ACA), right and left internal carotid arteries (Rt, Lt-ICA), basilar artery (BA), and right and left middle cerebral arteries (Rt, Lt-MCA). Ejection fraction (EF), cardiac output (CO), and stroke volume (SV) were also measured using standard echography. The velocity levels in the ACA, Rt-MCA, and Lt-MCA in VA ECMO were lower than those in VV ECMO, while those in the Lt-ICA and BA in VA ECMO were higher than those in VV ECMO. The EF, CO, and SV were lower in cases of VA ECMO than in VV ECMO. In cases of VA ECMO, there were no differences between groups 1 and 2 in velocities in the ACA, Rt-ICA, or Lt-ICA. However the velocities in group 2 in the BA, Rt-MCA, and Lt-MCA were lower than those in group 1 before and during ECMO. Similarly, the EF, CO, and SV were lower in group 2 (12.0%-31.0%, 0. 10-0.32 l/min, and 0.66-1.55 ml, respectively) than in group 1 (29. 5%-49.3%, 0.25-0.63 l/min, and 2.15-3.85 ml) during ECMO. However, in the infants on VV ECMO the CBF was either maintained or gradually increased before and during ECMO. Their cardiac parameters were: EF 46.1%-53.0%, CO 0.43-0.52 l/min, and SV 2.72-3.84 ml during ECMO. It is concluded that in VA ECMO CBF velocities, particularly in infants who developed ICH, decreased after the onset of ECMO in association with poor cardiac function, while in VV ECMO they were stable, probably due to normal systemic hemodynamics and cardiac function.

Elevated expression levels of the 14-3-3 family of proteins in lung cancer tissues.

Immunochemical staining of lung cancer sections with a murine monoclonal anti-14-3-3 antibody showed a sharp discrimination of the cancer tissue from neighboring normal counterparts in 88 of 121 primary lung cancer tissue specimens of all four major lung cancer histologies; specifically, 32 of 48 adenocarcinomas, 36 of 44 squamous cell carcinomas, 10 of 13 large cell carcinomas, and 10 of 16 small cell carcinomas, respectively, were stained positively. Sets of the 10,000 x g supernatants of normal and cancerous lung tissue homogenates, each set prepared from surgically dissected tissues of the cancer and its surrounding normal part, were assayed for 14-3-3 proteins by the sandwich enzyme-linked immunosorbent assay using two different monoclonal antibodies to 14-3-3 proteins. The results of the assay demonstrated 7.2 times higher 14-3-3 protein content in the lung cancer tissue (378 +/- 200 ng ml-1) as compared with the normal lung (54 +/- 35 ng ml-1). These results indicate that the 14-3-3 family of proteins can be an effective marker for lung cancer diagnosis such as sputum cytodiagnosis and that 14-3-3 proteins might be involved in the development of lung cancers.

Cytokeratin 8 and 19 as antigens recognized by adenocarcinoma-reactive human monoclonal antibody AE6F4.

The human monoclonal antibody (MAb) AE6F4 is secreted by a human-human hybridoma line established from the in vitro immunization of normal human peripheral blood lymphocytes with the human lung adenocarcinoma cell line, A549. This MAb is strongly reactive to lung cancer tissues. In the previous study, the antigens recognized by the MAb AE6F4 were purified from A549 cells and identified as 14-3-3 protein and 31 kDa cytosolic phospholipase A2 (cPLA2). The MAb AE6F4 also binds two kinds of antigens (53 kDa and 40 kDa), which are not related to 14-3-3 protein or 31 kDa cPLA2, in the human breast adenocarcinoma cell line, MCF-7. We purified a 38 kDa antigen, which is a degradation product of 53 kDa antigen from breast adenocarcinoma MCF-7 cells using ion-exchange and hydroxyapatite column chromatography. Two partial amino acid sequences of the purified 38 kDa antigen showed 95-100% homology to human cytokeratin 8 (CK8). Two-dimensional gel electrophoresis and immunoblot analysis of intermediate filament fraction separated from MCF-7 cells demonstrated that the 53 kDa and 40 kDa antigens were CK8 and CK19, respectively. Antigenic determinants on CK8 and CK19 recognized by the MAb AE6F4 were resistant to sodium periodate treatment, although antigenic determinant on 31 kDa antigen (14-3-3 protein and(or) cPLA2) was sensitive to this treatment. These results suggest that the MAb AE6F4 reacts with both carbohydrate and peptide antigenic determinants.

Immunocytochemical detection of lung cancer cells with monoclonal antibodies to 14-3-3 proteins.

Murine monoclonal antibodies were raised against 14-3-3 proteins, the antigen of human monoclonal antibody AE6F4 which had been shown potentially useful for the immunochemical diagnosis of lung cancer via sputum cytology. Enzyme-linked immunosorbent assays of the murine anti-14-3-3 monoclonal antibodies with isolated bovine brain 14-3-3 isoforms showed that the antibodies were classified into four different profiles of isoform reactivity. The comparison of 14-3-3 isoform and lung cancer tissue on the reactivity with murine monoclonal antibodies indicated that beta isoform can be responsible for cancer recognition, whereas human monoclonal antibody AE6F4 showed preferential binding to zeta isoform. No murine monoclonal antibody of the same isoform specificity as human monoclonal antibody AE6F4 was obtained. Since murine monoclonal antibodies with different isoform specificities could immunostain lung cancer cells in sputum successfully, the combination use of murine monoclonal anti-14-3-3 antibodies with human monoclonal antibody AE6F4 is potentially useful for facilitating the sputum cytodiagnosis of lung cancer.

The 14-3-3 protein as the antigen for lung cancer-associated human monoclonal antibody AE6F4.

Human monoclonal antibody (MAb) AE6F4, which had been shown potentially useful for the immunocytological detection of lung cancer cells in sputum, was characterized for its antigen(s). Of the three MAb-reacting materials found in A549 cells by the immunoblotting analysis, the cytoplasmic 31-kDa protein extractable with phosphate-buffered saline was evidenced as the most plausible antigen by its highest content and outstanding affinity to the MAb AE6F4-derivatized Sepharose 4B column. This 31-kDa protein was identified by the amino acid sequence analysis of the CNBr-cleaved fragment as the 14-3-3 family of proteins, the members of which are known to play important physiological roles such as in the regulation of neurotransmitter levels and intracellular signal transduction. The purified 14-3-3 protein from bovine brain showed a comparable MAb-reacting activity to that of the 31-kDa protein from A549 cells in the enzyme-linked immunosorbent assay (ELISA). The significant reactivity of bovine 14-3-3 protein by MAb AE6F4, shown by the cross inhibition of antibody binding to the coated 31-kDa antigen in ELISA as well as by the inhibition of immunostaining with lung cancer tissues, consistently demonstrated that the antigen(s) recognized by the MAb was involved in the 14-3-3 protein family. It was found that the expression of the 14-3-3 protein was significantly enhanced in lung cancer tissues compared with the neighboring normal part of the lung as examined by the immunoblotting method. These results implicated that some member(s) of the 14-3-3 protein family can be the tumor marker(s), providing a rational basis for the immunocytological diagnosis of lung cancer with this human MAb.

Specific cholinergic destruction in the basal magnocellular nucleus and impaired passive avoidance behavior of rodents.

A nerve growth factor (NGF)-diphtheria toxin conjugate (NGDT) was found to selectively abolish or depress the activity of NGF receptor-bearing cholinergic neurons of the basal magnocellular nucleus (BMN). Bilateral cortical injections of NGDT impaired the retention of passive avoidance behavior in mice. A memory deficit was also revealed when cortical injections of NGDT were administered after the acquisition of a passive avoidance response. Thus, retrograde destruction of BMN cholinergic neurons by the cortical injection of NGDT interfered with both learning and memory processes. The animal model outlined here should be useful in analyzing the pathogenesis of Alzheimer's disease and the functions of the cholinergic system in the BMN.

Incorporation of glutamic acid into protein by a soluble system.

A heat-labile, non-dialyzable factor(s) in soluble fractions from Escherichia coli strains and Bacillus subtilis was found to incorporate the radioactivity of [14C]glutamic acid into 95 degrees C CCl3COOH-insoluble fraction. Incorporation catalyzed by a partially purified factor from E. coli B required ATP, Mg2+, tRNA, casein, carbonate, and 2-mercaptoethanol. A mixture of nineteen amino acids other than glutamic acid had no effect on the incorporation. Heparin, spermine and monovalent cations were inhibitory. Incorporation proceeded via glutamyl-tRNA. The incorporation from [14C]glutamyl-tRNA required Mg2+, casein, carbonate, and 2-mercaptoethanol, and there was no incorporation from [14C]aspartyl-tRNA. The reaction product was identified as protein. The incorporated moiety was the glutamyl moiety of glutamic acid and it retained a free alpha-amino group in the product protein. The incorporating factor of E. coli B was demonstrated to be glutamyl-tRNA synthetase.