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.

Long-term results of double-door laminoplasty for cervical stenotic myelopathy.

STUDY DESIGN: A retrospective study of the long-term results from double-door laminoplasty (Kurokawa's method) for patients with myelopathy caused by ossification of the posterior longitudinal ligament and cervical spondylosis was performed. OBJECTIVE: To know whether the short-term results from double-door laminoplasty were maintained over a 10-year period and, if not, the cause of late deterioration. SUMMARY OF BACKGROUND DATA: There are few long-term follow-up studies on the outcome of laminoplasty for cervical stenotic myelopathy. METHODS: In this study, 35 patients with cervical myelopathy caused by ossification of the posterior longitudinal ligament in the cervical spine and 25 patients with cervical spondylotic myelopathy, including 5 patients with athetoid cerebral palsy, underwent double-door laminoplasty from 1980 through 1988 and were followed over the next 10 years. The average follow-up period was 153 months (range, 120-200 months) in patients with ossification of the posterior longitudinal ligament and 156 months (range, 121-218 months) in patients with cervical spondylotic myelopathy. Neurologic deficits before and after surgery were assessed using a scoring system proposed by the Japanese Orthopedic Association (JOA score). Patients who showed late deterioration received further examination including computed tomography scan and magnetic resonance imaging of the cervical spine. RESULTS: In 32 of the patients with ossification of the posterior longitudinal ligament and 23 of the patients with cervical spondylotic myelopathy, myelopathy improved after surgery. The improvement of Japanese Orthopedic Association scores was maintained up to the final follow-up assessment in 26 of the patients with ossification of the posterior longitudinal ligament and 21 of the patients with cervical spondylotic myelopathy. Late neurologic deterioration occurred in 10 of the patients with ossification of the posterior longitudinal ligament an average of 8 years after surgery, and in 4 of the patients with cervical spondylotic myelopathy, including the 3 patients with athetoid cerebral palsy, an average of 11 years after surgery. The main causes of deterioration in patients with ossification of the posterior longitudinal ligament were a minor trauma in patients with residual cervical cord compression caused by ossification of the posterior longitudinal ligament and thoracic myelopathy resulting from ossification of the yellow ligament in the thoracic spine. CONCLUSIONS: The short-term results of laminoplasty for cervical stenotic myelopathy were maintained over 10years in 78% of the patients with ossification of the posterior longitudinal ligament, and in most of the patients with cervical spondylotic myelopathy, except those with athetoid cerebral palsy. Double-door laminoplasty is a reliable procedure for individuals with cervical stenotic myelopathy.

The gene for component-II of botulinum C2 toxin.

The gene encoding component-II of the Clostridium botulinum C2 toxin was cloned from the chromosomal DNA of C. botulinum type C strain (C)-203U28, and the nucleotide sequence was determined. The gene (bc2h) encodes a protein with 721 amino acid residues and is located at 247 bp downstream of the gene for component-I. The N-terminal 16 amino acids were identical to those obtained by analysis of the purified component-II toxin. The ORF for bc2h had only 39% homology at the amino acid level with the C.perfringens iota-Ib protein and an ATP/GTP binding site which is present in the iota-Ib protein is missing from the protein encoded by bc2h. Both genes had a homologous region that predicts a transmembrane segment.

Characterization of component-I gene of botulinum C2 toxin and PCR detection of its gene in clostridial species.

Botulinum C2 toxin is composed of two nonlinked protein components, component-I (light chain) and component-II (heavy chain). It is produced by Clostridium botulinum types C and D, and is thought to play a lethal pathogenic role. These biological activities of C2 toxin may be due to the ADP-ribosylation of non-muscle actin by component-I of the toxin. We were able to isolate two overlapping gene fragments encoding component-I from the chromosomal DNA of Clostridium botulinum type C strain (C)-203U28, and determine the complete nucleotide sequence of component-I gene. The gene for component-I, bc21, consists of one open reading frame (ORF) encoding 431 amino acid residues (1293 nucleotides) without signaling peptide sequence. The molecular mass calculated from the deduced amino acid sequence was 49400.37 Da. Mono-ADP-ribosyltransferase activity was demonstrated in the lysate from E. coli transformed by the recombinant plasmid, pGEM-C2 encompassing whole component-I gene with its own promoter.

Inhibition of phagocytosis by rainbow trout (Oncorhynchus mykiss) macrophages by botulinum C2 toxin and its trypsinized component II.

Botulinum C2 toxin (C2T) is composed of two nonlinked protein components, components I and II. The toxin reconstituted with component I and trypsinized component II inhibited phagocytosis of rainbow trout (Oncorhynchus mykiss) and mouse macrophages. Inhibition in both cell types was observed over a range of toxin concentrations that were not toxic to the cells. Because cytoplasmic action of rainbow trout macrophages is ADP-ribosylated by component I, the inhibition of phagocytosis in trout cells by C2T is probably due to inactivation of cytoplasmic actin. Moreover, phagocytosis by trout cells was also inhibited in a dose-dependent manner by trypsinized component II alone, and did not cause cell death. The present results show that the macrophages of aquatic vertebrates are susceptible to C2T, and that trypsinized component II elicits a novel biological activity by binding to the cell membrane of the macrophages.

Growth-factor-like substance in amniotic fluid in the rat: effect on the development of fetal colonic goblet cells.

Extra- and intrauterine fetuses were studied to explore the effect of amniotic fluid on the development of colonic goblet cells. Significant differences in the density of goblet cells in developing colon were observed during fetal days 19-22 between these two groups, suggesting that amniotic fluid affects the development of the colonic goblet cells, especially during the period between days 19 and 21 when rat fetuses began to swallow amniotic fluid, as demonstrated by injecting India ink into the amniotic cavity. By immunological dot blot analysis, the amniotic fluid and gastric juice from intrauterine fetuses were positive for epidermal growth factor (EGF), whereas maternal abdominal serous fluid and gastric juice from extrauterine fetus were negative for EGF. The present results indicate that the amniotic fluid during late gestation greatly affects the development of the colonic goblet cells in rat fetuses and that the trophic factors for the cells seems to be EGF or an EGF-like substance in the amniotic fluid.

ADP-ribosylation of rainbow trout (Oncorhynchus mykiss) actin by botulinum C2 toxin.

Intracellular actin of rainbow trout macrophages was ADP-ribosylated by botulinum C2 toxin, which is composed of two nonlinked protein components, component I and trypsinized component II. The actin in the supernatants of various tissue homogenates of the trout was also directly ADP-ribosylated by component I of C2 toxin, indicating that fish actin other than those of land vertebrates is susceptible to enzymatic modification by component I of C2 toxin.

Serum hemolytic activity of Babesia gibsoni-infected dogs: the difference in the activity between self and nonself red blood cells.

The serum hemolytic activity of Babesia gibsoni-infected dogs varied when assayed with nonself red blood cells from different dogs, whereas it did not vary when assayed with red blood cells, irrespective of self or nonself, from a particular dog throughout the experiment. The variety in activity determined with nonself red blood cells was not related to the type of red blood cell by DEA, D and J systems. Serum hemolytic activity with self red blood cells was different in the course of infection from that with nonself red blood cells, especially in the late stage of infection, when the activity with self red blood cells decreased more rapidly than that with nonself red blood cells. The results indicate that the serum hemolytic activity of B. gibsoni-infected dogs determined with self red blood cells probably reflects the in vivo activity, suggesting that the rapid decrease in activity in the late stage of infection is a way of acquired resistances for the host to recover from hemolytic anemia in the infection. The facts that the hemolytic activity increased by heating the serum at 56 degrees C, that the osmotic fragility of red blood cells remained almost on the same during the course of infection and that Coombs' test for red blood cells of the infected animal was negative suggest that the immune-mediated hemolytic anemia is not a possible mechanism for the progressive and severe anemia in B. gibsoni-infection. The present results support the previous notion that the increased serum hemolytic activity is at least one of the causes of anemia in canine B. gibsoni-infection.

Dirofilaria immitis: detection of parasite-specific antigen by monoclonal antibodies in glomerulonephritis in infected dogs.

For the identification of circulating parasite antigens associated with immune-complex glomerulonephritis in dogs infected with Dirofilaria immitis, monoclonal antibodies (mAbs) were generated against adult worms. A total of 11 mAbs were selected for cloning because of their high productivity and their lack of cross-reactivity with Toxocara canis in indirect immunofluorescence tests. The ability of mAbs to detect circulating antigens was examined using an antigen-capture enzyme-linked immunosorbent assay. Of the 11 mAbs, only NAK-1, an IgG2a mAb, was capable of detecting circulating antigens in 75% of infected dogs. However, this mAb was highly species-specific in its detection of circulating antigens, since sera from dogs infected with other nematode parasites were negative. Furthermore, the mAb detected antigens at the same glomerular sites in which IgG and/or C3 were deposited. The antigen deposits were observed along capillaries and/or in mesangial cells. The epitope recognized by this mAb is probably a carbohydrate, as it remained stable for 1 h at 100 degrees C and was sensitive to periodate treatment. Two bands of 62 and 26 kDa, respectively, were detected on Western blots by the mAb when sera from dogs infected with D. immitis were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted.

Visualizations of binding and internalization of two nonlinked protein components of botulinum C2 toxin in tissue culture cells.

Binding and internalization of two nonlinked components of botulinum C2 toxin were visualized in tissue culture cells with components directly labeled with fluorescence. The binding of both untrypsinized and trypsinized component II (UT-II and T-II, respectively) to common specific sites on the cell membrane was evidenced by competitive binding between fluorescence-labeled and unlabeled components. The distribution patterns of fluorescence-labeled T-II and UT-II after binding to cells at 37 degrees C were different; T-II clustered on the cell membrane and entered the cells in endosomes, whereas UT-II entered the cells inefficiently and not in vesicles and was distributed on the nuclear surface. The difference may be due to the multivalent property of T-II, which is not shared with UT-II. Fluorescence-labeled component I, which binds only to cells bound with T-II, entered cells by the same route as T-II did; both colocated on the same clusters on the cell membrane and also in the same vesicles in the cytoplasm. The present results suggest that component I of C2 toxin, which ADP-ribosylates cytoplasmic actin, directly binds to T-II but not to UT-II on the cell membrane and is internalized into cells together with T-II in the same endosomes.

Purification and characterization of heterologous component IIs of botulinum C2 toxin.

Botulinum C2 toxin (C2T) elaborated by certain strains of Clostridium botulinum types C and D is composed of separate and dissimilar two proteins, components I and II. Previous studies have shown that these two components of C2T produced by type C and D strains were immunologically heterologous and that C2T-producers were classified into three groups depending on the difference in molecular characteristics of the components I and II. In the present study, the heterologous component IIs of C2T were purified from three representative strains of the groups and the molecular characteristics of the components were compared. Immunological analyses by agar gel double immunodiffusion test showed that the component IIs purified from the three strains have the specific epitope(s) in addition to the common one(s). The biological activity of C2Ts reconstituted with component I purified from a fixed strain and component II each from the three strains differed depending on the source of the component II. These results indicate that the component II of C2T produced by C. botulinum types C and D differs in molecular structure, which reflects on the difference in the biological activity of the toxin. The present study suggests that the pathophysiological activity of C2T, which possibly causes a necrotic enteritis, is dependent on the C2T-producing bacteria infected.

Comparison of pertussis toxin (PT)-neutralizing activities and mouse-protective activities of anti-PT mouse monoclonal antibodies.

An assay of neutralizing activity against CHO cell-clustering activity, one of the most common assays for anti-pertussis toxin antibodies, did not correlate well with an assay of the protective activities of monoclonal antibodies. A better correlation between neutralization of leukocytosis-promoting or islet-activating activity and mouse protection against aerosol challenge was seen.

Immunological treatment of cough occurring in dogs with dirofilariosis.

A persistent, spasmic and productive cough known as filarial cough often occurs in dogs with dirofilariosis, and has been considered to be the consequence of an allergic response to Dirofilaria immitis. Twenty-one dogs with filarial cough were subcutaneously injected with worm antigen (200 micrograms of protein concentration) extracted from adult D. immitis once a day for 5 days. These injections were effective for 17 (81%) of the dogs, resulting in a complete cure for 7 dogs and marked improvement for 10 dogs.

Antigenic identification of excretory-secretory products of adult Dirofilaria immitis.

Excretory-secretory (ES) products collected from adult Dirofilaria immitis cultured in vitro were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. ES products of male (M-ES) and female (F-ES) worms were separated into 16 and 21 bands by SDS-PAGE with Coomassie blue and silver staining, respectively. The antigenic bands were then analyzed by immunoblotting, using pooled sera from dogs that had naturally contracted D. immitis. Sera from dogs with microfilaremic infection showed 7 bands in M-ES and 10 bands in F-ES, while those from dogs with occult infection revealed 3 bands in M-ES and 10 bands in F-ES. Among these bands, those of 14, 18, 21, 22, 29, and 32 kilodaltons (Kd) were common to M-ES and F-ES, those of 39 and 44 Kd were specific to M-ES, and those of 20, 38, 43, 53, 63, 90, 110, 125 and 136 Kd were specific to F-ES.

Serum hemolytic activity in dogs infected with Babesia gibsoni.

The hemolytic activity in serum of Babesia gibsoni-infected dogs was examined. When the activity was assayed in a reaction system consisting of similar concentrations of the serum and canine red blood cells to those in blood, significant hemolysis was observed. The activity of the serum of B. gibsoni-infected dogs, either naturally or experimentally, was always higher than that of uninfected animals. Moreover, in the experimental infection with B. gibsoni, the change in serum hemolytic activity was parallel to those of anemia and parasitemia, whereas it was inversely parallel to that of the hematocrit value. The present study revealed the presence of a hemolytic factor(s) in the serum of B. gibsoni-infected dogs, suggesting that the progressive anemia was due to hemolysis by the factor(s).

Histopathological and immunopathological evaluation of filarial glomerulonephritis in Dirofilaria immitis infected dogs.

Filarial glomerulonephritis was studied using Dirofilaria immitis infected dogs. Of 34 infected dogs examined, 15 dogs (44.1%) had histopathological lesions in the kidney. These lesions included an increased number of mesangial cells and increased thickness of the matrix, the infiltration of the small round and plasma cells into the interstitium and thickening of the basement membrane. Deposits of IgG were demonstrated in the infected dogs, whereas C3 deposits were found in all dogs. Combined immunoglobulin and complement deposits were not always found in the dogs with histopathological lesions. The mean concentration (expressed as absorbance) of circulating immune complexes (CIC) was 0.675 +/- 0.517 in infected dogs, and 0.132 +/- 0.092 in uninfected dogs. Although there was significant difference in the level of CIC between infected and uninfected dogs (P less than 0.001), 11 dogs (32.4%) in infected group were negative. Otherwise, the CIC levels were correlated to the adult worm burden (r = 0.848; P less than 0.001) but not to the number of circulating microfilariae (mf) (r = 0.398; P less than 0.05). Transfer of mf to 7 naive dogs was performed to clarify the role of mf in the pathogenesis of filariasis. Antibodies to crude mf antigen became detectable two weeks after the transfer. Neither pathologic findings nor deposits of IgG and C3 in the kidney were found in dogs examined 20 days or 70 days after transfer. There was no evidence that histopathological lesions were induced by live mf, suggesting that adult worm burdens may be more closely related to filarial nephropathy.

ADP-ribosylation of nonmuscle actin by component I of botulinum C2 toxin inactivates the ability to interact with unmodified actin.

Botulinum C2 toxin, elaborated by Clostridium botulinum types C and D, is composed of two dissimilar unassociated proteins, designated components I and II. Component I catalyzes ADP-ribosylation of nonmuscle beta- and gamma-actins but not of muscle alpha-actin. The maximal levels of ADP-ribosylation of the actin were about 1.0 mol of ADP-ribose/mol of actin. Sedimentation velocity analysis showed that ADP-ribosylated actin remained in a monomeric state even under polymerization conditions. In addition to the inactivation of self-polymerization ability, the ADP-ribosylated actin affected neither the initial rate nor the final extent of polymerization of unmodified actin as monitored by the increase in fluorescence intensity of N-pyrenyliodoacetamide-labeled actin. Electron microscopy revealed that no filaments or particles were formed from ADP-ribosylated actin in the conditions favorable for polymerization of unmodified actin; moreover, actin filaments produced from unmodified actin in the presence of ADP-ribosylated actin were not distinguishable from those from unmodified actin alone. These results indicate that the introduction of one ADP-ribose residue into the beta/gamma-actin molecule by component I inactivated the actin, preventing not only the self-assembly of the modified actins but also the interaction with unmodified actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of immunosuppressants against Dirofilaria immitis infection in dogs.

Two immunosuppressive drugs, azathiopurine (AZP) and prednisolone (PDS), were examined on dogs experimentally infected with Dirofilaria immitis in order to estimate the involvement of immunological assail in rejecting the parasite by the host. AZP was orally administered to 3 dogs daily at a dosage of 1-10 mg/kg for a period from 3 days before infection until the end of the experiment. The dose was then varied and transiently ceased according to the severity of the side effects. PDS was subcutaneously administered daily to 2 dogs. They were administered 10 mg/kg of PDS from 3 days before infection to day 15 and 5.0-8.5 mg/kg from day 60 after infection to day 70. The serum D. immitis-specific antibody level assessed by an indirect hemagglutination test was steadily decreased in the AZP-medicated dogs. However, in the PDS-medicated dogs, the antibody titer was decreased until day 32 and, thereafter, was recovered. When all dogs were sacrificed between days 145-148, an average recovery rate of worms in both the AZP- and PDS-medicated dogs was 52.5% and 49.6%, respectively, while the controls showed 42.5%. The statistical analysis revealed no significant difference among the groups, indicating that the administration of AZP and PDS was not effective in protecting the larvae from the host's immune attack.