Seasonal changes in sites of immunostaining of steroidogenic enzymes were examined in testes of the Japanese black bear, Ursus thibetanus japonicus. In addition, serum concentrations of testosterone and estradiol-17β were investigated by radioimmunoassay, and the seasonal changes were compared with the results of immunostaining. On the basis of morphological observations of spermatogenic activity, the reproductive cycle was divided into five periods: an active period in May and June; a degenerative period in November; a resting period in January; an early-resumptive period in March; and a late-resumptive period in April. Serum concentrations of testosterone differed with season accompanied by differences in spermatogenic activity, with baseline levels in November and January, increasing levels in March and April, and high levels in May, and April and June of the next year. Immunoreactivities specific for cholesterol side-chain cleaving cytochrome P450, 17α-hydroxylase cytochrome P450 and 3β-hydroxysteroid dehydrogenase (3βHSD) were observed in Leydig cells throughout the year. Only the percentages of Leydig cells immunopositive for 3βHSD exhibited seasonal differences that correlated with serum concentrations of testosterone. Aromatase cytochrome P450 (P450arom) was immunolocalized in Leydig and Sertoli cells throughout the year, in spermatids in May, and April and June of the next year and in myoid cells in January and March. The percentages of Leydig cells immunopositive for this enzyme increased in May, and January, March and June of the next year. On the other hand, no pattern of seasonal change in serum estradiol-17β concentration was observed. These results suggest that 3βHSD is a key enzyme in the regulation of the testosterone production in Leydig cells. Furthermore, estrogen derived from Leydig and myoid cells seems to play a role in the regulation of Leydig cells by negative feedback as a paracrine and/or autocrine mediator.
The skulls of Japanese wolf (Canis hodophilax) were osteometrically examined and compared with those of Akita-Inu. The skull total length was not statistically different between two species. However, significant differences were demonstrated between two species in some ratios concerning the frontal bone. CT examination was carried out in the Japanese wolf skull. The data indicated that the frontal sinus is not be largely developed and compressed in the dorso-ventral direction in parasagittal area. The narrow frontal sinus fitted to external shape of the frontal bone. The cribriform plate had a well-developed complicated structure in a caudal part of the ethmoid bone. These data will be useful to examine the respiratory function and the olfactory sense in the Japanese wolf.
The distribution of desmin and fibronectin in the chick embryo gonad in and after sexual differentiation was investigated immunohistochemically. In the undifferentiated gonad (stage 27), fibronectin was detected in the dorsal mesentery, basement membrane and part of the parenchyma, while desmin was not recognized. After sexual differentiation (stages 31 to 34), desmin-positive cells gradually increased in number with the progress of development. They were obviously seen in developing interstitial regions between testicular cords. However, fibronectin was hardly observed at the same stages. At stage 37, desmin-positive cells were present only in a part of the Leydig cells and myoid cells, while fibronectin was discontinuous in the basement membrane surrounding testicular cords. In addition, the expressions of desmin and fibronectin were simultaneously and adjacently recognized. These results suggest that myoid cells may produce fibronectin protein and that fibronectin mainly contributes to the formation of testicular cord basement membrane in chick embryos. Thus, the cell-to-cell and cell-to-matrix interactions may be important in testicular morphogenesis of chick embryos.
Interactions between endocrine cells and epithelial cells, mediated by neurotensin, have been proposed in the chicken thymus. In this study, other neuropeptide candidates acting as mediators in the chicken thymus were examined immunohistochemically. Endocrine cells being oval, elongated or triangular in shape were immunoreactive with antibodies against methionine-enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide. These findings suggest that 4 neuropeptides may be involved in cell-to-cell interactions in the chicken thymus.
The purpose of this study was to establish the normal range of serum apolipoprotein B-100 (APO B-100) concentration in clinically normal cattle, and to assess its abnormalities with clinical diseases. We measured the serum concentration of APO B-100 in cattle of varying ages, breeds and sex, maintained under normal field conditions. Blood samples were obtained from 735 apparently healthy cattle and 146 cows with various diseases. The concentration of serum APO B-100 in cattle was assayed by the single radial immunodiffusion method. The concentration of serum APO B-100 in healthy adult breeding bulls (mean ± SD: Holstein; 101 ± 46 μg/ml, Japanese Black; 106 ± 46 μg/ml) was significantly (P<0.001) lower than that in cows (Holstein; 259 ± 63, Japanese Black; 210 ± 46 μg/ml), while that of APO B-100 in steers (Holstein; 290 ± 86 μg/ml, Japanese Black; 302 ± 90 μg/ml) was similar to the level in cows. The concentration of serum APO B-100 in cattle varied with sex and breed. APO B-100 concentration in cattle was decreased in association with metabolic disorders such as ketosis, displaced abomasum and fatty liver. From these results, it is assumed that the level of serum APO B-100 will be applied to diagnosis of metabolic diseases in cattle.
Laboratory data in 47 dogs with caval syndrome (CS) surviving after surgical HW removal compared with those in 15 dogs with CS which died after the treatment. The number of HWs removed in surviving dogs was significantly greater than in nonsurviving dogs. The Ht value was significantly higher in nonsurviving dogs than in surviving dogs. Plasma enzyme activities ranged widely from normal to extremely high levels, and there were no significant differences in plasma enzyme activities between the surviving and nonsurviving dogs. Serum total protein, and plasma triglyceride, creatinine, glucose, calcium, sodium (Na) and chloride (Cl) levels were much similar between the HW-free and surviving dogs, but significantly different between the HW-free and nonsurviving dogs. Plasma urea nitrogen, uric acid and potassium levels were higher, and plasma Na and Cl levels were lower in nonsurviving dogs than in surviving dogs.
This investigation was performed to determine whether primary cultures of mammary cells from lactating cows would sustain production of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF), and express mRNA for cytokines interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, interferon (INF)-τ, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by the reverse transcription-polymerase chain reaction (RT-PCR) in vitro. Cryopreserved mammary epithelial cells collected from cows at 1 week post calving were plated in collagen-coated 24-well culture plates (250,000 cells/well). IL-1 and IL-6 productions were measured using a A375 cell growth inhibition assay and a 7TD1 hybridoma proliferation assay, respectively. Production of IL-1 was demonstrated in mammary epithelial cells cultured with unsupplemented medium, but was not produced by cells cultured in medium supplemented with fetal bovine serum. IL-6 production in the conditioned medium was continued at steady level until day 14, whereas IL-6-like bioactivity was not detected in medium alone. TNF-like activity was not detectable in any experiments. This study also demonstrated the expression of mRNA for multiple cytokines including IL-1α, IL-1β, IL-6, IL-10, TNF-α, and GM-CSF by RT-PCR in mammary cell cultures. The results indicate that bovine mammary epithelial cells of lactating cows produce IL-1 and IL-6 and have gene expression for multiple cytokines. This in vitro model will be useful to investigate the function and regulation of IL-1 and IL-6 in the lactating mammary gland.
Domon L, a heat-treated component of gram-negative bacterium Achromobacter stenohalis, is used for the treatment of infectious diseases of animals. Here, we investigated the immunopotentiating potential of Domon L. In vitro studies showed that Domon L enhanced nitric oxide (NO) formation from murine macrophage RAW264.7 cells in concert with interferon (IFN)-γ. The effect of Domon L on NF-κB activation was investigated, in order to understand the molecular mechanisms of enhanced NO formation by Domon L. Domon L induced translocation of NF-κB to the nucleus in RAW264.7 cells. Induction of NF-κB dependent gene expression by Domon L was further confirmed using a transfectant containing an NF-κB-luciferase reporter gene. In vivo injection of Domon L elevated both serum IL-6 and mucoprotein, whose gene expression is partly under the control of NF-κB. The spleen cells of rats treated with Domon L produced much more NO when stimulated with LPS + IFNg than spleen cells of untreated rats. These results suggest that Domon L acts as an anti-infectious agent via NF-κB activation.
The renal clearance test was carried out in 6 normal male cats and 12 male cats with chronic renal failure. The average concentrations of creatinine (Cr), urea, sodium (Na), and potassium (K) in the serum of the cats with chronic renal failure were 5.09, 136.7 (mg/100 ml), 143.9 and 3.71 (mEq/l) respectively, and the specific gravity of urine was 1.009. The renal clearances of Cr, urea , Na, and K (ml/min/kg of body weight) were 2.639 ± 0.217, 1.034 ± 0.110, 0.024 ± 0.007 and 0.266 ± 0.028, respectively in normal cats, and were 0.789 ± 0.407, 0.358 ± 0.211, 0.095 ± 0.084 and 0.872 ± 0.204 in cats with chronic renal failure. Clearance of Cr and urea was significantly lower in cats with chronic renal failure than in normal cats, while the values of Na and K were significantly higher in cats with chronic renal failure. The glomerular filtration of Cr and urea and the urinary excretion of these 4 substances were significantly higher in cats with chronic renal failure. The tubular reabsorption rates of Na and K were significantly lower in cats with chronic renal failure compared to those in normal cats, but there was no significant difference in urea and creatinine.
Benazepril (BP), an angiotensin convertive enzyme inhibitor, was administered orally once daily for 4 weeks to 31 dogs with mild to moderate (NYHA functional classes II and III) congestive heart failure caused from mitral insufficiency (MI). There were no significant changes in clinical signs, electrocardiogram findings, radiographical observations and plasma biochemical results in 11 dogs treated with placebo for 4 weeks. In 31 dogs treated with BP, appetite increased, and mean scores of heart failure signs, such as activity, exercise tolerance, cough and respiratory effort, were significantly improved. No dog displays signs suggesting systemic hypotension. One dog died suddenly on the 26th day of treatment with BP. This dog had good vigor and appetite till the evening before the death, and cough and exercise tolerance had been gradually improving. The heart rate and ECG parameters of BP treated dogs did not change significantly, but length of long axis of the heart decreased. In plasma biochemical tests, plasma urea nitrogen (UN) levels did not change significantly, and plasma creatinine (CRE) levels increased slightly within the normal ranges during BP trial. Two dogs had higher plasma UN levels with slightly higher plasma CRE levels, but had normal general condition and other biochemical results. Plasma ACE activity decreased to 57.3% of pre-treatment level at 4 weeks after BP treatment. It is concluded that BP monotherapy was efficacious at least in dogs with relatively low grade congestive heart failure caused by MI.
We studied the relationship between the degree of mitral protrusion and degree of mitral regurgitation in an experimental model in which the degree of mitral protrusion could be adjusted. The model was developed by dissecting the dorsal papillary muscle through a left atriotomy in 5 dogs and re-attaching the papillary muscle to the original site using a single mattress suture threading through the epicardium under cardiopulmonary bypass. By manipulating the suture from a position outside the epicardium, the degree of mitral protrusion could be adjusted. The long-axis view of the mitral valve was imaged by B-mode echocardiography with the transducer placed directly over the surface of the right ventricular outflow tract. The height (H) from the coaptation point or tip of the protruded cusp in relation to the mitral annular plane was measured as an index of mitral protrusion. Mitral regurgitation as a result of the mitral protrusion decreased the left ventricular systolic pressure, and increased the heart rate, mean left atrial pressure (LAPm), and ratio of left ventricular end-diastolic dimension to body weight (LVEDD/BW). H was negatively correlated to LAPm and LVEDD/BW (r= -0.723 and -0.697, respectively). Our results indicated that H expresses not only the degree of mitral protrusion but also the degree of mitral regurgitation, and were in agreement with the previous findings obtained on dogs with spontaneous mitral regurgitation.
The present study was carried out to confirm whether arthropathy in juvenile dogs induced by ofloxacin, a new quinolone antibacterial agent, may be diagnosed by magnetic resonance (MR) imaging. Three-month-old male beagle dogs were orally administered ofloxacin at 20 mg/kg once daily for 7 consecutive days. On day 8, MR images were obtained with a 4.7-tesla (T) super-conductive high magnetic field strength unit. An irregular cartilage surface and dissecans changes in the distal femoral condyle were observed. These MR findings were essentially consistent with pathologic observation showing multifocal blisters on the articular cartilage with an increased amount of turbid synovial fluid in the joint. The results demonstrate that occurrence of ofloxacin arthropathy in juvenile dogs can be clearly diagnosed by use of MR imaging.
A plasmid pLTR-DT which contained a gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (BLV-LTR) in the multicloning site of pUC-18 was entrapped in cationic liposomes composed of N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dioleoyl phosphatidylethanolamine (DOPE) and dilauroyl phosphatidylcholine (DLPC) (1:2:2, molar ratio) (TMAG-liposome), and their antitumor effect on BLV-infected tumor cells was examined in vivo. The cationic TMAG-liposome containing pLTR-DT was successively injected into the tumor transplanted to nude mice. The growth of tumor was significantly inhibited by the injection of cationic TMAG-liposome containing pLTR-DT. On the other hand, TMAG-liposome containing pUC18 plasmids showed no such effect. These results suggest that a DT-A expression plasmid under the control of BLV-LTR is highly toxic to the BLV-infected tumor cells, and that the cationic liposomes, such as TMAG-liposome, may be efficient transfection reagent for BLV-infected tumor cells and can be utilized for DT-A gene delivery into BLV-infected tumor cells in vivo.
Five cases of acute toxoplasmosis in caged squirrel monkeys (Saimiri sciureus) were reviewed. The early stages of systemic toxoplasmosis were present in all cases. In the liver, there were multiple foci of hepatocellular necrosis. The lung had diffuse interstitial pneumonia. Tachyzoites were mainly seen associated with lesions in the liver, lung, and spleen. Immunohistochemically, tachyzoites reacted with T. gondii antibody. Electron-microscopically, intracellular tachyzoite was surrounded by a thin wall and was in a membrane-bound parasitophorus vacuole.
Brain-stem auditory evoked potentials (BAEPs) were recorded in 20 common marmosets (Callithrix jacchus) to investigate the effects of click frequency up to 99 kHz, in consideration of the higher hearing range of the marmoset, and intensity on wave forms and peak latencies. According to the results of BAEP recordings at frequencies of 4, 32, and 99 kHz, the number of components recorded was affected by the stimulus intensity and the clicks at an intensity of 80 dB peak equivalent sound pressure level (pe SPL) had the maximum number of clear components. Therefore, it was indicated that click stimulations at an intensity of 80 dB pe SPL over a broad range of frequencies appears to be useful for recording the maximum number of components in marmosets and may increase the information obtainable from BAEPs. BAEP latencies were prolonged as the stimulus intensity decreased from 100 to 50 dB pe SPL. The effects of stimulus frequency on the wave latencies and amplitudes in response to 80 dB pe SPL at frequencies between 0.5 and 99 kHz revealed various changes: the amplitude of wave I increased at 16 and 32 kHz, but that of waves III and V increased at 4-8 and 64-99 kHz. These increases in amplitudes of the waves may correlate with higher synchronous activity of the peripheral or central auditory pathways.
The expression of inhibin a-subunit mRNA in equine fetal gonads during pregnancy (Days 90 to 300) was examined by means of Northern blot analysis. In all samples examined, a single species of transcript was detected at the size of 1.5 kb. A digoxigenin-labeled antisense cRNA probe specific to equine inhibin α-subunit was synthesized and in situ hybridization analysis to locate the inhibin α-subunit mRNA positive cells was performed using frozen tissue sections of equine fetal ovary (day 150 of pregnancy) and equine fetal testis (day 180 of pregnancy). In the fetal ovary, positive cells were seen throughout the interstitial area but did not show any particular localization. In the fetal testis, on the other hand, the antisense cRNA hybridized almost exclusively to the interstitial cells surrounding developing seminiferous cords and Sertoli cells within the cords. Positive signals were also detected in a limited number of the interstitial cells located away from the cords. These results suggest that in equine fetal gonads, inhibin and/or inhibin α-subunit related molecules such as the monomeric form are produced and these molecules may have a paracrine/autocrine role within the gonads.
The airway responsiveness to bradykinin (0.1, 1 and 10 μg/kg, i.v.) was examined in two lines of guinea pigs, BHS (bronchial hypersensitive) and BHR (bronchial hyposensitive) lines, with different airway sensitivity to inhalation of acetylcholine (ACh)-aerosol. Normal Hartley strain guinea pigs were used as a control group. The airway contraction was measured by recording intratracheal pressure (PIT) and respiratory airflow (V) under the condition of artificial ventilation in anesthetized guinea pigs. The results show airway responsiveness to bradykinin in BHS guinea pigs to be significantly greater than in BHR and normal Hartley strain guinea pigs.
The incidence and numbers of enterotoxigenic Clostridium perfringens in the intestinal contents of cattle, swine and broiler chickens were determined and compared with those of total (enterotoxigenic and nonenterotoxigenic) C. perfringens. The method used for the enumeration of enterotoxigenic C. perfringens consisted of a combination of the most probable number (MPN) method and a nested polymerase chain reaction after enrichment culture of the sample. Enterotoxigenic C. perfringens was found in 26% (4.0 × 10-4.3 × 102 MPN/100 g), 22% (4.0 × 10-2.3 × 103 MPN/100 g) and 40% (4.0 × 10-2.4 × 104 MPN/100 g) of intestinal contents of 50 head each of cattle, swine and broiler chickens, respectively. Whereas, total C. perfringens was found in 76% (9.0 × 10-7.5 × 106 MPN/100 g), 44% (7.0 × 10-4.3 × 106 MPN/100 g) and 80% (4.3 × 10 2-9.3 × 107 MPN/100 g) of intestinal contents of 50 head each of cattle, swine and broiler chickens, respectively, by the conventional MPN method. In all cases, enterotoxigenic cells were not dominant in the population of C. perfringens: a small number of enterotoxigenic cells of C. perfringens co-existed with a large number of nonenterotoxigenic cells in the same sample. The ratios of enterotoxigenic C. perfringens cells to total C. perfringens cells were 1/10-1/105.
The effects of natural-type human tumor necrosis factor (nh-TNF) on tumor endothelial cells of experimental brain tumors were investigated electron microscopically. Tumor vessels with hypertrophic endothelial cells were observed 12 and 24 hr after an intralesional administration of 5,000 U of nh-TNF. Increased biosynthetic organelles such as the Golgi complex and rough endoplasmic reticulum were evident in the plump cytoplasms. These endothelial cells resembled those in high endothelial venules (HEV) functionally characterized by the high permeability of leukocytes. In addition, close interactions between these endothelial cells and leukocytes were observed. Our findings indicated that nh-TNF could promote the morphological change in tumor endothelial cells into HEV-like cells.
Replication of porcine reproductive and respiratory syndrome (PRRS) virus in swine alveolar macrophages (AM) and cell population in broncho-alveolar lavage fluid (BALF) obtained from PRRS virus-infected pigs were investigated. BALF samples were periodically collected from 6 pigs infected with PRRS virus and 3 non-inoculated control pigs by means of fiber-optic bronchoscope between post-inoculation day (PID) 0 and 56. The mean ratio of macrophages in BALF collected from infected group was 92.7 ± 3.2% before inoculation and gradually decreased from PID 14. On the other hand, the ratio of lymphocytes was 4.8 ± 3.2% before inoculation and increased from PID 21 and indicated 41.8 ± 9.1% on PID 28. After that, they decreased gradually and that of macrophages correspondingly increased. The ratio of neutrophils maintained between 0.7% and 5.1%. The ratios of macrophages, lymphocytes and neutrophils collected from control group were almost stable through the examination. Intracellular PRRS virus antigens in AM were detected from PID 2 by indirect immunofluorescence assay (IIFA). PRRS virus was first isolated from BALF samples collected from inoculated group between PID 2 and 49. From serum, virus was isolated between PID 2 and 21. Antibodies in sera measured by IIFA to PRRS virus were first detected on PID 14 and the antibody titer rose to 1:640 or 1: 1,280. The results suggested that PRRS virus replicates in swine AM for a relatively long period.
PiD-10 and piD-11 cells that have been established from persistently infected DBT cells with the JHM strain of MHV (JHMV) were resistant to infection with JHMV. There was no significant difference in the amount of adsorbed virus among piD-10, piD-11 and DBT cells. When an expression of mRNA of the MHV receptor in piD-10 and piD-11 cells was analyzed by the RT-PCR method, no significant difference was observed in the intensities of the amplified products among piD-10, piD-11 and DBT cells. Treatment of virus-adsorbed cells with PEG, which induces fusion of the cellular membrane with the viral envelop, causes entry of virus particles into cells. There was no significant difference in the yields of virus between PEG-treated and PEG-untreated cells. The titers of infectious virus internalized into piD-10 and piD-11 cells were the same as those in DBT cells. When piD-10 and piD-11 cells were fused with PEG and infected with JHMV, the yields of infectious virion particles from the fused cells between piD-10 and piD-11 cells were significantly lower than those from the fused cells between DBT and piD-10 or piD-11 cells. The present study showed that resistance of piD-10 and piD-11 cells to JHMV infection is not due to an inhibition of JHMV entry into the cells.