The distribution patterns of the intensity of negative charge on the free surfaces (glycocalyx of the plasma membrane) of endo-thelial cells (ECs) in blood vessels and reticular cells (RCs) in the splenic cord of the rat spleen were studied by an electron microscopic cytochemical method using polyethyleneimine (PEI) as a cationic probe. Spleens from adult male rats were perfusion-fixed with 0.5% glutaraldehyde - 4 % paraformaldehyde containing 0.05% cetylpyridinium chloride and then perfused with 0.5% PEI at pH 7.4. On the free surfaces (glycocalyx of the plasma membrane) of the ECs examined, distinct PEI-positive reactions were observed in blood vessels, such as trabecular arteries, central arteries, arterial capillaries, pulp veins and trabecular veins. These PEI-positive electron-dense substances in the trabecular arteries, central arteries, and trabecular veins took the shape of a band of 170-250 nm in thickness. On the other hand, the corresponding ultrastructure of the ECs lining the splenic sinuses and the RCs in the splenic cord showed exceedingly weak PEI reactions. The PEI-reactive deposits were significantly thinner than those in the above blood vessels. As the thickness of the electron-dense substances can be related to the density of the negative charge, these results suggest that there is a high intensity of negative charge on the free surfaces (glycocalyx of the plasma membrane) of ECs in blood vessels where blood cells and plasma pass into the red pulp or are discharged from the red pulp. In contrast, the splenic sinuses and RCs, which are the main components of the red pulp, contain weakly negative-charged sites. This may contribute to the microcirculation of the splenic blood vessels and elucidate the possible physiological functions of the spleen, such as blood storage.
The in vitro susceptibilities of 76 isolates of Actinobacillus pleuropneumoniae collected from pigs with pleuropneumonia were tested with 12 commonly used antimicrobial drugs by an agar dilution minimal inhibitory concentration procedure according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines. Field isolates had low MICs for ceftiofur, danofloxacin and penicillin. No correlation of antimicrobial resistance was related to serotype.
A total of 90 strains of Staphylococcus intermedius isolated from dogs were examined for antimicrobial susceptibility. There were no significant differences in the distribution patterns of MICs between strains from 1982 to 1985 and those from 1999, and between strains from healthy dogs and those from diseased dogs. All of the strains were susceptible to ABPC, DMPPC, CEX, TDM, ERFX, BFLX, and FF at concentrations of 0.05 to 6.25 μg/ml. The MICs of OTC, KM, EM, AIV-TS, and LCM were distributed in a broad range of 0.1 to >100 μg/ml, indicating the existence of resistant as well as susceptible populations of S. intermedius. Thirty-three strains (36.7%) were resistant to one or more anitmicrobial agents such as OTC (n=32), KM (n=9), EM (n=7), AIV-TS (n=7), and LCM (n=7).
Apolipoprotein (apo) C-III is a low molecular mass protein mainly distributed in the high-density lipoprotein (HDL) fraction. In cows with postparturient diseases such as ketosis, concentrations of cholesterol, phospholipids and apoA-I and the activity of lecithin:cholesterol acyltransferase, which are mainly distributed in or functionally associated with HDL, are reduced. The purpose of the present study was to examine whether the serum concentration of apoC-III was similarly decreased in the postparturient diseases. Compared with healthy controls, the apoC-III concentration was significantly (P<0.01) decreased in cows with fatty liver, ketosis, left displacement of the abomasum, milk fever and retained placenta. Concentrations of apoC-III in the HDL fractions from diseased cows were also lower than in controls. Of the diseased cows, the decreased apoC-III concentration was particularly distinct in cows with milk fever. Increased nonesterified fatty acid and reduced free cholesterol, cholesteryl ester and phospholipid concentrations were observed in cows with milk fever, as in the other diseased cows. The decrease in the apoC-III concentration is suggested to be closely associated with the postparturient disorders, in particular with milk fever.
Chronic stimulation of the β3-adrenergic receptor (AR) in obese animals resulted in a reduced adiposity associated with an increased expression of thermogenic uncoupling protein (UCP)1 in adipose tissues. In this study, the mRNA expression of newly cloned UCP isoforms (UCP2 and UCP3) were examined in obese yellow KK and C57BL control mice. UCP2 mRNA was found in all tissues examined, with higher levels in adipose tissues and skeletal muscle of the obese mice. UCP3 mRNA was expressed in skeletal muscle, heart and brown adipose tissue similarly in the two mouse strains. Daily injection of a selective β3-adrenergic agonist, CL316,243 (0.1 mg/kg), for 10 days resulted in a marked reduction of white fat pad weight and 1.8~4.8-fold increase in the mRNA levels of UCP2 and UCP3 in skeletal muscle of obese mice. No noticeable change in the UCP2 and 3 mRNA levels was found in brown and white adipose tissues. It was also found that CL316,243 injection produced a marked and sustained elevation of the plasma free fatty acid level. These results, together with our previous findings of the fatty acid-induced UCP expression in a myocyte cell line in vitro, suggest that the β3-AR agonist-induced UCP expression in skeletal muscle may be mediated through the elevated plasma free fatty acids. It was also suggested that anti-obesity effect of β3-AR agonists is attributable to increased thermogenesis not only by UCP1 but also by UCP2 and UCP3.
Association between exogenous atrial natriuretic peptide (ANP) and hemodynamic changes was ascertained in 3 dogs with overt congestive heart failure (CHF(+)) and 3 dogs without congestive heart failure (CHF(-)) caused by experimental mitral regurgitation (MR). The hemodynamic measurements were recorded in all dogs during and after 1 hr infusion of ANP at the rate of 0.1 (low dose), 0.5 (medium dose) and 1.0 (high dose) μg/kg/min, respectively. Heart rate, mean arterial pressure, pulmonary capillary wedge pressure (PCWP) and systemic vascular resistance decreased significantly during and after ANP infusion even with low dose in the CHF(+). Stroke volume, stroke volume index and cardiac output in the CHF(+) during and after ANP infusion showed an increasing trend as compared with the CHF(-). Double product, an indicator of myocardial oxygen consumption, significantly decreased during and after ANP administration at all doses in the CHF(+). These findings indicate that even at low dose, exogenous ANP improves cardiac performance and reduces myocardial oxygen consumption in the CHF(+), and suggest that ANP has beneficial effects in the treatment of dogs with overt congestive heart failure resulting from MR.
The expression of Kit, the receptor for stem cell factor (SCF), on bovine peripheral blood cells (PBCs) was examined by using monoclonal antibodies against the bovine Kit protein. Flow cytometric analysis showed that approximately 1.5% of PBCs expressed Kit. In cytospin preparations, the morphology of most Kit+ PBCs was similar to that of large lymphocytes. Subsets of Kit+ PBCs coexpressed CD3, IgM, and/or CD11b but not CD14 or G1. SCF did not induce the proliferation of Kit+ PBCs in vitro. These results indicate that Kit is expressed on subsets of lymphocytes in bovine peripheral blood, but the ligand of Kit, SCF, does not directly induce the proliferation of this cell population.
To examine in vivo effects of egg white derivatives (EWD), the numbers of peripheral blood cells and neutrophil phagocytosis were evaluated in cats injected intramuscularly with cyclophosphamide (CPA). There were no changes in the number of red blood cells (RBC) or packed cell volume (PCV) values regardless of oral administration of EWD or injection of CPA, but the numbers of platelets, white blood cells (WBC) and neutrophils in cats administered EWD significantly increased (p<0.05 to 0.01) when compared with those in control cats which received saline solution. In addition, the administration of EWD resulted in a significant enhancement in the phagocytic activity of neutrophils (p<0.01) when compared to control cats, suggesting that EWD has a stimulating effect on leukocyte progenitors. The numbers of platelets, WBC and neutrophils, and the phagocytic activity of neutrophils in cats injected with CPA alone were significantly lower (p<0.05 to 0.01) than those in control cats. However, co-administration of EWD to cats injected with CPA resulted in a significant increase in the numbers of platelets, WBC and neutrophils (p<0.05 to 0.01), and in the phagocytic response of neutrophils (p<0.01) when compared to cats injected with CPA alone. Therefore, these results suggest that co-administration of EWD may be effective in reducing some possible side effects in animals treated with immunosuppressive or antitumor agents.
Effects of salivary gland extract (SGE) from Rhipicephalus sanguineus on immunoglobulin class productivity of canine peripheral blood lymphocytes (PBL) in vitro were studied. The detectable limit of the ELISA for canine total immunoglobulin, IgM and A was at least 1, 1 and 15 ng/ml, respectively, and it seems to be useful for the evaluation of non-specific immunoglobulin class productivity in vitro. SGE from R. sanguineus suppressed pokeweed mitogen- or lipopolysaccharide-induced total immunoglobulin and IgA productivity of canine PBL although IgM productivity was not suppressed. These results suggested that the suppression was caused partly by the direct effect of SGE on B lymphocytes.
Feeding dry foods supplemented with urine acidifier (D,L-methionine (Met) or ammonium chloride) decreased urinary pH and struvite activity product in clinically normal cats. As a result, the number of struvite crystals in urine was greatly reduced. Supplementation with 3% Met but not 1% Met caused decrease in the urinary concentration of sediment, which resulted from a reduction in the HCl-soluble fraction. The concentration of HCl-insoluble sediment was not affected by supplementation with the urine acidifier.
An enzyme-linked immunosorbent assay was developed for the determination of canine β2-microglobulin (β2-m) in plasma and urine. The detectable sensitivity for pure canine β2-m was 0.05 μg/l and the analytical range was 0.1 to 50 μg/l. The mean analytical recovery when pure canine β2-m was added to normal plasma was 101.9%. The mean analytical recovery in the urine was 102.1%. The intra-day variation coefficient was 3.1% in plasma, 4.3% in serum and 1.9% in urine. No difference was found between the concentration of β2-m in plasma and serum (n=17). The concentration of β2-m in the plasma of normal dogs was 1.82 ± 0.57 mg/l (n=31). The mean excretion in 24 hr urine collected from normal dogs was 17.6 ± 9.2 μg/l, 0.22 ± 0.12 μg/kg of body weight or 14.2 ± 9.4 μg/g of urine creatinine. The β2-m creatinine index of random urine samples was 23.5 ± 16.6 μg/g (n=26). There was a close correlation between the β2-m creatinine index of 24 hr urine samples and that of random urine samples (r= 0.872).
Pneumocystis carinii pneumonia was diagnosed by postmortem examination of a one-year-old Cavalier King Charles Spaniel with four-week history of dyspnea. Cytologic and histologic examination of lung tissues revealed numerous P. carinii trophozoites and cysts, and P. carinii specific DNA was detected by polymerase chain reaction. The dog showed hypogammagloblinemia and extremely low levels of serum IgG. It was considered that P. carinii pneumonia in this case was associated with an immunodeficient condition which has already been reported in Miniature Dachshunds.
In our previous study, when rasH2 mice and non-transgenic (non-Tg) littermates were injected intraperitoneally with 1,000 mg/kg of urethane once or three times at two-day intervals, the incidence of lung proliferative lesions in rasH2 mice given triple doses of urethane was significantly increased, compared to that in rasH2 mice given a single dose of urethane, and the mutation frequency of the transgene in lung tumors in rasH2 mice given triple doses was lower than that in rasH2 mice given a single dose of urethane. In the present study, differential immunohistochemical expressions of Cyclin D1 and PCNA, that lead to abnormal cell proliferation and tumor development due to uncontrolled G1-S transition in the cell cycle, as well as p53 tumor suppressor gene in pulmonary proliferative lesions obtained from our previous study were investigated. Over-expression of Cyclin D1 in hyperplasias in rasH2 mice given triple doses was significantly increased, compared to that in the single-injection group, but no significant differences in Cyclin D1 between the single and triple injection groups were observed in hyperplasias in non-Tg mice or lung tumors in either rasH2 or non-Tg mice. There were no differences in the PCNA labeling index of hyperplasias in rasH2 or non-Tg mice between the triple-injection and single-injection groups, while the PCNA labeling index tended to be increased in the tumor, compared with that in hyperplasias. There was neither mutation of p53 nor an increase in immunoreactivity of wild type p53 in these proliferative lesions. These results suggest that cyclin D1 over-expression in alveolar/bronchiolar hyperplasias in rasH2 mice in the triple-injection group is not only indicative of a high cell proliferation rate but also of an important role in the process of malignant transformation.
Diffuse mesangial sclerosis (DMS) is one of the hereditary glomerular diseases and histologically characterized by severe glomerulosclerosis and subsequent tubulo-interstitial fibrosis (TIF). In DMS patients, renal dysfunction correlates well with TIF, rather than with glomerular lesions. Thus, molecular mechanisms whereby TIF in DMS progresses should be addressed. Previously, we found that nephrotic ICGN mice manifest DMS-like lesions and develop renal dysfunction in accordance with onset of TIF. In the present study, we investigated fibrogenic events involved in the progression of TIF after DMS manifestation, using the DMS mouse model. Immunohistochemistry revealed that expression of transforming growth factor-beta (TGF-β) was rare in the interstitial cells of the nephrotic mice at the early-stage of DMS, while the TGF-β expression became evident in the late-stage DMS mice. Platelet-derived growth factor (PDGF) was mildly expressed in the distal tubules of the early-stage DMS mice, whereas the PDGF expression markedly increased at the late-stage of DMS. As a result, α-actin-positive myofibroblastic cells were found dominant in the interstitial spaces of the late-stage DMS mice. Finally, TIF became severe in accordance with the overexpressions of these molecules. Our results suggest that in our murine model: 1) persistent proteinuria leads to over-expression of TGF-β and PDGF in non-glomerular areas; 2) these cytokines provoke interstitial myofibroblast accumulation; and 3) the myofibroblasts produce fibrotic matrix proteins in the interstitial spaces. This process may possibly contribute to the development of TIF in DMS patients.
A cDNA coding for feline liver xanthine dehydrogenase(XDH, EC22.214.171.124) was amplified by RT-PCR and cloned for determining the sequence. The clones contained an open reading frame of 4002 base pairs encoding 1333 amino acid residues. The calculated molecular weight and isoelectric point were approximately 146 kDa and 7.0. Comparison of the deduced amino acid sequences indicated remarkable high homology, i.e., the amino acid residues of feline XDH shared approximately 90%, 87%, 87% and 86% identity with those of human, bovine, rat and mouse, respectively. The anino acid sequences of two putative iron-sulfur centers, one NAD binding site and one molybdenum binding site were well conserved among mammalian animals.
Control of cryptosporidiosis is important in public health. Rivers that are polluted with Cryptosporidium and drinking water that is treated for drinking water production from polluted rivers could result in the waterborne disease of cryptosporidiosis. We carried out an epidemiological study of natural water supplies in Hokkaido, one of the largest dairy prefectures in Japan. To detect Cryptosporidium oocysts in environmental water, the filtration method was used for 28 samples, which were collected from 10 rivers. A method adapted from the United States Environmental Protection Agency (U.S. EPA) filtration method using a cartridge filter has been used for the collection of samples. Oocysts were separated from a pellet by discontinuous sucrose gradient method. Twelve samples were collected from 10 rivers and parasites were purified by iron (III) flocculation method. Cryptosporidium parvum oocysts were identified with the immunofluorescence antibody technique using DIF kit (Cellabs Pty. Ltd., Sydney/Australia). We detected Cryptosporidium oocysts in 6 out of 10 rivers sampled. Fifty percentage (14/28) of the samples were Cryptosporidium-positive. The average number of Cryptosporidium oocysts was 16.73/100 L (max. 80 /100 L).
To determine the infectivity of Cryptosporidium to hosts in slight infections, we examined the infectivity and oocyst output patterns of Cryptosporidium muris in mice inoculated with small numbers of oocysts. One of the 25 ICR mice inoculated with 2.4 × 101 oocysts and 19 of the 25 mice inoculated with 2.4 × 102 oocysts shed oocysts in the feces after inoculation. Four of the 50 mice inoculated with 2.4 × 101 oocysts for 10 consecutive days also shed oocysts and their OPG values were similar to that of the mice which received 2.4 × 10 2 oocysts. Consequently, it is clear that less than 10% of the mice which received 2.4 × 101C. muris oocysts for 10 consecutive days.
A monoclonal antibody (B9) was generated by using a rat malignant fibrous histiocytoma (MFH)-derived cloned cell line (MT-8) as the immunogen. Immunohistochemically, B9 reacted specifically with a cytoplasmic antigen of MT-8 cells. Furthermore, B9 immunolabeled another MFH-derived cloned cells (MT-9) and histiocytic sarcoma cells, as well as macrophages/histiocytes in normal and diseased tissues of rats. These findings suggest the presence of a common antigen recognized by B9 between MFH cells and macrophages/histiocytes. This suggests that MFH cells may express histiocytic nature.
Although the injection of bee venom (BV) has been reported to evoke tonic pain and hyperalgesia, there is conflicting evidence in the literature indicating that BV can also exert an anti-inflammatory and antinociceptive effects on inflammation. In this regard, BV has been traditionally used in Oriental medicine to relieve pain and to treat chronic inflammatory diseases such as rheumatoid arthritis. The present study was designed to test the hypothesis that BV induces acute nociception under normal conditions, but that it can serve as a potent anti-inflammatory and antinociceptive agent in a localized inflammatory state. The experiments were designed to evaluate the effect of BV pretreatment on carrageenan (CR)-induced acute paw edema and thermal hyperalgesia. In addition, spinal cord Fos expression induced by peripheral inflammation was quantitatively analyzed. In normal animals subcutaneous BV injection into the hindlimb was found to slightly increase Fos expression in the spinal cord without producing detectable nociceptive behaviors or hyperalgesia. In contrast pretreatment with BV (0.8 mg/kg) 30 min prior to CR injection suppressed both the paw edema and thermal hyperalgesia evoked by CR. In addition, there was a positive correlation between the percent change in paw volume and the expression of Fos positive neurons in the spinal cord. These results indicate that BV pretreatment has both antinociceptive and anti-inflammatory effects in CR-induced inflammatory pain. These data also suggest that BV administration may be useful in the treatment of the pain and edema associated with chronic inflammatory diseases.
Accumulating evidences indicate that an endotoxin originating from intestinal gram-negative bacteria may be involved in alcohol-induced liver injury including fatty liver. Therefore, whether immunization against intestinal bacterial endotoxin blocked fatty liver induced by chronic alcohol and diet including much-unsaturated fatty acid was investigated in rats. The titer of antibody against the endotoxin increased significantly after 13 weeks of continuous immunization. Daily alcohol treatment was initiated at 12 weeks and continued for 4 weeks. Plasma glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) and triglyceride (TG) levels increased significantly in non-immunized rats receiving alcohol, but not in immunized rats. Continuous alcohol treatment gradually decreased the survival rate to 60% from 13 days after beginning administration in non-immunized, but not immunized, rats. A histochemical study revealed that continuous treatment with alcohol and unsaturated fatty acids caused fatty liver in non-immunized, but not immunized, rats. This study strongly supports the hypothesis that alcohol-induced fatty liver is due to a circulating endotoxin, and suggests that immunization for endotoxin prevent the alcoholic fatty liver.
To identify which region of staphylococcal enterotoxin A (SEA) is responsible for the emetic activity, twelve synthetic peptides corresponding to the entire SEA amino acid sequence and their respective anti-peptide antibodies were prepared and tested. The anti-peptide antibodies were tested for neutralization of SEA-induced emesis in Suncus murinus (Shrew mouse). The results indicate that SEA-induced emesis was neutralized by the mixture of three anti-peptide antibodies to A-7 (corresponding to amino acid residues 121-140), A-8 (141-160) and A-9 (160-180). These findings suggest that the regions corresponding to residues 121-180 may be the epitopes responsible for the emetic activity of SEA.
The adsorption property of activated charcoal on verotoxin (VT)-producing Escherichia coli (VTEC) was examined using E. coli O157:H7. In the present study, E. coli O157:H7 strains were effectively adsorbed by activated charcoal. Adsorption was dose-dependent, and the maximum adsorption occurred within 5 min. At 10 mg of activated charcoal, bacteria tested were completely adsorbed. Activated charcoal also had the capacity to adsorb toxin (verotoxin 2) activity from the bacterial extract. Furthermore, the adsorption efficiency of activated charcoal for the normal bacterial flora in the intestine was assessed using Enterococcus faecium, Bifidobacterium thermophilum, and Lactobacillus acidophilus. Activated charcoal showed lower binding capacity to the normal bacterial flora tested than that to E. coli O157:H7 strains. These results suggest that activated charcoal could be a good adsorbent system for the removal of VTEC and verotoxin.
Medical and racing records of 155 Thoroughbred racehorses that underwent arthroscopic surgery for carpal chip fractures were investigated. Articular damage for 98.4% of the fractures was classified as G1 or G2 using McIlwraith's criteria. The rate of return to racing after surgery was 82.6%. Evaluation of racing performance after surgery was attempted using a placing index (PI) based on race finish position. There was no significant difference in the PI distribution between horses that underwent surgery and other healthy horses.
Fecal progesterone assays were conducted in 3 captive female sun bears (Helarctos malayanus) segregated from males to characterize the species-specific reproductive pattern in their original distribution area in Sarawak, Malaysia. Peaks of fecal progesterone concentrations were observed once annually, and lactation was observed after increasing progesterone concentrations in all females without mating stimulus. These results suggest that sun bears in Sarawak, Malaysia, may have a seasonal reproductive pattern and ovulation was noted to occur spontaneously, followed by pseudopregnancy.
Castrated goats were treated with dihydrotestosterone (DHT) for four weeks. Skin samples were collected from the head and the rump regions before and after the DHT treatment. The primer pheromone activities of these samples were assessed neurophysiologically by recording electrophysiological manifestations of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator activity. Pheromone activity was detected in both the head and rump skin samples following the DHT treatment, although the development of sebaceous glands was limited to the head region. Taken together with our previous finding that testosterone treatment results in the appearance of primer pheromone activity in the skin sample of the head region but not of the rump region, these observations suggests that the regional difference of pheromone production would be ascribed to intrinsic expression levels of 5α-reductase, an enzyme converting testosterone to DHT.
Exposure and ecological risks to heavy metals (copper, zinc, manganese, iron) at Lochnivar and Blue Lagoon National Parks in wildlife dependent on the Kafue river contaminated with mining waste was evaluated. Samples included water, fish, grasses and Kafue Lechwe (Kobus leche kafuensis) liver. At both parks copper ranged from 0.03-0.04 mg/l; 3.0-6.0 mg/kg; 11.0-44.0 mg/kg; trace -199.0 mg/kg; while zinc was 0.01 mg/l; 32.0-82.0 mg/kg; 15.0-21.0 mg/kg; and 52.0-138.0 mg/kg; in water, fish, grasses and lechwe, respectively. Manganese ranges were 0.15-0.16 mg/l; 7.0-18.0 mg/kg; 51.0-145.0 mg/kg; and 40.0-53.0 mg/kg while iron ranges were 0.13-0.14 mg/l; 26.0-134.0 mg/kg; 1766.0-1797.0 mg/kg; and131.0-856.0 mg/kg; in water, fish, grasses and lechwe, respectively. Levels in all samples except water were high indicating potential for adverse effects.