Partial purification of latent persimmon fruit polyphenol oxidase

Persimmon fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning with Triton X-114 and ammonium sulfate fractionation between 50 and 75%. The enzyme, which showed both monophenolase and diphenolase activities, was partially purified in a latent form and could be optimally activated by the presence of 1 mM sodium dodecyl sulfate (SDS) with an optimum pH of 5.5. In the absence of SDS, the enzyme showed maximum activity at acid pH. SDS-PAGE showed the presence of a single band when L-DOPA was used as substrate. The apparent kinetic parameters of the latent enzyme were determined at pH 5.5, the V(m) value being 15 times higher in the presence of SDS than in its absence, whereas the K(M) was the same in both cases, with a value of 0.68 mM. The effect of several inhibitors was studied, tropolone being the most active with a K(i) value of 0.45 microM. In addition, the effect of cyclodextrins (CDs) was studied, and the complexation constant (K(c)) between 4-tert-butylcatechol (TBC) and CDs was calculated using an enzymatic method. The value obtained for K(c) was 15580 M(-1).     

Promotion of the cycling of diet-enhancing nutrients by african grazers

Experiments in Serengeti National Park, Tanzania, provide direct evidence that large, free-ranging mammalian grazers accelerate nutrient cycling in a natural ecosystem in a way that enhances their own carrying capacity. Both nitrogen and sodium were at considerably higher plant-available levels in soils of highly grazed sites than in soils of nearby areas where animal density is sparse. Fencing that uncoupled grazers and soils indicated that the animals promote nitrogen availability on soils of inherently similar fertility and select sites of higher sodium availability as well as enhancing that availability.     

IMAGING DIAGNOSIS—TEMPOROMANDIBULAR JOINT DYSPLASIA IN A BASSET HOUND

A 5‐month‐old intact male Basset Hound presented for evaluation of pain and crepitation during manipulation of the temporomandibular joint, worse on the right side. A computed tomography (CT) scan of the head was performed. The CT images demonstrated the osseous features of temporomandibular joint dysplasia and facilitated a 3D reconstruction, which allowed better visualization of the dysplastic features. The patient responded to conservative management with a tape muzzle with no recurrence reported by the owner 6 months after presentation.     

Feline paraneoplastic syndrome associated with thymoma

CASE HISTORY: A 6-year-old, spayed, female, domestic short-haired cat presented with severe erythroderma and scaling skin. She showed disturbed gait and mild behavioural changes.

CLINICAL FINDINGS: The cat had a generalised, erythematous, scurfy dermatitis with marked, multifocal crusting and skin thickening. The skin was painful and contracted, which appeared to prevent normal freedom of movement.

DIAGNOSIS: The cat was suspected to have a paraneoplastic syndrome. A mediastinal mass was located and histologically confirmed as thymoma. The cat was diagnosed with a thymoma-associated cutaneous paraneoplastic syndrome.

CLINICAL RELEVANCE: This is a rare condition with few reports in the literature. The skin changes, both grossly and histologically, were considered to be different from those described in cases of paraneoplastic dermatosis associated with pancreatic neoplasia. The clinical presentation was characteristic and more cases may occur in practice than are recognised. In this case, as in previous reports, the tumour was grossly resectable, which could lead to cure of the clinical condition.     

Cardiovascular effects of butorphanol in isoflurane-anesthetized alpacas

Butorphanol has been used clinically to provide analgesia in alpacas, but cardiovascular effects have not been reported. Using a randomized cross‐over design, eight healthy, young adult female alpacas (3 ± 1 SD years) weighing 64 ± 9 SD kg were anesthetized with isoflurane by mask followed by tracheal intubation and maintenance of anesthesia with 1.75% et (isoflurane) in oxygen. Two treatments, butorphanol (0.1 mg kg^–1 IV) and control (saline, IV) were assigned to the animals in a randomized manner allowing a minimum of two weeks between treatments. While anesthetized, animals were instrumented for measurement of cardiovascular variables including systolic, diastolic, and mean arterial blood pressure, pulmonary arterial pressure, pulmonary capillary wedge pressure, central venous pressure, cardiac output (CO) and pulmonary temperature (TEMP). CO was measured via thermodilution using 5 mL of iced 5% dextrose and recording the average of three replicate measurements. Cardiac index, systemic vascular resistance (SVR) and pulmonary vascular resistance were also calculated. Arterial and mixed venous blood samples were collected for blood gas analysis [pH, pO₂, pCO₂, (HCO₃^?), BE, Hb_sat]. Variables were collected at baseline (time 0) and at 5, 10, 15, 30, 45, and 60 minutes following injection. Variables were analyzed by anova for repeated measures with post‐hoc differences between means identified using the Bonferroni comparison (p < 0.05). SVR decreased five minutes after administration of butorphanol (Huynh Feldt corrected p = 0.045) and remained decreased for 60 minutes. TEMP decreased with time in both groups (Huynh Feldt corrected p = 0.000027), but groups were not different between each other. Other cardiovascular and blood gas variables were not different between groups. We conclude that butorphanol (0.1 mg kg^–1 IV) had minimal effects on the cardiovascular system of these alpacas, causing a mild decrease in SVR.     

Luteolytic Effect of Prostaglandin F2α on Bovine Corpus Luteum Depends on Cell Composition and Contact

Prostaglandin F_2α (PGF_2α) is a main luteolytic factor in vivo; however, its direct luteolytic influence on steroidogenic cells of bovine corpus luteum (CL) is controversial and not fully understood. The aim of the study was to clarify PGF_2α action on bovine CL in different in vivo and in vitro conditions and to examine whether the contact among all main types of CL cells is necessary for luteolytic PGF_2α action. In experiment 1, the bovine CL (day 15 of the oestrous cycle) was perfused using in vivo microdialysis system with dinoprost (an analogue of PGF_2α) for 0.5 h. Dinoprost caused a short‐time increase in progesterone (P4), whose concentration decreased thereafter (at 6‐, 10‐, 12‐ and 24‐h after treatment). In experiment 2, the direct effect of PGF_2α on P4 accumulation in CL steroidogenic cells cultured in monolayer (day 15 of the cycle) was determined. PGF_2α after 24 h of incubation increased P4 accumulation in steroidogenic CL cells. In experiment 3 steroidogenic, endothelial CL and immune cells (day 15 of the cycle) were incubated with PGF_2α in cocultures for 24 h in glass tubes and the levels of P4, stable metabolites of nitric oxide (NO) and leukotriene (LT) C₄ were determined. Although PGF_2α treatment increased P4 secretion in homogeneous steroidogenic CL cell culture, the decrease in P4 secretion in cocultures of all types of CL cells was observed. The secretion of NO and LTC₄ increased after the treatment of PGF_2α both in pure cultures of CL cells and in cocultures. The interactions between endothelial and immune cells with steroidogenic CL cells are needed for luteolytic PGF_2α action within the bovine CL. Our results indicate that the cell coculture model, including the main types of CL cells, is the most approximate to study PGF_2α role in vitro.     

Transmission studies of Hendra virus (equine morbilli-virus) in fruit bats, horses and cats

Objective To determine the infectivity and transmissibility of Hendra virus (HeV). Design A disease transmission study using fruit bats, horses and cats. Procedure Eight grey-headed fruit bats (Pteropus poliocephalus) were inoculated and housed in contact with three uninfected bats and two uninfected horses. In a second exper iment, four horses were inoculated by subcutaneous injection and intranasal inoculation and housed in contact with three uninfected horses and six uninfected cats. In a third experi ment, 12 cats were inoculated and housed in contact with three uninfected horses. Two surviving horses were inoculated at the conclusion of the third experiment: the first orally and the second by nasal swabbing. All animals were necropsied and examined by gross and microscopic pathological methods, immunoperoxidase to detect viral antigen in formalin-fixed tissues, virus isolation was attempted on tissues and SNT and ELISA methods were used to detect HeV-specific antibody. Results Clinical disease was not observed in the fruit bats, although six of eight inoculated bats developed antibody against HeV, and two of six developed vascular lesions which contained viral antigen. The in-contact bats and horses did not seroconvert. Three of four horses that were inoculated devel oped acute disease, but in-contact horses and cats were not infected. In the third experiment, one of three in-contact horses contracted disease. At the time of necropsy, high titres of HeV were detected in the kidneys of six acutely infected horses, in the urine of four horses and the mouth of two, but not in the nasal cavities or tracheas. Conclusions Grey-headed fruit bats seroconvert and develop subclinical disease when inoculated with HeV. Horses can be infected by oronasal routes and can excrete HeV in urine and saliva. It is possible to transmit HeV from cats to horses. Transmission from P poliocephalus t o horses could not be proven and neither could transmission from horses to horses or horses to cats. Under the experimental conditions of the study the virus is not highly contagious.     

Absence of Sperm Factors as in the Parthenogenesis Does Not Interfere on Bovine Embryo Sensitiveness to Heat Shock at Pre‐Implantation Stage

Oocyte has been considered the major contributor for embryo thermo‐tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo‐tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo‐sensitiveness at early stages. In vitro fertilized (IVF) and parthenogenetic (PA) embryos at 44 h post‐insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat‐shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p < 0.05) for IVF embryos and at day eight (p < 0.01) for both IVF and PA embryos. Total cell number was not affected by heat shock in IVF and PA blastocysts, but there was an increased proportion (p < 0.05) of apoptotic cells in heat‐shocked embryos when compared to controls. There was no interaction (p > 0.05) between method of activation (IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock.     

Effects of Intrauterine Devices in Mares: A Histomorphological and Immunohistochemical Evaluation of the Endometrium

Oestrous suppression by intrauterine devices (IUDs) is caused by prolongation of luteal function, but the biological mechanism is unknown. The aim of the study was to investigate mechanisms which could explain the action of IUDs. Thirty mares were age‐matched and either inseminated (AI, n = 15) or fitted with an IUD (IUD, n = 15) and subsequently divided into four groups: AI‐P, pregnant (n = 8); AI‐N, non‐pregnant (n = 7); IUD‐P, prolonged luteal phase (n = 7); and IUD‐N, normal luteal phase (n = 8). The median ages were 5.5 and 7 years in AI‐P and IUD‐P groups and 14 and 11 years in AI‐N and IUD‐N groups, respectively. On Day 15 after ovulation, an endometrial biopsy was obtained to study histomorphological and immunohistochemical expression patterns of uterine proteins (uteroferrin, UF; uterocalin, UC; uteroglobin, UG), oestrogen and progesterone receptors (ER, PR), proliferation marker Ki‐67 and content of inflammatory cells. Expression of UF was higher in IUD mares; the difference between pregnant and IUD‐P mares was significant. Mares exhibiting a prolonged luteal phase (AI‐P, IUD‐P) showed only mild angiosclerosis and lower expression of both ER and PR than mares with a normal luteal phase (AI‐N, IUD‐N). No significant differences were detected in the numbers of inflammatory cells, with the exception of macrophages, which were more numerous in AI‐P than AI‐N mares. Although inflammatory cells were not detected in IUD mares, increased UF levels may indicate chronic inflammation. Young age and normality of the endometrial blood vessels may improve the efficacy of IUDs.