Investigation of diagnostic use of serum anti-Müllerian hormone concentration in dioestrus and anoestrus bitches before and after ovariohysterectomy and the relationship with ovarian follicle numbers.

The aims of the study were to (1) compare the serum concentration of anti-Müllerian hormone (AMH) with the number of follicles in ovaries and (2) determine the serum AMH con- centration before and after ovariohysterectomy in dioestrus and anoestrus bitches. Sixteen bitches were divided into two groups: Group I (n=8) consisted of dioestrus and group II (n=8) anoestrus bitches. The blood samples for AMH assesment were taken before ovariohysterectomy (day 0) and on day 1, 5 and 10. Both in group I and II, serum AMH concentrations on day 1 and 5 were significantly different compared to day 0 (p⟨0.05). However, the concentrations at day 10 were under the minimum detectable concentration (1.0 ng/mL) and this finding revealed that ovaries are the only source of AMH synthesis. Follicle counts were not statistically different between the groups (p>0.05). Significantly positive correlation in serum AMH with secondary follicle num- bers (r=.942, p⟨0.01), as well as negative correlation with antral follicle numbers (r=-.765, p⟨0.05) were determined in the group I. In the group II, positive correlations between serum AMH concentration and secondary follicle numbers (r=.960, p⟨0.01) and early antral follicles (r=.726, p⟨0.05) were noted. Assesment of AMH concentration seems to not only provide the diagnosis of the presence of ovaries but also correlate with the number of secondary follicles in young dioestrus and anoestrus bitches.

Immunohistochemical detection of autophagy-related microtubule-associated protein 1 light chain 3 (LC3) in the cerebellums of dogs naturally infected with canine distemper virus.

We investigated the expression of microtubule-associated protein 1 light chain 3 (LC3) protein in the cerebellums of dogs infected with canine distemper virus (CDV) using immunohistochemistry to detect autophagy. The cerebellums of 20 dogs infected with CDV were used. Specimens showing demyelination of white matter were considered to have an acute infection, whereas specimens showing signs of severe perivascular cuffing and demyelination of white matter were classified as having chronic CDV. Cerebellar sections were immunostained with CDV and LC3 antibodies. The cytoplasm of Purkinje cells, granular layer cells, motor neurons in large cerebellar ganglia and some neurons in white matter were positive for the LC3 antibody in both the control and CDV-infected dogs. In the infected cerebellums, however, white matter was immunostained more intensely, particularly the neurons and gemistocytic astrocytes in the demyelinated areas, compared to controls. Autophagy also was demonstrated in CDV-positive cells using double immunofluorescence staining. Our findings indicate that increased autophagy in the cerebellum of dogs naturally infected with CDV may play a role in transferring the virus from cell to cell.

Synthesis and anti-inflammatory activities of novel 5-(3,4-dichlorophenyl)-3-[(4-substitutedpiperazin-1-yl)methyl]-1,3,4-oxadiaxole-2(3H)-thiones.

To synthesize a new series of 5-(3,4-dichlorophenyl)-3-[(4-substitutedpiperazin-1-yl)methyl]-1,3,4-oxadiaxole-2-thiones for their anti-inflammatory activity.The title compounds synthesized by Mannich reaction of prepared 5-(3,4-dichloro-phenyl)-1,3,4-oxadiazole-2-thione and appropriate substituted piperazine derivatives. The structures of the compounds were confirmed by IR, NMR and elemental analyses. The newly synthesized compounds were evaluated for their anti-inflammatory and ulcerogenic activities.Most of the compounds were found to have significant anti-inflammatory profile in carrageenan footpad edema test. The compounds showed anti-inflammatory activity ranging from 30.6% to 57.8% inhibition.The compounds 5b, 5f, 5i, 5p and 5r showed most prominent anti-inflammatory activity with low gastric ulceration incidence than the reference drug indomethacine. These compounds could serve as lead molecules for further modifications.

Cytotoxic activities of some novel benzhydrylpiperazine derivatives.

This study presents the synthesis of nineteen 1-(substitutedbenzoyl)-4-benzhydrylpiperazine and 1-[(substitutedphenyl)sulfonyl]-4-benzhydrylpiperazine derivatives. In vitro cytotoxic activities of the compounds were screened against hepatocellular (HUH-7), breast (MCF-7) and colorectal (HCT-116) cancer cell lines by sulphorhodamine B assay. Among the test compounds, benzamide derivatives had high cytotoxic activity whereas sulfonamide derivatives showed variable 50% growth inhibition (GI50).

Immunohistochemical characterization of peroxisome proliferator-activated receptors in canine normal testis and testicular tumours.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. Recent studies have demonstrated that PPARs regulate lipid metabolism and are expressed in various cancers. The aim of the present study was to investigate the expression of PPAR-α, -ß and -γ in normal canine testicular tissue and canine testicular tumours (CTTs). Expression of PPAR-α, -ß and -γ was greater (P <0.05) than in normal testicular tissue. PPARs were therefore induced in CTTs and they may play a role in the biology of these tumours.

Synthesis and cytotoxic activity of novel 3-methyl-1-[(4-substitutedpiperazin-1-yl)methyl]-1H-indole derivatives.

A series of novel 3-methyl-1-[(4-substitutedpiperazin-1-yl)methyl]-1H-indoles (3a-l) were synthesized and their cytotoxicities were analyzed against 3 different human cell lines, including liver (HUH7), breast (MCF7) and colon (HCT116). The Mannich reaction of 3-methylindole (1) with 4-substitutedpiperazines (2) and formaldehyde resulted to the 3-methyl-1-[(4-substitutedpiperazin-1-yl)methyl]-1H-indoles (3a-l) in 38-69% yields. The investigation of anticancer screening revealed that the tested compounds showed comparable activity to the reference drug 5-fluorouracil and compounds 3g, 3h, 3i and 3k, had lower 50% inhibition (IC50) concentration than reference drug. Moreover, the cytotoxic effect of the most potent compound 3h on HUH7 and MCF7 cells through apoptosis was visualized by Hoechst staining and compared with paclitaxel, which is a mitotic inhibitor acting on microtubules. The morphological features of apoptosis were observed as condensed and fragmented nuclei that are similar to paclitaxel.

Persistent cloaca, fused kidneys, female pseudohermaphroditism and skeletal anomalies in a simmental calf.

A 5-day-old Simmental calf was referred to our department for atresia ani and postural abnormalities caused by skeletal deformities. The calf had a short and deviated tail and a bowed hind limb. The calf appeared like a male because of the prepuce and penis located just near the teats and the absence of female external genitalia. During the necropsy, a horseshoe kidney, single ureter that originated from the kidney, and bilateral uterine horns with one ovary each were detected. The ureter, blind-ended large intestine, and bilateral uterine horns were connected to a dilated cloaca having two sacs, which were filled with a yellowish brown viscous fluid admixed with meconium and urine. Skeletal deformities found included scoliosis, partial synostosis of vertebrae, deviation of rudimentary sacrum and coccygeal vertebrae, and narrowed pelvic cavity. This is the first report of an anomalous combination including urogenital, large intestinal, and skeletal deformities in cattle.

Platelet-rich plasma improves healing of tympanic membrane perforations: experimental study.

OBJECTIVE: The aim of this study was to investigate the effect of local application of platelet-rich plasma to perforated rat tympanic membranes, in terms of healing time and histopathological outcome. METHODS: Eighty-eight tympanic membranes of 44 rats were given a standard 3 mm perforation, and platelet-rich plasma was applied to the right tympanic membrane perforations. The left tympanic membranes were left to heal spontaneously, as controls. The 44 rats were divided into two groups. In group one, comprising 20 rats, daily otomicroscopic examination of the tympanic membrane perforations was performed. The 24 rats in group two were subdivided into four subgroups of six rats each; these subgroups were sacrificed sequentially on days three, seven, 14 and 28 for histopathological examination, regardless of tympanic membrane healing stage. RESULTS: In group one, the mean tympanic membrane healing times for tympanic membrane perforations receiving platelet-rich plasma and controls were respectively 10.2 +/- 2.1 and 13.0 +/- 2.9 days (mean +/- standard deviation). This difference was statistically significant (p < 0.001). In group two, histopathological evaluation of tympanic membrane perforation healing at days three, seven, 14 and 28 did not reveal any statistically significant difference, individually or within the four groups as a whole. CONCLUSION: These findings suggest that earlier healing of tympanic membrane perforations occurred in the platelet-rich plasma group compared with the control group. These findings suggest that platelet-rich plasma is effective in accelerating tympanic membrane perforation healing, and that it may be effective in human subjects, particularly as it is an autologous material.

The Efficacy of Platelet-rich Plasma Gel and Topical Estradiol Alone or in Combination on Healing of Full-thickness Wounds.

 This randomized study was designed to establish the efficacy of platelet-rich plasma (PRP) gel and topical estradiol alone, or in combination, on healing of full-thickness wounds created on the trunk of rabbits. Fifteen New Zealand white female rabbits and 60 wounds were studied. Four 2.25-cm² full-thickness wounds were created using a template and treatments including a sterile saline solution that were assigned randomly to the wounds. Wounds were bandaged, dressed intermittently, and assessed by wound measurements and a collection of samples at 7, 14, and 21 days to evaluate healing. Variables of interest were hydroxyproline concentration and scored gross and microscopic morphologic characteristics reflective of wound healing. PRP gel + estradiol-treated wounds completely healed faster than wounds given other treatments. Granulation tissue growth to the skin level was faster (P < 0.05) in the PRP gel + estradiol group than the other groups. Only the PRP gel + estradiol group had a significantly lower collagen level than the sterile saline solutions on day 7 (P < 0.05). The PRP gel + estradiol group and estradiol group led to an increase in angiogenesis on day 14 (P < 0.05). Complete epithelization was observed only in the PRP gel + estradiol group compared with the other groups on day 7. PRP gel + estradiol-treatment enhanced healing in full-thickness wounds by reducing the contraction rate with a trend toward accelerating both epithelial migration and the angiogenic response.

Cytochrome P450 aromatase expression in canine nervous tissue: an immunohistochemical study.

The enzyme cytochrome P450 aromatase is responsible for conversion of androgens to estrogens. Estrogens have been implicated in neurophysiology and neuropathology. The present study investigated the presence of aromatase immunoreactivity in the temporal, parietal and occipital cortices, olfactory bulb, cerebellum, and choroid plexus of the normal dog. Aromatase immunoreactivity was localized exclusively in neurons in the cortices and olfactory bulb. Immunoreactivity was also present in a small number of astrocytes in the substantia alba of the cerebellum. In the cortical regions, immunoreactive neurons, morphologically identified as pyramidal cells, were found throughout Layer II down to Layer VI, but not all pyramidal neurons were immunoreactive. In the olfactory bulb, immunoreactive neurons were mainly observed in mitral cells and inner granular cell layers. In the cerebellum, immunoreactivity was present in neurons of the deep cerebellar nuclei and in some neurons of the molecular and granular cell layers. Immunoreactivity was also present in endothelial cells of the subarachnoid vessels and those adjacent to ventricles in the cortex. The presence of well defined cytoplasmic aromatase immunoreactivity in neurons, some astrocytes, and endothelial cells suggests estrogen involvement in CNS physiology and function in the dog. The presence of aromatase in ependymal cells lining cerebral ventricles and choroid epithelial cells suggests that these cells may be partially responsible for estrogen concentration in the cerebrospinal fluid.

Myocarditis associated with foot-and-mouth disease virus type O in lambs.

The present study describes the pathogenetic mechanisms of myocarditis in 9 lambs that died in a foot-and-mouth disease outbreak in Samsun, Turkey. In all the heart samples tested, ELISA and sequencing for phylogenetic analyses showed that the virus, namely O/TUR/Samsun/05, was associated with the PanAsia pandemic strain of foot-and-mouth disease virus (FMDV) type O. The lambs had myocardial lesions but no typical vesicular lesions. In situ reverse transcription showed that many cardiomyocytes and some interstitial cells were positive for FMDV type O. Inflammatory infiltration, hyaline degeneration, and necrosis of sheets of myocytes were observed. The cellular infiltrates were mononuclear cells, including many lymphocytes, macrophages, a few plasma cells, and neutrophils. Major histocompatibility complex Class II+ dendritic and mononuclear cells, gammadelta T cells, CD172A+ and CD14+ macrophages and monocytes, and IgM+ B cells were detected mainly in the infected hearts. Inducible nitric oxide synthetase (iNOS) was seen mostly in areas of inflammation infiltrated by large numbers of cells. Of the 2 alpha-subunits of integrin known to be used as receptors by FMDV in epithelial tissues, CD49e (integrin alpha5) was detected in the membranes of cardiac myocytes with intercalated discs, but CD51 (integrin alphaV) was not detected in cardiac myocytes from infected or normal lambs. Interstitial and inflammatory cells were positive for both integrin subunits. The terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL)-positive signal was detected in the nuclei of both cardiac myocytes and interstitial cells from infected lambs. These findings suggest that the iNOS expressed by inflammatory cells in lesions may have a deleterious effect on cardiac myocytes in these lesions.

The influence of growth regulators on shoot proliferation and rooting of in vitro propagated caper.

The objectives of this study were to examine the effect of TDZ and BAP on shoot proliferation and to develop a satisfactory rooting procedure for in vitro propagation of caper (Capparis spinosa L). TDZ was found to be more effective than BAP in shoot production. The highest shoot number per explant (45.7) was achieved on the media that contained 4.54 microM TDZ in the absence of IAA. TDZ at 4.54 microM alone or in combination with 0.57 microM IAA did not inhibit shoot elongation, although they produced the most shoots. Dipping the caper shoots in the solution of IBA at 24.6 microM for 10 minutes before planting on MS media, improved the rooting percentage up to 80.5% under a 16 hr photoperiod.

Retrobulbar rhabdomyosarcoma in a budgerigar (Melopsittacus undulatus).

This report describes a retrobulbar rhabdomyosarcoma in a 7-year-old male budgerigar (Melopsittacus undulatus). The bird was presented with orbital distension and exophthalmia of the left eye and died during surgery. Necropsy revealed a tan to grey retrobulbar mass compressing all components of the left eye with obscured vision. In histopathological examination, the mass was composed of sheets of spindle-shaped cells with pleomorphic nuclei, numerous bizarre mitotic figures and mononucleated or multinucleated giant cells and also typically strap cells. Neoplastic cells had no cross-striations in sections stained by phosphotungstic acid haematoxylin. The tumour cells did not invade surrounding tissues, including the retina, and there was no metastasis to other organs. Immunohistochemically, tumour cells were positive for desmin, vimentin and alpha-smooth muscle actin, but were negative for S-100 protein and pancytokeratin.

Immunohistochemical investigation of cerebellum in dogs infected with canine distemper virus.

The cerebella of 21 dogs with canine distemper virus (CDV) infection and four normal dogs were examined histopathologically and immunohistochemically. Cerebella of CDV-infected dogs showed nonsuppurative demyelinating encephalomyelitis, classified as acute, subacute or chronic. Immunolocalisation of CDV antigen also confirmed the infection. Tissues were examined for co-localisation of the CDV antigen with either an astrocyte-specific marker, glial fibrillary acidic protein (GFAP), or an oligodendrocyte-specific marker, galactocerebroside (GalC). Immunoreactive cells were counted in demyelinating areas of the white matter. The number of astrocytes (GFAP positive) was significantly (p < 0.05) higher in CDV-infected dogs compared to controls. In contrast, the number of oligodendrocytes (GalC positive) was significantly (p < 0.001) lower in CDV-infected dogs and was much lower in chronic cases (p < 0.05). Approximately 41% of astrocytes and 17% of oligodendrocytes were immunoreactive for CDV. The ratio of CDV-infected oligodendrocytes and astrocytes remained almost constant during the progression of the disease (P > 0.05). In conclusion, CDV infects both astrocytes and oligodendrocytes. The gradual loss of oligodendrocytes is most likely responsible for the progressive demyelination in CDV infection. Astrocytosis in CDV infection should be further investigated if it occurs to stimulate oligodendrocytes for myelin production to compensate for the loss or to induce oligodendrocyte degeneration.

Neurosteroidogenesis in oligodendrocytes and Purkinje neurones of cerebellar cortex of dogs.

The cerebellum is a steroidogenic organ that expresses steroidogenic enzymes and produces neurosteroids. Purkinje neurones appear to be the most active steroidogenic cells in the cerebellar cortex. These neurones express 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), P450 side-chain cleavage (P450scc), 17 alpha-hydroxylase/c17, 20lyase (P450c17), P450 aromatase (P450arom) and produce pregnenolone, progesterone, dehydroepiandrosterone, androstenedion, oestradion and oestrone. Oligodendrocytes are predominantly the producer of myeline protein. The oligodendrocytes were identified by immunohistochemistry using a monoclonal antibody against myeline 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a myeline specific enzyme. In this study we have examined the distribution of 3 beta-HSD and CNPase by immunohistochemistry using monoclonal antibody in canine cerebellar cortex. The localization of oligodendrocytes within the cerebellar cortex was determined to be close to Purkinje neurones. This result suggests that endogenous progesterone synthesized de novo in the Purkinje neurone can promote myeline protein synthesis in oligodentrocytes.

Distribution of 3beta-hydroxysteroid dehydrogenase in the cerebellum in canine distemper virus infection.

The cerebella of eight dogs naturally infected with canine distemper virus (CDV) and two normal dogs were examined immunohistochemically for glial fibrillary acidic protein (GFAP) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). The clinical diagnosis of canine distemper was confirmed histopathologically and by the immunohistochemical demonstration of CDV antigen. In all dogs (healthy and infected), the Purkinje cells of the cerebellum were immunolabelled for 3beta-HSD activity. In infected dogs, 3beta-HSD labelling was prominent in astrocytes (particularly in areas of astrocytosis) whereas in healthy dogs such immunolabelling was weak. Double immunolabelling demonstrated that all GFAP-positive cells (especially in demyelinating areas) were also positive for 3beta-HSD. The results suggest that 3beta-HSD expression by astrocytes is associated with demyelination in CDV infection.

4-aryl-6,6-dimethyl-1,2,3,4,5,6,7,8-octahydroquinazoline-2,5-diones: synthesis, chromatographic resolution and pharmacological activity.

In this study, a series of 4-aryl-6,6-dimethyl-1,2,3,4,5,6,7,8- octahydroquinazoline-2,5-diones were synthesized by condensing urea with 4,4-dimethyl-1,3-cyclohexanedione and appropriate aromatic aldehydes according to the Biginelli reaction. The structures of the compounds were characterized by spectroscopic methods. The racemic compounds were resolved into the enantiomers by HPLC on amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD). The compounds were tested in vitro for their calcium antagonistic activities. BaCl2-induced contractions of rat ileum were inhibited dose-dependently. Compounds 3 and 5 exerted weak calcium antagonistic activity on smooth muscles compared with the standard, nicardipine.

Studies on synthesis, chromatographic resolution, and antiinflammatory activities of some 2-thioxo-1,2,3,4-tetrahydropyrimidines and their condensed derivatives.

In this study, the synthesis of some new 2-thioxo-1,2,3,4-tetrahydropyrimidines and their condensed derivatives, thiazolo[3,2-a]pyrimidines, are described. The structures of the compounds were confirmed by 1R, 1H-NMR, 13C-NMR, and mass spectroscopy. The direct high-performance liquid chromatographic separation of the compounds on derivatized cellulose chiral stationary phases such as cellulose tris(3,5-dimethylphenylcarbamate) (OD), cellulose tris(4-methylphenylcarbamate) (OG), and cellulose tris(4-methylbenzoate) (OJ) was studied. All of the compounds were screened for their antiinflammatory activity and also investigated histopathologically. Compounds 3 and 1a were found to be the most promising antiinflammatory agents in this group.