Mapping of somatostatin-28 (1-12) in the alpaca diencephalon

Using an immunocytochemical technique, we report for the first time the distribution of immunoreactive cell bodies and fibers containing somatostatin-28 (1-12) in the alpaca diencephalon. Somatostatin-28 (1-12)-immunoreactive cell bodies were only observed in the hypothalamus (lateral hypothalamic area, arcuate nucleus and ventromedial hypothalamic nucleus). However, immunoreactive fibers were widely distributed throughout the thalamus and hypothalamus. A high density of such fibers was observed in the central medial thalamic nucleus, laterodorsal thalamic nucleus, lateral habenular nucleus, mediodorsal thalamic nucleus, paraventricular thalamic nucleus, reuniens thalamic nucleus, rhomboid thalamic nucleus, subparafascicular thalamic nucleus, anterior hypothalamic area, arcuate nucleus, dorsal hypothalamic area, around the fornix, lateral hypothalamic area, lateral mammilary nucleus, posterior hypothalamic nucleus, paraventricular hypothalamic nucleus, suprachiasmatic nucleus, supraoptic hypothalamic nucleus, and in the ventromedial hypothalamic nucleus. The widespread distribution of somatostatin-28 (1-12) in the thalamus and hypothalamus of the alpaca suggests that the neuropeptide could be involved in many physiological actions.     

Mapping of tyrosine hydroxylase in the alpaca (Lama pacos) brainstem and colocalization with CGRP

The distribution of tyrosine hydroxylase (TH) in the brainstem of alpaca (Lama pacos) has been analysed using immunohistochemical methods. The following catecholaminergic cell nuclei have been detected: A1, C1, A2, C2 and area postrema in the medulla oblongata; A5, A6d, A7sc and A7d in the pons; as have several mesencephalic groups: A8, A9l, A9m, A9v, A9pc, A10, A10c, A10d and A10dc. This nuclear parcellation differs from that found in rodents, but agrees with the results reported in other members of the Artiodactyla order, such as giraffe or pig, and with the catecholaminergic distribution detected in species of other mammalian orders. Thus, these findings support the hypothesis that the animals included in the same order show the same nuclear complement in the neuromodulatory systems. In addition, it seems that other species share the same catecholaminergic groups as the alpaca, suggesting that a specific nuclear disposition was important and worth maintaining throughout evolution. Moreover, the distribution of TH has been compared with that of CGRP by double immunohistochemistry. Double-labelled neurons were very isolated and observed only in a few catecholaminergic groups: A1 and C2 in the medulla oblongata, A6d, A7sc and A7d in the pons, and A9l in the mesencephalon. However, interaction between TH and CGRP may be possible in more brainstem regions, particularly the area postrema. This interaction may prove important in the regulation of the specific cardiovascular control of alpacas given their morphological characteristics.     

Mapping of CGRP in the alpaca (Lama pacos) brainstem

In this study, we demonstrate the presence of immunoreactive structures containing calcitonin gene-related peptide in the alpaca brainstem. This is the first time that a detailed mapping of the cell bodies and fibers containing this neuropeptide in the alpaca brainstem has been carried out using an immunocytochemical technique. Immunoreactive cell bodies and fibers were widely distributed throughout the alpaca brainstem. A high density of calcitonin gene-related peptide-immunoreactive perikarya was found in the superior colliculus, the dorsal nucleus of the raphe, the trochlear nucleus, the lateral division of the marginal nucleus of the brachium conjunctivum, the motor trigeminal nucleus, the facial nucleus, the pons reticular formation, the retrofacial nucleus, the rostral hypoglossal nucleus, and in the motor dorsal nucleus of the vagus, whereas a high density of fibers containing calcitonin gene-related peptide was observed in the lateral division of the marginal nucleus of the brachium conjunctivum, the parvocellular division of the alaminar spinal trigeminal nucleus, the external cuneate nucleus, the nucleus of the solitary tract, the laminar spinal trigeminal nucleus, and in the area postrema. This widespread distribution indicates that the neuropeptide studied might be involved in multiple functions in the alpaca brainstem.     

Immunohistochemical mapping of pro-opiomelanocortin- and pro-dynorphin-derived peptides in the alpaca (Lama pacos) diencephalon

Using an indirect immunoperoxidase technique, we studied the distribution of cell bodies and fibres containing non-opioid peptides (adrenocorticotropin hormone (ACTH), alpha-melanocyte-stimulating hormone) and opioid peptides (beta-endorphin (1–27), alpha-neo-endorphin, leucine-enkephalin) in the alpaca diencephalon. No immunoreactive cell bodies containing ACTH were found. Perikarya containing the other four peptides were observed exclusively in the hypothalamus and their distribution was restricted. Perikarya containing alpha-melanocyte-stimulating hormone or alpha-neo-endorphin showed a more widespread distribution than those containing leucine-enkephalin or beta-endorphin (1–27). Cell bodies containing pro-opiomelanocortin-derived peptides were observed in the arcuate nucleus, anterior and lateral hypothalamic areas and in the ventromedial and supraoptic hypothalamic nuclei, whereas perikarya containing alpha-neo-endorphin (a pro-dynorphin-derived peptide) were found in the arcuate nucleus, dorsal and lateral hypothalamic areas, and in the paraventricular, ventromedial and supraoptic hypothalamic nuclei. Immunoreactive cell bodies containing leucine-enkephalin were found in the lateral hypothalamic area and in the paraventricular hypothalamic nucleus. Immunoreactive fibres expressing pro-opiomelanocortin-derived peptides were more numerous than those expressing pro-dynorphin-derived peptides. A close anatomical relationship was observed: in all the diencephalic nuclei in which beta-endorphin (1–27)-immunoreactive fibres were found, fibres containing alpha-melanocyte-stimulating hormone or alpha-neo-endorphin were also observed. Fibres containing beta-endorphin (1–27), alpha-melanocyte-stimulating hormone or alpha-neo-endorphin were widely distributed throughout the diencephalon, but fibres containing ACTH or leucine-enkephalin showed a moderate distribution. The distribution of the five peptides studied here is also compared with that reported previously in other mammalian species. The widespread distribution observed indicates that both the pro-dynorphin and the pro-opiomelanocortin systems are involved in multiple physiological actions (e.g., food intake, thermoregulation, neuroendocrine and reproductive mechanisms) in the alpaca diencephalon.     

Distribution of somatostatin-28 (1-12) immunoreactivity in the diencephalon and the brainstem of the dog

The term somatostatin refers to a family of peptides, mainly somatostatin-14, somatostatin-28 and somatostatin-28 (1-12), which are the cleavage products of a single 116 amino acid-long preprosomatostain molecule. The production of antibodies to these peptides allows their localization in a number of neuronal populations throughout the entire neuroaxis in many mammals. The dog has been pointed out as an extremely useful animal model for studying age-related cognitive dysfunction and other neuronal changes associated with aging in which somatostatin appears to be involved. However, only very scanty information is available with regard to the distribution of somatostatin in the brain of the dog. In the present work we have determined the pattern of the distribution of somatostatin-28 (1-12) immunoreactivity in the diencephalon and the brainstem of the dog. High to moderate densities of labeled perikarya were found in the anterior periventricular and arcuate hypothalamic nuclei, the reticular thalamic nucleus, in delimited parts of the nucleus of the brachium inferior colliculus, the retrorubral area, the dorsal raphe nucleus, the myelencephalic reticular formation and the dorsal motor nucleus of the vagus. Less dense population of somatostatin cells were localized in other diencephalic and brainstem nuclei. The distribution of labeled fibers was even broader as in addition to those above mentioned there were a number of areas that appeared devoid of labeled perikarya. Many of the findings were similar to those reported in earlier works while others underlined the existence of inconsistencies in the distribution pattern of this peptide in the brain of mammals.     

Presence of enamel on the incisors of the llama (Lama glama) and alpaca (Lama pacos)

Attempts have been made to define the relationships among the South American camelids, the guanaco, llama, alpaca, and vicuna, by comparing the morphology of their incisors. The alpaca has been reported to have an incisor morphology similar to the vicuna, lacking enamel on the lingual surface. The llama and guanaco are said to have enamel on both the labial and lingual surface of their incisor teeth. These comparisons have been based on gross morphological observations and not on histologic analysis. This study was undertaken to determine whether or not alpaca teeth have enamel on the lingual surface. The cross-sectional histologic anatomy of the incisor teeth was compared in two closely related South American camelid species, the llama (Lama glama), and the alpaca (Lama pacos). Thick sections (300 μm) and scanning electron microscopy were the techniques utilized. The mandibular first, second, and third incisors were examined in four llamas and five alpacas. A substantial layer of enamel was present on all surfaces of all llama incisors. The enamel layer on the labial surface of the alpaca incisors closely resembled that found in the llama. The enamel layer on the lingual surface of the alpaca incisors, although greatly reduced, was distinctly present. Alpacas may be more closely related to guanacos and llamas than to vicunas. A histologic study of vicuna incisors would help to better define the relationships of the four camelids. Anat Rec. 249:441–449, 1997. © 1997 Wiley-Liss, Inc.     

Listeria monocytogenes septicaemia and concurrent clostridial infection in an adult alpaca (Lama pacos)

A 10-year-old alpaca with a history of anorexia, weight loss and diarrhoea was humanely destroyed and shown to have a multifocal necrotizing hepatitis, splenitis and colitis, as well as an ulcerative to diphtheroid ileitis. Immunohistochemical examination revealed Listeria monocytogenes antigen in the liver and ileum. In addition, L. monocytogenes and Listeria sp.-specific gene fragments were detected by the polymerase chain reaction. L. monocytogenes was isolated from liver and small intestine and Clostridium perfringens type A with beta(2) toxin was found in the small intestine. It is suggested that the infection with C. perfringens type A facilitated the systemic spread of L. monocytogenes.     

Immunoreactive somatostatin-28(1-12) is increased in Huntington's disease

Somatostatin-28(1-12) concentrations were measured in Huntington's disease (HD) postmortem tissue using a specific radioimmunoassay. Concentrations of immunoreactive somatostatin-28(1-12) were significantly increased in the caudate and putamen but were unchanged in cortical areas A9 and A17. Since somatostatin-28(1-12) terminates with the amino acids Arg-Glu-OH, we examined whether this dodecapeptide compound might exert a neurotoxic effect. Injections of somatostatin-28(1-12) into rat striatum showed no evidence of histologic damage.     

Dalmeny disease in an alpaca (Lama pacos): sarcocystosis, eosinophilic myositis and abortion.

Disseminated eosinophilic myositis was diagnosed in an alpaca that had been imported to the USA from Peru 5 years earlier. The myositis was associated with macroscopically visible large sarcocysts that were characterized histologically by septate compartments containing bradyzoites, and ultrastructurally by cyst walls composed of anastomosing villous protrusions. Two hours before death, the alpaca aborted an 8-month-gestation fetus, but no lesions were found in the uterus, placenta or fetus. Additional macroscopical findings included haemoabdomen and myofibre haemorrhage, degeneration and necrosis. It is believed that this is the first described case of clinical disease associated with a Sarcocystis sp. (probably S. aucheniae) in camelids.     

Occlusal Development and Masseter Activity in Alpacas (Lama pacos)

Tooth eruption and the development of occlusion are significant ontogenetic changes in the masticatory apparatus of mammals. Here, we test the hypothesis that changes in masseter activity are correlated with increased occlusal contacts at major stages of dental development in the alpaca, Lama pacos. We compare electromyographic data from the superficial and deep masseter in infant and juvenile alpacas prior to and following m1 occlusion and from adults with full permanent dentitions. The pre‐m1 and post‐m1 occlusion groups exhibit similar masseter activity durations, chewing cycle durations, and with the exception of the balancing‐side deep masseter, similar timing differences between the jaw muscles. On average, the balancing‐side deep masseter fires significantly later in the post‐m1 occlusion group. The m2–m3 group exhibits significantly longer chewing cycle length and an even later firing balancing‐side deep masseter. Increased occlusion is also associated with an increase in the relative amount of working‐side superficial and deep masseter muscle activity when compared with the balancing side muscles. Although the development of occlusal relations in infant and juvenile alpacas are associated with minor changes in masseter activation patterns, additional molar occlusal contacts increase chewing cycle duration resulting in concomitant changes in masseter recruitment patterns. Currently, we cannot rule out that musculoskeletal development influences masseter activity as demonstrated in other mammals. However, the data presented here indicate that alpacas have a relatively delayed onset of the adult motor pattern that may be correlated with changes in occlusal relations due to tooth eruption. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Immunohistochemical characterization of lymphosarcoma in two alpacas (Lama pacos)

Species cross-reactive anti-peptide antibodies were assessed in formalin-fixed tissue for use in immunophenotyping of lymphosarcoma in two alpacas. Diagnosis of lymphosarcoma was made by routine histopathological examination. Primary antibodies used for immunophenotyping were anti-human CD3 and anti-human CD5 for T cells; and anti-human CD79a and anti-human CD79b for B cells/plasma cells. In one case, most of the neoplastic cells were labelled with both anti-CD3 and anti-CD79b, and smaller numbers were labelled with anti-CD79a. The other case was classified as a B-cell tumour on the basis of labelling of the majority of neoplastic cells with anti-CD79b and anti-CD79a. This is the first recorded attempt at immunophenotyping lymphosarcoma in alpacas and, to our knowledge, the first record of presumptive co-expression of T- and B-cell-associated molecules in lymphosarcoma in the veterinary literature.     

Comparative NMR studies of diffusional water permeability of red blood cells from different species. X. Camel (Camelus dromedarius) and alpaca (Lama pacos)

The diffusional water permeability (P _d) of camel and alpaca red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition withp-chloromercuribenzene sulphonate (PCMBS). The values ofP _d were, in the case of alpaca RBC≈4.6×10^?3 cm/s at 25°C, 5.4×10^?3 cm/s at 30°C, 6.6×10^?3 cm/s at 37°C and 7.7×10^?3 cm/s at 42°C. In case of camel RBC the values ofP _d where ≈4.2×10^?3 cm/s and 9.0×10^?3 cm/s at 42°C. Systematic studies on the effects of PCMBS on water diffusion in camel RBC indicated that the maximal inhibition was reached in 45 min with 1–2 mm PCMBS. The values of maximal inhibition were around 47% at 25°C and 68% at 30°C for alpaca RBC and around 62% at 25°C and 56% at 37°C for camel RBC. The basal permeability to water of alpaca RBC was estimated at around 2.6×10^?3 cm/s at 25°C, 1.7×10^?3 cm/s at 30°C and of camel RBC as 1.8×10^?3 cm/s at 25°C and 3.0×10^?3 cm/s at 37°C. The values of the activation energy of water diffusion (E _{a, d}) were around 23 kJ/mol for camel and 34 kJ/mol for alpaca RBC. This suggests that in addition to the number of transport channels other features of the pathways might be important for defining the temperature dependence of the water permeability.     

Comparative distribution of NADPH-diaphorase activity and tyrosine hydroxylase immunoreactivity in the diencephalon and mesencephalon of the domestic chicken (Gallus domesticus)

We described the distribution of NADPH-diaphorase-containing neurons in relation to tyrosine hydroxylase immunoreactivity in the diencephalon and mesencephalon of the chicken. In the diencephalon, both markers were found in the lateral hypothalamus, dorsal hypothalamic area, hypothalamic periventricular nucleus, paraventricular nucleus and mamillary area. A close examination showed that the fine distribution of these markers differed slightly, so that they were never observed in the same neurons. In the mesencephalon, NADPH-diaphorase and tyrosine hydroxylase immunoreactivity were found in the ventral pedunculopontine area (nucleus tegmenti pedunculopontinus pars compacta, adjacent areas surrounding the quintofrontal tract and the nucleus mesencephalicus profundus ventralis), the coeruleus complex (locus coeruleus, ventral and dorsal subcoeruleus nuclei), the ventral tegmental area and the central gray. The majority of these neurons contained either diaphorase or tyrosine hydroxylase. Nevertheless, in a few cases both markers appeared to colocalize in the same neuron, typically in large perikarya of the ventral pedunculopontine area.     

Comparative distribution of NADPH-diaphorase activity and tyrosine hydroxylase immunoreactivity in the diencephalon and mesencephalon of the domestic chicken (Gallus domesticus)

We described the distribution of NADPH-diaphorase-containing neurons in relation to tyrosine hydroxylase immunoreactivity in the diencephalon and mesencephalon of the chicken. In the diencephalon, both markers were found in the lateral hypothalamus, dorsal hypothalamic area, hypothalamic periventricular nucleus, paraventricular nucleus and mamillary area. A close examination showed that the fine distribution of these markers differed slightly, so that they were never observed in the same neurons. In the mesencephalon, NADPH-diaphorase and tyrosine hydroxylase immunoreactivity were found in the ventral pedunculopontine area (nucleus tegmenti pedunculopontinus pars compacta, adjacent areas surrounding the quintofrontal tract and the nucleus mesencephalicus profundus ventralis), the coeruleus complex (locus coeruleus, ventral and dorsal subcoeruleus nuclei), the ventral tegmental area and the central gray. The majority of these neurons contained either diaphorase or tyrosine hydroxylase. Nevertheless, in a few cases both markers appeared to colocalize in the same neuron, typically in large perikarya of the ventral pedunculopontine area.Abbreviations AVT Ventral tegmental area of Tsai - DAB diaminobenzidine - DHA dorsal hypothalamic area - DMA anterior dorsomedial thalamic nucleus - DMP posterior dorsomedial thalamic nucleus - DSV ventral supraoptic decussation - GCt central gray - ICH intercalated hypothalamic nucleus - IH inferior hypothalamic nucleus - LHy lateral hypothalamic area - LoC locus coeruleus - ML lateral mamillary nucleus - MM medial mamillary nucleus - MPv ventral part of the deep mesencephalic nucleus - nI interstitial nucleus of Rendahl - NADPH-d NADPH-diaphorase - NGS normal     

Alteration of tyrosine hydroxylase activity in PC12 cells infected with herpes simplex virus type 1

Summary During infection with herpes simplex virus type 1 (HSV-1) the activity of tyrosine hydroxylase (TH) in PC 12 pheochromocytoma cells was initially depressed reaching a nadir at 6 hours post-inoculation, but recovered rapidly with a return to baseline activity by 8 to 9 hours post-inoculation. Subsequently, TH activity again fell with a second more variable rise in activity occurring at 24 hours post-inoculation. Studies with metabolic inhibitors and 2 temperature-sensitive viral mutants indicated that these alterations of TH activity were dissociated from morphological cytopathology and likely required expression of late viral gene products. Immunotitration using anti-TH antibody suggested that early depression of TH activity resulted principally from loss of enzyme protein rather than simple enzyme inactivation, and that reconstitution of activity at 9 hours was related to augmented enzyme synthesis. These observations illustrate the complexity of perturbed cellular metabolism during HSV-1 infection and suggest involvement of two unexpected processes: alteration of a specialized cell function as a result of viral genes expressed late in the replicative cycle, and augmented synthesis of a cell-coded gene product during the course of infection.With 6 Figures     

Co-localization of tyrosine hydroxylase (TH)- and serotonin (5-HT)-immunoreactive innervation in the rat pituitary gland

Adult male Sprague-Dawley rats were examined for possible co-localization of tyrosine hydroxylase (TH) and serotonin (5-HT) within innervation of the pituitary neurointermediate lobe. Use of sheep antiserum to TH and a secondary antibody coupled to fluorescein isothiocyanate (FITC), followed by rabbit anti-5-HT, then rhodamine-coupled second antibody, produced co-existent staining in some, but not all fibers of both the neural and intermediate lobes, using paraffin embedded tissues. Application of a combination of the two primary antibodies followed by sequential application of secondary antibody also resulted in co-localized antigens in selected fibers. Multiple 'classic' neurotransmitters within the same nerve terminals may modulate selected areas of endocrine tissue to control hormone release.     

干细胞因子及其受体c-KIT对羊驼毛囊黑色素细胞增殖与分化的影响及其机制

目的 探讨SCF/c-KIT信号通路对羊驼毛囊黑色素细胞分化、增殖和定位的作用及羊驼丰富毛色性状形成的细胞学机制。 方法 选取8只成年雄性羊驼（4只有色被毛,4只白色被毛）,采用免疫组织化学方法研究SCF和c-KIT受体在羊驼皮肤组织中的表达定位;采用实时定量PCR方法分析SCF和c-KIT基因在羊驼皮肤组织中的表达水平。 结果 SCF和c-KIT受体在不同毛色皮肤组织中均有表达,但在不同被毛颜色羊驼皮肤组织中的表达量和表达部位存在差异;ΔΔCt法统计分析SCF和c-KIT基因在不同颜色被毛羊驼皮肤组织中的表达情况显示,SCF基因在有色被毛皮肤组织中的相对表达量是白色被毛皮肤组织的2.41倍,而c-KIT基因在有色被毛皮肤组织中的相对表达量是白色被毛组织的1.20倍。 结论 SCF/c-KIT信号通路参与调节黑色素细胞在皮肤组织中的增殖、分化;成熟黑色素细胞的数量及其在毛囊组织中的分布位置决定羊驼被毛颜色的形成;SCF信号调节不同分化程度黑色素细胞在毛囊组织中的分布。     

Effects of repeated systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal tyrosine hydroxylase activity in vitro and tyrosine hydroxylase content

We examined both in vitro tyrosine hydroxylase (TH) activity and TH content determined by a new enzyme immunoassay in the mouse striatum after repeated systemic injection of MPTP. Repeated systemic administration of MPTP to mice (30 mg/kg per day, subcutaneously for 8 days) caused an approximately 65% decrease of both TH activity and TH content in the striatum. The intensity of immunohistochemical staining of TH protein in the striatum was also reduced in MPTP-treated mice. These results indicate that the reduction of TH activity in vitro after the repeated administration of MPTP is due to reduction of TH protein as a result of nerve degeneration.     

IDDM2 and the polymorphism of the human tyrosine hydroxylase (hTH) gene in African Americans with type-1 diabetes

In this study, we investigate the polymorphic microsatellite repeat (TCAT)n, in the insulin gene region that has been associated with susceptibility to type-1 diabetes in some Caucasian populations. The microsatellite repeat polymorphism begins at base pair 1,170 in intron 1 of the hTH gene, which is located on the short arm of chromosome 11. This study is the first to investigate the association of this microsatellite repeat polymorphism in African-American type-1 diabetes patients and controls. The predicted amplified sequence was 254 bp. We found five alleles among African Americans in the Washington, DC area. The alleles were labeled K5 (244 bp), K4 (248 bp), K3 (252 bp), K2 (256 bp), and K1 (260 bp), and heterozygosity was greater than 0.75. The most frequent allele of the hTH microsatellite repeats was K5 (248 bp) with a frequency 0.62 in controls and 0.66 in type-1 diabetes patients, which did not differ significantly. Although the largest allele was more frequent in controls, the difference was not statistically significant. The five alleles of the hTH microsatellite generated 15 different genotypes. The most frequent genotype in controls and patients was K5/K4, whose frequencies were 0.19 and 0.17, respectively. No significant differences in genotype frequencies were found between type-1 diabetes patients and controls. This data shifts the focus from hTH to the VNTR at the insulin gene for IDDM2, the second major candidate gene for type-1 diabetes.     

Histamine secretion in leukocyte incubates of patients with allergic hyperreactivity induced by somatostatin-14 and somatostatin-28

Somatostatin is a potent histamine secretagogue found not only in rat mast cells but also in human leukocyte preparations. In concentrations 5 mol/l, somatostatin-14 induces histamine release, which correlates with the basophilic blood cell count, as shown in samples from allergic patients suffering from slight basophilia. Somatostatin-14 is twice as effective as somatostatin-28 on a molar basis, and acylating the tetradecapeptide withN-hydroxysuccinimidyl-p-hydroxy-phenyl-propionate decreases significantly the potency of histamine release.