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Many organs of Viviparus viviparus were explanted into a culture medium of low osmotic pressure, adapted for fresh water molluscs. The development in vitro of the explants differed according to the particular organ. The best results were obtained with the heart, which was still contracting after 2 months in culture, and with the tentacle, which remained excitable for a period of 6 to 7 weeks. The central nervous system survived well for 18 to 20 days. The fragments of digestive gland died rapidly, in about 1 week. Fragments of intestine retained a normal appearance for 2 weeks. The period of survival of male or female gonads was also 2 to 3 weeks. Vitellogenesis did not proceed very far in the ovary, but the young oocytes remained healthy. In the testis, only spermatogonia persisted and continued to divide during the culture period: further stages would appear to require a hormonal supplement. [ABSTRACT FROM AUTHOR]

A bioassay was developed in Helix aspersa to study the effect of known endocrine centers on the twomonth-old gonad, using organ culture. The incorporation ofC leucine andH fucose in the juvenile gonad almost doubled over control levels, in the presence of the brain and the dorsal bodies. [ABSTRACT FROM AUTHOR]

The Scrg1 gene was initially discovered as one of the genes upregulated in transmissible spongiform encephalopathies (TSE). Scrg1 encodes a highly conserved, cysteine-rich protein expressed principally in the central nervous system. The protein is targeted to the Golgi apparatus and large dense-core vesicles/secretory granules in neurons. We have recently shown that the Scrg1 protein is widely induced in neurons of scrapie-infected mice, suggesting that Scrg1 is involved in the host response to stress and/or the death of neurons. At the ultrastructural level, Scrg1 is associated with dictyosomes of the Golgi apparatus and autophagic vacuoles of degenerative neurons. It is well known that apoptosis plays a major role in the events leading to neuronal cell death in TSE. However, autophagy was identified in experimentally induced scrapie a long time ago and was recently reevaluated as a possible cell death program in prion diseases. The consistent association of Scrg1 with autophagic structures typical of scrapie is in agreement with the recruitment of Golgi-specific proteins in this degradation process and we suggest that Scrg1 might be used as a specific probe to identify neuronal autophagy in TSE.

Rat quiescin/sulphydryl oxidase (rQSOX) introduces disulphide bridges into peptides and proteins with the reduction of molecular oxygen to hydrogen peroxide. Its occurrence has been previously highlighted in a wide range of organs by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses, methods that have provided information concerning its expression in whole organs but that do not reveal the cell types expressing this enzyme. In this report, in addition to RT-PCR and Western blot experiments, the cell-specific localization of rQSOX has been investigated in a wide range of male and female adult rat tissues by using in situ hybridization and immunohistochemistry. Labelling was detected in most organs and systems including the immune, endocrine and reproductive systems, the respiratory, digestive and urinary tracts and the skin. No labelling was observed in the heart, blood vessel endothelium, liver or smooth and skeletal muscles. rQSOX expression was mainly localized in epithelial cells specialized in secretion, strengthening the hypothesis that QSOX enzymes play an important role in the mechanism of secretion, notably in the folding of secreted proteins. The intracellular patterns of immunolabelling indicate that the protein usually follows the secretory pathway, which is in accordance with its secreted nature and its presumed involvement in the elaboration of the extracellular matrix. In seminiferous tubules, where a high level of expression was noticed, QSOX might play an important physiological role in sperm function and serve as a marker for the diagnosis of male infertility.

We have previously identified Scrg1, a gene with increased cerebral mRNA levels in transmissible spongiform encephalopathies (TSE) such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. In this study, Scrg1-immunoreactive cells, essentially neurons, were shown to be widely distributed throughout the brain of scrapie-infected mice, while only rare and weakly immunoreactive cells could be detected in the brain of non-infected normal mice. Induction of the protein was confirmed by Western blot analysis. At the ultrastructural level, Scrg1 protein was associated with dictyosomes of the Golgi apparatus and autophagic vacuoles in the central neurons of the scrapie-infected mice. These results suggested a role for Scrg1 in the pathological changes observed in TSE. We have generated transgenic mice specifically expressing Scrg1 in neurons. No significant differences in the time course of the disease were detected between transgenic and non-transgenic mice infected with scrapie prions. However, tight association of Scrg1 with autophagic vacuoles was again observed in brain neurons of infected transgenic mice. High levels of the protein were also detected in degenerating Purkinje cells of Ngsk Prnp 0/0 mice overexpressing the Prnd gene coding for doppel, a neurotoxic paralogue of the prion protein. Furthermore, induction of Scrg1 protein was observed in the brain of mice injured by canine distemper virus or gold thioglucose treatment. Taken together, our results indicate that Scrg1 is associated with neurodegenerative processes in TSE, but is not directly linked to dysregulation of prion protein.

Mammalian Augmenter of Liver Regeneration protein (ALR) was first identified as a secondary growth factor involved in liver regeneration. Its sulfhydryl oxidase activity and involvement in iron homeostasis have been recently demonstrated. ALR is expressed in a broad range of peripheral organs, and initial experiments gave also evidence for the occurrence of this protein in brain. In the present study, we investigated in detail the expression of ALR in rat brain sections and determined its cellular and subcellular localizations using biomolecular and immunohistochemical procedures. As shown by Northern blot, ALR is differentially expressed throughout the rat brain, with the highest mRNA levels in the cerebellum and diencephalon. High protein levels were also detected in the brain and cerebellum by Western blot. ALR immunoreactivity was found in neurons and glial cells throughout brain rostrocaudal extent. Labeled astrocytes were particularly abundant in the white matter, and immunoreactive neurons were observed in several regions including the olfactory bulb, isocortex, hippocampal formation, amygdala, thalamus, hypothalamus, some nuclei of the brainstem and cerebellum. In neurons, immunoelectron microscopy showed the protein in the nucleus and mainly in mitochondria. These subcellular localizations may correlate with the occurrence of two ALR protein isoforms in the brain. In the central nervous system, the enzyme might be of importance in heavy metal homeostasis whose dysregulation can induce neurodegenerative disorders.

The spatiotemporal pattern of distribution of the sulfhydryl oxidase QSOX throughout ontogeny was mapped in rat brain using immunohistochemistry. The enzyme was detected on embryonic day (E) 12 in the dawning mantle layer, but the adult-like pattern was acquired postnatally around day 30 (P30). Throughout ontogenesis, rQSOX was detected in immature and mature neurons, but not in glial cells. The rQSOX developmental pattern can be divided into four periods: on E12 the enzyme was detected in the brainstem, more precisely in motoneurons; later (E16), rQSOX-positive cells were also observed in the forebrain, in the caudoputamen, and the subventricular zone. During late embryogenesis (E18-20), the amount of rQSOX cells considerably increased throughout the brain; they initially appeared in the hippocampus, then in the isocortex. From birth onwards, complex modifications of the rQSOX distribution occurred leading to the adult pattern by P30. Although rQSOX exhibits an overall increasing spatiotemporal pattern of distribution, different expression strategies were distinguished depending on the cell type or brain area. By comparing the rQSOX ontogeny with data on neurogenesis and brain histogenesis, we hypothesize that the enzyme could play a role in guiding migrating cells, their settling, and neuronal maturation, e.g., during outgrowth and synaptogenesis.

Neurons producing melanin-concentrating hormone (MCH) are involved in a large array of functions. Some of these functions may be mediated by specific subpopulations. One such subpopulation was characterized by the expression of the neurokinin 3 receptor and the 'cocaine- and amphetamine-regulated transcript' (CART) peptide, while another expresses neither one of these two molecules. MCH+/CART+ axons were traced throughout the brain and showed a strikingly different pattern of distribution than that of MCH+/CART- axons. Particularly, many MCH+/CART+ axons are observed in the telencephalon, while MCH+/CART- projections are mostly directed toward the brainstem. Calbindin, a protein involved in calcium homeostasis, has been largely used in many structures of the brain for the identification of neuronal phenotypes. However, few MCH neurons were labeled for this protein. On the other hand, neurons producing the peptides hypocretins (Hcrt), and codistributed with the MCH neurons, were all labeled for calbindin. Thus, at least two subpopulations of MCH neurons can be distinguished on the basis of neuronal phenotypes and connections. These neurons may be involved in distinct circuitry and in distinct functions.

Neurotropic viruses are involved in pathologies of the central nervous system, triggering transient or irreversible disorders, such as neurological diseases or homeostasis imbalance. In experimental animals, viruses have been shown to cause obesity, a complex disease depending on multiple factors, including genetic susceptibility and environmental components. Using a mouse model of virally induced obesity following brain infection by the Canine Distemper Virus (CDV), a morbillivirus closely related to the human measles virus, we investigated the modulation of expression of several hypothalamic neuropeptides known to intervene in the regulation of body weight and energy expenditure, both during the acute and late stages of infection. During the acute stage, while viral replication occurs, we found a dramatic decrease of expressions of neuropeptides, in particular neuropeptide Y, melanin-concentrating hormone (MCH), hypocretin, vasopressin and tachykinins, the magnitude of which seemed to be linked to the viral burden and the individual susceptibility. The effect of the virus, however, varied with the hypothalamic nucleus and neuropeptide involved, suggesting that certain circuits were affected while others remained intact. During the late stage of infection, marked recovery to the initial hypothalamic levels of peptide expression was seen in a number of lean animals, suggesting recovery of homeostasis equilibrium. Interestingly, some neuropeptidergic systems remained disturbed in mice exhibiting obese phenotype, arguing for their involvement in triggering/maintaining obesity. Even though our data could not fully explain the viral-induced obesity, they may be helpful in understanding the molecular events associated with obesity and in investigating therapeutic alternatives.

The distribution of the sulfhydryl oxidase QSOX in the rat brain was mapped using immunohistochemistry. QSOX is specifically expressed by neurons throughout the rostrocaudal extent of the brain as well as in the spinal cord. Although a majority of neurons express QSOX, different intensities of labeling were observed depending on the area: the strongest labeling was observed in the olfactory bulbs, isocortex, hippocampus, basal telencephalon, several thalamic and hypothalamic nuclei, cerebellum, and numerous brainstem nuclei. This study also describes the ultrastructural localization of QSOX in neuronal cells and demonstrates that the enzyme is associated with the Golgi apparatus. Finally, selected double immunohistochemistry showed that in the hypothalamus the highest levels of QSOX labeling were colocalized in neuron populations that express disulfide-bounded neuropeptides. These observations are consistent with a role of the enzyme in secreted peptide/protein folding. Data presented herein will serve as a basis for further investigations of the physiological function of QSOX in the central nervous system., (Copyright 2004 Wiley-Liss, Inc.)