Enrico Bastianelli

Hippocalcin in rat retina. Comparison with calbindin-D28k, calretinin and neurocalcin.

The post-natal developmental expression in rat retina of four calcium-binding proteins belonging to the calmodulin-troponin-C family was investigated by immunohistochemistry using anti-calbindin-D28k, anti-calretinin, anti-hippocalcin and anti-neurocalcin polyclonal antibodies on paraffin sections from Wistar rat retinae aged from post-natal days 1 (P1), 5 (P5), 10 (P10), 20 (P20) to adulthood (8 weeks). Immunoblot using anti-hippocalcin and homogenates proteins from retina, cerebellar cortex, hippocampus and cerebellum was also performed. Hippocalcin immunoreactivity in adult rat retina was demonstrated by both immunohistochemistry and Western blot. During post-natal development, calbindin-D28k, calretinin and neurocalcin immunoreactivity were detected at P1 in ganglion cells, whereas hippocalcin immunoreactivity was seen later at P5 in this cell layer. In the amacrine cell layer, neurocalcin immunoreactivity was detected at P5 and hippocalcin at P10. Calbindin-D28k was labelling the immature horizontal cell, calretinin was detected in nearly all ganglion cells and in some amacrine cells since P1. These three calcium-binding proteins do not seem to play a role in synaptogenesis which takes place later. We confirmed that calbindin-D28k appeared to be a good marker for horizontal cells. The presence of hippocalcin, a myristoylated calcium-binding protein belonging to the recovering subfamily and previously localized in few brain areas has been detected for the first time in retina.

Neurocalcin immunoreactivity in rat olfactory bulb

Neurocalcin, a newly discovered calcium-binding protein belonging to the recoverin-like superfamily, was detected immunohistochemically in tufted cells from the rat olfactory bulb. More precisely, only periglomerular tufted cells and some tufted cells from the external plexiform layer were expressing neurocalcin. Western blot analysis has confirmed the presence of neurocalcin in rat olfactory bulb. Lack of neurocalcin immunoreactivity in mitral cells and periglomerular cells favor a different phylogenic origin between tufted and mitral or periglomerular cells.

Decreased pool of mesenchymal stem cells is associated with altered chemokines serum levels in atrophic nonunion fractures

Nonunion fractures can cause severe dysfunction and are often difficult to treat mainly due to a poor understanding of their physiopathology. Although many aspects of impaired fracture healing have been extensively studied, little is known about the cellular and molecular mechanisms leading to atrophic nonunion. Therefore, the aim of the present study was to assess the pools and biological functions of bone marrow-derived mesenchymal stem cells (hMSCs) and circulating endothelial progenitor cells (EPCs) in atrophic nonunion patients compared to healthy subjects, and the systemic levels of growth factors involved in the recruitment, proliferation and differentiation of these cells. In nonunions, the pool of hMSCs was decreased and their proliferation delayed. However, once committed, hMSCs from nonunions were able to proliferate, differentiate into osteoblastic cells and mineralize in vitro as efficiently as hMSCs from healthy subjects. In parallel, we found altered serum levels of chemokines and growth factors involved in the chemotaxis and proliferation of hMSCs such as leptin, interleukin-6 (IL-6) and its soluble receptor, platelet-derived growth factor-BB (PDGF-BB), stem cell factor (SCF) and insulin-like growth factor-1 (IGF-1). Moreover, we showed that the number of EPCs and their regulating growth factors were not affected in nonunion patients. If nonunion is generally attributed to a vascular defect, our results also support a role for a systemic mesenchymal and osteogenic cell pool defect that might be related to alterations in systemic levels of factors implicated in their chemotaxis and proliferation.

Transient expression of calretinin during development of chick cerebellum. Comparison with calbindin-D28k.

Calcium ions play a critical role in neural development. Insights into the ontogeny of Ca2+ homeostasis were gained by investigating the developmental expression of two E-F hand calcium-binding proteins. Calretinin and calbindin were monitored through their immunoreactivity in the developing chick cerebellum (from E6 to E20). Calbindin was detected from E13 and in Purkinje cells only. Intensity of labelling increased with Purkinje cell development. Calretinin presented a transitory immunoreactivity between E11 and E20 in the internal granular cell layer. This cell layer contains cells which will differentiate into Golgi and granular cells which are calretinin-negative in adult chick cerebellum. Calretinin immunoreactivity presented a peak (both in number of cells and in intensity) at E15 and fell dramatically after E20 while calbindin immunoreactivity was restricted to the Purkinje cells and increased with the development of these cells.

Calmodulin, calbindin-D28k, calretinin and neurocalcin in rat olfactory bulb during postnatal development.

Odorant stimulation of receptor cells results in a calcium influx that activates the transduction pathway. The olfactory neurons extend axons to the olfactory bulb where they synapse onto mitral cells. Ca(2+)-acceptors also may participate in subsequent processing of olfactory information. The present study describes the distribution of calmodulin, calretinin, calbindin-D28k and neurocalcin during rat main olfactory bulb development. From postnatal day 1 (P1) we observed in the olfactory nerve layer a thin external bundle containing calbindin and calretinin whereas calmodulin was present in a large internal bundle. In tufted cells, neurocalcin immunoreactivity was detected at P10 and increased until P20. In mitral cells calmodulin was intensively immunoreactive at P1 but decreased during development to disappear at adulthood whereas calretinin was weakly labelled at P1 but raised in intensity until P20. In granule cells calbindin-D28k and calretinin were detected from P1. Giant neurons were positive for both calretinin and calbindin-D28k from postnatal day 20.

Calbindin-D28k, calretinin, and recoverin immunoreactivities in developing chick pineal gland

Bastianelli E, Pochet R. Calbindin‐D28k, calretinin, and recover in immunoreactivities in developing chick pineal gland. J. Pineal Res. 1994: 17: 103–111.

Cerebellar spongiform degeneration induced by acute lithium intoxication in the rat.

Cerebellar syndrome has been described after acute lithium intoxication in human. Neuropathological studies have demonstrated neuronal loss and spongiosis in the cerebellum. We describe an animal model of acute lithium-induced cerebellar degeneration. Five hours following administration of lithium chloride (250 mg/kg, i.p.), the cerebellar white matter of seven rats out 14 exhibited extensive spongiform changes. Microdialysis study in the rat cerebellar cortex demonstrated basal concentrations of dopamine (DA), hydroxy-3-methoxyphenylacetic acid (HVA) and 5-hydroxy-3-indolacetic acid (5-HIAA). These metabolites were unaffected by acute lithium intoxication suggesting that the cerebellar toxicity is not due to a modification of dopaminergic or serotoninergic neurotransmission.

Calbindin-D28k, calretinin, and S-100 immunoreactivities in rat pineal gland during postnatal development

Abstract: Profound morphological modifications occur during postnatal development of the rat pineal gland. We have immunohistochemically followed those events from postnatal day 1 to 20 by using three cytoarchitectonic markers (S‐100, calbindin‐D28k, and calretinin) that belong to the calmodulin/troponin C calcium‐binding protein family. In the developing rat pineal, anticalbindin‐D28k antibody labels three cell types: immature and mature astrocytes and perivascular type II pinealocytes. During development, calbindin‐D28k positive cells migrate from the base of the pineal stalk into the superficial part of the pineal. Calbindin‐D28k, usually used as a neuronal marker in the central nervous system, recognizes in rat pineal precursor astrocytes 5 days before S‐100 and labels a subpopulation somewhat different from S‐100 positive astrocytes. Calretinin im‐munoreactivity appeared in the postero‐superior part of the pineal and was abundant until postnatal day 5, then its density dramatically felt to leave, after postnatal day 20, an occasional population of cells whose morphology is compatible with neuron‐like cells.

Distribution of calmodulin, calbindin-D28k and calretinin among rat olfactory nerve bundles

Calmodulin, calbindin-D28k and calretinin are calcium-binding proteins largely distributed in the bipolar olfactory receptor cells. In the olfactory epithelium their distribution seemed to be random. Using immunohistochemistry we have examined their localization in rat olfactory axons extending to the olfactory bulb. Sections were analyzed both horizontally and vertically. Almost all fibers were immunoreactive for one of the three intracellular calcium-binding proteins whose distribution was not random among the bundles. Three different subclasses of fibers could be detected: calbindin-D28k and calretinin immunoreactivities were restricted to external fibers whereas calmodulin immunoreactivity was intense, abundant and largely distributed throughout the internal portion of the olfactory nerve. This additional degree of organization detected in the olfactory axons might play a role in odor discrimination.

Calmodulin, calbindin-D28K and calretinin in rat and chicken pineal glands: Immunocytochemical and immunoblotting analysis

In pineal gland, melatonin is synthesized in pinealocytes. Pharmacological studies using calmodulin antagonists suggested that melatonin synthesis was regulated through calmodulin. However, immunohistochemical studies showed that calmodulin could only be detected in pineal glial cells, and not in pinealocytes. To further investigate this discrepancy, we have tried to detect calmodulin not seen by immunohistochemical methods. We have used rat and chicken pineal homogenate supernatants and Triton X-100-treated pellets denatured by sodium dodecyl sulfate, subjected to electrophoresis and immunoblotting using anti-calmodulin antibodies. Two different IgG (#465 and #860) purified from anti-calmodulin sera were used. In rat pineal homogenate supernatants, calmodulin could be detected by immunoblotting using both antibodies. Some calmodulin could also be detected in the Triton-treated pellet fractions, but no additional cross-reacting bands were detected. However, in both chicken pineal homogenate supernatants and Triton-extracted pellets, in addition to a calmodulin immunoreactive band, two other proteins with approximate molecular masses (M(r)) of 56 kDa and 60 kDa were detected using anti-calmodulin #465. For comparison, similar immunoblot experiments were performed for detection of calbindin-D28K and calretinin, two other calcium binding proteins expressed in different pineal cell populations. Interestingly, Triton extraction of chicken pineal pellets revealed additional bands cross-reacting with each antibody. Anti-calbindin-D28K cross-reacted strongly with a M(r) = 68 kDa protein and weakly with a M(r) = 56 kDa protein. Anti-calretinin cross-reacted strongly with a M(r) = 93 kDa protein and weakly with a M(r) = 56 kDa protein.