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P Castano

Publications and source records attributed to P Castano.

At least 19 recordsLinked to original sources
PubMed2001

Pattern of expression of c-Myc, Max and Bin1 in human anagen hair follicles.

BACKGROUND: We recently reported the presence of c-Myc immunoreactivity in two distinct regions of the inner root sheath (IRS) of human anagen hair follicles; they corresponded to the regions where keratinocytes of Henle's and Huxley's layers enter the terminal differentiation phase that will lead to their exfoliation in the pilary canal. These regions were denoted lower (LR) ring and upper ring (UR). OBJECTIVES: To extend these observations to other genes connected to c-Myc and specifically to Max and Bin1. Max is the best known heterodimeric partner of c-Myc, interacting with its C-terminal domain, and Bin1 is an adaptor protein interacting with its N-terminal domain. METHODS: Human anagen hair follicles were processed for c-Myc, Max and Bin1 immunohistochemistry and immunofluorescence. The presence of different isoforms of Bin1 was evaluated by Western blot analysis. RESULTS: Analysis of sections cut in several planes, including tangential, demonstrated the presence of a third ring of c-Myc-positive cells (intermediate ring; IR) in the cuticle of the IRS corresponding to the region where this thin layer undergoes keratinization. Max immunoreactivity was observed in the three layers of the IRS starting in the lower bulbar region and ending in each of them at the level of the corresponding c-Myc-positive ring. Bin1 immunoreactivity was clearly distinguished only in Huxley's layer and in the cuticle, starting in some cells below the UR and terminating at the level of the latter. The companion layer of the outer root sheath was also labelled up to the infundibular region. Max and Bin1 immunostaining were less consistently observed in other skin adnexae and in the epidermis. CONCLUSIONS: The results indicate that the asynchronous differentiation along the axis of the hair follicle of the different layers of the IRS and of the companion layer involves the expression of different genes that are interrelated in the so-called 'Myc network'. The specific localization of c-Myc in the IRS only at the level of the discrete and limited regions of the three rings appears to be the hallmark of the switch from differentiation to terminal differentiation/cell deletion.

Adaptor Proteins, Signal Transducing↗
PubMed2000

c-Myc expression in human anagen hair follicles.

The hair follicle represents a very attractive organ system for studying the precise balance between cell proliferation, growth, differentiation, and death of cells, because it periodically and regularly regenerates, retaining its morphogenetic signals throughout its life. One of the most intriguing oncogenes which is able to induce both cell growth and apoptosis, depending upon the environmental conditions, is c-myc. The aim of the present study was to investigate its presence and localization in human hair follicles by immunohistochemistry and immunofluorescence. Our observations demonstrated the consistent presence of two clusters of c-Myc-expressing cells in anagen follicles, located in two annular regions of the inner root sheath, at the border between cells characterized by putative trichohyalin granules and cells which are keratinized. The lower group belongs to Henle's layer, while the upper group belongs to Huxley's layer. c-Myc oncoprotein seems to favour apoptosis/differentiation and may be a marker for terminal differentiation of trichocytes, at least in the inner root sheath. Our findings agree with the interpretation that the complex morphology of the hair follicle reflects its complex function; the extrusion of a highly organized multicellular structure, the hair shaft, driven by another highly organized multicellular structure, the inner root sheath.

Apoptosis↗
PubMed1995

A simple method for overcoming some problems when observing thick reflective biological samples with a confocal scanning laser microscope.

A simple device is described, which allows the range of depth of scanning to be reduced when observing thick reflecting biological samples with a confocal scanning laser microscope (CSLM). Thick histological sections of human skin and rat brain stem were mounted between two coverslips ('sandwich' style) and the optical tomography was performed from both sides by turning the 'sandwich' upside-down. The samples were impregnated using standard Golgi-Cox, 'rapid Golgi' or other silver methods. The ability to turn the 'sandwich' upside-down is particularly useful when the reflective structure inspected is deep inside the section, i.e., near the lower surface of the specimen, or when it is opaque to the laser beam or excessively reflective.

Animals↗
PubMed1995

A comparision between rapid Golgi and Golgi-Cox impregnation methods for 3-D reconstruction of neurons at the confocal scanning laser microscope.

We utilized two widely used impregnation methods, the silver "rapid Golgi" and the mercuric Golgi-Cox methods, for three-dimensional (3-D) reconstruction of neurons at the confocal scanning laser microscope (CSLM), to determine which of them was more suitable for this application. The Golgi-Cox method is the most consistent arid the cleanest procedure with respect to the "rapid Golgi" one which always produces samples with scattered reflective granules that interfere with the image formation at the CSLM. The interneuronal tissue in the case of Golgi-Cox impregnated specimens (i.e. the non-impregnated tissue among impregnated neurons) contributes less to the decrease of reflected light during z-sectioning than in the case of "rapid Golgi" impregnation, but the mercury impregnated samples reflect less than the silver impregnated ones. Owing to the necessity during deep z-scanning to adjust the sensitivity of the CLSM detector the acquisition of images from the deeper planes of the sample may be difficult. In our opinion the "sandwich" mounting of the specimen between two coverslips is indispensable in order to make it possible to scan it from both sides and, thus reduce the penetration in the sample and the consequent distortion of the image. Neither of the impregnation methods used is completely suitable for CLSM observations due both to their intrinsic limitations and to those imposed by the sample thickness.

Animals↗
PubMed1995

Three-dimensional reconstruction of the Meissner corpuscle of man, after silver impregnation and immunofluorescence with PGP 9.5 antibodies using confocal scanning laser microscopy.

The 3-dimensional organisation of the neural component of the human Meissner corpuscle was studied after silver impregnation and following immunofluorescence for protein gene product 9.5 (PGP 9.5) by confocal scanning laser microscopy. The morphology of the Meissner corpuscle was found to show consistent differences depending on the labelling method used. After silver impregnation by the Winkelmann technique the branches of the afferent nerve fibres of the corpuscle showed both thin regions and varicose elements, the latter probably corresponding to the portions rich in mitochondria observed by transmission electron microscopy. The bulkier elements were never more than 5-6 microns in diameter. After immunolabelling for PGP 9.5 the nerve fibre branches in the corpuscle always presented flattened and discoidal expansions with a diameter of up to 30 microns. On the basis of what is known as to the mechanism of action of silver impregnations it is considered that the black precipitate preferentially labels the parts of neurons that are rich in neurofilaments. In any case the precipitate is deposited throughout the neuronal cytoplasm except in the mitochondria and the nucleus. Accordingly, in the varicosities of the Meissner corpuscles that are rich in mitochondria, there is little space for the formation of the precipitate. The use of antiserum against PGP 9.5, which labels the larger proteinaceous component of the axoplasm, demonstrates the complete architecture of the neural component of the Meissner corpuscle, and visualises the discoidal and flattened expansions which are absent in the impregnated corpuscles. It is concluded that immunostaining provides images of the corpuscles, and of peripheral neural structures that are in general closer to reality.

Antibodies, Monoclonal↗
PubMed1994

Central and peripheral nervous structures as seen at the confocal scanning laser microscope.

Central neurons and peripheral nervous structures, e.g. cutaneous free endings, perifollicular nets, Meissners corpuscles and intramuscular fibres, were studied using various impregnation methods. The confocal scanning laser microscopes (CSLMs) used were equipped with different laser sources, in order to evaluate their limitations and advantages with these techniques and to contribute to a better understanding of the general morphology of the nervous system. When staining with silver sections with clouds of tiny silver granules which are beyond the resolution power of the conventional light microscope but which show a high reflectivity with the CSLM are obtained. Golgi-Cox mercuric impregnation, however, provides specimens which are precipitate-free, thus ensuring the reliability of information obtained. It does, however, have the disadvantage of being applicable only to the central nervous system. In all cases it is an advantage for the instrument to be fitted with different lasers (e.g. Ar and He-Ne), so as to optimize the images of samples impregnated with different methods. Notwithstanding the possibility that artefacts may distort the geometry of the sample and reduce the resolution, the images presented in this paper show that with careful selection of optical sectioning distances, the use of a suitable stack of sections and, if necessary, the aid of false electronic colours and of partial or complete rotation, it is possible to achieve a more precise interpretation of the morphology and organization of complex structures, such as those of the nervous system.

Animals↗
PubMed1993

Differential localization of the 5 alpha-reductase and the 3 alpha-hydroxysteroid dehydrogenase in neuronal and glial cultures.

The activities of the 5 alpha-reductase [the enzyme that converts testosterone into dihydrotestosterone (DHT)] and 3 alpha-hydroxysteroid dehydrogenase [the enzyme that converts DHT into 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol)] have been evaluated in primary cultures of neurons, oligodendrocytes, and type 1 and 2 astrocytes obtained from fetal or neonatal rat brain. All cultures were used on the fifth day. The formation of DHT and 3 alpha-diol was evaluated by incubating the different cultures with [14C]testosterone or [14C]DHT as substrates. The results obtained indicate that the formation of DHT takes place preferentially in neurons; however, type 2 astrocytes and oligodendrocytes also possess considerable 5 alpha-reductase activity, while type 1 astrocytes show a much lower enzymatic activity. A completely different localization was observed for 3 alpha-hydroxysteroid dehydrogenase. The formation of 3 alpha-diol appears to be mainly, if not exclusively, present in type 1 astrocytes. 3 alpha-Diol is formed in very low yields by neurons, type 2 astrocytes and oligodendrocytes. The compartmentalization of two strictly correlated enzymes (5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase) in separate central nervous system cell populations suggests the simultaneous participation of neurons and glial cells in the 5 alpha-reductive metabolism of testosterone and possibly other hormonal steroids (e.g. progesterone, corticoids, etc.).

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Intracellular signalling systems controlling the 5 alpha-reductase in glial cell cultures.

Glial cells are able to metabolize testosterone into DHT through the action of the enzyme 5 alpha-reductase. DHT may be further processed to 3 alpha-diol by the 3 alpha-hydroxysteroid-dehydrogenase. The aim of this study was to analyze if a modulation of two second messenger systems might be able to modify the 5 alpha-reductase and the 3 alpha-hydroxysteroid-dehydrogenase activities present in glial cells. To this purpose, the formation of DHT has been measured in rat glial cell cultures after different time of exposure to TPA, 4 alpha-Ph, an active and an inactive phorbol ester respectively, and 8-Br-cAMP. The results obtained indicate that the formation of DHT is not modified by the addition of phorbol esters. On the contrary, a statistically significant increase of 5 alpha-reductase activity, over control levels, has been observed after 6, 12, and 24 h of incubation with 8-Br-cAMP (10(-3) M). The effect of the cAMP analogue appears to be specific for the 5 alpha-reductase, since the 3 alpha-hydroxysteroid-dehydrogenase did not show any variation after exposure to the drug. In conclusion, the present data suggest that proteinkinase A (PKA) might be involved in the control of the 5 alpha-reductase in glial cells. It is postulated that nervous inputs utilizing cAMP as the second messenger might modify the activity of this enzyme in glial cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Is the 5 alpha-reductase-3 alpha-hydroxysteroid dehydrogenase complex associated with the myelin in the peripheral nervous system of young and old male rats?

The formation of the 5 alpha-reduced metabolites of testosterone, 5 alpha-androstan-17 beta-ol-3-one (dihydroxytestosterone, DHT) and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol) was evaluated in sciatic nerve fragments and homogenates and in the myelin purified from the sciatic nerve of adult (60-90-day-old) male rats. The effects of aging on the metabolism of testosterone in the sciatic nerve was also evaluated using 20-month-old animals. In the sciatic nerve, tissue homogenization and myelin purification are associated with a marked decrease in the 5 alpha-reductase activity; these data do not appear to be consistent with a possible association of the enzyme with myelin membranes, as it occurs in the CNS. However, the results may be subject to other interpretations which are presented in the discussion. The formation of 3 alpha-diol is higher in sciatic nerve fragments than in homogenates or in the purified myelin. This occurs when testosterone is used as the substrate. In aged animals, the same pattern is observed but at a lower level. In the incubations in which the formation of 3 alpha-diol was evaluated using DHT as the substrate in fragments, homogenates and purified myelin of the sciatic nerve of either adult or old animals, it was found that the yields of this metabolite are higher than those obtained following incubation with testosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

[Transverse mesocolon herniation. Description of a clinical case].

A case of herniation of small bowel through a defect of the transverse meso-colon with secondary herniation through the gastrocolic ligament and re-entry into the greater peritoneal cavity is reported. This form of lesser sac hernia is rare and only one hundred half cases have been reported in literature.

Aged↗
PubMed1991

Unmyelinated nerve fibers associated with Meissner's corpuscle in the green monkey (Cercopithecus aethiops L.).

The ultrastructure and organization of free nerve fibers occurring in dermal papillae containing Meissner's corpuscles were studied in the fingertips of the Green Monkey (Cercopithecus aethiops L.). The course of the thin unmyelinated fibers leads in the vicinity of Meissner's corpuscles in the connective tissue of its sheaths, between the epidermis and the peripheral fibroblast layer; they never come into contact with the nervous component of the receptor. Consequently, neither a "pericorpuscular reticulum" nor an "apparatus of Timofeew" is formed. The presence of thin axons containing dense-cored vesicles is evident, but they are not autonomous fibers, as indicated by the negative results of the Falck-Hillarp test for catecholamines. The nerve fibers occasionally seem to bear a close resemblance to the "open" and "plain" endings described by other authors in papillae devoid of corpuscles. Despite failure for close mutual contact to be established between the Meissner's corpuscle and the unmyelinated fibers ramifying in the connective tissue surrounding it, the hypothesis that the two may cooperate to form a "multimodal sensory package" is not without interest.

Animals↗
PubMed1990

5 alpha-reductase activity in isolated and cultured neuronal and glial cells of the rat.

The distribution of the 5 alpha-reductase, the enzyme which converts testosterone into its 'active' metabolite dihydrotestosterone (DHT), has been studied in neurons, astrocytes and oligodendrocytes isolated from the brain of male rats by density gradient ultracentrifugation and in neurons and glial cells grown in cultures. Purity of cellular preparations was examined by electron and light microscopy. Purified neurons, astrocytes and oligodendrocytes, obtained from the brain of adult male rats, are all able to form DHT from testosterone and consequently possess a 5 alpha-reductase activity. Among the 3 cell types studied, neurons appear to be more active than oligodendrocytes and astrocytes. Moreover, between the two population of glial cells, the oligodendrocytes seem to possess a slightly higher enzymatic activity than that present in the astrocytes. Neurons appeared more active in metabolizing testosterone than glial cells also in cell culture experiments. It is presently believed that the 5 alpha-reduction of testosterone to DHT provides one of the mechanisms through which the hormone becomes effective in the CNS. This is supported by the present findings, which indicate that neurons are the cell population in which the 5 alpha-reductase is more concentrated. However, the presence of a considerable 5 alpha-reductase activity in glial cells indicates that also non-neuronal cells might participate in androgen-mediated events occurring in the brain.

Animals↗
PubMed1990

Eccrine sweat glands of rat fingertips. Scanning electron microscope observations after enzymatic digestion of dermal connective tissue.

By removing epidermis with EDTA and a subsequent enzymatic digestion of dermis, eccrine sweat glands of rat fingertips were exposed and examined by scanning electron microscopy (SEM). Different protocols were tested to remove as much connective tissue as possible, while minimizing damage to other structures, and to expose the epithelial surface of secretory tubules in order to display vascular and nervous networks. SEM observations gave detailed information on the relationship between epithelial secretory cells and myoepithelial cells, as well as on the vascular and nervous networks which surround the glomeruli of glands.

Animals↗
PubMed1988

Ontogenetic development of the 5 alpha-reductase in the rat brain: cerebral cortex, hypothalamus, purified myelin and isolated oligodendrocytes.

In the central nervous system of the rat, the 5 alpha-reductase, the enzyme which converts testosterone into dihydrotestosterone, appears to be concentrated in the white matter and in particular to be associated with myelin. In order to verify whether a temporal correlation might exist between the formation of myelin membranes and the variations of the 5 alpha-reductase activity observed in the brain, the enzymatic activity was studied in the cerebral cortex and in the hypothalamus of male rat in the age range of 3-60 days, in myelin purified from animals of 15-60 days of life and in oligodendrocytes (i.e. in the cells responsible for the formation of the myelin) isolated from the brain of adult and very young rats (7th day of life, when the myelination process is not yet initiated). The results show that the formation of 5 alpha-androstane-17 beta-ol-3-one (DHT) in the cerebral cortex and in the hypothalamus has a peak activity in the first two weeks of life, before the beginning of the myelination process; purified myelin has an enzymatic activity always much higher than that present in the cerebral cortex and in the hypothalamus and shows a peak in the formation of DHT in the first period of myelinogenesis, on the third week of life. Finally the oligodendrocytes of young rats possess a much higher ability to convert testosterone into the 5 alpha-reduced metabolites than the oligodendrocytes of adult animals. A possible involvement of this enzyme in the myelin function may be hypothesized.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1988

Testosterone 5 alpha-reductase activity in the rat brain is highly concentrated in white matter structures and in purified myelin sheaths of axons.

Previous results obtained in this laboratory indicate that in the rat brain the 5 alpha-reductase, the enzymatic activity involved in metabolizing testosterone into 5 alpha-androstan-17 beta-ol-3-one (dihydrotestosterone), is particularly concentrated in the white matter. In the present experiments, this enzymatic activity was studied in the following white matter structures, which were microdissected using the punch technique of Palkovits: anterior commissure (CA), fornix (FX), habenulo-interpeduncular tract (HP), corpus callosum (CC), stria medullaris (SM), optic chiasm (CO), fimbria of the hippocampus (FI), cerebral peduncle (PC), pontine fibers (FP), cerebellar medulla (CMD) and corticospinal tract (TCS). Moreover brain myelin was isolated and purified by sucrose density gradient ultracentrifugation. The results obtained confirm that, in the rat brain, the enzymes involved in testosterone 5 alpha-reduction are preferentially localized in the white matter. However, clearcut differences in the metabolic activity exist between the different structures examined so far. DHT formation increases rostro-caudally, so that the highest activity has been recorded in the white matter structures punched at the level of pons (FP), medulla oblungata (TCS) and cerebellum (CMD). The high metabolic activity associated with the white matter structures appears to be linked to the presence of myelin, since the specific activity of the enzyme is particularly elevated in purified preparations of myelin sheaths.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1984

Electron microscope study on neurons occurring in the common peroneal nerve of rat and pigeon.

During an electron microscopical study of the common peroneal nerve of albino rats and pigeons, neurons were found along the course of the nerve. Their ultrastructural morphology was studied. On the basis of observed features, such as the occurrence of bare areas of the neurilemma, of thick processes of the perikarya (dendrites), and of synapses on the soma, it appears that these cells are aberrant sympathetic neurons which have migrated to a more distal position than usual.

Animals↗