Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix

Aim of this study was to investigate the distribution of versican proteoglycan within the human dentine organic matrix by means of a correlative immunohistochemical analysis with field emission in-lens scanning electron microscope (FEI-SEM), transmission electron microscope (TEM), fluorescence microscope (FM) and biochemical assay. Specimens containing dentine and predentine were obtained from non carious human teeth and divided in three groups: 1) FEI-SEM group: sections were exposed to a pre-embedding immunohistochemical procedure; 2) TEM group: specimens were fixed, demineralised, embedded and submitted to a post-embedding immunohistochemical procedure; 3) FM group: sections mineralised and submitted to a pre-embedding immunohistochemical procedure with fluorescence labelling. Specimens were exposed to two different antibodies to assay distribution of versican fragments and whole versican molecule. Western Blotting analysis of dentine and pulp extracts was also performed. The correlative FEI-SEM,TEM and FM analysis revealed positive immunoreaction for versican fragments both in predentine and dentine, while few gold particles identifying the whole versican molecule were found in predentine only under TEM. No labelling of versican whole molecule was detected by FEI-SEM and FM analysis. The immunoblotting analysis confirmed the morphological findings. This study suggests that in fully developed human teeth versican fragments are significant constituents of the human dentine and predentine organic matrix, while versican whole molecule can be visualised in scarce amount within predentine only. The role of versican fragments within human dentine organic matrix should be further elucidated.Key words: versican, dentine matrix, immunohistochemistry, TEM, FEISEM, fluorescence microscope.The human dentine organic matrix is composed by a large complex of macromolecules capable of self-assembly. The dentine matrix is represented predominantly by type I collagen and completed by non collagenous glycoproteins, elastin, hyaluronan and proteoglycans (PGs). While type I collagen is the backbone of the dentine with a predominant structural role, non-collagenous proteins, and in particular PGs, are believed to play fundamental functional roles during odontogenesis, mineralization and homeostasis of dentine.The process of odontogenesis appears to be controlled by a precise sequential expression of a pool of extracellular non-collagenous proteins that induces modifications within the extracellular environment of the predentine leading to the formation of the dentine matrix (Embery et al., 2001). Similarly, dentine mineralization involves a dynamic transition from the unmineralised predentine to the mineralised mature dentine, in which the role of specific regulative mineralisation proteins appears to be pivotal in the precipitation of the minerals and in the formation of apatite crystals (Embery et al., 2001). In particular, PGs has been shown to play crucial role in the mineralisation processes of dentine (Embery et al., 2001; Waddington et al., 2003).PGs are macro-molecules where, at least, one glycosaminoglycan side chain (GAGs) is covalently attached to the protein core of the molecules.Their size and structure can change and can be differentially found intracellulary, on the cell surface, or within the extracellular matrix.The majority of PGs have been identified by their antigenic and structural properties suggesting numerous biological functions (Embery et al., 2001). Biochemical, histochemical and immunohistochemical studies on PGs of dentine and predentine have yielded sufficient information to indicate that the predominant PGs belong to the small leucine-rich interstitial family (SLRP) (Fisher et al., 1983; Yoshiba et al., 1996). They include decorin and biglycan (Waddington et al., 2003; Orsini et al., 2007), which bear one or two chondroitin/dermatan sulphate GAGs, lumican, fibromodulin and osteoadherin that bear keratan sulphate GAGs chains (Iozzo et al., 1997, 1999; Neame et al., 2000). A second pool of PGs belongs to the large aggregation chondroitin/keratan sulphate family named hyaluronan-binding (HA), including aggrecan, versican, brevican and neurocan (Yamauchi et al., 1997).Versican was firstly isolated in chicken mesenchymal tissue, and it has been found to be expressed also in keratinocytes, smooth muscle cells of the vessels, brain and mesangial cells of the kidney. Similar PGs have been found in other connective tissues (Zimmermann et al., 1989; Shinomura et al., 1990; Zimmermann et al., 1994; Landolt et al.,1995) and recent studies have shown that, within the dental tissues, versican has been localised in gingival fibroblasts culture, dental pulp complex (Yamauchi et al., 1997; Bartold et al., 1995; Shibata et al., 2000; Shibata et al., 2002; Robey et al., 1993; Ababneh et al., 1999; Cheng et al., 1999), dentine Waddington et al., 2003), cementum (Ababneh et al., 1999; Cheng et al., 1999) and periodontal ligament (Sato et al., 2002).Within the dentine organic matrix versican can be detected either as fragments or as whole molecule. Waddington et al. (2003) reported that versican is mainly present as its degradation products (fragments), whereas the whole molecule has been isolated by Shibata et al. (1999; 2000) in rat dental pulp tissue.The aim of this study was to localise versican PG in human mature dentine by an immunohistochemical technique using a monoclonal antibody anti-versican (towards the whole molecule) and a polyclonal antibody anti-versican fragments, under high resolution field emission in-lens scanning electron microscope (FEI-SEM), electron transmission microscope (TEM) and fluorescence microscope (FM) and to confirm the morphological findings by a biochemical assay.     

Effects of castration on the expression of the NGF and TrkA in the vas deferens and accessory male genital glands of the rat

Nerve Growth Factor (NGF) is a member of the neurotrophin family. Neurotrophins exert their effects by binding to corresponding receptors, which are formed by the tyrosine protein kinases TrkA, TrkB, and TrkC, and the low affinity p75NTR receptor. The role of neurotrophins in the biology of male genital organs is far from clear. In particular, little is known about the influence of sex hormones on the expression of neurotrophins and their receptors. In the present study, using immunohistochemistry and real time RT-PCR, we investigated the expression of NGF and TrkA in the vas deferens and accessory male genital glands in normal and castrated rats.In normal rats, both NGF- and TrkA-immunoreactivities (IR) were localized in the epithelial layer of the vas deferens. NGF-IR was also found in the stroma and epithelium of the vesicular gland and prostate. TrkA-IR was distributed in the epithelial cells of vesicular and prostate glands. The nerves were weakly immunoreactive in all the examined organs. After castration the immunoreactivities increased. Real-time RT-PCR experiments indicated that NGF and TrkA mRNA levels increased significantly after castration. These results suggest that NGF and TrkA are expressed in the internal male genital organs of the rat and that their expression is downregulated by androgen hormones. We hypothesize NGF and TrkA play a role in the processes that regulate the involution of these organs under conditions of androgen deprivation.Key words: androgen hormones, stromal cells, immunohistochemistry, real-time RT-PCR, prostate.Nerve growth factor (NGF) is a member of the neurotrophin family, a family of neurotrophic factors that also includes Brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and neurotrophins 4/5 (NT4/5). Neurotrophins have essential roles in the survival, development and differentiation of neurons in the central and peripheral nervous systems (Levi-Montalcini, 1987; Ernfors et al., 1994; Snider, 1994; Barbacid, 1995; Huang and Reichart, 2001; Murer et al., 2001). Furthermore, recent data show that neurotrophins are involved in a variety of biological processes in nonneuronal tissues (Yamamoto et al., 1996; Sariola, 2001; Leon et al., 1994; Rosenbaum et al., 1998; Tessarollo, 1998). The biological effects of neurotrophins are mediated by tyrosine kinase receptors encoded by the trk protooncogene family, known as TrkA,TrkB and TrkC (Barbacid, 1995; Lewin and Barde, 1996; Patapoutian and Reichart, 2001). The Trk receptors are specific for their ligands; NGF is the preferred ligand for TrkA, BDNF and NT-4/5 are preferred ligands for TrkB and NT-3 is the preferred ligand for TrkC. In addition, all neurotrophins are recognized by a more widely expressed low-affinity receptor known as panneu-rotrophinreceptor p75NTR, which is a member of the tumor necrosis factor (TNF) receptor family (Teng &Hempstead, 2004).The presence of neurotrophins in the accessory male genital tissues has been well documented. NGF and large quantities of NGF have been found in the vesicular and prostate glands and are related to the rich sympathetic innervation of these organs (Harper et al., 1979, 1982; Harper and Thoenen, 1980; Hofmann and Unsicker, 1982).NGF and its receptors (TrkA, p75NTR) have been immunohistochemically expressed in the reproductive organs of the adult male rats (Li et al., 2005). In the prostate, NGF and NGF precursor have been immunohistochemically localized in the glandular epithelium, suggesting that secretory epithelial cells are the site of production of this factor (Shikata et al., 1984; MacGrogan et al., 1991; Paul et al., 1996). Paracrine neurotrophin synthesis by stromal cells has also been postulated (Pflug et al,. 1995; Dalal and Djakiew, 1997; Weeraratna et al., 2000). High- and low-affinity neurotrophin receptors have been recognized in the nerves and epithelial cells of the prostate (Weeraratna et al., 2000; Graham et al., 1992; MacGrogan et al., 1992; Paul and Habib, 1998; Guate et al., 1999), thus indicating that neurotrophins play a role as growth-regulating factors in this gland.The exact role of neurotrophins in the biology of male genital organs, however, is far from clear. Recently, in the vas deferens and accessory male genital glands of the rat, the expression of the BDNF and its receptors (TrkB and p75NTR) has been reported to be regulated by androgen hormones (Mirabella et al., 2006; Mirabella et al., 2008). In castrated rats, moreover, BDNF has been hypothesized to regulate, via interacting p75NTR, the castration-induced regression of the sympathetic innervation (Mirabella et al., 2006).The present study has, therefore, been undertaken to elucidate the presence and localization of NGF and TrkA in the vas deferens and accessory male genital glands of the rat. In addition, the expression of these proteins and their mRNAs have been determined after castration in order to evaluate whether this neurotrophin and its specific receptor are under the control of androgens.     

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle.As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90⁺ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.Key words: CD90, mesenchymal stem cells, hair follicle, dog.The hair follicle represents an important stem cell niche in the skin. It contains dermal and epithelial stem populations that display distinct properties and localization. While epithelial stem cells reside in the middle region of the hair follicle outer root sheath (Schneider et al., 2009; Lyle et al., 1998; Cotsarelis et al., 1990), dermal stem cells are located in the dermal sheath (DS) (Jahoda, 2003; Jahoda and Reynolds, 2001).The dermal sheath, or fibrous root sheath, is a layer of dense connective tissue that extends along the hair follicle, from the bulb up to the infundibulum. In the anagen hair follicle, it is comprised of mesenchymal cells located among collagen and elastic fibres.The cells are flat and elongated while collagen fibres form a circular inner layer and a longitudinal outer layer in the lower part of hair follicle (VonTscharner and Suter, 1994; Jahoda et al., 1992). At the base of the hair follicle, the DS is connected to the dermal papilla (Scott et al., 2000). The basement membrane, or glassy membrane, separates the DS from the epithelial component of the hair follicle (Scott et al., 2000).Follicular dermal stem cells have a mesenchymal origin and share many properties common to bone marrow-derived mesenchymal stem cells (MSCs) (Hoogduijn et al., 2006). They express the MSC cell-surface marker CD90, show a high colony forming unit ability and can differentiate into several mesenchymal lineages, such as osteoblasts, adipocytes, chondrocytes and myocytes (Hoogduijn et al., 2006; Jahoda et al., 2003). They also express neuroprogenitor markers (Hoogduijn et al., 2006) and, finally, they can repopulate the haematopoietic system (Lako et al., 2002). In the literature, we can find different information about stem cell localization: the whole dermal sheath, the peri-bulbar dermal sheath, the dermal papilla (Hoogduijn et al., 2006, McElwee et al., 2003, Gharzi et al., 2003, Jahoda et al., 2003.)CD90 (Thy-1) is a small GPI-anchored protein localized in the outer leaflet of the cell membrane (Low and Kincade, 1985). This protein is present in a large number of tissues and cells, even if a great species variation has been described (Mansour Haeryfar, 2004; Tokugawa et al., 1997; McKenzle and Fabre, 1981). CD90 plays a role in cell-cell interaction events, including intracellular adhesion and cell recognition during development (Saalbach et al., 2000; Morris, 1985), and is considered an important stem cell marker; for this last reason it is commonly used to identify mesenchymal stem cells in vitro (Kern et al., 2007; Yoshimura et al., 2006; Le Blanc and Ringdén, 2006; Pittenger et al., 1999). Furthermore, it has been identified in other kinds of stem cells such as haematopoietic progenitor cells (Craig et al., 1993) and hepatic progenitor cells in the human fetal liver (Masson et al., 2006).The hair follicle is the focus of increasing interest because it contains well defined stem cell populations that exhibit various developmental properties. We retain that in dogs, as already demonstrated in other species (Hoogduijn et al., 2006; Zhang et al., 2006; Jahoda et al., 2003; Lako et al., 2002), this organ may be a suitable and accessible source for both epithelial and mesenchymal stem cells that may be isolated and in vitro cultured. Since it is possible to take skin samples without injuring the patient, we chose the hair follicle to study and identify stem cells with the future purpose of using them in regenerative medicine.Dogs are affected by several skin diseases and some of them may be related to alterations of somatic stem cells. We retain that the study of hair follicle stem cell biology may improve our knowledge of etiology and pathogenesis of these skin diseases.In previous works we investigated the stem cells in dog hair follicles; we identified the location of putative epithelial stem cells at the isthmus and described the bulge-like region (Pascucci et al., 2006; Mercati et al., 2008). To the authors’ knowledge, there are no data available neither concerning the localization of DS stem cells nor concerning the expression of CD90 in the hair follicle as regards the canine species. Therefore, in this study, we described the morphological characteristics of DS cells and examined the immunohistochemical localization of CD90 protein in dog hair follicles with both light and transmission electron microscopy. The aim of our study is to observe the dermal sheath cells encompassing the hair follicle and to determine where CD90⁺ cells reside. CD90 is one of the main markers used to identify mesenchymal stem cells and it has been observed in stem cells isolated from the dermal sheath of hair follicles (Hoogduijn et al.,2006). For this reason, we suppose that CD90 protein can help us to identify the hair follicle dermal stem compartment in dog.     

Identification of the novel localization of tenascinX in the monkey choroid plexus and comparison with the mouse

Tenascin-X (Tn-X) belongs to the tenascin family of glycoproteins and has been reported to be significantly associated with schizophrenia in a single nucleotide polymorphism analysis in humans. This finding indicates an important role of Tn-X in the central nervous system (CNS). However, details of Tn-X localization are not clear in the primate CNS. Using immunohistochemical techniques, we found novel localizations of Tn-X in the interstitial connective tissue and around blood vessels in the choroid plexus (CP) in macaque monkeys. To verify the reliability of Tn-X localization, we compared the Tn-X localization with the tenascin-C (Tn-C) localization in corresponding regions using neighbouring sections. Localization of Tn-C was not observed in CP. This result indicated consistently restricted localization of Tn-X in CP. Comparative investigations using mouse tissues showed equivalent results. Our observations provide possible insight into specific roles of Tn-X in CP for mammalian CNS function.Key words: tenascin-X, choroid plexus, monkey, mouse, Ehlers-Danlos syndrome, schizophrenia.The tenascins (Tn) are a family of four glyco-protein members – tenascin-C (Tn-C), tenascin-R (Tn-R), tenascin-W (Tn-W) and tenascin-X (Tn-X) – found diversely in the extra-cellular matrix of vertebrate organs (Hsia and Schwarzbauer, 2005; Tucker and Chiquet-Ehrismann, 2009). Important functions of Tn have been investigated in developmental cell adhesion modulation and pathological conditions such as wound healing and tumourigenesis (Adams and Watt, 1993; Hsia and Schwarzbauer, 2005; Tucker and Chiquet-Ehrismann, 2009). Tn-C and Tn-R are prominent in the nervous system and play a role in the development of neurite outgrowth and postnatal synaptic plasticity (Yamaguchi, 2000; Chiquet-Ehrismann and Tucker, 2004; Dityatev and Schachner, 2006). Tn-W is found abundantly in the developing bone and stroma of certain tumours (Chiquet-Ehrismann and Tucker, 2004; Tucker and Chiquet-Ehrismann, 2009). Tn-X is the first tenascin member shown to be clearly associated with the human connective tissue disorder Ehlers–Danlos syndrome (EDS; Burch et al., 1997). Patients with a Tn-X deficiency suffer from skin hyperextensibility, joint hypermobility and poor wound healing ability (Bristow et al., 2005). These symptoms are caused by the occurrence of abnormal irregular collagen fibres. Tn-X plays a role in collagen fibrillogenesis by directly binding to collagen (Mao et al. 2002; Minamitani et al. 2004). Mice with a Tn-X deficiency also showed skin symptoms comparable with those of EDS (Mao et al., 2002).Interestingly, in an analysis of human single nucleotide polymorphisms, Tn-X was reported to be significantly associated with schizophrenia (Wei and Hemmings, 2004; Tochigi et al., 2007). However, thus far, there have been no neuroanatomical reports on the involvement of Tn-X in schizophrenia. In the mammalian central nervous system (CNS), Tn-X mRNA expression has only been shown in the rat meninges of the olfactory bulb (Deckner et al., 2000). Recently, we found novel Tn-X localizations in the adult mouse leptomeninges trabecula in the cerebral cortex and in the connective tissue in the lateral ventricle choroid plexus (CP; Imura and Sato, 2008). Our finding of Tn-X localization in CP, which produces cerebrospinal fluid (CSF), might be a key factor in the investigation of the association between CSF metabolism and enlarged ventricles in schizophrenia. Enlarged ventricles are typical structural abnormalities associated with schizophrenia (Staal et al., 1999). Furthermore, CP secretes biologically active molecules into the CSF for brain development, activity and protection (Strazielle and Ghersi-Egea, 2000; Brown et al., 2004; Thouvenot et al., 2006; Johanson et al., 2008). In these molecules, for instance, there is a brain-derived neurotrophic factor (BDNF), the gene expression level and polymorphism of which have been analysed in relation to the pathogenesis of schizophrenia (Buckley et al., 2007). One study reported that BDNF is able to stimulate Tn-X expression in vitro (Takeda et al., 2005).The validity and limitations of animal models (rodents and monkeys) for use in the study of schizophrenia have been discussed (Tordjman et al., 2007). The authors concluded that monkeys appear to be an interesting social interaction model, more so than rodents, because of their complex well-organized social structure. In addition to differences in social structure, the dopaminergic system of rats and monkeys is quite different (García-Cabezas et al., 2009), and dysfunction of the dopaminergic system is related to schizophrenia (Wang et al. 2008).The CSF outflow system has been studied in some animal models (Kapoor et al., 2008). An anatomical difference in arachnoid granulations has been shown between rodents and monkeys (Krisch, 1988). Arachnoid granulations in monkeys are structurally similar to those in humans (Cooper, 1958; Krisch, 1988). In contrast, arachnoid granulations in rodents are similar to those of cats and dogs (Krisch, 1988). It is possible that Tn-X localization in CP is different between rodents and monkeys.Therefore, details concerning Tn-X localization in monkey CP need to be clarified. In the present study, we compared the immunohistochemistry of Tn-X in monkey CP with that in mouse CP. Subsequently, to verify the reliability of Tn-X localization, we compared it with Tn-C localization in corresponding regions using neighbouring sections.     

Urocortin-like immunoreactivity in the primary lymphoid organs of the duck (Anas platyrhynchos)

Urocortin (UCN) is a 40 aminoacid peptide which belongs to corticotropin-releasing factor (CRF) family. This family of peptides stimulates the secretion of proopiomelanocortin (POMC)-derived peptides, adrenocorticotropic hormone (ACTH), β-endorphin and melanocyte-stimulating hormone (MSH) in the pituitary gland. In the present study, using Western blotting and immunohistochemistry, the distribution of UCN in the primary lymphoid organs of the duck was investigated at different ages. In the cloacal burse and thymus, Western blot demonstrated the presence of a peptide having a molecular weight compatible with that of the mammalian UCN. In the cloacal burse, immunoreactivity was located in the medullary epithelial cells and in the follicular associated and corticomedullary epithelium. In the thymus, immunoreactivity was located in single epithelial cells. Double labelling immunofluorescence studies showed that UCN immunoreactivity completely colocalised with cytokeratin immunoreactivity in both the thymus and cloacal burse. Statistically significant differences in the percentage of UCN immunoreactivity were observed between different age periods in the cloacal burse. The results suggest that, in birds, urocortin has an important role in regulating the function of the immune system.Key words: cloacal burse, thymus, cytokeratin, medullary reticular epithelial cells, CRFUrocortin (UCN) is a 40-amino acid peptide belonging to the mammalian corticotropin- releasing hormone (CRH) family which was first discovered in the rat midbrain (Vaughan et al., 1995). On the basis of its selective ability to bind CRH-receptor type 2 (CRH-R2), different types of UCN, i.e. UCN1, UCN2 and UCN3, have been identified (Lewis et al., 2001; Reyes et al., 2001). In mammals, UCN has been found in the central nervous (Vaughan et al., 1995), digestive (Muramatsu et al., 2000) and immune systems (Bamberger et al., 1998; Kageyama et al., 1999; Baigent et al., 2000), and in genital organs (Petraglia et al., 1996). UCN has also been found to play a role in regulating some CRH-receptor-mediated effects (Turnbull et al., 1999). While UCN2 and UCN3 selectively bind to CRH-R2, UCN1 binds to both CRH-R1 and CRH-R2 and shows a greater affinity to CRH-R2 than CRH alone (Chalmers et al., 1996). Despite the ability to interact with the same receptors, different functions are attributed to UCN and CRH. CRH is the primary neuroregulator of the vertebrate stress response in so far as it has been shown to be the major hypothalamic releasing factor for pituitary adrenocorticotropic hormone, whereas UCN seems not to be involved in the activation of the hypothalamus- hypophysis-adrenal axis (Turnbull et al., 1999). Conversely, UCN influences the function of the cardiovascular and nervous systems by increasing anxiety, decreasing appetite and influencing behavioral activity (Latchman, 2001). In non-mammalian vertebrates, few data have been reported on the presence and the role of UCN.The molecule, however, may have been conserved during vertebrate evolution, given that it has also been detected in amphibians and birds (Kozicz et al., 2002; Cavani et al., 2003; Boorse et al., 2005; Calle et al., 2005). In amphibians, UCN and CRH receptors have been found in the brain as well as in many other organs and tissues, including the pituitary gland, heart, kidney and alimentary canal (Kozicz et al., 2002; Boorse et al., 2005, 2006); thus suggesting a potential role for diverse actions in tissue maintenance and function. In Xenopus laevis, UCN injected in the third ventricle has been found to suppress food intake (Boorse et al. 2005). Moreover, it has been found to act as a cytoprotective factor in tadpole tail during metamorphosis (Boorse et al. 2006). In birds, UCN-ir has been found in neurons of the pigeon paramedian subgriseal mesencephalon which appear to be part of the brain circuitry involved in sympathetic nervous system-mediated behavioral responses to stress (Cavani et al. 2003; Cunha et al., 2007). Intracerebroventricular administered UCN, moreover, has been reported to decrease food intake in the chicken (Zhang et al., 2001). Up until now, however, no data are available regarding the presence and role of UCN in tissues and organs of birds outside the central nervous system (CNS). Since UCN and its receptors have been reported to be extensively expressed in immune tissues and addressed to play important roles in the regulation of the immune response (Baigent, 2001), the present study has investigated the presence and distribution of UCN in the primary lymphoid organs of the duck by means of Western blotting and immunohistochemistry. In addition, in order to verify if UCN also plays a role in the maturation of bird primary lymphoid organs, UCN expression was evaluated at different age periods.     

Immunohistochemical localization and functional characterization of somatostatin receptor subtypes in a corticotropin releasing hormone-secreting adrenal phaeochromocytoma: review of the literature and report of a case

Somastostatin receptors are frequently expressed in phaeochromocytoma but data on somatostatin receptor subtyping are scanty and the functional response to the somatostatin analogue octretide is still debated.We report an unusual case of pheochromocytoma, causing ectopic Cushing’s syndrome due to CRH production by the tumour cells, in a 50-yr-old woman. Abdominal computed tomography revealed an inhomogeneous, 9-cm mass in the right adrenal gland, and [¹¹¹In-DTPA⁰] octreotide scintigraphy showed an abnormal uptake of the radiotracer in the right perirenal region, corresponding to the adrenal mass. The patient underwent laparoscopic surgery and formalin-fixed and paraffin-embedded samples were studied. The tumour was extensively characterized by immunohistochemistry and somatostatin receptor (SSTRs) subtypes expression was analyzed. Histological and immunohistochemical examination of the surgical specimens displayed a typical pheochromocytoma, which was found to be immunoreative to S-100, chromogranin A and neurofilaments. Immunostaining for SSTR subtypes showed a positive reaction for SSTR₁, SSTR_2A, SSTR_2B, antisera on tumour cells. The intense and diffuse immunostaining for corticotropin releasing hormone (CRH) antiserum indicated that Cushing’s disease was dependent on CRH overproduction by the pheochromocytoma, in which no immunostaining for adrenocorticotropic hormone was found. Our report confirms the heterogeneity of the pattern of SSTR expression in pheochromocytomas, and provide further evidence for functional SSTR subtype SSTR_2a in a subgroup of pheochromocytomas, suggesting that these tumours may represent potential target for octreotide treatment.Key words: phaeochromocytoma, neuroendocrine tumours, somatostatin receptors, octreotide, corticotropin releasing hormone.Phaeochromocytomas are tumours derived from the chromaffin cells of the sympathoadrenal system, generally associated with cathecolamine overproduction. They represent a rare condition, occurring in less than 0.2% of patients with hypertension. The diagnosis of sporadic phaeochromocytoma is based on clinical history and features characterized by the triad episodic headache, sweating, and tachycardia, but an increasing number of these tumours are diagnosed in patients without classical symptoms (Pacak et al., 2001). Ectopic Cushing’s syndrome is one of the possible, albeit unusual, expression of pheochromocytoma. Up to date, there are few reports of pheochromocytomas producing adrenocorticotropic hormone (ACTH) and/or ACTH precursors (O’Brien T et al., 1992; Chen et al., 1995; White et al., 2000), and even more limited is the number of cases in which pheochromocytoma secrete corticotropin releasing hormone (CRH) (Eng et al., 1999; Bayraktar et al., 2006).Similar to other neuroendocrine tumours, pheochromocytomas often express somatostatin receptors (SSTR) (De Herder and Hofland, 2004), but data on the specific SSTRs subtypes expressed within the tumours are thus far sparse and conflicting and the real therapeutic effectiveness of somatostatin analogue in these tumours is still debated (Reubi et al., 1992; Kubota et al., 1994; Epelbaum et al., 1995; Hofland et al., 1999; Mundschenk et al., 2003; Unger et al., 2004; Ueberberg et al., 2005; Unger et al., 2007).     

Ribonuclear inclusions as biomarker of myotonic dystrophy type 2, even in improperly frozen or defrozen skeletal muscle biopsies

Myotonic dystrophy type 2 (DM2) is a dominantly inherited disorder caused by a CCTG repeat expansion in intron 1 of ZNF9 gene. The size and the somatic instability of DM2 expansion complicate the molecular diagnosis of DM2. In situ hybridization represents a rapid and sensitive method to obtain a definitive diagnosis in few hours, since it allows the direct visualization of the mutant mRNA foci on skeletal muscle sections. This approach makes the muscle biopsy an important tool for definitive diagnosis of DM2. Consequently, a rapid freezing at ultra cold temperature and a good storage of muscle specimens are essential to avoid morphologic alterations and nucleic acids degradation. However incorrect freezing or thawing may accidentally occur. In this work we report that fluorescence in situ hybridization may be applied on improperly frozen or inappropriately stored muscle biopsies since foci of mutant mRNA are well preserved and can still be detected in muscle sections no more useful for histopathological evaluation.Key words: myotonic dystrophy type 2, defrozen muscle biopsy, fluorescence, in situ hybridization, ribonuclear inclusions.Myotonic dystrophy type 2 (DM2) is a neuromuscular disorder due to the unstable (CCTG)n repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene on chromosome 3q21.3 (Liquori et al. 2001). Mutant ZNF9 pre-mRNA is spliced and polyadenylated, and the mRNA is exported to the cytoplasm where normal levels of ZNF9 protein expression occur (Botta et al., 2006; Margolis et al. 2006); however, the expanded repeats remain in cell nuclei as ribonuclear inclusions (Liquori et al. 2001). The DM2 ribonuclear inclusions contain only the CCUG repeat sequence derived from intron 1 but with no detectable flanking intronic RNA (Margolis et al. 2006). CCUG-containing mutant mRNAs form double-stranded hairpin loop structures that bind specific RNA-binding proteins such as muscle-blind-like proteins (MBNLs) that colocalize with ribonuclear inclusions in myonuclei (Mankodi et al., 2001; Fardaei et al., 2002). Sequestration of these proteins which are regulators of alternative splicing, alters the splicing of several pre-mRNA (reviewed by Osborne and Thornton, 2006) such as the insulin receptor (IR) and the chloride channel (ClC1) (Savkur et al., 2004; Charlet et al., 2002; Mankodi et al., 2002). Alterations in IR splicing leads to insulin insensitivity and predisposition to diabetes (Savkur et al. 2004) and alterations in ClC1 splicing results in electrical myotonia (Charlet et al., 2002; Mankodi et al., 2002). Conventional Southern blot analysis is not adequate for a definitive molecular diagnosis in DM2 due to the extremely large size and somatic instability of the expansion mutation (Liquori et al., 2001; Bachinski et al., 2003). The extraordinary somatic instability complicates the analysis of genotype-phenotype correlations including those in the effect of the gender of transmitting parents and anticipation. The copy number of DM2 CCTG is below 30 in phenotypically normal individuals and up 11.000 in patients (Day and Ranum, 2005). A complex genotyping diagnostic procedure is now commonly used consisting of a three-step molecular protocol (Day et al., 2003; Udd et al., 2003). However, a more practical tool to obtain a definitive diagnosis in few hours is represented by in situ hybridization which detects ribonuclear inclusions in cell nuclei of muscle fibers (Cardani et al., 2004; Sallinen et al., 2004). This approach makes muscle biopsy an essential tool for DM2 diagnosis. For this reason muscle specimens should be sent fresh, for rapid freezing, from the operating room to the pathology laboratory.To avoid RNA degradation, biopsies require special precautions with handling of material, such as immediate freezing of fresh tissues, because retrospective genetic analysis is impaired by conventional tissue processing techniques. However, many small hospitals are ill-equipped for snap freezing which requires access to liquid nitrogen or dry ice; thus, frequently outside hospitals provide specimens that are obscured with freeze artefacts because they either were submitted incorrectly or were improperly frozen, at the point of origin prior to shipment. Moreover, an accidental tissue thawing and refreezing may occur (for example power failure of the freezer) causing severe tissue damages and possible RNA degradation.Here we report our experience on DM2 muscle biopsies improperly preserved: these were no more useful for a histopathological analysis since they showed evident morphologic artefacts, but they proved to be still suitable for diagnosis by fluorescence in situ hybridization (FISH) since ribonuclear inclusions were preserved and still detectable on muscle sections.     

Human non-CG methylation: Are human stem cells plant-like?

Non-CG methylation is well characterized in plants where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in mammals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in mammals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.Key words: non-CG methylation, stem cells, Dnmt1, Dnmt3a, human methylomeIn plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.¹ DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.²Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.³ Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,^4,5 or transposons and repetitive sequences such as the human L1 retrotransposon⁶ in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.⁶Non-CG methylation has also been reported at origins of replication^7,8 and a region of the human myogenic gene Myf3.⁹ The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.¹⁰ The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.¹⁰ The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.¹¹ Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.¹² Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.¹³ Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.¹³ This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.The recent report by Lister et al.¹⁴ confirmed a much earlier report by Ramsahoye et al.¹⁵ suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,¹⁵ thus global methylation patterning was assessed. Lister et al.¹⁴ extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They report¹⁴ that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,¹⁴ one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.¹⁴Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,^14,15 raises the possibility that cancer stem cells¹⁶ carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencing⁸ or ligation mediated PCR¹⁷ methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint cluster¹⁸ or human breast cancer DNA¹³ treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strand¹⁸ or at a symmetrical CWG site.¹³ In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.¹³In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,^14,15 a DNA methyltransferase with similarities to the plant DRM methyltransferase family¹⁹ and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.¹⁵ DRM proteins however, possess a unique permuted domain structure found exclusively in plants¹⁹ and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable published^20–23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studies²⁴ or find that CG methylation seen in plants but not non-CG methylation is detected.^21,22,25,26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,²⁷ and is consistent with the recent report that cultured stem cells are epigenetically unstable.²⁸The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.^9,10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genes²⁹ even though the promoters were symmetrically de novo methylated at ^mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.^9,10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.¹⁴ Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,²⁹ however stem cell characteristics were not studied.Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.^30–33 However, depletion of CWG sites is not observed in the human genome.³⁴ Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylome¹⁴ where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.The extensive detail on non-CG methylation in the H1 methylome¹⁴ raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.¹⁴ This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.¹⁴By analogy with the well known methylated DNA binding proteins specific for CG methylation,³⁵ methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.^36–38 While Dnmt1 has been proposed to function stoichiometrically³⁹ and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,⁴⁰ a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer design^41,42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.^14,40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCR⁴³ the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell niche^44,45 is very low.The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,⁴⁶ COMPARE-MS⁴⁷) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIP⁴⁸) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.¹⁴In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function.     

Immunohistochemical localization of glucagon and pancreatic polypeptide on rat endocrine pancreas: coexistence in rat islet cells

We used immunofluorescence double staining method to investigate the cellular localization of glucagon and pancreatic polypeptide (PP) in rat pancreatic islets. The results showed that both A-cells (glucagon-secreting cells) and PP-cells (PP-secreting cells) were located in the periphery of the islets. However, A-cells and PP-cells had a different regional distribution. Most of A-cells were located in the splenic lobe but a few of them were in the duodenal lobe of the pancreas. In contrast, the majority of PP-cells were found in the duodenal lobe and a few of them were in the splenic lobe of the pancreas. Furthermore, we found that 67.74% A-cells had PP immunoreactivity, 70.92% PP-cells contained glucagon immunoreactivity with immunofluorescence double staining. Our data support the concept of a common precursor stem cell for pancreatic hormone-producing cells.Key words: glucagon, pancreatic polypeptide, rat, pancreas, Immunofluorescence double staining histochemistry.The pancreatic islet is comprised of numerous cell types that synthesize and secrete distinct peptide hormones. Four major cell types are recognized in pancreatic islets of many mammalian species including rat, A-cells which contain glucagon, B-cells which contain insulin, D-cells which contain somatostatin, and PP-cells which contain the pancreatic polypeptide (PP) (Erlandsen, 1980; Reddy et al., 1988).Previous studies have revealed coexistence of glucagon- and PP-like immunoreactivity in endocrine pancreas cells of frog, rat, baboon, murine, monkey, and fish (Kaung and Elde, 1980; Kaung, 1985a, 1985b; Wolfe-Coote et al., 1988; Herrera et al., 1991; Lozano et al., 1991; Park and Bendayan, 1992; Louw et al., 1997). However, those experiments were performed by staining adjacent ultrathin sections with anti-glucagon serum and anti-PP serum respectively by peroxidase antiperoxidase (PAP) or immuno-gold labeling or avidin-biotin-peroxidase method, and the situation of two kinds of positive cells were compared.It is still not clear whether one cell type contains two or more peptides. Therefore, we used immunofluorescence double staining to identify the peptides secreted by single specific cells.This is the first time that coexistence of glucagon and PP in rat islet cells has been detected by an immunofluorescence double staining method.     

Activation of PKC-ε counteracts maturation and apoptosis of HL-60 myeloid leukemic cells in response to TNF family members

Protein kinase C (PKC)-ε, a component of the serine/threo-nine PKC family, has been shown to influence the survival and differentiation pathways of normal hematopoietic cells. Here, we have modulated the activity of PKC-ε with specific small molecule activator or inhibitor peptides. PKC-ε inhibitor and activator peptides showed modest effects on HL-60 maturation when added alone, but PKC-ε activator peptide significantly counteracted the pro-maturative activity of tumor necrosis factor (TNF)-α towards the monocytic/macrophagic lineage, as evaluated in terms of CD14 surface expression and morphological analyses. Moreover, while PKC-ε inhibitor peptide showed a reproducible increase of TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis, PKC-ε activator peptide potently counteracted the pro-apoptotic activity of TRAIL. Taken together, the anti-maturative and anti-apoptotic activities of PKC-ε envision a potentially important proleukemic role of this PKC family member.Key words: acute myeloid leukemia, surface antigens, HL-60 cells, apoptosis, maturation.Activation of all protein kinase C (PKC) family of serine and threonine isoenzymes is associated with binding to the negatively charged phospholipids, phosphatidylserine, while different PKC isozymes have varying sensitivities to Ca²⁺ and lipid-derived second messengers such as diacylglycerol (Gonelli et al., 2009). Upon activation, PKC isozymes translocate from the soluble to the particulate cell fraction, including cell membrane, nucleus and mitochondria (Gonelli et al., 2009). PKC primary sequence can be broadly separated into two domains: the N-terminal regulatory domain and the conserved C-terminal catalytic domain.The regulatory domain of PKC is composed of the C1 and C2 domains that mediate PKC interactions with second messengers, phospholipids, as well as inter and intramolecular protein-protein interactions. Differences in the order and number of copies of signaling domains, as well as sequence differences that affect binding affinities, result in the distinct activity of each PKC isozyme (Gonelli et al., 2009).In recent years, a series of peptides derived from PKC have been shown to modulate its activity by interfering with critical protein-protein interactions within PKC and between PKC and PKC-binding proteins (Brandman et al., 2007, Souroujon and Mochly-Rosen, 1998). Focusing on PKC-ε isozyme and using a rational approach, one C2-derived peptide that acts as an isozyme-selective activator (Dorn et al., 1999) and another that acts as a selective inhibitor (Johnson et al., 1996) of PKC-ε, have been identified.These findings are particularly interesting since besides being involved in the physiology of normal cardiac (Braun and Mochly-Rosen, 2003, Johnson et al., 1996, Li et al., 2006), hematopoietic (Gobbi et al., 2009, Mirandola et al., 2006, Racke et al., 2001), and neuronal (Borgatti et al., 1996) cell models, mounting experimental evidences have linked altered PKC-ε functions to solid tumor development (Okhrimenko et al., 2005, Gillespie et al., 2005, Lu et al., 2006). Therefore, taking advantage of the recent availability of small molecule peptides able to activate or inhibit specifically PKC-ε by disrupting protein/protein interactions (Dorn et al., 1999, Johnson et al., 1996), which open important therapeutic perspectives, we have investigated the effects of both PKC-ε activator and PKC-ε inhibitor peptides on the maturation and survival of leukemic cells, using as a model system the HL-60 myeloblastic leukemia cell line, which can be induced to undergo terminal differentiation or apoptotic cell death by a variety of chemical and biological agents (Breitman et al., 1980, Zauli et al., 1996).     

Neurotensin receptor 1 immunoreactivity in the peripheral ganglia and carotid body

In the present study we investigated, through immunohistochemistry, the presence and location of neurotensin receptor 1 (NTR1) in the peripheral ganglia and carotid body of 16 humans and 5 rats. In both humans and rats, NTR1 immunostained ganglion cells were found in superior cervical ganglia (57.4±11.6% and 72.4±11.4%, respectively, p<0.05), enteric ganglia (51.9±10.4% and 64.6±6.1%, p<0.05), sensory ganglia (69.2±10.7% and 73.0±13.1%, p>0.05) and parasympathetic ganglia (52.1±14.1% and 59.4±14.0%, p>0.05), supporting a modulatory role for NT in these ganglia. Positivity was also detected in 45.6±9.2% and 50.8±6.8% of human and rat type I glomic cells, respectively, whereas type II cells were negative. Our findings suggest that NT produced by type I cells acts in an autocrine or paracrine way on the same cell type, playing a modulatory role on chemoception.Key words: neurotensin receptor 1, carotid body, autonomic ganglia, sensory ganglia, immunohistochemistry.Neurotensin (NT) is a tridecapeptide which was first isolated from bovine hypothalamus (Carraway and Leeman, 1973) and is widely distributed in the nervous system and intestine. In the nervous system, neurotensin acts as a neurotransmitter and neuromodulator (Dobner, 2006); in the periphery, as a paracrine or endocrine factor (Mazzocchi et al., 1997; Malendowicz, 1998). It also acts as a growth factor on various cell types (Malendowicz, 1993; Markowska et al., 1994a, 1994b; Evers, 2006).Three different NT receptors, termed NTR1, NTR2 and NTR3, have been identified and cloned to date. NTR1 and NTR2 are, respectively, high- and low-affinity seven trans-membrane domain G protein-coupled receptors. NTR3 is a high-affinity single trans-membrane domain type 1 receptor, with 100% homology with the sorting protein, gp95/sortilin (Kitabgi, 2006; Mazella et al., 1998). NTR3 can also form heterodimers with NTR1 in the plasma membrane (Martin et al., 2002). Nuclear internalization of the NTR1 has been reported and has been suggested to play a role in the production of long-term genomic effects (Feldberg et al., 1998; Laduron, 1992). It has also been reported that NTR2, but not NTR1, returns to the plasma membrane after NT-induced sequestration (Mazella and Vincent, 2006).In the peripheral nervous system, pregangliar fibers containing NT have been found in sympathetic, parasympathetic and enteric ganglia, and functional studies also suggest the expression of NTRs in ganglion cells. However, direct evidence of NTR1 protein expression in the different cell types of the ganglia has not yet been provided for human and rat. Only in rat dorsal root ganglia has evidence of NTR1 expression been given through hybridization in situ (Zhang et al., 1995), but there are no data on protein location or internalization.The carotid body is an arterial chemoreceptor, sensitive to reductions in partial blood oxygen pressure and pH and to increases in partial CO2 pressure, the stimulation of which induces increases in ventilatory frequency and volume.The carotid body is situated at the carotid bifurcation, and is composed of parenchymal lobules separated by connective tissue, in which afferent fibers of the glossopharyngeal nerve, arising from the petrosal ganglion, occur (Porzionato et al., 2005).Two different cell populations are present in the carotid body: type I cells, in turn separated into light, dark and pyknotic, and type II cells, at the edges of the clusters. Post-ganglionic sympathetic nerve fibers from the superior cervical ganglion are present, innervating blood vessels and type I cells, and preganglionic parasympathetic and sympathetic fibers reaching ganglion cells near the glomic cells. NT has been detected in glomic cells (Heath et al., 1988; Heym and Kummer, 1989; Smith et al., 1990) but the presence of the corresponding receptors in the various glomic cell types has not yet been investigated.Thus, the aim of the present study was to investigate, through immunohistochemistry, the presence and location of NTR1 in the peripheral ganglia and carotid body of both human and rat, with particular reference to the different cell types.     

Mesenchymal stem cells-derived vascular smooth muscle cells release abundant levels of osteoprotegerin

Although several studies have shown that the serum levels of osteoprotegerin (OPG) are significantly elevated in patients affected with atherosclerotic lesions in coronary and peripheral arteries, the cellular source and the role of OPG in the physiopathology of atherosclerosis are not completely defined. Therefore, we aimed to investigate the potential contribution of mesenchymal stem cells in the production/release of OPG. OPG was detectable by immunohistochemistry in aortic and coronary atherosclerotic plaques, within or in proximity of intimal vascular smooth muscle cells (SMC). In addition, bone marrow mesenchymal stem cell (MSC)-derived vascular SMC as well as primary aortic SMC released in the culture supernatant significantly higher levels of OPG with respect to MSC-derived endothelial cells (EC) or primary aortic EC. On the other hand, in vitro exposure to full-length human recombinant OPG significantly increased the proliferation rate of aortic SMC cultures, as monitored by bromodeoxyuridine incorporation. Taken together, these data suggest that OPG acts as an autocrine/paracrine growth factor for vascular SMC, which might contribute to the progression of atherosclerotic lesions.Key words: osteoprotegerin, mesenchymal stem cells, smooth muscle cells, atherosclerosis.A therosclerosis is a form of chronic low-grade inflammation resulting from interaction between modified lipoproteins, monocyte-derived macrophages and vascular smooth muscle cells (SMC) (Libby, 2002). Although the prevalent view is that intimal vascular SMC found in atherosclerotic plaques derive from cells migrating from the tunica media of the same artery (Libby, 2002), accumulating data indicate that also bone marrow (BM) mesenchymal stem cells (MSC), also known as multipotent stromal cells, have the potential to migrate in sites of vascular injury or inflammation and to differentiate into vascular SMC (Hillebrands et al., 2001, Li et al., 2001).Several studies have clearly demonstrated that the serum levels of the soluble member of the TNF-receptor super-family osteoprotegerin (OPG) are elevated in patients with coronary or carotid artery disease, especially those with clinically unstable atherosclerotic plaques (Jono et al., 2002, Schoppet et al., 2003, Secchiero et al., 2006a, Shin et al., 2006, Abedin et al., 2007, Avignon et al., 2007, Gulbiken et al., 2007, Kadoglu et al., 2008a, Omland et al., 2008). Despite the fact that neither the cellular source nor the physiological and pathological effects of elevated serum levels of OPG are well understood, a possible pathogenic link between elevated levels of OPG and inflammation has been suggested by recent in vitro studies of our and others research groups (Zauli et al., 2007, Mangan et al. 2007).Therefore, in order to assess the potential contribution of MSC in the pathogenesis of atherosclerosis, we have evaluated the release of OPG in the culture supernatants of BM-derived MSC differentiating along the vascular SMC or endothelial cell (EC) lineages. In addition, we have investigated the effect of recombinant human OPG on aortic SMC proliferation.     

Morphology and function of human Leydig cells in vitro. Immunocytochemical and radioimmunological analyses

The aim of our study was to show whether the cells isolated from testes of patients underwent bilateral orchiectomy for prostatic cancer are able to grown in vitro, and if so, are functionally active. Immuncytochemistry was performed to show the functional status of human cultured cells. In detail, immunolocalization of luteinizing hormone receptors (LHR), mitochondria, and cytoskeletal elements was demonstrated. Moreover, radioimmunological assay was used to measure testosterone secretion by cultured Leydig cells. Using Nomarski interference contrast and fine immunofluorescence analysis the positive immunostaining for LHR was observed in almost all Leydig cells, however it was of various intensity in individual cells. Testosterone measurement revealed significant difference between testosterone secretion by hCG-stimulated and unstimulated Leydig cells (p<0.05). Moreover, testosterone levels were significantly higher in 24- and 48-hour-cultures than in those of 72 hrs (p<0.05). Morphological analysis of Leydig cells in culture revealed the presence of mononuclear and multinucleate cells. The latter cells occurred in both hCG-stimulated and unstimulated cultures. In Leydig cells labeled with a molecular marker MitoTtracker, an abundance of mitochondria and typical distribution of microtubules and microfilaments were observed irrespective of the number of nuclei within the cell, suggesting no functional differences between mono- and multinucleate human Leydig cells in vitro. Since the percentage of multinucleate cells was similar in both hCG-stimulated and unstimulated cultures (23.70% and 22.80%), respectively, the appearance of these cell population seems to be independent of hormonal stimulation.Key words: human Leydig cells, LH receptors, primary culture, hCG-stimulation, immunocytochemistry, testosterone secretion, multinucleate cells, multicolor staining.It is well established that testosterone biosynthesis depends on the existence of mature Leydig cells in the testicular interstitium. Human Leydig cells arise from mesenchymal cells or fibroblast-like precursor cells through a hormonally regulated differentiation process (Chemes, 1996). Production of testosterone in human and mammalian Leydig cells is dependent on LH stimulation in vivo and on LH/hCG stimulation in in vitro conditions; to respond to hormonal regulation the cells are equipped with functional receptors for LH (Amador and Bartke, 1987; Simpson et al., 1987; Mendis-Handagama et al.,1990; Cooke, 1996; Ramadoss et al., 2006). In man, the Δ⁵-metabolic pathway is the major pathway for the metabolism of pregnenolone to testosterone (Rommerts, 1990). According to Hammar and Petersson (1986) in human testis from young and elderly men with prostatic carcinoma also the 5-ene pathway is preferred. For optimal steroidogenic function a number of neuroendocrine and neuronal markers have been demonstrated in human Leydig cells in vivo by the group of Holstein (Middendorff et al., 1993; 1995). Moreover, production of testosterone in Leydig cells, requires the presence of functionally active enzymes acting within mitochondria and the smooth endoplasmic reticulum (Payne and O’Shaughnessy, 1996; for review see Haider, 2004).Recent studies have shown that Leydig cells become hypofunctional with age. In the rat, aged Leydig cells produce less testosterone than Leydig cells from young adult rats (Luo et al., 1996; for review Zirkin et al., 1997). A detailed characteristics of aged rat Leydig cells in vivo, including reduced testosterone biosynthesis and reduced cell volume has been described by Ewing and Zirkin (1983). Now, there is evidence from in vitro studies that reactive oxygen species can result in the inhibition of testosterone production in mouse Leydig cells by affecting steroidogenic enzymes (Stocco et al. 1993; Peltola et al., 1996; Cao et al., 2004).Considering human samples as a very rare and valuable biological material, the aim of this study was to show whether Leydig cells obtained from testes of elderly patients who underwent orchiecto-my for prostatic cancer are able to grown in vitro, and if so, are functionally active. For this purpose localization of luteinizing hormone receptors (LHR) and visualization of mitochondria and cytoskeletal elements in both hCG-stimulated and unstimulated Leydig cell cultures were performed, as well as testosterone secretion by cultured Leydig cells was measured. It is worth noting that the effect of LH and an involvement of cytoskeletal proteins in steroidogenesis of mouse Leydig cells in vitro have been demonstrated by our own (Bilinska, 1989) and mitochondria have been described as integrally involved in Leydig cell steroidogenesis (Bilinska 1994; Kotula-Balak et al., 2001).     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.