A strategy for construction of highly sensitive glycosyl imprinted electrochemical sensor based on sandwich-like multiple signal enhancement and determination of neural cell adhesion molecule.

A novel electrochemical sensor for a neural cell adhesion molecule (CD56) was constructed by glycosyl imprinting. A sandwich-like multi-signal generation strategy was first proposed in glycosyl imprinting sensors via boric acid affinity. Glycosyl-imprinted polymers were formed by electro-polymerization with poly-sialic acid (PolySia) as a template molecule and p-aminobenzeneboronic (p-ABA) acid as a functional monomer. Methods such as scanning electron microscope (SEM), Fourier transform infrared spectrum (FT-IR), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used to characterize the successful formation of imprinted membranes. Confirmed by both simulation calculation and experimental results, a signal-amplified effect based on macromolecules was introduced for the first time. After re-absorption, aminobenzene borate was linked to the surface of the sensor by boric acid affinity due to the rich hexadoxyl structure of the CD56-terminal chain as a signal probe. Under optimal conditions, the detection limit of the sensor is as low as 0.47 ng/L, and it can be successfully applied to the detection of CD56 in human serum.

Differential expression of neurexin genes in the mouse brain.

Synapses, highly specialized membrane junctions between neurons, connect presynaptic neurotransmitter release sites and postsynaptic ligand-gated channels. Neurexins (Nrxns), a family of presynaptic adhesion molecules, have been characterized as major regulators of synapse development and function. Via their extracellular domains, Nrxns bind to different postsynaptic proteins, generating highly diverse functional readouts through their postsynaptic binding partners. Not surprisingly given these versatile protein interactions, mutations and deletions of Nrxn genes have been identified in patients with autism spectrum disorders, intellectual disabilities, and schizophrenia. Therefore, elucidating the expression profiles of Nrxns in the brain is of high significance. Here, using chromogenic and fluorescent in situ hybridization, we characterize the expression patterns of Nrxn isoforms throughout the brain. We found that each Nrxn isoform displays a unique expression profile in a region-, cell type-, and sensory system-specific manner. Interestingly, we also found that αNrxn1 and αNrxn2 mRNAs are expressed in non-neuronal cells, including astrocytes and oligodendrocytes. Lastly, we found diverse expression patterns of genes that encode Nrxn binding proteins, such as Neuroligins (Nlgns), Leucine-rich repeat transmembrane neuronal protein (Lrrtms) and Latrophilins (Adgrls), suggesting that Nrxn proteins can mediate numerous combinations of trans-synaptic interactions. Together, our anatomical profiling of Nrxn gene expression reflects the diverse roles of Nrxn molecules.

A Modular Organization of LRR Protein-Mediated Synaptic Adhesion Defines Synapse Identity.

Pyramidal neurons express rich repertoires of leucine-rich repeat (LRR)-containing adhesion molecules with similar synaptogenic activity in culture. The in vivo relevance of this molecular diversity is unclear. We show that hippocampal CA1 pyramidal neurons express multiple synaptogenic LRR proteins that differentially distribute to the major excitatory inputs on their apical dendrites. At Schaffer collateral (SC) inputs, FLRT2, LRRTM1, and Slitrk1 are postsynaptically localized and differentially regulate synaptic structure and function. FLRT2 controls spine density, whereas LRRTM1 and Slitrk1 exert opposing effects on synaptic vesicle distribution at the active zone. All LRR proteins differentially affect synaptic transmission, and their combinatorial loss results in a cumulative phenotype. At temporoammonic (TA) inputs, LRRTM1 is absent; FLRT2 similarly controls functional synapse number, whereas Slitrk1 function diverges to regulate postsynaptic AMPA receptor density. Thus, LRR proteins differentially control synaptic architecture and function and act in input-specific combinations and a context-dependent manner to specify synaptic properties.

Expression of E-cadherin, Slug and NCAM and its relationship to tumor invasiveness in patients with acromegaly

Pituitary adenomas account for 10-15% of primary intracranial tumors. Growth hormone (GH)-secreting adenomas account for 13% of all pituitary adenomas and cause acromegaly. These tumors can be aggressive, invade surrounding structures and are highly recurrent. The objective of this study was to evaluate E-cadherin, Slug and neural cell adhesion molecule (NCAM) expression in GH-secreting pituitary adenomas and its relationship to tumor invasiveness. A cross-sectional study of patients who underwent hypophysectomy due to GH-secreting pituitary adenoma from April 2007 to December 2014 was carried out. The medical records were reviewed to collect clinical data. Immediately after surgery, tumor samples were frozen in liquid nitrogen and stored in a biofreezer at -80°C for assessment of E-cadherin 1 (CDH1), SLUG (SNAI2), and NCAM (NCAM1) by real-time PCR. The samples were fixed in formalin and embedded in paraffin for immunohistochemical analysis of E-cadherin and NCAM. Thirty-five patients with acromegaly were included in the study. Of these, 65.7% had invasive tumors. Immunohistochemically, E-cadherin was expressed in 96.7% of patients, and NCAM in 80% of patients. There was no statistically significant relationship between tumor grade or invasiveness and immunohistochemical expression of these markers. Regarding gene expression, 50% of cases expressed CDH1, none expressed SNAI2, and 53.3% expressed NCAM1. There was no statistically significant relationship between tumor grade or invasiveness and gene expression of CDH1, SNAI2, and NCAM1. The absence of Slug overexpression and of E-cadherin and NCAM suppression suggests that expression of these markers is not associated with tumor invasiveness in GH-secreting pituitary adenomas.

Expression of E-cadherin, Slug and NCAM and its relationship to tumor invasiveness in patients with acromegaly.

Pituitary adenomas account for 10-15% of primary intracranial tumors. Growth hormone (GH)-secreting adenomas account for 13% of all pituitary adenomas and cause acromegaly. These tumors can be aggressive, invade surrounding structures and are highly recurrent. The objective of this study was to evaluate E-cadherin, Slug and neural cell adhesion molecule (NCAM) expression in GH-secreting pituitary adenomas and its relationship to tumor invasiveness. A cross-sectional study of patients who underwent hypophysectomy due to GH-secreting pituitary adenoma from April 2007 to December 2014 was carried out. The medical records were reviewed to collect clinical data. Immediately after surgery, tumor samples were frozen in liquid nitrogen and stored in a biofreezer at -80°C for assessment of E-cadherin 1 (CDH1), SLUG (SNAI2), and NCAM (NCAM1) by real-time PCR. The samples were fixed in formalin and embedded in paraffin for immunohistochemical analysis of E-cadherin and NCAM. Thirty-five patients with acromegaly were included in the study. Of these, 65.7% had invasive tumors. Immunohistochemically, E-cadherin was expressed in 96.7% of patients, and NCAM in 80% of patients. There was no statistically significant relationship between tumor grade or invasiveness and immunohistochemical expression of these markers. Regarding gene expression, 50% of cases expressed CDH1, none expressed SNAI2, and 53.3% expressed NCAM1. There was no statistically significant relationship between tumor grade or invasiveness and gene expression of CDH1, SNAI2, and NCAM1. The absence of Slug overexpression and of E-cadherin and NCAM suppression suggests that expression of these markers is not associated with tumor invasiveness in GH-secreting pituitary adenomas.

The Sorting Receptor SorCS1 Regulates Trafficking of Neurexin and AMPA Receptors.

The formation, function, and plasticity of synapses require dynamic changes in synaptic receptor composition. Here, we identify the sorting receptor SorCS1 as a key regulator of synaptic receptor trafficking. Four independent proteomic analyses identify the synaptic adhesion molecule neurexin and the AMPA glutamate receptor (AMPAR) as major proteins sorted by SorCS1. SorCS1 localizes to early and recycling endosomes and regulates neurexin and AMPAR surface trafficking. Surface proteome analysis of SorCS1-deficient neurons shows decreased surface levels of these, and additional, receptors. Quantitative in vivo analysis of SorCS1-knockout synaptic proteomes identifies SorCS1 as a global trafficking regulator and reveals decreased levels of receptors regulating adhesion and neurotransmission, including neurexins and AMPARs. Consequently, glutamatergic transmission at SorCS1-deficient synapses is reduced due to impaired AMPAR surface expression. SORCS1 mutations have been associated with autism and Alzheimer disease, suggesting that perturbed receptor trafficking contributes to synaptic-composition and -function defects underlying synaptopathies.

The clinical role of epithelial-mesenchymal transition and stem cell markers in advanced-stage ovarian serous carcinoma effusions.

We recently identified gene signatures that allow classification of ovarian carcinoma into 5 distinct clinically relevant groups. In the present study, we investigated the clinical role of 10 protein products of the discriminating genes, with focus on epithelial-mesenchymal transition and stem cell markers. Expression of E-cadherin, N-cadherin, P-cadherin, Zeb1, HMGA2, Rab25, CD24, NCAM (CD56), Sox11, and vimentin was assessed in 100 advanced-stage (International Federation of Gynecology and Obstetrics stages III-IV) serous ovarian carcinoma effusions using immunohistochemistry. Results were analyzed for association with clinicopathological parameters, including chemotherapy response, and survival. All 10 proteins were frequently expressed in carcinoma cells. HMGA2 expression was related to older age (P = .015). HMGA2 and NCAM expression was related to stage III disease (P = .011 and P = .023, respectively), and NCAM was overexpressed in peritoneal compared with pleural effusions (P = .001). Vimentin and Zeb1 expression was significantly related to poor chemotherapy response at diagnosis (P = .005 and P = .017, respectively). The associations between NCAM and peritoneal localization and of vimentin and poor chemoresponse were retained after Bonferroni correction. NCAM expression was associated with a trend for shorter overall survival in univariate survival analysis (P = .187), but emerged as an independent prognosticator in Cox multivariate analysis (P = .042). This study identifies vimentin and Zeb1 as markers of poor chemoresponse in metastatic serous ovarian carcinoma effusions and suggests NCAM as potential prognostic marker in metastatic disease. The generally limited prognostic role of the studied markers emphasizes the difficulty in applying data obtained in studies of primary ovarian carcinomas to analyses of ovarian carcinoma effusions, reflecting the unique biology of the latter.

Polysialic acid: biosynthesis, novel functions and applications.

As an anti-adhesive, a reservoir for key biological molecules, and a modulator of signaling, polysialic acid (polySia) is critical for nervous system development and maintenance, promotes cancer metastasis, tissue regeneration and repair, and is implicated in psychiatric diseases. In this review, we focus on the biosynthesis and functions of mammalian polySia, and the use of polySia in therapeutic applications. PolySia modifies a small subset of mammalian glycoproteins, with the neural cell adhesion molecule, NCAM, serving as its major carrier. Studies show that mammalian polysialyltransferases employ a unique recognition mechanism to limit the addition of polySia to a select group of proteins. PolySia has long been considered an anti-adhesive molecule, and its impact on cell adhesion and signaling attributed directly to this property. However, recent studies have shown that polySia specifically binds neurotrophins, growth factors, and neurotransmitters and that this binding depends on chain length. This work highlights the importance of considering polySia quality and quantity, and not simply its presence or absence, as its various roles are explored. The capsular polySia of neuroinvasive bacteria allows these organisms to evade the host immune response. While this "stealth" characteristic has made meningitis vaccine development difficult, it has also made polySia a worthy replacement for polyetheylene glycol in the generation of therapeutic proteins with low immunogenicity and improved circulating half-lives. Bacterial polysialyltransferases are more promiscuous than the protein-specific mammalian enzymes, and new studies suggest that these enzymes have tremendous therapeutic potential, especially for strategies aimed at neural regeneration and tissue repair.

Micromorphology of the dental pulp is highly preserved in cancer patients who underwent head and neck radiotherapy.

INTRODUCTION: Teeth are often included in the radiation field during head and neck radiotherapy, and recent clinical evidence suggests that dental pulp is negatively affected by the direct effects of radiation, leading to impaired sensitivity of the dental pulp. Therefore, this study aimed to investigate the direct effects of radiation on the microvasculature, innervation, and extracellular matrix of the dental pulp of patients who have undergone head and neck radiotherapy. METHODS: Twenty-three samples of dental pulp from patients who finished head and neck radiotherapy were analyzed. Samples were histologically processed and stained with hematoxylin-eosin for morphologic evaluation of the microvasculature, innervation, and extracellular matrix. Subsequently, immunohistochemical analysis of proteins related to vascularization (CD34 and smooth muscle actin), innervation (S-100, NCAM/CD56, and neurofilament), and extracellular matrix (vimentin) of the dental pulp was performed. RESULTS: The morphologic study identified preservation of the microvasculature, nerve bundles, and components of the extracellular matrix in all studied samples. The immunohistochemical analysis confirmed the morphologic findings and showed a normal pattern of expression for the studied proteins in all samples. CONCLUSIONS: Direct effects of radiotherapy are not able to generate morphologic changes in the microvasculature, innervation, and extracellular matrix components of the dental pulp in head and neck cancer patients.

Disialic, oligosialic and polysialic acids: distribution, functions and related disease.

Sialic acids (Sia) are involved in many biological activities and frequently exist as monosialyl residues at the non-reducing terminal end of glycoconjugates. Occasionally, polymerized structures in the form of disialic acid (diSia), oligosialic acid (oligoSia) and polysialic acid (polySia) are also found in glycoconjugates. In particular, polySia, which is an evolutionarily conserved epitope from sea urchin to humans, is one of the most biologically important glycotopes in vertebrates. The biological functions of polySia, especially on neural cell adhesion molecules, have been well studied and an in-depth body of knowledge concerning polySia has been accumulated. However, considerably less attention has been paid to glycoproteins containing di- and oligoSia groups. However, advances in analytical methods for detecting oligo/polymerized structures have allowed the identification and characterization of an increasing number of glycoproteins containing di/oligo/polySia chains in nature. In addition, sophisticated genetic techniques have also helped to elucidate the underlying mechanisms of polySia-mediated activities. In this review, recent advances in the study of the chemical properties, distribution and functions of di-, oligo- and polySia residues on glycoproteins are described.

IgSF8: a developmentally and functionally regulated cell adhesion molecule in olfactory sensory neuron axons and synapses.

Here, we investigated an Immunoglobulin (Ig) superfamily protein IgSF8 which is abundantly expressed in olfactory sensory neuron (OSN) axons and their developing synapses. We demonstrate that expression of IgSF8 within synaptic neuropil is transitory, limited to the period of glomerular formation. Glomerular expression decreases after synaptic maturation and compartmental glomerular organization is achieved, although expression is maintained at high levels within the olfactory nerve layer (ONL). Immunoprecipitations indicate that IgSF8 interacts with tetraspanin CD9 in the olfactory bulb (OB). CD9 is a component of tetraspanin-enriched microdomains (TEMs), specialized microdomains of the plasma membrane known to regulate cell morphology, motility, invasion, fusion and signaling, in both the nervous and immune systems, as well as in tumors. In vitro, both IgSF8 and CD9 localize to puncta within axons and growth cones of OSNs, consistent with TEM localization. When the olfactory epithelium (OE) was lesioned, forcing OSN regeneration en masse, IgSF8 was once again able to be detected in OSN axon terminals as synapses were reestablished. Finally, we halted synaptic maturation within glomeruli by unilaterally blocking functional activity and found that IgSF8 did not undergo exclusion from this subcellular compartment and instead continued to be detected in adult glomeruli. These data support the hypothesis that IgSF8 facilitates OSN synapse formation.

Expression of NCAM and OCIAD1 in well-differentiated thyroid carcinoma: correlation with the risk of distant metastasis.

AIMS: The biomarkers representing the metastatic potential of well-differentiated thyroid carcinoma remain to be established. A study was undertaken to find whether the expression status of neural cell adhesion molecule (NCAM) and/or ovarian cancer immunoreactive antigen domain containing 1 (OCIAD1) is associated with the metastatic potential of differentiated thyroid carcinoma. METHODS: NCAM and OCIAD1 were analysed by immunohistochemistry on tissue microarrays. RESULTS: Among 214 well-differentiated thyroid carcinomas, 68 patients had distant metastases. Immunohistochemical analyses showed that the majority of benign thyroid lesions expressed NCAM while a significant proportion of thyroid carcinomas lost or had reduced NCAM expression. Both follicular and papillary carcinomas with distant metastasis had a significantly higher frequency of preserving NCAM expression. Hierarchical clustering analysis showed that OCIAD1 had significant differential expression between benign and malignant thyroid lesions. The overall metastatic-to-localised tumour ratio was higher in NCAM-expressing clusters, but the difference between ratios of OCIAD1-positive and OCIAD1-negative subclusters was not significant. CONCLUSIONS: These analyses suggest that the preservation of NCAM expression in well-differentiated thyroid carcinoma is an indicator for a higher risk of distant metastasis. OCIAD1 is a potential biomarker of thyroid carcinoma but had no significant additive effect on the risk of distant metastasis. Further elucidation of the molecular mechanisms underlying the NCAM-mediated cellular processes will be beneficial for the development of effective treatments against the metastasis of thyroid carcinoma.

N-CAM exhibits a regulatory function in pathological angiogenesis in oxygen induced retinopathy.

BACKGROUND: Diabetic retinopathy and retinopathy of prematurity are diseases caused by pathological angiogenesis in the retina as a consequence of local hypoxia. The underlying mechanism for epiretinal neovascularization (tuft formation), which contributes to blindness, has yet to be identified. Neural cell adhesion molecule (N-CAM) is expressed by Müller cells and astrocytes, which are in close contact with the retinal vasculature, during normal developmental angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Notably, during oxygen induced retinopathy (OIR) N-CAM accumulated on astrocytes surrounding the epiretinal tufts. Here, we show that N-CAM ablation results in reduced vascular tuft formation due to reduced endothelial cell proliferation despite an elevation in VEGFA mRNA expression, whereas retinal developmental angiogenesis was unaffected. CONCLUSION/SIGNIFICANCE: We conclude that N-CAM exhibits a regulatory function in pathological angiogenesis in OIR. This is a novel finding that can be of clinical relevance in diseases associated with proliferative vasculopathy.

Melatonin controls photoperiodic changes in tanycyte vimentin and neural cell adhesion molecule expression in the Djungarian hamster (Phodopus sungorus).

The Djungarian hamster displays photoperiodic variations in gonadal size synchronized to the seasons by the nightly secretion of the pineal hormone melatonin. In short photoperiod (SP), the gonads regress in size, and circulating sex steroids levels decline. Thus, the brain is subject to seasonal variations of both melatonin and sex steroids. Tanycytes are specialized glial cells located in the ependymal lining of the third ventricle. They send processes either to the meninges or to blood vessels of the medio-basal hypothalamus. Furthermore, they are known to locally modulate GnRH release in the median eminence and to display seasonal structural changes. Seasonal changes in tanycyte morphology might be mediated either through melatonin or sex steroids. Therefore, we analyzed the effects of photoperiod, melatonin, and sex steroids 1) on tanycyte vimentin expression by immunohistochemistry and 2) on the expression of the neural cell adhesion molecule (NCAM) and polysialic acid as markers of brain plasticity. Vimentin immunostaining was reduced in tanycyte cell bodies and processes in SP. Similarly, tanycytes and their processes contained lower amounts of NCAM in SP. These changes induced by SP exposure could not be restored to long photoperiod (LP) levels by testosterone supplementation. Likewise, castration in LP did not affect tanycyte vimentin or NCAM expression. By contrast, late afternoon melatonin injections mimicking a SP-like melatonin peak in LP hamsters reduced vimentin and NCAM expression. Thus, the seasonal changes in vimentin and NCAM expression in tanycytes are regulated by melatonin independently of seasonal sex steroid changes.

Angiogenesis and blood vessel stability in inflammatory arthritis.

OBJECTIVE: To assess blood vessel stability in inflammatory synovial tissue (ST) and to examine neural cell adhesion molecule (NCAM), oxidative DNA damage, and hypoxia in vivo. METHODS: Macroscopic vascularity and ST oxygen levels were determined in vivo in patients with inflammatory arthritis who were undergoing arthroscopy. Vessel maturity/stability was quantified in matched ST samples by dual immunofluorescence staining for factor VIII (FVIII)/alpha-smooth muscle actin (alpha-SMA). NCAM and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were examined by immunohistochemistry. Angiogenesis was assessed in vitro, using human dermal endothelial cells (HDECs) in a Matrigel tube formation assay. RESULTS: A significant number of immature vessels (showing no pericyte recruitment) was observed in tissue from patients with inflammatory arthritis (P < 0.001), in contrast to osteoarthritic and normal tissue, which showed complete recruitment of pericytes. Low in vivo PO(2) levels in the inflamed joint (median [range] 22.8 [3.2-54.1] mm Hg) were inversely related to increased macroscopic vascularity (P < 0.04) and increased microscopic expression of FVIII and alpha-SMA (P < 0.04 and P < 0.03, respectively). A significant proportion of vessels showed focal expression of NCAM and strong nuclear 8-oxodG expression, implicating a loss of EC-pericyte contact and increased DNA damage, levels of which were inversely associated with low in vivo PO(2) (P = 0.04 for each comparison). Circulating cells were completely negative for 8-oxodG. Exposure of HDEC to 3% O(2) (reflecting mean ST in vivo measurements) significantly increased EC tube formation (P < 0.05). CONCLUSION: Our findings indicate the presence of unstable vessels in inflamed joints associated with hypoxia, incomplete EC-pericyte interactions, and increased DNA damage. These changes may further contribute to persistent hypoxia in the inflamed joint to further drive this unstable microenvironment.

Estudio piloto de marcadores inflamatorios en el sindrome de apnea obstructiva del sueño / Pilot study of inflammatory markers in the syndrome of obstructive sleep apnea

El síndrome de Apnea Obstructiva del Sueño (SAOS), consiste en la aparición repetida de episodios de obstrucción faríngea durante el sueño como consecuencia de un colapso de la vía respiratoria. La respuesta fisiológica a la hipoxia intermitente crónica es la generación de una respuesta inflamatoria local y sistémica. Se han evidenciado cambios importantes a nivel cardiovascular en pacientes con SAOS; sin embargo, se desconocen cuáles marcadores séricos y genéticos pudieran ser de utilidad. En el presente estudio, se presentan 15 marcadores séricos y 3 genéticos (IL-6, IL-1β y TNF-α) en un grupo de cinco pacientes para determinar cuáles pueden ser los marcadores de interés en la aparición y en el desarrollo de esta patología respiratoria. Se proponen como marcadores los niveles séricos: proteína C reactiva, TNFα, IL-6, el receptor soluble de TNF I, sCD62, sCD154, nitrotirosina y anti-oxLDL. Los niveles de IL-1 β, el receptor de TNF soluble II, sCD25, sCD54, nitritos y nitratos no parecieran ser buenos marcadores en SAOS. Los estudios genéticos no fueron concluyentes.

Obstructive sleep apnea syndrome (OSAS) is a repeated sequences of pharynx obstruction during sleep as a consequence of airway collapse. The physiological response to the desaturation is the generation of a local and systemic inflammatory immune response. Important changes at cardiovascular levels in patients with OSAS have been observed; however, it is not know which serum or genetic parameters could be useful. In the present study, we present 15 serum and 3 genetic (IL-6, IL-1β y TNF-α) markers in a group of five patients in order to determine which marker could be useful to study the genesis and progression of this respiratory pathology. The proposed serum markers are C reactive proteín, TNFα, IL-6, soluble de TNF receptor I, sCD62, sCD154, nitrotirosine and anti-oxLDL. The levels of IL-1 β, soluble TNF receptor II, sCD25, sCD54, nitrite y nitrate do not seem to be good markers for OSAS. The genetic studies were not conclusive.

Differential gene expression in the perichondrium and cartilage of the neonatal mouse temporomandibular joint.

Our goal was to discover genes differentially expressed in the perichondrium (PC) of the mandibular condylar cartilage (MCC) that might enhance regenerative medicine or orthopaedic therapies directed at the tissues of the temporomandibular joint. We used targeted gene arrays (osteogenesis, stem cell) to identify genes preferentially expressed in the PC and the cartilaginous (C) portions of the MCC in 2-day-old mice. Genes with higher expression in the PC sample related to growth factor ligand-receptor interactions [FGF-13 (6.4x), FGF-18 (4x), NCAM (2x); PGDF receptors, transforming growth factor (TGF)-beta and IGF-1], the Notch isoforms (especially Notch 3 and 4) and their ligands or structural proteins/proteoglycans [collagen XIV (21x), collagen XVIII (4x), decorin (2.5x)]. Genes with higher expression in the C sample consisted mostly of known cartilage-specific genes [aggrecan (11x), procollagens X (33x), XI (14x), IX (4.5x), Sox 9 (4.4x) and Indian hedgehog (6.7x)]. However, the functional or structural roles of several genes that were expressed at higher levels in the PC sample are unclear [myogenic factor (Myf) 9 (9x), tooth-related genes such as tuftelin (2.5x) and dentin sialophosphoprotein (1.6x), VEGF-B (2x) and its receptors (3-4x) and sclerostin (1.7x)]. FGF, Notch and TGF-beta signalling may be important regulators of MCC proliferation and differentiation; the relatively high expression of genes such as Myf6 and VEGF-B and its receptors suggests a degree of unsuspected plasticity in PC cells.

New multiple labelling method for improved satellite cell identification in human muscle: application to a cohort of power-lifters and sedentary men.

Presently applied methods to identify and quantify human satellite cells (SCs) give discrepant results. We introduce a new immunofluorescence method that simultaneously monitors two SC markers (NCAM and Pax7), the basal lamina and nuclei. Biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects were re-examined. Significantly different results from those with staining for NCAM and nuclei were observed. There were three subtypes of SCs; NCAM(+)/Pax7(+) (94%), NCAM(+)/Pax7(-) (4%) and NCAM(-)/Pax7(+) (1%) but large individual variability existed. The proportion of SCs per nuclei within the basal lamina of myofibres (SC/N) was similar for all groups reflecting a balance between the number of SCs and myonuclei to maintain homeostasis. We emphasise that it is important to quantify both SC/N and the number of SCs per fibre. Our multiple marker method is more reliable for SC identification and quantification and can be used to evaluate other markers of muscle progenitor cells.