The natural involution of the sheep proximal sesamoidean ligament is due to depletion of satellite cells and simultaneous proliferation of fibroblasts: Ultrastructural evidence.

The rapid involution that happens in some muscles of ungulate fetlock joints has never been investigated at an ultrastructural level. In this study, the proximal sesamoidean ligament (PSL) of sheep was chosen as a model to investigate, at the cellular level, the transition from muscle to connective structures that occurs during early development. In particular, we were interested in observing the presence of satellite cells and fibroblasts, detecting fluctuations in their numbers in the postnatal developing PSL, and evaluating putative apoptotic mechanisms. Interestingly, some features were shared by both PSL involution and muscle ageing; the most relevant being the significant and rapid decrease in the number of satellite cells together with a quick proliferation of fibroblasts in the muscle-connective transitional area (MCT-TA). Electron microscopy and immunohistochemical analyses revealed putative cellular mechanisms that led to a progressive involution of the muscle portion of the PSL during postnatal growth. Our findings showed a fast transition from muscle to connective tissue due to the depletion of satellite cells, apoptosis of some muscle fibres, and simultaneous proliferation of fibroblasts originating from mesenchymal progenitors or from differentiation of satellite cells typically located at the border between muscle and connective tissue of the PSL.

Overexpression of histidine-rich calcium binding protein in equine ventricular myocardium.

Histidine-rich calcium binding protein (HRC) is a high capacity, low affinity Ca(2+) binding protein, specifically expressed in striated muscles of mammals. In rabbit skeletal and cardiac muscles, HRC binds to sarcoplasmic reticulum (SR) membranes via triadin, a junctional SR protein. Recently, a potential role in heart failure and arrhythmogenesis has been assigned to HRC due to its activity as regulator of SR Ca(2+) uptake and Ca(2+) release. HRC might play a particularly relevant role in the equine heart, given its slower resting heart rate (20-35 beats/min) and longer action potential duration (APD) (0.6-1.0 s) than are found in other mammals. The results from this study showed for the first time direct evidence that HRC protein in equine cardiac muscle was expressed in association with the SR membranes and that HRC transcriptional activity was three times higher in the ventricles compared to the atria. The predominance of HRC mRNA up-regulation in ventricular myocardium was specific to the horse heart, since a more even distribution between atria and ventricles was found in animals of similar body size or species, such as cattle or domestic donkeys.

Canine adipose-derived-mesenchymal stem cells do not lose stem features after a long-term cryopreservation.

Adult stem cells are nowadays used for treating several pathologies. A putative stem cell population was found in the adipose tissue of mammals and canine adipose tissue-derived-mesenchymal stem cells (cA-MSC) have been shown to possess the capacity to differentiate into several lineages. The main goal of our research was to fully characterize cA-MSC and examine the effects of cryopreservation on their stemness features. Each sample of cA-MSC was analyzed immediately and then again after being frozen in liquid nitrogen for one year. After the cryopreservation period cells conserved their fibroblast-like morphology, alkaline phosphatase positivity and CD expression but showed a lower proliferation ratio and a lower telomerase activity in comparison with fresh cells. Finally, the cryopreservation protocol did not change the cA-MSC adipogenic, osteogenic and myogenic differentiative potential. Our data demonstrate that stored cA-MSC might represent a promising type of progenitor cell for autologous cellular-based therapies in veterinary medicine.

Glial cell line-derived neurotrophic factor expression in the brain of adult zebrafish (Danio rerio).

In mammals, glial cell line-derived neurotrophic factor (GDNF) is a growth factor of many neuronal populations in the central, peripheral and autonomous nervous system. GDNF may also function as a morphogen during kidney development and may regulate spermatogonial differentiation. GDNF has been characterised in zebrafish embryos and was demonstrated experimentally to be critical for the development of the enteric nervous system. However, in adult zebrafish, no data exist regarding GDNF expression and localisation in the brain and in different organs. Thus, the aim of the present study was to investigate the expression of GDNF in the brain of adult zebrafish (Danio rerio). Transcripts of GDNF mRNA were observed in brain extracts by a standard RT-PCR. The presence of the protein in the brain homogenates was confirmed by SDS-PAGE electrophoresis and Western blotting analysis. Immunohistochemistry and in situ hybridization experiments showed that GDNF protein and mRNA were localised in various nuclei of the telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata of the zebrafish brain. In conclusion, this study showed that the expression of GDNF was not restricted to developmental periods but it seems that this factor might be involved in adult zebrafish brain physiology, as observed in mammals.

Expression of the paired box domain Pax7 protein in myogenic cells isolated from the porcine semitendinosus muscle after birth.

The paired box domain gene Pax7 plays a pivotal role in satellite cell physiology and may represent one of the candidate genes influencing the dynamic stages of early post-natal growth observed in pig. Quiescent satellite cells express Pax7 and, when activated, they co-express the myogenic bHLH protein MyoD. The aims of this study were to investigate, by immunohistochemistry, the putative differential expression of Pax7 and to ascertain the amount of activated satellite cells (Pax7(+)/MyoD(+)) in myogenic cells isolated at different post-natal time points and in adults. Our results indicate that Pax7(+) cells represent between 10 and 15% of the whole myogenic cell population found at birth indicating that these cells provide a modest contribution to the development of new fibres. The number of activated satellite cells (Pax7(+)/MyoD(+)) was scarce after birth but it was higher respect to adults. An interesting result was that at 1 month after birth the number of Pax7(+) cells had increased within the pool of myogenic cells with respect to myogenic cells extracted at birth. We speculate that Pax7 might be one of the molecules involved in controlling the proliferation/differentiation ratio in the pool of satellite cells present in post-natal porcine skeletal muscles.

Morphometric study of the interproximal unit in the esthetic region to correlate anatomic variables affecting the aspect of soft tissue embrasure space.

BACKGROUND: The presence of a normal papilla is crucial to avoid the unpleasant esthetic defects that are of major concern to periodontists, restorative dentists, and patients. During the course of progressive periodontitis and following periodontal treatment, it is not uncommon to have a partial loss of the interdental papilla. This loss can lead to an unesthetic gingival appearance. This study evaluated different anatomic variables in an effort to determine their role in the papillary appearance of maxillary incisors. METHODS: A total of 178 interdental embrasures in 58 patients were selected randomly for examination. For each patient, a digital photograph and a modified periapical radiograph of the interdental embrasure of the four maxillary incisors were taken by using a special metric device fixed to a centrator as a reference marker. Clinical and radiographic data were obtained for the distance from the contact point to the alveolar crest and for the interradicular distance. We used a classification system with regard to peri-implant soft tissue based on esthetic assessments related to the space between reference lines through the highest gingival curvature of the crown-tooth margin and the contact point. RESULTS: In the group of interdental sites with an interradicular distance of less than approximately 2.4 mm, an increase in the distance between the contact point and the bone crest corresponded to a marked increase in the interdental black triangle's dimensions and, therefore, a less esthetic smile. In particular, when the interradicular distance was >2.4 mm, we statistically estimated that the other anatomic variable considered, the distance from the contact point to the alveolar crest, lost its influence on whether the interdental papilla would be present or absent. CONCLUSION: The interradicular distance and the distance between the contact point and the alveolar crest have independent and combined effects on the presence or absence of the interdental papilla.

Expression of eight distinct MHC isoforms in bovine striated muscles: evidence for MHC-2B presence only in extraocular muscles.

This study aimed to analyse the expression of myosin heavy chain (MHC) isoforms in bovine muscles, with particular attention to the MHC-2B gene. Diaphragm, longissimus dorsi, masseter, several laryngeal muscles and two extraocular muscles (rectus lateralis and retractor bulbi) were sampled in adult male Bos taurus (age 18-24 months, mass 400-500 kg) and analysed by RT-PCR, gel electrophoresis and immunohistochemistry. Transcripts and proteins corresponding to eight MHC isoforms were identified: MHC-alpha and MHC-beta/slow (or MHC-1), two developmental isoforms (MHC-embryonic and MHC-neonatal), three adult fast isoforms (MHC-2A, MHC-2X and MHC-2B) and the extraocular isoform MHC-Eo. All eight MHC isoforms were found to be co-expressed in extrinsic eye muscles, retractor bulbi and rectus lateralis, four (beta/slow, 2A, 2X, neonatal) in laryngeal muscles, three (beta/slow, 2A and 2X) in trunk and limb muscles and two (beta/slow and alpha) in masseter. The expression of MHC-2B and MHC-Eo was restricted to extraocular muscles. Developmental MHC isoforms (neonatal and embryonic) were only found in specialized muscles in the larynx and in the eye. MHC-alpha was only found in extraocular and masseter muscle. Single fibres dissected from masseter, diaphragm and longissimus were classified into five groups (expressing, respectively, beta/slow, alpha, slow and 2A, 2A and 2X) on the basis of MHC isoform electrophoretical separation, and their contractile properties [maximum shortening velocity (v(0)) and isometric tension (P(0))] were determined. v(0) increased progressively from slow to fast 2A and fast 2X, whereas hybrid 1-2A fibres and fibres containing MHC-alpha were intermediate between slow and fast 2A.

Myosin heavy chain 2B isoform is expressed in specialized eye muscles but not in trunk and limb muscles of cattle.

Myosin heavy chain isoforms (MHC) of adult skeletal muscles are codified by four genes named: slow, or type 1, and fast types 2A, 2X and 2B. The slow, 2A and 2X isoforms have been found expressed in all mammalian species studied so far whereas there is a large inter-species variability in the expression of MHC-2B. In this study histochemistry (m-ATPase), immunohistochemistry with the use of specific monoclonal antibodies and RT-PCR were combined together to assess whether the MHC-2B gene is expressed in bovine muscles. ATPase staining and RT-PCR experiments showed that three MHC isoforms (1, 2A, 2X) were expressed in trunk and limb muscles. Slow or type 1 expression was confirmed using a specific antibody (BA-F8) whereas the detection of fast MHC isoforms were validate by means of BF-35 antibody although not by the SC-71 antibody. MHC-2B was absent in limb and trunk muscles, but was present in specialized eye muscles (rectus lateralis and retractor bulbi) as consistently showed by RT-PCR and reactivity with a specific antibody (BF-F3). Interestingly, a cardiac isoform, MHC-a-cardiac was found to be expressed not only in extraocular muscles but also in masticatory muscles as masseter.

Characteristics of fungal phytases from Aspergillus fumigatus and Sartorya fumigata.

Aspergillus fumigatus phytase has previously been identified as a phytase with a series of favourable properties that may be relevant in animal and human nutrition, both for maximising phytic acid degradation and for increasing mineral and amino acid availability. To study the natural variability in amino acid sequence and its impact on the catalytic properties of the enzyme, we cloned and overexpressed the phytase genes and proteins from six new purported A. fumigatus isolates. Five of these phytases displayed < or= 2 amino acid substitutions and had virtually identical stability and catalytic properties when compared with the previously described A. fumigatus ATCC 13073 phytase. In contrast, the phytase from isolate ATCC 32239 ( Sartorya fumigata, the anamorph of which was identified as A. fumigatus) was more divergent (only 86% amino acid sequence identity), had a higher specific activity with phytic acid, and displayed distinct differences in substrate specificity and pH-activity profile. Finally, comparative experiments confirmed the favourable stability and catalytic properties of A. fumigatus phytase.

Neurotransmitters, neuromodulators, and neurotrophin receptors in the gut of pantex, a hybrid sparid fish (Pagrus major x Dentex dentex). Localizations in the enteric nervous and endocrine systems.

The gut of Pantex, a sparid hybrid fish (Pagrus major x Dentex dentex) with a great potential importance for the Italian aquaculture, was histochemically and immunohistochemically investigated in order to evidence components of the intramural nervous and diffuse endocrine systems. The general structural aspects of the intramural nervous system were shown by the Nissl-thionin staining. As in most other fish, it was only organized in the myenteric plexus. Acetylcholinesterase (AChE) activity was observed in both nerve cell bodies and terminals all along the gut. The NADPH-diaphorase reactivity too, possibly linked to the synthesis and release of nitric oxide, was present in nerve cell bodies and nerve terminals of the oesophagus, stomach and intestine. In addition, the intramural nervous system was shown to contain Trk (tyrosinekinase) receptors for neurotrophin, as evidenced by Trk A-, Trk B- and Trk C-like immunoreactivities, thus suggesting an involvement of neurotrophin in the function of this system. Trk B- and Trk C-like immunoreactivities were detected in epithelial endocrine cells, too. The additional presence of serotonin- and metenkephalin-like immunoreactivities in numerous endocrine cells in the epithelial layers of the stomach and intestine was showed.

Characterization of the myostatin gene in the gilthead seabream (Sparus aurata): sequence, genomic structure, and expression pattern.

We report on the sequence and expression analysis of the myostatin gene (MSTN) in the gilthead seabream Sparus aurata. A 2189-bp transcript was isolated, encoding an open reading frame (385 amino acids) that showed 74% to 60% protein similarity with other vertebrate myostatins. Phylogenetic analysis of MSTN and other related genes confirmed the evolutionary relationships of the isolated sequence. The complete sequences of two introns were also determined. Intron-exon boundaries were conserved when compared with those of mammalian MSTN genes, whereas intron size was smaller. Reverse transcriptase polymerase chain reaction on total RNA extracted from different tissues and developmental stages revealed MSTN expression in the skeletal muscle, but also in other tissues. The observed expression profile differed from that in mammals, suggesting possible additional functions of myostatin in the teleost fish.

Neurotransmitters and putative neuromodulators in the gut of Anguilla anguilla (L.). Localizations in the enteric nervous and endocrine systems.

The gut of silver eels (Anguilla anguilla L.) was investigated in order to describe both the cholinergic and adrenergic intramural innervations, and the localization of possible accessory neuromediators. Histochemical reactions for the demonstration of nicotinamide adenine dinucleotide phosphate, reduced form-(NADPH-)diaphorase and acetylcholinesterase (AChEase) were performed, as well as the immunohistochemical testing of tyrosine hydroxylase, met-enkephalin, substance P, calcitonin gene-related peptide (CGRP), bombesin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), somatostatin, cholecystokinin-octapeptide (CCK-8), serotonin, cholineacetyl transferase. The results evidenced a different pattern in comparison with other vertebrates, namely mammals, and with other fish. Both NADPH-diaphorase and AChEase activities were histochemically detected all along the gut in the myenteric plexus, the inner musculature and the propria-submucosa. Tyrosine hydroxylase immunoreactivity was observed in the intestinal tract only, both in the myenteric plexus and in the inner musculature. Several neuropeptides (metenkephalin, CGRP, bombesin, substance P, VIP, NPY, somatostatin) were, in addition, detected in the intramural innervation; some of them also in epithelial cells of the diffuse endocrine system (met-enkephalin, substance P, NPY, somatostatin). Serotonin was only present in endocrine cells. Tyrosine hydroxylase immunoreactivity was present in localizations similar to those of NADPH-diaphorase-reactivity, and in the same nerve bundles in which substance P- and CGRP-like-immunoreactivities were detectable in the intestinal tract. In addition, NADPH-diaphorase-reactive neurons showed an anatomical relationship with AChEase-reactive nerve terminals, and a similar relationship existed between the latter and substance P-like immunoreactivity.

Ultrastructural features of the gut in the white sturgeon, Acipenser transmontanus.

Electron-microscopic examinations of the sturgeon gut were performed. Oesophageal goblet cells were abundant in the stratified epithelium. The ultrastructural features of the secretory granules of the oesophageal and intestinal goblet cells were quite similar to those of other vertebrates. Lobules of multilocular adipose tissue were observed in the deep tunica propriasubmucosa of the oesophagus, in close association with vasculature and large fibre bundles of myelinated and unmyelinated axons. Similarly composed nerve fibre bundles were observed in the cardiac stomach, too. The presence of myelinated axons is an unusual feature in the vertebrate enteric nervous system. Cardiac and fundic zones of the stomach showed an epithelium with columnar ciliated and non-ciliated cells, the latter equipped with fuzzy microvilli. Cells lining the tubular gastric proper glands were markedly granulated. Intestinal superficial epithelium was columnar and contained ciliated, as well as non-ciliated and goblet cells. In the tunica propria all over the intestine, the presence and ultrastructure of granulated cells was in addition described. Intraepithelial granulated leukocytes were seen throughout the alimentary canal. Various types of endocrine cells were seen both in the stomach and in the intestine, the size of their granules was measured and their ultrastructure described and compared to that of mammalian cell types.

Morphological and histochemical peculiarities of the gut in the white sturgeon, Acipenser transmontanus.

The gut of adult sturgeon was examined. The oesophageal mucosa contained numerous caliciform cells, synthesizing both neutral and acidic glycoconjugates, the latter of the sialylated type. The deep tunica propria-submucosa contained lobules of multilocular adipose tissue, specially abundant during the cold season. The oesophageal tunica muscularis was made up of a large sheath of striated muscle fibres, arranged orthogonally to a thin, subserous smooth muscle layer. The siphon-shaped stomach showed a ciliated epithelium in cardiac and gastric proper gland zones, where tubular glands were present in the tunica propria. The columnar cells which composed the superficial epithelium and gastric pits were demonstrated to synthesize almost exclusively neutral glycoconjugates. Appendices pyloricae constituted a glandular body equipped with intestinal mucosa. The intestinal mucosa was organized in folds, containing numerous caliciform cells which synthesized neutral or acidic glycoconjugates, the latter either of the sialylated and sulphated type. The sulphoglycoconjugates were more abundant in the caliciform cells of the distal intestinal tracts. The tunica propria-submucosa of the spiral valve (medium intestine) contained lymphatic tissue and large lymphatic follicles. A muscularis mucosae was present only in the rectum, where in addition a peculiar granular cell type was present in the superficial tunica propria-submucosa, possibly related to defensive properties. The subserous connective tissue contained pancreatic lobules all along the stomach and intestine. The enteric nervous system showed some special aspects, the most intriguing of which was the presence of large, longitudinally oriented nerve bundles in the t. propria-submucosa of oesophagus and cardiac stomach. The nerve bundles contained, near unmyelinated nerves, some myelinated nerves, as well as neuronal bodies. Both these aspects are exceptional in vertebrates and obscure in their significance. The structural and histochemical aspects we here describe are in part different from those described for other fish. Some of these special features are possibly related with special functional roles, others require a deeper insight and different approaches to clarify them functionally.

Muscle growth in response to changing demands of functions in the teleost Sparus aurata (L.) during development from hatching to juvenile.

Growth of laterarl muscle in the teleost fish Sparus aurata (L.) was examined from hatching to juvenile by a basic morphofunctional approach that takes into account structural and ecophysiological aspects and combines in vivo observations and LM and TEM microscopic analysis. As shown in most teleost fishes, muscle growth proceeds by a double mechanism of hyperplasia and hypertrophy that contribute differentially to the overall development of the lateral muscle, giving rise in each myomere to a typical pattern of structurally and functionally different fibre types (slow-red and fast-white fibres, plus pink intermediate fibres) in a nerve-dependent process. During larval life the muscle growth takes place mainly due to hyperplastic growth at the level of specific proliferative zones of the myomeres, from which slow, pink and white muscle fibres are derived. In those species that reach a large adult size a new typical hyperplastic process disseminated throughout the fast white muscle layer takes place during post-larval life. In contrast, hypertrophic growth occurs in all stages, but is the dominant mechanism of muscle growth only in juvenile and adult. The suitable recruitment of the different fibre types enables the fish to optimize its performances according to specific functional and metabolic requirements related to the swimming behaviour and hydrodynamic regimes. The different mechanisms of growth are here analysed in their detailed structural and ultrastructural aspects in order to interpret their adaptive significance in the light of the fish life cycle, with particular reference to locomotion and feeding behaviour.

Different putative neuromodulators are present in the nerves which distribute to the teleost skeletal muscle.

The presence of putative neuromodulators in the nerve fibres was investigated in white skeletal muscle of two teleost fish not taxonomically correlated and showing different patterns of innervation (multiple versus focal innervation). Cryostat sections of epaxial, hypaxial and adductor mandibulae (AM) muscles of Sparus aurata and Anguilla anguilla were stained histochemically for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Other sections were used for indirect immunohistochemistry (streptavidin-biotin and rhodamine immunofluorescence methods), employing antibodies specific for putative excitatory or inhibitory peptides, including CGRP, substance P, met-enkephalin, bombesin, and VIP. In addition, ultrastructural observations were performed in order to describe the morphology of the motor endplates. A strong immunoreactivity for CGRP and substance P was found in many nerve terminals. Met-enkephalin, bombesin and VIP immunoreactivities were less frequently observed. No immunoreactivity was observed to CCK, NPY or 5-HT. NADPH-diaphorase was identified in nerve fibres of the AM complex only of A. anguilla. Electron microscopy observations evidenced more than one type of synaptic vesicle in motor endplates. Some differences in putative neuromodulator distributions were observed in the two species and muscle complexes, which may be related to the different taxonomical position as well as the different pattern of innervation of white muscle fibres.

Regeneration of skeletal muscle in two teleost fish: Sparus aurata and Brachydanio rerio.

Regeneration of skeletal muscle was studied in the sea bream Sparus aurata, in which extensive post-larval muscle hyperplasia contributes to its large adult size, and in the zebrafish Brachydanio rerio, which shows little post-larval hyperplasia and reaches only a small adult size. Small mechanical lesions of body wall muscle were made under general anaesthesia, and the progress of subsequent regeneration was assessed at various intervals by histology and electron microscopy (for general morphology), by immunostaining for desmin and myosin isoforms (to identify the phenotype of new fibres), and by 5'-bromo-2'-deoxyuridine (BrdU) incorporation (to identify proliferating cells). Despite the difference in normal growth-related hyperplasia in these fish, a vigorous regeneration occurred in both species, giving rise to new fibres with an initial myosin composition that differed from that in mature fast-white fibres. However, species differences in myosin expression in these fibres suggest that they may have derived from different myoblast populations. In sea bream, myosin expression in regenerating fibres resembled that seen in new fibres produced in post-larval white muscle, whereas in the zebrafish it resembled that of the primitive monolayer fibres formed during embryonic development. Subsequently, most regenerating fibres gradually transformed into the mature fast-white phenotype in both species.