Activated satellite cells in medial rectus muscles of patients with strabismus

PURPOSE. The goal of this study was to determine whether the medial rectus muscles of patients with a history of medial rectus underaction or overaction show alterations in the process of satellite cell activation when compared with normal age-matched control muscles. METHODS. Medial rectus muscles were obtained with consent from adult patients undergoing surgical resection due to medial rectus underaction or overaction and were prepared for histologic examination by fixation and paraffin embedding. Control muscles were obtained from cornea donor eyes of adults who had no history of strabismus or neuromuscular disease. Cross sections were obtained and stained immunohistochemically for the presence of activated satellite cells, as identified by MyoD immunoreactivity, and the presence of the total satellite cell population, as identified by Pax7 immunoreactivity. The percentages of MyoD- and Pax7-positive satellite cells per 100 myofibers in cross section were calculated. RESULTS. As predicted from results in the literature, MyoD-positive satellite cells, indicative of activation, were present in both the control and resected muscles. In the underacting medial rectus muscles, the percentages of MyoD- and Pax7-positive satellite cells, based on the number of myofibers, were approximately twofold higher than the percentages in the control muscles. In the overacting medial rectus muscles, the percentage of MyoD- positive satellite cells was twofold less than in the control muscles, whereas the percentage of Pax7-positive satellite cells significantly increased compared with that in the control specimens. CONCLUSIONS. The presence of an increased number of activated satellite cells in the resected underacting medial rectus muscles and the decreased numbers of activated satellite cells in the overacting muscles was unexpected. The upregulation in the number of MyoD- positive satellite cells in underacting muscles suggests that there is potential for successful upregulation of size in these muscles, as the cellular machinery for muscle repair and regeneration, the satellite cells, is retained and active in patients with medial rectus underaction. The decreased number of activated satellite cells in overacting MR muscle suggests that factors as yet unknown in these overacting muscles are able to affect the number of satellite cells and/or their responsiveness compared with normal age-matched control muscles. These hypotheses are currently being tested.

Involvement of Eukaryotic Translation Initiation Factor 5A (eIF5A) in Skeletal Muscle Stem Cell Differentiation

The eukaryotic translation initiation factor 5A (eIF5A) contains a special amino acid residue named hypusine that is required for its activity, being produced by a post-translational modification using spermidine as substrate. Stem cells from rat skeletal muscles (satellite cells) were submitted to differentiation and an increase of eIF5A gene expression was observed. Higher content of eIF5A protein was found in satellite cells on differentiation in comparison to non-differentiated satellite cells and skeletal muscle. The treatment with NI-guanyl- 1,7-diaminoheptane (GC7), a hypusination inhibitor, reversibly abolished the differentiation process. In association with the differentiation blockage, an increase of glucose consumption and lactate production and a decrease of glucose and palmitic acid oxidation were observed. A reduction in cell proliferation and protein synthesis was also observed. L-Arginine, a spermidine precursor and partial suppressor of muscle dystrophic phenotype, partially abolished the GC7 inhibitory effect on satellite cell differentiation. These results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle regeneration.

MAMMALIAN TARGET OF RAPAMYCIN COMPLEX 1 IS INVOLVED IN DIFFERENTIATION OF REGENERATING MYOFIBERS IN VIVO

This work was undertaken to provide further insight into the role of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle regeneration, focusing on myofiber size recovery. Rats were treated or not with rapamycin, an mTORC1 inhibitor. Soleus muscles were then subjected to cryolesion and analyzed 1, 10, and 21 days later. A decrease in soleus myofiber cross-section area on post-cryolesion days 10 and 21 was accentuated by rapamycin, which was also effective in reducing protein synthesis in these freeze-injured muscles. The incidence of proliferating satellite cells during regeneration was unaltered by rapamycin, although immunolabeling for neonatal myosin heavy chain (MHC) was weaker in cryolesion+rapamycin muscles than in cryolesion-only muscles. In addition, the decline in tetanic contraction of freeze-injured muscles was accentuated by rapamycin. This study indicates that mTORC1 plays a key role in the recovery of muscle mass and the differentiation of regenerating myofibers, independently of necrosis and satellite cell proliferation mechanisms. Muscle Nerve 42: 778-787,2010

Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo

Pires-Oliveira M, Maragno AL, Parreiras-E-Silva LT, Chiavegatti T, Gomes MD, Godinho RO. Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo. J Appl Physiol 108: 266-273, 2010. First published November 19, 2009; doi:10.1152/japplphysiol.00490.2009.-Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. In the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. In addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.

Effects of Recession versus Tenotomy Surgery without Recession in Adult Rabbit Extraocular Muscle

PURPOSE. Surgical recession of an extraocular muscle (EOM) posterior to its original insertion is a common form of strabismus surgery, weakening the rotational force exerted by the muscle on the globe and improving eye alignment. The purpose of this study was to assess myosin heavy chain (MyHC) isoform expression and satellite cell activity as defined by Pax7 expression in recessed EOMs of adult rabbits compared with that in muscles tenotomized but not recessed and with that in normal control muscles. METHODS. The scleral insertion of the superior rectus muscle was detached and sutured either 7 mm posterior to its original insertion site (recession surgery) or at the same site (tenotomy). One day before euthanatization, the rabbits received bromodeoxyuridine (BrdU) injections. After 7 and 14 days, selected EOMs from both orbits were examined for changes in fast, slow, neonatal, and developmental MyHC isoform expression, Pax7 expression, and BrdU incorporation. RESULTS. Recession and tenotomy surgery resulted in similar changes in the surgical EOMs. These included a decreased proportion of fast MyHC myofibers, an increased proportion of slow MyHC myofibers, and increased BrdU-positive satellite cells. Similar changes were seen in the non-operated contralateral superior rectus muscles. The ipsilateral inferior rectus showed reciprocal changes to the surgical superior rectus muscles. CONCLUSIONS. The EOMs are extremely adaptive to changes induced by recession and tenotomy surgery, responding with modulations in fiber remodeling and myosin expression. These adaptive responses could be manipulated to improve surgical success rates. (Invest Ophthalmol Vis Sci. 2010;51:5646-5656) DOI:10.1167/iovs.10-5523

Effect of arginine on the development of the pectoralis muscle and the diameter and the protein:deoxyribonucleic acid rate of its skeletal myofibers in broilers

This work aimed at evaluating the effects of the supplementation of starter diet with Arg on breast muscle development in broilers and the activation of satellite cells and the aggregation of myofibrillar protein. Male Cobb chicks (n = 990) were randomly assigned to 1 of 5 treatments in a complete random design. Measurements of 33 chicks per treatment were made in 6 repetitions. The treatments consisted of a basal diet with 1.390% digestible Arg (without supplementation) and 4 dietary levels of Arg (1.490, 1.590, 1.690, and 1.790%) with Arg:Lys ratios of 1.103, 1.183, 1.262, 1.341, and 1.421, respectively. Arginine supplementation was used only in the starter phase (1 to 21 d). Dietary supplementation with Arg had a positive effect (P < 0.05) on breast and breast fillet weight on d 7 and 21 and on myofiber diameter on d 14 and 21. However, no effect was observed (P > 0.05) on the protein: DNA ratio, which demonstrates that Arg does not interfere with the mitotic activity of the satellite cells. Independently from mechanism, Arg affected muscle growth in the starter phase positively. Dietary supplementation with Arg in the starter phase had no effect (P > 0.05) on the carcass yield of broilers on d 42. Diet supplementation with Arg at levels above the ones recommended for the starter phase may be necessary for improved muscle development in broilers.

Chromosome polymorphism in Astyanax fasciatus (Teleostei, Characidae). 2-Chromosomal location of a satellite DNA

Studies about composition of repetitive sequences and their chromosomal location have been helpful to evolutionary studies in many distinct organisms. In order to keep on assessing the possible relationships among different cytotypes of Astyanax fasciatus (Teleostei, Characiformes) in the Mogi-Guacu River (Sao Paulo State, Brazil), C-banding, chromomycin A 3 staining, and fluorescent in situ hybridization with a repetitive DNA sequence (As51) isolated from Astyanax scabripinnis were performed in the present work. The constitutive heterochromatin was distributed in terminal regions on long arms of submetacentric, subtelocentric, and acrocentric chromosomes and in the terminal region on short arms of a pair of submetacentric chromosomes in both standard cytotypes. This latter heterochromatic site was also GC-rich, as revealed by chromomycin A(3) staining, corresponding to the nucleolar organizer region (NOR), as shown by previous studies. The sites of the satellite As51 DNA were located in terminal regions on long arms of several chromosomes. Some variant karyotypic forms, which diverge from the two standard cytotypes, also presented distinctive chromosomes carrying As51 satellite DNA. It is possible that the standard 2n = 46 cytotype represents an invader population in the Mogi-Guacu River able to interbreed with the resident standard 2n = 48 cytotype. Therefore, the variant karyotypes would be related to a possible viable offspring, where complementary chromosomal rearrangements could favor new locations of the satellite DNA analyzed. Copyright (C) 2008 S. Karger AG, Basel

Effects of a novel calcium aluminate cement on the early events of the progression of osteogenic cell cultures

The present study evaluated the progression of osteogenic cell cultures exposed to a novel calcium aluminate cement (CAC+) in comparison with the gold standard mineral trioxide aggregate (MTA). Cells were enzimatically isolated from newborn rat calvarial bone, plated on glass coverslips containing either CAC+ or a control MTA samples in the center, and grown under standard osteogenic conditions. Over the 10-day culture period, roundening of sample edges was clearly noticed only for MTA group. Although both cements supported osteogenic cell adhesion, spreading, and proliferation, CAC+-exposed cultures showed significantly higher values in terms of total cell number at days 3 and 7, and total protein content and alkaline phosphatase activity at day 10. The present in vitro results indicate that the exposure to CAC+ supports a higher differentiation of osteogenic cells compared with the ones exposed to MTA. Further experimental studies should consider CAC+ as a potential alternative to MTA when the repair of mineralized tissues is one of the desired outcomes in endodontic therapy.

HOXB5 expression in oral squamous cell carcinoma

Human HOX genes encode transcription factors that act as master regulators of embryonic development. They are important in several processes such as cellular morphogenesis and differentiation. The HOXB5 gene in particular has been reported in some types of neoplasm, but not in oral cancer. OBJECTIVE: The present study investigated the expression of HOXB5 in oral squamous cell carcinoma (SCC) and in non-tumoral adjacent tissues, focusing on verifying its possible role as a broad tumor-associated gene and its association with histopathological and clinical (TNM) characteristics. MATERIAL AND METHODS: RT-PCR was performed to amplify HOXB5 mRNA in 15 OSCCs and adjacent non-tumoral epithelium. A possible association with TNM and histopathologic data was verifed by the chi-square and post-hoc t-test. RESULTS: HOXB5 was amplifed in 60% non-tumoral epithelium and in 93.3% carcinomas. No statistically signifcant differences were found regarding the HOXB5 mRNA expression and TNM or histological grade. CONCLUSION: HOXB5 is expressed in OSCCs and its role in cancer progression should be further investigated.

Vimentin expression and the influence of Matrigel in cell lines of head and neck squamous cell carcinoma

Vimentin is a cytoeskeletal intermediate filament protein commonly observed in mesenchymal cells; however, it can also be found in malignant epithelial cells. It is demonstrated in several carcinomas, such as those of the cervix, breast and bladder, in which it is widely used as a marker of the epithelial to mesenchymal transition that takes place during embryogenesis and metastasis. Vimentin is associated with tumors that show a high degree of invasiveness, being detected in invasion front cells. Its expression seems to be influenced by the tumor microenvironment. The aim of this study was to evaluate vimentin expression in head and neck squamous cell carcinoma (HNSCC) cell lines, and to investigate the contribution of the microenvironment to its expression. HNSCC cell lines (HN6, HN30 and HN31) and an immortalized nontumorigenic cell line (HaCaT) were submitted to a three-dimensional assay with Matrigel. Cytoplasmatic staining of the HN6 cell line cultured without Matrigel and of the HN30 and HN31 cell lines cultured with Matrigel was demonstrated through immunohistochemistry. Western Blotting revealed a significant decrease in vimentin expression for the HN6 cell line and a significant increase for the HN30 and HN31 cell lines cultured with Matrigel. The results suggest that vimentin can be expressed in HNSCC cells and its presence is influenced by the microenvironment of a tumor.

Immunohistochemical expression of p53, p16 and hTERT in oral squamous cell carcinoma and potentially malignant disorders

Oral carcinogenesis is a multi-step process. One possible step is the development of potentially malignant disorders known as leukoplakia and erytroplakia. The objective of this study was to use immunohistochemistry to analyze the patterns of expression of the cell-cycle regulatory proteins p53 and p16INK4a in potentially malignant disorders (PMD) of the oral mucosa (with varying degrees of dysplasia) and in oral squamous cell carcinomas (OSCC) to correlate them with the expression of telomerase (hTERT). Fifteen PMD and 30 OSCC tissue samples were analyzed. Additionally, 5 cases of oral epithelial hyperplasia (OEH) were added to analyze clinically altered mucosa presenting as histological hyperplasia without dysplasia. p53 positivity was observed in 93.3% of PMD, in 63.3% of OSCC and in 80% of OEH. Although there was no correlation between p53 expression and the grade of dysplasia, all cases with severe dysplasia presented p53 suprabasal immunoexpression. p16INK4a expression was observed in 26.7% of PMD, in 43.3% of OSCC and in 2 cases of OEH. The p16INK4a expression in OEH, PMD and OSCC was unable to differentiate non-dysplastic from dysplastic oral epithelium. hTERT positivity was observed in all samples of OEH and PMD and in 90% of OSCC. The high hTERT immunoexpression in all three lesions indicates that telomerase is present in clinically altered oral mucosa but does not differentiate hyperplastic from dysplastic oral epithelium. In PMD of the oral mucosa, the p53 immunoexpression changes according to the degree of dysplasia by mechanisms independent of p16INK4a and hTERT.

Histologic grading and nucleolar organizer regions in oral squamous cell carcinomas

OBJECTIVE: The purposes of this study were to histologically assess different types of oral squamous cell carcinoma and the silver-binding nucleolar organizer region (AgNOR) morphology in neoplastic cells, as well as to quantify the number of AgNORs in each type of carcinoma in order to relate AgNOR count and histologic grading. MATERIAL AND METHODS: Twenty-eight cases of oral squamous cell carcinoma were divided into 4 groups, namely well-differentiated, moderately differentiated, poorly differentiated, and undifferentiated. For NOR study, 3-µm-thick sections were stained with 50% aqueous silver nitrate solution. The predominant microscopic pattern of NORs was determined. Quantitative analyses of NORs were obtained of all cells present on each histological field using a 0.025 mm² eyepiece graticule. Different histological fields were analyzed until the total number of NORs was 120 cells for each tumor. Kruskall-Wallis test was applied to compare the groups of sample data at a significance level of p=0.05. RESULTS: The mean number of AgNORs per nucleus was 3.20 for the well-differentiated group, 5.33 for the moderately differentiated one, 8.27 for the poorly differentiated one, and 10.08 for the undifferentiated one. AgNOR count was significantly different (p<0.05) among all of the studied groups. CONCLUSION: AgNOR staining technique seems to be a useful diagnostic tool since differences in AgNOR numeric values can be identified in the different types of oral squamous cell carcinoma. This technique is easy to handle and inexpensive, thus justifying its large use in histopathology.

Composition, functional properties and sensory characteristics of Mozzarella cheese manufactured from different somatic cell counts in milk

In the present study, composition, functional properties and sensory characteristics of Mozzarella cheese produced from milk with somatic cell counts (SCC) at low (<200,000 cells/mL), intermediate (≈400,000 cells/mL) and high (>800,000 cells/mL) levels were investigated. Three batches of cheese were produced for each SCC category. The cheeses were vacuum packed in plastic bags and analysed after 2, 9, 16, 23 and 30 days of storage at 4ºC. SCC level did not affect the moisture, fat, total protein and ash content, mesophilic and psychrotrophic bacteria, and sensory parameters of Mozzarella cheese. However, meltability increased in cheese manufactured from high SCC milk. Results indicated that raw milk used to produce Mozzarella cheese should not contain high SCC (>800,000 cells/mL) in order to avoid changes in the functional properties of the Mozzarella cheese.

Protocols for obtainment and isolation of two mesenchymal stem cell sources in sheep

PURPOSE: To evaluate different protocols to isolate stem cells from ovine umbilical cord blood and adipose tissue. METHODS: There were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. There were used 3 protocols for obtainment and culture of umbilical cord blood stem cells and 4 protocols for ovine adipose tissue stem cells. RESULTS: It was possible to observe only one successful protocol for the obtainment of umbilical cord blood stem cells. When analyzing the techniques used to obtain adipose tissue stem cells, only one of the methods was effective as well. Through colony forming unit assay, there were obtained 58 colonies of cells after seven days in culture. Flow citometry tests revealed the cells were positive to CD44 and exhibited negative reaction to CD38, CD45, CD41/61. These cells showed a growth curve with very well defined phases LOG, LAG and PLATEAU. This phases are typically seem in mesenchymal stem cells growth curves. CONCLUSIONS: The isolation and culture of mesenchymal stem cells from ovine umbilical cord blood are complex and request more detailed assays. Stem cells from fat tissue sheep showed mesenchymal characteristics, according to their cell growth curve, ability to origin colonies of fibroblastoid cells and positive reactivity with the antibody CD44 by flow citometry.

Correlation of meta 1 expression with culture stage, cell morphology and infectivity in Leishmania (Leishmania) amazonensis promastigotes

The parasitic protozoan Leishmania (Leishmania) amazonensis alternates between mammalian and insect hosts. In the insect host, the parasites proliferate as procyclic promastigotes andthen differentiate into metacyclic infective forms. The meta 1 gene is preferentially expressed during metacyclogenesis. Meta 1 expression profile determination along parasite growth curves revealed that the meta 1 mRNA level peaked at the early stationary phase then decreased to an intermediate level. No correlation was observed between meta 1 expression and infectivity. Conversely, infectivity correlated with the increase of apoptotic cells in the late stationary phase.