Increased Viability of Odontoblast-Like Cells Subjected to Low-Level Laser Irradiation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE) x S(SM)(EK) x S(SM)(SSRs)). Clustering analyses showed a mean of 9 +/- 12.4 isolates per cluster (3.8 +/- 8 isolates/taxon) for MLEE, 6.2 +/- 4.9 isolates per cluster (4 +/- 4.5 isolates/taxon) for SSRs, and 4.1 +/- 2.3 isolates per cluster (2.6 +/- 2.3 isolates/taxon) for EK. A total of 45 (13%), 39(11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (Si) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented with similarity and grouping analysis. Therefore, the use of genotyping systems that give results which offer minimum disparity, or the combination of the results of these systems, can provide greater security and consistency in the determination of strains and their genetic relationships. (C) 2010 Elsevier B.V. All rights reserved.

Ischaemia and reperfusion effects on skeletal muscle tissue: morphological and histochemical studies

This was a study on the oxidative stress due to ischaemia (I) and reperfusion (R) in skeletal muscle tissue. Using a tourniquet, groups of rats were submitted to ischaemia for 4 h, followed by different reperfusion periods. The animals were divided in four groups: control; 4 h of ischaemia (IR); 4 h of ischaemia plus 1 h reperfusion (IR-1 h); 4 h of ischaemia plus 24 h reperfusion (IR-24 h); and 4 h of ischaemia plus 72 h reperfusion (IR-72 h). At the end of the procedures, samples of soleus muscle were collected and frozen in n-hexane at -70 degrees C. Cryostat sections were submitted to haematoxylin-eosin, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) stains. An additional muscle sample was processed for electron microscopy. No alterations were found in control animals. IR group showed fibres had normal aspect besides some round, acidophilic and hypertrophic fibres. There were several fibres with angular outlines and smaller diameters in this group compared with control group. NADH-TR/SDH reaction was moderately intense in most fibres. In some fibres, cytoplasm showed areas without activity and other fibres had very intense reactivity. IR-1 h group showed oedema hypercontracted fibres with disorganized myofibrils, mitochondria with focal lesions and dilated sarcoplasmic reticulum. NADH-TR/SDH reaction was moderate to weak. IR-24 h showed intense inflammatory infiltrate in the endomysium and perimysium. NADH-TR/SDH reaction was similar to IR-1 h. IR-72 h showed necrotic fibres, areas with inflammatory infiltrate, reduced muscle fibres at different stages of necrosis and phagocytosis, and many small round and basophilic fibres characterizing a regeneration process. NADH-TR/SDH reaction was weak to negative. Our results suggest that ischaemia and the subsequent 1-, 24- and 72-h reperfusions induced progressive histological damage. Although progressive, it may be reversible because there were ultrastructural signs of recovery after 72-h reperfusion. This recovery could in part be due to the low oxidative stress identified by the morphological and histochemical analysis.

PURIFICATION AND PROPERTIES OF SHIKIMATE DEHYDROGENASE FROM CUCUMBER (CUCUMIS-SATIVUS L)

Shikimate dehydrogenase (SDH, EC 1.1.1.25) extracted from cucumber pulp (Cucumis sativus L.) was purified 7-fold by precipitation with ammonium sulfate and elution from columns of Sephadex G-25, DEAE-cellulose, and hydroxyapatite. Two activity bands were detected on polyacrylamide gel electrophoresis at the last purification step. pH optimum was 8.7, and molecular weight of 45 000 was estimated on a Sephadex G-100 column. SDH was inhibited competitively by protocatechuic acid with a K(i) value of 2 x 10-4 M. K(m) values of 6 x 10-5 and 1 x 10-5 M were determined for shikimic acid and NADP+, respectively. The enzyme was completely inhibited by HgCl2 and p-(chloromercuri)benzoate (PCMB). NaCl and KCl showed partial protection against inhibition by PCMB. Heat inactivation between 50 and 55-degrees-C was biphasic, and the enzyme was completely inactivated after 10 min at 60-degrees-C. Incubation of SDH with either NADP+ or shikimic acid protected the enzyme against heat inactivation.

Crescimento pós-natal do coelho Norfolk: correlação entre parâmetros somáticos e área dos tipos de fibras musculares

Skeletal muscle fibers areas, the weight of body, carcass and muscle, the gastric content and the length of intestines and caecum of Norfolk rabbit from neonate to 26th week were measured. Types of fibers and areas were studied in -70-degrees-C frozen blocks of flexor carpo radialis muscle. Some sections were stained with Haematoxylin and Eosin and others were submitted to SDH, NADH-TR and m-ATPase, followed by acid and alkali preincubations. All parameters revealed a high degree of correlation. Carcass weight/body weight ratio reached 55,1% at 10th week. From neonate to 26th week, the areas of SO, FOG and FG muscle fibers increased of x9.1, x12.4 and x22.2. While SO and FOG fibers tend to stabilize their growth at 10th week, FG fibers were active at this age. Due to early stabilization of SO and FOG fibers growth, the additional increase in muscle tissue in attributed mainly to hypertrophy of FG fibers.

FIBER TYPES DISTRIBUTION IN THE DIGASTRIC MUSCLE OF TUFTED CAPUCHIN MONKEY (CEBUS-APELLA)

Fiber types distribution in the diagastric muscle of tufted capuchin monkey was studied by means of NADH-TR, myosin-ATPase, after alkaline and acid preincubations and SDH histochemical reactions. Three different types of fibers were found presenting an equal distribution. The percentage and types of fibers were as follow: 18.2 % SO (Slow Oxydative), 38.4 % FOG (Fast Oxydative Glycolytic) and 43.4 % FG (Fast Glycolytic). FG fibers revealed the largest area. The relatively high concentration of fast twitch (81.2 %) seems to indicate this muscle is involved with the acceleration and fast speed of jaw movements. Aerobic metabolism represented by SO + FOG fibers (56.6 %) suggests that this muscle possesses an additional role than that related to the lowering of the jaw.

Características histoquímicas e estruturais do testículo do morcego hematófago (Desmodus rotundus rotundus, Geoffrey, 1810).

The testicular stroma of the vampire bat including the testicular capsula and the lamina propria of the seminiferous tubuli, was strongly PAS-positive. This observation was a possible indication of great amounts of structural glycogen and other glycoconjugates at the level of smooth muscle cells; elongated contractile cells and/or collagen frameworks of the tunica albuginea and tubular lamina propria. In the last the basement membranes of the seminiferous tubules were particularly strongly PAS positive, as an indication of their neutral mucosubstances structural composition, previously described (Malmi et al., 1987). The epithelium lining from the cavitary and surface rete testis complex showed low reactivities to mucosubstances; total proteins and lipids and oxidative enzymes studied. Although the apical granulation at the rete testis epithelium showed an intense PAS reactivity with hypothesis of glycoprotein secretion, through the rete. The PAS, Sudan Black B, NADH, MDH and LDH reactions of the testicular interstitium seem correlate to steroid metabolism (biosynthesis and secretion), at the Leydig cells level. The seminiferous epithelium generally had low reactions to all the histochemical studies realized. Particularly in the adbasal compartment the histochemical localizations of NADH diaphorase and LDH were possible related to glycolytic activities and general carbohydrates metabolism, both enzymes, and hydrogen transport, the NADH. The strong PAS, diastase and PAS, and alcian blue pH 2.5 and PAS reactions observed in the adluminal seminiferous epithelium compartment were directly related to the spermatids acrosomal glycoconjugates structuration. Also the SDH localization at this level seems to be related to the mitochondrial activities at the middle piece level in the late spermatids.

Histoenzymology and morphometry of the masticatory muscles of tufted capuchin monkey (Cebus apella Linnaeus, 1758)

Samples of the anterior and posterior regions of the masseter and temporal muscles and of the anterior belly of the digastric muscle of 4 adult male tufted capuchin monkeys (Cebus apella) were removed and stained with HE and submitted to the m-ATPase reaction (with alkaline and acid preincubation) and to the NADH-TR and SDH reactions. The results of the histoenzymologic reactions were similar, except for acid reversal which did not occur in fibers of the fast glycolytic (FG) type in the mandibular locomotor muscles. FG fibers had a larger area and were more frequent in all regions studied. No significant differences in frequency or area of each fiber type were detected, considering the anterior and posterior regions of the masseter and temporal muscles. The frequency of fibers of the fast oxidative glycolytic (FOG) and slow oxidative (SO) types and of FOG area differed significantly between the anterior belly of the digastric muscle and the mandibular locomotor muscle. The predominance of fast twitch (FG and FOG) fibers and the multipenniform and bipenniform internal architecture of the masseter and temporal muscles, respectively, are characteristics that permit the powerful bite typical of tufted capuchin monkeys.

Localização histoquímica de algumas hidrolases no epidídimo do gerbilo

This study mainly showed that alkaline phosphatase expression had been present in the proximal regions of the epididymidis ductus of the gerbil which comprised the initial segment and proximal caput. The reactivities of acid phosphatase and ATPase were strong in the proximal and distal regions of the epididymidis ductus at the level of the apical cytoplasm and epithelium, except at the corpus level, a very thin isthmus located between the caput and cauda epididymidis, and as a general rule a low enzymatic reactive region of the epididymis of gerbil. SDH revealed also low activities in all the regions and regional structures of the duct, except into the luminal content formed by storaged spermatozoa, prior on the cauda level. The enzymes presented in the epididymis were correlated to some histophysiological roles such as the enzymatic mediation of endocytosis, secretion, absorption and active transport concerning to phosphatases and ATPase and a possible mitochondrial role of SDH could occur at the spermatozoa level in which the middle pieces were formed by a great amount of mitochondria.

Morphological and histochemical analysis of the human vestibular fold

A morphological and histochemical study of the human vestibular fold was carried out using routine histological techniques. Seven μm-thick histological sections stained with hematoxylin-eosin (HE) and Calleja showed the presence of elastic collagen fibers and seromucous glands in the vestibular fold. Muscle fibers forming the ventricular muscle were also identified. Ultrastructural analyses of the epithelial layer by scanning electron microscopy (SEM) revealed ciliated cells and gland ducts opening on the epithelial surface. Histochemical analyses were performed on ventricular muscles submitted to nicotinamide-adenine-dinucleotide tetrazolium reductase (NADH-TR), succinate dehydrogenase (SDH), and myofibrillar adenosine triphosphatase (mATPase) reactions. Based on these reactions, it was observed that the muscle is formed by three types of muscle fibers: slow-twitch oxidative (SO), fast-twitch oxydative glycolytic (FOG) and fast-twitch glycolytic (FG) fibers distributed in a mosaic pattern. The fiber frequency was 22.7%, 69.9% and 7.4%, respectively. The higher frequency of SO and FOG fibers characterized the muscle as having aerobic metabolism and resistance to fatigue. The ventricular muscle was considered fast. The study of the neuromuscular junctions performed after nonspecific esterase reaction showed that they are of the en-plaque type and have multiple occurrences in the ventricular muscle.

Effects of zoledronic acid on odontoblast-like cells

The aim of the study was to evaluate the effects of a highly potent bisphosphonate, zoledronic acid (ZOL), on cultured odontoblast-like cells MDPC-23. The cells (1.5 × 104 cells/cm2) were seeded for 48 h in wells of 24-well dished. Then, the plain culture medium (DMEM) was replaced by fresh medium without fetal bovine serum. After 24 h, ZOL (1 or 5 μM) was added to the medium and maintained in contact with the cells for 24 h. After this period, the succinic dehydrogenase (SDH) enzyme production (cell viability-MTT assay), total protein (TP) production, alkaline phosphatase (ALP) activity, and gene expression (qPCR) of collagen type I (Col-I) and ALP were evaluated. Cell morphology was assessed by SEM. Five μM ZOL caused a significant decrease in SDH production. Both ZOL concentrations caused a dose-dependent significant decrease in TP production and ALP activity. ZOL also produced discret morphological alterations in the MDPC-23 cells. Regarding gene expression, 1 μM ZOL caused a significant increase in Col-I expression. Although 5 μM ZOL did not affect Col-I expression, it caused a significant alteration in ALP expression (ANOVA and Tukey's test, p < 0.05). ZOL presented a dose-dependent cytotoxic effect on the odontoblast-like cells, suggesting that under clinical conditions the release of this drug from dentin could cause damage to the pulpo-dentin complex. © 2012 Elsevier Ltd.

In vitro transdentinal effect of low-level laser therapy

Low-level laser therapy (LLLT) has been used for the treatment of dentinal hypersensitivity. However, the specific LLL dose and the response mechanisms of these cells to transdentinal irradiation have not yet been demonstrated. Therefore, this study evaluated the transdentinal effects of different LLL doses on stressed odontoblast-like pulp cells MDPC-23 seeded onto the pulpal side of dentin discs obtained from human third molars. The discs were placed in devices simulating in vitro pulp chambers and the whole set was placed in 24-well plates containing plain culture medium (DMEM). After 24 h incubation, the culture medium was replaced by fresh DMEM supplemented with either 5% (simulating a nutritional stress condition) or 10% fetal bovine serum (FBS). The cells were irradiated with doses of 15 and 25 J cm-2 every 24 h, totaling three applications over three consecutive days. The cells in the control groups were removed from the incubator for the same times as used in their respective experimental groups for irradiation, though without activating the laser source (sham irradiation). After 72 h of the last active or sham irradiation, the cells were evaluated with respect to succinic dehydrogenase (SDH) enzyme production (MTT assay), total protein (TP) expression, alkaline phosphatase (ALP) synthesis, reverse transcriptase polymerase chain reaction (RT-PCR) for collagen type 1 (Col-I) and ALP, and morphology (SEM). For both tests, significantly higher values were obtained for the 25 J cm-2 dose. Regarding SDH production, supplementation of the culture medium with 5% FBS provided better results. For TP and ALP expression, the 25 J cm-2 presented higher values, especially for the 5% FBS concentration (Mann-Whitney p < 0.05). Under the tested conditions, near infrared laser irradiation at 25 J cm -2 caused transdentinal biostimulation of odontoblast-like MDPC-23 cells. © 2013 Astro Ltd.

Transdentinal cytotoxicity of experimental adhesive systems of different hydrophilicity applied to ethanol-saturated dentin

The aim of this study was to evaluate the transdentinal cytotoxicity of experimental adhesive systems (EASs) with different hydrophilicity and dentin saturation solutions on odontoblast-like cells. One hundred 0.4-mm-thick dentin discs were mounted in in vitro pulp chambers and assigned to 10 groups. MDPC-23 cells were seeded onto the pulpal side of the discs, incubated for 48 h. The EASs with increasing hydrophilicity (R1, R2, R3 and R4) were applied to the occlusal side after etching and saturation of etched dentin with water or ethanol. R0 (no adhesive) served as controls. R1 is a non-solvated hydrophobic blend, R2 is similar to a simplified etch-and-rinse adhesive system and R3 and R4 are similar to self-etching adhesives. After 24 h, cell metabolism was evaluated by MTT assay (n = 8 discs) and cell morphology was examined by SEM (n = 2 discs). Type of cell death was identified by flow cytometry and the degree of monomer conversion (%DC) was determined by infrared spectroscopy (FTIR) after 10 s or 20 s of photoactivation. Data were analyzed by the Kruskal-Wallis and Mann-Whitney tests (α = 0.05). Dentin saturation with ethanol resulted in higher necrotic cell death ratios for R2, R3 and R4 compared with water saturation, although R2 and R3 induced higher SDH production. Photoactivation for 20 s significantly improved the %DC of all EASs compared with 10 s. A significant positive correlation was observed between the degree of hydrophilicity and %DC. In conclusion, except for R1, dentin saturation with ethanol increased the cytotoxicity of EASs, as expressed by the induction of necrotic cell death. © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Efeito transdentinário do LED em diferentes comprimentos de onda sobre células odontoblastóides

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação de sistemas de manejo em Nitossolo Vermelho distroférrico e na cultura do girassol

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)