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Canine Leishmaniasis is widespread in various Mexican states, where different species of Leishmania have been isolated from dogs. In the present study, we describe the detection of L. braziliensis, L. infantum, and L. mexicana in serum of dogs from the states of Yucatan and Quintana Roo in the Yucatan Peninsula (Mexico). A total of 412 sera were analyzed by ELISA using the total extract of the parasite and the iron superoxide dismutase excreted by different trypanosomatids as antigens. We found the prevalence of L. braziliensis to be 7.52%, L. infantum to be 6.07%, and L. mexicana to be 20.63%, in the dog population studied. The results obtained with ELISA using iron superoxide dismutase as the antigen were confirmed by western blot analysis with its greater sensitivity, and the agreement between the two techniques was very high.

Prosthetic rehabilitation of the edentulous posterior maxilla with implant-supported prostheses frequently presents a challenge for the oral surgeon because of the lack of bone due to alveolar ridge resorption or maxillary sinus pneumatization. To overcome these problems, different solutions were proposed over the years. Maxillary sinus membrane elevation is a common surgical technique for increasing bone height in the posterior maxilla prior to dental implant placement. However, the biological nature of bone regeneration in maxillary sinus membrane remains largely unidentified. The authors present a clinical case and literature review to understand the fundamental of bone formation in osteotomy sinus floor elevation., (Copyright 2019 Biolife Sas. www.biolifesas.org.)

In recent years various studies about the biostimulatory effects of the laser therapy in orthodontics have been carried out. This study investigates the potential advantages obtainable using the Low-level Laser Therapy during orthodontic treatment and the most efficient clinical protocols. Recently published randomized controlled trials (RCTs) have been obtained through a search on electronic databases (Cochrane Library and Pubmed). Clinical studies in humans in which Low-level Laser Therapy was applied during orthodontic treatment were included. In conclusion, 14 relevant clinical studies were identified. This study shows the possibility to obtain an increase in tooth movement between 31% and 100% depending on the laser therapy considered and the time interval for measuring the value. In addition, there is a potential impact in reducing orthodontic pain limited to the day following the application of laser therapy when orthodontic therapy includes canine retraction, and during a period not exceeding five days from the placement of fixed orthodontic appliances in the others clinical cases.Low-level Laser Therapy is considered effective both to increase the movement of the dental elements and to reduce pain during orthodontic therapy. Different clinical protocols have been identified depending on the orthodontic cases considered. Both an LED device and an AlGaAs diode device can be used. In the future paying more attention to the therapeutic possibilities offered by laser devices with greater power is recommended. A greater energy density directed to the target tissues has been proven to provoke more significant therapeutic effects., (Copyright 2019 Biolife Sas. www.biolifesas.org.)

The aim of our study was to evaluate the properties of a laser-modified titanium surface, specifically the promotion of a faster differentiation of human Mesenchymal Stem Cells (hMSCs) into osteoblasts and a more stable connection between differentiated cells and titanium, compared to machined and sand-blasted surfaces. Furthermore, we wanted to assess if the titanium alone could be a sufficient factor in the induction of the differentiation towards the osteogenic lineage., Materials and Methods: we harvested stem cells from an individual (under his consensus) and cultivated them into dishes containing titanium disks presenting three different surfaces: machined (M), sand-blasted (S) and laser-modified (L). In the test group, cells were cultivated in an osteogenic medium, while in the control group, cells were seeded in a standard DMEM. Evaluations of the degree of differentiation were made with Alizarin coloration after 28, 38, 42, 49, 56 and 63 days from induction., Results: no signs of differentiation were evident in the control group, while in the test group there was a statistically significant differentiation, evident since the fourth week. Laser-modified and sand-blasted surfaces showed similar values, higher than the machined surface., Discussion: on the laser-modified surface the differentiation reached its peak on the sixth week, while on the seventh week for the other two surfaces. After the peak, the differentiation showed a slow decrease for the laser-modified surface and a rapid decrease for the other two., Conclusions: titanium alone can't be considered enough to induce differentiation of human Mesenchymal Stem Cells into osteoblasts. Still, the laser-modified once induced a faster differentiation of stem cells and a more stable connection between osteoblasts and titanium., (Copyright 2019 Biolife Sas. www.biolifesas.org.)

Hereditary Hemorrhagic Telangiectasia or Rendu-Osler-Weber syndrome is an incomplete penetrance dominant autosomal transmission disease which determines microcirculatory beds alterations (capillary and venules), caused by the loss of the support tissues that usually enclose blood vessels, and hemorrhage potentially in every organ. The syndrome clinical manifestations are multiple telangiectasia of small proportions on the skin or on the mucous membranes (e.g. of the gastrointestinal tract or other organs), in association with recurring bleedings of the affected areas and external and internal melena. The treatment is a supportive one so to prevent complications. This study reports a case of a patient affected by this syndrome in need of a dental implant following the fracture of a tooth. Furthermore, a bibliographical review of etiopathogenesis, clinical manifestations and therapy options has been made., (Copyright 2019 Biolife Sas. www.biolifesas.org.)

Several factors compete for both the achievement and the long-term maintenance of osseointegration; among these, of importance is the width and integrity of the peri-implant soft tissue. Many authors already underlined the importance for implant-prosthesis procedures to maintain a good biological seal together with a low bacterial cell surface charge (this is also valid for a natural tooth with an undamaged periodontium). The aim of this work is to present, through a clinical case, a new technique that focuses on the regeneration of soft tissue around a post-extractive implant. For the case reported, a post-extractive implant surgery of an inferior molar of the fourth quadrant with a buccal bone resorption of 3mm in the mesial section of the root, three dimensional collagen matrices (Bioteck) and a blend of equine spongy bone granules (OX Bioteck) were used, combined with aPDT without dye (Rey Protocol). With an easy and not invasive surgery, this technique allows the recreation of new gingiva around the implant., (Copyright 2019 Biolife Sas. www.biolifesas.org.)

Since 1991 the measurement of 3 biochemical parameters for the risk assessment of trisomy 21 pregnancy (AFPplus, Alpha-check) is also offered by private laboratories in Switzerland. This work aimed at the proof of reliability of this risk analysis in the hands of practitioners and private laboratories. The investigated cohort included 2,641 pregnant women. An anonymous questionnaire on the outcome of pregnancy furnished informations in 97% of cases (2,561 cases). At a trisomy 21 risk cutoff of one to 380, 9% of the pregnant women had conspicuous AFPplus results (trisomy 21-risk > 1 to 380; age distribution at term: 17 to 45 years; median 29.4 years). Among the conspicuous cases were 8 with trisomy 21, 1 with trisomy 18, 5 spontaneous abortions and 3 special cases. Seven of a total of 9 trisomies were detected by the screening. From these data the following diagnostic parameters for AFPplus have been calculated: specificity 91.0%, sensitivity 77.8%, positive predictive value 99.9%, false positive rate 9.0%, false negative rate 22.2%. The use of AFPplus by a practitioner cooperating with a private laboratory thus reveals a detection rate comparable to that known from larger centers. AFPplus by a practitioner cooperating with a private laboratory thus reveals a detection rate comparable to that known from larger centers. AFPplus allows to individualize the statistical age-related risk of a pregnant women before age 35. Above age 35 AFPplus may support the indication for cases suitable for cytogenetic investigation. Thorough personal information of the patient before the 16th pregnancy week by the practitioner or by genetic counseling has without any doubt first priority.

A water soluble protein, a major component of the cytosolic fraction of rat heart cells, was purified using either reverse phase HPLC or antibodies affinity chromatography procedures and characterized. The protein has an apparent Mr of 24 k, as judged by SDS-gel electrophoresis. Under non-denaturing conditions, however, the protein occurs as a homomultimer (Mr between 400 and 650 k) of the monomeric 24 kDa species and could be selectively enriched by fractionation of the cytosolic fraction on 10 to 40% sucrose gradients. Polyclonal antibodies, raised against the denatured 24 kDa protein, were used to investigate its tissue distribution. Besides the heart, where it is very abundant, the 24 kDa protein is expressed also in other red muscles and in kidneys, but was not detectable in stomach, thymus, liver, and brain. The amino acid composition of the protein and the partial amino acid sequence of various proteolytic fragments was determined. A search for homologies of the primary structure of known proteins has shown that the 24 kDa protein is strikingly similar, if not identical to alpha-B-crystallin. In fact, the two proteins were found to be indistinguishable also by immunological criteria. This study demonstrates that the lens protein alpha B-crystallin is a major cytosolic component of heart cells.

The Na(+)-Ca2+ exchanger of the cardiac sarcolemma can rapidly transport Ca2+ during excitation-contraction coupling. To begin molecular studies of this transporter, polyclonal antibodies were used to identify a complementary DNA (cDNA) clone encoding the Na(+)-Ca2+ exchanger protein. The cDNA hybridizes with a 7-kilobase RNA on a Northern blot and has an open reading frame of 970 amino acids. Hydropathy analysis suggests that the protein has multiple transmembrane helices, and a small region of the sequence is similar to that of the Na(+)- and K(+)-dependent adenosine triphosphatase. Polyclonal antibodies to a synthetic peptide from the deduced amino acid sequence react with sarcolemmal proteins of 70, 120, and 160 kilodaltons on immunoblots. RNA, synthesized from the cDNA clone, induces expression of Na(+)-Ca2+ exchange activity when injected into Xenopus oocytes.

Rat hearts were perfused in the working heart or Langendorff mode and then subjected to total normothermic ischemia. The content of alpha-crystallin in the water soluble protein fraction obtained from these hearts diminished in a time-dependent manner during ischemia. The protein was recovered in the low g pellet of the homogenate. The redistribution was dramatic, selective for alpha-crystallin and irreversible. Large crystallin clumps formed also when exposing the soluble protein fraction of control hearts to slightly acidic pH (6.5-7.0). Electron microscopic analysis showed that aggregation of the globular homo-oligomeric units of crystallin occurred. The aggregates probably represented denatured protein and were similar in appearance to lenticular alpha H-crystallin. In purified form, however, cardiac crystallin particles did not cluster at pH 6.5. Aggregation only occurred in the presence of other protein components (including, probably, cytosolic actin) of the soluble fraction. A direct and selective interaction between actin and cardiac crystallin could be demonstrated using actin-Sepharose affinity chromatography procedures. The results suggest that large aggregates of cardiac crystallin form very early during ischemia, due to acidification of the cytosol. Cardiac crystallin is highly homologous to stress proteins and is localized on the Z-disks, where it plays probably a structural or protective role. Its rapid and complete denaturation could be involved in the genesis of the irreversible structural damages occurring during ischemia.

A major component of the soluble fraction of rat heart is a homopolymer (Mr about 400-650 k) of a small protein (Mr about 20 k). This cardiac protein, which is highly homologous to alpha-B-crystallin, was isolated in its native state and visualized by electron microscopy. A homogeneous population of globular particles with an average diameter of about 14-16 nM could be seen using either negative staining or rotary shadowing procedures. The structures were globular in nature with a central depression (torus-like structures). Polyclonal antibodies, raised against the cardiac crystallin, were used for the immunocytochemical localization of the macromolecular complexes. Using fluorescent secondary antibodies, a clear and sharp striation of fixed and permeabilized rat heart myocytes could be observed, similar to that observed with anti-desmin antibodies and characteristic for the central region of the I-band. Cardiac crystallin was not found associated with F-actin in preparations of rat heart myofibrils. On the other hand, it was a major contaminant of cardiac desmin preparations. These observations suggest that cardiac crystallin is involved in the organization of cytoskeletal filaments of the Z-lines.

The Na+/Ca2+ exchanger of calf heart sarcolemma has been identified in solubilized membrane preparations with the help of specific antibodies as a molecule of approximate Mr of 30 KDa. The conclusion supports the previous proposal by Soldati et al. (J. Biol. Chem. 260, 13321-13327, 1985) that the exchanger is a molecule of Mr about 33 KDa. Antibodies (IgG) were raised in rabbits by injecting proteins electroeluted from different regions of preparative SDS gels of solubilized heart sarcolemma. After purification the IgG against the proteins of the 30 KDa region recognized the 33 KDa component but also proteins of Mr about 70 and 140 KDa. Conversely, antibodies against the 140 KDa protein(s) also recognized the 70 and the 33 KDa proteins. However, if the solubilized sarcolemma extract was treated with DTT prior to the transfer to nitrocellulose the 140 KDa protein was not seen. Both the antibodies against the 30 KDa and those against the 140 KDa proteins inhibited the Na+/Ca2+ exchange activity of sarcolemma vesicles. It is proposed that the basic unit of the Na+/Ca2+ exchanger of heart sarcolemma is a monomer of Mr about 33 KDa, the functionally active exchanger being a tetramer in which the four 33 KDa subunits are held together by disulfide bonds. In the monomer-tetramer transition an intermediate dimeric state of Mr 70 KDa is also formed.

Eukaryotic plasma membranes contain three Ca-transporting systems: a Ca channel, an ATPase, and an Na/Ca exchanger. The ATPase is high-affinity, low-capacity system, which continuously pumps Ca out of cells. The Na/Ca exchanger is a low-affinity, high-capacity system, which is particularly active in excitable cells. The exchanger probably functions in both the Ca efflux and influx directions. The Ca-ATPase is a single polypeptide of Mr 138 kD, which is activated by calmodulin or, in its absence, by acidic phospholipids, polyunsaturated fatty acids, and limited proteolytic treatments. Trypsin produces a number of fragments, some of which (Mr 90, 85, and 81 kD) function as ATPases and transport Ca across reconstituted bilayer membranes. Trypsin proteolysis in the presence of different effectors has permitted us to locate the calmodulin-interacting domain of the enzyme in a 9-kD peripheral sequence that consists of a 4-kD calmodulin-binding subdomain and a subdomain of Mr 5 kD, which is essential for the expression of calmodulin stimulation. The Na/Ca exchanger of plasma membranes has not yet been identified with certainty. On the basis of purification attempts using different approaches, probable Mr's of 82, 70, or 33 kD have been proposed. Antibodies raised against the 33-kD protein partially inhibit the exchange activity of heart sarcolemma vesicles. They interact with the 33-kD protein, but also, under nonreducing conditions, with proteins of Mr approximately 70 and approximately 140 kD. Under reducing conditions, the reactivity with the latter component disappears. It is suggested that the monomeric Mr of the exchanger is 33 kD, and that intermolecular disulfide bridges associate monomers into dimeric and tetrameric forms.

We have used fractionation procedures to enrich solubilized cardiac sarcolemma in the Na+-Ca2+ exchange protein. Sarcolemma is extracted with an alkaline medium to remove peripheral proteins and is then solubilized with decylmaltoside. Next, the exchanger is applied to DEAE-Sepharose and eluted with high salt. The DEAE fraction is applied to WGA-agarose, and a small fraction of protein, enriched in the exchanger, can be eluted by changing the detergent to Triton X-100. This fraction is reconstituted into asolectin proteoliposomes for measurement of Na+-Ca2+ exchange activity and gel electrophoresis. The purified fraction has a Na+-Ca2+ exchange activity of 600 nmol Ca2+/mg of protein per s at 10 microM Ca2+ and a purification factor of about 30 as compared with control reconstituted sarcolemmal vesicles. Ca2+-Ca2+ exchange and Na+-Ca2+ exchange activities were both present in the same final reconstituted vesicles indicating that the same protein is responsible for both transport activities. SDS-PAGE reveals two prominent protein bands at 70 and 120 kDa. After mild chymotrypsin treatment (1 microgram/ml), there is no loss of exchange activity, but the 120 kDa band disappears and the 70 kDa band becomes more dense. This suggests that the 70 kDa band is due to an active proteolytic fragment of the 120 kDa protein. Under non-reducing gel conditions, only a single protein band is seen with an apparent molecular weight of 160 kDa. Antibodies to the purified exchanger preparation are able to immunoprecipitate exchange activity and confirm that the 70 kDa protein derives from the 120 kDa protein. We propose that both the 70 and 120 kDa proteins are associated with the Na+-Ca2+ exchanger.

Effects of peripheral benzodiazepine receptor modulating drugs, Ro 5-4864 and PK 11195, on tension induced by K+ and the calcium agonist SDZ 202 791 (S isomer), were studied in rat caudal arteries. A significant reduction of tonic phase tension occurred with 30 nM PK 11195 or 3 microM Ro 5-4864, but decreases of the initial (first 3 min), phasic contraction were detected only at the highest concentrations of Ro 5-4864 and PK 11195. Protoporphyrin IX, the putative endogenous ligand of the peripheral benzodiazepine receptor, (at 10-100 nM) markedly increased the effectiveness of Ro 5-4864 and PK 11195 in reducing phasic contraction. Intracellular calcium localization and distribution in fura-2 loaded single vascular cells were quantitated using a high sensitivity, two-stage microchannel plate, photon-counting (PMI-VIM) camera. Peripheral benzodiazepines reduced intracellular calcium release from centrally located calcium pools, and this decrease of calcium release was potentiated by protoporphyrin IX. The decrease in intracellular calcium activity, which was more pronounced in the central regions where sarcoplasmic reticular elements are numerous, was probably the major mechanism of these vasodilator properties. Measurements of soluble guanylate cyclase activity also supported the intracellular Ca2+ release mechanism. Under conditions where protoporphyrin IX did not significantly stimulate guanylate cyclase, Ro 5-4864 alone or more effectively in combination with protoporphyrin IX stimulated cGMP production and caused relaxation. Guanylate cyclase forms a possible target for these benzodiazepine modulators, a hypothesis that merits further investigation.

The cardiac sarcolemma Na+/Ca2+ exchanger has been solubilized in Triton X-100 and reconstituted into asolectin liposomes by removing the detergent with Amberlite XAD-2 beads. The specific Na+/Ca2+ exchange activity of the proteoliposomes was increased about 10 times with respect to the original sarcolemma. The exchange activity was strongly dependent on the amount of acidic phospholipids present in the liposomes. Phosphatidylserine and phosphatidylinositol increased the initial rate of the reconstituted exchanger. Fractionation of the solubilized sarcolemma by rate zonal centrifugation produced fractions in which the specific activity of the exchanger was enriched up to 130-fold with respect to the original membranes. The fractionation scheme used has shown that the exchange activity correlated best with a 33-kDa protein present in the fractionated extract. No correlation has been found between exchange activity and other proteins of higher molecular mass (70 or 82 kDa) which have previously been suggested to correspond to the Na+/Ca2+ exchanger. Thus, the 33-kDa protein may represent the Na+/Ca2+ exchanger of cardiac sarcolemma.

Injection of Xenopus laevis oocytes with rabbit heart poly(A)+RNA results in expression of Na+ inside (Nai+)-dependent Ca2+ uptake activity. The activity was measured by first loading the oocytes with Na+ using nystatin and then incubating the oocytes in K+ or Na+ medium containing 45Ca. The expressed Na+ gradient-dependent Ca2+ uptake was five to eight times that observed with water-injected oocytes or with poly(A)+RNA-injected oocytes for which the Na+ load step had been omitted. Induced activity was related to the amount of RNA injected and was insensitive to nifedipine. Fractionation of the poly(A)+RNA on a sucrose gradient determined that the active message had a size range between 3 and 5 kb. The properties of the Na+ gradient-dependent Ca2+ uptake indicated that Na+-Ca2+ exchange activity had been expressed in X. laevis oocytes. The result may be useful for cloning and identifying the molecular component responsible for Na+-Ca2+ exchange.

Inhibition of the cardiac Na+-K+-ATPase with cardiac glycosides causes a rise in internal Na+ and a subsequent increase in cellular Ca2+ due to the sarcolemmal Na+-Ca2+ exchange mechanism. We investigated the adaptation of cultured cardiac cells to prolonged elevation of internal Ca2+ after exposure to ouabain. Cultured neonatal rat heart cells were treated with 100 microM ouabain for 4-48 h. This ouabain concentration inhibited Na+-K+-ATPase activity by approximately 45% and caused modest cellular Ca2+ loading. We found that cells adapted to ouabain treatment by increasing the amount of sarcolemmal Na+-Ca2+ exchange activity by 50-90% over a 24-h period. Kinetic and immunological data indicate that the increase was due to increased numbers of functional exchangers. Neither total cellular nor total sarcolemmal protein content was affected by the ouabain treatment. These results may be relevant toward understanding the effects of therapeutic use of cardiac glycosides.