Liquid-liquid extraction of proteases from fermented broth by PEG/citrate aqueous two-phase system

This work deals with the use of an aqueous two-phase system (ATPS) of PEG/citrate to remove proteases from a Clostridium perfringens fermentation broth. To plan the experimental tests and evaluate the corresponding results, three successive experimental designs were employed, for which the PEG molar mass (M-PEG) and concentration (C-PEG), the citrate concentration (C-C) and the pH were selected as independent variables, while the purification factor (PF), the partition coefficient (K), the activity yield (Y) and the selectivity (S) were selected as responses. PF of proteases in the top phase was shown to increase with increasing MPEG and decreasing Cc, whereas a completely opposite trend was observed for K. On the other hand, Y was favored by simultaneous decreases in both these variables, while S decreased with increasing Cc. Therefore, selecting a simultaneous increase in PF and Y as the most desirable result, the best performance of the system was obtained using M-PEG = 10-000 g/mol C-PEG = 22% (w/w) and C-c = 8.0% (w/w) at pH 8.5. Under these conditions, the activity yield was very high (131 %) but the purification factor (4.2) and the selectivity (4.3) were lower than those ensured by more selective purification methods. According to these results, the ATPS seems to be an interesting alternative primary concentration/decontamination step for vaccine preparation from C. perfringens fermented broth. (C) 2007 Elsevier B.V. All rights reserved.

Suramin inhibits macrophage activation by human group IIA phospholipase A(2), but does not affect bactericidal activity of the enzyme

Suramin is a polysulphonated napthylurea antiprotozoal and anthelminitic drug, which also presents inhibitory activity against a broad range of enzymes. Here we evaluate the effect of suramin on the hydrolytic and biological activities of secreted human group IIA phospholipase A(2) (hsPLA(2)GIIA). The hsPLA(2)GIIA was expressed in E. coli, and refolded from inclusion bodies. The hydrolytic activity of the recombinant enzyme was measured using mixed dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) liposomes. The activation of macrophage cell line RAW 264.7 by hsPLA(2) GIIA was monitored by NO release, and bactericidal activity against Micrococcus luteus was evaluated by colony counting and by flow cytometry using the fluorescent probe Sytox Green. The hydrolytic activity of the hsPLA(2) GIIA was inhibited by a concentration of 100 nM suramin and the activation of macrophages by hsPLA(2) GIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA(2) GIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 mu M. These results demonstrate that suramin selectively inhibits the activity of the hsPLA(2) GIIA against macrophages, whilst leaving the anti-bacterial function unchanged.

Purification and Biochemical Characterization of Thermostable Alkaline Phosphatases Produced by Rhizopus microsporus var. rhizopodiformis

The biochemical properties of the alkaline phosphatases (AIPs) produced by Rhizopus micro-sporus are described. High enzymic levels were produced within 1-2 d in agitated cultures with 1% wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3x, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn(2+), Na(+) and Mg(2+) Stimulated the activity, while Al(3+) and Zn(2+) activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 degrees C and pH 8.0, respectively. The enzymes were stable at 50 degrees C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K(m) 0.28 and 0.22 mmol/L, with upsilon(lim) 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.

Incorporation of antigenic GPI-proteins from Leishmania amazonensis to membrane mimetic systems: Influence of DPPC/cholesterol ratio

The reconstitution of membrane proteins into liposomes is a useful tool to prepare antigenic components that induce immunity. We have investigated the influence of the dipalmitoylphosphatidylcholine (DPPC)/cholesterol molar ratio on the incorporation of a GPI-protein from Leishmania amazonensis on liposomes and Langmuir monolayers. The latter system is a well behaved and practical model, for understanding the effect of variables such as surface composition and lipid packing on protein incorporation. We have found that the DPPC/cholesterol molar ratio significantly alters the incorporation of the GPI-protein. In the absence of cholesterol, reconstitution is more difficult and proteoliposomes cannot be prepared, which we correlated with disruption of the DPPC layer. Our results provide important information that Could be employed in the development of a vaccine system for this disease or be used to produce other GPI-systems for biotechnological application. (c) 2009 Elsevier Inc. All rights reserved.

A Statistical Study of the Association of Seven Dental Anomalies in the Brazilian Population

The objective of this study was to show the association patterns among seven types of dental anomalies (second pre-molar agenesis, upper side incisive reduced in size, lower first molar infra-ochlesis, enamel hypoplasia, first molar ectopic eruption, supra numerous teeth and upper canine ectopic eruption) in a population sample without dental treatment ranging in age from 7 to 14. A total of 172 patients were attended and underwent the clinical examination at the Clinica Infantil da Fundacao Educacional de Barretos. Eleven patients from this total were selected according to a first dental anomaly diagnosis and submitted to panoramic radiography. A significant association (p < 0.05) was detected among six pairs of anomalies (second pre-molar agenesis x first pre-molar ectopic eruption; second pre-molar agenesis x lower first molar infra-ochlesis; second pre-molar agenesis x upper side incisive reduced in size; supra numerous teeth x reduced size upper side incisive; first pre-molar ectopic eruption x enamel hypoplasia; lower first molar infra-ochlesis x upper side incisive reduced in size) suggesting a common genetic origin for these conditions. The association was not significant in only one case where there was anomaly sharing by the patients. The existence of an anomaly is clinically relevant for early diagnosis of a possible association and an anomaly can indicate an increased risk of other anomalies.

Sol-gel preparation of near-infrared broadband emitting Er(3+)-doped SiO(2)-Ta(2)O(5) nanocomposite films

This article reports a study on the preparation, densification process, and structural and optical properties of SiO(2)-Ta(2)O(5) nanocomposite films obtained by the sol-gel process. The films were doped with Er(3+) and the Si:Ta molar ratio was 90:10. Values of refractive index, thickness and vibrational modes in terms of the number of layers and thermal annealing time are described for the films. The densification process is accompanied by OH group elimination, increase in the refractive index, and changes in film thickness. Full densification of the film is acquired after 90 min of annealing at 900 degrees C. The onset of crystallization and devitrification, with the growth of Ta(2)O(5) nanocrystals occurs with film densification, evidenced by high-resolution transmission electron microscopy. The Er(3+)-doped nanocomposite annealed at 900 degrees C consists of Ta(2)O(5) nanoparticles, with sizes around 2 nm, dispersed in the SiO(2) amorphous phase. The main emission peak of the film is detected at around 1532 nm, which can be assigned to the (4)I(13/2)->(4)I(15/2) transition of the Er(3+) ions present in the nanocomposites. This band has a full width at half medium of 64 nm, and the lifetime measured for the (4)I(13/2) levels is 5.4 ms, which is broader compared to those of other silicate systems. In conclusion, the films obtained in this work are excellent candidates for use as active planar waveguide. (C) 2010 Elsevier B.V. All rights reserved.

The effect of cholesterol on the reconstitution of alkaline phosphatase into liposomes

Tissue-nonspecific alkaline phosphatase (TNAP), present on the surface of chondrocyte- and osteoblast-derived matrix vesicles (MVs), plays key enzymatic functions during endochondral ossification. Many studies have shown that MVs are enriched in TNAP and also in cholesterol compared to the plasma membrane. Here we have studied the influence of cholesterol on the reconstitution of TNAP into dipalmitoylphosphatidylcholine (DPPC)-liposomes, monitoring the changes in lipid critical transition temperature (T(c)) and enthalpy variation (Delta H) using differential scanning calorimetry (DSC). DPPC-liposomes revealed a T(c) of 41.5 degrees C and Delta H of 7.63 Kcal mol(-1). The gradual increase in cholesterol concentration decrease Delta H values, reaching a Delta H of 0.87 Kcal mol(-1) for DPPC: cholesterol system with 36 mol% of cholesterol. An increase in T(c), up to 47 degrees C for the DPPC:cholesterol liposomes (36 mol% of Chol), resulted from the increase in the area per molecule in the gel phase. TNAP (0.02 mg/mL) reconstitution was done with protein:lipid 1:10,000 (molar ratio), resulting in 85% of the added enzyme being incorporated. The presence of cholesterol reduced the incorporation of TNAP to 42% of the added enzyme when a lipid composition of 36 mol% of Chol was used. Furthermore, the presence of TNAP in proteoliposomes resulted in a reduction in Delta H. The gradual proportional increase of cholesterol in liposomes results in broadening of the phase transition peak and eventually eliminates the cooperative gel-to-liquid-crystalline phase transition of phospholipids bilayers. Thus, the formation of microdomains may facilitate the clustering of enzymes and transporters known to be functional in MVs during endochondral ossification. (C) 2010 Elsevier B.V. All rights reserved.

Local delivery of EGF-liposome mediated bone modeling in orthodontic tooth movement by increasing RANKL expression

Aims: It has long been demonstrated that epidermal growth factor (EGF) has catabolic effects oil bone. Thus. we examined the role of EGF in regulating mechanically induced bone modeling in a rat model of orthodontic tooth movement. Main methods: The maxillary first molars of rats were moved mesially using an orthodontic appliance attached to the maxillary incisor teeth. Rats were randomly divided into 4 groups: (G1) administration of PBS (Phosphate buffer saline Solution (n = 24); (G2) administration of empty liposomes (it = 24): (Q) administration 20 rig of EGF Solution (n = 24): and (G4) 20 ng of EGF-liposomes Solution (it = 24). Each Solution was injected in the mucosa of the left first molar adjacent to the appliance. At days 5, 10, 14 and 2 1 after drug administration. 6 animals of each group were sacrificed. Histomorphometric analysis was used to quantify osteoclasts (Tartrate-resistant acid phosphatase (TRAP) + cells) and tooth movement. Using immunohistochemistry assay we evaluated the RANKL (receptor activator of nuclear factor kappa B ligand) and epidermal growth factor receptor (EGFR) expression. Key findings: The EGF-liposome administration showed an increased tooth movement and osteoclast numbers compared to controls (p<0.05). This was correlated with intense RANKL expression. Both osteoblasts and osteoclasts expressed EGFR. Significance: Local delivery of EGF-liposome stimulates, osteoclastogenesis and tooth movement. (C) 2009 Elsevier Inc. All rights reserved.

Combined glucosamine and chondroitin sulfate provides functional and structural benefit in the anterior cruciate ligament transection model

Evidence that combined glucosamine sulfate and chondroitin sulfate (Gluchon) or isolated glucosamine (Glu) modifies joint damage in osteoarthritis (OA) is still lacking. We studied joint pain and cartilage damage using the anterior cruciate ligament transection (ACLT) model. Wistar rats were subjected to ACLT of the right knee ( OA) or sham operation. Groups received either Glu (500 mg/kg), Gluchon (500 mg/kg glucosamine +400 mg/kg chondroitin) or vehicle (non-treated-NT) per os starting 7 days prior to ACLT until sacrifice at 70 days. Joint pain was evaluated daily using the rat-knee joint articular incapacitation test. Structural joint damage was assessed using histology and biochemistry as the chondroitin sulfate ( CS) content of cartilage by densitometry (microgram per milligram dried cartilage), comparing to standard CS. The molar weight (Mw) of the CS samples, used as a qualitative biochemical parameter, was obtained by comparing their relative mobility on a polyacrylamide gel electrophoresis to standard CS. Gluchon, but not Glu, significantly reduced joint pain (P<0.05) compared to NT. There was an increase in CS content in the OA group (77.7 +/- 8.3 mu g/mg) compared to sham (53.5 +/- 11.2 mu g/mg) (P<0.05). The CS from OA samples had higher Mw (4:62 +/- 0:24 x 10(4) g/mol) compared to sham (4:18 +/- 0:19 x 10(4) g/mol) (P<0.05). Gluchon administration significantly reversed both the increases in CS content (54.4 +/- 12.1 mu g/mg) and Mw (4:18 +/- 0:2 x 104 g/mol) as compared to NT. Isolated Glu decreased CS content though not reaching statistical significance. Cartilage histology alterations were also significantly prevented by Gluchon administration. Gluchon provides clinical (analgesia) and structural benefits in the ACLT model. This is the first demonstration that biochemical alterations occurring in parallel to histological damage in OA are prevented by Gluchon administration.

Randomized Study of Surgical Prophylaxis in Immunocompromised Hosts

Although prophylaxis is current practice, there are no randomized controlled studies evaluating preoperative antimicrobial prophylaxis in dental procedures in patients immunocompromised by chemotherapy or organ transplants. To evaluate prophylaxis in dental-invasive procedures in patients with cancer or solid organ transplants, 414 patients were randomized to receive one oral 500-mg dose 2 hours before the procedure (1-dose group) or a 500-mg dose 2 hours before the procedure and an additional dose 8 hours later (2-dose group). Procedures were exodontia or periodontal scaling/root planing. Follow-up was 4 weeks. No deaths or surgical site infections occurred. Six patients (1.4%) presented with use of pain medication > 3 days or hospitalization during follow-up: 4 of 207 (2%) in the 1-dose group and 2 of 207 (1%) in the 2-dose group (relative risk, 2.02; 95% confidence interval, 0.37-11.15). In conclusion, no statistically significant difference occurred in outcome using 1 or 2 doses of prophylactic amoxicillin for invasive dental procedures in immunocompromised patients.

Protonectin (1-6): A novel chemotactic peptide from the venom of the social wasp Agelaia pallipes pallipes

Peptides constitute the largest group of Hymenoptera venom toxins; some of them interact with GPCR, being involved with the activation of different types of leukocytes, smooth muscle contraction and neurotoxicity. Most of these toxins vary from dodecapeptides to tetradecapeptides, amidated at their C-teminal amino acid residue. The venoms of social wasps can also contains some tetra-, penta-, hexa- and hepta-peptides, but just a few of them have been structurally and functionally characterized up to now. Protonectin (ILG-TILGLLKGL-NH(2)) is a polyfunctional peptide, presenting mast cell degranulation, release of lactate dehydrogenase (LDH) from mast cells, antibiosis against Gram-positive and Gram-negative bacteria and chemotaxis for polymorphonucleated leukocytes (PMNL), while Protonectin (1-6) (ILGTIL-NH(2)) only presents chemotaxis for PMNL However, the mixture of Protonectin (1-6) with Protonectin in the molar ratio of 1:1 seems to potentiate the biological activities dependent of the membrane perturbation caused by Protonectin, as observed in the increasing of the activities of mast cell degranulation, LDH releasing from mast cells, and antibiosis. Despite both peptides are able to induce PMNL chemotaxis, the mixture of them presents a reduced activity in comparison to the individual peptides. Apparently, when mixed both peptides seems to form a supra-molecular structure, which interact with the receptors responsible for PMNL chemotaxis, disturbing their individual docking with these receptors. In addition to this, a comparison of the sequences of both peptides suggests that the sequence ILGTIL is conserved, suggesting that it must constitute a linear motif for the structural recognition by the specific receptor which induces leukocytes migration. (C) 2010 Elsevier Ltd. All rights reserved.

Otitis media with effusion and dental occlusion: is there any relationship?

Aim Auditory tube dysfunction is one of the aetiological causes of otitis media. Studies suggest a correlation between otitis media with effusion and dental malocclusion. Our goal was to determine any correlation between dental malocclusion and otitis media with effusion in children with chronic upper airway obstruction due to tonsil and adenoid enlargement. Materials and methods This prospective study evaluated 52 children with otitis media with effusion and 48 without, aged 4.2 to 10.8 years. A questionnaire was answered by the parents about breast or bottle-feeding and bad oral habits. Malocclusion was diagnosed according to Angle`s classification for molar relationships in Classes I, II and III, posterior and anterior cross bite, open and deep bite, and overjet. Results and conclusion The results suggested no correlation between dental malocclusion and otitis media with effusion. Other potential confounders, such as breast or bottle-feeding and oral habits were also not correlated.

The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination. (C) 2009 Elsevier B.V. All rights reserved.

Structure activity relationship, acute toxicity and cytotoxicity of antimycobacterial neolignan analogues

Objectives The study`s aims were to evaluate the antimycobacterial activity of 13 synthetic neolignan analogues and to perform structure activity relationship analysis (SAR). The cytotoxicity of the compound 2-phenoxy-1-phenylethanone (LS-2, 1) in mammalian cells, such as the acute toxicity in mice, was also evaluated. Methods The extra and intracellular antimycobacterial activity was evaluated on Mycobacterium tuberculosis H37Rv. Cytotoxicity studies were performed using V79 cells, J774 macrophages and rat hepatocytes. Additionally, the in-vivo acute toxicity was tested in mice. The SAR analysis was performed by Principal Component Analysis (PCA). Key findings Among the 13 analogues tested, LS-2 (1) was the most effective, showing promising antimycobacterial activity and very low cytotoxicity in V79 cells and in J774 macrophages, while no toxicity was observed in rat hepatocytes. The selectivity index (SI) of LS-2 (1) was 91 and the calculated LD50 was 1870 mg/kg, highlighting the very low toxicity in mice. SAR analysis showed that the highest electrophilicity and the lowest molar volume are physical-chemical characteristics important for the antimycobacterial activity of the LS-2 (1). Conclusions LS-2 (1) showed promising antimycobacterial activity and very weak cytotoxicity in cell culture, as well as an absence of toxicity in primary culture of hepatocytes. In the acute toxicity study there was an indication of absence of toxicity on murine models, in vivo.

Age and gender influence on maximal bite force and masticatory muscles thickness

The present study aimed investigate the age and gender influence on maximal molar bite force and at outlining the criteria for normal masticatory muscle development in a sample of 177 Brazilian Caucasian dentate individuals aged 7-80 years divided into five age groups: I(7-12 years), II (13-20 years), III (21-40 years), IV (41-60 years), and V (61-80 years). Except for Group V, which comprised nine women and eight men, all groups were equally divided in respect to gender (20 M/20 F). Bite force was recorded with a mouth-adapted 1000 N dynamometer and the highest out of three records was regarded as the maximal bite force. The data were submitted to multivariate statistical analysis (SPSS 17.0 p < 0.05). Effects of group and gender were found, but no interactions between them. The ANOVA showed significant differences between groups bilaterally. Bonferroni`s test showed that group I had significantly lower bite force means at both sides as compared to all groups, except group V. No differences were found between the left and right sides. In all the groups, gender was found to be a significant factor associated with maximal bite force. A global comparison including all the subjects and measures showed that the means of men were approximately 30% higher than those of women, within-group comparisons yielded similar results in all groups. Muscle thickness was measured with a SonoSite Titan ultrasound tool using a high-resolution real-time 56 mm/10 MHz linear-array transducer. Three ultrasound images were obtained from the bilateral masseter and temporal muscles at rest and at maximal voluntary contraction. The means of the three measures in each clinical condition were analyzed with multivariate statistical analysis (SPSS 17.0 p < 0.05). A gradual increase in thickness of the masseter and temporal muscles was found both at rest and maximal voluntary contraction for groups I to IV, whereas a decrease in muscle thickness was observed in group V. Multivariate analysis showed that in both conditions there was an effect of group and gender. The study of the development of the stomatognathic system in relation to age and gender can provide useful data for the identification of normal and impaired functioning patterns. The results of this study indicate that age and gender are associated with structural and functional alterations in the muscles of the stomatognathic system. (C) 2010 Elsevier Ltd. All rights reserved.