Determination and diagnostic value of CA9 mRNA in peripheral blood of patients with oral leukoplakia

Background: Oral leukoplakia is one of the most common oral premalignant disorder. The classical evaluation through tissue biopsy is not always valid to evaluate the risk of malignization.

Material and methods: RT-qPCR was performed on 47 blood samples (21 patients with leukoplakia, 2 with oral squamous cell carcinoma (OSCC), and 24 healthy patients) and on 11 tissue samples (3 leukoplakia, 4 OSCC, and 4 samples of healthy tissue).

Results: There are significant differences in expression between the different groups (F?=?4.057, p?=?.006). The Duncan post hoc test shows that the only group that differentiates is the tumour tissue. Using Wilcoxon test, different covariables of patients with leukoplakia were analysed with respect to the group of healthy patients and no significant differences were observed.

Conclusions: The diagnostic route through liquid biopsy has not been conclusive in this study, but there are significant differences in the levels analysed in the different tissue samples.     

Purification,characterization and partial sequence of a pro‐inflammatory lectin from seeds of Canavalia oxyphylla Standl. & L. O. Williams

Recent studies have shown that lectins are promising tools for use in various biotechnological processes, as well as studies of various pathological mechanisms, isolation, and characterization of glycoconjugates and understanding the mechanisms underlying pathological mechanisms conditions, including the inflammatory response. This study aimed to purify, characterize physicochemically, and predict the biological activity of Canavalia oxyphylla lectin (CoxyL) in vitro and in vivo. CoxyL was purified by a single‐step affinity chromatography in Sephadex® G‐50 column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the pure lectin consists of a major band of 30 kDa (α‐chain) and two minor components (β‐chain and γ‐chain) of 16 and 13 kDa, respectively. These data were further confirmed by electrospray ionization mass spectrometry, suggesting that CoxyL is a typical ConA‐like lectin. In comparison with the average molecular mass of α‐chain, the partial amino acid sequence obtained corresponds to approximately 45% of the total CoxyL sequence. CoxyL presented hemagglutinating activity that was specifically inhibited by monosaccharides (D‐glucose, D‐mannose, and α‐methyl‐D‐mannoside) and glycoproteins (ovalbumin and fetuin). Moreover, CoxyL was shown to be thermostable, exhibiting full hemagglutinating activity up to 60°C, and it was pH‐sensitive for 1 h, exhibiting maximal activity at pH 7.0. CoxyL caused toxicity to Artemia nauplii and induced paw edema in rats. This biological activity highlights the importance of lectins as important tools to better understand the mechanisms underlying inflammatory responses. Copyright © 2014 John Wiley & Sons, Ltd.     

Structural and functional characterization of myotoxin, Cr-IV 1, a phospholipase A2 D49 from the venom of the snake Calloselasma rhodostoma

A new D49 PLA(2) was purified from the venom of Calloselasma rhodostoma after two chromatographic steps. Molecular exclusion chromatography was done through a Protein-Pack 300 SW column (0.78 cm x 30 cm), eluting with 0.25 M ammonium bicarbonate, pH 7.9, at a flow rate of 0.3 ml/min. Reverse-phase HPLC was then performed on mu-Bondapack C-18. The sample was determined to have a molecular mass of 13,870.94 Da MALDI-TOF by mass spectrometry, and the amino acid composition showed that Cr-IV 1 presented a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). Cr-IV 1 presented a sequence of 122 amino acid residues: DLWEFGQMILKETGSLPFPY YTTYGCYCGV GGRGGKPKDA TDRCCFVHDC CYGKLTGCPK TNDRYSYSRL DYTIVCGEGG PCKQICECDK AAAVCFRENL RTYNKKYRYHLKPFCKEPAE TC and a calculated pI value of 8.0. Cr-IV 1 had PLA(2) activity in the presence of a synthetic chromogenic substrate (4-nitro-3-(octanoyloxy)benzoic acid) and showed a rapid cytolytic effect on mouse skeletal muscle myoblasts and myotubes in culture. In mice, Cr-IV 1 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr-IV 1 was determined to be 0.07 mg/k body weight by intracerebroventricular (i.c.v.) injection. The combination of structural and functional information obtained herein classifies Cr-IV 1 as a new member of the D49 PLA(2) family, as it presents the typical behavior of a phospholipase A(2) from this family.     

Induction of Th17 differentiation by corneal epithelial‐derived cytokines

This study was to explore a potential role of epithelium‐derived cytokines in Th17 differentiation. Th17 induction was evaluated by murine CD4⁺ T cells treated with different combinations of five inducing cytokines, or conditioned media of human corneal epithelial cells (HCECs) exposed to a variety of stimuli. Th17 differentiation was determined by measuring Th17 associated molecules, IL‐17A, IL‐17F, IL‐22, CCL‐20, and STAT3 at mRNA and protein levels, and numbers of IL‐17‐producing T cells by real‐time PCR, and cytokine immunobead and ELISPOT assays, respectively. IL‐23 was the strongest inducer for expanding Th17 cells in the presence of TGF‐β1 + IL‐6; and IL‐1β was the strongest Th17 amplifier in the presence of TGF‐β1 + IL‐6 + IL‐23. These inducing cytokines were found to be significantly stimulated in HCECs challenged by hyperosmotic media (450 mOsM), microbial components (polyI:C, flagellin, R837, and other TLR ligands) and TNF‐α. Interestingly, when incubated with conditioned media of HCECs irritated by polyI:C or TNF‐α, CD4⁺ T cells displayed increased mRNA levels of IL‐17A, IL‐17F, IL‐22, CCL‐20, and STAT3, increased IL‐17 protein in the supernatant, and increased numbers of IL‐17‐producing T cells (Th17 cells). These findings demonstrate for the first time that Th17 differentiation can be promoted by cytokines produced by corneal epithelium that are exposed to hyperosmotic, microbial, and inflammatory stimuli. J. Cell. Physiol. 222:95–102, 2010. © 2009 Wiley‐Liss, Inc.     

Antioxidant properties and total phenolic contents of some tropical seaweeds of the Brazilian coast

Many types of macroalgae contain a wide range of bioactive compounds that have antioxidant potential. However, in contrast to terrestrial plants, only a few studies have reported the antioxidant activity of seaweeds. Therefore, extracts from 26 marine macroalgae species from the south and southeast coasts of Brazil were evaluated for their antioxidant activity, using the 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) method and β-carotene/linoleic acid assay, and their total phenolic contents, through Folin–Ciocalteu method. Padina gymnospora, Sargassum vulgare, and Osmundaria obtusiloba presented the highest values of total phenolic content. Using β-carotene bleaching assay, Colpomenia sinuosa, Dictyota sp., Dichotomaria marginata, Ganonema farinosum, and Spyridia clavata presented up to 65 % of antioxidant activity. Some of the extracts showed more than 60 % of inhibition of DPPH in the lowest concentration (0.01 mg/mL), including Amansia sp., Bostrychia tenella, Cryptonemia seminervis, Hypnea musciformis, Plocamium brasiliense (1), and S. clavata. Both Amansia sp., and C. seminervis presented the most relevant antioxidant potential, with percentage of inhibition greater than 70 % in the three tested concentrations. These two species were then analyzed by nuclear magnetic resonance spectroscopy (NMR) and were selected for guided fractionation bioassay. They both presented lipid compounds, fatty acids, esters of fatty acids, triglycerides, and sterols as major components. The fractionation of extracts revealed that the organic fractions were responsible for the antioxidant activity. The results obtained through this work indicate that the analyzed seaweeds are a promising source of compounds with high antioxidant potential.     

The use of ASB-14 in combination with CHAPS is the best for solubilization of human brain proteins for two-dimensional gel electrophoresis.

Protein extraction is the most important step to reveal a proteome by Two-Dimensional Gel Electrophoresis. Usually, the urea/thiourea based standard protein extraction buffer (SB) is combined with detergents with the aim of achieving better resolution and solubilization of different classes of proteins. In order to produce better gels and achieve the greatest spot resolution of Human Brain Proteins, comparisons using 2-DE of extracted proteins from Human Brain Frontal Cortex with SB constituents (7M Urea, 2M Thiourea and 100mM DTT) were made, using different detergent compositions in the buffer. SB preparations in combination with CHAPS and ASB-14 as well as with ASB-16 (reported for the first time in 2-DE experiments) have been tested. Our results confirm that the most efficient solubilizing solution for 2-DE analysis of cytosolic and membrane Human Brain Proteins is SB combined with 4% CHAPS and 2% ASB-14.     

Isolation and characterization of a new trypsin inhibitor from Crotalaria paulina seeds

A new trypsin inhibitor (CPTI) has been isolated from Crotalaria paulina seeds. Purification of the inhibitor was carried out by gel filtration, ion-exchange chromatography, and subsequent reversed-phase HPLC. The presence of a single polypeptide chain, with a molecular mass of 20 kDa and isoelectric point 4.0, was detected. The trypsin inhibitor had a Ki value of 4.5 x 10(-8) M and was capable of acting on human, bovine, and porcine trypsin and weakly on bovine chymotrypsin. Amino acid analysis showed that CPTI has a high content of aspartate, glutamate, leucine, serine, and glycine, having 177 amino acid residues in its composition. These data suggest that the protein belongs to the Kunitz-type trypsin inhibitors.     

Glutamine synthetase gene expression during the regeneration of the annelid Enchytraeus japonensis

Enchytraeus japonensis is a highly regenerative oligochaete annelid that can regenerate a complete individual from a small body fragment in 4–5 days. In our previous study, we performed complementary deoxyribonucleic acid subtraction cloning to isolate genes that are upregulated during E. japonensis regeneration and identified glutamine synthetase (gs) as one of the most abundantly expressed genes during this process. In the present study, we show that the full-length sequence of E. japonensis glutamine synthetase (EjGS), which is the first reported annelid glutamine synthetase, is highly similar to other known class II glutamine synthetases. EjGS shows a 61–71% overall amino acid sequence identity with its counterparts in various other animal species, including Drosophila and mouse. We performed detailed expression analysis by in situ hybridization and reveal that strong gs expression occurs in the blastemal regions of regenerating E. japonensis soon after amputation. gs expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. Furthermore, in the elongated blastema, gs expression was detectable also in the presumptive regions of the brain, ventral nerve cord, and stomodeum. In the fully formed intact head, gs expression was also evident in the prostomium, brain, the anterior end of the ventral nerve cord, the epithelium of buccal and pharyngeal cavities, the pharyngeal pad, and in the esophageal appendages. In intact E. japonensis tails, gs expression was found in the growth zone in actively growing worms but not in full-grown individuals. In the nonblastemal regions of regenerating fragments and in intact worms, gs expression was also detected in the nephridia, chloragocytes, gut epithelium, epidermis, spermatids, and oocytes. These results suggest that EjGS may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion, macromolecule synthesis, and gametogenesis.     

Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye

During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl2, MgCl2, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl2 and was recovered by adding a chelator. Ca2+ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl2. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl2. Ruthenium red, which binds to Ca(2+)-binding sites, readily stained AFPs in the absence of Ca2+, but less stain was visible after freezing and thawing AFPs in CaCl2. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca(2+)-binding sites. Once Ca2+ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca2+ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca2+ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca(2+)-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.