Effects of Cellulose Nanofibers Filling and Palmitic Acid Emulsions Coating on the Physical Properties of Fish Gelatin Films

In this preliminary study, fish gelatin films with improved strength and water resistance were prepared from a dispersion of fish gelatin and carboxylated cellulose nanofibrils (CNF) by using the casting method, followed by subsequent coating with palmitic acid emulsion. The surface topography displayed a uniform distribution of the CNF particles in the gelatin films, but aggregation occurred at a CNF dosage of 4 wt% or higher. Due to the reinforcing effect of CNF, a dosage-dependent increase in the Young’s modulus and tensile strength was observed for the CNF-reinforced films. The addition of CNF also led to an obvious increase in thermal stability. Via surface coating, the emulsion at the 60:40 (w/w) ratio of palmitic acid to water showed excellent layer-forming and high adhesion properties, contributing to the significant improvement of water resistance. The enhanced properties of these fish gelatin films would promote their practical applications in edible packaging.     

The effect of pH,bile and calcium on the adhesion ability of probiotic enterococci of animal origin to the porcine jejunal epithelial cell line IPEC-J2

Examination of adhesion ability using a quantitative assay based on radiolabelled bacteria showed that 10 Enterococcus strains exhibited adhesion ability from 2 to 4%. Enterococcus faecium EF2019 (isolate from rabbit faeces, deponed to Czech Culture of Microorganisms in Brno, CCM 7420) showed the highest adhesion ability (4.0 ± 0.4%). With regard to survival, all strains displayed good resistance towards 0.3% oxgall and HCl (pH 3.0). Pretreatment of strains with HCl (pH 3.0) significantly reduced their adhesion. Pretreatment of strains by oxgall significantly reduced the adhesion capacity of E. faecium EF2019, EF1839 and EF319 strains, while the adhesion ability of E. faecium EE3 (isolate from canine feed) slightly increased. Furthermore, addition of calcium (200 mmol/l) significantly increased (P < 0.001) the adhesion ability for all strains tested. The adhesion ability of the isolates from rabbits, EF1839 and EF529, as well as the isolate EE3 (strain from canine feed) increased from 2–3% up to 50–55% upon calcium addition. Despite, in general low adhesive properties, strains can survive passage through the gastrointestinal tract.     

Degradation of Chrysanthemum (Dendranthema grandiflora) wastes by Pleurotus ostreatus for the production of reducing sugars

In Colombia, a great amount of waste is generated during the cut-off and harvest stages in flowers culture. This study examines the possibility of degrading Chrysanthemum wastes by using Pleurotus ostreatus, Trametes versicolor, and Phanerochaete chrysosporium cultures; this has not been studied previously. The initial effect of fungi on the degradation of Chrysanthemum wastes were studied individually and in co-cultures. The highest degradation was by P. ostreatus. After that, the influence of pH and waste, copper, and manganese concentrations on reducing sugars concentration were determined in a submerged culture in 100 mL Erlenmeyer flasks. There was a significant effect of manganese and waste concentrations on sugar concentration, while the effect of copper concentration and pH were not significant. Following, the process was carried out in a 1.5 L reactor at the optimal values of the variables studied in Erlenmeyer flask but varying air injection from 0 to 2 vvm. The highest concentration of sugars was 21.2 g/L with 78% of glucose content at 6.3% w/v of waste, 7.5 mM of Mn and Cu and 2 vvm of air injection. Finally, laccase, Manganese peroxidase, endo-1,4-β-glucanase, exo-1,4-β-glucanase and 1,4-β-glucosidase were detected in the extract obtained under these conditions. The highest activities were obtained for laccase (4,694 U/L) and 1,4-β-glucosidase (9,513 U/L).     

Remarkable disparity in mechanical response among the extracellular domains of type I and II cadherins

Cadherins, a large family of calcium-dependent adhesion molecules, are critical for intercellular adhesion. While crystallographic structures for several cadherins show clear structural similarities, their relevant adhesive strengths vary and their mechanisms of adhesion between types I and II cadherin subfamilies are still unclear. Here, stretching of cadherins was explored experimentally by atomic force microscopy and computationally by steered molecular dynamics (SMD) simulations, where partial unfolding of the E-cadherin ectodomains was observed. The SMD simulations on strand-swapping cadherin dimers displayed similarity in binding strength, suggesting contributions of other mechanisms to explain the strength differences of cell adhesion in vivo. Systematic simulations on the unfolding of the extracellular domains of type I and II cadherins revealed diverse pathways. However, at the earliest stage, a remarkable similarity in unfolding was observed for the various type I cadherins that was distinct from that for type II cadherins. This likely correlates positively with their distinct adhesive properties, suggesting that the initial forced deformation in type I cadherins may be involved in cadherin-mediated adhesion.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:25     

Comparing submerged and solid-state fermentation of agro-industrial residues for the production and characterization of lipase by Trichoderma harzianum

Lipase production by Trichoderma harzianum was evaluated in submerged fermentation (SF) and solid-state fermentation (SSF) using a variety of agro-industrial residues. Cultures in SF showed the highest activity (1.4 U/mL) in medium containing 0.5 % (w/v) yeast extract, 1 % (v/v) olive oil and 2.5 C:N ratio. This paper is the first to report lipase production by T. harzianum in SSF. A 1:2 mixture of castor oil cake and sugarcane bagasse supplemented with 1 % (v/w) olive oil showed the best results among the cultures in SSF (4 U/g ds). Lipolytic activity was stable in a slightly acidic to neutral pH, maintaining 50 % activity after 30 min at 50 °C. Eighty percent of the activity remained after 1 h in 25 % (v/v) methanol, ethanol, isopropanol or acetone. Activity was observed with vegetable oils (olive, soybean, corn and sunflower) and long-chain triacylglycerols (triolein), confirming the presence of a true lipase. The results of this study are promising because they demonstrate an enzyme with interesting properties for application in catalysis produced by fermentation at low cost.     

Inhibition of Cancer Cell Adhesion,Migration and Proliferation by a Bispecific Antibody that Targets two Distinct Epitopes on αv Integrins

Members of the αv family of integrins regulate activation of transforming growth factor beta (TGFβ) and are directly involved in pro-tumorigenic phenotypes. Thus, αv integrins may be therapeutic targets for fibrosis and cancer, yet the isolation of selective inhibitors is currently a challenge. We generated synthetic antibodies selective for αv integrins by phage display selections on cell lines that displayed integrin heterodimers. We identified antibodies that targeted two distinct epitopes on cell-surface αv integrins and partially inhibited cell adhesion mediated by interactions between integrins and the latency-associated peptide, part of the pro-form of TGFβ. Using the isolated antibody paratope sequences we engineered a bispecific antibody capable of binding to both epitopes simultaneously; this antibody potently and completely inhibited cell adhesion mediated by integrins αvβ1, αvβ3 and αvβ5. In addition, the bispecific antibody inhibited proliferation and migration of lung carcinoma lines, where the highest and lowest potencies observed correlated with integrin-αv cell surface expression levels. Taken together, our results demonstrate that phage display selections with live cells can yield high quality anti-integrin antibodies, which we used as biparatopic building blocks to construct a bispecific antibody that strongly inhibited integrin function and may be a therapeutic candidate for cancer and fibrosis.     

Enhancing the activity and thermostability of thermostable β-glucosidase from a marine Aspergillus niger at high salinity

To evaluate the effect of salinity on the catalyzing ability of β-glucosidase in the marine fungus Aspergillus niger, the thermodynamic parameters of the β-glucosidase were investigated at different salinities. At the optimum salinity of 6% NaCl (w/v) solution, the optimum temperature and pH of the β-glucosidase activity was 66 °C and 5.0, respectively. Under these conditions, the β-glucosidase activity increased 1.46 fold. The half-life of denaturation in 6% NaCl (w/v) solution was approximately twice as long as that in NaCl free solution. The Gibb's free energy for denaturation, ΔG, was 2 kJ/mol higher in 6% NaCl (w/v) solution than in NaCl free solution. The melting point (68.51 °C) in 6% NaCl (w/v) solution was 1.71 °C higher than that (66.80 °C) in NaCl free solution. Similarly, the activity and thermostability of the pure β-glucosidase increased remarkably at high salinity. The thermostable β-glucosidase, of which the activity and the thermostability are remarkably enhanced at high salinity, is valuable for industrial hydrolyzation of cellulose in high salinity environments.     

Production of extracellular alkaline proteases by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis>

Summary The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37 °C. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25 °C for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH₄NO₃ showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.     

Expression of the Mucus Adhesion Gene Mub, Surface Layer Protein Slp and Adhesion-Like Factor EF-TU of Lactobacillus acidophilus ATCC 4356 Under Digestive Stress Conditions, as Monitored with Real-Time PCR

Expression of the mucus adhesion gene Mub, surface layer protein Slp and adhesion-like factor EF-Tu by Lactobacillus acidophilus ATCC 4356 grown in the presence of mucin, bile and pancreatin and at low pH was studied using real-time PCR. None of the genes were up-regulated under increasing concentrations of mucin, while Slp and EF-Tu were up-regulated in the presence of bile and pancreatin at normal concentrations (0.3%, w/v) and under stress conditions (1.0%, w/v).     

Periodate oxidation and alkaline degradation of heparin-related glycans

Heparin, heparan sulphate, and various derivatives thereof have been oxidised with periodate at pH 3.0 and 4° and at pH 7.0 and 37°. Whereas oxidation under the latter conditions destroys all of the nonsulphated uronic acids, treatment with periodate at low pH and temperature causes selective oxidation of uronic acid residues. The reactivity of uronic acid residues depends on the nature of neighbouring 2-amino-2-deoxyglucose residues. d-Glucuronic acid residues are susceptible to oxidation when flanked by N-acetylated amino sugars, but resistant when adjacent residues are either unsubstituted or N-sulphated. L-Iduronic acid residues in their natural environment (2-deoxy-2-sulphoamino-d-glucose) are resistant to oxidation, whereas removal of N-sulphate groups renders a portion of these residues periodate-sensitive. Oxidised uronic acid residues in heparin-related glycans may be cleaved by alkali, producing a series of oligosaccharide fragments. Thus, periodate oxidation-alkaline elimination provides an additional method for the controlled degradation of heparin.     

The Impact of Desiccation on the Adhesion of Barnacles Attached to Non-stick Coatings

Fouling-release coatings prevent fouling of ships' hulls through hydrodynamic forces generated as the ship moves through the water. The effectiveness of such coatings may be evaluated by measuring the adhesion strength of settled organisms, e.g. barnacles. The influence of desiccation of the barnacle adhesive on such measurements was investigated. Shear forces required to remove barnacles of the genus Balanus increased during the course of desiccation up to the point when the barnacles suddenly self-detached. The increase was thought to be due to the rising cohesive strength of the adhesive. Growing tensile forces within the weakly cross-linked adhesive, however, are suggested to have led to self-detachment. The shear forces required to remove barnacles of the genus Elminius were generally low and did not differ significantly during the course of desiccation. The different results may be attributed to specific base morphologies. It was concluded that measuring the adhesion strength of members of the Balanidae on non-stick surfaces in air could produce flawed results due to the influence of desiccation of the barnacle adhesive. The investigations have also provided new insights into the characteristics of barnacle adhesive.     

Inulinase production by the marine yeast Cryptococcus aureus G7a and inulin hydrolysis by the crude inulinase

The marine yeast strain G7a isolated from sediment of China South Sea was found to secrete a large amount of inulinase into the medium. This marine yeast strain was identified to be a strain of Cryptococcus aureus according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine yeast showed the highest activity at pH 5.0 and 50 °C. The optimal medium for inulinase production was artificial seawater containing inulin 4.0% (w/v), K₂HPO₄ 0.3% (w/v), yeast extract 0.5% (w/v), KCl 0.5% (w/v), CaCl₂ 0.12% (w/v), NaCl 4.0% (w/v) and MgCl₂·6H₂O 0.6% (w/v), while the optimal cultivation conditions for inulinase production were pH 5.0, a temperature of 28 °C and a shaking speed of 170 rpm. Under the optimal conditions, over 85.0 U/ml of inulinase activity was produced within 42 h of fermentation at shake flask level. This is very high level of inulinase activity produced by yeasts. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase.     

Comparative analysis of the intergenic spacer regions and population structure of the species complex of the pathogenic yeast Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic basidiomycete responsible for the high incidence of cryptococcosis in patients with AIDS and in other immune-compromised individuals. This study, which focused on the molecular structure and genetic variability of the two varieties in the C. neoformans and Cryptococcus gattii species complex, employed sequence analysis of the intergenic spacer regions, IGSI and IGSII. The IGS region is the most rapidly evolving region of the rDNA families. The IGSI displayed the most genetic variability represented by nucleotide base substitutions and the presence of long insertions/deletions (indels). In contrast, the IGSII region exhibited less heterogeneity and the indels were not as extensive as those displayed in the IGSI region. Both intergenic spacers contained short, interspersed repeat motifs, which can be related to length polymorphisms observed between sequences. Phylogenetic analysis undertaken in the IGSI, IGSII and IGSI +5S rRNA + IGSII regions revealed the presence of six major phylogenetic lineages, some of which segregated into subgroups. The major lineages are represented by genotypes 1 (C. neoformans var. grubii), genotype 2 (C. neoformans var. neoformans), and genotypes 3, 4, 5 and 6 represented by C. gattii. Genotype 6 is a newly described IGS genotypic group within the C. neoformans species complex. With the inclusion of IGS subgenotypic groups, our sequence analysis distinguished 12 different lineages. Sequencing of clones, which was performed to determine the presence of multiple alleles at the IGS locus in several hybrid strains, yielded a single IGS sequence type per isolate, thus suggesting that the selected group of cloned strains was mono-allelic at this locus. IGS sequence analyses proved to be a powerful technique for the delineation of the varieties of C. neoformans and C. gattii at genotypic and subgenotypic levels.     

A novel triterpenoid 16-hydroxy betulinic acid isolated from Mikania cordata attributes multi-faced pharmacological activities

The aerial parts of extensively used ethnomedicinal plant Mikania cordata (Burm. f.) Robinson growing wild in Bangladesh were investigated to isolate and characterize compounds responsible for the bioactivities of the plant. In the present study, a new derivatives of betulinic acid, 16-hydroxy betulinic acid [3β,16-dihydroxy-lup-20(29)-en-28-oic] was isolated and the structure of the compound was determined by NMR spectroscopic means and comparing with available literature data. The isolated compound was then investigated for different pharmacological activities including antibacterial, antifungal, analgesic, anti-inflammatory and antipyretic potential employing different methods. The compound showed potent antibacterial activity with inhibition zone of diameter ranging from 12.0 to 17.5?mm and antifungal activity with mycelial growth inhibition ranging from 37.6 to 54.5%. The MIC values for antibacterial and antifungal activities ranged from 31.5–125 and 250–1000?μg/mL respectively. The compound (50 and 100?mg/kg body weight) showed potent peripheral and central analgesic activity with 55.19% and 41% of writhing inhibition at 90?min after administration of the compound and the highest 55.98%, 79.18% elongation of reaction time, respectively. In anti-inflammatory activity screening, the compound (100?mg/kg b.w.) revealed the highest 77.08% edema inhibition at 4?h after administration of carrageenan. In antipyretic assay, 16-hydroxy betulinic acid displayed a strong antipyretic effect in yeast-induced rats. From the present study it is apparent that 16-hydroxy betulinic acid might play vital role to establish M. cordata as ethnomedicinal plant to treat wound, cuts and fever.     

RNA aptamer therapy for vaso-occlusion in sickle cell disease

Patients with sickle cell disease (SCD) often suffer painful vaso-occlusive episodes caused in part by the adhesion of sickle erythrocytes (SS-RBC) to the vascular endothelium. To investigate inhibition of SS-RBC adhesion as a possible treatment for vaso-occlusion, 2 adhesion molecules, α(v)β(3) and P-selectin, were targeted by high-affinity RNA aptamers. An in vitro flow chamber assay was used to test the antiadhesion activity of α(v)β(3) aptamer clone 17.16. Human SS-RBC were passed across a confluent monolayer of thrombin-stimulated human umbilical vein endothelial cells (HUVEC) at a constant rate. α(v)β(3) aptamer reduced SS-RBC adhesion to activated endothelial cells to the level seen with untreated HUVEC. An aptamer reactive with complement component 8 was used as a negative control and exerted no inhibition, confirming the specificity of α(v)β(3) aptamer (P=0.04). At 2?dyn/cm(2) shear stress, 30?nM α(v)β(3) aptamer showed maximal effect in decreasing SS-RBC adhesion to HUVEC. The antiadhesive activity of the P-selectin aptamer clone PF377 was also tested using HUVEC pretreated with IL-13 to upregulate expression of P-selectin as seen in activated endothelial cells. At 1?dyn/cm(2) shear stress, 60?nM of P-selectin aptamer had antiadhesion activity similar to heparin, a known inhibitor of SS-RBC adhesion to P-selectin. A negative control did not prevent adhesion (P=0.05). These data show the potential utility of aptamers to block endothelial adhesion molecules to prevent or treat vaso-occlusion in SCD.     

Src, PKCα, and PKCδ are required for αvβ3 integrin-mediated metastatic melanoma invasion

Background

Integrins, cell-surface receptors that mediate adhesive interactions between cells and the extracellular matrix (ECM), play an important role in cancer progression. Expression of the vitronectin receptor αvβ3 integrin correlates with increased invasive and metastatic capacity of malignant melanomas, yet it remains unclear how expression of this integrin triggers melanoma invasion and metastasis.

Results

Two melanoma cell lines C8161.9 and M14 both express high levels of αvβ3 integrin and adhere to vitronectin. However, only the highly metastatic C8161.9 cells are capable of invading vitronectin-enriched Matrigel in an αvβ3-depenent manner. Elevated levels of PKCα and PKCδ, and activated Src were detected specifically in the highly metastatic melanoma cells, but not in the low metastatic M14 cells. Inhibition of Src or PKC activity suppressed αvβ3-dependent invasion. Furthermore, over expression of Src or PKCα and PKCδ was sufficient to confer αvβ3-dependent invasiveness to M14 cells. Stress fiber formation and focal adhesion formation were almost completely absent in C8161.9 cells compared to M14 cells. Inhibition of Src signaling was sufficient to restore normal actin architecture, and resulted in decreased p190RhoGAP phosphorylation and enhanced RhoA activity. Src had no effect on Rac activity. Loss of PKCα expression, but not PKCδ, by siRNA inhibited Rac and PAK activity as well as invasiveness. Loss of PKCα restored focal adhesion formation and partially restored stress fiber formation, while loss of PKCδ primarily restored stress fibers.

Conclusion

The misregulated expression of PKCα and PKCδ and elevated Src activity in metastatic melanoma cells is required for efficient αvβ3-mediated invasion. PKCα and Src enhance αvβ3-mediated invasion in part by increasing the GTPase activity of Rac relative to RhoA. PKCα influences focal adhesion formation, while PKCδ controls stress fibers.     

Dynamic adhesion of umbilical cord blood endothelial progenitor cells under laminar shear stress

Late outgrowth endothelial progenitor cells (EPCs) represent a promising cell source for rapid reendothelialization of damaged vasculature after expansion ex vivo and injection into the bloodstream. We characterized the dynamic adhesion of umbilical-cord-blood-derived EPCs (CB-EPCs) to surfaces coated with fibronectin. CB-EPC solution density affected the number of adherent cells and larger cells preferentially adhered at lower cell densities. The number of adherent cells varied with shear stress, with the maximum number of adherent cells and the shear stress at maximum adhesion depending upon fluid viscosity. CB-EPCs underwent limited rolling, transiently tethering for short distances before firm arrest. Immediately before arrest, the instantaneous velocity decreased independent of shear stress. A dimensional analysis indicated that adhesion was a function of the net force on the cells, the ratio of cell diffusion to sliding speed, and molecular diffusivity. Adhesion was not limited by the settling rate and was highly specific to α₅β₁ integrin. Total internal reflection fluorescence microscopy showed that CB-EPCs produced multiple contacts of α₅β₁ with the surface and the contact area grew during the first 20 min of attachment. These results demonstrate that CB-EPC adhesion from blood can occur under physiological levels of shear stress.     

Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen

A serine protease was purified 6.7-fold and with 35% recovery from the seeds Solanum dubium Fresen by a simple purification procedure that combined ammonium sulfate fractionation, cation exchange and gel filtration chromatographies. The enzyme, named dubiumin, has a molecular mass of 66 kDa as estimated by gel filtration and SDS-PAGE. Carbohydrate staining established the existence of a carbohydrate moiety attached to the enzyme. Inhibition of enzyme activity by serine protease inhibitors such as PMSF and chymostatin indicated that the enzyme belongs to the chymotrypsin-like serine protease class. Dubiumin is a basic protein with pI value of 9.3, acts optimally at pH 11.0, and is stable over a wide range of pH (3.0-12.0). The enzyme is also thermostable retaining complete activity at 60 °C after 1 h and acts optimally at 70 °C for 30 min. Furthermore, it is highly stable in the presence of various denaturants (2.0% SDS, 7.0 M urea and 3.0 M guanidine hydrochloride) and organic solvents [CH₃CN-H₂O (1:1, v/v) and MeOH-H₂O (1:1, v/v)] when incubated for 1 h. The enzyme showed a high resistance to autodigestion even at low concentrations.     

Microalgal proteins: a new source of raw material for production of plywood adhesive

Microalgae have attracted increasing interests due to their potential as an alternative to land crops to produce renewable fuels, chemicals, foods, and personal care products. In this study, we demonstrate the feasibility of producing type II plywood adhesive using total proteins extracted from Spirulina platensis and Chlamydomonas reinhardtii. Denaturation with NaOH and chemical cross-linking improved tensile strength and water resistance of the adhesive. Among the three aldehydes tested, glyoxal was found to be the best cross-linker. The optimum concentration of NaOH was approximately 50 mM and of glyoxal was 2 % (w/w). Glyoxal (2 % w/w) improved the tensile strength of plywood samples up to 55, 270, and 650 % of dry, soak/dry, and soak/dry (60 °C), respectively, for S. platensis proteins. Increase in hot pressing temperature and time also improved tensile strength. The optimum hot pressing conditions were 120 °C for 5 min after 10 min assembling time. Of the two algae sources tested, C. reinhardtii UTEX 2337 proteins had better adhesive strength and water resistance than S. platensis proteins and showed comparable adhesive properties to soy proteins. Notably, bioadhesives made from both algal proteins had lower viscosity than soy proteins. This feature should allow easier spreading of adhesive on wood surfaces and deeper penetration into veneers. Our results suggest that algal proteins are a promising resource for the production of bioadhesive for type II plywood.     

Phenolic hydroxyl ionization in calotropins from Calotropis gigantea latex

The ionization of the phenolic hydroxyl groups in calotropins DI and DII isolated from the latex of Calotropis gigantea has been studied by spectrophotometric titration at 295 nm in the pH range 6–13.2. Of the 12 tyrosine residues of calotropin DI and 13 tyrosine residues of calotropin DII, only four residues were ionized reversibly in the pH range 8.9–10.7 with the apparent pK of 9.7. The remaining tyrosine residues were ionized irreversibly in the pH range 11.2–13.2 with the apparent pK of 11.5. Both calotropins showed time-dependent ionization of phenolic groups at 295 nm in the pH range 11.5–12.0. Chemical modification with tetranitromethane suggested the presence of three tyrosine residues on the surface of each calotropin molecule.