Purification of c-phycocyanin from Spirulina fusiformis and its effect on the induction of urokinase-type plasminogen activator from calf pulmonary endothelial cells

c-Phycocyanin (c-pc), a blue coloured, fluorescent protein was purified from blue-green alga, Spirulina fusiformis and its effect on fibrinolytic system in vascular endothelial cells was investigated.The c-pc consisted of two subunits, alpha and beta, whose molecular masses were 16 and 17 kDa, respectively. N-terminal sequences of both subunits were well conserved compared with other blue green algal phycobiliproteins. Fibrinolytic activity in the medium conditioned by calf pulmonary arterial endothelial cells was measured by the fibrin plate method.The c-pc increased the fibrinolytic activity in dose- and time-dependent manners. Fibrin zymographic studies indicated that c-pc-induced urokinase-type plasminogen activator in the cells. These in vitro results suggest that c-pc from S. fusiformis is a potent profibrinolytic protein in the vascular endothelial system.     

Antibiofilm and Antimicrobial Efficacy of DispersinB®-KSL-W Peptide-Based Wound Gel Against Chronic Wound Infection Associated Bacteria

The medical importance of bacterial biofilms has increased with the recognition of biofilms as one of the major contributors to the slow or non-healing chronic wounds such as diabetic foot ulcers, venous leg ulcers, and pressure ulcers. Being a protected community of microorganisms, biofilms are notoriously refractory to antibiotic treatments. As the conventional treatment modalities have proven ineffective, this study provides the in vitro evidence to support the use of a novel combination of DispersinB^® antibiofilm enzyme that inhibits biofilm formation and disperses preformed biofilm, and thus making the biofilm bacteria more susceptible to a broad-spectrum KSL-W antimicrobial peptide. The combination of DispersinB^® and KSL-W peptide showed synergistic antibiofilm and antimicrobial activity against chronic wound infection associated biofilm-embedded bacteria such as Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Coagulase-negative Staphylococci (CoNS), and Acinetobacter baumannii. In addition, the wound gel formulation comprising DispersinB^®, KSL-W peptide, and a gelling agent Pluronic F-127 showed a broad-spectrum and enduring antimicrobial activity against test organisms. Furthermore, as compared to commercial wound gel Silver-Sept?, DispersinB^®-KSL-W peptide-based wound gel was significantly more effective in inhibiting the biofilm-embedded MRSA, S. epidermidis, CoNS, Vancomycin-resistant Enterococci, A. baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa (P < 0.05). Thus, this study provides promising evidence for the potential application of antibiofilm-antimicrobial DispersinB^®-KSL-W wound gel in chronic wound management.     

Aspergillus parasiticus growth and aflatoxin production on black and white pepper and the inhibitory action of their chemical constituents.

Aspergillus parasiticus Speare NRRL 2999 growth and aflatoxin production in black and white pepper and the penetration of the fungus in black pepper corn over various incubation periods were studied. Also, the effects of piperine and pepper oil on growth and aflatoxin production were studied. Under laboratory conditions, black and white pepper supported aflatoxin production (62.5 and 44 ppb (ng/g), respectively) over 30 days of incubation. Fungal growth measured in terms of chitin was considerably less in white pepper than in black pepper. A histological study of black pepper corn showed the fungus penetrating up to the inner mesocarp and establishing itself in the middle mesocarp. Piperine and pepper oil were found to inhibit fungal growth and toxin production in a dose-dependent manner. Thus, both black and white pepper could be considered as poor substrates for fungal growth and aflatoxin production.     

Characterization of the poly-β-1,6-N-acetylglucosamine polysaccharide component of Burkholderia biofilms

We demonstrated the production of poly-β-1,6-N-acetylglucosamine (PNAG) polysaccharide in the biofilms of Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia cepacia, and Burkholderia cenocepacia using an immunoblot assay for PNAG. These results were confirmed by further studies, which showed that the PNAG hydrolase, dispersin B, eliminated immunoreactivity of extracts from the species that were tested (B. cenocepacia and B. multivorans). Dispersin B also inhibited biofilm formation and dispersed preformed biofilms of Burkholderia species. These results imply a role for PNAG in the maintenance of Burkholderia biofilm integrity. While PNAG was present in biofilms of all of the wild-type test organisms, a ΔpgaBC mutant of B. multivorans (Mu5) produced no detectable PNAG, indicating that these genes are needed for Burkholderia PNAG formation. Furthermore, restoration of PNAG production in PNAG negative E. coli TRXWMGΔC (ΔpgaC) by complementation with B. multivorans pgaBCD confirmed the involvement of these genes in Burkholderia PNAG production. While the confocal scanning laser microscopy of untreated wild-type B. multivorans showed thick, multilayered biofilm, Mu5 and dispersin B-treated wild-type biofilms were thin, poorly developed, and disrupted, confirming the involvement of PNAG in B. multivorans biofilm formation. Thus, PNAG appears to be an important component of Burkholderia biofilms, potentially contributing to its resistance to multiple antibiotics and persistence during chronic infections, including cystic fibrosis-associated infection.     

A microplate spectrofluorometric assay for bacterial biofilms

A spectrofluorometric assay was developed for quantification of bacterial biofilms grown on a microtiter plate. The method involved staining biofilms formed by gram-negative and gram-positive bacteria with wheat germ agglutinin-Alexa Fluor 488 conjugate, which selectively binds to N-acetylglucosamine residues in biofilms. The fluorescence of stained biofilms was measured with a fluorescent plate reader. This method was compared with a widely used microplate colorimetric assay involving crystal violet staining of biofilms formed by both gram-negative and gram-positive bacteria. A strong linear association existed between the two methods (r ²=0.99/0.94). Being more sensitive and specific as compared to colorimetric method, the spectrofluorometric assay provides a better alternative for quantification and characterization of bacterial biofilms.     

Pigment production in Spirulina fussiformis in different photophysical conditions

The present investigation makes a comparative investigation of individual light source on the different commercially important pigments in Spirulina fussiformis in photobioreactor culture condition. Continuous culture system was carried out throughout the experimental condition. Initially, seed culture, corresponding to 0.2 g/L on dry weight basis was cultivated in Zarrouks medium with different colored light source in reactor. Maximum daily biomass productivity, 0.8 g/L, 0.75 g/L and 0.69 g/L in white light (WL), blue light (BL) and green light (GL), respectively, conditions was noticed. Pigment content during WL treatment showed the highest accumulation (5.5 microg/mL) of chlorophyll whereas, other pigments roughly remained constant without much change, implying WL intensity is better for chlorophyll synthesis. On the other hand, chlorophyll and phycocyanin content gradually increased up to 7 microg/mL and 2 mg/mL, respectively, at BL intensity. The response to GL was negative to all pigments studied except for phycocyanin; in this case a highest production (2.5 mg/mL) was seen during 18 days experimental period. Additionally, when yellow light (YL) treatment experiments were conducted, the rate of production gradually decreased from 6th day onward in all pigments demonstrating the photobleaching effect of YL. The average rate of pigments production did not show significant accumulation in red light (RL) light treatment except phycoerythrin which showed an increasing trend of production. It is worth to mention here that higher light intensity is better for production of phycocyanin and phycoerythrin in Spirulina.     

Oleic acid prevents apoptotic cell death induced by <Emphasis Type="Italic">trans</Emphasis>10, <Emphasis Type="Italic">cis</Emphasis>12 isomer of conjugated linoleic acid via p38 MAP kinase dependent pathway

Our previous study indicated that oleic acid prevented apoptotic cell death induced by trans10, cis12 (t10, c12)-conjugated linoleic acid in rat hepatoma dRLh-84 cells. The intracellular mechanism of action oleic acid is still unknown. Here, we showed that p38 mitogen-activated protein kinase (MAPK) inhibition using its specific inhibitor SB203580 cancelled the ameliorative effect of oleic acid on the cytotoxicity of t10, c12-conjugated linoleic acid. In addition, SubG1 cell population analysis showed that p38 MAPK played an essential role in the prevention of apoptotic cell death by oleic acid. In fact, p38 phosphorylation level was upregulated in cells treated with oleic acid irrespective of t10, c12-conjugated linoleic acid stimulation. Interestingly, t10, c12-conjugated linoleic acid increased intracellular triglyceride accumulation. However, oleic acid completely inhibited this effect. These observations indicated the involvement of blockade of a p38 MAPK pathway in the ameliorative effect of oleic acid on apoptosis induced by t10, c12-conjugated linoleic acid.