Scale-up of naringinase production process based on the constant oxygen transfer rate for a novel strain of Bacillus methylotrophicus

Naringinase bioprocess based on Bacillus methylotrophicus was successfully scaled up based on constant oxygen transfer rate (OTR) as the scale-up criterion from 5-L bioreactor to 20-L bioreactor. OTR was measured in 5 and 20-L bioreactor under various operating conditions using dynamic method. The operating conditions, where complete dispersion was observed were identified. The highest OTR of 0.035 and 0.04?mMol/L/s was observed in 5 and 20-L bioreactor, respectively. Critical dissolved oxygen concentration of novel isolated strain B. methylotrophicus was found to be 20% of oxygen saturation in optimized medium. The B. methylotrophicus cells grown on sucrose had maximum oxygen uptake rate of 0.14?mMol/L/s in optimized growth medium. The cells produced the maximum naringinase activity of 751 and 778?U/L at 34?hr in 5 and 20-L bioreactors, respectively. The maximum specific growth rate of about 0.178/hr was observed at both the scales of operations. The maximum naringinase yield of 160 and 164?U/g biomass was observed in 5 and 20-L bioreactors, respectively. The growth and production profiles at both scales were similar indicating successful scale-up strategy for B. methylotrophicus culture.     

Apoptosis,cytokine and chemokine induction by non-structural 1 (NS1) proteins encoded by different influenza subtypes

Background

Influenza pandemic remains a serious threat to human health. Viruses of avian origin, H5N1, H7N7 and H9N2, have repeatedly crossed the species barrier to infect humans. Recently, a novel strain originated from swine has evolved to a pandemic. This study aims at improving our understanding on the pathogenic mechanism of influenza viruses, in particular the role of non-structural (NS1) protein in inducing pro-inflammatory and apoptotic responses.

Methods

Human lung epithelial cells (NCI-H292) was used as an in-vitro model to study cytokine/chemokine production and apoptosis induced by transfection of NS1 mRNA encoded by seven infleunza subtypes (seasonal and pandemic H1, H2, H3, H5, H7, and H9), respectively.

Results

The results showed that CXCL-10/IP10 was most prominently induced (> 1000 folds) and IL-6 was slightly induced (< 10 folds) by all subtypes. A subtype-dependent pattern was observed for CCL-2/MCP-1, CCL3/MIP-1α, CCL-5/RANTES and CXCL-9/MIG; where induction by H5N1 was much higher than all other subtypes examined. All subtypes induced a similar temporal profile of apoptosis following transfection. The level of apoptosis induced by H5N1 was remarkably higher than all others. The cytokine/chemokine and apoptosis inducing ability of the 2009 pandemic H1N1 was similar to previous seasonal strains.

Conclusions

In conclusion, the NS1 protein encoded by H5N1 carries a remarkably different property as compared to other avian and human subtypes, and is one of the keys to its high pathogenicity. NCI-H292 cells system proves to be a good in-vitro model to delineate the property of NS1 proteins.

     

The effects of phorbol ester, diacylglycerol, phospholipase C and Ca2+ ionophore on protein phosphorylation in human and sheep erythrocytes.

Treatment of human or sheep erythrocytes with PMA (phorbol myristate acetate) enhanced [32P]phosphate labelling of membrane polypeptides of approx. 100, 80 and 46 kDa. The 80 kDa and 46 kDa polypeptides coincided with bands 4.1 and 4.9 respectively on Coomassie-Blue-stained gels. Similar but smaller effects were obtained by treating human cells with 1-oleoyl-2-acetyl-rac-glycerol (OAG), exogenous bacterial phospholipase C or ionophore A23187 + Ca2+, each of which treatments would be expected to raise the concentration of membrane diacylglycerol. In contrast, sheep cells, which do not increase their content of diacylglycerol when treated with phospholipase C or A23187 + Ca2+, only showed enhanced phosphorylation with OAG. Neither human nor sheep cells showed any enhanced [32P]phosphate labelling of phosphoproteins when treated with 1-mono-oleoyl-rac-glycerol. It is concluded that diacylglycerol from a variety of sources can activate erythrocyte protein kinase C, but that the most effective diacylglycerol is that derived from endogenous polyphosphoinositides. In contrast with bacterial phospholipase C and A23187, which stimulate synthesis of phosphatidate by increasing the cell-membrane content of diacylglycerol in human erythrocytes, PMA, OAG or 1-mono-oleoyl-rac-glycerol caused no change in phospholipid metabolism.     

Neurotoxic,cytotoxic, apoptotic and antiproliferative effects of some marine algae extracts on the NA2B cell line

Oxidative stress contributes to cancer pathologies and to apoptosis. Marine algae exhibit cytotoxic, antiproliferative and apoptotic effects; their metabolites have been used to treat many types of cancer. We investigated in culture extracts of Petalonia fascia, Jania longifurca and Halimeda tuna to determine their effects on mouse neuroblastoma cell line, NA2B. NA2B cells were treated with algae extracts, and the survival and proliferation of NA2B cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of algae extracts on oxidative stress in NA2B cells also were investigated using nitric oxide synthase (NOS) immunocytochemistry and apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling. We observed significant neurite inhibition with moderate damage by the neurotoxicity-screening test (NST) at IC₅₀ dilutions of the extracts. MTT demonstrated that J. longifurca extracts were more toxic than P. fascia and H. tuna extracts. We found an increase of endothelial and inducible NOS immunostaining for oxidative stress and TUNEL analysis revealed increased apoptosis after application of extract. Our findings suggest that the algae we tested may have potential use for treatment of cancer.     

Fermentation of raw glycerol to 1,3-propanediol by new strains ofClostridium butyricum

Industrial glycerol obtained through the transesterification process using rapeseed oil did not support growth of several strains ofClostridium butyricum obtained from bacterial culture collections. Ten new strains ofC. butyricum were obtained from mud samples from a river, a stagnant pond, and a dry canal. These new isolates fermented the commercial glycerol and produced 1,3-propanediol as a major fermentation product with concomitant production of acetic and butyric acids. Four of the ten isolates were able to grow on industrial glycerol obtained from rapeseed oil. One strain,C. butyricum E5, was very resistant to high levels of glycerol and 1,3-propanediol. Using fed-batch fermentation, 109 g L^–1 of industrial glycerol were converted into 58 g of 1,3-propanediol, 2.2 g of acetate and 6.1 g of butyrate per liter.     

Regulators of complement activity mediate inhibitory mechanisms through a common C3b‐binding mode

Regulators of complement activation (RCA) inhibit complement‐induced immune responses on healthy host tissues. We present crystal structures of human RCA (MCP, DAF, and CR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologous CCP domains (i–iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations in CCP domains explain the diverse C3b‐binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease‐related mutations and immune evasion.