Alkaline proteases and thermostable α-amylase co-produced by Bacillus licheniformis NH1: Characterization and potential application as detergent additive

Alkalophilic Bacillus licheniformis NH1 strain produced at least five major extracellular proteases and a unique amylase as showed by zymography technique. The optimum pH and temperature for the proteolytic activity were 10.0 and 70 °C, respectively, while those of amylolytic activity were 6.5 and 90 °C, respectively. The alkaline proteases and thermostable α-amylase showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40 °C, and relative stability towards oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various solid and liquid detergents. Wash performance analysis revealed that the NH1 crude enzyme could effectively remove a variety of stains, such as blood, chocolate and barbecue sauce. Considering its promising properties, B. licheniformis NH1 crude enzyme containing both α-amylase and proteases activities may be considered a potential candidate for future use in detergent processing industries.     

Inhibition of the calcium release-activated calcium (CRAC) current in Jurkat T cells by the HIV-1 envelope protein gp160.

The HIV-1 envelope glycoprotein gp120/160 has pleiotropic effects on T cell function. We investigated whether Ca(2+) signaling, a crucial step for T cell activation, was altered by prolonged exposure of Jurkat T cells to gp160. Microfluorometric measurements showed that Jurkat cells incubated with gp160 had smaller (approximately 40%) increases in [Ca(2+)](i) in response to phytohemagglutinin and had a reduced Ca(2+) influx (approximately 25%). gp160 had similar effects on Jurkat cells challenged with thapsigargin. We used the patch clamp technique to record the Ca(2+) current, which is responsible for Ca(2+) influx and has properties of the calcium release-activated Ca(2+) current (I(CRAC)). gp160 reduced I(CRAC) by approximately 40%. The inhibitory effects of gp160 were antagonized by staurosporine (0.1 microm), an inhibitor of protein-tyrosine kinases and protein kinase Cs (PKCs), and by G? 6976 (5 microm), an inhibitor acting especially on PKC alpha and PKC beta I. 12-O-Tetradecanoyl phorbol 13-acetate (16 nm), a PKC activator, reproduced the effects of gp160 in untreated cells. A Western blotting analysis of PKC isoforms alpha, beta I, delta, and zeta showed that only the cellular distribution of PKC alpha and -beta I were significantly modified by gp160. In addition, gp160 was able to modify the subcellular distribution of PKC alpha and PKC beta I caused by phytohemagglutinin. Therefore the reduction in I(CRAC) caused by prolonged incubation with gp160 is probably mediated by PKC alpha or -beta I.     

Elastokine-mediated up-regulation of MT1-MMP is triggered by nitric oxide in endothelial cells

Membrane-type I matrix metalloproteinase (MT1-MMP) has been previously reported to be up-regulated in human microvascular endothelial cell-1 line (HMEC) by elastin-derived peptides (elastokines). The aim of the present study was to identify the signaling pathways responsible for this effect. We showed that elastokines such as (VGVAPG)₃ peptide and kappa elastin induced nitric oxide (NO) production in a time-, concentration- and receptor-dependent manner as it could be abolished by lactose and a receptor-derived competitive peptide. As evidenced by the use of NO synthase inhibitors, elastokine-mediated up-regulation of MT1-MMP and pseudotube formation on Matrigel required NO production through activation of the PI₃-kinase/Akt/NO synthase and NO/cGMP/Erk1/2 pathways. Elastokines induced both PI₃-kinase p110γ sub-unit, Akt and Erk1/2 activation, as shown by a transient increase in phospho-Akt and phospho-Erk1/2, reaching a maximum after 5 and 15 min incubation, respectively. Inhibitors of PI₃-kinase and MEK1/2 suppressed elastokine-mediated MT1-MMP expression at both the mRNA and protein levels, and decreased the ability of elastokines to accelerate pseudotube formation. Besides, elastokines mediated a time- and concentration-dependent increase of cGMP, suggesting a link between NO and MT1-MMP expression. This was validated by the use of a guanylyl cyclase inhibitor, a NO donor and a cGMP analog. The guanylyl cyclase inhibitor abolished the stimulatory effect of elastokines on MT1-MMP expression. Inversely, the cGMP analog, mimicked the effect of both elastokines and NO donor in a concentration- and time-dependent manner. Overall, our results demonstrated that such elastokine properties through NO and MT1-MMP may be of importance in the context of tumour progression.     

3-D structure modelling of the Staphylococcus simulans lipase: conformational changes, substrate specificity and novel structural features

We have modelled, using the CHARMM27 energy force field, the structures of closed and open forms of Staphylococcus simulans lipase (SSL) on the basis of the crystal structures of Bacillus stearothermophilus and Staphylococcus hyicus lipases, respectively. The models suggested the presence of a main lid and a second lid that may act with the former as a double door to control the access to the active site. Superimposition of both closed and open forms of SSL allowed us to determine the hinge regions allowing the movements of the main and the second lid upon lipase activation. The flexibility of these hinge regions was checked by molecular dynamics simulations. The SSL models also allowed us to identify key residues involved in binding substrates, calcium or zinc ions.     

Preparation and testing of Sardinella protein hydrolysates as nitrogen source for extracellular lipase production by Rhizopus oryzae

Various fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were used as nitrogen sources for the production of extracellular lipase by the filamentous fungus Rhizopus oryzae. The best results were obtained with defatted meat–fish protein hydrolysates (DMFPH), indicating the presence in the lipid fraction of some constituents which may repress lipase synthesis. Furthermore, it was found that the extensive hydrolysis of fish proteins resulted in a higher lipase production. The use of 40 g DMFPH l^–1 for the growth of Rhizopus oryzae in medium R1 resulted in a lipase production of 394 U ml^–1, higher than the yield obtained with standard soy peptone as nitrogen source (373 U ml^–1). The most appropriate medium for the growth and the production of lipase is composed only of 24 g DMFPH l^–1 and 10 g glucose l^–1, indicating that the strain can obtain its nitrogen and salts requirements directly from fish substrate.     

Contribution of protein kinase A and protein kinase C pathways in ultraviolet B-induced IL-8 expression by human keratinocytes

We have previously demonstrated that treatment of the human keratinocyte cell line NCTC 2544 with a UVB dose equivalent to 1h exposure (100 mJ/cm2) results in a significant increase of IL-8 production. In this study, we use specific inhibitors to investigate the role of both PKA- and PKC-mediated pathways in the regulation of UVB-induced IL-8 expression in NCTC 2544 cell line. We show here that the treatment of irradiated human keratinocytes with PKA inhibitors [H89 and PKA inhibitor (PKAi)] induced a significant decrease of IL-8 production at both mRNA and protein levels. However, the regulation of IL-8 production seems to be mediated via a cAMP-independent PKA pathway, since drugs known to enhance cAMP concentrations [PGE2, cholera toxin and dibutyryl cAMP] decrease IL-8 production in irradiated cells by down-regulating NF-kappa B activation in response to UVB radiation. Using PMA (a potent pharmacological activator of PKC) and calphostin C (a specific PKC inhibitor), we demonstrated an up-regulation of IL-8 in NCTC 2544 cells and a down-regulation of the cytokine in UVB-irradiated cells, respectively. We also observed that in our experimental conditions, staurosporine, an inhibitor of both PKC and PMA-stimulated cellular responses, does not involve PKC inhibition in irradiated cells and significantly decreased NF-kappa B activity in response to UVB radiation. Finally, we concluded that a cAMP-independent PKA activation and a PKC-associated pathway are probably involved in the regulation of UVB-induced IL-8 synthesis in human keratinocytes.     

Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains

Non-nodulating Agrobacterium-like strains identified among root nodule isolates of common bean were labeled with gusA, a reporter gene encoding beta-glucuronidase (GUS). Bean plants were then co-inoculated with an infective Rhizobium strain and labeled transconjugants of Agrobacterium-like strains. Blue staining of nodules showed that Agrobacterium-like strains were able to colonize these symbiotic organs. Isolation and characterization by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes revealed a mixed population of Rhizobium and Agrobacterium-like strains in all nodules showing GUS activity. PCR amplification of the nifH gene and nodulation tests did not show any evidence of acquisition of symbiotic gene by lateral transfer from Rhizobium to Agrobacterium-like strains. Moreover, these strains were able to invade mature nodules. Based on sequencing of the 16S rRNA gene, one of these Agrobacterium-like strains showed 99.4% sequence similarity with Agrobacterium bv. 1 reference strains and 99% similarity with an Agrobacterium bv. 1 strain isolated from Acacia mollisima in Senegal. Agrobacterium tumefaciens C58 and the disarmed variant AT123 did not show any ability to colonize nodules. Co-inoculation of bean seeds with Agrobacterium and Rhizobium strains did not enhance nodulation and plant yield under controlled conditions.     

Influence of oxygen supply on the stability of recombinant plasmid pTG201 in immobilized E. coli cells

Summary Cells of Escherichia coli K12, carrying the recombinant plasmid pTG201, were immobilized in -carrageenan gel in order to improve the following plasmid parameters: (i) maintenance of a high level of plasmid copy number, (ii) good plasmid stability and (iii) good expression of plasmid encoded gene. The experiments were carried out on LB medium without antibiotic selection in continuous and batch cultures supplied with air or pure oxygen. Parallel experiments with free cells were also performed. In all the cases immobilized cells presented better plasmid stability parameters than free cells. Best results were obtained with immobilized cells supplied with pure oxygen. In this case, an average plasmid copy number of 60 and a value of plasmid-carrying cells close to 100% were maintained with little change during more than 200 generations. In addition, an optical microscopy analysis is proposed to allow the quantitation of cell growth in gel beads.     

Behavior of aldehyde moieties involved in the activation of suppressor cells by sodium periodate

The treatment of mouse spleen cells with periodate at the optimal mitogenic concentration (1 mM) induces the activation of suppressor cells of the in vitro antibody response and leads to the formation of aldehydes on the carbohydrate termini of the surface sialoglycoconjugates. These aldehyde moieties are found on the C8 (N-AN 8) and the C7 (N-AN 7) derivatives of sialic acid. Immediate borohydride reduction prevents the activation of the suppressor cells. Data from this work show that borohydride reduction must be performed within the first 6 hr to prevent the generation of suppressor cells; 18 hr after the initial periodate oxidation, borohydride treatment did not reverse the in vitro suppressive activity of periodate-treated cells. The kinetics of the disappearance of aldehydes from the cell surface were studied by using [3H]borohydride labeling and chromatographic analysis of sialic acid derivatives. About 70 to 80% of the aldehyde moieties were found to be present 6 hr after periodate oxidation. After 18 hr, 50 to 70% of the aldehyde had disappeared from the lymphocyte membrane. Oxidized sialyl residues disappear completely after 60 hr of culture. This period corresponds to the de novo synthesis of sialic acid residues on the surface of periodate-activated cells. The two classes of oxidized sialyl-glycoconjugates were found to behave in different ways. In effect, our data showed that the aldehydes remaining at 18 hr are mainly located on the gangliosides, whereas the aldehyde moieties located on high m.w. glycoproteins disappear from the cell surface between 9 and 18 hr. This would suggest that the remaining aldehydes located on gangliosides are not directly involved in the expression of suppressive activity.