Blockade of maitotoxin-induced endothelial cell lysis by glycine and L-alanine

The maitotoxin (MTX)-induced cell deathcascade in bovine aortic endothelial cells (BAECs) is a model foroncotic/necrotic cell death. The cascade is initiated by an increase incytosolic free Ca²⁺ concentration([Ca²⁺]_i), which is followed by the biphasicuptake of vital dyes. The initial phase of dye entry reflectsactivation of large pores and correlates with surface membrane blebformation; the second phase reflects cell lysis. In the present study,the effect of the cytoprotective amino acid glycine was examined.Glycine had no effect on MTX-induced change in[Ca²⁺]_i or on the first phase of vital dyeuptake but produced a concentration-dependent (EC₅₀ ~1mM) inhibition of the second phase of dye uptake. No cytoprotectiveeffect was observed with L-valine, L-proline,or D-alanine, whereas L-alanine wasequieffective to glycine. Furthermore, glycine had no effect onMTX-induced bleb formation. To test the hypothesis that glycinespecifically blocks formation of a lytic "pore," the loss offluorescence from BAECs transiently expressing GFP and concatemers ofGFP ranging in size from 27 to 162 kDa was examined using time-lapsevideomicroscopy. MTX-induced loss of GFP was rapid, correlated with thesecond phase of dye uptake, and was relatively independent of molecularsize. The MTX-induced loss of GFP from BAECs was completely blocked byglycine. The data suggest that the second "lytic" phase ofMTX-induced endothelial cell death reflects formation of a novelpermeability pathway that allows macromolecules such as GFP or LDH toescape, yet can be prevented by the cytoprotective agents glycine andL-alanine.

     

Ionic-strength-dependent substrate inhibition of the lysis of Micrococcus luteus by hen egg-white lysozyme

The kinetics of lysis of Micrococcus luteus by hen egg-white lysozyme in dilute buffer media is characterized by pronounced substrate inhibition. This effect occurs within the complete pH range where lysozyme activity is detectable. The electrostatic potential of the negatively charged cell-wall proteoglycan increases with decreasing ionic strength, resulting in an enhanced affinity between proteoglycan and lysozyme and probably favouring multipoint substrate attachment. For the lysozyme-catalyzed hydrolysis of cell-wall proteoglycan three plausible mechanisms of substrate inhibition can be postulated. Two out of the three models fit our experimental data, the simplest of the two providing the most rigorous information on the kinetic parameters Km, V and Ki. Three graphical methods consistent with the chosen model were applied for preliminary parameter estimation and the constants obtained were compared to those from nonlinear least-squares analysis. If substrate inhibition is neglected it is shown that serious bias is imposed upon the parameters.     

Resistance of corynebacterial strains to infection and lysis by corynephage BFK 20

AIMS: Defence mechanisms of the corynebacterial strains against corynephage BFK 20, which causes lysis of Brevibacterium flavum CCM 251. METHODS AND RESULTS: We tested adsorption of the phage BFK 20 to the corynebacterial cell surface. We observed strong adsorption ranging from ca 79 to 93% on the cells of B. flavum ATCC strains, but only ca 76% for B. flavum CCM 251. Minor adsorption for Brevibacterium lactofermentum BLOB (ca 13%) and no adsorption for Corynebacterium glutamicum RM3 were determined. BFK 20 infection had no significant effect on growth and viability of C. glutamicum and B. lactofermentum, but significantly influenced growth and viability of B. flavum ATCC 21127, 21128 and 21474. Cell growth stopped in short time after infection but with no lysis. Brevibacterium flavum CCM 251 cell growth was arrested too and lysis occurred. The Southern hybridization confirmed the presence of significant amount of BFK 20 DNA in samples from B. flavum CCM 251 and B. flavum ATCC strains after BFK 20 infection. Only weak hybridization signal was detected for DNA from infected cells of B. lactofermentum BLOB and no signal for C. glutamicum RM3. CONCLUSIONS: Based on the above results we suggest presence of a mechanism leading to abortive infection in B. flavum ATCC 21127, 21128 and 21474. In B. lactofermentum BLOB and C. glutamicum RM3 the adsorption barrier is more likely. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increases the knowledge on defence mechanisms of corynebacteria against bacteriophages.