Reduction In Rate Of Inactivation Research Articles

The impact of bacterial cell aggregation on UV inactivation kinetics

Reconditioning of food processing water streams for reuse is an increasingly common water management practice in the food industry and UV disinfection is often employed as part of the water treatment. Several factors may impact the effect of UV radiation. Here, we aim to assess the impact of cell aggregation on UV inactivation kinetics and investigate if UV exposure induces aggregation. Three strains, isolated from food processing water reuse lines (Raoultella ornithinolytica, Pseudomonas brenneri, Rothia mucilaginosa) and both an aggregating and a non-aggregating strain of Staphylococcus aureus were exposed to UVC light at 255 nm using UV LED equipment. Total Viable Count and phase-contrast microscopy, coupled with image analysis, were used to compare the UV inactivation kinetics with the average particle size for a range of UV doses. Tailing effect, seen as a strong reduction in inactivation rate, was observed for all strains at higher UV doses (industrial strains ≥ 50 or 120 mJ/cm2, S. aureus strains ≥ 40 or 60 mJ/cm2). The naturally aggregating strains were more UV tolerant, both within and between species. When aggregates of S. aureus were broken, UV tolerance decreased. For the processing water isolates, the lowest applied UV dose (25 mJ/cm2) significantly increased the average particle size. Application of higher UV doses obtained with longer exposure times did not further increase the particle size compared with untreated samples. For the S. aureus strains, however, no consistent change in average particle size was observed due to UV. Our results demonstrate that aggregating strains have a higher degree of protection and that UV radiation induces aggregation in some, but not all bacteria. A better understanding of the mechanisms governing microbial aggregation and survival during UV treatment could help to improve UV applications and predictions of microbial inactivation.

Stabilizing effect of Ca2+ on myosin and myofibrils of squid mantle muscle as affected by heating conditions

The suppressive effects of Ca2+ on the thermal denaturation of myosin and myofibrils of squid mantle muscle were compared. The stabilization effect on myosin was smaller than that on myofibrils and was not affected by KCl concentration. The stabilizing effect of Ca2+ on myosin decreased as the heating temperature dropped, showing no stabilization at 20°C, while the effect on myofibrils was the same at all temperatures tested. The stabilizing effect of Ca2+ on myosin disappeared even at 30°C in the presence of sorbitol, where a small inactivation rate was found, while the effect of Ca2+ on myofibrils was equally detected irrespective of the reduction in inactivation rate in the presence of sorbitol. Stabilization of myosin by Ca2+ again appeared even in the presence of sorbitol when the heating temperature was raised to 38°C. It was suggested that Ca2+ confers stabilization on myosin only when myosin is under unstable conditions. The stabilization effect of Ca2+ on myosin was enhanced upon F-actin binding: Ca2+-bound myosin was more significantly stabilized by F-actin binding, and the effect was no longer affected by the conditions for heating.

A reappraisal of the Ca2+ dependence of fast inactivation of Ca2+ release in frog skeletal muscle

Two procedures to inhibit Ca(2+) release designed to differentiate between local and common pool mechanisms for the Ca(2+) dependent, fast inactivation of Ca(2+) release in skeletal muscle of the frog were compared. Inhibition by voltage dependent inactivation of Ca(2+) release, without modification of the single channel current of the Ryanodine Receptor (RyR) and the [Ca(2+)] close to the open pore, produced a reduction in the rate of inactivation linearly related to the reduction in the peak of Ca(2+) release flux. Linear fits in the individual fibers were performed, giving average values (+/-SEM, N = 8) of the best fit parameters of 5.75 x 10(-3) +/- 7.35 x 10(-4 )microM(-1) for the slope and 0.07 +/- 0.015 ms(-1) for the ordinate intercept. Inhibition of Ca(2+) release by reducing the Ca content of the sarcoplasmic reticulum (SR) involves reduction of the Ca(2+) current through the single RyR. The reduction in rate of inactivation also followed linearly the reduction in Ca(2+) peak release flux. The average values (+/-SEM) of the best fit parameters of linear fits were 14 x 10(-3) +/- 3.76 x 10(-3 )microM(-1) and 0.019 +/- 0.006 ms(-1) (N = 7) for slope and ordinate intercept respectively. The differences between both parameters were statistically significant (by t test, at P = 0.05). The extent of inactivation, measured by the peak/final Ca(2+) release flux ratio, was differentially affected by the two procedures. Inhibition by voltage dependent inactivation, despite slowing down the fast inactivation, increased the peak/final Ca(2+) release flux ratio. In contrast, depletion of the SR reticulum reduced it. If the fast inactivation is driven by the high [Ca(2+)] attained locally, close to the open pore of the RyR, the inhibition of Ca(2+) release due to voltage dependent inactivation should not modify the rate of inactivation while inhibition by SR Ca(2+) depletion should reduce it. A process driven by [Ca(2+)] in a common pool should depend on the overall Ca(2+) release independently of how it was modified. In this case both inhibitory procedures should reduce the inactivation rate similarly. Our findings are generally consistent with a common pool process. The differences between the two protocols could be understood if the organization of RyR in junctional and parajunctional release units is considered.

Rare Missense Mutations in the Calcium Channel β2 Subunit of Autistic Patients

Calcium channels are crucially involved in brain development and neuronal function. Mutations in the pore-forming Cav subunits of high- and low-voltage dependent calcium channels (VDCC) have been found in patients with autistic syndromes (Splawski et al., Cell 2004;119:19-31; Splawski et al., PNAS 2005;102:8089-96; Splawski et al., JBC 2006;281:22085-91). In Timothy syndrome, the G406R mutation of Cav1.2 results in a reduction of inactivation rate. Such biophysical effects can likewise be induced by the influence of auxiliary VDCC β subunits. For instance, we demonstrated a novel mechanism of β subunit modulation: the inactivation of VDCC is under length-dependent control of the β2 subunit N terminus (Herzig et al., FASEB J 2007;21:1527-38). A similar mechanism operates with the β1 subunit (Jangsangthong et al., Pflugers Arch 2009, in press).Therefore, the β2-subunit gene was screened for mutations in 155 patients with Autistic Spectrum Disorder (ASD). We detected several new variations and compared the genotypes with 375 matching controls. (Male to female ratio for both groups is 1:4.). Statistical analysis of preselected variations showed two significant SNPs in functional intronic regions (χ2 p = 5x10−6 and χ2 p =9x10−3).Furthermore, we also identified several rare ASD-specific missense mutations at the gene locus of the β2 subunit. These mutations occur in highly conserved domains and may lead to alterations in the β2 subunit function, e.g. by interfering with subunit phosphorylation. The affected amino acids are highly conserved among species, suggesting an importance for topology and function of the subunit. We will clone these variations into expression vectors and characterize their functional effects by electrophysiological studies. These studies may provide new insights into molecular mechanisms leading to ASD.

Inactivation of wheat-germ aspartate transcarbamoylase by the triazinyl dye, Procion Red HE3B

Aspartate transcarbamoylase from wheat germ is irreversibly inactivated by the triazinyl dye Procion Red HE3B. Since triazinyl dyes may mimic nucleotides, and UMP is a known allosteric modifier of this enzyme, the reaction was studied to elucidate whether the dye is an ‘affinity label’ for the enzyme. The reaction is apparently first order in the first 5–10 min, but is more complex in the longer term and does not go to completion. Kinetic analysis of the initial phase suggests that there are two parallel reactions, one saturable (dye binds reversibly before reaction) and one non-saturable (bimolecular). The apparent rate constant Kapp (i.e. the sum of the rate constants for the parallel reactions) varies only slightly over the pH range 7–10. In the presence of a number of active centre ligands, as well as the allosteric ligand UMP, there is a clear increase in Kapp. This finding is contrary to the reduction in rate of inactivation (protection) normally provided by ligands against active-site directed reagents, suggesting that in the saturable reaction, there is a conformational change upon dye-binding that increases the exposure of the essential residue(s) with which the dye reacts. These results show that, although it probably inactivates by reaction with specific amino-acid residues, the dye is not bound at the substrate-binding or allosteric sites, i.e. it is not an affinity-labelling reagent in the usual sense.

J. Mar. Biol. Assoc., U.K. : Southward A.J., 1962. The distribution of some plankton animals in the English Channel and approaches. II. Surveys with the Gulf III high-speed sampler, 1958–1960 42 (2): 275–375

Reconditioning of food processing water streams for reuse is an increasingly common water management practice in the food industry and UV disinfection is often employed as part of the water treatment. Several factors may impact the effect of UV radiation. Here, we aim to assess the impact of cell aggregation on UV inactivation kinetics and investigate if UV exposure induces aggregation. Three strains, isolated from food processing water reuse lines (Raoultella ornithinolytica, Pseudomonas brenneri, Rothia mucilaginosa) and both an aggregating and a non-aggregating strain of Staphylococcus aureus were exposed to UVC light at 255 nm using UV LED equipment. Total Viable Count and phase-contrast microscopy, coupled with image analysis, were used to compare the UV inactivation kinetics with the average particle size for a range of UV doses. Tailing effect, seen as a strong reduction in inactivation rate, was observed for all strains at higher UV doses (industrial strains ≥ 50 or 120 mJ/cm2, S. aureus strains ≥ 40 or 60 mJ/cm2). The naturally aggregating strains were more UV tolerant, both within and between species. When aggregates of S. aureus were broken, UV tolerance decreased. For the processing water isolates, the lowest applied UV dose (25 mJ/cm2) significantly increased the average particle size. Application of higher UV doses obtained with longer exposure times did not further increase the particle size compared with untreated samples. For the S. aureus strains, however, no consistent change in average particle size was observed due to UV. Our results demonstrate that aggregating strains have a higher degree of protection and that UV radiation induces aggregation in some, but not all bacteria. A better understanding of the mechanisms governing microbial aggregation and survival during UV treatment could help to improve UV applications and predictions of microbial inactivation.