Inactivation Of Group Research Articles

Inactivation of Group I and Group II Clostridium botulinum spores by ultraviolet irradiation in water

Spores of Clostridium botulinum are widely distributed in the environment, including in foods. Prevention of foodborne botulism relies on the inhibition of spore germination and subsequent growth and toxin production, or the destruction of viable spores in food and beverages. This study examined the lethality of 254 nm UV radiation (UV-C) to spores of Group I and Group II C. botulinum. Spores of C. botulinum were inactivated by UV-C, with doses required for incremental log reduction (D10) values calculated using linear regression ranging from 2.87 to 3.70 mJ/cm2 for Group I strains and 4.46 to 6.15 mJ/cm2 for Group II strains. The measured D10 value for spores of C. sporogenes ATCC 19404 was 8.27 mJ/cm2 indicating it was more resistant than the strains of C. botulinum used in this study. Calculation of dose per log using a Weibull model resulted in higher D10 values of 6.67 to 8.81 mJ/cm2 for Group I strains and 9.24 to 10.7 mJ/cm2 for Group II strains. Spores of C. sporogenes possessed a D10 value of 14.4 mJ/cm2. The higher values for the Weibull model indicate the Weibull model to be more conservative as a result as it factors in the lag prior to inactivation and the tailing observed with very low numbers of survivors. Spores of both Group I and Group II C. botulinum strains tended to form large aggregates, visible with phase contrast microscopy, that resulted in severe tailing. Disruption of aggregates by ultrasonication was necessary to obtain linear destruction curves extending beyond 5 log reduction. All strains from Group I and Group II required <55 mJ/cm2 to achieve 5 log inactivation. The strain of C. sporogenes used in this work can therefore be a conservative non-pathogenic surrogate, having higher UV-C resistance than the C. botulinum strains used in this study. Overall, this study is the first detailed study to demonstrate UV-C as an effective treatment method to inactivate C. botulinum spores in a suspending medium. In addition, the study paves the way for further studies towards the applications of this technology to inactivate C. botulinum spores in beverages or other liquids.

Inactivation of group 2 \u03c3 factors upregulates production of transcription and translation machineries in the cyanobacterium Synechocystis sp. PCC 6803

We show that the formation of the RNAP holoenzyme with the primary σ factor SigA increases in the ΔsigBCDE strain of the cyanobacterium Synechocystis sp. PCC 6803 lacking all group 2 σ factors. The high RNAP-SigA holoenzyme content directly induces transcription of a particular set of housekeeping genes, including ones encoding transcription and translation machineries. In accordance with upregulated transcripts, ΔsigBCDE contain more RNAPs and ribosomal subunits than the control strain. Extra RNAPs are fully active, and the RNA content of ΔsigBCDE cells is almost tripled compared to that in the control strain. Although ΔsigBCDE cells produce extra rRNAs and ribosomal proteins, functional extra ribosomes are not formed, and translation activity and protein content remained similar in ΔsigBCDE as in the control strain. The arrangement of the RNA polymerase core genes together with the ribosomal protein genes might play a role in the co-regulation of transcription and translation machineries. Sequence logos were constructed to compare promoters of those housekeeping genes that directly react to the RNAP-SigA holoenzyme content and those ones that do not. Cyanobacterial strains with engineered transcription and translation machineries might provide solutions for construction of highly efficient production platforms for biotechnical applications in the future.

The regulation and role of neuronal gap junctions during neuronal injury

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. In a recent study with the use of in vivo and in vitro models of cortical ischemia in mice, we have demonstrated that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluR). Specifically, we found that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein), whereas inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. Using the analysis of neuronal death, we also established that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemic neuronal death in vitro and in vivo. Similar results were obtained using in vitro models of TBI and epilepsy. Our study demonstrated that mechanisms for the injury-mediated increase in neuronal gap junction coupling are part of the mechanisms for glutamate-dependent neuronal death.

Neuronal gap junctions play a role in the secondary neuronal death following controlled cortical impact

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. Recent studies in mice showed a critical role for neuronal gap junctions in NMDA receptor-mediated excitotoxicity and ischemia-mediated neuronal death. Here, using controlled cortical impact (CCI) in adult mice, as a model of TBI, and Fluoro-Jade B staining for analysis of neuronal death, we set to determine whether neuronal gap junctions play a role in the CCI-mediated secondary neuronal death. We report that 24h post-CCI, substantial neuronal death is detected in a number of brain regions outside the injury core, including the striatum. The striatal neuronal death is reduced both in wild-type mice by systemic administration of mefloquine (a relatively selective blocker of neuronal gap junctions) and in knockout mice lacking connexin 36 (neuronal gap junction protein). It is also reduced by inactivation of group II metabotropic glutamate receptors (with LY341495) which, as reported previously, control the rapid increase in neuronal gap junction coupling following different types of neuronal injury. The results suggest that neuronal gap junctions play a critical role in the CCI-induced secondary neuronal death.

Neuronal Gap Junction Coupling Is Regulated by Glutamate and Plays Critical Role in Cell Death during Neuronal Injury

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI), and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. We report here that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluRs). Specifically, using electrotonic coupling, Western blots, and siRNA in the mouse somatosensory cortex in vivo and in vitro, we demonstrate that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36) (neuronal gap junction protein), and inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. We also show that the regulation is via cAMP/PKA (cAMP-dependent protein kinase)-dependent signaling and posttranscriptional control of Cx36 expression and that other glutamate receptors are not involved in these regulatory mechanisms. Furthermore, using the analysis of neuronal death, we show that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemia-mediated neuronal death in vitro and in vivo. Similar results are obtained using in vitro models of TBI and epilepsy. Our results indicate that neuronal gap junction coupling is a critical component of glutamate-dependent neuronal death. They also suggest that causal link among group II mGluR function, neuronal gap junction coupling, and neuronal death has a universal character and operates in different types of neuronal injuries.

Metaplasticity of Hypothalamic Synapses following In Vivo Challenge

Neural networks that regulate an organism's internal environment must sense perturbations, respond appropriately, and then reset. These adaptations should be reflected as changes in the efficacy of the synapses that drive the final output of these homeostatic networks. Here we show that hemorrhage, an in vivo challenge to fluid homeostasis, induces LTD at glutamate synapses onto hypothalamic magnocellular neurosecretory cells (MNCs). LTD requires the activation of postsynaptic alpha2-adrenoceptors and the production of endocannabinoids that act in a retrograde fashion to inhibit glutamate release. In addition, both hemorrhage and noradrenaline downregulate presynaptic group III mGluRs. This loss of mGluR function allows high-frequency activity to potentiate these synapses from their depressed state. These findings demonstrate that noradrenaline controls a form of metaplasticity that may underlie the resetting of homeostatic networks following a successful response to an acute physiological challenge.

Hemorrhage induced inactivation of presynaptic group III mGluRs controls metaplasticity in circuits regulating fluid balance

In response to perturbations in body fluid homeostasis, noradrenaline (NA) is released in the paraventricular nucleus (PVN) of the hypothalamus to modulate neuroendocrine output. Here, we use whole‐cell patch clamp recordings from magnocellular neurosecretory cells in the PVN to show that bath application of NA (5 min, 100 μM) induces a robust, fast‐acting, long‐term depression (LTD) at glutamatergic synapses. This LTD is accompanied by a loss of presynaptic signaling at group III mGluRs. Depressed, but not naïve glutamatergic synapses exhibit a rapid presynaptic potentiation in response to high frequency stimulation. To determine if a physiological stressor that activates noradrenergic fibers to the PVN could induce similar changes in synaptic strength, slices were taken from rats that were hemorrhaged (under anesthesia, removal of 25% blood volume via cardiac puncture). In hemorrhaged rats, glutamatergic synapses displayed a reduced release probability and were unresponsive to the mGluR agonist, L‐AP4. High frequency stimulation of these synapses induced a rapid LTP. The ability of synapses to switch between depressed and potentiated states may be an important mechanism for filtering irrelevant activity (i.e. emotional inputs) from activity critical to controlling cardiovascular homeostasis. This work was supported by the Canadian Institutes of Health Research.

Synaptically stimulated induction of group i metabotropic glutamate receptor-dependent long-term depression and depotentiation is inhibited by prior activation of metabotropic glutamate receptors and PROTEIN KINASE C

We have investigated metaplasticity of the group I metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) and depotentiation (DP) induced by physiological synaptic stimulation in the medial perforant path of the dentate gyrus in vitro. Group I mGluR-LTD/DP was inhibited by prior preconditioning brief high frequency stimulation (HFS) if the preconditioning HFS induced long-term potentiation (LTP) or if the induction of LTP was inhibited by an NMDA receptor antagonist. The inhibitory effect of the preconditioning HFS on LTD/DP was dependent upon activation of mGluRs, as it was blocked by the presence of the mGluR antagonist ( S)-α-methyl-4-carboxyphenylglycine during the preconditioning stimulation. The inhibitory effect of the preconditioning HFS involved stimulation of PKC, as the presence of the PKC inhibitor bisindolylmaleimide (BIS) during the preconditioning stimulation prevented the inhibitory effect of such preconditioning stimulation. Activation of PKC was also necessary for the induction of mGluR-LTD itself, as the PKC inhibitor BIS prevented the induction of the mGluR-LTD. We suggest that the physiological stimulation of mGluRs by the preconditioning stimulation produces a PKC-dependent inactivation of subsequent group I mGluR functioning and thereby an inhibition of induction of group I mGluR-dependent LTD/DP induction.

Functional elements of the strong psbAI promoter of Synechococcus elongatus PCC 7942.

The psbAI gene of the cyanobacterium Synechococcus elongatus PCC 7942 is one of three psbA genes that encode a critical photosystem II reaction center protein, D1. Regulation of the gene family in response to changes in the light environment is complex, occurs at transcriptional and posttranscriptional levels, and results in an interchange of two different forms of D1 in the membrane. Expression of psbAI is downregulated under high-intensity light (high light) in contrast to induction of the other two family members. We show that, in addition to a known accelerated degradation of the psbAI message, promoter activity decreases upon exposure to high light. Unlike the other psbA genes, additional sequences upstream of the psbAI -35 element are required for expression. Mutagenizing the atypical psbAI -10 element from TCTCCT to TATAAT increased the magnitude of expression from both psbAI::lacZ and psbAI::luxAB fusions but did not affect downregulation under high light. Inactivation of group 2 sigma factor genes rpoD2 and sigC, in both wild-type and -10-element mutagenized backgrounds, resulted in elevated psbAI::luxAB expression but did not alter the response to high light. The results are consistent with redundancy of promoter recognition among cyanobacterial group 2 sigma factors. Electrophoretic mobility shift assays showed that the DNA sequence corresponding to the untranslated leader of the psbAI message binds one or more proteins from an S. elongatus extract. The corresponding region of psbAII efficiently competed for this binding activity, suggesting a shared regulatory factor among these disparately regulated genes.

The mouse Ulnaless mutation deregulates posterior HoxD gene expression and alters appendicular patterning.

The semi-dominant mouse mutation Ulnaless alters patterning of the appendicular but not the axial skeleton. Ulnaless forelimbs and hindlimbs have severe reductions of the proximal limb and less severe reductions of the distal limb. Genetic and physical mapping has failed to separate the Ulnaless locus from the HoxD gene cluster (Peichel, C. L., Abbott, C. M. and Vogt, T. F. (1996) Genetics 144, 1757-1767). The Ulnaless limb phenotypes are not recapitulated by targeted mutations in any single HoxD gene, suggesting that Ulnaless may be a gain-of-function mutation in a coding sequence or a regulatory mutation. Deregulation of 5' HoxD gene expression is observed in Ulnaless limb buds. There is ectopic expression of Hoxd-13 and Hoxd-12 in the proximal limb and reduction of Hoxd-13, Hoxd-12 and Hoxd-11 expression in the distal limb. Skeletal reductions in the proximal limb may be a consequence of posterior prevalence, whereby proximal misexpression of Hoxd-13 and Hoxd-12 results in the transcriptional and/or functional inactivation of Hox group 11 genes. The Ulnaless digit phenotypes are attributed to a reduction in the distal expression of Hoxd-13, Hoxd-12, Hoxd-11 and Hoxa-13. In addition, Hoxd-13 expression is reduced in the genital bud, consistent with the observed alterations of the Ulnaless penian bone. No alterations of HoxD expression or skeletal phenotypes were observed in the Ulnaless primary axis. We propose that the Ulnaless mutation alters a cis-acting element that regulates HoxD expression specifically in the appendicular axes of the embryo.

Effects of pentoxifylline on in vivo leukocyte function and clearance of group B streptococci from preterm rabbit lungs

Pentoxifylline was evaluated for its ability to enhance inactivation of group B streptococci in lungs of prematurely born rabbits. Mechanisms associated with intrapulmonary streptococcal clearance and the pharmacodynamics of pentoxifylline were also investigated. Randomized, controlled animal trial. University research laboratory. A total of 123 New Zealand rabbits were delivered prematurely by cesarean section and were used for clearance studies. Twenty-three preterm pups were additionally utilized to study the pharmacodynamics of pentoxifylline. Preterm rabbits were infected with group B streptococcal aerosols and given intraperitoneal injections of either pentoxifylline (25, 12.5, and 12.5 mg/kg) or placebo at 0, 6, and 12 hrs after infection. At 0, 4, and 24 hrs, the numbers of streptococci were determined in the left lung, while the right lung underwent bronchoalveolar lavage to quantify intra-alveolar leukocytes, phagocytosis of inhaled bacteria, and concentrations of lysozyme and tumor necrosis factor. In a separate experiment, blood and bronchoalveolar fluid from infected animals were analyzed for pentoxifylline content. Streptococcal proliferation was less in pentoxifylline-treated animals than in controls at 24 hrs (p < .01). Pulmonary macrophages and polymorphonuclear leukocytes recovered from bronchoalveolar lavage fluid did not differ in numbers or phagocytic activity. Pentoxifylline-treated animals had lower levels of lysozyme (p < .02) and tumor necrosis factor (p < .005) in bronchoalveolar lavage fluid compared with placebo-treated pups. Therapeutic levels of pentoxifylline were achieved in blood and bronchoalveolar lavage fluid. Despite lowering lysozyme and tumor necrosis factor content in epithelial lining fluid, pentoxifylline improves the inactivation of group B streptococci in preterm rabbit lungs. These findings suggest that increased group B streptococcal clearance was coincident with an anti-inflammatory effect due to pentoxifylline. We conclude pentoxifylline may be clinically useful as an adjunctive therapy for group B streptococcal pneumonia in newborns.

The inactivation of Group B arthropod-borne animal viruses by proteases

Trypsin, chymotrypsin, and papain inactivate both the hemagglutinin and the infectivity of Group B viruses but not of Group A viruses under the conditions employed. Evidence has been presented that the inactivation is the result of a proteolytic action on the virus particles.

On the Identity of the “Virulence Enhancement” Factor in Gastric Mucin

Summary Purified blood group A substance isolated from hog gastric mucin possesses the property of enhancing markedly the virulence of S. typhosa for mice. Inactivation of blood group A substance by methods which result in the loss of its ability to inhibit A isohemagglutination also results in loss of its virulence enhancement activity. The virulence-enhancing action of blood group A substance is considerably augmented by the inorganic fraction of mucin. Thus, 1.5 per cent of blood group A substance combined with 0.5 per cent mucin ash enhances the virulence of S. typhosa to a degree which compares favorably with that of 3 per cent blood group A substance or 5 per cent mucin. It is suggested that the general phenomenon of virulence enhancement by gastric mucin may be attributed to blood group A substance augmented by inorganic salts.