Clostridium perfringens invasiveness is enhanced by effects of theta toxin upon PMNL structure and function: The role of leukocytotoxicity and expression of CD11/CD18 adherence glycoprotein

Abstract Clostridium perfringens infections are characterized by the lack of an inflammatory response at the site of infection and rapidly progressive margins of tissue necrosis. Studies presented here investigated the role of theta toxin from C. perfringens in the pathophysiology of these events. Mice passively immunized with neutralizing monoclonal antibody against theta toxin and challenged with an LD₁₀₀ of log phase C. perfringens had significantly less mortality than untreated controls. Intramuscular injection of killed, washed C. perfringens in mice induced a massive time-dependent influx of polymorphonuclear leukocytes (PMNL) into tissue; injection of either viable, washed C. perfringens or killed organisms plus theta toxin dramatically attenuated PMNL influx although PMNL accumulated in adjacent vessels. The anti-inflammatory effects could not be attributed to an absence of chemoattractants since C. perfringens proteins had chemotactic factor activity, and killed bacilli generated serum-derived chemotactic factors. Scanning and transmission electron microscopy demonstrated the dramatic leukocidal effects of high doses of theta toxin on PMNL. In contrast, sublethal concentrations of theta toxin primed PMNL chemiluminescence, disrupted PMNL cytoskeletal actin polymerization/disassembly, and stimulated functional upregulation of CD11b/CD18 adherence glycoprotein. In summary, these results demonstrate that theta toxin is an important virulence factor in C. perfringens infection. In a concentration-dependent fashion, theta toxin contributes to the pathogenesis of clostridial gangrene by direct destruction of host inflammatory cells and tissues, and by promoting dysregulated PMNL/endothelial cell adhesive interactions.     

Priming of associative long-term depression in the dentate gyrus by theta frequency synaptic activity.

Associative long-term synaptic depression (LTD) was investigated utilizing negatively correlated activity patterns in the medial and lateral perforant path inputs to the dentate gyrus in anesthetized rats. Normally only nonassociative, or heterosynaptic, LTD is elicited in naive pathways. We report here, however, that associative LTD in the lateral path is readily induced after being "primed" by a brief period of lateral path synaptic activity at a theta rhythm frequency (5 Hz). Priming of associative LTD lasts at least 2 hr and is not seen following priming activity at non-theta frequencies (1 and 15 Hz). N-methyl-D-aspartate receptor activation is critical for establishing the priming effect, but not for the subsequent induction of the associative LTD. These data suggest that theta rhythm activity in the dentate gyrus may predispose the system to a specific form of synaptic plasticity, associative LTD.     

High-frequency component of hippocampal theta activity

The signal corresponding to the second frequency group in spectra of the theta activity of a rat (a twofold theta-frequency harmonic with adjoning frequency components) and its regulation were studied. The level of the signal of the second frequency group was high in the theta activity diring natural orienting behavior and in desynchronized EEG during waking immobility. In some rats, the EEGs segments recorded during orienting behavior evoked by reticular stimulation contained an enhanced signal of the second frequency group (as compared to the signal of the first frequency group). The second frequency group could be also observed in the absence of the first. Estimation of signal dimension revealed two independent components in the theta activity with the high-level signal of the second frequency group. Time correlation between variations of signals of the first and second frequency groups in the theta activity accompanying natural orienting behavior was absent. These signals changed in different ways during transition from awake immobility of an animal to orienting behavior. It was concluded that in addition to the main component of the hippocampal theta activity, there exists the independently regulated twofold theta-freduency component.     

Effect of transection of afferent pathways on evoked potentials,theta rhythm,and neuronal activity in the hippocampus

The effect of destruction of septo-hippocampal and subiculo-hippocampal connections on the electrical activity in the hippocampus was studied in tubocurarized rabbits. The theta rhythm and the so-called "intracellular theta rhythm" [7] were found to disappear on destruction of the septo-hippocampal connections, other consequences being the loss of neuronal capacity for responding to sciatic nerve stimulation, loss of neuronal inhibitory responses, and total or partial suppression of the negative phase of the evoked potential (EP). Destruction of subiculo-hippocampal connections entails a decrease in amplitude of the theta rhythm recorded from the hippocampal surface, retention of the "intracellular theta rhythm," and a slight decrease in amplitude of both EP phases. The number of neurons failing to respond to sciatic stimulation is increased; the character of cellular response remains unchanged. It is hypothesized that hyperpolarization of hippocampal pyramidal neurons may be responsible for generating the negative phase of the EP, and that this phase and the hippocampal theta rhythm may be of a common origin.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 4, pp. 439–444, July–August, 1970.     

Substance P Protects Against Desensitization of the Nicotinic Response in Isolated Adrenal Chromaffin Cells

Substance P, a peptide endogenous to the splanchnic nerve, is known to inhibit the acetylcholine-and nicotine-induced release of catecholamines from isolated adrenal chromaffin cells. In the present study the effect of substance P on desensitization of catecholamine release from these cells was examined. Substance P (10(-5) M) completely protected against desensitization of catecholamine release produced by acetylcholine at 37 degrees C or 23 degrees C and by nicotine at 23 degrees C; substance P also afforded appreciable protection against nicotine-induced desensitization at 37 degrees C. The peptide had no effect on K+-induced desensitization of catecholamine release. Like substance P, d-tubocurarine also prevented nicotinic desensitization. Substance P prevented both of two components of nicotinic desensitization, i.e. the Ca2+-dependent component and the Ca2+-independent, depletion-independent component of desensitization. Substance P had little effect on subsequent catecholamine uptake, indicating that substance P's protection against desensitization is a result of facilitation of catecholamine release rather than inhibition of catecholamine reuptake. Nicotine-induced catecholamine release and nicotinic desensitization of catecholamine release were Na+-independent, although substance P's inhibition of nicotine-induced catecholamine release was reduced by extracellular Na+. These in vitro studies suggest a similar role for substance P in vivo: substance P's protection against nicotinic desensitization may ensure a maintained output of adrenal catecholamines during stress, when the splanchnic nerve releases large amounts of acetylcholine.     

Cold-labile hemolysin produced by limited proteolysis of theta-toxin from Clostridium perfringens

A nicked toxin whose hemolytic activity is temperature dependent was obtained by limited proteolysis of theta-toxin (Mr 54,000) with subtilisin. The nicked toxin (C theta) is a complex of two fragments: the N-terminal fragment (Mr 15,000) with basic isoelectric point and the C-terminal fragment (Mr 39,000) with the single cysteinyl residue of the toxin whose reduced form is essential for the hemolytic activity. C theta hemolyzes erythrocytes only at temperatures above 25 degrees C, whereas the native toxin hemolyzes them even at 10 degrees C. At temperatures below 25 degrees C, C theta does not hemolyze them although it does bind to membrane cholesterol and although no distinct difference was observed between the secondary structure of C theta and that of native toxin. It was found that C theta binds to the cells only in a reversible manner at low temperature, while the native one binds irreversibly to the cells within 10 min, which explains the cold lability of C theta on hemolysis. The structural basis of the cold lability was discussed through comparison of C theta with another nicked derivative of theta-toxin that was also obtained.     

The importance of timing in segregated theta phase-coupling for cognitive performance

Functional cortical circuits for central executive functions have been shown to emerge by theta (~6 Hz) phase-coupling of distant cortical areas. It has been repeatedly shown that frontoparietal theta coupling at ~0° relative phase is associated with recognition, encoding, short-term retention, and planning; however, a causal link has not been demonstrated so far. Here we used transcranial alternating current stimulation simultaneously applied at 6 Hz over left prefrontal and parietal cortices with a relative 0° ("synchronized" condition) or 180° ("desynchronized" condition) phase difference or a placebo stimulation condition, whereas healthy subjects performed a delayed letter discrimination task. We show that exogenously induced frontoparietal theta synchronization significantly improves visual memory-matching reaction times as compared to placebo stimulation. In contrast, exogenously induced frontoparietal theta desynchronization deteriorates performance. The present findings provide for the first time evidence of causality of theta phase-coupling of distant cortical areas for cognitive performance in healthy humans. Moreover, the results demonstrate the suitability of transcranial alternating current stimulation to artificially induce coupling or decoupling of behaviorally relevant brain rhythms between segregated cortical regions.     

Amino acid substitutions in alphaA and alphaC of Cyt2Aa2 alter hemolytic activity and mosquito-larvicidal specificity

Cyt2Aa2 produced by Bacillus thuringiensis subsp. darmstadiensis exhibits in vitro cytolytic activity against broad range of cells but shows specific in vivo toxicity against larvae of Dipteran insects. To investigate the role of amino acids in alphaA and alphaC of this toxin, 3 single-point mutants (A61C, S108C and V109A) were generated. All 3 mutant proteins were highly produced as inclusion bodies that could be solubilized and activated by proteinase K similar to that of the wild type. Hemolytic activity of A61C and S108C mutants was significantly reduced whereas the V109A mutant showed comparable hemolytic activity to the wild type. Interestingly, the A61C mutant exhibited high larvicidal activity to both Aedes aegypti and Culex quinquefasciatus. S108C and V109A mutants showed low activity against C. quinquefasciatus but relatively high toxicity to A. aegypti. These results demonstrated for the first time that amino acids in alphaA and alphaC are involved in the selectivity of the Cyt toxin to the targeted organism.     

Differential shaping of EEG theta rhythms

Heart rate, EEG, frontal EMG, and forearm EMG were recorded in 20 subjects for 3 baseline, 8 feedback, and 2 postbaseline sessions in order to compare two biofeedback methods of teaching subjects to increase theta EEG activity. Subjects were divided into high- and low-EMG groups. Five high-EMG subjects, and 5 low-EMG subjects then received 8 sessions of strictly theta feedback. The remaining 10 subjects, 5 from the high-EMG group, and 5 from the low-EMG group, received a “graduated” training which involved shaping the target response. This procedure consisted of 4 initial sessions of EMG feedback, followed by a second phase consisting of 4 sessions of theta feedback. Results showed a clear relationship between subjects' baseline frontal EMG levels and the effect of the training methods. Although subjects with high-EMG baseline increased their theta output only with the two-phase training, subjects with low-EMG baseline levels performed better when given theta feedback only. This result shows not only that amounts of theta can be reliably increased, but that training techniques should be adapted to the physiological characteristics of the individual—in this case, baseline levels of frontal EMG levels.     

Septal deafferentation produces continuous rhythmic slow activity (theta) in the rat hippocampus

Hippocampal EEG was recorded in behaving rats in which the brain stem afferents to the septal region were previously damaged. In these animals rhythmic slow activity (RSA or theta) was continuously present, including drinking and immobility. The average frequency of RSA, however, was significantly higher during running (8-9 Hz) than during drinking or awake motionless state (6-8 Hz). In normal rats irregular sharp waves, rather than RSA were present during drinking and immobility. The results suggest that brain stem afferents are necessary to suppress the rhythmic firing of septal "pacemaker" cells.     

Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase

Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 °C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 °C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40–60 °C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.     

PKC theta activity maintains normal quantal size in chromaffin cells

Protein kinase C (PKC) activity mediates multiple neurosecretory processes, but these are poorly understood due in part to the existence of at least 12 PKC isoforms. Using amperometry to record quantal catecholamine release from chromaffin cells, we found that both broad spectrum PKC antagonists and rottlerin, a selective inhibitor of the novel isoforms PKC θ and PKC δ, decreased quantal size and the number of secretory events recorded per stimulus. In contrast, drugs that selectively inhibit the atypical and conventional PKC isoforms had no effect on these parameters. While both PKC θ and δ were expressed in chromaffin cells, mice deficient for PKC θ, but not for PKC δ, exhibited lower quantal size than wild-type and were insensitive to rottlerin. Finally, an inhibitory PKC θ pseudosubstrate produced rottlerin-like responses in wild-type mice, indicating that the lack of rottlerin response in the PKC θ mutants was not the result of a form of compensation. These findings demonstrate neurosecretory regulation by a novel PKC isoform, PKC θ, and should contribute to defining mechanisms of activity-dependent regulation of neurosecretion.     

Locomotion-related oscillatory body movements at 6-12 Hz modulate the hippocampal theta rhythm

The hippocampal theta rhythm is required for accurate navigation and spatial memory but its relation to the dynamics of locomotion is poorly understood. We used miniature accelerometers to quantify with high temporal and spatial resolution the oscillatory movements associated with running in rats. Simultaneously, we recorded local field potentials in the CA1 area of the hippocampus. We report that when rats run their heads display prominent vertical oscillations with frequencies in the same range as the hippocampal theta rhythm (i.e., 6-12 Hz). In our behavioral set-up, rats run mainly with speeds between 50 and 100 cm/s. In this range of speeds, both the amplitude and frequency of the "theta" head oscillations were increasing functions of running speed, demonstrating that the head oscillations are part of the locomotion dynamics. We found evidence that these rhythmical locomotor dynamics interact with the neuronal activity in the hippocampus. The amplitude of the hippocampal theta rhythm depended on the relative phase shift with the head oscillations, being maximal when the two signals were in phase. Despite similarity in frequency, the head movements and LFP oscillations only displayed weak phase and frequency locking. Our results are consistent with that neurons in the CA1 region receive inputs that are phase locked to the head acceleration signal and that these inputs are integrated with the ongoing theta rhythm.     

Factors Affecting Activity and Stability of the Kluyveromyces lactis Killer Toxin

A number of physical parameters determining the activity and stability of the killer toxin produced by the yeast Kluyveromyces lactis have been investigated. The toxin was active over a relatively narrow pH range of 4.4 to 5.8, with a maximum at the lower end of the range. However, it was stable up to at least pH 8.0 but appeared to be irreversibly inactivated below pH 4.4. The toxin was stable at 40°C but rapidly inactivated at 50°C. Strong agitation caused the inactivation of the toxin in one medium but not another; this seemed to be due to oxygen-mediated disulfide bond formation, which could be prevented by sulfhydryl protecting agents.     

Mutagenesis of Trichoderma reesei endoglucanase I: impact of expression host on activity and stability at elevated temperatures

Background

Trichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60–70°C.

Results

A B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50–70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in T_m) and improved half-life at 60°C (t_1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t_1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes.

Conclusion

Structure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0118-z) contains supplementary material, which is available to authorized users.     

Alteration of phase–phase coupling between theta and gamma rhythms in a depression-model of rats

Alterations in oscillatory brain activity are strongly correlated with cognitive performance in various physiological rhythms, especially the theta and gamma rhythms. In this study, we investigated the coupling relationship of neural activities between thalamus and medial prefrontal cortex (mPFC) by measuring the phase interactions between theta and gamma oscillations in a depression model of rats. The phase synchronization analysis showed that the phase locking at theta rhythm was weakened in depression. Furthermore, theta-gamma phase locking at n:m (1:6) ratio was found between thalamus and mPFC, while it was diminished in depression state. In addition, the analysis of coupling direction based on phase dynamics showed that the unidirectional influence from thalamus to mPFC was diminished in depression state only in theta rhythm, while it was partly recovered after the memantine treatment in a depression model of rats. The results suggest that the effects of depression on cognitive deficits are modulated via profound alterations in phase information transformation of theta rhythm and theta-gamma phase coupling.     

Presynaptic NMDARs in the hippocampus facilitate transmitter release at theta frequency

A rise in [Ca(2+)](i) provides the trigger for neurotransmitter release at neuronal boutons. We have used confocal microscopy and Ca(2+) sensitive dyes to directly measure the action potential-evoked [Ca(2+)](i) in the boutons of Schaffer collaterals. This reveals that the trial-by-trial amplitude of the evoked Ca(2+) transient is bimodally distributed. We demonstrate that "large" Ca(2+) transients occur when presynaptic NMDA receptors are activated following transmitter release. Presynaptic NMDA receptor activation proves critical in producing facilitation of transmission at theta frequencies. Because large Ca(2+) transients "report" transmitter release, their frequency on a trial-by-trial basis can be used to estimate the probability of release, p(r). We use this novel estimator to show that p(r) increases following the induction of long-term potentiation.     

Enzyme-Linked Immunosorbent Assay of Substance P: A Study in the Eye

A solid phase enzyme-linked immunosorbent assay for quantitation of substance P is presented. The assay measures the capacity of soluble substance P to compete with the solid phase antigen for a limited quantity of specific substance P antibody. The solid-phase antigen consists of a synthetic substance P.poly-D-glutamic acid conjugate coated to polystyrene micro-ELISA plate wells. Soluble substance P and antibodies to substance P are first preincubated together and then added to the wells containing solid-phase antigen. Subsequently the wells are incubated with anti-antibodies conjugated to alkaline phosphatase. The wells are finally incubated with p-nitrophenyl phosphate an the absorbance is read in a spectrophotometer 16--24 hr after the start of the assay. The threshold for detection of substance P was 5--10 pg per well (0.25 ml). Substance P was extracted from rabbit eyes and the values obtained with the present method are compared with previously reported values based on radioimmunoassay.     

Cation-dependent substance P activation of the enzyme guanylate cyclase

Substance P enhanced guanylate cyclase (E.C.4.6.1.2) two- to fourfold in pancreas, small intestine, cerebellum, liver, kidney, and lung. Dose response relationship revealed that substance P caused a maximal augmentation of guanylate cyclase activity at concentration of 1 micromolar. Increasing substance P's concentration to the millimolar range caused no further increase in activity. There was an absolute cation requirement for substance P's enhancement of guanylate cyclase activity. Substance P could increase guanylate cyclase activity with either calcium or manganese in the incubation medium but more augmentation was observed with manganese. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of substance P.