C. tetani Toxin Light Chain

A new series of pyrano[4,3-b]pyran 4a-i and pyrano[3,2-c]chromene 6a-r derivatives bearing a 2-thiophenoxyquinoline nucleus were synthesized by reaction of 2-(4-(un)-substituted thiophenoxy)quinoline-3-carbaldehydes 2a-i with 6-methyl-4-hydroxypyran-2-one 3 and 4-hydroxy-6-(un)-substituted-2H-chromen-2-one 5a-b respectively and malononitrile at room temperature in the presence of KOH as a basic catalyst. All the compounds were screened against three Gram-positive bacteria (Streptococcus pneumoniae, Bacillus subtilis, Clostridium tetani), three Gram-negative bacteria (Salmonella typhi, Escherichia coli, Vibrio cholerae) and two fungi (Candida albicans, Aspergillus fumigatus) using the broth microdilution MIC (minimum inhibitory concentration) method. Upon antimicrobial screening, it was observed that the majority of the compounds were found to be active against Bacillus subtilis, Clostridium tetani and Candida albicans as compared to standard drugs.

1. The contribution of intracellular pH (pH(i)) to the failure of Ca2+ release and inhibition of contractile proteins observed during fatigue was assessed in single intact mouse muscle fibres at 22 degrees C. Fatigue was induced by repeated tetani at intensities designed to induce different levels of intracellular acidosis. Force and either intracellular free Ca2+ concentration ([Ca2+](i); measured using indo-1) or pH(i) (measured using SNARF-1) were recorded in fibres fatigued at two different intensities. 2. Intensity was varied by the repetition rate of tetani and quantified by the duty cycle (the fraction of time when the muscle was tetanized). Stimulation at the low intensity (duty cycle similar to 0.1) reduced force to 30% of initial values in 206 +/- 21 s (60 +/- 7 tetani); at the high intensity (duty cycle similar to 0.3) force was reduced to 30% in 42 +/- 7 s (43 +/- 7 tetani) (P < 0.05; n=14). 3. When force was reduced to 30% of initial values, tetanic [Ca2+](i) had fallen from 648 +/- 87 to 336 +/- 64 nM (48% decrease) at the low intensity but had only fallen from 722 +/- 84 to 468 +/- 60 nM (35% decrease) at the higher intensity (P < 0.05 low vs, high intensity; n = 7). 4. Fatigue resulted in reductions in Ca2+ sensitivity of the contractile proteins which were greater at the high intensity (pre-fatigue [Ca2+](i) required for 50% of maximum force (Ca-50) = 354 +/- 23 nM; post-fatigue Ca-50 = 421 +/- 48 nM and 524 +/- 43 nM for low and high intensities, respectively). Reductions in maximum Ca2+-activated force (F-max) were similar at the two intensities (pre-fatigue F-max = 328 +/- 22 mu N; post-fatigue F-max = 271 +/- 20 and 265 +/- 19 mu N for low and high intensities, respectively). 5. Resting pH(i) was 7.15 +/- 0.05. During fatigue at the low intensity, pH(i) was reduced by 0.12 +/- 0.02 pH units and at the high intensity pH(i) was reduced by 0.34 +/- 0.07 pH units (P < 0.05; n = 5). 6. Our results indicate that the more rapid fall in force at a high intensity is due to a reduction in Ca2+ sensitivity of the contractile proteins, probably related to the greater acidosis. Our data also indicate that the failure of Ca2+ release and reduced maximum Ca2+-activated force observed during fatigue are not due to reductions in intracellular pH.