Agonist interactions at the calcium pools in skinned and unskinned canine tracheal smooth muscle

We studied the functionally discrete calcium sources used by acetylcholine, 5-hydroxytryptamine, histamine and high K+ in the dog tracheal smooth muscle. The extracellular calcium dependence of their responses was assessed by altering the calcium and by pretreatment with the calcium antagonist, nifedipine. The intracellular calcium pool was assessed by studying the interactions between caffeine and the agonists in both skinned and unskinned preparations. The extent of overlap for the different calcium pools between the various agonists was determined by studying the dose-response relationships of these agents before and after pretreatment with another agonist, i.e., the conditioning agonist, in zero calcium conditions. The rank order of sensitivity to calcium removal and to nifedipine was histamine greater than KCl greater than 5-hydroxytryptamine greater than acetylcholine. Caffeine-induced atenuation of the agonist responses was predominantly through physiological antagonism. However, the caffeine responses in unskinned fibres were augmented by pretreatment with the agonists through both nifedipine-sensitive (as with KCl) and -insensitive (as with acetylcholine) mechanisms. The responses to acetylcholine and caffeine were inhibited by theophylline and forskolin. In the skinned muscle fibres, the pCa-tension relationship suggested high calcium sensitivity, a significant caffeine-sensitive calcium pool, and no evidence of calcium release by exogenous inositol trisphosphate. The results are consistent with multiple extracellular and intracellular calcium sources for the agonist responses. We observed considerable overlap of the calcium sources used by these agonists. Of the four agonists studied, histamine appeared to inhibit the release and sequestration of calcium utilized by the other agonists most effectively.     

Crystal structure of an efficacious gonococcal adherence inhibitor: An enolase from Lactobacillus gasseri

Enolases are highly conserved metalloenzymes ubiquitous to cellular metabolism. While these enzymes share a large degree of sequence and structural similarity, they have been shown to possess a wide range of moonlighting functions. Recent studies showed that an enolase from Lactobacillus gasseri impedes the ability of Neisseria gonorrhoeae to adhere to epithelial cells. We present the crystal structure of this enolase, the first from Lactobacillus, with one of its Mg²⁺ cofactors. Determined using molecular replacement to 2.08 Å, the structure has a flexible and surface exposed catalytic loop containing lysines, and may play a role in the inhibitory function.     

Dose to swallowing structures and dysphagia in head and neck Intensity Modulated Radiation Therapy – A long term prospective analysis

AimTo analyse the long term swallowing function in head and neck cancer patients and correlate with the dose to midline swallowing structures.BackgroundThe use of concurrent chemo radiation (CRT) as the present standard of care resulted in high rates of early and late toxicities. Dysphagia, aspiration, and xerostomia are early as well as late effects of radiation. Not many studies on the dysphagia scores during radiation and follow-up period have correlated dose to the swallowing structures, hence this study.Materials and MethodsHistologically proven head and neck cancer patients treated with intensity modulated radiation therapy were accrued in this study. The pharyngeal constrictors, larynx and cervical oesophagus were contoured and labelled as midline swallowing structures. The volume of the midline swallowing structures which were outside the PTV was delineated separately and was given a mean dose constraint of 45 Gy. Dysphagia was assessed at baseline, weekly intervals during irradiation and follow-up at six years. The dose to the structures for swallowing was correlated with degree of dysphagia.ResultsThere was a gradual increase in the dysphagia grade during the course of radiation. There was a significant recovery of late dysphagia compared to dysphagia during the completion of radiation therapy in patients who received <45 Gy to the swallowing structures (p < 0.0001).ConclusionGiving a constraint to the swallowing structure and limiting it to <45 Gy resulted in earlier recovery of swallowing function resulted in good physical, mental and social well being of the patients when compared to those who received >45 Gy.     

Structural Basis for the Inhibition of Histone Deacetylase 8 (HDAC8), a Key Epigenetic Player in the Blood Fluke Schistosoma mansoni

The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/β HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.