Regulatory Elements within the Prodomain of Falcipain-2, a Cysteine Protease of the Malaria Parasite Plasmodium falciparum

Falcipain-2, a papain family cysteine protease of the malaria parasite Plasmodium falciparum, plays a key role in parasite hydrolysis of hemoglobin and is a potential chemotherapeutic target. As with many proteases, falcipain-2 is synthesized as a zymogen, and the prodomain inhibits activity of the mature enzyme. To investigate the mechanism of regulation of falcipain-2 by its prodomain, we expressed constructs encoding different portions of the prodomain and tested their ability to inhibit recombinant mature falcipain-2. We identified a C-terminal segment (Leu155–Asp243) of the prodomain, including two motifs (ERFNIN and GNFD) that are conserved in cathepsin L sub-family papain family proteases, as the mediator of prodomain inhibitory activity. Circular dichroism analysis showed that the prodomain including the C-terminal segment, but not constructs lacking this segment, was rich in secondary structure, suggesting that the segment plays a crucial role in protein folding. The falcipain-2 prodomain also efficiently inhibited other papain family proteases, including cathepsin K, cathepsin L, cathepsin B, and cruzain, but it did not inhibit cathepsin C or tested proteases of other classes. A structural model of pro-falcipain-2 was constructed by homology modeling based on crystallographic structures of mature falcipain-2, procathepsin K, procathepsin L, and procaricain, offering insights into the nature of the interaction between the prodomain and mature domain of falcipain-2 as well as into the broad specificity of inhibitory activity of the falcipain-2 prodomain

Mutagenesis of the NaChBac sodium channel discloses a functional role for a conserved S6 asparagine

Asparagine is conserved in the S6 transmembrane segments of all voltage-gated sodium, calcium, and TRP channels identified to date. A broad spectrum of channelopathies including cardiac arrhythmias, epilepsy, muscle diseases, and pain disorders is associated with its mutation. To investigate its effects on sodium channel functional properties, we mutated the simple prokaryotic sodium channel NaChBac. Electrophysiological characterization of the N225D mutant reveals that this conservative substitution shifts the voltage-dependence of inactivation by 25 mV to more hyperpolarized potentials. The mutant also displays greater thermostability, as determined by synchrotron radiation circular dichroism spectroscopy studies of purified channels. Based on our analyses of high-resolution structures of NaChBac homologues, we suggest that the side-chain amine group of asparagine 225 forms one or more hydrogen bonds with different channel elements and that these interactions are important for normal channel function. The N225D mutation eliminates these hydrogen bonds and the structural consequences involve an enhanced channel inactivation

A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C. elegans

Endoplasmic-reticulum associated degradation (ERAD) is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY*) was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy

Molecular mechanism for 3:1 subunit stoichiometry of rod cyclic nucleotide-gated ion channels

Molecular determinants of ion channel tetramerization are well characterized, but those involved in heteromeric channel assembly are less clearly understood. The heteromeric composition of native channels is often precisely controlled. Cyclic nucleotide-gated (CNG) channels from rod photoreceptors exhibit a 3:1 stoichiometry of CNGA1 and CNGB1 subunits that tunes the channels for their specialized role in phototransduction. Here we show, using electrophysiology, fluorescence, biochemistry, and X-ray crystallography, that the mechanism for this controlled assembly is the formation of a parallel 3-helix coiled-coil domain of the carboxy-terminal leucine zipper region of CNGA1 subunits, constraining the channel to contain three CNGA1 subunits, followed by preferential incorporation of a single CNGB1 subunit. Deletion of the carboxy-terminal leucine zipper domain relaxed the constraint and permitted multiple CNGB1 subunits in the channel. The X-ray crystal structures of the parallel 3-helix coiled-coil domains of CNGA1 and CNGA3 subunits were similar, suggesting that a similar mechanism controls the stoichiometry of cone CNG channels

Impact of e-learning and role play-based training on psychology students’ communication skills:a feasibility study

Background: Given the importance of communication skills in the psychologist-patient relationship, several training programs have been proposed. Cumulative microtraining (CMT) has shown positive impacts on communication skills in previous studies. Methods: The aim of this naturalistic pre–post study was to test the feasibility of a hybrid CMT program and obtain preliminary data on its impact on communication skills in French-speaking third-year psychology students. The training included an e-learning curriculum and role plays. Pre–post measures included recorded peer-to-peer role plays and self-assessments by participants themselves using the Calgary Cambridge Grid (n = 38) and assessed by an independent rater (n = 29) with a checklist focused on objective behaviors and the CARE questionnaire measuring perceived empathy. Results: The results showed increases in most communication skills at different levels. Summarizing, paraphrasing, and structuring skills were significantly increased after training (all P ≤ 0.001), as were self-reported measurements (all P &lt; 0.001), and empathy and confidence assessed by an independent rater (all p &lt; 0.05). Conclusion: This study provides new evidence on the impact of CMT, including e-learning and role plays, on both self-rated assessments and assessments by an independent rater who measured communication and empathy in a population of French-speaking students. These findings highlight the importance of implementing such instruction in initial training despite the cost involved. It demonstrates the feasibility of its inclusion in university curriculum, facilitated by the adaptation of theoretical aspects of teaching in e-learning.</p

Submental intubation for cancrum oris: A case report

Cancrum oris (Noma) is a devastating gangrenous disease that leads to severe tissue destruction in the face. We describe the anesthetic management of a 12-year-old girl with cancrum oris sequelae in a Rural Secondary level Hospital in Central India (Padhar Hospital). She presented with a large defect in her upper lip on the left side that extended into the columella and the floor of the left nostril. She was scheduled to undergo reconstructive surgery and the surgeons planned to use an Abbé flap based on the lower lip. For this, access to both the mouth and the nose was required. We considered a tracheostomy but decided to attempt the submental route for orotracheal intubation. Following intravenous induction the patient's trachea was intubated with a cuffed oral tracheal tube. This was passed through the submental incision and then reconnected. The surgery proceeded uneventfully and the patient was extubated before transfer. She made a satisfactory recovery and the submental scar healed without complication or scarring. We describe briefly the features of cancrum oris and review the technique of submental intubation (described in adults with midfacial trauma). The use of submental intubation in children and for cancrum oris sequelae has not been previously reported. © 2005 Blackwell Publishing Ltd.link_to_subscribed_fulltex