The SM protein Sly1 accelerates assembly of the ER–Golgi SNARE complex

Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins constitute the core of an ancient vesicle fusion machine that diversified into distinct sets that now function in different trafficking steps in eukaryotic cells. Deciphering their precise mode of action has proved challenging. SM proteins are thought to act primarily through one type of SNARE protein, the syntaxins. Despite high structural similarity, however, contrasting binding modes have been found for different SM proteins and syntaxins. Whereas the secretory SM protein Munc18 binds to the ‟closed conformation” of syntaxin 1, the ER–Golgi SM protein Sly1 interacts only with the N-peptide of Sed5. Recent findings, however, indicate that SM proteins might interact simultaneously with both syntaxin regions. In search for a common mechanism, we now reinvestigated the Sly1/Sed5 interaction. We found that individual Sed5 adopts a tight closed conformation. Sly1 binds to both the closed conformation and the N-peptide of Sed5, suggesting that this is the original binding mode of SM proteins and syntaxins. In contrast to Munc18, however, Sly1 facilitates SNARE complex formation by loosening the closed conformation of Sed5.In eukaryotic cells, material is transported in vesicles that pinch off of one set of membranes and move along microtubule tracks to the next compartment, where they specifically fuse. Key players in the fusion of a vesicle with its acceptor membrane are the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins. Heterologous sets of SNARE proteins drive the fusion of two membranes by zippering into a tight four-helix bundle structure. Distinct sets of SNARE proteins carry out different vesicle fusion steps in the cell. An essential SNARE protein for transport into and across the Golgi is Sed5/syntaxin 5 (1). Besides SNARE complexes, Sed5 exists also in a 1:1 complex with the Sec1/Munc18 (SM) protein Sly1 that is essential for ER–Golgi and intra-Golgi trafficking (2). Distinct types of SM proteins are thought to function together with the respective SNARE complex, specifically its syntaxin (reviewed in refs. 3–8).The very N-terminal region, the so-called N-peptide, of Sed5 binds with nanomolar affinity to the outer surface of Sly1 (9–12). This mode of interaction is consistent with the notion that Sly1 can stay bound during SNARE complex formation and that it might even be actively involved in this reaction (13). This idea was strengthened by the observation that Sec1, the SM protein essential for secretion in yeast, interacts with the assembled SNARE complex but not with its isolated syntaxin (14). Unfortunately, for the interaction of Sec1 with its SNARE unit, no definitive structural foundation exists so far, and it remains uncertain whether Sec1 can be considered as a model for SM protein function. Fortunately, the animal counterpart of Sec1, Munc18-1, has been studied in more detail. However, the results of numerous biochemical studies on Munc18-1 appear not to fit into the concept of SM proteins being factors that promote SNARE assembly. On the contrary, initial studies found that Munc18-1 strongly interferes with the ability of its cognate syntaxin 1 to form a SNARE complex (15, 16). This inhibition is difficult to reconcile with an essential role of Munc18-1 during neurotransmitter release (17). The structure of the Munc18-1/syntaxin 1a complex revealed that the central cavity of Munc18-1 wraps around syntaxin in the so-called “closed conformation” (18). In this conformation, the three-helix bundle formed by syntaxin’s N-terminal Habc domain folds back onto its SNARE motif (19), restricting the availability of syntaxin for its SNARE partners. Thus, although they share a similar structure, Munc18-1 and Sly1 appear to bind to their cognate syntaxins in different modes.New light on this discrepancy was shed when it was discovered that Munc18-1 is able to bind simultaneously to a second, spatially separated binding site on syntaxin 1 (15). This second site involves the N-peptide region of syntaxin 1 that, similar to the Sed5 N-peptide (12), binds to the outer surface of Munc18-1. Interestingly, both binding sites are also present in the Munc18/syxtaxin 1 complex of the choanoflagellate Monosiga brevicollis (20). This suggests that the binding mode involving two different sites is evolutionarily conserved. A comparable binding mode was also described for the SM protein Vps45, which regulates trans-Golgi network trafficking. Vps45 binds tightly to the N-peptide of its cognate syntaxin Tlg2/syntaxin 16 (21). It was shown later that Vps45 is also able to interact with the remainder of its Qa-SNARE, possibly in a closed conformation (15, 22).Not all SM proteins are known to bind to the N-peptide of their cognate syntaxin. For example, in the SM protein Vps33, which plays an essential role in the degradation pathway, the N-peptide binding pocket is blocked (23, 24). Vps33 is part of a multisubunit tethering complex known as the homotypic fusion and protein sorting complex (25). Nevertheless, the structure of Vps33 is very similar to other SM protein types despite having low sequence similarity (23, 24). It would be surprising if such structures were not preserved to maintain similar molecular functions. This raises the question of whether there are missing pieces to our understanding of the molecular role of SM proteins.With the idea of a conserved molecular role of SM proteins in mind, we aimed here at a more thorough comparison of Sly1 and Munc18, which are still thought to represent two examples of SM proteins with contrasting syntaxin binding modes. So far nothing is known about a second binding site in the Sly1/Sed5 complex, but the presence of a homologous N-peptide binding site in Munc18 and Sly1 reveals a certain similarity of the two SM protein types. It is debated whether binding of Sly1 to the N-peptide of Sed5 is essential for Golgi trafficking while biochemically less is known (11, 26–28). Neither the effect of Sly1 on SNARE complex assembly has been determined rigorously, nor is it clear whether Sed5 can adopt a closed conformation that interferes with its ability to form a SNARE complex. We therefore sought to determine whether Sly1, in addition to its tight interaction with the N-peptide of Sed5, interacts with the remaining part of Sed5 and, if so, whether this interaction would have an impact on the ability of Sed5 to form a SNARE complex. We found that Sed5 adopts a closed conformation. Comparable to Munc18-1, Sly1 binds simultaneously to both the closed conformation and the N-peptide region of Sed5, although the latter is the major contributor to its affinity to the complex. Remarkably, in contrast to Munc18-1, which blocks SNARE complex assembly, Sly1 was found to assist Sed5 in forming a SNARE complex.     

An outpatient clinical study of dissociative disorder not otherwise specified

The relatively high prevalence of the diagnosis of dissociative disorder not otherwise specified is frequently considered to be disproportionate. The disproportionate rate of this diagnosis is thought to be related to nosologic and/or diagnostic issues in dissociative identity disorder. We sought to investigate and compare the symptom patterns of these two clinical entities. We conducted a cross-sectional study involving 1314 participants who were screened with the Dissociative Experience Scale (DES) and the Somatoform Dissociation Questionnaire (SDQ). Of the participants, 272 who scored above the cut-off points for the screening questionnaires (DES score > 30 and/or SDQ score > 40 points) were invited to complete a structured interview using the Dissociative Disorders Interview Schedule (DDIS); of this subsample, only 190 participants agreed to participate in the second phase of the study. The mean score for the DES was 18.55 ± 17.23, and the mean score for the SDQ was 30.19 ± 13.32. Of the 190 participants, 167 patients were diagnosed as having a dissociative disorder (87.8%). We found that DD-NOS was the most prevalent category of dissociative disorder.     

Human tooth germ stem cell response to calcium-silicate based endodontic cements

Objective

The aim of this study was to compare the cytotoxic effects of endodontic cements on human tooth germ stem cells (hTGSCs). MTA Fillapex, a mineral trioxide aggregate (MTA)-based, salicylate resin containing root canal sealer, was compared with iRoot SP, a bioceramic sealer, and AH Plus Jet, an epoxy resin-based root canal sealer.

Material and Methods

To evaluate cytotoxicity, all materials were packed into Teflon rings (4 mmµ3 mm) and co-cultured with hTGSCs with the aid of 24-well Transwell permeable supports, which had a pore size of 0.4 µm. Coverslips were coated with MTA Fillapex, iRoot SP and AH Plus Jet and each coverslip was placed onto the bottom of one well of a six-well plate for scanning electron microscopy (SEM) analysis. Before the cytotoxicity and SEM analysis, all samples were stored at 37ºC and at 95% humidity and 5% CO₂ for 24 hours to set. The cellular viability was analyzed using MTS test (3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium). The cytotoxic effects and SEM visualization of the tested materials were analyzed at 24-hour, 72-hour, one-week and two-week periods.

Results

On the 1^st day, only MTA Fillapex caused cytotoxicity compared to negative control (NC) group (p<0.008). No significant difference was observed between the other tested materials at this period (p>0.05). After 14 days of incubation with the test materials, MTA Fillapex exhibited significantly higher cytotoxicity compared with iRoot SP, AH Plus Jet and the NC group (P<0.008). In the SEM analysis, the highest levels of cell attachment were observed for iRoot SP and the control group. After 24 hours, MTA Fillapex reduced the number of cells attached to the surface.

Conclusions

Within the limitations of this study, sealers exerted different cytotoxic effects on hTGSCs. Although all materials have exerted cellular toxicity, iRoot SP and AH Plus Jet may promote better attachment to hTGSCs.     

Pepsin levels and oxidative stress markers in exhaled breath condensate of patients with gastroesophageal reflux disease

Aim

To evaluate the pepsin and oxidative stress markers in exhaled breath condensate (EBC) in patients with gastroesophageal reflux disease (GERD).

Patients and Method

Patients with a presumptive diagnosis of GERD with recurrent respiratory and gastrointestinal problems aged between 2 and 14 years were included in the study. All patients underwent pH monitoring. Patients with a reflux index (RI) ≥ 4 were assessed as the reflux group, and those with an RO < 4 were assessed as the non-reflux group. Pepsin levels and oxidative stress markers [NO metabolites (NOX) and total sulphydrile (TSH) levels] were measured in the EBC.

Results

There were 24 patients in the reflux group [RI 17.6 (6.6–46.4)] [median, interquartile range] and 23 in the non-reflux group [RI 0.8 (0.5–1.9) (p < 0.001). Pepsin levels in the EBC were below the level of detection. The median levels of NOx in the EBC of children with reflux [13.7 μmol/L (7.3–24.5)] were lower in than non-reflux group [21.0 μmol/L (14.0–25.2)] (p = 0.034). There was a negative correlation between reflux index and NOX levels in EBC (rs: − 0.331, p = 0.023). In contrast, there was no difference in TSH levels between the reflux and non-reflux groups [37.4 μmol/L (30.2–44.6) vs 40.1 μmol/L (37.4–44.9), respectively, (p > 0.05)].

Conclusion

Decreased levels of NOX in patients with GER disease suggest increased oxidative stress in airways of these patients.     

Acute Renal Failure and Mortality After Open-Heart Surgery in Infants

Acute renal failure (ARF) is a major complication in infants who undergo cardiac surgery. The aim of this investigation was to identify possible risk factors for ARF and mortality in this patients group. Out of 64 patients, 21 (32.8%) cases developed acute renal failure and overall mortality rate was 25%. The mortality rate was higher in the infants who developed ARF than those who did not (66.7% and 4.7%, respectively, p < 0.05). Also, ARF was positively correlated with mortality (r:0.70, p < 0.0001). The nonsurvivors had lower mean serum albumin than did the survivors (p < 0.05), and serum albumin level was negatively correlated with mortality (r = ? 0.34, p < 0.05). For the patients with serum albumin level < 3.5 g/dL, the unadjusted odds ratio for mortality was 4.3 (CI 95%:1.05 ? 17.86). Total bypass time and aorta clamping time were significantly longer in the nonsurvivor group than in the survivor group (p < 0.05 for both). In conclusion, the significant risk factors for mortality in these patients were development of ARF, low serum albumin level, and long total bypass and aorta clamping times, which may be predictive of poor prognosis.     

nNOS expression in the brain of rats after burn and the effect of the ACE inhibitor captopril

Objective

To investigate the role of endogenous neuronal nitric oxide synthase (nNOS) on brain injury after burn and the effects of the captopril.

Methods

Wistar albino rats (200–250 g) were exposed on the dorsal surface to 90 °C (burn) or 25 °C (sham) water for 10 s. The ACE group was treated with intraperitoneal 10 mg/kg captopril immediately after burn and this treatment was repeated twice daily. At the end of the 24 h brain samples were taken. nNOS was studied in brain areas by immunohistochemistry.

Results

There was no difference between the cerebellar and hypothalamic areas the nNOS expression of all groups. nNOS expression increased in the frontal cortex, striatum and midbrain in the burn group compared to the control group. In the frontal cortex, nNOS expression significantly decreased after ACE inhibitor treatment (p < 0.05). The striatal nNOS of the ACE group significantly increased when compared to the control group (p = 0.001). In the midbrain of the animals, nNOS decreased in the ACE group. Hippocampal nNOS expression did not change after burn and significantly increased after ACE inhibitor therapy (p < 0.05).

Conclusions

Our data showed that the pathophysiological events following burn appear to be related to an acute inflammatory reaction which is associated with nNOS in the frontal cortex, striatum and midbrain, and captopril treatment abrogates the nNOS response in the frontal cortex and midbrain.     

Anger in women with premenstrual dysphoric disorder: Its relations with premenstrual dysphoric disorder and sociodemographic and clinical variables

Purpose

The aim of the present study was to investigate anger and anger levels in women meeting the criteria of Premenstrual Dysphoric Disorder and to determine the relation between anger levels and the severity of PMDD and other variables.

Methods

50 women meeting the criteria of Premenstrual Dysphoric Disorder and 50 healthy controls were included in the study. Sociodemographic, familial and reproductive period characteristics of the women participating in the study were recorded. All subjects were administered the State–Trait Anger Scale and Premenstrual Syndrom Scale scales.

Results

A significant difference was found between the Premenstrual Dysphoric Disorder group and the healthy control group in terms of Premenstrual Syndrom Scale scores and anger sub scores. When the state trait anger scale scores were examined, it was seen that subscles had higher scores compared to healthy women. In Premenstrual Dysphoric Disorder group; there was a positive correlation between Premenstrual Syndrom Scale scores and trait anger, anger-in and anger control scores.

Conclusions

Anger appears to be an important problem that makes life more difficult for subjects with Premenstrual Dysphoric Disorder. Wide-scale further studies focused on anger and its relation with Premenstrual Dysphoric Disorder are needed to develop ways of coping with anger in Premenstrual Dysphoric Disorder.