Neuronal apoptosis-inhibitory protein does not interact with Smac and requires ATP to bind caspase-9.

The neuronal apoptosis-inhibitory protein (NAIP) is the founding member of the mammalian family of inhibitor of apoptosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro. The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three baculoviral inhibitory repeat (BIR) domains, a central nucleotide binding oligomerization domain (NOD), and a carboxyl-terminal leucine-rich repeat (LRR) domain. The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family. In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac. Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9. In contrast, the full-length protein did not, suggesting some form of structural autoregulation. However, the association of the wild type full-length protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP. Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9. Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains. Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal IAP binding motif (IBM). Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains. These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition

Frozen embryo transfer: Endometrial preparation by letrozole versus hormone replacement cycle: A randomized clinical trial

Background: The endometrial preparation with stimulating natural cycles for frozen embryo transfer (FET) have benefits like lower cost and ease of use. Objective: Comparing the clinical outcome of letrozole versus hormone replacement (HR) for endometrial preparation in women with normal menstrual cycles for FET in artificial reproduction techniques. Materials and Methods: A total of 167 participants who had frozen embryos and regular ovulatory cycles were randomly divided into two groups for endometrial preparation. One group (82 women) was stimulated with letrozole 5mg/day and the other group (85 women) was hormonally stimulated by oral estradiol valerate (2 mg three times a day). All participants were followed serially by ultrasonography. Any patient who did not reach optimal endometrial thickness was excluded from the study. Implantation, biochemical and clinical pregnancy and abortion rate were reported. Results: There was no significant difference in the mean age, duration, and primary or secondary infertility, cause of the infertility, number, and quality of transferred embryos between the groups. The mean estradiol level on the day of transfer was 643 ± 217 in the HR group and 547 ± 212 in the letrozole group (P = 0.01), which was significantly different. The clinical pregnancy rate was 38.7 in the letrozole group, higher than the HR group (25.3) but not significantly different (P=0.06). Conclusion: For endometrial preparation in women with a normal cycle, letrozole yields higher pregnancy rate although it is not significant; due to its cost, ease in use, and lower side effects, letrozole is a good choice. Key words: Letrozole, Hormone replacement, Endometrial, Preparation, Frozen, Embryo

Mechanism of stabilization of Bacillus circulans xylanase upon the introduction of disulfide bonds

The introduction of disulfide bonds has been used as a strategy to enhance the stability of Bacillus circulans xylanase. The transition temperature of the S100C/N148C (DS1), V98C/A152C (DS2), and A1GC/G187,C188 (cXl) in comparison to the wild type was increased by 5.0, 4.1 and 3.8 \ub0C, respectively. Interestingly, a combination of two disulfide bonds of DS1 and cXl (cDS1, circular disulfide 1) led to a 12 \ub0C increase in the transition temperature. Importantly, an increase in the melting point and \u394\u394G values of the cDS1 mutant was cooperative. These results suggest that the mechanism of stabilization by disulfide bonds under irreversible denaturation condition is achieved through: (1) a change in the rate-limiting step on the denaturation pathway; (2) destabilizing the unfolded state without affecting the relative rate constants on the denaturation pathway (like cXl mutant); and (3) or combination of the two (cDS1 mutant).NRC publication: Ye

Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.yesNRC publication: Ye

Seroepidemiologic Survey of Hepatitis A in Mazandarani Pilgrims before Going to Karbala in Arbaeen Pilgrimage in 2018

Background and purpose: Recently, due to the reduction of hepatitis A in Iran there are people with low or no Hepatitis A Virus- Antibody (HAV-Ab), therefore, people at Arbaeen Pilgrimage are at risk for hepatitis A. This study aimed at examining HAV-Ab in pilgrims traveling from Mazandaran province, Iran to Karbala in 2018. Materials and methods: This cross-sectional study was performed in 200 people selected from 40,000 pilgrims in Mazandaran province. Serum samples were evaluated for presence of HAV-Ab and data for demographic characteristics and history of hepatitis A immunity were recorded. Data analysis was performed in SPSS V16. Results: The participants were 137 (68.5%) males and 63 (31.5%) females. Positive and negative HAV-Ab were seen in 156 (78%) and 44 (22%), respectively. Mean ages of people with positive and negative HAV-Ab were almost similar (50 years and 47 years, respectively). HAV-Ab status was found to be significantly associated with job (P0.05). Conclusion: Twenty-two percent of people who were travelling to Karbala had no HAV-Ab, which is significant because the risk of developing fulminant hepatitis increases in adults, therefore, vaccination of Karbala pilgrims for hepatitis A virus is suggested

Role of the salt bridge between glutamate 546 and arginine 907 in preservation of autoinhibited form of Apaf-1

Apaf-1, the key element of apoptotic mitochondrial pathway, normally exists in an auto-inhibited form inside the cytosol. WRD-domain of Apaf-1 has a critical role in the preservation of auto-inhibited form; however the underlying mechanism is unclear. It seems the salt bridges between WRD and NOD domains are involved in maintaining the inactive conformation of Apaf-1. At the present study, we have investigated the effect of E546-R907 salt bridge on the maintenance of auto-inhibited form of human Apaf-1. E546 is mutated to glutamine (Q) and arginine (R). Over-expression of wild type Apaf-1 and its E546Q and E546R variants in HEK293T cells does not induce apoptosis unlike - HL-60 cancer cell line. In vitro apoptosome formation assay showed that all variants are cytochrome c and dATP dependent to form apoptosome and activate endogenous procaspase-9 in Apaf-1-knockout MEF cell line. These results suggest that E546 is not a critical residue for preservation of auto-inhibited Apaf-1. Furthermore, the behavior of Apaf-1 variants for in vitro apoptosome formation in HEK293T cell is similar to exogenous wild type Apaf-1. Wild type and its variants can form apoptosome in HEK293T cell with different procaspase-3 processing pattern in the presence and absence of exogenous cytochrome c and dATP. (C) 2015 Elsevier B.V. All rights reserved.Funding Agencies|Research Council of University of Tehran; Tarbiat Modares University; Linkoping University; Integrative Regenerative Medicine Center (IGEN); Cancerfonden [2013/391]; VR-NanoVision [K2012-99X-22325-01-5]</p

Efficient synthesis, biological evaluation, and docking study of isatin based derivatives as caspase inhibitors

In this paper, a new series of isatin-sulphonamide based derivatives were designed, synthesised and evaluated as caspase inhibitors. The compounds containing 1-(pyrrolidinyl)sulphonyl and 2-(phenoxymethyl)pyrrolidin-1-yl)sulphonyl substitution at C5 position of isatin core exhibited better results compared to unsubstituted derivatives. According to the results of caspase inhibitory activity, compound 20d showed moderate inhibitory activity against caspase-3 and −7 in vitro compared to Ac-DEVD-CHO (IC50 = 0.016 ± 0.002 μM). Among the studied compounds, some active inhibitors with IC50s in the range of 2.33–116.91 μM were identified. The activity of compound 20d was rationalised by the molecular modelling studies exhibiting the additional van der Waals interaction of N-phenylacetamide substitution along with efficacious T-shaped π-π and pi-cation interactions. The introduction of compound 20d with good caspase inhibitory activity will help researchers to find more potent agents