Impact of micronised progesterone and medroxyprogesterone acetate in combination with transdermal oestradiol on cardiovascular markers in women diagnosed with premature ovarian insufficiency or an early menopause: a randomised pilot trial.

OBJECTIVE: To compare the difference between micronised progesterone (MP) and medroxyprogesterone acetate (MPA) in combination with transdermal oestradiol (t-E2) on cardiovascular disease (CVD) risk markers in women diagnosed with an early menopause and premature ovarian insufficiency (EMPOI). BACKGROUND: The European Society for Cardiology has identified carotid femoral pulse wave velocity (cfPWV) as the gold standard cardiogenic biomarker for risk stratification of arterial disease. Menopause has been shown to augment the age-dependent increase in arterial stiffness, with hormone replacement therapy (HRT) being the mainstay of management of women diagnosed with EMPOI. STUDY DESIGN: A pilot randomised prospective open-label trial. Women were randomised to either cyclical MP (Utrogestan® 200mg) or MPA (Provera® 10mg) in conjunction with t-E2 (Evorel® Patches 50mcg/day) for 12 months. Seventy-one subjects were screened, and baseline data are available for 57 subjects. MAIN OUTCOME MEASURE: Carotid-femoral pulse wave velocity (cfPWV). RESULTS: PWV did not significantly change from baseline in either treatment arm. MP + t-E2 demonstrated a positive effect on traditional CVD markers, with a significant improvement seen in cardiac output (CO) (0.71±1.01mL/min, 95% CI 0.20 to 1.21) and reduction in diastolic blood pressure (DBP) (-3.43±6.31mmHg, 95% Cl -6.57 to -0.29) and total peripheral resistance (TPR) (-0.15±0.19mmHg⋅min⋅mL-1, 95% CI -0.24 to -0.05) after 12 months. MPA + t-E2, in contrast, did not demonstrate significant changes from baseline in traditional haemodynamic parameters. CONCLUSION: The positive changes in traditional markers were not reflected in the cardiogenic biomarker, cfPWV, which has demonstrated a higher positive predictive value for cardiovascular events than traditional measurements.

The effect of micronized progesterone and medroxyprogesterone acetate in combination with transdermal estradiol on hemostatic biomarkers in postmenopausal women diagnosed with POI and early menopause: a randomized trial.

OBJECTIVE: To compare the impact of micronized progesterone (MP) or medroxyprogesterone acetate (MPA) in combination with transdermal estradiol (t-E2) on traditional coagulation factors and thrombin generation parameters in postmenopausal women diagnosed with premature ovarian insufficiency or early menopause. METHOD: Randomized prospective trial conducted in women diagnosed with premature ovarian insufficiency or early menopause and an intact uterus, recruited over 28 months. All participants were prescribed t-E2 and randomized to either cyclical MP or MPA using a web-based computer randomization software, Graph Pad. Thrombin generation parameters were measured at baseline and repeated after 3-months. Traditional hemostatic biomarkers were measured at baseline and repeated after 3, 6, and 12-months. Seventy-one participants were screened for the study, of whom 66 met the inclusion criteria. In total, 57 participants were randomized: 44 completed the thrombin generation assessment arm of the study, whilst 32 completed 12-months of the traditional coagulation factor screening component of the trial. RESULTS: Thrombin generation parameters did not significantly change from baseline after 3-months duration for either progestogen component when combined with t-E2, unlike the traditional coagulation factors. Protein C activity, free Protein S, and Antithrombin III levels decreased with time in both treatment arms. CONCLUSION: Fluctuations in traditional hemostatic biomarkers were not reproduced by parallel changes in thrombin generation parameters that remained neutral in both groups compared with baseline. The absence of statistically significant changes in thrombin generation for the first 3-months of hormone therapy use is reassuring and would suggest a neutral effect of both progestogens on the global coagulation assay.

B Cell Subset Analysis and Gene Expression Characterization in Mid-Luteal Endometrium.

The human endometrium is the innermost mucosal membrane of the uterus and is the first point of contact for an implanting blastocyst. A wide variety of immune cells are found amongst the endometrial epithelial layers and stromal cells which both provide host immune responses against pathogens and also assist with placentation and pregnancy establishment, however, B cells have not been characterized, despite being a vital player in both adaptive and mucosal immunity. Through analysis of mid-luteal endometrial biopsies, we find 1-5% of endometrial immune cells are B cells, the majority were naïve or memory B cells, with few plasma cells. Compared with circulating B cells, endometrial B cells had an activated phenotype, with increased expression of CD69, HLA-DR, CD74, and CD83, and IL-10 production capacities. PD1+CXCR5+ICOS+ T follicular helper-like cells and FAS+IgD-BCL6+ germinal center B cells were also present in the endometrium, which may indicate that endometrial B cells are playing an active role through germinal center reactions in the human endometrial environment.

Is the clinician an independent variable in embryo transfer outcomes under standardized direct and indirect supervision? A 5-year observational cohort study.

Objective: To compare the cumulative pregnancy rate (CPR) for experienced clinicians and trainees naive to the skill of embryo transfer (ET) during an assisted reproductive treatment (ART) cycle. To establish the minimum number of procedures required to achieve consistent outcomes. Method: A non-interventional retrospective observational cohort study looking at all consecutive ETs undertaken over a 5-year study period. The CPR was determined by a self-reported urinary home pregnancy test undertaken 16 days after oocyte retrieval. Results: The CPR did not differ between an experienced clinician (39%) and trainee (45%) for the first 50 (p=0.41) and last 50 (40.7% versus 42.7%) (p=0.81) ET procedures. The CPR for the individuals remained consistent with their peaks and troughs mirroring the overall success rate of the unit. This pattern continued when the data was further stratified for co-variables (age [≤37 years of age], catheter type [soft] and embryo quality [expanded blastocyst of grade ≥2]): CPRs for experienced clinicians was 65.7% (first 50 transfers) and 40.9% (last 50 transfers); CPR for trainees was 66.7% (first 50 transfers) and 53.6% (last 50 transfers); p=0.95 and p=0.37, respectively. The trainees, however, were more likely to use a stylet catheter with a 2-step transfer technique, with a cost over clinical implication. Furthermore, patients expressed a preference for an experienced clinician to perform their procedure, despite being informed that the grade of the clinician had no impact on the cycle outcome after an analysis of the unit's data. Conclusion: The clinician's grade and duration of service have not been shown to significantly impact the outcome of the ART cycle. The findings, however, should be interpreted with caution, as they reflect the culture of training in the unit, where there is a strong emphasis on adequate direct and indirect supervision. Furthermore, the relationship between the volume of work and outcomes is established in postgraduate medical education, with the exact number required to achieve clinical competence being dependent on the procedure and intensity of the workload.

The correlation between raised body mass index and assisted reproductive treatment outcomes: a systematic review and meta-analysis of the evidence.

BACKGROUND: Public funding for fertility services within the United Kingdom is limited, and therefore, strict guidance exists regarding who can be offered treatment under the National Health Service (NHS). Body mass index (BMI) is a universal criteria adopted by both the public and private sector. This study addresses an important aspect of the impact of a raised BMI on fertility treatment outcomes. We standardise the analysis of the data by only including studies incorporating the WHO BMI criteria; the current reference point for clinicians and clinical commissioning groups in ascertaining which group of patients should receive treatment. This study is an update of the previous systematic review performed in 2010, with the inclusion of a larger number of cycles from central databases such as the Society for Assisted Reproductive Technology (SART). METHODS: An electronic literature search was conducted through the Cochrane, Medline and Embase libraries. Data extraction for each outcome measure was pooled and expressed as an odds ratio with 95% confidence intervals. Where clinical heterogeneity was evident, the random effects model was used to calculate the risk ratio and a fixed effects model was used for the remaining studies. A p value < 0.05 was considered statistically significant. RESULTS: A total of 49 studies have been identified and included in this systematic review. Overweight and obese (BMI ≥ 25 kg/m2) women have a statistically significant lower live birth rate (OR 0.81, 95% CI 0.74-0.89, p < 0.00001) following Assisted Reproductive Technology (ART) when comparisons are drawn to women with a normal BMI. An increase is also demonstrated in the number of miscarriages experienced by women with a BMI ≥ 30 kg/m2 (OR 1.52, 95% CI 1.28-1.81, p < 0.00001). CONCLUSION: Although this review concludes that a clear impact of BMI on ART outcomes is demonstrated, there remains questions as to the pathophysiology underlying these differences. This review supports the government's stringent criteria regarding BMI categories under which NHS funding is made available for ART, through a clear description of poor reproductive outcomes in women with a BMI ≥ 30 kg/m2.

Potential role of Plasmodium falciparum exported protein 1 in the chloroquine mode of action.

In the human malaria parasite Plasmodium falciparum, membrane glutathione S-transferases (GST) have recently emerged as potential cellular detoxifying units and as drug target candidates with the artemisinin (ART) class of antimalarials inhibiting their activity at single-digit nanomolar potency when activated by iron sources such as cytotoxic hematin. Here we put forward the hypothesis that the membrane GST Plasmodium falciparum exported protein 1 (PfEXP1, PF3D7_1121600) might be directly involved in the mode of action of the unrelated antimalarial 4-aminoquinoline drug chloroquine (CQ). Along this line we report potent biochemical inhibition of membrane glutathione S-transferase activity in recombinant PfEXP1 through CQ at half maximal inhibitory CQ concentrations of 9.02 nM and 19.33 nM when using hematin and the iron deficient 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, respectively. Thus, in contrast to ART, CQ may not require activation through an iron source such as hematin for a potent inhibition of membrane GST activity. Arguably, these data represent the first instance of low nanomolar inhibition of an essential Plasmodium falciparum enzyme through a 4-aminoquinoline and might encourage further investigation of PfEXP1 as a potential CQ target candidate.

Structural and biochemical characterization of ligand recognition by CysB, the master regulator of sulfate metabolism.

CysB, a member of LysR-type transcriptional regulators, up-regulates the expression of genes associated with sulfate metabolism and cysteine biosynthesis. CysB is activated under sulfur limiting conditions by O-acetylserine (OAS) and N-acetylserine (NAS), but the activation mechanism of CysB remain unknown. Here, we report four crystal structures of ligand binding domains of CysB (CysB-LBD) in apo form and in complex with sulfate, OAS, and NAS. Our results show that CysB has two distinct allosteric ligand binding sites; a sulfate and NAS specific site-1 and a second, NAS and OAS specific site-2. All three ligands bind through the induced-fit mechanism. Surprisingly, OAS remodels the site-1 by binding to site-2, suggesting that site-1 and site-2 are coupled allosterically. Using DNA binding and site-directed mutagenesis approach, we show that OAS enhances NAS mediated activation and mutation at site-1 has no effect on site-2 mediated OAS activation. Results indicate that inducer binding triggered signals from OAS-Specific site-2 are relayed to DBD through site-1. Together, results presented here suggest that induced-fit binding and allosteric coupling between two ligand binding sites and DBD underlie the key feature of CysB activation. Further, this study provides first structural glimpse into recognition of inducer ligands by CysB and provides a general framework to understand how LTTR family regulators respond to dual activators.

Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism.

By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4-6 log-fold (104-106) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT-OASS (cysteine regulatory complex) follows a noncanonical "facilitated dissociation" mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that "competitive allostery" is the general feature of noncanonical "facilitated/accelerated dissociation" mechanisms. Further understanding of the mechanistic framework of "competitive allosteric" mechanism may allow us to design a new family of "competitive allosteric drugs/small molecules" that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

Investigation of calcium-dependent activity and conformational dynamics of zebra fish 12-lipoxygenase.

BACKGROUND: A 12-lipoxygenase in zebra fish (zf12-LOX) was found to be required for normal embryonic development and LOXs are of great interest for targeted drug designing. In this study, we investigate the structural-functional aspects of zf12-LOX in response to calcium. METHODS: A soluble version of zf12-LOX was created by mutagenesis. Based on multiple sequence alignment, we mutated the putative calcium-responsive amino acids in N-PLAT domain of soluble zf12-LOX. Using a series of biophysical methods, we ascertained the oligomeric state, stability, structural integrity and conformational changes of zf12-LOX in response to calcium. We also compared the biophysical properties of soluble zf12-LOX with the mutant in the absence and presence of calcium. RESULTS: Here we provide a detailed characterization of soluble zf12-LOX and the mutant. Both proteins exist as compact monomers in solution, however the enzyme activity of soluble zf12-LOX is significantly increased in presence of calcium. We find that the stimulatory effect of calcium on zf12-LOX is related to a change in protein structure as observed by SAXS, adopting an open-state. In contrast, enzyme with a mutated calcium regulatory site has reduced activity-response to calcium and restricted large re-modeling, suggesting that it retains a closed-state in response to calcium. Taken together, our study suggests that Ca2+-dependent regulation is associated with different domain conformation(s) that might change the accessibility to substrate-binding site in response to calcium. GENERAL SIGNIFICANCE: The study can be broadly implicated in better understanding the mode(s) of action of LOXs, and the enzymes regulated by calcium in general.

Kinetic investigation of human 5-lipoxygenase with arachidonic acid.

Human 5-lipoxygenase (5-LOX) is responsible for the formation of leukotriene (LT)A4, a pivotal intermediate in the biosynthesis of the leukotrienes, a family of proinflammatory lipid mediators. 5-LOX has thus gained attention as a potential drug target. However, details of the kinetic mechanism of 5-LOX are still obscure. In this Letter, we investigated the kinetic isotope effect (KIE) of 5-LOX with its physiological substrate, arachidonic acid (AA). The observed KIE is 20±4 on kcat and 17±2 on kcat/KM at 25°C indicating a non-classical reaction mechanism. The observed rates show slight temperature dependence at ambient temperatures ranging from 4 to 35°C. Also, we observed low Arrhenius prefactor ratio (AH/AD=0.21) and a small change in activation energy (Ea(D)-Ea(H)=3.6J/mol) which suggests that 5-LOX catalysis involves tunneling as a mechanism of H-transfer. The measured KIE for 5-LOX involves a change in regioselectivity in response to deuteration at position C7, resulting in H-abstraction form C10 and formation of 8-HETE. The viscosity experiments influence the (H)kcat, but not (D)kcat. However the overall kcat/KM is not affected for labeled or unlabeled AA, suggesting that either the product release or conformational rearrangement might be involved in dictating kinetics of 5-LOX at saturating conditions. Investigation of available crystal structures suggests the role of active site residues (F421, Q363 and L368) in regulating the donor-acceptor distances, thus affecting H-transfer as well as regiospecificity. In summary, our study shows that that the H-abstraction is the rate limiting step for 5-LOX and that the observed KIE of 5-LOX is masked by a change in regioselectivity.

Molecular basis of peptide recognition in metallopeptidase Dug1p from Saccharomyces cerevisiae.

Dug1p, a M20 family metallopeptidase and human orthologue of carnosinase, hydrolyzes Cys-Gly dipeptide, the last step of glutathione (GSH) degradation in Saccharomyces cerevisiae. Molecular bases of peptide recognition by Dug1p and other M20 family peptidases remain unclear in the absence of structural information about enzyme-peptide complexes. We report the crystal structure of Dug1p at 2.55 Å resolution in complex with a Gly-Cys dipeptide and two Zn(2+) ions. The dipeptide is trapped in the tunnel-like active site; its C-terminus is held by residues at the S1' binding pocket, whereas the S1 pocket coordinates Zn(2+) ions and the N-terminus of the peptide. Superposition with the carnosinase structure shows that peptide mimics the inhibitor bestatin, but active site features are altered upon peptide binding. The space occupied by the N-terminus of bestatin is left unoccupied in the Dug1p structure, suggesting that tripeptides could bind. Modeling of tripeptides into the Dug1p active site showed tripeptides fit well. Guided by the structure and modeling, we examined the ability of Dug1p to hydrolyze tripeptides, and results show that Dug1p hydrolyzes tripeptides selectively. Point mutations of catalytic residues do not abolish the peptide binding but abolish the hydrolytic activity, suggesting a noncooperative mode in peptide recognition. In summary, results reveal that peptides are recognized primarily through their amino and carboxyl termini, but hydrolysis depends on the properties of peptide substrates, dictated by their respective sequences. Structural similarity between the Dug1p-peptide complex and the bestatin-bound complex of CN2 suggests that the Dug1p-peptide structure can be used as a template for designing natural peptide inhibitors.

A cross-sectional national questionnaire survey assessing the clinical attitudes of members of the British Menopause Society to the management of women with premature ovarian insufficiency.

OBJECTIVE: To explore the current clinical attitudes of members of the British Menopause Society to the management of premature ovarian insufficiency. DESIGN: An electronic cross-sectional questionnaire survey. SETTING: Members of the British Menopause Society. POPULATION: All members of the British Menopause Society with a valid email address. METHOD: Completion of an electronic survey. MAIN OUTCOME MEASURES: Investigations and treatment options and preferences for the management of women with premature ovarian insufficiency. RESULTS: A total of 130 questionnaires were returned and analysed. The majority of responses were from Hospital Consultants (n = 55/130; 42.3%). A total of 53/124 (42.7%) clinicians routinely performed a bone density scan. A total of 73/130 (56.2%) clinicians would prescribe hormone replacement therapy in preference to combined ethinyl estradiol and progesterone (COC; 27/130, 20.8%). A total of 44/108 (40.7%) routinely prescribed oral estradiol in preference to transdermal administration (62/108, 57.4%). A total of 26/128 (20.3%) prescribed oral micronised progesterone, 31/128 (24.2%) oral progestogens, while 42/128 (32.8%) preferred the intra-uterine system. Fertility concerns remain an important aspect of care, with 33.9% (n = 39/115) of clinicians indicating that more than 50% of their patients had a concern regarding their fertility. CONCLUSION: The majority of clinicians indicated a preference for hormone replacement therapy instead of the COC as their choice of hormone replacement in women with premature ovarian insufficiency. However, there was a significant variation in practices. This information can be useful in counselling women and in guiding clinical practitioners. The results highlight the need for further research to determine the optimal regimens for the management of women with premature ovarian insufficiency.

Fertility desires, choice of hormone replacement and the effect of length of time since menopause on bone density in women with premature ovarian insufficiency: a review of 223 consecutive new referrals to a tertiary centre.

Premature ovarian insufficiency can have significant implications for the affected women. This review assesses the fertility desires, choice of hormone replacement, and the effect of time since menopause on the bone density of these women. This is a retrospective analysis of 223 consecutive new referrals. The average age (mean [± standard deviation]) of the women was 37.35 (± 5.88) years, with 24.1% (n = 19/79) presenting within 12 months of the onset of symptoms, most commonly, vasomotor type symptoms (n = 98/223; 43.9%). Of the women included, 58.7% (n = 131/223) took hormone replacement therapy (HRT), most commonly, an oral (n = 90/131; 68.7%) sequential preparation (n = 91/131; 69.5%), with a significant number of women >40 years of age preferring the transdermal route (n = 26/54; 48.1%; p<0.01). A total of 37.7% (n = 84/223) of the women expressed concerns regarding their future fertility, more notable in women ≤ 40 years (n = 72/142; 50.7%; p < 0.01). Of these, 41.7% (n = 35/84) took HRT, most commonly, a sequential regimen (n = 26/35; 74.3%) with oral estradiol (n = 30/35; 85.7%); 69.5% (n = 155/223) of the women had had a bone densitometry scan performed, with 66.5% (n = 103/155) showing normal bone mineral density (BMD), but a greater likelihood of having reduced BMD the greater the time delay in presentation. No difference was seen for the three broad categories of BMD when further analysed for the cause of premature ovarian insufficiency, but a significant difference was noted for the spinal Z-scores, whereby women who underwent a surgically induced menopause were noted to have lower BMD compared with the other causes (p < 0.01). These findings can be useful in counselling women and guiding clinicians in their management of women with premature ovarian insufficiency.

A randomised controlled trial comparing the effects of micronized progesterone to medroxyprogesterone acetate on cardiovascular health, lipid metabolism and the coagulation cascade in women with premature ovarian insufficiency: study protocol and review of the literature.

Premature ovarian insufficiency (POI) can have significant health implications for the affected patient population, but remains a largely under researched area. There is lack of evidence from randomised controlled trials to guide clinical practice, regarding the optimal hormone replacement therapy regimens, dose and route of administration. Furthermore, little research has addressed the effect of the various progestogens used on health parameters in women with POI. Here we describe an ongoing randomised clinical trial looking at the effects of micronized progesterone and medroxyprogesterone acetate, both used in combination with transdermal oestradiol on the cardiovascular system, lipid profile and coagulation cascade in women with POI as a step towards better understanding of the implications of hormone treatment in this cohort of women.

IVF twins: buy one get one free?

There has been an overall increase in the incidence of multiple pregnancies and assisted reproduction technology is largely responsible for this rise. Although twins may appeal to couples undergoing in vitro fertilisation (IVF), they have been associated with serious health consequences to the babies, their mothers and the family unit, as well as having massive financial implications for the National Health Service. Transfer of more than one embryo during IVF is mainly responsible for IVF twins, and elective transfer of a single embryo at a time with cryopreservation of surplus embryos for later transfer has been shown to be an effective strategy to minimise the risk of twins without compromising IVF success rates. Factors that will impact on the success of the policy of elective single embryo transfer (eSET) include improvement in embryo selection for transfer, better cryopreservation techniques and adequate state funding for IVF. However, in implementing the policy of eSET it is important that each case is assessed on an individual basis since in some situations (e.g. in older women) the transfer of two embryos may be more cost effective. Adequate and continuous education of all stakeholders is essential if the policy of eSET is to be successful in the UK.

Equilibrium binding and kinetic characterization of putative tetracycline repressor family transcription regulator Fad35R from Mycobacterium tuberculosis.

Fatty acids play critical role in the survival and virulence of Mycobacterium tuberculosis (Mtb). Activation of fatty acids by acyl-CoA synthetases (Fad) into fatty acyl-CoA is the first and one of the crucial steps in fatty acid metabolism. Mtb possesses 36 fatty acyl-CoA synthetases, unlike Escherichia coli, which has single enzyme. However, the mechanisms by which the expression of these multiple Fad genes is regulated remain uncharacterized. We characterized the DNA- and ligand-binding properties of a putative tetracycline repressor family regulator, named Fad35R, located upstream of the Fad35 gene and ScoA-citE operon. We identified a palindromic regulatory motif upstream of Fad35 and characterized the binding of Fad35R to this motif. Equilibrium binding studies show that Fad35R binds to this motif with high affinity (K(d) ∼ 0.033 µm) and the specificity of binding was confirmed by an electromobility gel shift assay. Kinetic studies indicate that faster association (k(a,avg) ∼ 5.4 × 10(4) m(-1) · s(-1)) and slower dissociation rates (k(d,avg) ∼ 5.84 × 10(-4) s(-1)) confer higher affinity. The affinity for the promoter is maximum at 300 mm NaCl but decreases rapidly beyond this range. Ligand-binding studies indicate that Fad35R binds specifically to tetracycline and also binds to fatty acid derivatives. The promoter-binding affinity is decreased significantly in the presence of palmityl-CoA, suggesting that Fad35R can sense the levels of activated fatty acids and alter its DNA-binding activity. Our results suggest that Fad35R may be the functional homologue of FadR and controls the expression of genes in a metabolite-dependent manner.

Amino-terminal extension present in the methionine aminopeptidase type 1c of Mycobacterium tuberculosis is indispensible for its activity.

BACKGROUND: Methionine aminopeptidase (MetAP) is a ubiquitous enzyme in both prokaryotes and eukaryotes, which catalyzes co-translational removal of N-terminal methionine from elongating polypeptide chains during protein synthesis. It specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged. The MetAP action for exclusion of N-terminal methionine is mandatory in 50-70% of nascent proteins. Such an activity is required for proper sub cellular localization, additional processing and eventually for the degradation of proteins. RESULTS: We cloned genes encoding two such metalloproteases (MtMetAP1a and MtMetAP1c) present in Mycobacterium tuberculosis and expressed them as histidine-tagged proteins in Escherichia coli. Although they have different substrate preferences, for Met-Ala-Ser, we found, MtMetAP1c had significantly high enzyme turnover rate as opposed to MtMetAP1a. Circular dichroism spectroscopic studies as well as monitoring of enzyme activity indicated high temperature stability (up to 50 °C) of MtMetAP1a compared to that of the MtMetAP1c. Modelling of MtMetAP1a based on MtMetAP1c crystal structure revealed the distinct spatial arrangements of identical active site amino acid residues and their mutations affected the enzymatic activities of both the proteins. Strikingly, we observed that 40 amino acid long N-terminal extension of MtMetAP1c, compared to its other family members, contributes towards the activity and stability of this enzyme, which has never been reported for any methionine aminopeptidase. Furthermore, mutational analysis revealed that Val-18 and Pro-19 of MtMetAP1c are crucial for its enzymatic activity. Consistent with this observation, molecular dynamic simulation studies of wild-type and these variants strongly suggest their involvement in maintaining active site conformation of MtMetAP1c. CONCLUSION: Our findings unequivocally emphasized that N-terminal extension of MtMetAP1c contributes towards the functionality of the enzyme presumably by regulating active site residues through "action-at-a-distance" mechanism and we for the first time are reporting this unique function of the enzyme.

Cys-Gly specific dipeptidase Dug1p from S. cerevisiae binds promiscuously to di-, tri-, and tetra-peptides: Peptide-protein interaction, homology modeling, and activity studies reveal a latent promiscuity in substrate recognition.

Dug1p is a recently identified novel dipeptidase and plays an important role in glutathione (GSH) degradation. To understand the mechanism of its substrate recognition and specificity towards Cys-Gly dipeptides, we characterized the solution properties of Dug1p and studied the thermodynamics of Dug1p-peptide interactions. In addition, we used homology modeling and ligand docking approaches to get structural insights into Dug1p-peptide interaction. Dug1p exists as dimer and the stoichiometry of peptide-Dug1p complex is 2:1 indicating each monomer in the dimer binds to one peptide. Thermodynamic studies indicate that the free energy change for Dug1p-peptide complex formation is similar (▵G(bind) âˆ¼ -7.0 kcal/mol) for a variety of peptides of different composition and length (22 peptides). Three-dimensional model of Dug1p is constructed and docking of peptides to the modeled structure suggests that hydrogen bonding to active site residues (E172, E171, and D137) lock the N-terminal of the peptide into the binding site. Dug1p recognizes peptides in a metal independent manner and peptide binding is not sensitive to salts (dlogK/dlog[salt] âˆ¼ 0) over a range of [NaCl] (0.02-0.5 M), [ZnCl(2)], and [MnCl(2)] (0-0.5 mM). Our results indicate that promiscuity in peptide binding results from the locking of peptide N-terminus into the active site. These observations were supported by our competitive inhibition activity assays. Dug1p activity towards Cys-Gly peptide is significantly reduced (∼ 70%) in the presence of Glu-Cys-Gly. Therefore, Dug1p can recognize a variety of oligopeptides, but has evolved with post-binding screening potential to hydrolyze Cys-Gly peptides selectively.

The pressure profile test is more sensitive and specific than Palmer's test in predicting correct placement of the Veress needle.

OBJECTIVES: Although the majority of laparoscopic complications result from improper Veress needle placement, the safety tests commonly used to determine correct placement are not always reliable. A prospective observational study (Canadian Task Force Classification II-2) was set up to determine the reliability of Palmer's and pressure profile tests in predicting the correct intraperitoneal placement of the Veress needle prior to insufflation. STUDY DESIGN: One hundred consecutive women undergoing gynaecological laparoscopic surgery between September 2006 and June 2007 were recruited. The operating surgeons conducted Palmer's and pressure profile tests in all 100 cases and recorded the ease with which these tests were performed and whether or not they felt that the needle placement was correct. They were also asked to comment on the saline drop test and double click acoustic test if appropriate. RESULTS: The overall sensitivity of Palmer's test was 0.92 while its specificity was 0.5. The overall sensitivity and specificity of the pressure profile test were 0.99 and 0.75, respectively, making this a more reliable test for predicting intraperitoneal placement of the Veress needle. CONCLUSIONS: The pressure profile test was a more reliable guide to confirming the correct placement of the Veress needle as a negative test is more likely to indicate failure to achieve intraperitoneal placement.