Enzymatic mechanism of creatine amidinohydrolase as deduced from crystal structures.

Crystal structures of the enzyme creatine amidinohydrolase (creatinase, EC 3.5.3.3) with two different inhibitors, the reaction product sarcosine and the substrate creatine, bound have been analyzed by X-ray diffraction methods. With the inhibitor carbamoyl sarcosine, two different crystal forms at different pH values have been determined. An enzymatic mechanism is proposed on the basis of the eight structures analyzed. The enzyme binds substrate and inhibitor in a distorted geometry where the urea resonance is broken. His232 is the general base and acid, and acts as a proton shuttle. It withdraws a proton from water 377 and donates it to the N(3) atom of the guanidinium group. OH- 377 adds to the C(1) atom of the guanidinium group to form a urea hydrate. Proton withdrawal by His232 leads to products. The reaction product sarcosine binds to the active site in a reverse orientation. The free enzyme was found to have a bicarbonate bound to the active site.     

Advances in endometrial cancer protein biomarkers for use in the clinic

Introduction: Endometrial cancer (EC) is the fourth most common cancer in women in developed countries. The identification of sensitive and specific biomarkers to improve early detection of EC is crucial for an appropriate management of this disease, in which 30% of patients are diagnosed only at advanced stages, which is associated with high levels of morbidity and mortality. Despite major efforts and investments made to identify EC biomarkers, no protein has yet reached the stage of clinical application.

Areas covered: This review gathers the numerous candidate biomarkers for EC diagnosis proposed in proteomic studies published from 1978 to 2017. Additionally, we summarize limitations associated with the proteomic technologies and study designs employed in those articles. Finally, we address new perspectives in EC biomarker research, including the comprehensive knowledge of previously suggested candidate biomarkers in conjunction with novel mass spectrometry-based proteomic technologies with enhanced sensitivity and specificity not yet applied to EC studies and a directed clinical perspective in the study design.

Expert commentary: These ingredients could be the recipe to accelerate the application of protein biomarkers in the clinic.     

Ecological role and historical trends of large pelagic predators in a subtropical marine ecosystem of the South Atlantic

Large pelagic predators occupy high positions in food webs and could control lower trophic level species by direct and indirect ecological interactions. In this study we aimed to test the hypotheses: (1) pelagic predators are keystone species, and their removals could trigger impacts on the food chain; (2) higher landings of pelagic predators could trigger fishing impacts with time leading to a drop in the mean trophic level of catches; and (3) recovery in the pelagic predators populations, especially for sharks, could be achieved with fishing effort reduction. We performed a food web approach using an Ecopath with Ecosim model to represent the Southeastern and Southern Brazil, a subtropical marine ecosystem, in 2001. We then calibrated the baseline model using catch and fishing effort time series from 2001 to 2012. Afterwards, we simulated the impact of fishing effort changes on species and assessed the ecological impacts on the pelagic community from 2012 to 2025. Results showed that the model was well fitted to landing data for the majority of groups. The pelagic predators species were classified as keystone species impacting mainly on pelagic community. The ecosystem was resilient and fisheries seem sustainable at that time. However, the temporal simulation, from 2001 to 2012, revealed declines in the biomass of three sharks, tuna and billfish groups. It was possible observe declines in the mean trophic level of the catch and in the mean total length of landings. Longline fisheries particularly affected the sharks, billfish and swordfish, while hammerhead sharks were mostly impacted by gillnet fishery. Model simulations showed that large sharks’ biomasses could be recovered or maintained only after strong fishing effort reduction.     

Validation of the determination of ΔF508 mutations of the cystic fibrosis gene in over 11000 mouthwashes

Mouthwashes can be used as a DNA resource for mutation detection and, because collection and DNA isolation is simple and cheap, they could in particular, be used for large numbers of samples. To determine the failure rate (the proportion of mouth samples in which no PCR product was obtained) and the specificity of buccal epithelial cell mutation detection in large numbers of samples, we collected mouthwashes and blood samples from 11413 blood donors and tested the mouthwashes for the F508 mutation, which has an estimated frequency of 75% among cystic fibrosis chromosomes in The Netherlands. Blood samples were tested for the F508 mutations only if the mutation was identified in the mouthwash or in the case of a failure to obtain PCR products. The sensitivity of the test was determined in mouthwashes of 75 F508 carriers known from earlier family studies. These samples were offered blindly between the mouthwashes of the blood donors. Both specificity and sensitivity of the mouthwash procedure were 100%. The overall failure rate was 5.6%. This large figure was caused mainly by insufficient rinsing of the mouth in one particular blood bank. Exclusion of the results of this blood bank reduced the failure rate to 1.8%. Our results also confirm that for a large number of samples the mouthwash procedure is suitable for mutation detection and, with proper instructions, can be used in community screening.     

Molecular structure of an A-DNA decamer d(ACCGGCCGGT)

The molecular structure of the DNA decamer d(ACCGGCCGGT) has been solved and refined by single-crystal X-ray-diffraction analysis at 0.20 nm to a final R-factor of 18.0%. The decamer crystallizes as an A-DNA double helical fragment with unit-cell dimensions of a = b = 3.923 nm and c = 7.80 nm in the space group P6(1)22. The overall conformation of this A-DNA decamer is very similar to that of the fiber model for A-DNA which has a large average base-pair tilt and hence a wide and shallow minor groove. This structure is in contrast to that of several A-DNA octamers in which the molecules all have low base-pair-tilt angles (8-12 degrees) resulting in an appearance intermediate between B-DNA and A-DNA. The average helical parameters of this decamer are typical of A-DNA with 10.9 base pairs/turn of helix, an average helical twist angle of 33.1 degrees, and a base-pair-tilt angle of 18.2 degrees. However, the CpG step in this molecule has a low local-twist angle of 24.5 degrees, similar to that seen in other A-DNA oligomers, and therefore appears to be an intrinsic stacking pattern for this step. The molecules pack in the crystal using a recurring binding motif, namely, the terminal base pair of one helix abuts the surface of the shallow minor groove of another helix. In addition, the GC base pairs have large propeller-twist angles, unlike those found most other A-DNA structures.     

A rapid two-step purification of rat cystatin C, one major inhibitor of cysteine proteinases

Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. Twentyfold concentrated urine was chromatographed by a rapid purification procedure. A two-step purification including affinity chromatography on carboxymethyl papain- Sepharose and high-resolution anion exchange chromatography was developed. The purified protein has an apparent molecular mass of 15 kDa and pI of 10.2; its aminoacid composition was similar to human cystatin C. As opposed to previous data, purified urinary rat cystatin C did not contain significant amounts of carbohydrate. Antisera against rat cystatin C, raised in rabbits, partially cross-reacted with human and mouse cystatin C, indicating their antigenic similarities. Like human cystatin C, native rat cystatin C, named slow form, is degraded into a more acidic form, called fast form, by a loss of N-terminal amino acids; fast form displayed a pI of 9.4.     

Kinetics of glutamate efflux in rat liver mitochondria

The transport of glutamate was studied in isolated rat liver mitochondria preloaded with glutamate in the presence of respiratory inhibitors. Glutamate efflux was initiated by dilution of the loaded mitochondria into a glutamate-free medium, and the rate of transport was measured by following the disappearance of glutamate from the mitochondrial matrix following rapid centrifugation through silicone oil. Glutamate efflux was inhibited extensively by bromcresol purple and partially by N-ethylmaleimide, compounds which are both known to inhibit mitochondrial glutamate uptake. The efflux process was stereospecific for L-glutamate and exhibited an activation energy of 19.2 kcal/mol. The rate of glutamate efflux was not affected by changes in the mitochondrial membrane potential. However, a good correlation was observed between the rate of glutamate efflux and the matrix pH, the efflux rate being stimulated by a decrease in matrix pH in the range from 8.0 to 7.2. In contrast, acidification of the incubation medium in the pH range 7.4 to 6.5 inhibited the rate of glutamate efflux. A kinetic analysis was made of the efflux reaction by a computer curve-fitting procedure which fits the experimental data to an integrated rate equation (Williamson, J.R., and Viale, R.O. (1979) Methods Enzymol. 56, 252-278). The results indicated that a fall in the matrix pH primarily caused a decrease in the K'm for matrix glutamate, with little change in V'max. In contrast, a low external pH had an effect on the V'max but not on the K'm for intramitochondrial glutamate. The results are in agreement with a symmetrical sequential model of glutamate transport where the glutamate anion binds to the protonated carrier.