Cryo-EM structure of bacterial nitrilase reveals insight into oligomerization, substrate recognition, and catalysis.

Many enzymes can self-assemble into higher-order structures with helical symmetry. A particularly noteworthy example is that of nitrilases, enzymes in which oligomerization of dimers into spiral homo-oligomers is a requirement for their enzymatic function. Nitrilases are widespread in nature where they catalyze the hydrolysis of nitriles into the corresponding carboxylic acid and ammonia. Here, we present the Cryo-EM structure, at 3 Å resolution, of a C-terminal truncate nitrilase from Rhodococcus sp. V51B that assembles in helical filaments. The model comprises a complete turn of the helical arrangement with a substrate-intermediate bound to the catalytic cysteine. The structure was solved having added the substrate to the protein. The length and stability of filaments was made more substantial in the presence of the aromatic substrate, benzonitrile, but not for aliphatic nitriles or dinitriles. The overall structure maintains the topology of the nitrilase family, and the filament is formed by the association of dimers in a chain-like mechanism that stabilizes the spiral. The active site is completely buried inside each monomer, while the substrate binding pocket was observed within the oligomerization interfaces. The present structure is in a closed configuration, judging by the position of the lid, suggesting that the intermediate is one of the covalent adducts. The proximity of the active site to the dimerization and oligomerization interfaces, allows the dimer to sense structural changes once the benzonitrile was bound, and translated to the rest of the filament, stabilizing the helical structure.

A Putative New Role of Tv-PSP1 Recognizes IRE and ERE Hairpin Structures from Trichomonas vaginalis.

To understand whether protein Tv-PSP1 from Trichomonas vaginalis recognizes mRNA parasite stem-loop structures, we conducted REMSA and intrinsic fluorescence assays. We found the recombinant Tv-PSP1 structure, determined with X-ray crystallography, showed unusual thermal stability of the quaternary structure, associated with a disulfide bridge CYS76-CYS104. To gain deeper insight into the Tv-PSP1 interaction with mRNA stem-loops (mRNAsl) and its relationship with thermal stability, we also used an integrated computational protocol that combined molecular dynamics simulations, docking assays, and binding energy calculations. Docking models allowed us to determine a putative contact surface interaction region between Tv-PSP1 and mRNAsl. We determined the contributions of these complexes to the binding free energy (ΔGb) in the electrostatic (ΔGelec) and nonelectrostatic (ΔGnon-elec) components using the Adaptive Poisson-Boltzmann Solver (APBS) program. We are the first, to the best of our knowledge, to show the interaction between Tv-PSP1 and the stem-loop structures of mRNA.

Effects of Primary Kidney Disease Etiology on Renal Osteodystrophy in Pediatric Dialysis Patients.

Congenital diseases of the kidney and urinary tract (CAKUT) and glomerulonephritis are the main causes of chronic kidney disease (CKD) in children. Although renal osteodystrophy (ROD) and indices of mineral metabolism have been characterized in dialyzed children, the impact of primary kidney disease on ROD is unknown. We performed a cross-sectional study of bone biopsies performed in 189 pediatric dialysis patients aged 12.6 ± 5.4 years. Patients were classified into three groups according to primary kidney disease: CAKUT (n = 82), hereditary (n = 22), or glomerular disease (n = 85). Serum concentrations of calcium, phosphate, alkaline phosphatase (ALP), parathyroid hormone (PTH), and 25(OH) vitamin D were measured at the time of biopsy. Fibroblast growth factor 23 (FGF23) levels were measured in a subset of 59 patients. Levels of calcium, phosphate, PTH, and 25(OH) vitamin D were similar across groups. CAKUT patients had higher serum ALP and lower C-terminal FGF23 levels. Bone turnover and bone volume parameters did not differ across groups. However, osteoid volume (OV/BV), osteoid surface (OS/BS), and osteoid maturation time (OMT) were highest in the CAKUT group and lowest in the hereditary group. Multiple regression analysis revealed that calcium, phosphate, ALP, and PTH were independently associated with OV/BV and osteoid thickness (O.Th). PTH was an independent factor affecting bone formation rate. The relationship between CKD etiology and bone histomorphometric variables was abrogated after adjustment for biochemical parameters in the multivariable models. Overall, bone histology differed according to CKD etiology in the unadjusted analysis; however, this association could not be confirmed independently of biochemical parameters. Although CAKUT patients had a greater mineralization defect with elevated serum ALP levels, longitudinal studies will be needed to elucidate mediation pathways that might be involved in the complex interplay of CKD-mineral bone disease (MBD). © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Effect of the Metal-Ligand Interface on the Chiroptical Activity of Cysteine-Protected Nanoparticles.

Gold, silver, and copper small nanoparticles (NPs), with average size ≈2 nm, are synthesized and afterward protected with l- and d-cysteine, demonstrating emergence of chiroptical activity in the wavelength range of 250-400 nm for all three metals with respect to the bare nanoparticles and ligands alone. Silver-cysteine (Ag-Cys) NPs display the higher anisotropy factor, whereas gold-cysteine (Au-Cys) NPs show optical and chiroptical signatures slightly more displaced to the visible range. A larger number of circular dichroism (CD) bands with smaller intensity, as compared to gold and silver, is observed for the first time for copper-cysteine (Cu-Cys) NPs. The manifestation of optical and chiroptical responses upon cysteine adsorption and the differences between the spectra corresponding to each metal are mainly dictated by the metal-ligand interface, as supported by a comparison with calculations of the oscillatory and rotatory strengths based on time-dependent density functional theory, using a metal-ligand interface motif model, which closely resembles the experimental absorption and CD spectra. These results are useful to demonstrate the relevance of the interface between chiral ligands and the metal surfaces of Au, Ag, and Cu NPs, and provide evidence and further insights into the origin of the transfer mechanisms and induction of extrinsic chirality.

AiiM Lactonase Strongly Reduces Quorum Sensing Controlled Virulence Factors in Clinical Strains of Pseudomonas aeruginosa Isolated From Burned Patients.

Pseudomonas aeruginosa is an opportunistic bacterium associated with healthcare infections in intensive care units (ICUs), ventilator-associated pneumonia (VAP), surgical site infections, and burns. This bacterium causes 75% of death in burned patients, since it can develop a persistent biofilm associated with infections, express several virulence factors, and antibiotic-resistance mechanisms. Some of these virulence factors are proteases such as elastase and alkaline protease, or toxic metabolites such as pyocyanin and is one of the few microorganisms able to produce cyanide, which inhibits the cytochrome oxidase of host cells. These virulence factors are controlled by quorum sensing (QS). In this work, 30 P. aeruginosa clinical strains isolated from burned patients from a tertiary hospital in Mexico City were studied. Antibiotic susceptibility tests were done, and virulence factors (elastase, alkaline protease, HCN, and pyocyanin) were determined in presence of an N-acylhomoserine lactonase, AiiM able to hydrolyze a wide range of acyl homoserine lactones. The treatment reduced significantly the activities of elastase and alkaline protease, and the production of pyocyanin and HCN in all producer strains but not the secretion of toxins through the type III secretion system. Our work suggests that AiiM treatment may be an effective therapy to combat P. aeruginosa infection in burn patients.

Effects of erythropoietin on fibroblast growth factor 23 in mice and humans.

BACKGROUND: Erythropoietin (EPO) has been reported as a novel determinant of fibroblast growth factor 23 (FGF23) production; however, it is unknown whether FGF23 is stimulated by chronic exposure to EPO or by EPO administration in nonpolycystic chronic kidney disease (CKD) models. METHODS: We analyzed the effects of chronic EPO on FGF23 in murine models with chronically high EPO levels and normal kidney function. We studied the effects of exogenous EPO on FGF23 in wild-type mice, with and without CKD, injected with EPO. Also, in four independent human CKD cohorts, we evaluated associations between FGF23 and serum EPO levels or exogenous EPO dose. RESULTS: Mice with high endogenous EPO have elevated circulating total FGF23, increased disproportionately to intact FGF23, suggesting coupling of increased FGF23 production with increased proteolytic cleavage. Similarly, in wild-type mice with and without CKD, a single exogenous EPO dose acutely increases circulating total FGF23 out of proportion to intact FGF23. In these murine models, the bone marrow is shown to be a novel source of EPO-stimulated FGF23 production. In humans, serum EPO levels and recombinant human EPO dose are positively and independently associated with total FGF23 levels across the spectrum of CKD and after kidney transplantation. In our largest cohort of 680 renal transplant recipients, serum EPO levels are associated with total FGF23, but not intact FGF23, consistent with the effects of EPO on FGF23 production and metabolism observed in our murine models. CONCLUSION: EPO affects FGF23 production and metabolism, which may have important implications for CKD patients.

Identification of a perchloric acid-soluble protein (PSP)-like ribonuclease from Trichomonas vaginalis.

A perchloric acid-soluble protein (PSP), named here tv-psp1, was identified in Trichomonas vaginalis. It is expressed under normal culture conditions according to expressed sequence tag (EST) analysis. On the other hand, Tv-PSP1 protein was identified by mass spectrometry with a 40% of identity to human PSP (p14.1). Polyclonal antibodies against recombinant Tv-PSP1 (rTv-PSP1) recognized a single band at 13.5 kDa in total protein parasite extract by SDS-PAGE and a high molecular weight band analyzed by native PAGE. Structural analysis of Tv-PSP1, using dynamic light scattering, size exclusion chromatography, and circular dichroism spectroscopy, showed a trimeric structure stable at 7 M urea with 38% α-helix and 14% ß-sheet in solution and a molecular weight of 40.5 kD. Tv-PSP1 models were used to perform dynamic simulations over 100 ns suggesting a stable homotrimeric structure. Tv-PSP1 was located in the nucleus, cytoplasm, and hydrogenosomes of T. vaginalis, and the in silico analysis by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) showed interactions with RNA binding proteins. The preliminary results of RNA degradation analysis with the recombinant Tv-PSP1 showed RNA partial deterioration suggesting a possible putative ribonuclease function.

Clinical experience with the use of ferric citrate as a phosphate binder in pediatric dialysis patients.

BACKGROUND: Ferric citrate, an iron-based phosphate binder, has been shown to improve both hyperphosphatemia and iron deficiency in adult chronic kidney disease patients, but its use in the pediatric dialysis population has not been described. METHODS: This is a retrospective analysis of 11 unselected pediatric dialysis patients who received ferric citrate as a phosphate binder between 2015 and 2017. Time-averaged laboratory values were compared pre- and post-ferric citrate initiation using the Wilcoxon signed-rank test. RESULTS: The median age of this cohort was 13 years old (range 4-17 years old). Five patients were on hemodialysis, and six patients were on peritoneal dialysis. The median duration of ferric citrate therapy was 214 days (range 39-654 days), with a median time-averaged ferric citrate dose of 3.5 tablets per day (range 1.5-8.4 tablets per day). Compared to the pre-ferric citrate period, ferric citrate treatment was associated with decreased serum phosphate (6.5 to 5.2 mg/dl, p = 0.014), decreased phosphate age-related standard deviation score (SDS) (2.3 to 0.9, p = 0.019), increased transferrin saturation (26 to 34%, p = 0.049), increased ferritin (107 to 230 ng/ml, p = 0.074), and maintenance of hematocrit. CONCLUSIONS: In pediatric dialysis patients, ferric citrate may be able to concurrently lower phosphate levels and treat iron deficiency. However, larger studies are needed to further evaluate safety and efficacy in the pediatric chronic kidney disease population.

Exploring interfacial water trapping in protein-ligand complexes with multithermal titration calorimetry.

In this work, we examine the hypothesis about how trapped water molecules at the interface between triosephosphate isomerase (TIM) and either of two phosphorylated inhibitors, 2-phosphoglycolate (2PG) or phosphoglycolohydroxamate (PGH), can explain the anomalous highly negative binding heat capacities (ΔCp,b) of both complexes, TIM-2PG and TIM-PGH. We performed fluorimetric titrations of the enzyme with PGH inhibitor under osmotic stress conditions, using various concentrations of either osmolyte: sucrose, ethylene glycol or glycine betaine. We also analyze the binding processes under various stressor concentrations using a novel calorimetric methodology that allows ΔCp,b determinations in single experiments: Multithermal Titration Calorimetry. The binding constant of the TIM-PGH complex decreased gradually with the concentration of all osmolytes, but at diverse extents depending on the osmolyte nature. According to the osmotic stress theory, this decrease indicates that the number of water molecules associated with the enzyme increases with inhibitor binding, i.e. some solvent molecules became trapped. Additionally, the binding heat capacities became less negative at higher osmolyte concentrations, their final values depending on the osmolyte. These effects were also observed in the TIM-2PG complex using sucrose as stressor. Our results strongly suggest that some water molecules became immobilized when the TIM-inhibitor complexes were formed. A computational analysis of the hydration state of the binding site of TIM in both its free state and its complexed form with 2PG or PGH, based on molecular dynamics (MD) simulations in explicit solvent, showed that the binding site effectively immobilized additional water molecules after binding these inhibitors.

Effects of acute kidney injury and chronic hypoxemia on fibroblast growth factor 23 levels in pediatric cardiac surgery patients.

BACKGROUND: Fibroblast growth factor-23 (FGF23) levels are elevated in cardiopulmonary bypass (CPB)-associated acute kidney injury (AKI); however, it is unknown how much of the circulating FGF23 is intact and bioactive. Hypoxia may induce FGF23 production, yet its impact in humans is unknown. Pediatric cardiac surgery patients have both a high incidence of CPB-associated AKI and a high prevalence of chronic hypoxemia. METHODS: We assessed the effects of hypoxemia and CPB-associated AKI on C-terminal FGF23 (cFGF23) and intact FGF23 (iFGF23) levels in 32 pediatric cardiac surgery patients with normal estimated glomerular filtration rate (eGFR). Plasma cFGF23 and iFGF23 were measured preoperatively and serially postoperatively. RESULTS: Despite normal renal and ventricular function, preoperative cFGF23 levels were high and elevated out of proportion to iFGF23 levels. Preoperative oxygen saturation measurements correlated inversely with FGF23 levels. Preoperative cFGF23 and oxygen saturation both predicted postoperative AKI. Postoperatively, cFGF23 and iFGF23 increased by 2 h postreperfusion; iFGF23 then returned to baseline, but cFGF23 remained elevated through 24 h postreperfusion. Group status (AKI vs. non-AKI) modified the effect of time on changes in iFGF23 levels but not cFGF23 levels. CONCLUSIONS: Preoperative cFGF23 may predict CPB-associated kidney dysfunction. Changes over time in cFGF23 and iFGF23 levels post-CPB differ. Chronic hypoxemia may affect FGF23 production in humans.

Complex kinetics and residual structure in the thermal unfolding of yeast triosephosphate isomerase.

BACKGROUND: Saccharomyces cerevisiae triosephosphate isomerase (yTIM) is a dimeric protein that shows noncoincident unfolding and refolding transitions (hysteresis) in temperature scans, a phenomenon indicative of the slow forward and backward reactions of the native-unfolded process. Thermal unfolding scans suggest that no stable intermediates appear in the unfolding of yTIM. However, reported evidence points to the presence of residual structure in the denatured monomer at high temperature. RESULTS: Thermally denatured yTIM showed a clear trend towards the formation of aggregation-prone, ß-strand-like residual structure when pH decreased from 8.0 to 6.0, even though thermal unfolding profiles retained a simple monophasic appearance regardless of pH. However, kinetic studies performed over a relatively wide temperature range revealed a complex unfolding mechanism comprising up to three observable phases, with largely different time constants, each accompanied by changes in secondary structure. Besides, a simple sequential mechanism is unlikely to explain the observed variation of amplitudes and rate constants with temperature. This kinetic complexity is, however, not linked to the appearance of residual structure. Furthermore, the rate constant for the main unfolding phase shows small, rather unvarying values in the pH region where denatured yTIM gradually acquires a ß-strand-like conformation. It appears, therefore, that the residual structure has no influence on the kinetic stability of the native protein. However, the presence of residual structure is clearly associated with increased irreversibility. CONCLUSIONS: The slow temperature-induced unfolding of yeast TIM shows three kinetic phases. Rather than a simple sequential pathway, a complex mechanism involving off-pathway intermediates or even parallel pathways may be operating. ß-strand-type residual structure, which appears below pH 8.0, is likely to be associated with increased irreversible aggregation of the unfolded protein. However, this denatured form apparently accelerates the refolding process.

Exploring the evolutionary route of the acquisition of betaine aldehyde dehydrogenase activity by plant ALDH10 enzymes: implications for the synthesis of the osmoprotectant glycine betaine.

BACKGROUND: Plant ALDH10 enzymes are aminoaldehyde dehydrogenases (AMADHs) that oxidize different ω-amino or trimethylammonium aldehydes, but only some of them have betaine aldehyde dehydrogenase (BADH) activity and produce the osmoprotectant glycine betaine (GB). The latter enzymes possess alanine or cysteine at position 441 (numbering of the spinach enzyme, SoBADH), while those ALDH10s that cannot oxidize betaine aldehyde (BAL) have isoleucine at this position. Only the plants that contain A441- or C441-type ALDH10 isoenzymes accumulate GB in response to osmotic stress. In this work we explored the evolutionary history of the acquisition of BAL specificity by plant ALDH10s. RESULTS: We performed extensive phylogenetic analyses and constructed and characterized, kinetically and structurally, four SoBADH variants that simulate the parsimonious intermediates in the evolutionary pathway from I441-type to A441- or C441-type enzymes. All mutants had a correct folding, average thermal stabilities and similar activity with aminopropionaldehyde, but whereas A441S and A441T exhibited significant activity with BAL, A441V and A441F did not. The kinetics of the mutants were consistent with their predicted structural features obtained by modeling, and confirmed the importance of position 441 for BAL specificity. The acquisition of BADH activity could have happened through any of these intermediates without detriment of the original function or protein stability. Phylogenetic studies showed that this event occurred independently several times during angiosperms evolution when an ALDH10 gene duplicate changed the critical Ile residue for Ala or Cys in two consecutive single mutations. ALDH10 isoenzymes frequently group in two clades within a plant family: one includes peroxisomal I441-type, the other peroxisomal and non-peroxisomal I441-, A441- or C441-type. Interestingly, high GB-accumulators plants have non-peroxisomal A441- or C441-type isoenzymes, while low-GB accumulators have the peroxisomal C441-type, suggesting some limitations in the peroxisomal GB synthesis. CONCLUSION: Our findings shed light on the evolution of the synthesis of GB in plants, a metabolic trait of most ecological and physiological relevance for their tolerance to drought, hypersaline soils and cold. Together, our results are consistent with smooth evolutionary pathways for the acquisition of the BADH function from ancestral I441-type AMADHs, thus explaining the relatively high occurrence of this event.

Potassium and ionic strength effects on the conformational and thermal stability of two aldehyde dehydrogenases reveal structural and functional roles of K⁺-binding sites.

Many aldehyde dehydrogenases (ALDHs) have potential potassium-binding sites of as yet unknown structural or functional roles. To explore possible K(+)-specific effects, we performed comparative structural studies on the tetrameric betaine aldehyde dehydrogenase from Pseudomonas aeruginosa (PaBADH) and on the dimeric BADH from spinach (SoBADH), whose activities are K(+)-dependent and K(+)-independent, respectively, although both enzymes contain potassium-binding sites. Size exclusion chromatography, dynamic light scattering, far- and near-UV circular dichroism, and extrinsic fluorescence results indicated that in the absence of K(+) ions and at very low ionic strength, PaBADH remained tetrameric but its tertiary structure was significantly altered, accounting for its inactivation, whereas SoBADH formed tetramers that maintained the native tertiary structure. The recovery of PaBADH native tertiary-structure was hyperbolically dependent on KCl concentration, indicating potassium-specific structuring effects probably arising from binding to a central-cavity site present in PaBADH but not in SoBADH. K(+) ions stabilized the native structure of both enzymes against thermal denaturation more than did tetraethylammonium (TEA(+)) ions. This indicated specific effects of potassium on both enzymes, particularly on PaBADH whose apparent T(m) values showed hyperbolical dependence on potassium concentration, similar to that observed with the tertiary structure changes. Interestingly, we also found that thermal denaturation of both enzymes performed in low ionic-strength buffers led to formation of heat-resistant, inactive soluble aggregates that retain 80% secondary structure, have increased ß-sheet content and bind thioflavin T. These structured aggregates underwent further thermal-induced aggregation and precipitation when the concentrations of KCl or TEACl were raised. Given that PaBADH and SoBADH belong to different ALDH families and differ not only in amino acid composition but also in association state and surface electrostatic potential, the formation of this kind of ß-sheet pre-fibrillar aggregates, not described before for any ALDH enzyme, appear to be a property of the ALDH fold.

Moonlighting peptides with emerging function.

Hunter-killer peptides combine two activities in a single polypeptide that work in an independent fashion like many other multi-functional, multi-domain proteins. We hypothesize that emergent functions may result from the combination of two or more activities in a single protein domain and that could be a mechanism selected in nature to form moonlighting proteins. We designed moonlighting peptides using the two mechanisms proposed to be involved in the evolution of such molecules (i.e., to mutate non-functional residues and the use of natively unfolded peptides). We observed that our moonlighting peptides exhibited two activities that together rendered a new function that induces cell death in yeast. Thus, we propose that moonlighting in proteins promotes emergent properties providing a further level of complexity in living organisms so far unappreciated.

The folding pathway of glycosomal triosephosphate isomerase: structural insights into equilibrium intermediates.

The guanidine hydrochloride-induced conformational transitions of glycosomal triosephosphate isomerase (TIM) were monitored with functional, spectroscopic, and hydrodynamic measurements. The equilibrium folding pathway was found to include two intermediates (N(2) ↔I(2) ↔2M↔2U). According to this model, the conformational stability parameters of TIM are as follows: ΔG(I2-N2) = 5.5 ± 0.6, ΔG(2M-I2) =19.6 ± 1.6, and ΔG(2U-2M) = 14.7 ± 3.1 kcal mol(-1) . The I(2) state is compact (α(SR) = 0.8); it is able to bind 8-anilinonaphthalene-1-sulfonic acid ANS and it is composed of ∼45% of α-helix and tertiary structure content compared with the native enzyme; however, it is unable to bind the transition-state analog 2-phosphoglycolate. Conversely, the 2M state lacks detectable tertiary contacts, possesses ∼10% of the native α-helical content, is significantly expanded (α(SR) = 0.2), and has low affinity for ANS. We studied the effect of mutating cysteine residues on the structure and stability of I(2) and 2M. Three mutants were made: C39A, C126A, and C39A/C126A. The replacement of C39, which is located at ß(2) , was found to be neutral. The I(2) -C126A state, however, was prone to aggregation and exhibited an emission maximum that was 3-nm red-shifted compared with the I(2) -wild type, indicating solvent exposure of W90 at ß(4) . Our results suggest that the I(2) state comprises the (ßα)(1-4) ß(5) module in which the conserved C126 residue located at ß(5) defines the boundary of the folded segment. We propose a folding pathway that highlights the remarkable thermodynamic stability of this glycosomal enzyme.

Thermal denaturation of ß-glucosidase B from Paenibacillus polymyxa proceeds through a Lumry-Eyring mechanism.

ß-glucosidase B (BglB), 1,4-ß-D: -glucanohydrolase, is an enzyme with various technological applications for which some thermostable mutants have been obtained. Because BglB denatures irreversibly with heating, the stabilities of these mutants are assessed kinetically. It, therefore, becomes relevant to determine whether the measured rate constants reflect one or several elementary kinetic steps. We have analyzed the kinetics of heat denaturation of BglB from Paenibacillus polymyxa under various conditions by following the loss of secondary structure and enzymatic activity. The denaturation is accompanied by aggregation and an initial reversible step at low temperatures. At T ≥ T ( m ), the process follows a two-state irreversible mechanism for which the kinetics does not depend on the enzyme concentration. This behavior can be explained by a Lumry-Eyring model in which the difference between the rates of the irreversible and the renaturation steps increases with temperature. Accordingly, at high scan rates (≥1 °C min(-1)) or temperatures (T ≥ T ( m )), the measurable activation energy involves only the elementary step of denaturation.

Binding thermodynamics of phosphorylated inhibitors to triosephosphate isomerase and the contribution of electrostatic interactions.

Electrostatic interactions have a central role in some biological processes, such as recognition of charged ligands by proteins. We characterized the binding energetics of yeast triosephosphate isomerase (TIM) with phosphorylated inhibitors 2-phosphoglycollate (2PG) and phosphoglycolohydroxamate (PGH). We determined the thermodynamic parameters of the binding process (K(b), ΔG(b), ΔH(b), ΔS(b) and ΔC(p)) with different concentrations of NaCl, using fluorimetric and calorimetric titrations in the conventional mode of ITC and a novel method, multithermal titration calorimetry (MTC), which enabled us to measure ΔC(p) in a single experiment. We ruled out specific interactions of Na(+) and Cl(-) with the native enzyme and did not detect significant linked protonation effects upon the binding of inhibitors. Increasing ionic strength (I) caused K(b), ΔG(b) and ΔH(b) to become less favorable, while ΔS(b) became less unfavorable. From the variation of K(b) with I, we determined the electrostatic contribution of TIM-2PG and TIM-PGH to ΔG(b) at I=0.06 M and 25 °C to be 36% and 26%, respectively. The greater affinity of PGH for TIM is due to a more favorable ΔH(b) compared to 2PG (by 19-24 kJ mol(-1) at 25 °C). This difference is compatible with PGH establishing up to five more hydrogen bonds with TIM. Both binding ΔC(p)s were negative, and less negative with increasing ionic strength. ΔC(p)s at I=0.06 M were much more negative than predicted by surface area models. Water molecules trapped in the interface when ligands bind to protein could explain the highly negative ΔCps. Thermodynamic binding functions for TIM-2PG changed more with ionic strength than those for TIM-PGH. This greater dependence is consistent with linked, but compensated, protonation equilibriums yielding the dianionic species of 2PG that binds to TIM, process that is not required for PGH.

Comportamiento clínico- epidemiológico de las ametropías / Clinical-epidemiological behavior of ametropia

Fundamento: en Cuba, las ametropías constituyen una causa frecuente de asistencia a las consultas oftalmológicas. La existencia de estos defectos se ha convertido en un problema que afecta a la sociedad, tanto por los daños orgánicos y físicos que puede provocar en el sistema visual, como por los trastornos psicológicos que se presentan en quienes los padecen, ya sea por cuestiones estéticas o de incapacidad de realizar determinadas actividades cotidianas. Objetivos: determinar el comportamiento clínico-epidemiológico de las ametropías en el policlínico Previsora del municipio Camagüey, en el mes de octubre de 2008. Método: se realizó un estudio observacional descriptivo de los pacientes que acudieron a la consulta de refracción, el universo y la muestra estuvo conformado por los doscientos pacientes diagnosticados como amétropes. Resultados: el grupo de edades más afectado es el de cuarenta a cuarenta y nueve años y el sexo femenino fue el que más incidió, el astigmatismo fue la ametropía más frecuente, y según el valor dióptrico predominó el grado leve. La mayoría de los casos presentó buena agudeza visual con corrección y sólo una cuarta parte aproximadamente fue regular o mala. Conclusiones: el glaucoma, la retinopatía miópica y la catarata fueron las enfermedades oftalmológicas que más se asociaron a pacientes miopes, mientras que la retinopatía diabética se encontró con más frecuencia en hipermétropes.

Background: In Cuba, ametropia constitutes a frequent cause of attendance in ophthalmologic consultations. The existence of these defects has become a problem that affects society, so much for organic and physical damages that it may cause in the visual system, as for psychological dysfunctions that are presented who suffer them, either for aesthetic matters or for inability of carrying out certain daily activities. Objective: to determine the clinical-epidemic behavior of ametropia at the Previsora polyclinic of Camagüey municipality, in October 2008. Method: a descriptive observational study of patients that assisted to the refraction consultation was carried out, the universe and the sample was comply with two hundred patients diagnosed with ametropia. Results: the most affected age group is the one from forty to forty-nine years and the female sex with the most occurrence, astigmatism was the most frequent ametropia, and according to the dioptric value, light grade prevailed. Most of the cases presented good visual acuity with correction and only a fourth part approximately was regular or bad. Conclusions: glaucoma, myopic retinopathy and cataract were ophthalmologic diseases most associate to myopic patients, while diabetic retinopathy was most frequent in hypermetrope patients.

Comportamiento clínico- epidemiológico de las ametropías / Clinical-epidemiological behavior of ametropia

En Cuba, las ametropías constituyen una causa frecuente de asistencia a las consultas oftalmológicas. La existencia de estos defectos se ha convertido en un problema que afecta a la sociedad, tanto por los daños orgánicos y físicos que puede provocar en el sistema visual, como por los trastornos psicológicos que se presentan en quienes los padecen, ya sea por cuestiones estéticas o de incapacidad de realizar determinadas actividades cotidianas. Objetivos: determinar el comportamiento clínico-epidemiológico de las ametropías en el policlínico Previsora del municipio Camagüey, en el mes de octubre de 2008. Método: se realizó un estudio observacional descriptivo de los pacientes que acudieron a la consulta de refracción, el universo y la muestra estuvo conformado por los doscientos pacientes diagnosticados como amétropes. Resultados: el grupo de edades más afectado es el de cuarenta a cuarenta y nueve años y el sexo femenino fue el que más incidió, el astigmatismo fue la ametropía más frecuente, y según el valor dióptrico predominó el grado leve. La mayoría de los casos presentó buena agudeza visual con corrección y sólo una cuarta parte aproximadamente fue regular o mala. Conclusiones: el glaucoma, la retinopatía miópica y la catarata fueron las enfermedades oftalmológicas que más se asociaron a pacientes miopes, mientras que la retinopatía diabética se encontró con más frecuencia en hipermétropes (AU)

In Cuba, ametropia constitutes a frequent cause of attendance in ophthalmologic consultations. The existence of these defects has become a problem that affects society, so much for organic and physical damages that it may cause in the visual system, as for psychological dysfunctions that are presented who suffer them, either for aesthetic matters or for inability of carrying out certain daily activities. Objective: to determine the clinical-epidemic behavior of ametropia at the Previsora polyclinic of Camagüey municipality, in October 2008. Method: a descriptive observational study of patients that assisted to the refraction consultation was carried out, the universe and the sample was comply with two hundred patients diagnosed with ametropia. Results: the most affected age group is the one from forty to forty-nine years and the female sex with the most occurrence, astigmatism was the most frequent ametropia, and according to the dioptric value, light grade prevailed. Most of the cases presented good visual acuity with correction and only a fourth part approximately was regular or bad. Conclusions: glaucoma, myopic retinopathy and cataract were ophthalmologic diseases most associate to myopic patients, while diabetic retinopathy was most frequent in hypermetrope patients (AU)

Tratamiento quirúrgico del Pterigion Primario con autoplastia conjuntival / Surgical treatment of the Primary Pterygium with conjunctival autoplasty

El Pterigion es una proliferación fibrovascular que avanza hacia la córnea provocando alteraciones en la visión; su tratamiento es netamente quirúrgico. Objetivo: determinar el comportamiento de la aplicación de la autoplastia conjuntival en el Pterigion Primario. Método: Se realizó un estudio explicativo para valorar el comportamiento de la aplicación de la autoplastia conjuntival en el Pterigion Primario en pacientes operados en el Servicio de Oftalmología en Camagüey durante el año 2007, los pacientes se distribuyeron según edad, sexo y tipo de Pterigion, se relacionó la exposición a agentes irritantes y se determinó la aparición de complicaciones y su relación con las variables anteriores. El universo en coincidencia con la muestra se constituyó por doscientos enfermos los que cumplieron los criterios de inclusión y exclusión, se aplicó una encuesta y tratamiento quirúrgico con la aplicación de la autoplastia conjuntival. Los pacientes se evolucionaron después del tratamiento quirúrgico hasta los seis meses para la determinación de las complicaciones. Resultados: predominó el sexo masculino y más de la mitad de los enfermos tenían entre treinta y seis a cincuenta y cinco años. El Pterigion grado II ocupó más de la mitad de la serie de estudio. La mayoría de los pacientes refirieron exposición al sol y al calor como agentes irritantes. Conclusiones: la frecuencia de recidiva fue significativamente baja y no se demostró relación con la edad, sexo, ni grado de Pterigion (AU)

The Pterygium is a fibrovascular proliferation that advances toward the cornea causing alterations in the vision; its treatment is highly surgical. Objective: to determine the behavior of the application of the conjunctival autoplasty in the Primary Pterygium. Method: a prospective study in patients operated in the Ophthalmology Service in Camagüey during the year 2007 was performed, patients were distributed according to age, sex and type of Pterygium, the exhibition to irritating agents was related and it was determined the appearance of complications and its relationship with the previous variables. The universe in coincidence with the sample was constituted by two-hundred sick persons which completed the inclusion and exclusion criteria, it was applied a survey and surgical treatment with the application of the conjunctival autoplasty. Patients were evolved after the surgical treatment until the six months for the determination of the complications. Results: the masculine sex prevailed and more than half of the sick persons had among thirty-six to fifty-five years. The grade II pterygium occupied more than half of the study series. Most of the patients referred sun and heat exposure like irritating agents. Conclusions: the recidivation frequency was significantly low and the relationship with age, sex, neither grade of pterygium was not demonstrated (AU)