Efectos de la mastectomía en los parámetros baropodométricos de pacientes con cáncer de seno / The effect of mastectomy on baropodometric parameters for breast cancer patients / Efeitos da mastectomia nos parâmetros baropodometricos de pacientes com câncer de mama

Objetivo: la mastectomía posterior al cáncer de seno produce diversas alteraciones posturales y biomecánicas en el plano frontal y sagital. Dichos cambios conducen a una cinemática alterada de la columna vertebral, desequilibrio muscular y alteración del soporte del peso del pie. Este estudio pretende describir las alteraciones de la baropodometría después de la mastectomía, con base en análisis baropodométricos de la presión media y la carga estática. Metodología: se realizó un estudio descriptivo correlacional. Las propiedades de carga del pie se identificaron en 17 mujeres mastectomizadas. Se utilizó una placa de presión Ecosanit Foot para medir la carga del pie en posición anatómica y con los pies juntos. Resultados: en el estudio participaron 17 mujeres mastectomizadas con una edad media de 54.16 años. Las pacientes reportaron una presión media significativamente mayor en la posición anatómica del lado dominante mastectomizado en comparación con el lado no dominante mastectomizado (227.2 ± 22.16 vs. 175.6 ± 14.95, p =0.05). No hubo diferencia significativa para la carga estática entre el lado dominante mastectomizado y el lado no dominante mastectomizado en la posición anatómica (52.43±4.069 vs. 49.69 ± 4.094, de forma respectiva). Conclusión: los resultados actuales de nuestro estudio evidenciaron la distribución desequilibrada del peso (vector carga en el pie) en pacientes después de la mastectomía. Por tanto, en este texto se describen las alteraciones posturales, musculares, y el desequilibrio estático y dinámico en pacientes con cáncer de seno.

Objective: mastectomy after breast cancer produces several postural and biomechanical alterations in the frontal and sagittal plane. Such changes lead to disturbed kinematics of the spine, muscle imbalance and altered foot weight bearing. This study aims to describe body balance alterations after mastectomy based on the baropodometric analysis of their mean pressure and static load. Methodology: a descriptive correlational research was carried out. Foot weight-bearing properties were identified in 17 patients who have undergone a mastectomy. An Ecosanit Foot pressure plate was used to measure foot load at anatomical position and keeping the feet together. Results: 17 female patients who had undergo mastectomy with a mean age of 54.16 years took part on the research. Patients who have had a dominant-side mastectomy demonstrated significant greater mean pressure at anatomical position when compared to those patients who have had a non-dominant side mastectomy (227.2 ± 22.16 versus 175.6 ± 14.95, p =0.05). There was no significant difference for static load between the patients who have had a dominant side mastectomy and patients who have had a non dominant side mastectomy at anatomical position (52.43 ± 4.069 versus 49.69 ± 4.094, respectively). Conclusion: the current results of our research showed the unbalanced weight distribution in patients after mastectomy. Consequently, it describes the postural and muscular alterations, and the static and dynamic imbalance in breast cancer patients.

Objetivo: A mastectomia após câncer de mama produz diversas alterações posturais e biomecânicas nos planos frontal e sagital. Tais mudanças levam a alteração da cinemática da coluna, desequilíbrio muscular e alteração do suporte de peso do pé. Este estudo tem como objetivo descrever as alterações baropodométricas após mastectomia, com base em análises baropodométricas de pressão média e carga estática. Metodologia: foi realizado estudo correlacional descritivo. Propriedades de carga nos pés foram identificadas em 17 mulheres mastectomizadas. Uma placa de pressão Ecosanit Foot foi usada para medir a carga do pé em posição anatômica e com os pés juntos. Resultados: participaram do estudo 17 mulheres mastectomizadas com idade média de 54,16 anos. Os pacientes relataram uma pressão média significativamente maior na posição anatômica do lado mastectomizado dominante em comparação ao lado mastectomizado não dominante (227,2 ± 22,16 vs. 175,6 ± 14,95, p =0,05). Não houve diferença significativa para a carga estática entre o lado mastectomizado dominante e o lado mastectomizado não dominante na posição anatômica (52,43 ± 4,069 vs. 49,69 ± 4,094, respectivamente). Conclusão: Os resultados atuais do nosso estudo mostraram distribuição de peso desequilibrada (carga vetorial no pé) em pacientes após mastectomia. Portanto, este texto descreve alterações posturais, musculares e desequilíbrio estático e dinâmico em pacientes com câncer de mama.

Semilleros de investigación como espacio de reconocimiento de personas con altas capacidades / Research hotbeds as a space for the recognition of gifted people / Semilleros de investigación como espaço de reconhecimento de pessoas com altas habilidades

Resumen (analítico) Se presentan los resultados de una investigación sobre semilleros de investigación como espacio de reconocimiento de personas con altas capacidades y talentos. Ejecutado a partir de la ruta fenomenológica y hermenéutica, utilizó como técnica la entrevista semiestructurada a cinco participantes con más de cinco años en semilleros de investigación, la cual fue interpretada a través de un análisis estructural. Se logra refigurar el concepto de semilleros de investigación y plantearlos como espacios de reconocimiento que configuran nuevas identidades y agenciamientos para la constitución de personas capaces, con apertura de sentidos hacia las posibilidades del reconocimiento mutuo y agenciamiento de nuevas prácticas en el ámbito académico, laboral y social. Las líneas de sentido son construidas por los semilleristas en contraposición a los procesos escolares; lo cual deja abierta la posibilidad de otros procesos de reconocimiento.

Abstract (analytical) The present study is the result of the research on research hotbeds (semilleros de investigación) as a space for the recognition of gifted and talented people. Using phenomenological and hermeneutical approaches, semi-structured interviews were used as a data collection technique with five participants who have more than 5 years of experience in research hotbeds. The data was interpreted using a structural analysis. It is possible to redefine the concept of research hotbeds and propose them as a space of recognition that configure new identities and methods for the constitution of capable people, opening up the possibilities of mutual recognition and developing new practices in academic, labor and social dimensions. Meanings are constructed by participants in the research hotbeds in contrast to school processes, creating the possibility of other forms of recognition.

Resumo (analítico) O presente trabalho é o resultado da pesquisa sobre o focos de pesquisa (semilleros de investigación) como espaço de reconhecimento de pessoas com altas capacidades e talentos. Executado a partir do percurso fenomenológico e hermenêutico, utilizando a entrevista semiestruturada como técnica para cinco participantes com mais de cinco anos em focos de pesquisa, interpretada através de uma análise estrutural. É possível reconfigurar o conceito de semilleros de investigación propôlos como um espaço de reconhecimento que configura novas identidades e agenciamentos para a constituição de pessoas capazes com abertura de sentidos para as possibilidades de reconhecimento mútuo e montagem de novas práticas no meio acadêmico, laboral e esferas sociais. As linhas de sentido são construídas pelas semilleristas em contraposição aos processos escolares. A possibilidade de outros processos de reconhecimento é deixada em aberto.

Loss of ABCA8B decreases myelination by reducing oligodendrocyte precursor cells in mice.

The myelin sheath, which is wrapped around axons, is a lipid-enriched structure produced by mature oligodendrocytes. Disruption of the myelin sheath is observed in several neurological diseases, such as multiple sclerosis. A crucial component of myelin is sphingomyelin, levels of which can be increased by ABCA8, a member of the ATP-binding cassette transporter family. ABCA8 is highly expressed in the cerebellum, specifically in oligodendroglia. However, whether ABCA8 plays a role in myelination and mechanisms that would underlie this role remain unknown. Here, we found that the absence of Abca8b, a mouse ortholog of ABCA8, led to decreased numbers of cerebellar oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes in mice. We show that in oligodendrocytes, ABCA8 interacts with chondroitin sulfate proteoglycan 4 (CSPG4), a molecule essential for OPC proliferation, migration, and myelination. In the absence of Abca8b, localization of CSPG4 to the plasma membrane was decreased, contributing to reduced cerebellar CSPG4 expression. Cerebellar CSPG4+ OPCs were also diminished, leading to decreased mature myelinating oligodendrocyte numbers and cerebellar myelination levels in Abca8b-/- mice. In addition, electron microscopy analyses showed that the number of nonmyelinated cerebellar axons was increased, whereas cerebellar myelin thickness (g-ratio), myelin sheath periodicity, and axonal diameter were all decreased, indicative of disordered myelin ultrastructure. In line with disrupted cerebellar myelination, Abca8b-/- mice showed lower cerebellar conduction velocity and disturbed locomotion. In summary, ABCA8 modulates cerebellar myelination, in part through functional regulation of the ABCA8-interacting protein CSPG4. Our findings suggest that ABCA8 disruption may contribute to the pathophysiology of myelin disorders.

Lipid efflux mechanisms, relation to disease and potential therapeutic aspects.

Lipids are hydrophobic and amphiphilic molecules involved in diverse functions such as membrane structure, energy metabolism, immunity, and signaling. However, altered intra-cellular lipid levels or composition can lead to metabolic and inflammatory dysfunction, as well as lipotoxicity. Thus, intra-cellular lipid homeostasis is tightly regulated by multiple mechanisms. Since most peripheral cells do not catabolize cholesterol, efflux (extra-cellular transport) of cholesterol is vital for lipid homeostasis. Defective efflux contributes to atherosclerotic plaque development, impaired ß-cell insulin secretion, and neuropathology. Of these, defective lipid efflux in macrophages in the arterial walls leading to foam cell and atherosclerotic plaque formation has been the most well studied, likely because a leading global cause of death is cardiovascular disease. Circulating high density lipoprotein particles play critical roles as acceptors of effluxed cellular lipids, suggesting their importance in disease etiology. We review here mechanisms and pathways that modulate lipid efflux, the role of lipid efflux in disease etiology, and therapeutic options aimed at modulating this critical process.

Therapeutic modulation of the bile acid pool by Cyp8b1 knockdown protects against nonalcoholic fatty liver disease in mice.

Bile acids (BAs) are surfactant molecules that regulate the intestinal absorption of lipids. Thus, the modulation of BAs represents a potential therapy for nonalcoholic fatty liver disease (NAFLD), which is characterized by hepatic accumulation of fat and is a major cause of liver disease worldwide. Cyp8b1 is a critical modulator of the hydrophobicity index of the BA pool. As a therapeutic proof of concept, we aimed to determine the impact of Cyp8b1 inhibition in vivo on BA pool composition and as protection against NAFLD. Inhibition of Cyp8b1 expression in mice led to a remodeling of the BA pool, which altered its signaling properties and decreased intestinal fat absorption. In a model of cholesterol-induced NAFLD, Cyp8b1 knockdown significantly decreased steatosis and hepatic lipid content, which has been associated with an increase in fecal lipid and BA excretion. Moreover, inhibition of Cyp8b1 not only decreased hepatic lipid accumulation, but also resulted in the clearance of previously accumulated hepatic cholesterol, which led to a regression in hepatic steatosis. Taken together, our data demonstrate that Cyp8b1 inhibition is a viable therapeutic target of crucial interest for metabolic diseases, such as NAFLD.-Chevre, R., Trigueros-Motos, L., Castaño, D., Chua, T., Corlianò, M., Patankar, J. V., Sng, L., Sim, L., Juin, T. L., Carissimo, G., Ng, L. F. P., Yi, C. N. J., Eliathamby, C. C., Groen, A. K., Hayden, M. R., Singaraja, R. R. Therapeutic modulation of the bile acid pool by Cyp8b1 knockdown protects against nonalcoholic fatty liver disease in mice.

ABCA8 Regulates Cholesterol Efflux and High-Density Lipoprotein Cholesterol Levels.

OBJECTIVE: High-density lipoproteins (HDL) are considered to protect against atherosclerosis in part by facilitating the removal of cholesterol from peripheral tissues. However, factors regulating lipid efflux are incompletely understood. We previously identified a variant in adenosine triphosphate-binding cassette transporter A8 (ABCA8) in an individual with low HDL cholesterol (HDLc). Here, we investigate the role of ABCA8 in cholesterol efflux and in regulating HDLc levels. APPROACH AND RESULTS: We sequenced ABCA8 in individuals with low and high HDLc and identified, exclusively in low HDLc probands, 3 predicted deleterious heterozygous ABCA8 mutations (p.Pro609Arg [P609R], IVS17-2 A>G and p.Thr741Stop [T741X]). HDLc levels were lower in heterozygous mutation carriers compared with first-degree family controls (0.86±0.34 versus 1.17±0.26 mmol/L; P=0.005). HDLc levels were significantly decreased by 29% (P=0.01) in Abca8b-/- mice on a high-cholesterol diet compared with wild-type mice, whereas hepatic overexpression of human ABCA8 in mice resulted in significant increases in plasma HDLc and the first steps of macrophage-to-feces reverse cholesterol transport. Overexpression of wild-type but not mutant ABCA8 resulted in a significant increase (1.8-fold; P=0.01) of cholesterol efflux to apolipoprotein AI in vitro. ABCA8 colocalizes and interacts with adenosine triphosphate-binding cassette transporter A1 and further potentiates adenosine triphosphate-binding cassette transporter A1-mediated cholesterol efflux. CONCLUSIONS: ABCA8 facilitates cholesterol efflux and modulates HDLc levels in humans and mice.

Cardiotrophin-1 eliminates hepatic steatosis in obese mice by mechanisms involving AMPK activation.

BACKGROUND & AIMS: Cardiotrophin-1 (CT-1) is a hepatoprotective cytokine that modulates fat and glucose metabolism in muscle and adipose tissue. Here we analyzed the changes in hepatic fat stores induced by recombinant CT-1 (rCT-1) and its therapeutic potential in non-alcoholic fatty liver disease (NAFLD). METHODS: rCT-1 was administered to two murine NAFLD models: ob/ob and high fat diet-fed mice. Livers were analyzed for lipid composition and expression of genes involved in fat metabolism. We studied the effects of rCT-1 on lipogenesis and fatty acid (FA) oxidation in liver cells and the ability of dominant negative inhibitor of AMP-activated protein kinase (AMPK) to block these effects. RESULTS: CT-1 was found to be upregulated in human and murine steatotic livers. In two NAFLD mouse models, treatment with rCT-1 for 10days induced a marked decrease in liver triglyceride content with augmented proportion of poly-unsaturated FA and reduction of monounsaturated species. These changes were accompanied by attenuation of inflammation and improved insulin signaling. Chronic administration of rCT-1 caused downregulation of lipogenic genes and genes involved in FA import to hepatocytes together with amelioration of ER stress, elevation of NAD(+)/NADH ratio, phosphorylation of LKB1 and AMPK, increased expression and activity of sirtuin1 (SIRT1) and upregulation of genes mediating FA oxidation. rCT-1 potently inhibited de novo lipogenesis and stimulated FA oxidation in liver cells both in vitro and in vivo. In vitro studies showed that these effects are mediated by activated AMPK. CONCLUSIONS: rCT-1 resolves hepatic steatosis in obese mice by mechanisms involving AMPK activation. rCT-1 deserves consideration as a potential therapy for NAFLD.

Cardiotrophin-1 determines liver engraftment of syngenic colon carcinoma cells through an immune system-mediated mechanism.

Cardiotrophin-1 (CT-1/CTF1) is a member of the interleukin-6 (IL-6) family of cytokines that stimulates STAT-3 phosphorylation in cells bearing the cognate receptor. We report that Ctf1(-/-) mice (hereby referred to as CT-1(-/-) mice) are resistant to the hepatic engraftment of MC38 colon carcinoma cells, while these cells engraft normally in the mouse subcutaneous tissue. Tumor intake in the liver could be enhanced by the systemic delivery of a recombinant adenovirus encoding CT-1, which also partly rescued the resistance of CT-1(-/-) mice to the hepatic engraftment of MC38 cells. Moreover, systemic treatment of wild-type (WT) mice with a novel antibody-neutralizing mouse CT-1 also reduced engraftment of this model. Conversely, experiments with Panc02 pancreatic cancer and B16-OVA melanoma cells in CT-1(-/-) mice revealed rates of hepatic engraftment similar to those observed in WT mice. The mechanism whereby CT-1 renders the liver permissive for MC38 metastasis involves T lymphocytes and natural killer (NK) cells, as shown by selective depletion experiments and in genetically deficient mice. However, no obvious changes in the number or cell killing capacity of liver lymphocytes in CT-1(-/-) animals could be substantiated. These findings demonstrate that the seed and soil concept to understand metastasis can be locally influenced by cytokines as well as by the cellular immune system.

Carbohydrate affinity for the glucose-galactose binding protein is regulated by allosteric domain motions.

Protein function, structure, and dynamics are intricately correlated, but studies on structure-activity relationships are still only rarely complemented by a detailed analysis of dynamics related to function (functional dynamics). Here, we have applied NMR to investigate the functional dynamics in two homologous periplasmic sugar binding proteins with bidomain composition: Escherichia coli glucose/galactose (GGBP) and ribose (RBP) binding proteins. In contrast to their structural and functional similarity, we observe a remarkable difference in functional dynamics: For RBP, the absence of segmental motions allows only for isolated structural adaptations upon carbohydrate binding in line with an induced fit mechanism; on the other hand, GGBP shows extensive segmental mobility in both apo and holo states, enabling selection of the most favorable conformation upon carbohydrate binding in line with a population shift mechanism. Collective segmental motions are controlled by the hinge composition: by swapping two identified key residues between RBP and GGBP we also interchange their segmental hinge mobility, and the doubly mutated GGBP* no longer experiences changes in conformational entropy upon ligand binding while the complementary RBP* shows the segmental dynamics observed in wild-type GGBP. Most importantly, the segmental interdomain dynamics always increase the apparent substrate affinity and thus, are functional, underscoring the allosteric control that the hinge region exerts on ligand binding.

Role of cardiotrophin-1 in obesity and insulin resistance.

Cardiotrophin-1 (CT-1) is a member of the gp130 family of cytokines. In a recent study we examined the metabolic features of ct-1 null mice and the effects on body composition, glucose and lipid metabolism of acute and chronic administration of recombinant CT-1. Our data revealed that CT-1 is a key regulator of energy metabolism with potential applications in the treatment of obesity and the metabolic syndrome. This commentary discusses the significance of these findings in the context of other key studies in the field of obesity and insulin resistance.

Structural, thermodynamic, and mechanistical studies in uroporphyrinogen III synthase: molecular basis of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is a rare autosomal disease ultimately related to deleterious mutations in uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the biosynthetic route of the heme group. UROIIIS catalyzes the cyclization of the linear tetrapyrrol hydroxymethylbilane (HMB), inverting the configuration in one of the aromatic rings. In the absence of the enzyme (or when ill-functioning), HMB spontaneously degrades to the by-product uroporphyrinogen I, which cannot lead to the heme group and accumulates in the body, producing some of the symptoms observed in CEP patients. In the present chapter, clinical, biochemical, and biophysical information has been compiled to provide an integrative view on the molecular basis of CEP. The high-resolution structure of UROIIIS sheds light on the enzyme reaction mechanism while thermodynamic analysis revealed that the protein is thermolabile. Pathogenic missense mutations are found throughout the primary sequence of the enzyme. All but one of these is rarely found in patients, whereas C73R is responsible for more than one-third of the reported cases. Most of the mutant proteins (C73R included) retain partial catalytic activity but the mutations often reduce the enzyme's stability. The stabilization of the protein in vivo is discussed in the context of a new line of intervention to complement existing treatments such as bone marrow transplantation and gene therapy.

Intracellular rescue of the uroporphyrinogen III synthase activity in enzymes carrying the hotspot mutation C73R.

A single mutation (C73R) in the enzyme uroporphyrinogen III synthase (UROIIIS) is responsible for more than one-third of all of the reported cases of the rare autosomal disease congenital erythropoietic porphyria (CEP). CEP patients carrying this hotspot mutation develop a severe phenotype of the disease, including reduced life expectancy. Here, we have investigated the molecular basis for the functional deficit in the mutant enzyme both in vitro and in cellular systems. We show that a Cys in position 73 is not essential for the catalytic activity of the enzyme but its mutation to Arg speeds up the process of irreversible unfolding and aggregation. In the mammalian cell milieu, the mutant protein levels decrease to below the detection limit, whereas wild type UROIIIS can be detected easily. The disparate response is not produced by differences at the level of transcription, and the results with cultured cells and in vitro are consistent with a model where the protein becomes very unstable upon mutation and triggers a degradation mechanism via the proteasome. Mutant protein levels can be restored upon cell treatment with the proteasome inhibitor MG132. The intracellularly recovered C73R-UROIIIS protein shows enzymatic activity, paving the way for a new line of therapeutic intervention in CEP patients.

Backbone chemical shifts assignments of D-allose binding protein in the free form and in complex with D-allose.

D-allose binding protein (ALBP) belongs to the family of perisplamic receptors of the bacterial ABC transporter system. ALBP experiences a significant conformational rearrangement upon binding to the sugar. Here, we report the sequential backbone assignment for the ALBP from Escherichia coli in the free form (BMRB no. 16982) and in complex with D-allose (BMRB no. 16984).

Halophilic enzyme activation induced by salts.

Halophilic archea (halobacteriae) thrive in hypersaline environments, avoiding osmotic shock by increasing the ion concentration of their cytoplasm by up to 3-6 M. To remain folded and active, their constitutive proteins have evolved towards a biased amino acid composition. High salt concentration affects catalytic activity in an enzyme-dependent way and a unified molecular mechanism remains elusive. Here, we have investigated a DNA ligase from Haloferax volcanii (Hv LigN) to show that K(+) triggers catalytic activity by preferentially stabilising a specific conformation in the reaction coordinate. Sodium ions, in turn, do not populate such isoform and the enzyme remains inactive in the presence of this co-solute. Our results show that the halophilic amino acid signature enhances the enzyme's thermodynamic stability, with an indirect effect on its catalytic activity. This model has been successfully applied to reengineer Hv LigN into an enzyme that is catalytically active in the presence of NaCl.

Structural basis for the aminoacid composition of proteins from halophilic archea.

Proteins from halophilic organisms, which live in extreme saline conditions, have evolved to remain folded at very high ionic strengths. The surfaces of halophilic proteins show a biased amino acid composition with a high prevalence of aspartic and glutamic acids, a low frequency of lysine, and a high occurrence of amino acids with a low hydrophobic character. Using extensive mutational studies on the protein surfaces, we show that it is possible to decrease the salt dependence of a typical halophilic protein to the level of a mesophilic form and engineer a protein from a mesophilic organism into an obligate halophilic form. NMR studies demonstrate complete preservation of the three-dimensional structure of extreme mutants and confirm that salt dependency is conferred exclusively by surface residues. In spite of the statistically established fact that most halophilic proteins are strongly acidic, analysis of a very large number of mutants showed that the effect of salt on protein stability is largely independent of the total protein charge. Conversely, we quantitatively demonstrate that halophilicity is directly related to a decrease in the accessible surface area.

Protein stabilization and the Hofmeister effect: the role of hydrophobic solvation.

Using the IGg binding domain of protein L from Streptoccocal magnus (ProtL) as a case study, we investigated how the anions of the Hofmeister series affect protein stability. To that end, a suite of lysine-to-glutamine modifications were obtained and structurally and thermodynamically characterized. The changes in stability introduced with the mutation are related to the solvent-accessible area of the side chain, specifically to the solvation of the nonpolar moiety of the residue. The thermostability for the set of ProtL mutants was determined in the presence of varying concentrations (0-1 M) of six sodium salts from the Hofmeister series: sulfate, phosphate, fluoride, nitrate, perchlorate, and thiocyanate. For kosmotropic anions (sulfate, phosphate, and fluoride), the stability changes induced by the cosolute (encoded in m(3)=deltaDeltaG(0)/deltaC(3)) are proportional to the surface changes introduced with the mutation. In contrast, the m(3) values measured for chaotropic anions are much more independent of such surface modifications. Our results are consistent with a model in which the increase in the solution surface tension induced by the anion stabilizes the folded conformation of the protein. This contribution complements the nonspecific and weak interactions between the ions and the protein backbone that shift the equilibrium toward the unfolded state.

Uroporphyrinogen III synthase mutations related to congenital erythropoietic porphyria identify a key helix for protein stability.

In the present study we have investigated deleterious mutants in the uroporphyrinogen III synthase (UROIIIS) that are related to the congenital erythropoietic porphyria (CEP). The 25 missense mutants found in CEP patients have been cloned, expressed, and purified. Their enzymatic activities have been measured relative to wild-type UROIIIS activity. All mutants retain measurable activity, consistent with the recessive character of the disease. Most of the mutants with a significant decrease in activity involve residues likely associated in binding. However, other mutants are fully active, indicating that different mechanisms may contribute to enzyme missfunction. UROIIIS is a thermolabile enzyme undergoing irreversible denaturation. The unfolding kinetics of wild-type UROIIIS and the suite of mutants have been monitored by circular dichroism. This analysis allowed the identification of a helical region in the molecule, essential to retain the kinetic stability of the folded conformation. C73R is found in one-third of CEP patients, and Cys73 is part of this helix. The integrated analysis of the enzymatic activity and kinetic stability data is used to gain insight in the relationship between defects in UROIIIS sequence and CEP.

Anion modulation of the 1H/2H exchange rates in backbone amide protons monitored by NMR spectroscopy.

The ability of three anionic cosolutes (sulfate, thiocyanate, and chloride) in modulating the (1)H/(2)H exchange rates for backbone amide protons has been investigated using nuclear magnetic resonance (NMR) for two different proteins: the IGg-binding domain of protein L (ProtL) and the glucose-galactose-binding protein (GGBP). Our results show that moderate anion concentrations (0.2 M-1 M) regulate the exchange rate following the Hofmeister series: Addition of thiocyanate increases the exchange rates for both proteins, while sulfate and chloride (to a less extent) slow down the exchange reaction. In the presence of the salt, no alteration of the protein structure and minimal variations in the number of measurable peaks are observed. Experiments with model compounds revealed that the unfolded state is modulated in an equivalent way by these cosolutes. For ProtL, the estimated values for the local free energy change upon salt addition (m (3,DeltaG )) are consistent with the previously reported free energy contribution from the cosolute's preferential interaction/exclusion term indicating that nonspecific weak interactions between the anion and the amide groups constitute the dominant mechanism for the exchange-rate modulation. The same trend is also found for GGBP in the presence of thiocyanate, underlining the generality of the exchange-rate modulation mechanism, complementary to more investigated effects like the electrostatic interactions or specific anion binding to protein sites.