Capecitabine-Related Thrombotic Microangiopathy.

PURPOSE: Renal injury is common in cancer patients and its etiology is multifactorial. Different patterns of renal histological lesions have been described in relation to oncologic treatments, notably acute tubular necrosis and tubulointerstitial nephritis, but also thrombotic microangiopathy (TMA). METHODS: We report a case of TMA secondary to capecitabine in an 82-year-old woman diagnosed with localized colon adenocarcinoma. RESULTS: The patient, with previous normal kidney function, presented with renal impairment during the fourth cycle of chemotherapy. After potential nephrotoxic factors were ruled out, capecitabine was discontinued and a kidney biopsy was performed, which displayed TMA lesions. An improvement in renal function was observed after definitive cessation of cytotoxic chemotherapy. Although rare, renal toxicity in the form of TMA may be associated with the use of cytotoxic agents such as gemcitabine, but there is no reported evidence of its association to capecitabine. Early withdrawal of the drug and nephrology consultation is necessary to prevent irreversible damage. CONCLUSION: We describe, to our knowledge, the first case reported in the literature regarding the possible association of TMA and capecitabine.

Singlet oxygen quenching as a probe for cytochrome c molten globule state formation.

Singlet oxygen refers to the nonradical metastable excited state of molecular oxygen that readily oxidizes various cellular components. Its behavior in different biological systems has been studied for many years. Recently, we analyzed the effect of singlet oxygen quenching by heme cofactor in cytochrome c (cyt c). Here, we have exploited this effect in the investigation of conformational differences in the molten globule states of cyt c induced by different sodium anions, namely sulfate, chloride and perchlorate. The high efficiency of heme toward quenching singlet oxygen enabled us to use this property for the analysis of the otherwise experimentally difficult-to-determine parameter of heme upon exposure to solvents as highly similar conformational states of cyt c in the molten globule states are induced by different salts at acidic pH. Our results from singlet oxygen quenching experiments correlate well with other spectroscopic methods, such as circular dichroism and fluorescence measurements, and suggest increasing availability of heme in the order: perchlorate < chloride < sulfate. Based on our findings we propose that singlet oxygen phosphorescence measurements are useful in determining the differences in the protein conformation of their heme regions, particularly regarding the relative heme exposure to the solvent.

Specific anion effect on properties of HRV 3C protease.

Specific salts effect is intensively studied from the prospective of modification of different physico-chemical properties of biomacromolecules. Limited knowledge of the specific salts effect on enzymes led us to address the influence of five sodium anions: sulfate, phosphate, chloride, bromide, and perchlorate, on catalytic and conformational properties of human rhinovirus-14 (HRV) 3C protease. The enzyme conformation was monitored by circular dichroism spectrum (CD) and by tyrosines fluorescence. Stability and flexibility of the enzyme have been analyzed by CD in the far-UV region, differential scanning calorimetry and molecular dynamics simulations, respectively. We showed significant influence of the anions on the enzyme properties in accordance with the Hofmeister effect. The HRV 3C protease in the presence of kosmotropic anions, in contrast with chaotropic anions, exhibits increased stability, rigidity. Correlations of stabilization effect of anions on the enzyme with their charge density and the rate constant of the enzyme with the viscosity B-coefficients of anions suggest direct interaction of the anions with HRV 3C protease. The role of stabilization and decreased fluctuation of the polypeptide chain of HRV 3C protease on its activation in the presence of kosmotropic anions is discussed within the frame of the macromolecular rate theory.

Purification of MBP fusion proteins using engineered DARPin affinity matrix.

Maltose binding protein (MBP) has a long history as an expression tag with the ability to increase the solubility of fused proteins. A critical step for obtaining a sufficient amount of the MBP fusion protein is purification. Commercially available amylose matrix for the affinity purification of MBP fusion proteins has two main issues: (i) low (micromolar) affinity and (ii) the limited number of uses due to the cleavage of polysaccharide matrix by the amylases, present in the crude cell extract. Here, we present a new affinity purification approach based on the protein-protein interaction. We developed the affinity matrix which contains immobilized Designed Ankyrin Repeat Protein off7 (DARPin off7) - previously identified MBP binder with nanomolar affinity. The functionality of the DARPin affinity matrix was tested on the purification of MBP-tagged green fluorescent protein and flavodoxin. The affinity purification of the MBP fusion proteins, based on the MBP-DARPin off7 interaction, enables the purification of the fusion proteins in a simple two-steps procedure. The DARPin affinity matrix - easy to construct, resistant to amylase, insensitive to maltose contamination, and reusable for multiple purification cycles - provides an alternative approach to commercially available affinity matrices for purification of proteins containing the MBP tag.

Heme is responsible for enhanced singlet oxygen deactivation in cytochrome c.

The deactivation of singlet oxygen, the lowest electronic excited state of molecular oxygen, by proteins is usually described through the interaction of singlet oxygen with certain amino acids. Changes in accessibility of these amino acids influence the quenching rate and the phosphorescence kinetics of singlet oxygen. In the cellular environment, however, numerous proteins with covalently bound or encapsulated cofactors are present. These cofactors could also influence the deactivation of singlet oxygen, and these have received little attention. To confront this issue, we used cytochrome c (cyt c) and apocytochrome c (apocyt c) to illustrate how the heme prosthetic group influences the rate constant of singlet oxygen deactivation upon acidic pH-induced conformational change of cyt c. Photo-excited flavin mononucleotide (FMN) was used to produce singlet oxygen. Our data show that the heme group has a significant and measurable effect on singlet oxygen quenching when the heme is exposed to solvents and is therefore more accessible to singlet oxygen. The effect of amino acids and heme accessibility on the FMN triplet state deactivation was also investigated.

Destabilization effect of imidazolium cation-Hofmeister anion salts on cytochrome c.

We have analyzed an effect of imidazolium cation-Hofmeister anion salts on stability of basic horse heart cytochrome c (cyt c) at pH4.5 (net charge +17). The effect of salts consisting of imidazolium cations, 1-ethyl-3-methylimidazolium (EMIm+) and 1-butyl-3-methylimidazolium (BMIm+), and five anions: chloride, bromide, iodide, nitrate, and thiocyanate on thermal and pH stability of cyt c was compared with the effect of corresponding sodium salts. Correlation between parameter of dTtrs/d [ion] (Ttrs; thermal midpoints) with surface tension changes of solvent in the presence of both imidazolium and sodium salts implies direct interaction between ions and proteins. Surprisingly, the imidazolium salts have more pronounced destabilization effect on highly positively charged cyt c than the corresponding sodium counterparts. Our analysis suggests the direct interaction of imidazolium cations with polypeptide chain, in analogy to guanidium cation, but the destabilization effect is significantly strengthened by decreased surface tension of imidazolium salt solvents. Comparison of an effect of imidazolium and sodium salts on acidic and alkaline transitions and to thermal transition of cyt c implies a role of hydrophobic interaction between imidazolium cation and polypeptide chain.

Synthesis and antimalarial activity of new heterocyclic hybrids based on chloroquine and thiazolidinone scaffolds.

A series of new 21 chloroquine heterocyclic hybrids containing either benzylamino fragment or N-(aminoalkyl)thiazolidin-4-one moiety were synthesized and screened for their antimalarial activity against chloroquine (CQ)-sensitive 3D7 and multidrug-resistance Dd2 strains of Plasmodium falciparum. Although no compounds more active than CQ against 3D7 was found; against Dd2 strain, six compounds, four of them with benzylamino fragment, showed an excellent activity, up to 3-fold more active than CQ. Non specific cytotoxicity on J774 macrophages was observed in some compounds whereas only two of them showed liver toxicity on HepG2 cells. In addition, all active compounds inhibited the ferriprotoporphyrin IX biocrystalization process in concentrations around to CQ. In vivo preliminary results have shown that at least two compounds are as active as CQ against Plasmodium berghei ANKA.

New 1-aryl-3-substituted propanol derivatives as antimalarial agents.

This paper describes the synthesis and in vitro antimalarial activity against a P. falciparum 3D7 strain of some new 1-aryl-3-substituted propanol derivatives. Twelve of the tested compounds showed an IC(50) lower than 1 microM. These compounds were also tested for cytotoxicity in murine J774 macrophages. The most active compounds were evaluated for in vivo activity against P. berghei in a 4-day suppressive test. Compound 12 inhibited more than 50% of parasite growth at a dose of 50 mg/kg/day. In addition, an FBIT test was performed to measure the ability to inhibit ferriprotoporphyrin biocrystallization. This data indicates that 1-aryl-3-substituted propanol derivatives hold promise as a new therapeutic option for the treatment of malaria.

A six-gene signature predicting breast cancer lung metastasis.

The lungs are a frequent target of metastatic breast cancer cells, but the underlying molecular mechanisms are unclear. All existing data were obtained either using statistical association between gene expression measurements found in primary tumors and clinical outcome, or using experimentally derived signatures from mouse tumor models. Here, we describe a distinct approach that consists of using tissue surgically resected from lung metastatic lesions and comparing their gene expression profiles with those from nonpulmonary sites, all coming from breast cancer patients. We show that the gene expression profiles of organ-specific metastatic lesions can be used to predict lung metastasis in breast cancer. We identified a set of 21 lung metastasis-associated genes. Using a cohort of 72 lymph node-negative breast cancer patients, we developed a 6-gene prognostic classifier that discriminated breast primary cancers with a significantly higher risk of lung metastasis. We then validated the predictive ability of the 6-gene signature in 3 independent cohorts of breast cancers consisting of a total of 721 patients. Finally, we show that the signature improves risk stratification independently of known standard clinical variables and a previously established lung metastasis signature based on an experimental breast cancer metastasis model.