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Biomedical subjects

Juan Cabezas-Herrera

Publications and source records attributed to Juan Cabezas-Herrera.

13 recordsLinked to original sources
PubMed2006

Antifolates as antimycotics? Connection between the folic acid cycle and the ergosterol biosynthesis pathway in Candida albicans.

The increased incidence of invasive mycoses and the emerging problem of antifungal drug resistance have encouraged the search for new antifungal agents or effective combinations of existing drugs. Infections due to Candida albicans are usually treated with azole antifungals such as fluconazole, ketoconazole or itraconazole. Whilst azoles may have little or no toxicity, they generally offer rather poor fungicidal activity. Even in the absence of resistance, treatment failures or recurrent infections are not uncommon, especially in immunocompromised individuals. Here we demonstrate that the non-classical antifolate pyrimethamine shows synergy with azole antifungal compounds and interferes with the ergosterol biosynthesis pathway in C. albicans. By disturbing folate metabolism in this fungus, pyrimethamine can inhibit ergosterol production. The molecular connection between the folic acid cycle and the ergosterol biosynthesis pathway is discussed and we show that the filamentous form of this fungus is more susceptible to methotrexate than the yeast form because the drug is more effectively transported through the membrane of the filamentous form. When used to treat the hyphal form, methotrexate showed synergy with other antifungals such as azoles and terbinafine. This finding could have important clinical applications, as a combination of azoles with antifolates and/or inhibitors of folic acid synthesis could represent an attractive alternative for the treatment of C. albicans infections.

Antifungal Agents↗
PubMed2006

Acetyl- and butyrylcholinesterase activities decrease in human colon adenocarcinoma.

Apart from the hydrolysis of acetylcholine (ACh), acetyl- (AChE) and butyrylcholinesterase (BChE), through noncatalytic mechanisms, intervene in hematopoiesis, morphogenesis, and neurogenesis (Layer and Willbold, 1995; Soreq and Seidman, 2001). Cholinesterase (ChE) molecules occur as globular (G1, G2, and G4) and asymmetric (A4, A8, and A12) forms (Legay, 2000; Massoulié, 2002). The G species might display amphiphilic (GA) or hydrophilic (GH) properties (Perrier et al., 2002). The involvement of ChEs in tumorigenesis is supported by the measurement of ChE activity in tumors (García-Ayllón et al., 2001; Ruiz-Espejo et al., 2003), the amplification of ChE genes in leukemias and ovarian tumors, and the relationship between the expression of AChE and the aggressiveness of astrocytomas(Perry et al., 2002). This research was undertaken to determine whether ChE activity is altered in gut carcinomas.

Acetylcholinesterase↗
PubMed2005

Acetylcholinesterase biogenesis is impaired in lung cancer tissues.

Studies cited by Cowan et al. [J. Appl. Toxicol. 23, 177 (2003)] indicate existence of inflammatory and cholinergic pathways in both nerve agents and sulfur mustard (HD) injury. Increase in AChE synthesis and neurite extension was noted after exposure to HD [K.W. Lanks et al., Exp. Cell Res. 355 (1975)]. Moreover, anti-inflammatory drugs reduce the dermal, respiratory and ocular damage caused by exposure to HD. On the other hand, recent studies have noted the involvement of neuro-inflammatory processes during exposure to the nerve agents sarin or soman [Cowan et al., 2003]. The use of various anti-inflammatory drugs in addition to the classical antidotal drugs (e.g. atropine and oximes) caused decrease in certain toxic symptoms and inflammation-induced brain damage. Our new bifunctional drugs (Scheme 1) are based on CNS-permeable molecular combination of pseudo-reversible AChE inhibitor (pyridostigmine, PYR) coupled via a hydrophobic spacer (octyl or decyl hydrocarbon chain) to a non-steroidal anti-inflammatory drug (NSAID) such as Ibuprofen or Diclofenac (Scheme 1). This study evaluates the efficacy of certain bifunctional compounds against HD and soman poisoning in mice in vivo.

Acetylcholinesterase↗
PubMed2005

Cholinesterase activity of human lung tumours varies according to their histological classification.

The probable involvement of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in cancer and the relevance of cholinergic responses for lung cancer growth prompted us to study whether cholinesterase activity of human lung is altered by malignancy. Surgical pieces of non-small lung carcinomas (NSLC) and their adjacent non-cancerous tissues (ANCT) were analysed for AChE and BChE activities. AChE activity in adenocarcinoma (AC) was 7.80 +/- 5.59 nmol of substrate hydrolysed per min and per mg of protein (mU/mg), the same as in their ANCT (8.83 +/- 4.72 mU/mg; P = 0.823); in large cell carcinoma (LCC), 7.52 +/- 3.32 mU/mg, approximately 50% less than in their ANCT (15.39 +/- 5.66 mU/mg; P = 0.043); and in squamous cell carcinoma (SCC), 1.39 +/- 0.58 mU/mg, 80% less than in ANCT (6.08 +/- 2.88 mU/mg; P = 0.003). BChE activity was 5.85 +/- 3.20 mU/mg in AC and 9.56 +/- 3.38 mU/mg in ANCT (P = 0.022); 2.94 +/- 2.01 mU/mg in LCC and 6.50 +/- 6.63 mU/mg in ANCT (P = 0.068); and 4.49 +/- 2.30 mU/mg in SCC and ANCT 6.56 +/- 4.09 mU/mg (P = 0.026). Abundant AChE dimers and fewer monomers were identified in lung and, although their distribution was unaffected by cancer, the binding with concanavalin A revealed changes in AChE glycosylation between SCC and their ANCT. The fall in BChE activity affected all molecules, with a strong decrease of the amphiphilic tetramers. Western blotting revealed protein bands with the expected mass of the principal AChE subunits, and the deeper intensity of the protein signal in SCC than in healthy lung, in lanes loaded with the same units of AChE activity, supported an augment in the amount of AChE protein/unit of AChE activity in SCC. The increased availability of acetylcholine in neoplastic lung, resulting from the fall of cholinesterase activity, may enhance cholinergic signalling and contribute to tumour progression.

Acetylcholine↗
PubMed2005

Real time PCR assay with fluorescent hybridization probes for genotyping intronic polymorphism in presenilin-1 gene.

BACKGROUND: Identification of all susceptibility loci for Alzheimer's disease has been a major goal in resolving the pathogenesis of this disease. METHODS: A PCR assay with fluorescently labeled oligonucleotide hybrinization probes with subsequent fluorescent probe melting point analysis was developed. RESULTS: Allelic discrimination of intronic polymorphism of presenilin-1 gene and the restriction fragment length polymorphism method yielded identical results, proving its usefulness for genotyping PS1 gene. CONCLUSIONS: This method provides excellent robustness, speed, and accuracy, and is well suited for determination of the polymorphism in both small and large numbers of samples. This assay could help to overcome the controversy regarding the association between the PS1 s165932 intronic polymorphism and Alzheimer's disease.

Alleles↗
PubMed2005

Kinetics of the inhibition of bovine liver dihydrofolate reductase by tea catechins: origin of slow-binding inhibition and pH studies.

Dihydrofolate reductase (DHFR) is the subject of intensive investigation since it appears to be the primary target enzyme for "antifolate" drugs, such as methotrexate and trimethoprim. Fluorescence quenching and stopped-flow fluorimetry show that the ester bond-containing tea polyphenols (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are potent and specific inhibitors of DHFR with inhibition constants (K(I)) of 120 and 82 nM, respectively. Both tea compounds showed the characteristics of slow-binding inhibitors of bovine liver DHFR. In this work, we have determined a complete kinetic scheme to explain the slow-binding inhibition and the pH effects observed during the inhibition of bovine liver DHFR by these tea polyphenols. Experimental data, based on fluorimetric titrations, and transient phase and steady-state kinetic studies confirm that EGCG and ECG are competitive inhibitors with respect to 7,8-dihydrofolate, which bind preferentially to the free form of the enzyme. The origin of their slow-binding inhibition is proposed to be the formation of a slow dissociation ternary complex by the reaction of NADPH with the enzyme-inhibitor complex. The pH controls both the ionization of critical catalytic residues of the enzyme and the protonation state of the inhibitors. At acidic pH, EGCG and ECG are mainly present as protonated species, whereas near neutrality, they evolve toward deprotonated species due to ionization of the ester-bonded gallate moiety (pK = 7.8). Although DHFR exhibits different affinities for the protonated and deprotonated forms of EGCG and ECG, it appears that the ionization state of Glu-30 in DHFR is critical for its inhibition. The physiological implications of these pH dependencies are also discussed.

Amino Acid Sequence↗
PubMed2005

The antifolate activity of tea catechins.

A naturally occurring gallated polyphenol isolated from green tea leaves, (-)-epigallocatechin gallate (EGCG), has been shown to be an inhibitor of dihydrofolate reductase (DHFR) activity in vitro at concentrations found in the serum and tissues of green tea drinkers (0.1-1.0 micromol/L). These data provide the first evidence that the prophylactic effect of green tea drinking on certain forms of cancer, suggested by epidemiologic studies, is due to the inhibition of DHFR by EGCG and could also explain why tea extracts have been traditionally used in "alternative medicine" as anticarcinogenic/antibiotic agents or in the treatment of conditions such as psoriasis. EGCG exhibited kinetics characteristic of a slow, tight-binding inhibitor of 7,8-dihydrofolate reduction with bovine liver DHFR (K(I) = 0.109 micromol/L), but of a classic, reversible, competitive inhibitor with chicken liver DHFR (K(I) = 10.3 micromol/L). Structural modeling showed that EGCG can bind to human DHFR at the same site and in a similar orientation to that observed for some structurally characterized DHFR inhibitor complexes. The responses of lymphoma cells to EGCG and known antifolates were similar, that is, a dose-dependent inhibition of cell growth (IC50 = 20 micromol/L for EGCG), G0-G1 phase arrest of the cell cycle, and induction of apoptosis. Folate depletion increased the sensitivity of these cell lines to antifolates and EGCG. These effects were attenuated by growing the cells in a medium containing hypoxanthine-thymidine, consistent with DHFR being the site of action for EGCG.

Animals↗
PubMed2005

Antifolate activity of epigallocatechin gallate against Stenotrophomonas maltophilia.

The catechin epigallocatechin gallate, one of the main constituents of green tea, showed strong antibiotic activity against 18 isolates of Stenotrophomonas maltophilia (MIC range, 4 to 256 microg/ml). In elucidating its mechanism of action, we have shown that epigallocatechin gallate is an efficient inhibitor of S. maltophilia dihydrofolate reductase, a strategic enzyme that is considered an attractive target for the development of antibacterial agents. The inhibition of S. maltophilia dihydrofolate reductase by this tea compound was studied and compared with the mechanism of a nonclassical antifolate compound, trimethoprim. Investigation of dihydrofolate reductase was undertaken with both a trimethoprim-susceptible S. maltophilia isolate and an isolate with a high level of resistance. The enzymes were purified using ammonium sulfate precipitation, gel filtration, and methotrexate affinity chromatography. The two isolates showed similar levels of dihydrofolate reductase expression and similar substrate kinetics. However, the dihydrofolate reductase from the trimethoprim-resistant isolate demonstrated decreased susceptibility to inhibition by trimethoprim and epigallocatechin gallate. As with other antifolates, the action of epigallocatechin gallate was synergistic with that of sulfamethoxazole, a drug that blocks folic acid metabolism in bacteria, and the inhibition of bacterial growth was attenuated by including leucovorin in the growth medium. We conclude that the mechanism of action of epigallocatechin gallate on S. maltophilia is related to its antifolate activity.

Anti-Bacterial Agents↗
PubMed2005

Use of a time-resolved immunofluorometric assay for determination of canine C-reactive protein concentrations in whole blood.

OBJECTIVE: To develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for measurement of C-reactive protein (CRP) in canine whole blood. ANIMALS: 12 healthy dogs and 35 dogs with inflammatory processes. PROCEDURE: CRP was isolated from acute-phase serum by affinity chromatography and used as a standard for calibration. Analytic and functional limit of detection and intra-assay and interassay precision were calculated. Accuracy was evaluated by recovery assays and by comparison with results of a commercial ELISA. Correlation between CRP concentrations in whole blood and corresponding plasma fractions was tested by use of TR-IFMA. Stability of blood samples at 4 degrees C was assessed during a 1-month period, and effects of anticoagulants were evaluated. Measurements of CRP in blood samples from 12 healthy dogs were compared with those of 35 dogs with inflammatory diseases. RESULTS: Analytic and functional limits of detection were 0.53 and 3.26 microg/mL, respectively. Intra-assay and interassay coefficients of variation varied between 2.1% to 8.9% and 8.0% to 12.3%, respectively. Mean recoveries of added CRP were 104% and 114%. Measurements of CRP by use of TR-IFMA and ELISA were highly correlated (R2 = 0.97). Measurements of CRP in whole blood and in corresponding plasma fractions by use of TR-IFMA were also highly correlated (R2 = 0.97). Neither storage nor use of anticoagulants disturbed measurement of CRP concentrations in whole blood. Concentrations of CRP in whole blood of dogs with inflammation were significantly higher than in healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Determination of CRP concentrations in whole blood may provide a diagnostic test for inflammation in dogs.

Animals↗
PubMed2003

Breast cancer metastasis alters acetylcholinesterase activity and the composition of enzyme forms in axillary lymph nodes.

Because of the probable involvement of cholinesterases (ChEs) in tumorigenesis, this research was addressed to ascertaining whether breast cancer metastasis alters the content of acetylcholinesterase (AChE) and/or butyrylcholinesterase (BuChE) in axillary lymph nodes (LN). ChE activity was assayed in nine normal (NLN) and seven metastasis-bearing nodes (MLN) from women. AChE and BuChE forms were characterised by sedimentation analyses, hydrophobic chromatography and western blotting. The origin of ChEs in LN was studied by lectin interaction. AChE activity dropped from 21.6 mU/mg (nmol of the substrate hydrolysed per minute and per milligram protein) in NLN to 3.8 mU/mg in MLN (p < 0.001), while BuChE activity (3.6 mU/mg) was little affected. NLN contained globular amphiphilic AChE dimers (G2A, 35%), monomers (G1A, 30%), hydrophilic tetramers (G4H, 8%), and asymmetric species (A4, 23%, and A8, 4%); MLN displayed only G2A (65%) and G1A (35%) AChE forms. NLN and MLN contained G4H (79%), G4A (7%), and G1H (14%) BuChE components. Neither the binding of ChE forms with lectins and antibodies nor the subunit size were altered by metastasis. The higher level of AChE in NLN than in brain and the specific pattern of AChE forms in NLN support its role in immunity. The different profile of AChE forms in NLN and MLN may be useful for diagnosis.

Acetylcholinesterase↗
PubMed2002

Muscular dystrophy with laminin deficiency decreases the content of butyrylcholinesterase tetramers in sciatic nerves of Lama2dy mice.

The Lama2dy mouse, a model of congenital muscular dystrophy (CMD) by merosin deficiency (MCMD), shows muscle degeneration and dysmyelination of peripheral nerves. Although it has been reported that MCMD reduces acetylcholinesterase (AChE) activity of mouse sciatic nerve, no information is available regarding its action on butyrylcholinesterase (BuChE). Amphiphilic BuChE monomers (G(1)(A), 39%), dimers (G(2)(A), 18%), and tetramers (G(4)(A), 33%), along with hydrophilic tetramers (G(4)(H), 10%), were identified in mouse sciatic nerve. It also contained abundant G(4)(A) (75%) and less G(1)(A), G(2)(A), G(4)(H) and A(12) AChE components. In dystrophic nerves, the BuChE activity increased 2-fold but the proportion of the G(4)(A) form dropped from 33% to 10%. AChE activity decreased and the composition of enzyme forms was unaffected. Lectin interaction studies showed that, in contrast to skeletal muscle, the defect of merosin did not greatly alter the glycosylation of nerve cholinesterases. The anomalous synthesis of BuChE forms in dystrophic nerve may be related with peripheral neuropathy of MCMD.

Acetylcholinesterase↗
PubMed2002

Cholinesterase activity and acetylcholinesterase glycosylation are altered in human breast cancer.

Increasing evidence supports the involvement of cholinesterases in tumorigenesis. Several tumour cells show ChE activity, while the acetyl- (AChE) and butyrylcholinesterase (BuChE) genes are amplified in leukemias, ovarian carcinoma and other cancers. ChE activity was measured in 31 samples of tumoral breast (TB) and 20 of normal breast (NB). Despite the wide variations observed, BuChE predominated over AChE both in TB and NB. The mean AChE activity in NB was 1.61 nmol of the substrate hydrolysed per minute and per miligram protein (mU/mg), which rose to 3.09 mU/mg in TB (p = 0.041). The BuChE activity dropped from 5.24 mU/mg in NB to 3.39 mU/mg in TB (p = 0.002). Glycolipid-linked AChE dimers and monomers and hydrophilic BuChE tetramers and monomers were identified in NB and TB, and their proportions were unmodified by the neoplasia. The amount of AChE forms reacting with wheat germ agglutinin (WGA) decreased in TB while that of BuChE species was unaffected, demonstrating that the glycosylation of AChE was altered in TB. The binding of AChE and BuChE with antibodies was unaffected by the neoplasia. The difference in lectin reactivity between erythrocyte and breast AChE, the lack of AChE in blood plasma, and the finding of monomeric BuChE in breast but not in plasma suggest that breast epithelial cells produce AChE for membrane attachment and hydrophilic BuChE for secretion. Several reasons are provided to explain the altered expression of ChEs in breast cancer.

Acetylcholinesterase↗