LOXL2-mediated H3K4 oxidation reduces chromatin accessibility in triple-negative breast cancer cells.

Oxidation of H3 at lysine 4 (H3K4ox) by lysyl oxidase-like 2 (LOXL2) generates an H3 modification with an unknown physiological function. We find that LOXL2 and H3K4ox are higher in triple-negative breast cancer (TNBC) cell lines and patient-derived xenografts (PDXs) than those from other breast cancer subtypes. ChIP-seq revealed that H3K4ox is located primarily in heterochromatin, where it is involved in chromatin compaction. Knocking down LOXL2 reduces H3K4ox levels and causes chromatin decompaction, resulting in a sustained activation of the DNA damage response (DDR) and increased susceptibility to anticancer agents. This critical role that LOXL2 and oxidized H3 play in chromatin compaction and DDR suggests that functionally targeting LOXL2 could be a way to sensitize TNBC cells to conventional therapy.

Análisis de los títulos aprobados de grado en farmacia / Study of the approved degrees in pharmacy

La implementación de los estudios universitarios al Espacio Europeo de Educación Superior (EEES), se ha regulado en España con la publicación del Real Decreto 1393/2007, que establece la ordenación de las enseñanzas universitarias oficiales de Grado, Master y Doctorado. En este contexto, el Ministerio de Educación ya ha aprobado los primeros estudios de Grado en Farmacia adaptados al EEES de algunas universidades españolas. El objetivo de nuestro trabajo es analizar los planes de estudio de los títulos de Grado en Farmacia que han sido aprobados. Hemos estudiado la planificación de las enseñanzas en cuanto a los Módulos requeridos para la verificación del título oficial, así como otros Módulos propios. También hemos analizado el porcentaje de carga lectiva de las materias en el plan de estudios, atendiendo a su carácter: básicas, obligatorias y optativas. Por último, hemos examinado la selección de las materias que constituyen los módulos. Podemos concluir que los planes de estudio de las universidades analizadas presentan una gran uniformidad en cuanto a los contenidos formativos. La mayor diversidad en la trayectoria curricular tiene lugar en la selección de las asignaturas optativas, así como en los Módulos de carácter humanístico, estos últimos frecuentes en las universidades privadas(AU)

To comply with the European Higher Education Area (EHEA) in Spain, a law was published (RD 1393/2007) which regulates official studies for degrees, masters and doctorates. Several new Pharmacy degrees have been approved by the Ministry for Education according to this law, both in public and private universities. The objective of our work was to analyse these Pharmacy degrees, paying attention to the content of the various modules, as well as to the relative percentage of teaching load within each topic in the curricula according to its nature: basic, obligatory and optional. We can conclude that the degrees of all universities analysed are very homogeneous, particularly in regard to the formative content of the established modules, in accordance to the general guidelines. The greater variation seems to be located in optional subjects as well as in Art and Humanities modules, the latter being quite common in private universities(AU)

¿Cómo valoran nuestros alumnos las estrategias docentes que utilizamos? / Student perception of our teaching strategies

La implantación de los nuevos grados, dentro del EEES, supone un aumento en la participación del alumno en su aprendizaje y la introducción de nuevas estrategias docentes. Debido a ello, nuestro objetivo ha sido valorar la opinión de los alumnos sobre tres aspectos: las estrategias docentes utilizadas en el proceso de enseñanza-aprendizaje, la metodología de evaluación y la importancia que otorgan a la adquisición de competencias transversales, como hablar en público o trabajar en equipo. Para ello, se ha realizado un estudio, a través de una encuesta voluntaria y anónima a los alumnos de tres asignaturas de Farmacia. De los datos obtenidos destacamos que un 93.1% afirmó que la asistencia a clase ayuda a comprender la asignatura y que un 82.8% consideró que la participación activa en el aula le ayuda a aprender. Respecto a las estrategias docentes, los alumnos eligieron las explicaciones del profesor y la pizarra como el método más útil para su aprendizaje, quedando en segundo lugar la realización de ejercicios, prácticas y en último lugar, las presentaciones en Power Point. Como conclusión, los alumnos valoran positivamente aquellas estrategias que fomentan su participación, así como el sistema de evaluación continua. Sin embargo, hemos de resaltar la alta valoración otorgada a las explicaciones del profesor en la pizarra. Por ello, los datos obtenidos deberían hacernos reflexionar sobre la importancia de incluir nuevas estrategias docentes que fomenten la participación del alumno, pero sin olvidar, el valor del que quizá sea el instrumento docente más antiguo, la pizarra y el profesor(AU)

The European Higher Education Area (EHEA) will change our teaching methodology with the implementation of new teaching strategies and greater participation of students in the learning process. The objective of our study is to evaluate the students’ views in three main areas: a) teaching strategies promoted by the lecturer for the teaching/learning process, b) evaluation methods and c) achieve good competence in other areas such as oral communication skills or team-work. Thus we designed a questionnaire which was filled by Pharmacy students from various years (n=118; 40.5% males and 59.5% females) registered in several subjects. Analysis of data revealed that 93.1% considered that attendance to lectures helped them to understand the topics covered. A good proportion (82.8%) was also of the view that participating in lectures was a good aid to learning. The students valued the teachers explanations and the use of the blackboard as the most useful for them to learn followed by practicals and exercises and lastly by PowerPoint presentations. Our data suggest that students value strategies that make them participate in their own learning process as well as a system of continuing evaluation. The results obtained highlight the importance of teaching strategies which stimulate student participation but also the high value the students allocate to the most classic and characteristic features of any classroom: teacher and blackboard(AU)

Allometric scaling of echocardiographic measurements in healthy Spanish foals with different body weight.

Scaling in biology is usually allometric, and therefore, the size of the heart may be expressed as a power function of body weight (BW). The present research analyses the echocardiographic measurements in 68 healthy Spanish foals weighed between 70 and 347kg in order to determine the correct scaling exponent for the allometric equation. The echocardiographic parameters measured were: left ventricular internal dimensions (LVID), free wall thickness (LVFWT), interventricular septum thickness (IVST) at systole (s) and diastole (d), EPSS (distance between the point E of the mitral valve and the interventricular septum), and aorta diameters at the level of the aortic valve (AOD), base of valve leaflets (ABS), sinus of Valsalva (ASV) and sino-tubular junction (AJT). Indices of left ventricular performance were calculated. It was found that LVIDd, IVSTs, AOD, and ASV have a relationship to BW raised to 0.300-0.368 power, whereas left ventricular end-diastolic volume and stroke volume scaled to BW raised to 0.731-0.712 power. With these data, appropriate values can be calculated for normal Spanish foals.

Ocular biometry in the adult anesthetized ferret (Mustela putorius furo).

A total of 44 eyes of 22 ferrets were examined by B-mode ultrasonography. Four dimensions were measured: the distance from the corneal surface to the anterior lens surface (M1), the axial lens thickness (M2), the distance from the posterior lens surface to the retina (M3) and the distance from the anterior cornea to the retina (M4). The values obtained were (mean, standard deviation, range): M1: 1.31 +/- 0.16 mm (1-1.7); M2: 3.42 +/- 0.15 mm (3.2-3.7); M3: 2.26 +/- 0.11 mm (2.1-2.5); M4: 7 +/- 0.24 mm (6.4-7.7). To our knowledge this is the first description of ocular biometric measurement in the ferret. These values show little variability of ocular dimensions in ferrets.

Effect of chemical enhancers on the in vitro percutaneous absorption of sumatriptan succinate.

The effects of percutaneous enhancers on the transdermal absorption of sumatriptan succinate were investigated by in vitro permeation studies. Pretreatment of porcine skin with ethanol (vehicle), polyethylene glycol 600, Span 20, oleic acid, R-(+)-limonene, alpha-bisabolol and 1,8-cineole (at 5% in ethanol, w/w) produced in all cases an increase in the flux of sumatriptan. The amount of sumatriptan retained in the skin was also determined. Ethanol has showed a low but significant increment on the drug transdermal flux. Treatment of the skin with alpha-bisabolol shows the same enhancer effect than ethanol. Span 20, oleic acid, and polyethylene glycol 600 have shown a moderate enhancing activity on transdermal flux of sumatriptan. R-(+)-limonene showed the greatest ability to enhance the flux of sumatriptan.

High-performance liquid chromatographic determination of sumatriptan after in vitro transdermal diffusion studies.

A simple, accurate, precise and rapid HPLC method with UV detection has been validated in order to determine the in vitro transdermal absorption of sumatriptan succinate. The HPLC method is a modification of that described by Nozal et al. [M.J. Nozal, J.L. Bernal, L. Toribio, M.T. Martin, F.J. Diez, J. Pharm. Biomed. Anal. 30 (2002) 285-291]. Separation was carried out on a 250 mm Kromasil C18 column at room temperature. The detector response, at 282.7 nm, was found to be linear in a concentration range between 0.145 and 145 microM. The limit of detection (LOD) was 0.019 microM and the limit of quantification (LOQ) was 0.145 microM.

Growth, morphological and biochemical changes in oxa-spermine derivative-treated MCF-7 human breast cancer cells.

The growth inhibitory properties of two oxa-spermine derivatives named compound 1 and compound 2, representatives of a novel type of polyamine derivatives, were studied. Dose-response growth inhibitory curves obtained after 48h drug exposure demonstrated the much higher cytotoxic activity of compound 1 towards MCF-7 human breast cancer cells. Further experiments with compound 1 showed that this oxa-spermine derivative exhibited considerable cytotoxicity with IC(50) values of 3.74 microM and 2.93 microM after 24h and 48h drug exposure respectively. In MCF-7 cells, after 8h drug (10 microM) exposure it caused shrinkage, chromatin condensation and nuclear fragmentation. However, no clear DNA laddering was detected in treated cells. Drug treatment provoked an increase in polyamine oxidase (PAO) activity. This enzyme is able to produce cytotoxic H(2)O(2) and 3-acetamidopropanal, catalyzing the oxidative deamination of N(1)-acetylated derivatives of spermine and spermidine to spermidine and putrescine respectively. Taken together these data demonstrate that the novel oxa-polyamine derivative compound 1 has considerable cytotoxic activity towards MCF-7 cells and indicate that an induction of PAO may be involved in its cytotoxic and apoptotic effects.

Oxidative damage and cytogenetic analysis in leukocytes of Parkinson's disease patients.

BACKGROUND: Postmortem studies suggest excessive free radical toxicity in the substantia nigra of patients with PD. Increased lipid peroxidation and oxidative DNA damage have been reported in the CNS. Markers of oxidative stress have been identified in the blood of patients with PD. OBJECTIVE: To assess the presence of spontaneous chromosome and primary or oxidative DNA damage in peripheral blood leukocytes of patients with untreated PD. METHODS: Patients with de novo PD (20) and control subjects (16), matched for age, sex, and smoking habits, underwent cytogenetic analysis using the human lymphocyte micronucleus assay coupled with the fluorescence in situ hybridization technique and the Comet assay. RESULTS: Compared with controls, patients with PD showed an increase in the incidence of spontaneous micronuclei (p < 0.001); single strand breaks (p < 0.001); and oxidized purine bases (p < 0.05). Fluorescence in situ hybridization analysis showed micronuclei harboring acentric fragments. CONCLUSIONS: There is chromosomal, primary DNA damage and oxidative DNA damage demonstrable in lymphocytes of patients with untreated PD.

Biochemical effects and growth inhibition in MCF-7 cells caused by novel sulphonamido oxa-polyamine derivatives.

The novel polyamine derivatives sulphonamido oxa-spermine (oxa-Spm) and sulphonamido oxa-spermidine (oxa-Spd) exhibited rapid cytotoxic action towards MCF-7 human breast cancer cells with IC50 values of 4.35 and 6.47 pM, respectively, after 24-h drug exposure. Neither compound is a substrate of serum amine oxidase. Both oxa-Spm and oxa-Spd caused cell shrinkage, as determined by phase-contrast microscopy. After incubation with 10 microM of either compound for 8 h, the cells underwent chromatin condensation and nuclear fragmentation. However, no clear DNA ladder was obtained by electrophoresis. The sulphonamido oxa-polyamine derivatives and especially oxa-Spd enhanced the activity of polyamine oxidase (PAO), an enzyme capable of oxidising N1-acetylated spermine and spermidine to spermidine and putrescine, respectively, generating cytotoxic H2O2 and 3-acetamidopropanal as by-products. The intracellular polyamine content was only marginally reduced in response to drug treatment. In conclusion, our data show that these novel sulphonamido oxa-polyamine derivatives possess high cytotoxic activity against MCF-7 cells and indicate that induction of PAO may mediate their cytotoxicity via apoptosis.

Chromosome and oxidative damage biomarkers in lymphocytes of Parkinson's disease patients.

As cancer development usually results from exposure to several environmental risk factors in interaction with the genetic susceptibility of the host, it could be of interest to investigate if neurodegeneration, as occurs in Parkinson's disease (PD) patients can be attributed at least partially, to environmental risk factors. There is growing evidence that oxidative stress could play a significant role as a risk factor in the aetiology and pathogenesis of neurodegenerative diseases, emphasising the need for new individual and human-based approaches. The aim of our research is to explore the relation between chromosome instability and oxidative stress biomarkers in Parkinson's disease using a variety of strategies. We determined peripheral markers for oxidative damage in PD by testing for spontaneous and induced chromosomal damage, DNA strand breaks, oxidised pyrimidines and altered purines both in peripheral blood and cultured lymphocytes. We also measured glutathione S-transferase activity in the plasma of patients and controls. Compared to healthy controls, PD patients show higher frequencies of micronuclei (17.2 +/- 4.8 vs. 9.0 +/- 3.4, p < 0.001) and a significant increase in the levels of single strand breaks (SSB). Significant differences were also obtained in the distribution of oxidised purine bases between the two groups. Preliminary data obtained by fluorescence in situ hybridization analysis showed that the percentage of centromere negative micronuclei is higher than that of centromere positive micronuclei. Glutathione S-transferase activity in plasma from PD patients and controls was also measured and the enzymatic activity in PD patients was lower than in healthy controls.

Cytotoxicity, DNA binding and localisation of novel bis-naphthalimidopropyl polyamine derivatives.

Bis-naphthalimidopropyl spermidine (BNIPSpd), spermine (BNIPSpm) and oxa-spermine (BNIPOSpm) showed high in vitro cytotoxicity against human breast cancer MCF-7 cells with IC(50) values of 1.38, 2.91 and 8.45 microM, respectively. These compounds were found to effectively displace the intercalating agent ethidium bromide bound to the calf thymus DNA using fluorimetric methods (C(50) 0.08-0.12 microM) and their apparent equilibrium binding constants (K(app)) were calculated to be in the range of 10.5-18 x 10(7) M(-1). Furthermore, strong stabilisation of calf thymus DNA duplex in the presence of bis-naphthalimidopropyl polyamine derivatives (BNIPSpd, BNIPSpm and BNIPOSpm) was observed by UV spectrophotometric analysis (T(m)=93.3-97 degrees C compared with 75 degrees C for calf thymus DNA without drug). Because of their inherent fluorescence, these compounds were localised preferentially inside the nucleus as evidenced by their direct observation under the fluorescence microscope. The results obtained suggest that the cytotoxic activity of the bis-naphthalimidopropyl polyamines may be in part, caused by their effects on DNA.

Induction, regulation, degradation, and biological significance of mammalian metallothioneins.

MTs are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homology. Mammalian MTs are 61 or 62 amino acid polypeptides containing 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biological demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 years of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biological role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biological role of MTs. This article reviews the current knowledge on the biochemistry, induction, regulation, and degradation of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biological roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.

Exposure of cultured human proximal tubular cells to cadmium, mercury, zinc and bismuth: toxicity and metallothionein induction.

The kidney, in particular the proximal convoluted tubule, is a major target site for the toxic effects of various metals. However, little is known about the early effects of these metals after acute exposure in man. In the present study we have evaluated the toxicity of several inorganic metal compounds (CdCl2, HgCl2, ZnCl2, and Bi(NO3)3) and the induction of metallothionein by these compounds in cultured human proximal tubular (HPT) cells for up to 4 days. The results showed that bismuth was not toxic even at the highest dose (100 microM) used, while zinc, cadmium and mercury exhibited varying degrees of toxicity, zinc being the least toxic and mercury the most potent. A significant degree of interindividual variation between the different isolates used in these experiments was also observed. All metals used in the present study induced MT, as revealed by immunocytochemistry. All metals showed maximal induction between 1 and 3 days after treatment. Although a certain amount of constitutive MT was present in the cultures, the intensity of the staining varied with time in culture and between the different isolates studied. No correlation could be made between the intensity of the staining in control cultures (indicating total amount of constitutive MT) and the susceptibility of a given isolate to metal toxicity. Furthermore, no correlation could be made between metal-induced MT and the susceptibility of a given isolate to that particular metal.

Single-strand breaks, cell cycle arrest and apoptosis in HL-60 and LLCPK1 cells exposed to 1,2-dibromo-3-chloropropane.

We investigated 1,2-dibromo-3-chloropropane (DBCP)-induced DNA damage, cell cycle alterations and cell death in two cell lines, the human leukemia HL-60 and the pig kidney LLCPK1, both of which are derived from potential target sites for DBCP-induced toxicity. DBCP (30-300 micromol/L) caused a concentration-dependent increase in the levels of DNA single-strand breaks in both cell lines as well as in cultured human renal proximal tubular cells. After extended DBCP exposure in LLCPK1 cells (100 micromol/L, 30 h), the level of DNA breaks returned almost to control values. Incubation for 48 h showed a clear reduction of growth with DBCP concentrations as low as 10 micromol/L. Flow cytometric analysis showed that DBCP (1-10 micromol/L) exposure for 24 h caused an accumulation of LLCPK1 cells in the G2/M-phase. In HL-60 cells the accumulation in G2/M-phase was less marked, and at higher concentrations the cells accumulated in S-phase. Flow cytometric studies of HL-60 and LLCPK1 cells exposed to 100-500 micromol/L DBCP showed increased number of apoptotic cells/bodies with a lower DNA content than that of the G1 cells. Microscopic studies revealed that there were increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominant mode of death in these cell lines, following exposure to DBCP. The characteristic ladder pattern of apoptotic cells was observed when DNA from DBCP-treated HL-60 cells and LLCPK1 cells was electrophoresed in agarose. The finding that DBCP can cause an accumulation of cells in G2/M-phase and induce apoptosis in vitro may be of importance for the development of DBCP-induced toxicity in vivo.

Glutathione S-transferases in human renal cortex and neoplastic tissue: enzymatic activity, isoenzyme profile and immunohistochemical localization.

1. Glutathione S-transferase (GST) activity in the cytosol of renal cortex and tumours from eight men and eight women was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. GST activities ranged from 685 to 2192 nmol/min/mg protein in cortex (median 1213) and from non-detectable (minimum 45) to 2424 nmol/min/mg protein in tumours (median 469). The activities in the tumours were lower than those in the normal cortices (p < 0.05). 2. In men, the activity in the cortical cytosol was in all cases higher than that measured in the corresponding tumours (p < 0.05). In women, the difference in activity between cortices and tumours was not significantly different (p > 0.05). 3. The age of the patients ranged from 42 to 81 years (median 62) and was not found to play a role in the levels of GST activity observed in cortex or in renal tumours from either sex. 4. Immunoblotting and immunohistochemical studies confirmed that GST-alpha was the predominant form expressed both in normal cortex and tumour and probably accounted for most of the GST activity present in these samples. GST-mu and GST-phi were expressed in both tumours and normal cortex and, while in some cases the level of expression in the cortices was higher than that found in the tumours, the reverse was also observed. Within the GST-mu class, GST M1/M2 was only detected in one sample (tumour), which showed the highest overall expression of GST-mu. GSTM3 was the predominant isoenzyme of the mu class in normal and tumour tissue, whereas GTM4 and GSTM5 were not detected. 5. These differences could have functional significance where xenobiotics or cytotoxic drugs are specific substrates for the different classes of GSTs.