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BACKGROUND: The beta-galactosidase from Kluyveromyces lactis is a protein of outstanding biotechnological interest in the food industry and milk whey reutilization. However, due to its intracellular nature, its industrial production is limited by the high cost associated to extraction and downstream processing. The yeast-system is an attractive method for producing many heterologous proteins. The addition of a secretory signal in the recombinant protein is the method of choice to sort it out of the cell, although biotechnological success is not guaranteed. The cell wall acting as a molecular sieve to large molecules, culture conditions and structural determinants present in the protein, all have a decisive role in the overall process. Protein engineering, combining domains of related proteins, is an alternative to take into account when the task is difficult. In this work, we have constructed and analyzed two hybrid proteins from the beta-galactosidase of K. lactis, intracellular, and its Aspergillus niger homologue that is extracellular. In both, a heterologous signal peptide for secretion was also included at the N-terminus of the recombinant proteins. One of the hybrid proteins obtained has interesting properties for its biotechnological utilization. RESULTS: The highest levels of intracellular and extracellular beta-galactosidase were obtained when the segment corresponding to the five domain of K. lactis beta-galactosidase was replaced by the corresponding five domain of the A. niger beta-galactosidase. Taking into account that this replacement may affect other parameters related to the activity or the stability of the hybrid protein, a thoroughly study was performed. Both pH (6.5) and temperature (40 degrees C) for optimum activity differ from values obtained with the native proteins. The stability was higher than the corresponding to the beta-galactosidase of K. lactis and, unlike this, the activity of the hybrid protein was increased by the presence of Ni2+. The affinity for synthetic (ONPG) or natural (lactose) substrates was higher in the hybrid than in the native K. lactis beta-galactosidase. Finally, a structural-model of the hybrid protein was obtained by homology modelling and the experimentally determined properties of the protein were discussed in relation to it. CONCLUSION: A hybrid protein between K. lactis and A. niger beta-galactosidases was constructed that increases the yield of the protein released to the growth medium. Modifications introduced in the construction, besides to improve secretion, conferred to the protein biochemical characteristics of biotechnological interest.

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Possible biological effects of mobile phone microwaves were investigated in vitro. In this study, which was part of the 5FP EU project REFLEX (Risk Evaluation of Potential Environmental Hazards From Low-Energy Electromagnetic Field Exposure Using Sensitive in vitro Methods), six human cell types, immortalized cell lines and primary cells, were exposed to 900 and 1800 MHz. RNA was isolated from exposed and sham-exposed cells and labeled for transcriptome analysis on whole-genome cDNA arrays. The results were evaluated statistically using bioinformatics techniques and examined for biological relevance with the help of different databases. NB69 neuroblastoma cells, T lymphocytes, and CHME5 microglial cells did not show significant changes in gene expression. In EA.hy926 endothelial cells, U937 lymphoblastoma cells, and HL-60 leukemia cells we found between 12 and 34 up- or down-regulated genes. Analysis of the affected gene families does not point towards a stress response. However, following microwave exposure, some but not all human cells might react with an increase in expression of genes encoding ribosomal proteins and therefore up-regulating the cellular metabolism.

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Several studies conducted in Rome have shown low levels of vaccination coverage in gypsy communities. Thus a vaccination campaign targeting to 2400 gypsy children aged 0-13 years, present in 32 settlements in Rome, was conducted in 2002. The campaign was related to vaccinations required and recommended in Italy: diphtheria, tetanus, hepatitis B, pertussis (whooping cough), haemophilus influenzae and measles. In the majority of cases it was decided to carry out the vaccinations directly in the gypsy settlements, in accordance with the methods of pulse immunisation. In the case of small settlements a strategy for reorienting the population to vaccination centres was adopted. Around 2000 children were vaccinated, equivalent to 80% of the paediatric population present during the period. The number of children who have never been vaccinated has decreased from 40% prior to the campaign to 9% after the third week. Vaccination coverage in medium- and small-sized settlements (<200 inhabitants) after the campaign shows values of over 70%; in the large settlements, more modest increases have been recorded and coverage has rarely exceeded 50%. This experience has highlighted the importance of networking between public healthcare institutions and non-profit organisations. The mobilisation of a wide range of competences has thus enabled the attainment of a high level of effectiveness.

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Vascular access failure is an important cause of morbidity and mortality for patients on haemodialysis. We have prospectively studied, with a 5 years follow up, a monitoring protocol of native vascular accesses, using the available methods in every haemodialysis unit. All the native vascular accesses, created from 1- 1998 to XII-2001, with a posterior follow up until XII-2002, were monitored. Monitoring was based on physical examination, dificulty for blood flow greater than 300 ml/min, and in a monthly basis: dynamic venous pressure, urea recirculation and urea kinetic model. Abnormalities suggestive of fistulogram were a priori defined. During the recruitment period, a total of 164 accesses were created in 144 patients. Of these only 3 were grafts, 28 native vascular accesses were never functioning (primary failure rate 17. 1%), and 127 native accesses created in 113 patients (age 63.3 +/- 12.4 years; 18 % diabetics), were monitored (83% cephalic vein). Monitoring findings indicated realization of fistulogram in 35% and percutaneus angioplasty in 25% of the accesses. In order to maintain patency, the surgical intervention rate was 0.03 procedures/patient/year, the radiological 0.10 and the total 0.13. During the 5 years of the study occurred 41 thrombosis episodes in 40 accesses (0.07 thrombosis/patient/year), with percutaneus repermeabilization in 30%. Primary (unassisted) survival was 30.3 months (Confidence Interval 95% 25.6, 35.0) and secondary (assisted) survival 42.8 months (Confidence Interval 95%: 38.7, 46.9). Logistic regression analysis showed that presence of a central catheter at the time of creating the vascular access posses a greater risk for thrombosis (Relative Risk 5.6 if in subclavian vein), whereas age, diabetes, time to canulation, number of previous accesses and anatomic type do not increase that risk. In conclusion, in an old haemodialysis population, with an important diabetes prevalence, it is possible to create functioning native vascular accesses in most of them. The monitoring protocol allowed the detection and posterior correction of stenosis in a great number of accesses. The main risk of thrombosis is the presence of a central catheter at the time of creating a vascular access.

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El fallo del acceso vascular es causa importante de morbimortalidad en los pacientesen hemodiálisis. Se presentan los resultados de un estudio prospectivo, durante5 años, aplicando un protocolo de monitorización de los accesos vasculares nativos.Se monitorizaron, utilizando métodos disponibles en cualquier Unidad de Diálisis,todos los accesos nativos realizados desde I-1998 hasta XII-2001, con un seguimientoposterior hasta XII-2002. La monitorización se basó en examen físico, dificultad paraflujo mayor de 300 ml/min, y mensualmente, de modo alterno: presión venosa dinámica,recirculación de urea y su modelo cinético. Se definieron a priori las alteracionesindicativas de realización de fistulografía.Durante el período de reclutamiento se realizaron 164 accesos a 144 pacientes. Deestos accesos sólo 3 fueron prótesis y 28 accesos nativos nunca funcionaron (fallo primario17,1%). Se monitorizaron 127 accesos nativos (83% vena cefálica) realizadosa 113 pacientes (edad 63,3 ± 12,4 años; 18% diabéticos).Indicadas por anomalías en la monitorización se realizaron fistulografías en el 35%de los accesos y angioplastia de estenosis en el 25%. Las tasas de intervención quirúrgicapara mantener la viabilidad fueron de 0,03 procedimientos/paciente/año, laradiológica de 0,10 y la total de 0,13. En los 5 años se produjeron 41 episodios detrombosis en 40 accesos (0,07 trombosis/paciente/año), con repermeabilización percutáneaen el 30%. El tiempo de supervivencia primaria fue de 30,3 meses (Intervalode Confianza al 95%: 25,6, 35,0) y el de la acumulativa 42,8 meses (Intervalo deConfianza al 95%: 38,7, 46,9).El análisis de regresión logística demostró que la presencia de un catéter central alcrear el acceso confiere un riesgo mayor de trombosis (Riesgo Relativo 5,6 si en subclavia),mientras que la edad, diabetes, tiempo de utilización, número de accesos vascularesprevios y tipo del mismo no lo incrementan.En conclusión, el protocolo de monitorización permitió detectar y corregir estenosisen numerosos accesos vasculares nativos. En un porcentaje elevado de trombosisse produjo la recanalización percutánea del acceso. El mayor riesgo de trombosisdetectado fue la presencia previa de un catéter en vena subclavia

Vascular access failure is an important cause of morbidity and mortality for patientson haemodialysis.We have prospectively studied, with a 5 years follow up, a monitoringprotocol of native vascular accesses, using the available methods in every haemodialysisunit.All the native vascular accesses, created from I-1998 to XII-2001, with a posteriorfollow up until XII-2002, were monitored. Monitoring was based on physical examination,dificulty for blood flow greater than 300 ml/min, and in a monthly basis: dynamicvenous pressure, urea recirculation and urea kinetic model. Abnormalities suggestiveof fistulogram were a priori defined.During the recruitment period, a total of 164 accesses were created in 144 patients.Of these only 3 were grafts, 28 native vascular accesses were never functioning (primaryfailure rate 17.1%), and 127 native accesses created in 113 patients (age 63.3 ±12.4 years; 18% diabetics), were monitored (83% cephalic vein).Monitoring findings indicated realization of fistulogram in 35% and percutaneusangioplasty in 25% of the accesses. In order to maintain patency, the surgical interventionrate was 0.03 procedures/patient/year, the radiological 0.10 and the total0.13. During the 5 years of the study occurred 41 thrombosis episodes in 40 accesses(0.07 thrombosis/patient/year), with percutaneus repermeabilization in 30%. Primary(unassisted) survival was 30.3 months (Confidence Interval 95% 25.6, 35.0)and secondary (assisted) survival 42.8 months (Confidence Interval 95%: 38.7,46.9).Logistic regression analysis showed that presence of a central catheter at the time ofcreating the vascular access posses a greater risk for thrombosis (Relative Risk 5.6 if insubclavian vein), whereas age, diabetes, time to canulation, number of previousaccesses and anatomic type do not increase that risk.In conclusion, in an old haemodialysis population, with an important diabetes prevalence,it is possible to create functioning native vascular accesses in most of them.The monitoring protocol allowed the detection and posterior correction of stenosis ina great number of accesses. The main risk of thrombosis is the presence of a centralcatheter at the time of creating a vascular access

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The present study investigates the cellular response to weak, sine wave, 0.5-MHz electric currents. The experimental exposure to identical signals at an intensity high enough as to significantly increase the temperature in target tissues, has provided positive responses in clinical treatments of tumors with capacitive electric transfer (CET) thermal therapy. The present results show that the in vitro exposure to CET signals at athermal doses causes cytotoxic effects in human neuroblastoma cells. Such a response seems to be due to signal-induced alterations in the cell cycle. As a whole, the results suggest that the potential therapeutic effects of the CET strategy could be due to the thermal response of the tissues to the currents, added to an athermal response of the cells to the electric current itself.

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OBJECTIVE: To determine the construct validity of FACES III in spanish (México). DESIGN: Cross-sectional study, descriptive, confirmatory. SETTING: Mexico City, South of Federal District, Tlalpan area, divided into geo- statistical zones of a primary care unit. PATIENTS AND OTHER PARTICIPANTS: A random sample of 270 families selected in their dwellings with proportional coverage based on 17 895 dwellings of the influence area. Measurements and main results. Domicile survey applying FACES III (Family Adaptability and Cohesion Evaluation Scales) in spanish, México. Twelve criteria for evaluate the construct validity of FACES III trough confirmatory factor analysis were established by the researchers. Eight criteria were surpassing appropriately and four do not surpass the minimum values established. CONCLUSIONS: FACES III in spanish (México) is an instrument with reliability of 70% with Cronbach alfa and validity measured using confirmatory Factor Analysis. Nevertheless, we found four limitations that should be analyzed and evaluated in the planning of future studies.

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Objetivo. Determinar la validez de constructo del cuestionario FACES III en español (México).Diseño. Estudio transversal, descriptivo, confirmatorio. Emplazamiento. Zona de influencia sectorizada en áreas geoestadísticas básicas (AGEB) de una unidad de atención primaria en la Delegación Tlalpan, distrito federal al sur de la Ciudad de México. Participantes. Un total de 270 familias elegidas aleatoriamente en sus viviendas de modo proporcional al tamaño de las AGEB con una base de 17.895 viviendas de la zona de influencia. Mediciones y resultados principales. Encuesta domiciliaria de la Escala de Evaluación de la Cohesión y la Adaptabilidad Familiar (FACES III) en español (México).Se establecieron doce criterios para evaluar la validez de constructo del cuestionario FACES III a través del análisis factorial confirmatorio. Ocho criterios fueron superados adecuadamente y cuatro no rebasaron los valores mínimos establecidos en el análisis factorial. Conclusiones. FACES III en español (México) es fiable (70 por ciento con el índice alfa de Cronbach) y válido. Sin embargo, se encontraron cuatro limitaciones que deben ser analizadas y evaluadas en la planeación de futuros estudios (AU)

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Gap junction communication is known to be involved in controlling cell proliferation and differentiation, and seems to play a crucial role in suppression of tumor promotion. Melatonin, a hormone secreted by the pineal gland, has putative oncostatic properties. Intercellular communication through gap junctions was assessed by microinjecting Lucifer yellow fluorescent dye into primary hepatocytes and visualizing the spread of the dye to adjacent neighboring cells using phase contrast/fluorescent microscopy. Treatment of primary hepatocyte cultures with a physiological range of melatonin concentrations for 24 h prior to microinjection resulted in significant enhancement in intercellular communication at 0.2 and 0.4 nmol/L but not at lower (0.1 nmol/L) or higher (0.8 or 1.0 nmol/L) concentrations. A time-dependent study showed that the changes in intercellular communication began 10 h after melatonin treatment and reached a maximum at 12 h of treatment. This nonlinear, functional gap junction response to melatonin occurred in the physiological concentration range detected in blood of mammals during nightly releases of the hormone by the pineal gland. These melatonin levels may affect the ability of gap junction communication to exert cell growth control in vivo. The uneven decline between individuals in nocturnal release of melatonin that occurs with age could identify potentially sensitive subpopulations susceptible to developing pathologies involving alterations in biological processes dependent on gap junction communication.

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Citrus trees have a long juvenile phase that delays their reproductive development by between 6 and 20 years, depending on the species. With the aim of accelerating their flowering time, we transformed juvenile citrus seedlings to constitutively express the Arabidopsis LEAFY (LFY) or APETALA1 (AP1) genes, which promote flower initiation in Arabidopsis. Both types of transgenic citrus produced fertile flowers and fruits as early as the first year, notably through a mechanism involving an appreciable shortening of their juvenile phase. Furthermore, expression of AP1 was as efficient as LFY in the initiation of flowers, and did not produce any severe developmental abnormality. Both types of transgenic trees flowered in consecutive years, and their flowering response was under environmental control. In addition, zygotic and nucellar derived transgenic seedlings had a very short juvenile phase and flowered in their first spring, demonstrating the stability and inheritance of this trait. These results open new possibilities for domestication, genetic improvement, and experimental research in citrus and other woody species.

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Objetivos: La Historia Clínica (HC) es una herramienta esencial para el trabajo médico y útil para el proceso y evaluación de control de calidad asistencial. Hemos estudiado el grado de cumplimentación y la concordancia entre los sistemas de registro de nuestro Servicio de Urgencias Extrahospitalario (HC y libro de registro). Método: Estudio retrospectivo descriptivo y comparativo. Muestra de 320 HC, escogidas de modo aleatorio de un total de 36.174 pacientes atendidos durante 1998. Criterio de inclusión: estar anotados en el libro de registro general de consulta para ser cotejados con las HC realizadas. Variables a estudio: Datos de Identificación: administrativos, clínicos y de identificación del facultativo. Variables dicotómicas (sí/no) y análisis estadístico de Epi-Info V 6-04, con proporciones, chi cuadrado, F de Snedecor, Índice de Kappa. Resultados: De los 320 registros seleccionados, 296 (92,5 por ciento) tenían HC y 24 (7,5 por ciento) no. La concordancia entre datos anotados en "libro de registro" y HC coincidieron en un 98,7 por ciento. El 0,61 por ciento de la muestra correspondió a días festivos y el resto laborales. El registro de Identificación Personal (IDP) fue incompleto en un 97,8 por ciento a expensas de la dirección, el teléfono y DNI del paciente (89,6 por ciento: p < 0,01). Las odontalgias eran las patologías que menos HC generaban (F = 18.004; p < 0,001). Conclusiones: La realización de la HC a los pacientes anotados en el libro general de registro muestra un porcentaje aceptable. El bajo número de registro de IDP fue a expensas de la dirección, teléfono y DNI, situación que se podría resolver con la informatización de dichos registros. Las constantes vitales, anamnesis y pruebas complementarias y su transcripción son de baja cumplimentación, lo que resta calidad a la HC, justificable por ser patologías más banales las observadas en estos centros. No se cumplimenta adecuadamente la identificación del médico (AU)

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OBJECTIVE: To determine through five methods (Cronbach's alpha, split, Guttman method, parallel and strict parallel) the confidence coefficient of FACES III, instrument validated on spanish version, and to evaluate it's consistency with these five statistical methods. DESIGN: Descriptive, cross-sectional. SETTING: Tlalpan Area, south of Federal District, México, divided into geo-statistical zones. PATIENTS AND OTHER PARTICIPANTS: A randomised sample of 270 dwellings with proportional coverage, based on 17,895 ordinary dwellings of the area. INTERVENTION: Domicile survey using FACES III (Family Adaptability and Cohesion Evaluation Scales); April-May 1995. MEASUREMENTS AND MAIN RESULTS: The confidence coefficient of FACES III spanish version was calculated using no standardised Cronbach's Alpha = 0.69; split I = 0.73 and split II = 0.66; Guttman method = 0.75; parallel = 0.69 and strict parallel = 0.53. CONCLUSIONS: Cronbach's coefficient demonstrated more advantages than the other statistical methods in relation with the ordinal measurement scale of FACES III. We suggest to take into consideration three important aspects for the correct confidence analysis of this kind of instruments: Variance analysis depending on the scale of items (F test, Friedman or Cochran) Inter-items interactions analysis (nonadditivity) and the use of the balance value as purge element of error' source. Analysis of Tukey estimate, coefficient of concordance (W) and Hotelling's T squared. It's necessary the evaluation and analysis of this aspects before the report of confidence's coefficient values whose can have hidden skew.

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PC-12 cells primed with nerve growth factor (NGF) were exposed to sinusoidal extremely-low-frequency (ELF) magnetic fields (MFs) selected to test the predictions of the ion parametric resonance (IPR) model under resonance conditions for a single ion (hydrogen). We examined the field effects on the neurite outgrowth (NO) induced by NGF using three different combinations of flux densities of the parallel components of the AC MF (Bac) and the static MF (Bdc). The first test examined the NO response in cells exposed to 45 Hz at a Bdc of 2.96 microT with resonant conditions for H+ according to the model. The Bac values ranged from 0.29 to 4.11 microT root-mean-square (rms). In the second test, the MF effects at off-resonance conditions (i.e., no biologically significant ion at resonance) were examined using the frequency of 45 Hz with a Bdc of 1.97 microT and covering a Bac range between 0.79 and 2.05 microT rms. In the third test, the AC frequency was changed to 30 Hz with the subsequent change in Bdc to 1.97 microT to tune for H+ as in the first test. The Bac values ranged from 0.79 to 2.05 microT rms. After a 23 h incubation and exposure to the MF in the presence of NGF (5 ng/ml), the NO was analyzed using a stereoscopic microscope. The results showed that the NGF stimulation of neurite outgrowth (NSNO) was affected by MF combinations over most of the Bac exposure range generally consistent with the predictions of the IPR model. However, for a distinct range of Bac where the IPR model predicted maximal ionic influence, the observed pattern of NSNO contrasted sharply with those predictions. The symmetry of this response suggests that values of Bac within this distinct range may trigger alternate or additional cellular mechanisms that lead to an apparent lack of response to the MF stimulus.

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There is strong evidence that pineal melatonin is involved in controlling neoplastic processes. We have reported that physiological, but not pharmacological or sub physiological, concentrations of melatonin enhance intercellular communication in normal C3H/10T/2 fibroblasts. Gap junctional intercellular communication intervenes in the control of cell proliferation and differentiation, and seems to play a crucial role in suppression of tumor promotion. A number of in vivo studies have shown that extremely low frequency (ELF) magnetic fields (MF) can act as cancer promoters or co-promoters. In vitro, 60 Hz MF have been reported to block melatonin-induced inhibition of cell proliferation in human breast cancer cells. The mechanisms responsible for the observed interactions of MF at the cellular level remain unknown. In the present study melatonin was added to confluent fibroblasts at a concentration of 10(-10) M. Twenty-seven hours later, a fluorescent dye was scrape-loaded into groups of cells and the transfer of the dye to adjacent cells through gap junctions was quantified. Under these conditions melatonin induced a significant increase of dye transfer; this increase was not observed when the cultures were exposed to the MF for 30 min before the previously reported results suggesting that the in vivo oncostatic action of melatonin could be exerted, in part, through modulation of the levels of gap junctional intercellular communication. Also, the data indicate that ELF-MF could counteract the melatonin-induced enhancement of junctional transfer.

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Gap junctional intercellular communication is known to be involved in controlling cell proliferation and differentiation, and seems to play a crucial role in suppression of tumor promotion. The pineal gland and its hormone, melatonin, are believed to intervene in the control of neoplastic processes. Several possible mechanisms have been suggested to be potentially responsible for melatonin's oncostatic action; however, the actual mechanisms involved in melatonin's effects at the cellular level remain unidentified. In the present study low-density cultures of C3H/10T1/2 mouse embryo fibroblasts were incubated until relatively quiescent monolayers were established (17-18 days). Gap junctional intercellular communication in control samples and in cells treated with 10(-12) to 10(-8) M melatonin was determined by the scrape-loading assay using the fluorescent dye Lucifer yellow. The results showed that concentrations of melatonin considered physiological (10(-11) and 10(-10) M) induced a significant increase in the transfer of the dye to adjacent cells through gap junctions; both higher and lower concentrations were ineffective. These results suggest that melatonin could exert its putative oncostatic action, in part, by modulating the levels of gap junctional intercellular communication.

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Several reports have shown that weak, extremely-low-frequency (ELF), pulsed magnetic fields (PMFs) can adversely affect the early embryonic development of the chick. In this study, freshly fertilized chicken eggs were exposed during the first 48 h of postlaying incubation to PMFs with 100 Hz repetition rate, 1.0 microT peak-to-peak amplitude, and 500 microseconds pulse duration. Two different pulse waveforms were used, having rise and fall times of 85 microseconds (PMF-A) or 2.1 microseconds (PMF-B). It has been reported that, with 2 day exposure, these fields significantly increase the proportion of developmental abnormalities. In the present study, following exposure, the eggs were allowed to incubate for an additional 9 days in the absence of the PMFs. The embryos were taken out of the eggs and studied blind. Each of the two PMF-exposed groups showed an excess in the percentage of developmental anomalies compared with the respective sham-exposed samples. This excess of anomalies was not significant for the PMF-A-treated embryos (P = 0.173), whereas it was significant for the PMF-B-exposed group (P = 0.007), which showed a particularly high rate of early embryonic death. These results reveal that PMFs can induce irreversible developmental alterations and confirm that the pulse waveform can be a determinant factor in the embryonic response to ELF magnetic fields. The data also validate previous work based on the study of PMFs' effects at day 2 of embryonic development under field exposure.

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We reviewed 55 consecutive cases of rhegmatogenous retinal detachment treated with pneumatic retinopexy as a primary procedure and followed up for a minimum of 1 year. 40 eyes were phakic, 11 aphakic and 4 pseudophakic. 0.6 ml of sulfur hexafluoride were injected into 42 eyes, while 0.3 ml of perfluoropropane were introduced into 13. Transconjunctival cryotherapy was performed in 51 eyes, while light coagulation was used as retinopexy after the retina was reattached in the remaining 4 cases. Of the 55 eyes undergoing treatment, 46 (83.6%) retinas were reattached with one operation. The benefits, complications and disadvantages of the procedure are reported.

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