On-line surface plasmon resonance biosensing of vascular endothelial growth factor signaling in intact-human hepatoma cell lines.

Surface plasmon resonance (SPR) monitoring of biorecognition events at intracellular levels is a valuable tool for studying the angiogenic response of carcinoma living cells during tumor growth and proliferation. We report here a comparative study of two different strategies to detect human hepatoma cell interactions between transmembrane vascular endothelial growth factor receptor (VEGFR2) and vascular endothelial growth factor (VEGF). To monitor VEGFR2 activation after VEGF stimulation, intact hepatocellular carcinoma HepG2 or Huh7 cells (2 × 10(5) cells per mL) were directly immobilized on the sensor chip. Distinguishable SPR sensorgrams were obtained for each cell line depending on the time required for VEGFR2 activation. SPR signals for VEGF-VEGFR2 binding were inhibited by the VEGFR inhibitor, CBO-P11. The SPR response after VEGF stimulation/inhibition was in good agreement with the results observed by immunoblotting analysis. In a second approach we used intact cell lines as analytes. SPR analysis was done by injecting HepG2 and HuH7 cell suspensions (2-4 × 10(4) cells per mL) onto a sensor surface previously immobilized with VEGF via a thiol self-assembled monolayer (SAM). Specificity and reproducibility were evaluated reusing the same chip surface over more than 60 complete regeneration cycles. Comparison between both methods yielded differences in terms of reliability, making the latter strategy more effective for the analysis of real samples. The investigation of VEGF signaling in intact human hepatoma living cells by SPR monitoring comprises a novel and promising design for the study of tumor angiogenesis via downregulation of VEGF and VEGFR2 pathways. Further investigation on VEGFR activation and vascular function could contribute to establish a robust and meaningful tool for early cancer diagnostics.

Effects of a low-fat diet with antioxidant supplementation on biochemical markers of multiple sclerosis long-term care residents.

INTRODUCTION: Multiple sclerosis (MS) treatment options are primarily limited to immunomodulatory therapies in MS non-progressive forms. Nutrition intervention studies suggest that diet may be considered as a complementary treatment to control disease progression. Therefore, dietary intervention may help to improve wellness and ameliorate symptoms of MS patients. OBJECTIVES: To assess the effect of a low-fat diet with antioxidant supplementation on biochemical markers of institutionalized patients with progressive forms of multiple sclerosis. METHODS: A randomized prospective placebo-controlled study involving 9 participants, 5 of them assigned to the intervention group (low-fat diet and antioxidant supplementation) and the other 4 to the placebo group (low-fat diet). The effect of the dietary intervention, involving diet modification and antioxidant supplementation, was examined for 42 days by measuring anthropometric, biochemical parameters and oxidative stress markers in blood at baseline (day 0), intermediate (day 15) and end (day 42) stages of the treatment. RESULTS: The intervention group obtained C reactive protein levels significantly lower than those observed in the corresponding placebo group at the end of the study. Oxidative stress and inflammatory markers isoprostane 8-iso-PGF2α and interleukine IL-6 values also diminished after dietary intervention in the intervention group. Catalase activity increased significantly in the intervention group prior antioxidant supplementation. No significant differences were observed in other oxidative stress markers. CONCLUSIONS: The results suggest that diet and dietary supplements are involved in cell metabolism modulation and MS-related inflammatory processes. Consequently, low fat diets and antioxidant supplements may be used as complementary therapies for treatment of multiple sclerosis.

Introducción: Las posibilidades de tratamiento de la esclerosis múltiple (EM) se encuentran limitadas principalmente a terapias con inmumoduladores en las formas no progresivas de EM. Los estudios de intervención nutricional sugieren que la dieta puede considerarse como un tratamiento alternativo para controlar la progresión de la enfermedad. Por esta razón, las intervenciones en la dieta pueden ayudar a mejorar el bienestar y mejorar los síntomas de los pacientes con EM. Objetivos: Valorar el efecto de una dieta pobre en grasas con suplementación de antioxidantes en los marcadores bioquímicos de pacientes institucionalizados que presentan formas progresivas de EM. Métodos: Se realizó un estudio prospectivo aleatorizado controlado por placebo con 9 participantes, 5 de los cuales se asignan al grupo de intervención (dieta baja en grasas y suplementación antioxidante) y los 4 restantes al grupo placebo (dieta baja en grasas). Se evaluó el efecto de la intervención dietética que supone modificación de la dieta e introducción de antioxidantes durante 42 días mediante valoraciones de parámetros antropométricos y bioquímicos y marcadores del estrés oxidativo en sangre y orina en las etapas inicial (día 0), intermedia (día 15) y final (día 42) del tratamiento. Resultados: Se obtuvieron niveles de proteína C reactiva significativamente inferiores en el grupo de intervención con respecto al grupo placebo al final del estudio. Los marcadores de estrés oxidativo e inflamación: isoprostanos 8-iso-PGF2e interleucina IL-6 también disminuyeron en el grupo de intervención después de la intervención dietética. La actividad de la enzima catalasa aumentó de forma significativa en el grupo de intervención antes de la suplementación con antioxidantes. No se observaron diferencias significativas en otros marcadores de estrés oxidativo. Conclusiones: Los resultados obtenidos sugieren que la dieta y los suplementos dietéticos están involucrados en la modulación del metabolismo celular y los procesos de inflamación de la EM. En consecuencia, las dietas bajas en grasas y los suplementos antioxidantes podrían ser utilizados como terapias alternativas en el tratamiento de la EM.

Composición físico-química del queso colonial (Brasil) / Physical and chemical composition of colonial cheese (Brasil)

Se ha llevado a cabo una evaluación físico-química del queso tipo Colonial, una variedad de queso elaborado a partir de leche cruda de vaca, de corta o ninguna maduración y que no se encuentra bajo normalización del Ministerio de Agricultura de Brasil. Fueron estudiadas cien (100) muestras del queso tipo Colonial, elaborado con leche cruda de vaca recogidas en zonas turísticas del Sur de Brasil. Los parámetros estudiados han sido: determinación de proteína, grasa, humedad/extracto seco, cenizas, cloruros, pH y actividad de água (Aw). (AU)

The physical and chemical composition of Colonial raw cow‘s milk cheese produced in Rio Grande do Sul ( South Brasil) were studied. Hundred samples of this soft and manufacturated cheese were collected in the two main touristic areas in South Brasil. The physical and chemical characteristics studied were: protein and fat content, humidity/ dry extract, salt concentration, pH and water activity (Aw) (AU)

Iron, folate and vitamins B12 & C dietary intake of an elderly institutionalized population in León, Spain.

Aging is associated with increased risk of developing anaemia and micronutrient deficiencies. The purpose of this study was to evaluate the daily intake of micronutrient whose deficient in diet could cause anaemia (iron, folic acid, vitamin B12) and vitamin C to establish the prevalence of anaemia in a group of institutionalized of 124 elderly subjects residing in five nursing homes in León (Spain). A precise weighing method was used to conduct the control of food intake covering seven days. Energy, alcohol, iron, folate, vitamin B12, and vitamin C intake were obtained. Weight, and Height also were measured. Serum iron, serum ferritin, haemoglobin and hematocrit were also measured. Average daily iron intake was higher than the 10 mg recommended by the National Academy of Science although significantly higher (p < 0.05) in males (17.0 +/- 7.4 mg) than in females (11.8 +/- 1.5 mg). Moreover, vitamin C intake in all subjects is high (118.8 +/- 43.7 mg) and higher than Spanish RDA (198%). Average intakes of folate and vitamin B12 in the present study exceeded the RDA, (103% and 144%). However, 45.83% of males and 5.97% of females showed deficiencies in vitamin B12 and 53.91% of the subjects showed deficiencies in folic acid. The average haemoglobin concentration (14.28 +/- 1.33 g/dL), hematocrit percentage (43.71 +/- 6.31), ferritin concentration (87.01 +/- 59.74 ng/mL) and serum iron (85.36 +/- 33.98 micrograms/dL) showed similar figures to the results obtained in other studies carried out on elderly populations. It would be necessary to adequately compose the menus given in nursing homes, decreasing energy contribution, and supplying micronutrient rich foods or fortified foods.

Daily intake of macronutrients in a group of institutionalized elderly people in León. Spain.

Monitoring of energy distribution of the three macronutrients of diet could be beneficial in order to improve the physiological status of elderly people. The objective of this study is to analyse total daily energy intake as well as the caloric contribution of the macronutrients and alcohol, which make up basic diet of five nursing homes in León (Spain). Dietary consumption was evaluated in a group of 107 elderly people, aged 65-98 years. A precise weighing method was used to conduct the control of food intake covering seven days. Protein, carbohydrates, fat, alcohol, dietary fiber and cholesterol intake were obtained. Weight, and Height also were measured. Total dietary energy intake was significantly higher in men (130.5%) than in women (115.6%), with regard to recommended value. Relative contribution of macronutrients to total energy intake is extremely unbalanced. Energy derived from protein was very high (16.7%), energy derived from fat was also very high, and significantly higher for females (39.6%) than for males (34.4%), whereas the proportion derived from carbohydrates was very low, although also significantly higher in females (41.5%) than in males (35.8%), due to the high energy percentage that make up the alcohol intake in males (9.1%). A review of the diet offered by nursing homes, not only directed at the adjustment of total energy intake but also with respect to alcohol intake and macronutrient content of foodstuffs used in the elaboration of the menus, would be required in order not to unbalance the caloric profile of the diet.

[Evaluation of diet and biochemical parameters for minerals in a group of elderly subjects in the province of Leon (Spain)]. / Evaluación dietética y parámetros bioquímicos de minerales en un colectivo de ancianos de la provincia de León (España).

The population of the elderly people is increasing in our society, and it is very frequently affected by undernutrition problems. It is mainly due to an inadequate intake of micronutrients, as well as to a higher incidence of chronic diseases, which negatively affect the nutritional status. The aim of this study is to assess the levels, in terms of minerals (Ca, Fe, I, Zn, Mg, Na and K), of a population of 124 elderly persons (60 males and 64 females), aged between 65 and 98 years, who are institutionalized in five institutions for elderly people in the province of León (Spain). The dietetics study was carried out recording the food intake throughout 7 days by the precise weighting method. Blood concentration in Mg, Fe, Ca and ferritin was determined for the biochemical study. Calcium intake was 813 +/- 182 mg in males and 792 +/- 173 mg in females, which are lower values than those of reference that are being discussed at present. Regarding the values of serum calcium, no significant differences were found between males and females (9.2 +/- 0.4 mg/dL vs 9.1 +/- 0.6 mg/dl, respectively), but all these individuals are situated at the lower limit of range of normality. Iron intake was high, and there were 98% of males and 89% of females whose iron intakes were higher than the recommendations. Iron serum concentration was 89.6 +/- 37.9 micrograms/dl vs 79.3 +/- 24.2 micrograms/dl, and blood ferritin concentration was 107 +/- 64 ng/ml vs 64 +/- 48 ng/ml, in males and females respectively. Despite the fact that there is a very high percentage of elderly people whose iodine intake is lower than the 80% of the recommended intake (96% in males and 75% in females), no old person participating in this study was diagnosed of hypothyroidism. In relation to zinc intake, no significant differences are found between sexes, recording in both cases very low mean intake values compared to the dietetic recommendation (61 +/- 10% I/RD).

Characterization of Staphylococcus aureus isolated from poultry meat in Spain.

Ninety-six Staphylococcus aureus isolates from retail chicken carcasses in Spain were characterized using cultural and biochemical tests. The strains were phage typed with the international bacteriophage set for typing S. aureus of human origin. Eighty-eight (91.7%) strains were of the poultry ecovar. Strains of human ecovar were not found. These facts are congruent with findings of other authors. Ninety (93.7%) strains were phage typeable. Lysis by phages of Group III was the most frequent with 66 (68.7%) sensitive strains. Twenty-eight (29.2%) strains were sensitive at 100 routine test dilution (RTD) and only 16 (16.7%) at RTD. By using reversed phage typing, we managed to increase the number of phage typeable strains by 46 (47.9%). More than one S. aureus phage type was detected in 14 (35%) carcasses, which emphasizes the convenience of subtyping several S. aureus isolates from the same food sample in epidemiological studies. Two phage patterns (75/84 and 6/1030/ W57) were the most common. The S. aureus isolates were closely related, as 78 strains showed the most common or indistinguishable (<2 phage reaction differences) phage patterns.

Comparison of the efficacy of different techniques, culture media, and sources of blood in determining the hemolytic activity of Listeria spp..

Hemolytic activity is a fundamental criterion for the differentiation of Listeria species; therefore, a simple and inexpensive procedure to clearly distinguish hemolytic strains from each other and from nonhemolytic strains would be of great aid. We compared the efficacy of several techniques, culture media, and types of blood in demonstrating the hemolysis of Listeria spp. The hemolytic activities of Listeria monocytogenes and Listeria seeligeri were more easily detected with a red blood cell top-layer (RBCTL) technique and with a microplate technique than when the strains were streaked on blood agar (BA). Listeria ivanovii produced a marked hemolysis regardless of the technique employed. In general, the hemolytic activity of these three species was stronger on media containing brain heart infusion (BHI) agar and (or) potassium tellurite (PT). However, Listeria innocua produced questionable hemolytic reactions when nonselective culture media with BHI and PT were utilized, limiting the advantages gained by employing the two compounds. The RBCTL and the BA techniques disclosed greater hemolytic activity for L. monocytogenes, L. seeligeri, and L. ivanovii with sheep and guinea pig blood than with horse and human blood. When the microplate technique was used, all four kinds of blood were equally effective.

Influence of strain and trisodium phosphate concentration on growth parameters of Listeria monocytogenes in vitro.

AIMS: The present study was conducted to determine the influence of strain and trisodium phosphate (TSP) concentration in the growth of Listeria monocytogenes in vitro. METHODS AND RESULTS: Three strains (ATCC 11916, 64d, isolated from chicken meat, and M2-5b, a clinical animal isolate) were inoculated in broth with 0, 0.5, 1 and 1.5% (w/v) of TSP. The shortest lag phase and highest maximum rate of growth (mu) were obtained in the presence of 0.5% TSP. In contrast, the highest lag phase and lowest mu were obtained with 1.5% TSP. For each TSP concentration, significant differences (P < 0.05) in lag phase and mu of the three L. monocytogenes strains were observed. CONCLUSION: The behaviour of L. monocytogenes is significantly influenced by both the origin of the strain and the salt concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate the importance of choosing an adequate TSP concentration for the decontamination of foods, because low concentrations could favour the growth of L. monocytogenes.

Occurrence of Listeria species in retail poultry meat and comparison of a cultural/immunoassay for their detection.

The prevalence of Listeria spp. on the skin of a hundred fresh chicken carcasses purchased from 20 retail stores in León was investigated using the routine test procedure recommended by the United States Department of Agriculture (USDA). PALCAM and Modified Oxford agar (MOX) were used for isolation. Listeria spp., Listeria monocytogenes, L. innocua, L. welshimeri, L. grayi and L. ivanovii were present in 95%, 32%, 66%, 7%, 4% and 2% of the samples, respectively. Next, an immunoassay test (Listeria Rapid Test; Oxoid, Unipath) and the routine test procedure (USDA) were compared for their ability to detect Listeria spp. on 40 chicken carcasses. When we used MOX for the isolations, the sensitivity of the immunoassay test was 100% and the specificity 85.7%. When we used PALCAM medium the sensitivity and specificity of the test was 94.29% and 80%, respectively.

Immunohistochemistry of the extracellular matrix of the normal equine lamina cribrosa.

Purpose To use immunohistochemical techniques to identify and localize the structural macromolecules of the extracellular matrix (ECM) of the normal adult equine lamina cribrosa in order to make comparisons to the extracellular matrix of the lamina cribrosa of horses with glaucoma. METHODS: Normal eyes of five adult horses between 5 and 10 years of age were fixed in 10% neutral buffered formalin and embedded in paraffin. Polyclonal rabbit-derived antibodies against human elastin, laminin, fibrillin-1, and collagen types I, III and IV, and polyclonal goat-derived antibodies against collagen type VI were used as primary antibodies. Transverse and longitudinal histologic sections of the optic nerve head and lamina cribrosa were stained using several dilutions of the primary antibodies, biotinylated link antibody, horseradish peroxidase-labeled streptavidin, and 3,3'-diaminobenzidine as a chromogen. The immunohistochemical staining patterns were qualitatively interpreted. RESULTS: The normal adult horse lamina cribrosa labeled positively for collagen types I, III and VI, laminin, elastin and fibrillin. Collagen type VI staining of the laminar ECM was most intense, followed by labeling for collagen types III and I, respectively. Laminar blood vessels were weakly positive for laminin and slightly positive for type IV collagen. The scleral ECM of the laminar insertion zone had more intense labeling for collagen types I and VI than did the laminar plates. CONCLUSIONS: The extracellular matrix of the laminar plates of the adult equine lamina cribrosa is similar to the dog as it consists of elastic and collagen fibers (with collagen types VI, III and I). Both the normal dog and horse lamina display more intense staining of collagen type VI than is found in the ECM of the normal human lamina cribrosa. The macromolecular structure of the equine lamina cribrosa suggests that it is a very resilient structure that may provide some protection to the optic nerve axons during episodes of elevated intraocular pressure.

The influence of manufacturing and drying conditions on the survival and toxinogenesis of Staphylococcus aureus in two Spanish dry sausages (chorizo and salchichón).

The effects of formulation, starter culture and fermentation temperature on growth and synthesis of toxin A (SEA) and TNase by Staphylococcus aureus during fermentation and drying of Spanish chorizo were investigated. Inhibitory factors able to inhibit SEA synthesis in culture media were unable to prevent SEA production in chorizo fermented at 20 and 30°C, though a lower temperature and starter culture SP318 (Lactobacillus sake, Pediococcus pentosaceous and Staphylococcus xylosus) decreased staphylococcal growth and SEA formation. Reduction and even disappearance of the SEA during ripening was observed. In most batches, TNase was a reliable indicator of staphylococcal growth and SEA production. Dextrose added to the salchichón formulation repressed S. aureus growth during drying. Lactobacillus curvatus in combination with dextrose was an effective anti-staphylococcal agent during fermentation.

Characterization and extracellular activity of psychrotrophic bacteria isolated from Villalón cheese (fresh variety of Spanish sheep's milk cheese).

The incidence of psychrotrophic bacteria was investigated in a Spanish fresh ewes' cheese (Villalón). Counts of mesophiles and psychrotrophs were (log cfu/g) 5.72 +/- 1.10 and 3.90 +/- 1.01, respectively, for factory cheeses made from pasteurized-milk. Figures for hand-made cheeses made from raw-milk were 7.35 +/- 0.48 and 6.94 +/- 0.65, respectively. A total of 59 representative psychrotrophic isolates were characterized and tested for protease and lipase production at 7 and 30 degrees C. The strains were assigned to Enterobacteriaceae (predominant in raw-milk cheese), heterofermentative lactic acid bacteria (dominant in pasteurized-milk cheese and absent in raw-milk cheese) and Pseudomonas. More than 73% of the Enterobacteriaceae produced both proteolytic and lipolytic enzymes at either 30 or 7 degrees C. This percentage is considerably higher than those previously reported. The seven isolates of pseudomonads investigated produced proteases at 7 degrees C and six were positive at 30 degrees C; lipolytic activity was shown by five of the isolates at both temperatures. Among the heterofermentative lactic acid bacteria seven of the ten isolates were proteolytic at 30 degrees C.

Mycotoxins in two Spanish cheese varieties.

Samples of cheeses naturally contaminated with moulds (12 samples of mouldy Manchego cheese and 10 of a naturally ripened blue cheese) were analysed for the presence of mycotoxins (aflatoxins BI and MI, sterigmatocystin, patulin, penicillic acid and mycophenolic acid in Manchego cheese, and mycophenolic acid and roquefortine in blue cheese). In addition, 24 Penicillium and Aspergillus strains isolated from the samples were assessed for their mycotoxigenicity. Four of Manchego cheese samples were positive to mycophenolic acid and one sample of blue cheese contained roquefortine. The rest of mycotoxins investigated were not found. One Aspergillus strain isolated from Manchego cheese showed the ability to produce aflatoxin MI. The rest of strains from these samples being no producers. In contrast, 7 out of 9 Penicillium (P. roqueforti) strains isolated from blue cheese were able to produce roquefortine, with one strain also producing mycophenolic acid.

[Incidence, behavior and control of Aeromonas hydrophila in meat and dairy products]. / Incidencia, comportamiento y control de Aeromonas hydrophila en productos cárnicos y lácteos.

This review deals with several aspects of Aeromonas hydrophila and other motile Aeromonas species associated with foodborne illness. Although it is mainly dedicated to the factors affecting growth and survival of this species in foods of animal origin, information on other topics is also provided. This paper includes sections on: Taxonomy, diseases caused by Aeromonas, virulence factors, reservoirs and prevalence in foods and water, factors affecting growth and survival, isolation and identification, and control measures.

Biovars of coagulase-positive staphylococci isolated from bovine mastitic milk.

Fifty strains of coagulase-positive staphylococci associated with bovine mastitis were biotyped. Of them, 20 were identified as biovar C (cattle & sheep), 17 as biovar B (poultry & swine), 2 as biovar D (hares) and 1 as biovar F (pigeon & fox). Of the remaining strains, 2 were closely related to human biovar A, 2 failed one property to be classified as biovar B, 3 shared properties of biovars B and D and 3 could not be identified. Bovine strains belonging to biovar C formed an heterogeneous group showing differences in crystal violet growth type and production of alpha haemolysin. Only strains associated with biovar A were Tween 80 positive. The highest incidence of lytic reactions amongst all biovars was with phages 42 E (III) and 102 (IV). Antibiotic resistance was most frequently found in biovar C (65%). The 2 strains in biovar D, one classed as biovar C and one as intermediate, were enterotoxigenic (C or D toxins).

Incorporation of (U-14C)-glucose into glycogen in normal rat pancreatic islets.

The incorporation of glucose into glycogen was determined in pancreatic islets isolated from normal rats and incubated with glucose (5 or 20 mM) and compounds known to affect glycogen metabolism in other tissues. Incubation of pancreatic islets with glucose (20 mM) induced a marked increase in radioactive glycogen. Exposure to epinephrine in the presence of glucose (20 mM) slightly increased incorporation of glucose into glycogen. In contrast the incorporation of glucose into glycogen was not affected when isolated islets were exposed to glucagon or insulin, whereas anti-insulin serum in the incubation medium decreased radioactive glycogen formation.