Alternative procedures for parent choice in a breeding program for the common bean (Phaseolus vulgaris L.)

Six common bean cultivars were crossed in diallel and the segregant populations were assessed in the F2 and F3 generations to compare methodologies for parental selection in a breeding program based on hybridization. The cultivars involved in the diallel were A 114, A 77, ESAL 686, Milionário, Carioca, and Flor de Mayo. The segregant F2 and F3 generations were assessed on the experimental campus of the Universidade Federal de Larvas, in July 1994. It was found that the cultivars differed in their general combining ability (GCA). Flor de Mayo, which belongs to the Durango race, had the largest positive GCA estimate for grain field, and the cultivars from the Mesoamerican race, Milionário and A 114, the smallest GCA estimates. For flowering, the cultivar that most contributed to reduced plant cycle was ESAL 686. There was agreement among the results obtained from the diallel and the estimates of the parameter m + a of the populations. However, it was evident that the estimate of genetic variance of the populations should be considered as a condition to identify the hybrid population that will produce a line with high performance.

Genetic control of characters associated with bean golden mosaic geminivirus resistance in Phaseolus vulgaris L.

Bean golden mosaic is the most important viral disease of the bean crop (Phaseolus vulgaris L.) in Latin America. The genetics of resistance to a Brazilian strain of bean golden mosaic virus (BGMV), was studied in a 4 x 4 diallel cross without reciprocals, among the parental genotypes DOR 303, EMGOPA 201 Ouro, Carnaval, and Redlands Greenleaf C. Seedlings of the four parents, six F1 hybrids, 12 backcrosses, and F2 generations for each combination were inoculated on the eighth day after sowing by exposure to a viruliferous whitefly (Bemisia tabaci Genn.) population for 24 h, in a glasshouse, prior to transplantation to field conditions. The full set of two parents, F1, F2 and respective backcrosses for each combination was considered to be a family. Data were recorded and analyzed for foliar yellowing, plant dwarfing, and pod malformation, using a randomized block design, with two replications. Weighted generation mean analysis was performed for each of the six families. An additive gene action model was significant for the three characteristics evaluated. On the other hand, non-additive gene action had greater absolute value in most cases. Resistance to foliar yellowing conferred by genes from DRO 303 was highly heritable and was expressed equally well in the different genetic backgrounds evaluated. Such resistance may be oligogenic. Broad- and narrow-sense heritabilities were relatively high for all response traits. The three traits studied were all positively correlated, indicating that they can be simultaneously selected for enhancement. The highest correlation coefficient was obtained for dwarfing x pod malformation.

Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase

The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ß-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ß-naphthylamide was eluted at 750 µS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10%) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/KM ratio for L-Leu-ß-naphthylamide. The enzyme activity was increased 40% by 0.15 M NaCl, inhibited 94% by 2.0 mM Zn2+, inhibited 91% by sodium p-hydroxymercuribenzoate and inhibited 45% by 0.7 mM o-phenanthroline and 30 µM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 µM), p-nitroaniline (0.25 mM) and ß-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 µM) inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4oC and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.

Serum cytokines in patients with Brazilian pemphigus foliaceus (fogo selvagem)

Pemphigus foliaceus (PF) is characterized by acantholysis determined by IgG4 binding to desmoglein I, a 160-kDa desmosomal glycoprotein. To investigate the immunopathological aspects of Brazilian PF, we determined levels of serum cytokines in patients with PF. Twenty-five patients with PF and a control group consisting of 10 healthy individuals were studied. Serum IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-gamma were measured in the two groups by ELISA. The median concentration of IL-2 was lower in PF patients compared to the control group (0.45 and 9.50 pg/ml, respectively), as also was the concentration of IL-4 (0.26 and 10.16 pg/ml, respectively). The same was observed for IL-5 (7.94 and 15.74 pg/ml, respectively) and for IFN-gamma (5.90 and 8.58 pg/ml, respectively). For IL-10 and IL-12, higher concentrations were observed in PF compared to the control group (IL-10: 24.76 and 20.92; IL-12: 2.92 and 1.17 pg/ml, respectively). Considering the Th1/Th2 paradigm, it seems that a Th2 profile, mainly represented by IL-10, predominates in PF.

Effect of an aqueous extract of Phaseolus vulgaris on the properties of tail tendon collagen of rats with streptozotocin-induced diabetes

Changes in the structural and functional properties of collagen caused by advanced glycation might be of importance for the etiology of late complications in diabetes. The present study was undertaken to investigate the influence of oral administration of aqueous pod extract (200 mg/kg body weight) of Phaseolus vulgaris, an indigenous plant used in Ayurvedic Medicine in India, on collagen content and characteristics in the tail tendon of streptozotocin-diabetic rats. In diabetic rats, collagen content (117.01 ± 6.84 mg/100 mg tissue) as well as its degree of cross-linking was increased, as shown by increased extent of glycation (21.70 ± 0.90 µg glucose/mg collagen), collagen-linked fluorescence (52.8 ± 3.0 AU/µmol hydroxyproline), shrinkage temperature (71.50 ± 2.50ºC) and decreased acid (1.878 ± 0.062 mg hydroxyproline/100 mg tissue) and pepsin solubility (1.77 ± 0.080 mg hydroxyproline/100 mg tissue). The alpha/ß ratio of acid- (1.69) and pepsin-soluble (2.00) collagen was significantly decreased in streptozotocin-diabetic rats. Administration of P. vulgaris for 45 days to streptozotocin-diabetic rats significantly reduced the accumulation and cross-linking of collagen. The effect of P. vulgaris was compared with that of glibenclamide, a reference drug administered to streptozotocin-diabetic rats at the dose of 600 µg/kg body weight for 45 days by gavage. The effects of P. vulgaris (collagen content, 64.18 ± 1.97; extent of glycation, 12.00 ± 0.53; collagen-linked fluorescence, 33.6 ± 1.9; shrinkage temperature, 57.0 ± 1.0; extent of cross-linking - acid-soluble collagen, 2.572 ± 0.080, and pepsin-soluble collagen, 2.28 ± 0.112) were comparable with those of glibenclamide (collagen content, 71.5 ± 2.04; extent of glycation, 13.00 ± 0.60; collagen-linked fluorescence, 38.9 ± 2.0; shrinkage temperature, 59.0 ± 1.5; extent of cross-linking - acid-soluble collagen, 2.463 ± 0.078, and pepsin-soluble collagen, 2.17 ± 0.104). In conclusion, administration of P. vulgaris pods had a positive influence on the content of collagen and its properties in streptozotocin-diabetic rats.

Dermal dendritic cell number correlates with serum autoantibody titers in Brazilian pemphigus foliaceus patients

Pemphigus foliaceus (PF) is an autoimmune bullous disease endemic in Brazil. Since serum IL-12 is increased in patients with PF and Langerhans cells (LC) produce IL-12, we titrated serum autoantibodies by indirect immunofluorescence, and quantified epidermal dendritic cells, known as LC, and dermal dendritic cells (DC). Biopsies of blistering lesions were obtained from 22 patients, 13 of whom were submitted to biopsy of both injured and of apparently healthy skin. The control groups consisted of skin from 8 cadavers and from 12 women submitted to breast plastic surgery. LC and DC were identified with anti-CD1a antibody and quantified by morphometric analysis. LC number in the lesion and in apparently healthy skin from PF patients was similar to that of both control groups. DC number in the injured skin (median = 0.94 DC/mm basement membrane) was higher than that of the cadaver group (median = 0.13 DC/mm basement membrane). In the 13 patients with biopsies of both injured and apparently healthy skin, LC and DC were present in larger numbers in the lesion. There was a direct correlation between DC number in the lesion of the PF group and serum autoantibody titers. This correlation was not observed for LC number. The increased number of DC in the lesion, as well as its direct correlation with serum autoantibody titers suggest the participation of DC in the pathogenesis of PF. The relationship between increased DC number and IL-12 in PF needs to be clarified.

Evidence for the participation of nitric oxide in pemphigus

Pemphigus is an inflammatory autoimmune disorder of the skin. Nitric oxide (NO) is an inflammatory mediator linked to a variety of physiological and pathophysiological phenomena that include skin tumors, psoriasis, urticaria, and atopic dermatitis. Inflammatory cells present in pemphigus lesions are important sources of NO production. We investigated whether NO is involved in pemphigus. A prospective cohort study was conducted at the Dermatology Service of the Hospital Universitário Walter Cantídio of the Federal University of Ceará. All patients seen at the outpatient clinic between August 2000 and July 2001, with a clinically and histologically confirmed diagnosis of pemphigus were included. The median age was 42.5 years (range: 12-69 years) with a male to female ratio of 3:2. Total serum nitrite levels, used as a marker for NO production, were determined by the Griess reaction. Skin biopsies from pemphigus and breast surgery (control) patients were used for the detection of the inducible NO synthase (iNOS) by immunohistochemistry. Twenty-two (22) patients with pemphigus and eight (8) controls who did not differ in demographic characteristics were included. Total serum nitrite levels were significantly higher (>7 µmol/L) in pemphigus patients compared to controls (<6 µmol/L), regardless of the severity of the clinical activity of pemphigus (P < 0.0001). All pemphigus biopsies presented increased immunostaining for iNOS that was not detected in normal skin samples. These data are the first to demonstrate that pemphigus patients display increased serum NO levels that are associated with increased iNOS expression in the affected skin.

Polymorphism of the promoter region and exon 1 of the CTLA4 gene in endemic pemphigus foliaceus (fogo selvagem)

Endemic pemphigus foliaceus (EPF) is an autoimmune bullous skin disease characterized by acantholysis and antibodies against a desmosomal protein, desmoglein 1. Genetic and environmental factors contribute to development of this multifactorial disease. HLA class II and some cytokine gene polymorphisms are the only genetic markers thus far known to be associated with susceptibility to or protection from EPF. The cytotoxic T-lymphocyte antigen-4 gene (CTLA4) encodes a key immunoreceptor molecule that regulates and inhibits T-cell proliferation. It participates in the regulatory process controlling autoreactivity and therefore has been considered a strong candidate gene in autoimmune diseases. In the search for genes that might influence EPF pathogenesis, we analyzed variants of the CTLA4 gene in a sample of 118 patients and 291 controls from a Brazilian population. This is the first study investigating the possible role of polymorphisms of the 2q33 chromosomal region in differential susceptibility to pemphigus foliaceus. Promoter region and exon 1 single nucleotide polymorphisms -318 (C,T) and 49 (A,G) were genotyped using sequence-specific oligonucleotide probes after amplification by the polymerase chain reaction. The allelic and genotypic frequencies did not differ significantly between the patient and the control groups (-318T: 9.8 and 10.9%, 49G: 33.0 and 35.2% were the allelic frequencies in patients and controls, respectively). In addition, no significant difference was found when the patient and control population samples were stratified by the presence of HLA-DRB1 alleles. We conclude that the CTLA4 -318 (C,T) and 49 (A,G) polymorphisms do not play a major role in EPF development.

Changes in the hormone and lipid profile of obese adolescent Saudi females with acne vulgaris

Acne vulgaris is a multifactorial disease affecting a majority of the adolescent population. The objective of this study was to test for a correlation between fasting serum lipid profiles and levels of testosterone, insulin, leptin, and interleukin 1-β (IL-1β) and the incidence of severe acne vulgaris in obese adolescent females. Four groups of adolescent females were studied: obese with acne, obese without acne, non-obese with acne, and non-obese without acne. Obese females with acne, compared to obese females without acne and non-obese subjects, had significantly higher serum triglycerides, low-density lipoprotein cholesterol and apolipoprotein-B (apo-B) (mean ± SD: 197 ± 13.7 vs 171 ± 11.5, 128 ± 8.3 vs 116 ± 7.7, 96 ± 13.7 vs 85 ± 10.3 mg/dL, respectively) but significantly lower high-density lipoprotein cholesterol and apo-A1 levels (40 ± 3.3 vs 33 ± 3.5 and 126 ± 12 vs 147 ± 13 mg/dL). Serum testosterone, insulin and leptin levels were significantly higher in obese subjects with or without acne compared to non-obese females with or without acne (3 ± 0.5 vs 2.1 ± 0.47, 15.5 ± 3.3 vs 11.6 ± 3, 0.9 ± 0.2 vs 0.6 ± 0.15 nmol/mL, respectively). Serum IL-1b was significantly elevated in obese and non-obese subjects with acne compared to subjects without acne; in those without acne, these levels were higher in obese than non-obese subjects (2.4 ± 0.2, 1.4 ± 0.1 vs 1.8 ± 0.12 and 1.3 ± 0.11 pg/mL, respectively). Our results indicate that there is a relationship between obesity (BMI >27) and acne. By early recognition, the etiology and treatment protocol of acne may prevent unwanted conditions.

Evidence for the endophytic colonization of Phaseolus vulgaris(common bean) roots by the diazotroph Herbaspirillum seropedicae

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium, which associates with important agricultural plants. In the present study, we have investigated the attachment to and internal colonization of Phaseolus vulgaris roots by the H. seropedicae wild-type strain SMR1 and by a strain of H. seropedicae expressing a red fluorescent protein (DsRed) to track the bacterium in the plant tissues. Two-day-old P. vulgaris roots were incubated at 30°C for 15 min with 6 x 10(8) CFU/mL H. seropedicae SMR1 or RAM4. Three days after inoculation, 4 x 10(4) cells of endophytic H. seropedicae SMR1 were recovered per gram of fresh root, and 9 days after inoculation the number of endophytes increased to 4 x 10(6) CFU/g. The identity of the recovered bacteria was confirmed by amplification and sequencing of the 16SrRNA gene. Furthermore, confocal microscopy of P. vulgaris roots inoculated with H. seropedicae RAM4 showed that the bacterial cells were attached to the root surface 15 min after inoculation; fluorescent bacteria were visible in the internal tissues after 24 h and were found in the central cylinder after 72 h, showing that H. seropedicae RAM4 is capable of colonizing the roots of the dicotyledon P. vulgaris. Determination of dry weight of common bean inoculated with H. seropedicae SMR1 suggested that this bacterium has a negative effect on the growth of P. vulgaris.

Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.

Relationship of macrophage migration inhibitory factor levels in PBMCs, lesional skin and serum with disease severity and activity in vitiligo vulgaris

Melanocyte loss in vitiligo vulgaris is believed to be an autoimmune process. Macrophage migration inhibitory factor (MIF) is involved in many autoimmune skin diseases. We determined the possible role of MIF in the pathogenesis of vitiligo vulgaris, and describe the relationship between MIF expressions and disease severity and activity. Serum MIF concentrations and mRNA levels in PBMCs were measured in 44 vitiligo vulgaris patients and 32 normal controls, using ELISA and real-time RT-PCR. Skin biopsies from 15 patients and 6 controls were analyzed by real-time RT-PCR. Values are reported as median (25th-75th percentile). Serum MIF concentrations were significantly increased in patients [35.81 (10.98-43.66) ng/mL] compared to controls [7.69 (6.01-9.03) ng/mL]. MIF mRNA levels were significantly higher in PBMCs from patients [7.17 (3.59-8.87)] than controls [1.67 (1.23-2.42)]. There was also a significant difference in MIF mRNA levels in PBMCs between progressive and stable patients [7.86 (5.85-9.13)vs 4.33 (2.23-8.39)] and in serum MIF concentrations [40.47 (27.71-46.79) vs 26.80 (10.55-36.07) ng/mL]. In addition, the vitiligo area severity index scores of patients correlated positively with changes of both serum MIF concentrations (r = 0.488) and MIF mRNA levels in PBMCs (r = 0.426). MIF mRNA levels were significantly higher in lesional than in normal skin [2.43 (2.13-7.59)vs 1.18 (0.94-1.83)] and in patients in the progressive stage than in the stable stage [7.52 (2.43-8.84)vs 2.13 (1.98-2.64)]. These correlations suggest that MIF participates in the pathogenesis of vitiligo vulgaris and may be useful as an index of disease severity and activity.

TEXTURA DE FEIJÃO-VAGEM (Phaseolus vulgaris L) PROCESSADO OU NÃO: EFEITO DA APLICAÇÃO DE CONCENTRAÇÕES CRESCENTES DE CÁLCIO VIA ABSORÇÃO RADICULAR

Esta investigação teve como objetivo estudar o efeito do cultivo de feijão-vagem com doses crescentes de cálcio na textura das vagens antes e após processo de enlatamento. O experimento foi desenvolvido com a cultivar UEL 1, utilizando-se areia grossa como substrato. Os tratamentos constaram das seguintes concentrações de cálcio em solução nutritiva: 0, 75, 150 e 300 ppm. Foram determinados os teores de cálcio das vagens in natura, substâncias pécticas e textura das vagens in natura e processada. As concentrações de cálcio nas vagens aumentaram em paralelo à elevação dos níveis de cálcio adicionados à solução nutritiva. Os teores de pectina solúvel nas vagens in natura não diferiram entre os tratamentos. Houve um acréscimo da quantidade de pectina insolúvel em função do aumento das doses de cálcio. Após o processamento térmico ocorreu uma solubilização das pectinas. A maior concentração de pectina solúvel nas vagens enlatadas foi observada nas vagens cultivadas sem adição de cálcio na solução nutritiva. As medidas de textura demonstraram uma tendência de aumento de firmeza das vagens concomitante à elevação nos teores de cálcio antes e após o processo de enlatamento.

Efeito da secagem em leito de jorro bidimensional sobre as propriedades físicas e tecnológicas do feijão preto (Phaseolus vulgaris, L.)

A secagem de feijão preto (Phaseolus vulgaris, L.) em leito de jorro bidimensional foi estudada num equipamento em escala piloto de 100cm de altura total, 10cm de largura e 40cm de comprimento. Um soprador centrífugo de 7cv foi utilizado para fornecer o ar responsável pela formação do jorro ao sistema e nenhum aquecimento adicional foi realizado, ficando a temperatura de operação em torno de 50ºC. Foi feito um estudo sobre a influência do tempo de secagem, da vazão de ar injetado no sistema e da carga de grãos em cada batelada sobre o tempo de cozimento, a capacidade de absorção, a densidade e o diâmetro da partícula. A taxa de secagem também foi estudada. Observou-se que em 6 horas a redução de umidade do grão é cerca de 60% e as condições operacionais estudadas não afetaram significativamente as propriedades físicas e tecnológicas do feijão.

Propriedades físico-químicas de polifenoloxidase de feijão (Phaseolus vulgaris L.)

As atividades de polifenoloxidases do feijão (Phaseolus vulgaris L.), cultivares: (Aruã, Aporé, A774, Carioca, Diamante Negro, Ouro Branco, Pérola, RAO 33 e Rudá) foram investigadas sob condições de cinética enzimática. O pH ótimo para o extrato bruto das nove cultivares foi em pH 7,2 utilizando catecol como substrato. Nenhuma atividade PPO foi observada na presença do ácido gálico ou D,L- DOPA.