Studies of dietary phytoestrogen from momordica charantia Linn. Seeds / Estudios de fitoestrógeno en la dieta a partir de semillas de momordica charantia Linn

Phytoestrogens are secondary plant metabolites produced by plants that are similar in structure of estrogen. Human consumption of these compounds has been associated with many health benefits. In this study, we investigated the potential phytoestrogen content of dietary Momordica charantia Linn. (Bitter melon) seeds were collected from the Hyderabad-Karnataka regions of India. A phenolic dietary phytoestrogen has been isolated from the crude ethanol extract of Bitter melonseeds. After preparative HPLC whitish amorphous compound was yielded. The HPLC purified compound is subjected to spectral analysis using IR, NMR and MS. The spectral data revealed that the phenolphthalein a phytoestrogenic molecule is present in the ethanol extract of dietary Bitter melonseeds.

Los fitoestrógenos son metabolitos vegetales secundarios, producidos por plantas que tienen una estructura similar al estrógeno. El consumo humano de estos compuestos se ha asociado con muchos beneficios para la salud. En este estudio, investigamos el contenido potencial de fitoestrógenos de Momordica charantia Linn en la dieta. Se recolectaron semillas (melón amargo) de las regiones de Hyderabad-Karnataka de la India. Se ha aislado un fitoestrógeno dietético fenólico del extracto de etanol crudo de semillas de melón amargo. Después de HPLC preparativa, se obtuvo un compuesto amorfo blanquecino. El compuesto purificado por HPLC se somete a análisis espectral usando IR, NMR y MS. Los datos espectrales revelaron que la fenolftaleína, una molécula fitoestrogénica, está presente en el extracto etanólico de las semillas de melón amargo en la dieta.

Proteic and phenolics compounds contents in Bacupari callus cultured with glutamine and nitrogen sources / Conteúdo de proteínas e compostos fenólicos em calos de Bacupari cultivados com glutamina e fontes de nitrogênio

Abstract In this study was evaluated the influence of glutamine supplementation on the endogenous content of amino acids, proteins, total phenolics, flavonoids and proanthocyanidins in Bacupari callus. The explants were inoculated in MS medium, MS with half concentration of the nitrogen salts (MS½) and nitrogen-free MS, supplemented with glutamine (5, 10, 30 and 60mM) named as Gln5, Gln10, Gln30 and Gln60. Amino acids and proteins were analyzed after 20, 80 and 140 days and the secondary metabolites on the 140th day. There was no difference in the amino acids on the 20th day. On the 80th day the treatments MS and MS½ presented the lowest levels. On the 140th day MS and MS½ presented the lowest amino acid concentration and Gln10 the highest. Concerning proteins, there was difference only on the 140th day, being the highest concentrations observed in Gln5, and the lowest in MS½ treatment. Total phenolics content was higher in the treatment Gln60 and lowest in MS. Treatments Gln5, Gln10, Gln30 and MS½ were statistically equal. For flavonoids, the highest values occurred in the treatments Gln30, Gln60 and MS½ and the lowest in Gln5, Gln10 and MS. Similarly, for the proanthocyanidins the highest concentrations were observed in treatment Gln60 and the lowest in Gln5 and MS. In conclusion, the treatment with 60mM of glutamine favors the protein accumulation and production of secondary metabolites in Bacupari callus.

Resumo Nesse estudo foi avaliado o efeito da suplementação com glutamina no conteúdo endógeno de aminoácidos, proteínas, fenólicos totais, flavonoides e proantocianidinas em calos de Bacupari. Os explantes foram inoculados em meio MS, meio MS com metade da concentração de dos sais de nitrogênio (MS½) e meio MS sem nitrogênio suplementado com glutamina (5, 10, 30 e 60mM) denominados como Gln5, Gln10, Gln30 e Gln60. Os aminoácidos e as proteínas foram analisados após 20, 80 e 140 dias e os metabólitos secundários no 140° dia. Não houve diferença nos aminoácidos no 20° dia. No 80° dia os tratamentos MS e MS½ apresentaram os menores níveis. No 140° dia, MS e MS½ apresentaram as menores concentrações de aminoácidos e o Gln10 as maiores. A respeito das proteínas, houve diferença apenas no 140° dia, sendo as maiores concentrações observadas nos tratamentos Gln, e as menores no MS½. O conteúdo de fenólicos totais foi maior no tratamento Gln60 e menor no MS. Os tratamentos Gln5, Gln10, Gln30 e MS½ foram estatisticamente iguais. Para os flavonóides, os maiores valores ocorreram nos tratamentos Gln30, Gln60 e MS½ e os menores no Gln5, Gln10 e MS. Da mesma forma, para as proantocianidinas, as maiores concentrações foram observadas no tratamento Gln60 os menores no Gln5 e MS. Em conclusão, o tratamento com 60 mM de glutamina favorece o acúmulo de proteínas e a produção de metabólitos secundários em calos de Bacupari.

Proteomic profile of Piper tuberculatum (Piperaceae) / Perfil proteômico e metabólico de Piper tuberculatum (Piperaceae)

Abstract Piper tuberculatum (Piperaceae) is a species that accumulates especially amides as secondary metabolites and several biological activities was previously reported. In this article, we report a proteomic study of P. tuberculatum. Bidimensional electrophoresis (2D SDS-PAGE) and mass spectrometry (ESI-Q-TOF) were used in this study. Over a hundred spots and various peptides were identified in this species and the putative functions of these peptides related to defense mechanism as biotic and abiotic stress were assigned. The information presented extend the range of molecular information of P. tuberculatum.

Resumo Piper tuberculatum (Piperaceae) é uma espécie que acumula especialmente amidas como metabólitos secundários e diversas atividades biológicas dessa espécie foram relatadas anteriormente. No presente artigo, relatamos um estudo proteômico dessa espécie. Eletroforese bidimensional (2D SDS-PAGE) e espectrometria de massas (ESI-Q-TOF) foram utilizadas nesse estudos. Mais de cem spots e vários peptídeos foram identificados nesta espécie e as funções putativas desses peptídeos relacionadas a mecanismo de defesa como estresse biótico e abiótico foram atribuídos. As informações apresentadas ampliam a gama de informações moleculares dessa espécie.

Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli

Background: The acidic subunit of amarantin (AAC)-the predominant amaranth seed storage protein-has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by inserting four VY antihypertensive peptides. This modified protein (AACM.4) was expressed in Escherichia coli. In addition, we also recombinantly expressed other derivatives of the amarantin protein. These include: unmodified amarantin acidic subunit (AAC); amarantin acidic subunit modified at the third VR with four VY peptides (AACM.3); and amarantin acidic subunit doubly modified, in the third VR with four VY peptides and in the fourth VR with the RIPP peptide (AACM.3.4). Results: E. coli BL21-CodonPlus (DE3)-RIL was the most favorable strain for the expression of proteins. After 6 h of induction, it showed the best recombinant protein titer. The AAC and AACM.4 were obtained at higher titers (0.56 g/L) while proteins modified in the third VR showed lower titers: 0.44 g/L and 0.33 g/L for AACM.3 and AACM.3.4, respectively. As these AAC variants were mostly expressed in an insoluble form, we applied a refolding protocol. This made it possible to obtain all proteins in soluble form. Modification of the VR 4 improves the thermal stability of amarantin acidic subunit; AAC manifested melting temperature (Tm) at 34°C and AACM.4 at 37.2°C. The AACM.3 and AACM.3.4 did not show transition curves. Conclusions: Modifications to the third VR affect the thermal stability of amarantin acidic subunit.

Características de las residencias de terapia intensiva pediátrica de la República Argentina: Encuesta nacional / Characteristics of pediatric intensive care residency programs in Argentina: A national survey

Introducción. La residencia de terapia intensiva pediátrica (TIP) tiene pocos años de desarrollo en nuestro país. Conocer su situación brinda la posibilidad de establecer estrategias para contribuir al desarrollo y capacitación de profesionales. Objetivos. 1) Describir las características de las residencias de TIP del país. 2) Evaluar si existen características que se relacionen con una mayor ocupación de las vacantes. 3) Explorar la inserción laboral en el hospital formador de los residentes. Diseño. Descriptivo, observacional. Encuesta nacional. Criterios de inclusión. Residencias de TIP funcionales entre el 1/4/2014 y el 31/5/2014. Resultados. Se analizaron 31 residencias. Solo 11/31 tenían volumen de internación anual >400 pacientes. No había normas y/o criterios de atención en 9/31. En 17/31, el programa estuvo adecuado al marco de referencia nacional. Hubo 13/31 que no contaban con jefe ni instructor de residentes. Fueron acreditadas por el Ministerio de Salud 5/31. Hubo 65 vacantes; el número aumentó en los últimos 4 años; la ocupación disminuyó de 59% en 2009 a 30% en 2013. El 60% de los residentes tuvo inserción laboral en la TIP formadora. El análisis de regresión logística multivariado identificó la variable ingresos anuales > 400 pacientes como predictora independiente de ocupación de vacantes > 60%. Conclusiones. 1) Hay un déficit en la ocupación de cargos. 2) El número de residencias acreditadas es escaso. 3) Las unidades de cuidados intensivos pediátricos con mayor número de ingresos se asociaron a una mayor cobertura de vacantes. 4) Más de la mitad de los residentes se insertaron laboralmente en la TIP formadora.

Introduction. Pediatric intensive care residency programs have been in place in Argentina for just a few years. Knowing their status offers the possibility to establish strategies to help with professional development and training. Objectives. 1) To describe the characteristics of pediatric intensive care residency programs across Argentina. 2) To assess whether certain characteristics are related to a higher vacancy filling rate. 3) To assess job placement in the hospital where residents are trained. Design. Descriptive, observational study. National survey. Inclusion criteria. Pediatric intensive care residency programs in place between April 1st, 2014 and May 31st, 2014. Results. Thirty-one residency programs were analyzed. Only 11/31 had an annual hospitalization volume >400patients. There were no guidelines and/or criteria for care in 9/31. The program suited the national reference frameworkin17/31. There was no head ofresidents or resident trainer in 13/31. Only 5/31 had been certified by the Ministry of Health. There were 65 vacancies; this number increased in the past four years; vacancy filling rate decreased from 59% in 2009 to 30% in 2013. Sixty percent of residents got a job in the pediatric intensive care unit where they were trained. A multivariate logistic regression analysis identified the outcome measure annual hospitalization volume >400 patients as an independent predictor of vacancy filling rate >60%. Conclusions. 1) Vacancy filling is deficient. 2) The number of certified residency programs is scarce. 3) Pediatric intensive care units with a higher number of hospitalizations were associated with a higher vacancy filling rate. 4) More than half of residents got a job in the pediatric intensive care unit where they were trained.

Historical review of clinical vaccine studies at Oswaldo Cruz Institute and Oswaldo Cruz Foundation - technological development issues

This paper presents, from the perspective of technological development and production, the results of an investigation examining 61 clinical studies with vaccines conducted in Brazil between 1938-2013, with the participation of the Oswaldo Cruz Institute (IOC) and the Oswaldo Cruz Foundation (Fiocruz). These studies have been identified and reviewed according to criteria, such as the kind of vaccine (viral, bacterial, parasitic), their rationale, design and methodological strategies. The results indicate that IOC and Fiocruz have accumulated along this time significant knowledge and experience for the performance of studies in all clinical phases and are prepared for the development of new vaccines products and processes. We recommend national policy strategies to overcome existing regulatory and financing constraints.

Estratégias para o enfrentamento do absenteísmo em consultas odontológicas nas Unidades de Saúde da Família de um município de grande porte: uma pesquisa-ação / Strategies for tackling absenteeism in dental appointments in the Family Health Units of a large municipality: action research

O objetivo deste trabalho foi analisar os motivos das faltas às consultas odontológicas em Unidades de Saúde da Família (USF) e implementar estratégias para sua redução por meio da pesquisa-ação. O estudo foi realizado em 12 USF de Piracicaba/SP, de 01 de janeiro a 31 de dezembro de 2010. A amostra se consistiu de 385 usuários, entrevistados por telefone, sobre os motivos das faltas, além de 12 cirurgiões-dentistas e 12 enfermeiras. Realizaram-se duas oficinas com os profissionais: uma para problematização dos dados coletados nas entrevistas e elaboração de estratégias; e outra após 4 meses, para avaliação. O maior motivo de faltas foi a coincidência do horário de funcionamento das unidades com o de trabalho dos usuários. Dentre as estratégias ressaltou-se a realização de palestras sobre saúde bucal, educação permanente nas reuniões de equipe, capacitação dos Agentes Comunitários de Saúde, participação em grupos terapêuticos e parcerias entre Equipe de Saúde Bucal e equipamentos sociais da comunidade. A adoção de prontuário único foi a estratégia desafiadora encontrada pelos profissionais. Concluiu-se que as estratégias implementadas levaram à diminuição das faltas em 66,6% e o caráter motivador das oficinas possibilitou a reflexão crítica para o redirecionamento da prática em saúde.

The aim of this study was to analyze the reasons for missed appointments in dental Family Health Units (FHU) and implement strategies to reduce same through action research. This is a study conducted in 12 FHUs in Piracicaba in the State of São Paulo from January, 1 to December, 31 2010. The sample was composed of 385 users of these health units who were interviewed over the phone and asked about the reasons for missing dental appointments, as well as 12 dentists and 12 nurses. Two workshops were staged with professionals: the first to assess the data collected in interviews and develop strategy, and the second for evaluation after 4 months. The primary cause for missed appointments was the opening hours of the units coinciding with the work schedule of the users. Among the strategies suggested were lectures on oral health, ongoing education in team meetings, training of Community Health Agents, participation in therapeutic groups and partnerships between Oral Health Teams and the social infrastructure of the community. The adoption of the single medical record was the strategy proposed by professionals. The strategies implemented led to a 66.6% reduction in missed appointments by the units and the motivating nature of the workshops elicited critical reflection to redirect health practices.

Effect of Cooking on Proximate, Phytochemical Constituents and Hematological Parameters of Tetracarpidium conophorum in Male Albino Rats.

Aims: To determine the effect of cooking on proximate, phytochemical constituents and their changes in hematological parameters. Study Design: Determination of proximate and quantitative phytochemical constituents of the cooked and raw T. conophorum (CTC and RTC respectively) nut and the effect of the nut on the hematological indices on male albino rats fed with the cooked and raw diet formulations of the nut for 30 days period. Methodology: Rats were divided into six groups of five rats each. Each feed and walnut was weighed and mixed in the ratio of 1:1 before administration. Group A: Normal animal feed, Group B: Mixture of animal feed and cooked nut (ratio of 1:1). Group C: Mixture of animal feed and the raw nut (ratio of 1:1), Group D: 100% of the cooked nut, Group E: 100% of the raw nut while Group F: Mixture of raw nut and cooked (ratio of 1:1). Result: The result showed that crude protein, carbohydrate and crude fibre contents of RTC were significantly higher (P<0.05) than the CTC. While the percentage moisture, fat and ash content of the CTC were significantly higher than the RTC. The quantitative phytochemical analysis revealed that there was no significant difference (P>0.05) between the alkaloid and flavonoid contents of RTC and CTC. Tannin, saponin, glycosides, hydrogen cyanide and steroid contents of RTC were significantly higher (P<0.05) than the CTC while terpenoid content of the CTC was significantly higher (P<0.05) than of the the RTC. The hemoglobin values showed no significant difference between the test groups and control group. The neutrophil values of group E and F were significantly higher (P<0.05) when compared with the control group. Rats in group E had a significant decrease (P<0.05) in lymphocyte value as against the control. Total WBC levels in group B, C and F recorded a significant decrease (P<0.05) when compared with the control. Conclusion: The study suggests that processing cooking affects some nutrient constituents and some hematological parameters.

Purification of a protease inhibitor from Dolichos biflorus using immobilized metal affinity chromatography.

Plant protease inhibitors (PIs) are generally small proteins which play key roles in regulation of endogenous proteases and may exhibit antifeedant, antifungal, antitumor and cytokine inducing activities. Dolichos biflorus (horse gram) is an unexploited legume, which is rich in nutrients and also has therapeutic importance. It contains a double-headed PI, which is an anti-nutritional factor. As there is no report available on its simultaneous removal and purification in single step, in this study, a double-headed PI active against both trypsin and chymotrypsin was purified from Dolichos biflorus to ~14-fold with ~84% recovery using an immobilized metal affinity chromatography (IMAC) medium consisting of Zn-alginate beads. The method was single-step, fast, simple, reliable and economical. The purified inhibitor showed a single band on SDS-PAGE corresponding to molecular mass of 16 kDa and was stable over a pH range of 2.0-12.0 and up to a temperature of 100°C for 20 min. The optimum temperature for trypsin and chymotrypsin inhibitor was observed to be 50°C and 37°C, respectively and pH optimum was pH 7.0 and 8.0, respectively. Thus, IMAC using Zn-alginate beads was useful in simultaneous purification and removal of an anti-nutritional factor from horse gram flour in single step. This procedure may also be employed for purification of other plant PIs in one step.

Oil, protein, antioxidants and free radical scavenging activity of stone from wild olive trees [Olea europaea L.]

The wild olive trees or oleaster [var. sylvestris] and the cultivated olive trees [var. europaea] constitute the two botanical varieties of Olea europaea L. from Mediterranean. In this study, a partial chemical profile was conducted including the total lipids, the fatty acid profiles, soluble proteins, polyphenols, flavanoids contents and antioxidants activities of stone from six oleaster trees. The comparison was made by two olive cultivars cultivated in the same region. The oleaster and cultivar stones were richer in oil content having an average of 8.99 and 7.38% dry weight basis [DW], respectively. Qualitatively, all studied oils have the same fatty acids profile with the oleic acid C18:1n-9 as the major fatty acid. The oleaster stone oils were richer in monounsaturated fatty acids having an average of 64.87%. They, also, richer in protein content with an average of 198.86 mg/g DW.The globulin is the major fraction, followed by the albumin, the prolamin and the glutemin fractions. The oleaster stone extracts contain polyphenols, flavonoids with an average of 151.14 and 11.91 mg gallic acid equivalent/100g of DW, respectively. The studied extracts showed antioxidant activity using the free radical scavenging activity determined by DPPH and ABTS. The unexploited oleaster stone seems to be a source of oil with good fatty acids balance, in protein and antioxidants metabolites and would be useful for the formulation of supplements and/or pharmaceutical ingredients

Chemical Genomics in Plant Biology.

Chemical genomics is a newly emerged and rapidly progressing field in biology, where small chemical molecules bind specifically and reversibly to protein(s) to modulate their function(s), leading to the delineation and subsequent unravelling of biological processes. This approach overcomes problems like lethality and redundancy of classical genetics. Armed with the powerful techniques of combinatorial synthesis, high-throughput screening and target discovery chemical genomics expands its scope to diverse areas in biology. The well-established genetic system of Arabidopsis model allows chemical genomics to enter into the realm of plant biology exploring signaling pathways of growth regulators, endomembrane signaling cascades, plant defense mechanisms and many more events.

Establecimiento de condiciones óptimas para obtener un aislado proteico de avellana chilena (Gevuina avellana, Mol)

Una alternativa para resolver el problema del déficit global de proteínas ha sido la utilización de tortas desgrasadas generadas por la extracción de aceite de fuentes vegetales tales como raps, soya, lupino, etc, proceso que al mismo tiempo incrementa el contenido proteico, siendo factible que sea usada para enriquecer algunos tipos de alimentos. Tal es el caso de la avellana chilena (Gevuina avellana, Mol), especie monotípica caracterizada por su elevado porcentaje de aceite (50%) y cuya torta desgrasada podría utilizarse para obtener un aislado proteico. Con este fin se optimizaron las condiciones de extracción de proteína utilizando para ello la metodología superficie de respuesta (MSR) seleccionando el diseño central compuesto, tres variables independientes: tiempo de contacto de la torta con el solvente, relación muestra/solvente y pH, con 5 niveles de trabajo cada uno y tres replicaciones en el punto central. Los datos fueron sometidos a un análisis de regresión y después de ANOVA. El primero para determinar la ecuación polinómica y el segundo para seleccionar los factores de control con efecto significativo sobre el rendimiento de la proteína. La mejor combinación de los factores resultó ser tiempo entre 30 y 40 minutos, pH entre 9 y 9,5 y la relación muestra/ solvente entre 1/15 a 1/16, con un rendimiento final de 76%. En cuanto a las características físicas esta fueron: densidad aparente 0,504 g/cm3, compactación 43,34% y color amarillo claro. El análisis proximal mostró una concentración de proteínas de 76%, hidratos de carbono 13%, fibra cruda 0,68% y aceite 1,29%. En cuanto a las propiedades funcionales destacó absorción de agua (320 g/100 g), absorción de aceite (410 g/100g) y capacidad espumante (221, 05 %).

Establishment of optimun conditions in order to obtain a protein isolate from Chilean Hazelnut. An alternative to solve the problem of the overall deficit of proteins has been the use of defatted cakes generated by the extraction of oil from vegetable sources such as rapeseed, soybean, lupin, etc. This process at the same time increases the protein content, making this feasible to be used to enrich some types of food. This is the case of the chilean hazelnut (Gevuina avellana, Mol), monotypic species characterized by their high percentage of oil (50%) and whose defatted cake isolated protein could be used to obtain an isolated protein. For this purpose optimized conditions of extraction of protein were carried out using the surface response methodology (SRM) and a central composite design with three independent variables: time of contact of the cake with the solvent, sample /solvent ratio and pH was used. All variables were controlled at five different levels. The data were subjected to an analysis of regression and ANOVA, the first to determine the polynomial equation and the second to select the control factors with significant effect on the extraction of the protein. The best combination of factors turned out to be: time between 30 and 40 minutes, pH between 9 and 9.5 and a relationship sample / solvent between 1/15 to 1/16 with a final yield of 76%. The physical characteristics were: density 0,504 g/cm3, compaction 43, 34 % apparent and pale yellow. Proximal analysis showed a concentration of protein of 76%, 13%, raw fiber carbohydrate 0.68% and oil 1.29%. With regard to the functional properties emphasized water absorption (320 g / 100 g), absorption of oil (410 g / 100 g) and foaming capacity (221 %).

Purification and characterization of Mn-peroxidase from Musa paradisiaca (banana) stem juice.

Mn-peroxidase (MnP), a biotechnologically important enzyme was purified for the first time from a plant source Musa paradisiaca (banana) stem, which is an agro-waste easily available after harvest of banana fruits. MnP was earlier purified only from the fungal sources. The enzyme was purified from stem juice by ultrafiltration and anion-exchange column chromatography on diethylamino ethylcellulose with 8-fold purification and purification yield of 65%. The enzyme gave a single protein band in SDS-PAGE corresponding to molecular mass 43 kDa. The Native-PAGE of the enzyme also gave a single protein band, confirming the purity of the enzyme. The UV/VIS spectrum of the purified enzyme differed from the other heme peroxidases, as the Soret band was shifted towards lower wavelength and the enzyme had an intense absorption band around 250 nm. The Km values using MnSO4 and H2O2 as the substrates of the purified enzyme were 21.0 and 9.5 μM, respectively. The calculated kcat value of the purified enzyme using Mn(II) as the substrate in 50 mM lactate buffer (pH 4.5) at 25°C was 6.7s-1, giving a kcat/Km value of 0.32 μM-1s-1. The kcat value for the MnP-catalyzed reaction was found to be dependent of the Mn(III) chelator molecules malonate, lactate and oxalate, indicating that the enzyme oxidized chelated Mn(II) to Mn(III). The pH and temperature optima of the enzyme were 4.5 and 25°C, respectively. The enzyme in combination with H2O2 liberated bromine and iodine in presence of KBr and KI respectively. All these enzymatic characteristics were similar to those of fungal MnP. The enzyme has the potential as a green brominating and iodinating agent in combination with KBr/KI and H2O2.

Molecular characterization and transcriptome profiling of expansin genes isolated from Calotropis procera fibers

The Calotropis procera seed fibers provide an excellent model system to study the genes involved in fiber elongation, fineness and strength. Expansins constitute one of the important gene families involved in plant cell expansion and other cell wall modification processes. Four homologs of Expansin A gene i.e. CpEXPA1, CpEXPA2, CpEXPA3 and CpEXPA4 were isolated from the cDNA library obtained from fast growing Calotropis procera fibers. These homologs represented typical Expansin A family. Each of them had two conserved domains including GH45 like domain and the putative polysaccharide binding domain. The deduced amino acid sequences of the homologs indicated three conserved motifs: i) eight cysteine residues at N-terminus, ii) four tryptophan residues at C-terminus and iii) a Histidine-Phenylalanine-Aspartate motif in the center of the sequence. The presence of N-terminal signal peptide consisting of hydrophobic amino acids and a transmembrane region in all these expansin isoforms suggests their cotranslational insertion into the endoplasmic reticulum and then transportation to the cell wall by secretory pathway. The relative quantification of the four expansins in root, stem, fiber and leave tissues indicated that the transcripts of CpEXPA1, CpEXPA2, CpEXPA3 and CpEXPA4 are variably transcribed in these tissues. The lowest transcription of all the four Expansin A isoforms was observed in elongating roots indicating that root tissue might be having specific expansins other than those confined to air grown organs.

Role of H2O2 and cell wall monoamine oxidases in germination of Vigna radiata seeds.

Plant cell wall expresses monoamine oxidases (MAOs) that catalyze oxidation of secreted amines and produce H2O2 in the process. The H2O2, so produced is used by cell wall peroxidases for lignification of cell wall or for plant defense. The natural substrates for these MAOs are elusive, but polyamines and certain catecholamines have been proposed as candidates. Reactive oxygen species are also known to act as signaling molecules controlling plant metabolism. Mungbean (Vigna radiata) has long served as the plant model of choice while studying molecular programs followed during germination and seed development. In this study, we tested the effect of externally added MAO substrates epinephrine and H2O2 on storage protein mobilization in germinating seeds of Vigna radiata. The seeds were imbibed in the presence of 50 M epinephrine and 10 M H2O2. These low concentrations of the two compounds were used to exclude direct effects on proteolysis and were arrived at after testing a range of the two and choosing the most effective concentration. These seeds showed 11% and 7% decrease in fresh weight respectively, indicating greater storage mobilization and a corresponding 19% and 46% increase in axis length as compared to untreated seeds. Soluble protein in seeds treated with epinephrine and H2O2 decreased significantly by 34% and 33% as compared to untreated seeds. Electrophoretic analysis of seed proteins revealed a startling and selective depletion of storage proteins in treated seeds. The results indicated a clear involvement of H2O2 in storage protein mobilization in the cotyledons. We propose that H2O2 generated within cell walls of seeds serves as a signaling molecule guiding germination events, including protein reserve mobilization.

Effect of mercury ion on the stability of the lipid-protein complex of isolated chloroplasts.

Mercury is known to interact with different parts of living systems causing serious biochemical and physiological disorder. In order to know the effect of mercury (Hg2+) ion on chloroplasts, the cell free organelle are incubated in an isotonic buffer medium in presence of mercury ion. The metal ion is found to induce membrane lipid peroxidation, loss of photosynthetic pigments and degradation of proteins. Such degradation brings about a drastic modification of lipid-protein organization of chloroplasts as reflected from a blue shift of absorption peaks and lowering of chlorophyll-a fluorescence intensity. The detrimental effect of Hg2+ ion has been explained in terms of direct binding with lipid-protein complex of photosynthetic membrane. Such a binding of metal ion exposes the lipid-protein complex for an easier entry and attack of reactive oxygen species (ROS) generated during incubation of chloroplasts in light and dark, thereby resulting in higher disorganization, which is evident from cation- induced changes in absorption and emission characteristics of the organelle.

Cloning and characterization of a novel cysteine protease gene (HbCP1) from Hevea brasiliensis.

The full-length cDNA encoding a cysteine protease,designated HbCP1, was isolated for the first time from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbCP1 contained a 1371 bp open reading frame encoding 457 amino acids.The deduced HbCP1 protein,which showed high identity to cysteine proteases of other plant species,was predicted to possess a putative repeat in toxin (RTX) domain at the N-terminal and a granulin (GRAN) domain at the C-terminal.Southern blot analysis indicated that the HbCP1 gene is present as a single copy in the rubber tree.Transcription pattern analysis revealed that HbCP1 had high transcription in laticifer,and low transcription in bark and leaf.The transcription of HbCP1 in latex was induced by ethylene and tapping.Cloning of the HbCP1 gene will enable us to further understand the molecular characterization of cysteine protease and its possible function in the rubber tree.

Cloning and expression of antiviral/ribosome-inactivating protein from Bougainvillea xbuttiana.

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP)from the leaves of Bougainvillea x buttiana was isolated.The cDNA consisted of 1364 nucleotides with an open reading frame (ORF)of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids.The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species.The deduced protein has been designated BBAP1 (Bougainvillea x buttiana antiviral protein1).The ORF was cloned into an expression vector and expressed in E.coli as a fusion protein of approximately 78 kDa.The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity,and imparted a high level of resistance against the tobacco mosaic virus (TMV).

Studies on glucose-metabolizing enzymes in cytosolic and bacteroidal fractions of mungbean (Vigna radiata L.) and lentil (Lens culinaris L.) nodules.

Nitrogen is exported in the form of ureides or amides from the nodules in pulse crops. In order to understand the carbon metabolism in ureide and amide exporting nodules, activities of enzymes involved in glucose metabolism were compared in cytosolic and bacteroidal fractions of mungbean (ureide exporter) and lentil (amide exporter) nodules during development. Activities of hexokinase, fructokinase, phosphoglucomutase, fructose-1,6-bisphosphatase, phosphohexose isomerase and UDP-glucose pyrophosphorylase were detected in cytosolic fraction of nodules of both the crops during development. Out of these enzymes, specific activity of phosphohexose isomerase was the highest in nodules of both the crops, in comparison with other enzymes. In comparison with mungbean, activities of various enzymes were less in cytosolic fraction of lentil. Activities of hexokinase, fructokinase, phosphoglucomutase were present only in cytosolic fraction of mungbean (Vigna radiata L.), however, low activity of these enzymes was also observed in lentil (Lens culinaris L.) bacteroids. Activities of phosphohexose isomerase and fructose-1,6-bisphosphatase were higher in bacteroids of lentil, as compared to mungbean during early nodule development, but this pattern was reversed with progress of crop development. Higher activities of phosphoglucomutase and fructose-1,6-phosphatase in mungbean cytosolic fraction could lead to increased flow of carbon towards pentose phosphate pathway.