Layer-by-layer assembly of multilayer films composed of streptavidin and biotinylated antibody by real-time biomolecular interaction analysis

Layer-by-layer assembly of multilayer films of streptavidin and biotinylated antibody was completed on the streptavidin coated surface. Real-time biomolecular interaction analysis (BIA) based on surface plasmon resonance technique was used to monitor the multilayer assembly in solution continuously. The results indicate that the uniform multilayer film can be fabricated successfully based on the strong interaction between streptavidin and biotin. The mean surface mass concentration of each adsorption layer is 1. 32 ng/mm(2) for biotinylated antibody, 2. 93 ng/mm(2) for streptavidin, according to the correlation of SPR response with surface concentration.

Real-time immunoassay of antibody activity in serum by surface plasmon resonance biosensor

A surface plasmon resonance biosensor has been used to determine antibody activity in serum. As a model system, the interaction of mouse IgG and sheep anti-mouse IgG polyclonal antibody was investigated in real time. The factors, including pH value, ionic strength, protein concentration, influencing electrostatic adsorption of mouse IgG protein onto carboxylated dextran-coated sensor chip surface, were studied. The procedures of mouse IgG protein immobilization and immune reaction were monitored in real time. The regeneration effect using the different elution reagents was also investigated. The same mouse IgG immobilized surface can be used for 100 cycles of binding and elution with only 0.38% loss per regeneration in reactivity. The results show that the surface plasmon resonance biosensor is a rapid, simple, sensitive, accurate and reliable detection technique for real-time immunoassay of antibody activity. The assay allows antibodies to be detected and studied in their native form without any purification. (C) 2000 Elsevier Science B.V. All rights reserved.

Studies on electrostatic adsorption of proteins on modified surface by real-time surface plasmon resonance technique

The elucidation of key influence factors for electrostatic adsorption is very important to control protein nonspecific adsorption on modified surfaces. In this study, real-time surface plasmon resonance technique is used to characterize the electrostatic adsorption of two proteins (mouse IgG and protein A) on carboxymethyldextran-modified surface. The results show that protein solution pH and ionic strength are key influence factors for efficient electrostatic adsorption. The influence of protein, solution pH on the amount of electrostatic adsorption depends on the type of the charge and the charge density of both protein and modified matrix on the surface. The electrostatic adsorption process involves a competition between the positively charged protein and other positively charged species in the buffer solution. A decrease of ionic strength leads to an increasing electrostatic adsorption. The kinetic adsorption constants of protein A at different pH values were also calculated and compared.

Electrostatic layer-by-layer assembly of polycation and DNA multilayer films by real-time surface plasmon resonance technique

The assembly of alternating DNA and positively charged poly(dimethyldiallylammonium chloride) (PDDA) multilayer films by electrostatic layer-by-layer adsorption has been studied. Real time surface plasmon resonance (BIAcore) technique was used to characterize and monitor the formation of multilayer films in solution in real time continuously. The results indicate that the uniform multilayer can be obtained on the poly(ethylenimine) (PEI) coated substrate surface. The kinetics of the adsorption of DNA on PDDA surface was also studied by real-time BIAcore technique, and the observed rate constant was calculated using a Langmuir model (k(obs) = (1.28 +/- 0.08) x 10(-2) s(-1)).

Expression profiles of penaeidin from Fenneropenaeus chinensis in response to WSSV and vibrio infection by real-time PCR

Penaeidin from Chinese shrimp (Fenneropenaeus chinensis) has proved to be one of the most important antimicrobial peptides in the bodies of animals. The relative quantitative real-time PCR method is developed to study through time, the mRNA expression profile of penaeidin in the muscle and haemocyte tissue of Chinese shrimp infected with vibrio (Vibrio anguillarum) and WSSV (white spot syndrome virus). Research results showed that the same pathogens infection experiments produced similar gene expression profile in different tissues while different expression profiles appeared in the same tissues infected by different exterior pathogens. In vibrio infection experiments, a "U" Re expression profile resulted. Expression levels of penaeidin increased and surpassed the non-stimulated level, indicating that penaeidin from Chinese shrimp has noticeable antimicrobial activities. In WSSV infection experiments, the expression profile appeared as an inverse "U" with the expression of penaeidin gradually decreasing to below baseline level after 24 h. The expression of antimicrobial peptides gene in mRNA level in response to virus infection in shrimp showed that international mechanisms of virus to haemocytes and microbial to haemocytes are completely different. Decline of penaeidins expression levels may be due to haemocytes being destroyed by WSSV or that the virus can inhibit the expression of penaeidins by yet undiscovered modes. The expression profiles of penaeidin in response to exterior pathogen and the difference of expression profiles between vibrio and WSSV infection provided some clues to further understanding the complex innate immune mechanism in shrimp.

Tandem duplication-random loss is not a real feature of oyster mitochondrial genomes

Duplications and rearrangements of coding genes are major themes in the evolution of mitochondrial genomes, bearing important consequences in the function of mitochondria and the fitness of organisms. Yu et al. (BMC Genomics 2008, 9: 477) reported the complete mt genome sequence of the oyster Crassostrea hongkongensis (16,475 bp) and found that a DNA segment containing four tRNA genes (trnK(1), trnC, trnQ(1) and trnN), a duplicated (rrnS) and a split rRNA gene (rrnL5') was absent compared with that of two other Crassostrea species. It was suggested that the absence was a novel case of "tandem duplication-random loss" with evolutionary significance. We independently sequenced the complete mt genome of three C. hongkongensis individuals, all of which were 18,622 bp and contained the segment that was missing in Yu et al.'s sequence. Further, we designed primers, verified sequences and demonstrated that the sequence loss in Yu et al.'s study was an artifact caused by placing primers in a duplicated region. The duplication and split of ribosomal RNA genes are unique for Crassostrea oysters and not lost in C. hongkongensis. Our study highlights the need for caution when amplifying and sequencing through duplicated regions of the genome.

Floral Organogenesis and Development of two Taxa in Tribe Hyoscyameae (Solanaceae)-Przewalskia tanguitca and Hyoscyamus niger

Floral organogenesis and development of Przeivalskia langutica Maxim, endemic to China and Hyoscyamus niger L. , which belong to the tribe Hyoscyameae (Solanaceae), were studied using scanning electron microscope. They have three common characters of floral organ initiation and development: 1) initia-tion of the floral organs in the two species follows Hofmeister's rule; 2) the mode of corolla tube development belongs to the "late sympetaly" type; 3) primordia of the floral appendages initiated in a pentameroua pattern and acropetal order. But initiation of the calyx-lobe primordia showed different modes in these two species. The calyx-lobe primordia of H, niger have simultaneously whorled initiation, while those of P, tangulica have helical initiation, but the five calyx-lobe primordia form a ring after all five calyx-lobe primordia occur. The systematic significance of the present results in the genera Hyoscyamus and Przeivalskia is discussed in this paper.

Floral organogenesis and development of two taxa of the Solanaceae - Anisodus tanguticus and Atropa belladonna

Floral organogenesis and development of two Solanaceae species, Anisodus tanguticus and Atropa belladonna, were studied by using scanning electron microscopy (SEM) as part of a project on systematics and evolution in the tribe Hyoscyameae. These two species share the following common characters of floral organ initiation and development: (1) initiation of the floral organs in the two species follows Hofmeister's rule; (2) the mode of corolla tube development belongs to the "late sympetaly" type, namely, petals are initiated separately and later become joined by fusion of their basal meristem, then rise together and form a corolla tube; (3) primordia of the floral appendages are initiated in a pentamerous pattern and acropetal order: sepals are initiated first, followed by the petals and stamens, and finally the carpels. The whorl of five stamen primordia forms almost simultaneously and originates opposite the sepal primordia, but initiation of the sepal primordia shows different modes in the two species. The sepal primordia of Anisodus tanguticus have simultaneously whorled initiation, while those of Atropa belladonna have helical initiation. The systematic significance of the present results in the genera Anisodus and Atropa is discussed in this paper.

New taxa, names, and combinations in Saxifraga (Saxifragaceae) for the Flora of China

The following new species, new variety, new name, and four new combinations are published for the forthcoming account of Saxifraga L. in the Flora of China, Volume 8: S. epiphylla Gornall & H. Ohba, sp. nov., S. gemmigera Engler var. gemmuligera (Engler) J. T. Pan & Gornall, comb. nov., S. heterotricha Marquand & Airy-Shaw var. anadena (H. Smith) J. T. Pan & Gornall, comb. et stat, nov., S. hypericoides Franchet var. aurantiascens (Engler & Irmscher) J. T. Pan & Gornall, comb. nov., S. hypericoides var. rockii (Mattfeld) J. T. Pan & Gornall, comb. et stat. nov., S. sinomontana J. T. Pan & Gornall, nom. nov., and S. sinomontana var. amabilis H. Smith ex J. T. Pan, var. nov. In addition, the names S. mengtzeana Engler & Irmscher and S. mengtzeana var. cordatifolia Engler & Irmscher are lectotypified here.

Diagnóstico da resistência do carrapato-do-boi a carrapaticidas em Mato Grosso do Sul.

No Brasil, casos de resistência do carrapato-do-boi [Rhipicephalus (Boophilus) microplus] aos acaricidas organofosforados têm sido relatados a partir do início dos anos de 1970 e, aos piretróides, no fi nal dos anos de 1980. Desde então, as reclamações de produtores quanto a esse problema vêm se avolumando em todas as regiões pecuárias. Por isso, torna-se importante conhecer bem a real situação de sua resistência e caracterizar adequadamente seu controle a fi m de torná-lo mais efi ciente e reduzir seu custo. Este estudo objetivou avaliar a suscetibilidade de populações de R. (B.) microplus em relação a acaricidas de distintas classes e caracterizar seu controle no Estado de Mato Grosso do Sul. Para tanto, foram realizados bioensaios toxicológicos com carrapatos obtidos em bovinos de propriedades de gado de corte e de leite em onze das principais regiões produtivas do Estado: Três Lagoas, Dourados, Alto Taquari, Iguatemi, Baixo Pantanal, Campo Grande, Bodoquena, Paranaíba, Aquidauana, Nova Andradina e Cassilândia. A escolha das propriedades amostradas priorizou aquelas com histórico de problemas críticos no controle químico dos carrapatos, mas, na ausência deles em condições de realizar os testes com tal precedente histórico, as fazendas foram escolhidas por consulta conforme a disponibilidade de carrapatos. Nos testes de suscetibilidade foi utilizada a técnica de imersão de teleóginas (cinco minutos), com posterior avaliação de parâmetros biológicos. Na imersão foram utilizados doze acaricidas comerciais (totalizando sete princípios ativos pertencentes a quatro classes distintas): amitraz; diazinon; cipermetrina; clorpirifós + cipermetrina + citronelal; diclorvós (DDVP) + clorfenvinfós; cimiazole + cipermetrina; etion + cipermetrina; DDVP + clorpirifós; clorpirifós + cipermetrina; cipermetrina + clorpirifós + butóxido de piperonila + citronelal e cipermetrina + clorfenvinfós. Após a imersão (lotes de no mínimo 10 teleóginas por produto), as teleóginas foram secas e mantidas em câmara climatizada por 30 dias para a avaliação de parâmetros reprodutivos, tais como: peso da postura (no 16o dia), taxa de eclosão e efi ciência reprodutiva (no 40o dia). Foram considerados eficazes os produtos cuja efi cácia foi igual ou superior a 95%, critério este estabelecido pelo Ministério da Agricultura, Pecuária e Abastecimento (Mapa) para o registro de novos produtos. Em todas as propriedades foi verificada resistência a pelo menos um produto carrapaticida, havendo diversas propriedades com resistência de até 100% a um ou mais princípios ativos. Constatou-se que a efi cácia dos produtos piretróides nas populações amostradas foi, em geral, inferior a 70%, não sendo recomendado seu uso nas propriedades visitadas. Dentre os doze produtos avaliados, apenas dois produtos, DDVP 60% + clorfenvinfós 20% (97,68%) e Cipermetrina 15% + clorpirifós 25% + butóxido de piperonila 15% + citronelal 1% (100%), apresentaram efi cácia média superior a 95% e devem controlar satisfatoriamente as infestações pelo carrapato em condições de campo. Paralelamente, observou-se que os pecuaristas utilizam, também, produtos não autorizados ou sem registro ofi cial e efetuam diferentes combinações de produtos a seu dispor, incluindo produtos caseiros ou específi cos para uso agrícola, em função da ausência de um programa nacional de controle do carrapato bovino.