Regulative specification of ectoderm in skeleton disrupted sea urchin embryos treated with monoclonal antibody to Pl-nectin

Pl-nectin is a glycoprotein first discovered in the extracellular matrix (ECM) of Paracentrotus lividus sea urchin embryo, apically located on ectoderm and endoderm cells. The molecule has been described as functioning as an adhesive substrate for embryonic cells and its contact to ectoderm cells is essential for correct skeletogenesis. The present study was undertaken to elucidate the biochemical characteristics of Pl-nectin and to extend knowledge on its in vivo biological function. Here it is shown that the binding of mesenchyme blastula cells to Pl-nectin-coated substrates was calcium dependent, and reached its optimum at 10 mM Ca2+. Perturbation studies using monoclonal antibody (McAb) to Pl-nectin, which prevent ectoderm cell-Pl-nectin contact, show that dorsoventral axis formation and ectoderm differentiation were retarded. At later stages, embryos recovered and, even if growth and patterning of the skeleton was greatly affected, the establishment of dorsoventral asymmetry was reached. Similarly, the expression of specific ectoderm and endoderm territorial markers was achieved, although occurring with some delay. Endoderm differentiation and patterning was not obviously affected. These results suggest that both endoderm and ectoderm cells have regulative capacities and differentiation of territories is restored after a lag period. On the contrary, failure of inductive differentiation of the skeleton cannot be rescued, even though the ectoderm has recovered.     

Synthesis and preliminary biochemical assessment of ethyl-terminated perfluoroalkylamine oxide surfactants

The synthesis and usefulness in membrane biochemistry of a new class of surfactants have been investigated. 1-Ethyl-2-dimethylamine oxide polar heads were grafted onto a hydrocarbon, a fluorocarbon or an ethyl-capped fluorocarbon hydrophobic tail. The ability of the resulting surfactants to extract and/or to stabilize in aqueous solution a test membrane protein, cytochrome b(6)f, was evaluated. While it is not a detergent, the hemifluorinated derivative efficiently kept purified cytochrome b(6)f soluble, native and functional. The data suggest that alkyl-capped fluorocarbon surfactants provide an interesting alternative to classical detergents for handling membrane proteins in aqueous solutions under non-dissociating conditions.     

OpenADAM: an open source genome-wide association data management system for Affymetrix SNP arrays

Background

The goal of DNA barcoding is to develop a species-specific sequence library for all eukaryotes. A 650 bp fragment of the cytochrome c oxidase 1 (CO1) gene has been used successfully for species-level identification in several animal groups. It may be difficult in practice, however, to retrieve a 650 bp fragment from archival specimens, (because of DNA degradation) or from environmental samples (where universal primers are needed).

Results

We used a bioinformatics analysis using all CO1 barcode sequences from GenBank and calculated the probability of having species-specific barcodes for varied size fragments. This analysis established the potential of much smaller fragments, mini-barcodes, for identifying unknown specimens. We then developed a universal primer set for the amplification of mini-barcodes. We further successfully tested the utility of this primer set on a comprehensive set of taxa from all major eukaryotic groups as well as archival specimens.

Conclusion

In this study we address the important issue of minimum amount of sequence information required for identifying species in DNA barcoding. We establish a novel approach based on a much shorter barcode sequence and demonstrate its effectiveness in archival specimens. This approach will significantly broaden the application of DNA barcoding in biodiversity studies.     

Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila

The glutamatergic neuromuscular junction (NMJ) in Drosophila adds new boutons and branches during larval development. We generated transgenic fruit flies that express a novel green fluorescent membrane protein at the postsynaptic specialization, allowing for repeated noninvasive confocal imaging of synapses in live, developing larvae. As synapses grow, existing synaptic boutons stretch apart and new boutons insert between them; in addition, new boutons are added at the ends of existing strings of boutons. Some boutons are added de novo, while others bud from existing boutons. New branches form as multiple boutons bud from existing boutons. Nascent boutons contain active zones, T bars, and synaptic vesicles; we observe no specialized growth structures. Some new boutons exhibit a lower level of Fasciclin II, suggesting that the levels of this synaptic cell adhesion molecule vary locally during synaptic growth.     

Enzyme reaction engineering: effect of methanol on the synthesis of antibiotics catalyzed by immobilized penicillin G acylase under isothermal and non-isothermal conditions

The effect of methanol on the kinetically controlled synthesis of cephalexin by free and immobilized penicillin G acylase (PGA) was investigated. Catalytic and hydrophobic membranes were obtained by chemical grafting, activation, and PGA immobilization on hydrophobic nylon supports. Butyl methacrylate (BMA) was used as graft monomer. Increasing concentrations of methanol were found to cause a greater deleterious effect on the activity of free than on that of the immobilized enzyme. Methanol, however, improved the kinetic stability of cephalexin synthesized by free PGA, resulting in higher maximum yields. By contrast, immobilized PGA reached 100% yields even in the absence of the cosolvent. Cephalexin synthesis by the catalytic membrane was also performed in a non-isothermal bioreactor. Under these conditions, a 94% increase of the synthetic activity and complete conversion of the limiting substrate to cephalexin were obtained. The addition of methanol reduced the non-isothermal activity increase. The physical cause responsible for the non-isothermal behavior of the hydrophobic catalytic membrane was identified in the process of thermodialysis.     

Raman detection and identification of normal and leukemic hematopoietic cells

The analysis of leukocytes of peripheral blood is a crucial step in hematologic exams commonly used for disease diagnosis and, typically, requires molecular labelling. In addition, only a detailed, laborious phenotypic analysis allows identifying the presence and stage of specific pathologies such as leukemia. Most of the biochemical information is lost in the routine blood tests. In the present study, we tackle 2 important issues of label‐free biochemical identification and classification of leukocytes using Raman spectroscopy (RS). First, we demonstrate that leukocyte subpopulations of lymphocytes (B, T and NK cells), monocytes and granulocytes can be identified by the unsupervised statistical approach of principal component analysis and classified by linear discriminant analysis with approximately 99% of accuracy. Second, we apply the same procedure to identify and discriminate normal B cells and transformed MN60 lymphocyte leukemic cell lines. In addition, we demonstrate that RS can be efficiently used for monitoring the cell response to low‐dose chemotherapy treatment, experimentally eliciting the sensitivity to a dose‐dependent cell response, which is of fundamental importance to determine the efficacy of any treatment. These results largely expand established Raman‐based research protocols for label‐free analysis of white blood cells, leukemic cells and chemotherapy treatment follow‐up.