Patty Mulder

Pancreatic-Type Ribonuclease 1 Gene Duplications in Rat Species

Mammalian pancreatic-type ribonucleases (RNases) 1 represent single-copy genes in the genome of most investigated mammalian species, including Mus musculus and other murid rodents. However, in six species belonging to the genus Rattus and closely related taxa, several paralogous gene products were identified by Southern blotting and PCR amplifications of genomic sequences. Phylogenies of nucleotide and derived amino acid sequences were reconstructed by several procedures, with three Mus species as outgroup. Duplications of the RNase 1 occurred after the divergence of Niviventer cremoriventer and Leopoldamys edwardsi from the other investigated species. Four groups of paralogous genes could be identified from specific amino acid sequence features in each of them. Low ratios of nonsynonymous-to-synonymous substitutions and the paucity of pseudogene features suggest functional gene products. One of the RNase 1 genes of R. norvegicus is expressed in the pancreas. RNases 1 were isolated from pancreatic tissues of R. rattus and R. exulans and submitted to N-terminal amino acid sequence analysis. In R. rattus, the orthologue of the expressed gene of R. norvegicus was identified, but in R. exulans, two paralogous gene products were found. The gene encoding for one of these had not yet been found by PCR amplification of genomic DNA. A well-defined group of orthologous sequences found in five investigated species codes for very basic RNases. Northern blot analysis showed expression of messenger RNA for this RNase in the spleen of R. norvegicus, but the protein product could not be identified. Evolutionary rates of RNase 1, expressed as nucleotide substitutions per site per 103 million years (Myr), vary between 5 and 9 in the lines leading to Mus, Niviventer, and Lepoldamys (on the basis of an ancestral date of mouse/rat divergence of 12.2 Myr) and between 20 and 50 in the lines to the other sequences after divergence from Niviventer and Leopoldamys (5.5 Myr).

Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus which are not present in the mature protein. The gene encoding hevamine contains no introns. Southern hybridization of Hevea brasiliensis DNA with a full-length hevamine cDNA probe showed that there are multiple copies of genes encoding hevamine or hevamine homologues in the Hevea brasiliensis genome. In the lutoid body (vacuolar) fraction of rubber latex only three hevamine components were detected with very similar primary structures, which are probably products of one single locus on the genome.

Development of small-volume, microfluidic chaotic mixers for future application in two-dimensional liquid chromatography

We report a microfluidic chaotic micromixer with staggered herringbone grooves having a geometry optimized for fast mobile-phase modification at the interface of a two-dimensional liquid chromatography system. The volume of the 300 μm mixers is 1.6 microliters and they provide mixing within 26 s at a flow rate of 4 μL min−1 and 0.09 s at a flow rate of 1000 μL min−1. Complete mixing is achieved within a distance of 3 cm along the 5 cm-long microchannel over the whole range of flow rates. The mixers can be used to mix aqueous phosphate-buffered saline solutions with methanol or acetonitrile at different ratios (1 : 2, 1 : 5 and 1 : 10). We also describe in detail an improved fabrication protocol for these mixers using a two-step soft photolithographic procedure. Mixers are made by replication in poly(dimethylsiloxane).

How microtechnologies enable organs-on-a-chip

Engineering cellular microenvironments that more accurately reflect the in vivo situation is now recognized as being crucial for the improvement of the in vitro viability and in vivo-like function of cells or tissues. Microfluidic technologies have been increasingly applied since the late 1990s for this purpose, with a growing number of examples of perfused cell and tissue cultures in microfluidic chambers and channels. More recently, additional microfabricated features have been implemented in microfluidic structures to achieve 3-D cell culture systems which mimic not only in vivo fluid flows, but also the structure, transport, and mechanical properties of tissue in, for example, the lung or the intestine. The ultimate challenge becomes the combination of different organ functions into single, linked-compartment devices - the body-on-the-chip.

Behaviour of Human Umbilical Vein Endothelial Cells (HUVEC) Cultivated in Microfluidic channels

Our long-term goal is to develop advanced tools for cell studies and analysis based on microfluidic systems. In this paper, we report on endothelial cell cultivation in microchannels and 96-well tissue plates, and compare cell phenotype and cellular status in the two environments This was done under both pro-inflammatory conditions (cell stimulation in the presence of the cytokine, Tumour Necrosis Factor, TNFalpha) and normal (medium only) conditions. In addition, we considered the behaviour of cells in a pro-inflammatory environment in the presence of an anti-inflammatory drug