Design and evaluation of a novel flow chamber for measuring cell adhesion to absorbable polymer films

There is great interest in improving cellular attachment to synthetic materials, particularly for developing small diameter tissue-engineered vascular grafts. However, limited research has been conducted to evaluate the adhesion characteristics of different cell types to absorbable substrates. Tissue engineered vessels typically fail as a result of delamination of the endothelial cell layer when exposed to fluid or blood flow. The focus of this research was to design and evaluate a flow chamber, using fibroblasts, smooth muscle cells, and endothelial cells, to probe the bounds of the system. A flow chamber was designed and fabricated to compare the relative adhesion characteristics of cells to absorbable polymer films. A preliminary investigation of mouse fibroblast (3T3M) adhesion to semicrystalline poly-L-lactide (PLL) films was conducted to determine general operating specifications. Cell coverage on films was evaluated using a live-dead assay and image analysis; following exposure to flow, tests were similarly conducted. Based on these results, additional studies were conducted to compare the adhesion of rat aortic smooth muscle cells (SMC) and endothelial cells (EC) on PLL films.     

An estimate of the fraction of belugas (<Emphasis Type="Italic">Delphinapterus leucas</Emphasis>) in the Canadian high Arctic that winter in West Greenland

Five belugas, or white whales (Delphinapterus leucas), were tracked by satellite from Creswell Bay, Somerset Island, in the Canadian high Arctic towards West Greenland in autumn 2001. After 1 October, three of the whales stayed in the North Water polynya and the other two whales moved to West Greenland. One of the whales that moved to Greenland migrated south along the west coast, following a route and timing similar to another beluga tracked in 1996. The belugas that moved towards West Greenland from Canada did so before or near 1 October. The movements of both these whales followed a similar timing and assumed migratory route of belugas hunted in autumn in West Greenland. In Greenland, the hunt begins in September, where the first whales are taken in the northernmost community of Qaanaaq. Hunting takes place farther south in Upernavik in October, and finally in November and December, belugas are taken even farther south in Uummannaq and Disko Bay. The whales that remain in the North Water after 1 October most likely do not contribute to the harvest in West Greenland. Based on the total number of belugas satellite-tracked in Canada between 1995 and 2001 with tags that lasted beyond 1 October, approximately 0.15 (95% CI 0.06-0.35; n=26) of the summering stock of belugas in the Canadian high Arctic move to West Greenland for the winter. Genetic studies have indicated that belugas moving east through Lancaster Sound are significantly differentiated from belugas taken in the autumn hunt in West Greenland. These conflicting results suggest molecular genetics cannot be solely relied on to reveal the stock identity of these belugas.     

Determination of the execution points of mutations in the nuclear replication cycle of Aspergillus nidulans.

Cultures of nuclear replication cycle mutants of Aspergillus nidulans were transferred to the nonpermissive temperature, and the fraction of nuclei still able to reach mitosis was determined. For the determinations, benomyl [methyl-1(butylcarbomoyl)benzimidazolecarbamate] was added to trap nuclei in mitosis, and these were detected by staining with aceto-orcein. The assumptions and controls required to relate the experimentally determined fractions to the points where a mutation blocks the nuclear cycle are discussed. Nine genetically distinct mutants were tested. Two of these were blocked early in the cycle, two in the middle, and five close to, or during, mitosis.     

Polar tube proteins of microsporidia of the family encephalitozoonidae

Encephalitozoonidae are microsporidia associated with human infections including hepatitis, encephalitis, conjunctivitis, and disseminated disease. Microsporidia produce a small resistant spore containing a polar tube which serves as a unique vehicle of infection. Polar tube proteins (PTPs) from Encephalitozoon hellem. Encephalitozoon (Septata) intestinalis, and Encephalitozoon cuniculi were purified to homogeneity by HPLC. By SDS-PAGE, the Mr of E. hellem PTP was 55 kDa, while the Mr of E. intestinalis and E. cuniculi PTP was 45 kDa. Polyclonal rabbit antiserum to these purified PTPs localized to polar filaments by immunogold electron microscopy and immunofluorescence, and demonstrated cross-reactivity by both immunoblotting and immunogold electron microscopy. These PTPs have similar solubility properties, hydrophobicity, and proline content to a 43-kDa PTP we have previously purified from Glugea americanus, a fish microsporidium. As the polar tube is critical in the transmission of this organism, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.     

Advantage of using inosine at the 3' termini of 16S rRNA gene universal primers for the study of microbial diversity

To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3' termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54 degrees C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50 degrees C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3' termini in studying the microbial diversity of environmental samples.     

Survival of Animal Tissue Cells in Primary Culture in the Absence of Serum

The ability of cells from tissues of several species of animals to survive in primary culture without serum was tested. Of the species tested, cells from the kidneys of Macaca mulatta (rhesus) and Cercopithecus aethiops (vervet) monkeys and chicken embryo cells not only survived under these conditions, but indeed developed into confluent monolayer cultures. The addition of either serum or its globulin or albumin fraction enhanced the development of cell monolayers and permitted those cells unable to survive in the absence of serum to do so. Certain specific protein trypsin-inhibitors not of serum origin were unable to provide conditions necessary for cell survival or growth when used in place of serum proteins.     

PFA: Program for the Quantitative Assessment of Cell Metabolism by Spectral Data Analysis

Assessment of mitochondrial oxidative metabolism has wide-ranging importance, from pharmacokinetic analysis to studies in cell viability and apoptosis. Here we present the Perfusion File Analyzer (PFA) application for the real-time analysis of spectral data to measure cytochrome c reduction, cytochrome a3 reduction, and other parameters important to cellular metabolism, which are collected during tissue perfusion experiments. Our current efforts are focused on quantitating changes in mitochondrial function by normalizing baseline drift of spectral data while addressing two major challenges: (1) a lack of real-time feedback from the system when aiming is compromised, and (2) an inability to adjust calculated data in the event of spectral shift. PFA has been developed to address these issues, and is currently used for quality assessment of human islets prior to clinical transplantation.     

Gene silencing by S-adenosylmethionine in muscle differentiation.

A well-characterised experimental system, the myogenin gene in C2C12 muscle cell culture, was chosen to better understand the methylation mechanism underlying the regulation of gene expression. We already demonstrated that demethylation dynamics of a specific CpG site in the 5'-flanking region of myogenin well correlates with gene expression and terminal differentiation. Here we demonstrate that S-adenosylmethionine-sulphate-p-toluenesulphonate (SAM) inhibits myogenin expression and myoblast differentiation by delaying the demethylation of specific CpG in differentiating myoblasts. These results suggest new perspectives in methylation mechanisms and the use of SAM in the partial silencing of gene expression, as it could be required in disease treatment.     

Phenotypic and Genetic Analysis of Clock, a New Circadian Rhythm Mutant in Drosophila Melanogaster

Clock is a semidominant X-linked mutation that results in shortening the period of Drosophila melanogaster's free-running locomotor activity rhythm from ca. 24.0 to ca. 22.5 hr. This mutation similarly shortened the phase response curve, determined by resetting activity rhythms with light pulses. Eclosion peaks for Clk cultures were separated by only 22.5 hr instead of the normal 24 hr. Clk was mapped close to, but separable from, another rhythm mutation--period01--by recombination. The estimated distance between these two mutations was short enough to suggest that Clk could be a per allele. If this is the case, the new mutant is unique in that it, unlike other per variants, is associated with essentially normal 1-min courtship song rhythms when Clk is expressed in males. Also, the new rhythm variant could not, in contrast to a short-period per mutation, have its effects on free-running activity rhythms uncovered by deletions. This result, and the lack of coverage of Clk's effects by duplications, suggest that it is not a simple hypomorphic or amorphic mutation.