Analysis of ribosomal DNA restriction patterns in the genusKluyveromyces

Riboprinting was used to determine the relationships among strains belonging to 15 species of the genusKluyveromyces. The small subunit ribosomal RNA gene (SSU rDNA) was amplified using the Polymerase Chain Reaction (PCR) and subjected to a battery of nine restriction enzymes. Similarity coefficients between strains were calculated based on shared and unique restriction fragments. Cluster analysis revealed three major groups that generally correlated with previously reported relationships based on other molecular data. Variations in SSU rDNA restriction fragments may be used for differentiation of theKluyveromyces strains included in this study.The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.     

An interference-free fluorescent assay of telomerase for the high-throughput analysis of inhibitors

We have developed a telomerase assay that can quickly and accurately rank the ability of molecules to inhibit telomerase activity. It is based on the method of Orlando and co-workers which utilizes PicoGreen to detect dsDNA formed during the polymerase chain reaction (PCR) amplification of telomerase products. PCR cycles were optimized to give as linear a signal as possible relative to telomerase products; 96-well streptavidin-coated PCR plates were used to isolate the preamplification telomerase products and to wash inhibitors away before the amplification step. The inhibitor removal step is critical to prevent false positives potentially caused by inhibition of Taq polymerase during amplification. Use of the streptavidin-coated PCR plate allows this step to be done much more rapidly than use of the liquid/liquid extraction adopted by others. We have demonstrated that this assay can correctly order the ability of four inhibitors to inhibit telomerase and reproduce within a factor of two the absolute IC(50) values determined by the more time-consuming direct assay. We have shown that the difference in IC(50) values determined in this assay versus the direct assay can be corrected for by using the standard curve appropriately. Using this method 96 compounds can be assessed in 3-5h.     

What's hot in animal biosafety?

In recent years, the emergence or re-emergence of critical issues in infectious disease and public health has presented new challenges and opportunities for laboratory animal care professionals. The re-emergence of bioterrorism as a threat activity of individuals or small groups has caused a heightened awareness of biosecurity and improved biosafety. The need for animal work involving high-risk or high-consequence pathogens and for arthropod-borne diseases has stimulated renewed interest in animal biosafety matters, particularly for work in containment. Application of these principles to animals retained in outdoor environments has been a consequence of disease eradication programs. The anticipated global eradication of wild poliovirus has prompted the promulgation of new biosafety guidelines for future laboratory and animal work. Increased concern regarding the use of biologically derived toxins and hazardous chemicals has stimulated a new categorization of facility containment based on risk assessment. Recognition that prion disease agents and other high-consequence pathogens require safe handling and thorough destruction during terminal decontamination treatment has led to the development of new biosafety guidelines and technologies. The implementation of these guidelines and technologies will promote state-of-the-art research while minimizing risk to laboratory animals, researchers, and the environment.     

An anatomical model to determine step height in step testing for estimating aerobic capacity

Physical fitness has been reported to be inversely related to coronary heart disease and other health related problems. One of the most valid means of assessing physical fitness is the test of aerobic capacity. Aerobic capacity is the greatest rate at which the body can consume oxygen and represents the most efficient integration of the various physiological processes which make up the oxygen transport system. However, direct measurement of aerobic capacity requires sophisticated laboratory equipment, and is adversive to subjects. Step tests are widely used to estimate aerobic capacity. Because the biomechanical efficiency and work rate is determined by step height, accommodation of step height to the subject's statute height should provide a better estimation of aerobic capacity. A hip angle of 73.3 degrees, when stepping, was found to give the best relationship of recovery heart rate of a step test to direct measurement of aerobic capacity. Using 73.3 degrees, the following equations were developed for determining the stepping height when using the step test: Hf = 0.189 Ih and Hf = 0.192 Ih for females and males respectively, where hf is the step height and Ih is the statute height of the subject. A correlation coefficient (r) of 0.93 was calculated between various hip angles and calculated foot height of 182 observations of 47 females while a correlation coefficient (r) of 0.96 was calculated from 208 observations of 53 males. Using these equations to determine step height, measurement of 30 females showed a mean hip angle of 73.3 degrees +/- 2.2 and measurement of 30 males showed a mean hip angle of 73.3 degrees +/- 2.1.(ABSTRACT TRUNCATED AT 250 WORDS)