Non-Hertzian approach to analyzing mechanical properties of endothelial cells probed by atomic force microscopy

Detailed measurements of cell material properties are required for understanding how cells respond to their mechanical environment. Atomic force microscopy (AFM) is an increasingly popular measurement technique that uniquely combines subcellular mechanical testing with high-resolution imaging. However, the standard method of analyzing AFM indentation data is based on a simplified "Hertz" theory that requires unrealistic assumptions about cell indentation experiments. The objective of this study was to utilize an alternative "pointwise modulus" approach, that relaxes several of these assumptions, to examine subcellular mechanics of cultured human aortic endothelial cells (HAECs). Data from indentations in 2- to 5-microm square regions of cytoplasm reveal at least two mechanically distinct populations of cellular material. Indentations colocalized with prominent linear structures in AFM images exhibited depth-dependent variation of the apparent pointwise elastic modulus that was not observed at adjacent locations devoid of such structures. The average pointwise modulus at an arbitrary indentation depth of 200 nm was 5.6+/-3.5 kPa and 1.5+/-0.76 kPa (mean+/-SD, n=7) for these two material populations, respectively. The linear structures in AFM images were identified by fluorescence microscopy as bundles of f-actin, or stress fibers. After treatment with 4 microM cytochalasin B, HAECs behaved like a homogeneous linear elastic material with an apparent modulus of 0.89+/-0.46 kPa. These findings reveal complex mechanical behavior specifically associated with actin stress fibers that is not accurately described using the standard Hertz analysis, and may impact how HAECs interact with their mechanical environment.     

The gene expression of adrenomedullin, calcitonin-receptor-like receptor and receptor activity modifying proteins (RAMPs) in CCl4-induced rat liver cirrhosis

This study was undertaken to determine AM expression in carbon tetrachloride (CCl4)-induced liver cirrhosis developed with peritoneal ascites. Sprague-Dawley rats received subcutaneous injections of CCl4 twice weekly in olive oil (1:1, 0.3 ml per kg body weight) for 6 or 12 weeks until ascites developed, or saline in olive oil as control. At 6 weeks, fibrosis developed and at 12 weeks cirrhosis developed with ascites formation. At both 6 and 12 weeks, increases in plasma renin and AM were evident, as was the gene expression of AM. At 12 weeks after CCl4 injection, the gene expression of calcitonin-like-receptor (CRLR) and receptor activity modifying proteins (RAMP1, RAMP2 and RAMP3) were all elevated when compared to the control. The results suggest that liver cirrhosis increases mRNA expressions of AM, CRLR and RAMP1, RAMP2 and RAMP3 and that the increase in AM gene expression precedes the development of cirrhosis. The increase in AM synthesis as reflected by an increase in AM gene expression, together with a lack of increase in AM peptide at both 6 and 12 weeks may suggest an elevation of AM release. Given the potent vasodilatory action of AM, the increase in the synthesis and release of AM in the cirrhotic liver may also contribute to peripheral vasodilatation in liver cirrhosis.     

Hybridization monitor: a method for identifying differences between complex genomes

We have developed a method to identify and amplify differential fragments between two complex genomes. This technique, named hybridization-monitored genome differential analysis (HMDA), incorporates a monitor system into a PCR-based solid subtraction hybridization that tracks the entire hybridization process. This is achieved by monitoring the subtraction progress using PCR analysis of the conserved sequence of 18S rDNA in the tester sample after each round of subtraction. Homologous fragments can then be eliminated when bound to the driver DNA immobilized on a solid membrane. The hybridization continues until the conserved DNA sequence of 18S rDNA can no longer be detected, and most of the unbound DNA fragments left in the liquid were mainly the tester-specific fragments, thus greatly decreasing the complexity of DNA template of PCR amplification, increasing the amplification efficiency of differences accordingly, and ensuring high positive efficiency and coverage across the tester genome. We have applied the technique in a comparison between the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe, which are two completely sequenced organisms. Results indicated that 95% of the subtracted clones have been confirmed to be different to the driver analyzed using the BLASTN homology alignment. With this technique, 240-fold enrichment of differences is obtained, and the coverage of the difference is up to 79%. These results indicate that HMDA can efficiently identify sequences that differ between two complex genomes.     

Regional and global concerns over wetlands and water quality

Water quality in many stream catchments and river basins is severely impacted by nutrient enrichment as a result of agriculture. Water-resource managers worldwide are considering the potential role of riparian zones and floodplain wetlands in improving stream-water quality, as there is evidence at the site scale that such wetlands are efficient at removing nutrients from through-flowing water. However, recent studies have highlighted disadvantages of such use of wetlands, including emissions of greenhouse gases and losses of biodiversity that result from prolonged nutrient loading. Here, we discuss the water purification function of wetlands at the site and catchment scale and suggest ways in which these disadvantages could be overcome.     

Expression of SK3-type dehydrin in transporting organs is associated with cold acclimation in Solanum species

The expression of a gene, encoding a dehydrin protein designated as DHN24 was analyzed at the protein level in two groups of Solanum species differing in cold acclimation ability. The DHN24 protein displays consensus amino acid sequences of dehydrins, termed K- and S-segments. The S-segment precedes three K-segments, classifying the protein into SK₃-type dehydrins. A group of Solanum species able to cold acclimation constituted by S. sogarandinum and S. tuberosum, cv. Aster, and a second one composed of a S. sogarandinum line, that lost ability to cold acclimation, and of S. tuberosum, cv. Irga, displaying low ability to cold acclimation were studied. Under control conditions, noticeable levels of the DHN24 protein was observed in stems, tubers, and roots of Solanum species. No protein was detected in leaves. During low temperature treatment the DHN24 protein level substantially increased in tubers, in transporting organs and in apical parts, and only a small increase was observed in leaves. The increase in protein abundance was only observed in the plants able to cold acclimate and was found to parallel the acclimation capacity. Upon drought stress, the DHN24 level decreased in stems and in leaves, but increased in apical parts. These results suggest that Dhn24 expression is regulated by organ specific factors in the absence of stress and by factors related to cold acclimation processes during low temperature treatment in collaboration with organ-specific factors. A putative function of the SK₃-type dehydrin proteins during plant growth and in the tolerance to low temperature is discussed.     

Physical mapping of the <Emphasis Type="Italic">Rf</Emphasis><Subscript><Emphasis Type="Italic">1</Emphasis></Subscript> fertility-restoring gene to a 100 kb region in cotton

Cytoplasmic male sterility (CMS) plays an important role in crop heterosis exploitation. Determining one or more nuclear genes that can restore male fertility to CMS is essential for developing hybrid cultivars. Genetic and physical mapping is the standard technique required for isolating these restoration genes. By screening 2,250 simple sequence repeat (SSR) primer pairs in cotton (Gossypium hirsutum L.), we identified five new SSR markers that are closely linked to the Rf ₁ gene, a fertility restorer gene of cotton for CMS-D2. Based on our previous fine mapping of the Rf ₁ gene and assemblage of three published STS markers, we constructed a high-resolution genetic map of Rf ₁ containing 13 markers in a genetic distance of 0.9 cM. The 13 molecular markers were used to screen a bacterial artificial chromosome (BAC) library from a restorer line 0-613-2R containing Rf ₁ gene, which yielded 50 single positive clones. There was an average of 3.8 clones ranging from 1 to 12 BAC clones per PCR marker. These 50 clones produced an average insert size of 120 kb (ranging between 80 and 225 kb). Thirty-five primer pairs were designed based on 38 sequences of BAC ends, and two new STS markers tightly linked to Rf ₁ gene have been tagged and integrated into this map. The physical map for the Rf ₁ gene was constructed by fingerprinting the positive clones digested with the HindIII enzyme. We were able to delimit the possible location of the Rf ₁ gene to a minimum of two BAC clones spanning an interval of approximately 100 kb between two clones designated 081-05K and 052-01N. Further work using these two BAC clones will lead to isolation of the Rf ₁ gene in cotton.     

Morphology and Phase Behavior of Two-Component Lipid Membranes

The stability and shapes of domains with different bending rigidities in lipid membranes are investigated. These domains can be formed from the inclusion of an impurity in a lipid membrane or from the phase separation within the membrane. We show that, for weak line tensions, surface tensions and finite spontaneous curvatures, an equilibrium phase of protruding circular domains or striped domains may be obtained. We also predict a possible phase transition between the investigated morphologies.