Resolution of the conformational distribution and dynamics of a flexible molecule using frequency-domain fluorometry

We report the first resolution of both the conformational distribution and end-to-end diffusion coefficient of a flexible molecule. This molecular information was recovered using only the donor intensity decay in a single solvent at a single viscosity, as observed by the technique of frequency-domain fluorometry. This technique can be extended to measurements of structural fluctuations of biological macromolecules.     

Unidirectional hybridization of kaluga <Emphasis Type="Italic">Acipenser dauricus</Emphasis> Georgi, 1775 and Amur sturgeon <Emphasis Type="Italic">Acipenser schrenckii</Emphasis> Brandt, 1869, inferred from the mitochondrial DNA genotyping of their natural hybrids

In 2009 through 2011, among 730 individuals of kaluga and Amur sturgeon collected in the lower reaches of the Amur River and the Amursky Liman, 17 morphologically intermediate individuals (hybrids) with the body length of 56 to 202 cm (median, 81 cm) were identified, including 11 individuals (4.6%) found in 2009, three individuals (1.6%) found in 2010, and three individuals (1.1%), in 2011. In 16 hybrids, 819 bp of the mtDNA control regions were sequences and 11 haplotypes were identified. Since all these haplotypes were from the mtDNA lineages of kaluga, it was concluded that hybridization occurred in one direction, kaluga (♀) × Amur sturgeon (♂). This asymmetry could be caused by the large size differences between these species. Since the earlier examined morphologically typical Amur sturgeons showed the absence of alien haplotypes (Shedko et al., 2015), the absence of the mtDNA introgression is claimed. This can be caused by low viability or sterility of the backcross females (kaluga (♀) × Amur sturgeon (♂)) × Amur sturgeon (♂). The samples of hybrids and typical kaluga individuals demonstrated no differences in the frequency spectra of the mtDNA haplotypes. However, haplotype and nucleotide diversity in the first sample was somewhat higher than in the second one (0.950 versus 0.927 and 0.0054 versus 0.0044, respectively). The data obtained will be useful for population monitoring of kaluga and Amur sturgeon, Amur River endemics, which are classified as critically endangered by the IUCN Red List of Threatened Species.     

A morphometric comparison of subcellular structures between primary breast tumors and their nodal metastases.

A morphometric study of subcellular structures in human primary breast tumors (of the ductal infiltrating type) and their synchronous lymph node metastases was carried out using planimetry and stereology. Each primary neoplasm was compared separately with its axillary metastatic growth in order to detect differences in the quantitative estimates of their subcellular components. A set of 24 morphometric parameters was used to quantitatively describe each neoplastic cell profile. Although some subcellular parameters showed significant differences between the cells of a primary tumor and its metastases, no parameter was found to be consistently altered in all cases. No consistent increase or decrease in the values of the affected parameters was observed in the metastases. More than being due to the expression of a biologically variant (more-or-less differentiated) metastatic phenotype, the differences in stereologic parameters detected in metastatic cells appear to reflect the existence of different functional states of the same cell type.     

Violation of the law of uniformity of the first generation of hybrids

Variability was revealed in the isozyme spectra in the seed progeny from crosses of two beet plants homozygous for contrasting alleles of marker enzyme loci.     

Selective inactivation of the Arg-Gly-Asp-Ser (RGDS) binding site in von Willebrand factor by site-directed mutagenesis.

In order to assess the requirement for the Arg-Gly-Asp-Ser (RGDS) consensus adhesion sequence in von Willebrand factor (vWF) for vWF binding to platelets and endothelial cells, point mutations were introduced into this sequence by site-directed mutagenesis. A glycine to alanine substitution yielded RADS-vWF, while an aspartate to glutamate substitution resulted in RGES-vWF. Recombinant RADS-vWF and RGES-vWF, purified from transformed Chinese hamster ovary cells, were compared with recombinant wild type vWF (WT-vWF) in functional assays with platelets and human umbilical vein endothelial cells (HU-VECs). High molecular weight RADS-vWF and RGES-vWF multimers inhibited binding of 125I-vWF to a mixture of insolubilized native type I and III collagen and competed effectively with 125I-vWF for binding to formalin-fixed platelets in the presence of ristocetin, indicating functional collagen and platelet glycoprotein Ib binding. However, RADS-vWF and RGES-vWF were unable to displace the binding of 125I-vWF to thrombin or ADP-activated platelets. The attachment of HUVECs to either RADS-vWF or RGES-vWF coated surfaces was reduced and spreading was almost completely inhibited, compared with WT-vWF. We conclude that point mutations of the RGDS sequence in vWF selectively impair binding to platelet glycoprotein IIb/IIIa and the HUVEC vitronectin receptor.     

Genetic diversity and molecular fingerprinting of Prunus cerasus var. austera from central Italy

In central Italy, Prunus cerasus var. austera is cultivated as small stands or scattered trees in marginal areas for the production of jam and wine. Thanks to the healthy attributes of its products and its ability to grow in different environmental conditions, this variety has gained new interest in the development of marginal areas. We assessed the level of the genetic variability of P. cerasus var. austera germplasm from central Italy and identified a ‘core collection’ representative of the present genetic diversity. A total of 161 trees, morphologically identified as var. austera, and one tree, identified as var. caproniana were collected and genotyped by 14 SSRs. Two individuals provided by a commercial plant nursery, one of P. cerasus var. caproniana and one of P. cerasus var. austera, were used as control. Thirteen SSRs presented private alleles in austera. Seven individuals morphologically identified as austera revealed private alleles specific to caproniana. The PCoA and Bayesian clustering analysis showed a main genetic group including var. austera, while a second group included all the caproniana-like genotypes. A core collection of 31 trees (46% of austera genotypes) was selected. This study can be considered as a starting point for future investigations on this variety.     

Formaldehyde fixation of cells does not greatly reduce the ability to amplify cellular DNA

Formaldehyde fixation of cells is routinely used to study DNA-protein interactions in vivo. In these studies, DNA is often analyzed using a polymerase chain reaction technique. Although it is known that formaldehyde can damage DNA, no studies have been performed so far to compare the efficiency of DNA amplification between normal and fixed cells. Here we show that formaldehyde fixation results in a 15% to 20% reduction in the ability to amplify cellular DNA. The loss of amplifiability is independent of the length of the amplification region and the degree to which DNA is compacted on packaging into chromatin.