Cell division and cell enlargement in isolated Cucurbita cotyledons grown in darkness and in light

The spatial and temporal patterns of post-embryonal cell growth and cell division were characterised in excised cotyledons of vegetable marrow (Cucurbita pepo L. var. giromontia Alef.) incubated in water. The concurrent roles of these two processes in cotyledon growth were determined using paradermal sections of the first palisade layer of developing cotyledons. Tissue specificity was observed in the pattern of cell division. The daughter cells derived from an initial cell, which had already differentiated before imbibition of the seeds, were tightly packed in a cluster, which enabled us to monitor cell division during early cotyledon development. Heterogeneity of cell size was recognised during the process of cell proliferation in the cluster, suggesting that cell division is uncoupled from control of cell size. There was significantly more cell division in the marginal part of the cotyledons than in other parts, suggesting high activity of the marginal meristem. Light enhanced cell and cotyledon enlargement, but had no effect on the number of divisions. This study elucidated the cellular basis of post-germinative Cucurbita cotyledon morphogenesis and development. Electronic Publication     

Anterior repression of a Drosophila stripe enhancer requires three position-specific mechanisms

The striped expression pattern of the pair-rule gene even skipped (eve) is established by five stripe-specific enhancers, each of which responds in a unique way to gradients of positional information in the early Drosophila embryo. The enhancer for eve stripe 2 (eve 2) is directly activated by the morphogens Bicoid (Bcd) and Hunchback (Hb). As these proteins are distributed throughout the anterior half of the embryo, formation of a single stripe requires that enhancer activation is prevented in all nuclei anterior to the stripe 2 position. The gap gene giant (gt) is involved in a repression mechanism that sets the anterior stripe border, but genetic removal of gt (or deletion of Gt-binding sites) causes stripe expansion only in the anterior subregion that lies adjacent to the stripe border. We identify a well-conserved sequence repeat, (GTTT)(4), which is required for repression in a more anterior subregion. This site is bound specifically by Sloppy-paired 1 (Slp1), which is expressed in a gap gene-like anterior domain. Ectopic Slp1 activity is sufficient for repression of stripe 2 of the endogenous eve gene, but is not required, suggesting that it is redundant with other anterior factors. Further genetic analysis suggests that the (GTTT)(4)-mediated mechanism is independent of the Gt-mediated mechanism that sets the anterior stripe border, and suggests that a third mechanism, downregulation of Bcd activity by Torso, prevents activation near the anterior tip. Thus, three distinct mechanisms are required for anterior repression of a single eve enhancer, each in a specific position. Ectopic Slp1 also represses eve stripes 1 and 3 to varying degrees, and the eve 1 and eve 3+7 enhancers each contain GTTT repeats similar to the site in the eve 2 enhancer. These results suggest a common mechanism for preventing anterior activation of three different eve enhancers.     

Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerases beta and eta

DNA polymerases beta (pol beta ) and eta (pol eta ) are the only two eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Frameshift errors are an important aspect of mutagenesis. We have compared the types of frameshifts that occur during translesion synthesis past cisplatin and oxaliplatin adducts in vitro by pol beta and pol eta on a template containing multiple runs of nucleotides flanking a single platinum-GG adduct. Translesion synthesis past platinum adducts by pol beta resulted in approximately 50% replication products containing single-base deletions. For both adducts the majority of -1 frameshifts occurred in a TTT sequence 3-5 bp upstream of the DNA lesion. For pol eta, all of the bypass products for both cisplatin and oxaliplatin adducts contained -1 frameshifts in the upstream TTT sequence and most of the products of replication on oxaliplatin-damaged templates had multiple replication errors, both frameshifts and misinsertions. In addition, on platinated templates both polymerases generated replication products 4-8 bp shorter than the full-length products. The majority of short cisplatin-induced products contained an internal deletion which included the adduct. In contrast, the majority of oxaliplatin-induced short products contained a 3' terminal deletion. The implications of these in vitro results for in vivo mutagenesis are discussed.     

Fluorescence data analysis on gel-based biochips

A series of biochip readers developed for gel-based biochips includes three imaging models and a novel nonimaging biochip scanner. The imaging readers, ranging from a research-grade versatile reader to a simple portable one, use wide-field objectives and 12-bit digital large-coupled device cameras for parallel addressing of multiple array elements. This feature is valuable for monitoring the kinetics of sample interaction with immobilized probes. Depending on the model and the label used, the sensitivity of these readers approaches 0.3 amol of a labeled sample per gel element. In the selective scanner, both the spot size of the excitation laser beam and the detector field of view match the size of the biochip array elements so that the whole row of the array can be read in a single scan. The portable version reads 50-mm long, 150-element, one-dimensional arrays in 5 s. With a dynamic range of 4000:1, a sensitivity of 1-5 amol of a labeled sample per gel element, and a data format facilitating online processing, the scanner is an attractive, inexpensive solution for biomedical diagnostics. Fluorophores for sample labeling were compared experimentally in terms of detection sensitivity, influence on duplex stability, and suitability for multilabel analysis and thermodynamic studies. Texas Red and tetracarboxyphenylporphyn proved to be the best choice for two-wavelength analysis using the imaging readers.     

Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota

Human DNA polymerase ι (polι) is a Y-family polymerase whose cellular function is presently unknown. Here, we report on the ability of polι to bypass various stereoisomers of benzo[a]pyrene (BaP) diol epoxide (DE) and benzo[c]phenanthrene (BcPh) DE adducts at deoxyadenosine (dA) or deoxyguanosine (dG) bases in four different template sequence contexts in vitro. We find that the BaP DE dG adducts pose a strong block to polι-dependent replication and result in a high frequency of base misincorporations. In contrast, misincorporations opposite BaP DE and BcPh DE dA adducts generally occurred with a frequency ranging between 2 × 10^–3 and 6 × 10^–4. Although dTMP was inserted efficiently opposite all dA adducts, further extension was relatively poor, with one exception (a cis opened adduct derived from BcPh DE) where up to 58% extension past the lesion was observed. Interestingly, another human Y-family polymerase, polκ, was able to extend dTMP inserted opposite a BaP DE dA adduct. We suggest that polι might therefore participate in the error-free bypass of DE-adducted dA in vivo by predominantly incorporating dTMP opposite the damaged base. In many cases, elongation would, however, require the participation of another polymerase more specialized in extension, such as polκ.     

Component and state separation in DMPC/DSPC lipid bilayers: a Monte Carlo simulation study

In this paper a two-state, two-component, Ising-type model is used to simulate the lateral distribution of the components and gel/fluid state acyl chains in dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) lipid bilayers. The same model has been successful in calculating the excess heat capacity curves, the fluorescence recovery after photobleaching (FRAP) threshold temperatures, the most frequent center-to-center distances between DSPC clusters, and the fractal dimensions of gel clusters (Sugar, I. P., T. E. Thompson, and R. L. Biltonen, 1999. Biophys. J. 76:2099-2110). Depending on the temperature and mole fraction the population of the cluster size is either homogeneous or inhomogeneous. In the inhomogeneous population the size of the largest cluster scales with the size of the system, while the rest of the clusters remain small with increasing system size. In a homogeneous population, however, every cluster remains small with increasing system size. For both compositional and fluid/gel state clusters, threshold temperatures-the so-called percolation threshold temperatures-are determined where change in the type of the population takes place. At a given mole fraction, the number of percolation threshold temperatures can be 0, 1, 2, or 3. By plotting these percolation threshold temperatures on the temperature/mole fraction plane, the diagrams of component and state separation of DMPC/DSPC bilayers are constructed. In agreement with the small-angle neutron scattering measurements, the component separation diagram shows nonrandom lateral distribution of the components not only in the gel-fluid mixed phase region, but also in the pure gel and pure fluid regions. A combined diagram of component and state separation is constructed to characterize the lateral distribution of lipid components and gel/fluid state acyl chains in DMPC/DSPC mixtures. While theoretical phase diagrams of two component mixtures can be constructed only in the case of first-order transitions, state and component separation diagrams can be constructed whether or not the system is involved in first-order transition. The effects of interchain interactions on the component and state separation diagrams are demonstrated on three different models. The influences of state and component separation on the in-plane and off-plane membrane reactions are discussed.     

Phylogeny of the endemic Baikalian Sergentia (Chironomidae,Diptera)

Fragments of two mitochondrial genes, cytochrome b (CytB) and Cytochrome c oxidase subunit I (COI) have been used as phylogenetic markers in Sergentia (Chironomidae, Diptera). The concatenated (1241 bp) sequences from both genes were used to infer the phylogenetic relationships among seven Sergentia species. Five of the species belong to the endemic fauna of Lake Baikal. Alignments of the nucleotide sequences were used for the construction of trees using Neighbor-Joining and maximum parsimony methods. Both methods yielded similar results. Monophyly of both Sergentia and the Baikalian endemic species was well supported. The date of origin of the endemic group of Sergentia was estimated as 25.7 MYA which closely coincides with the start of geological changes in the Baikal area. A cytological tree, based on 12 chromosomal characteristics, for the same set of Sergentia species showed a great similarity to the molecular phylogeny.     

Serial in vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes

New technologies are needed to characterize the migration, survival, and function of antigen-specific T cells in vivo. Here, we demonstrate that Epstein-Barr virus (EBV)--specific T cells transduced with vectors encoding herpes simplex virus-1 thymidine kinase (HSV-TK) selectively accumulate radiolabeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU). After adoptive transfer, HSV-TK+ T cells labeled in vitro or in vivo with [131I]FIAU or [124I]FIAU can be noninvasively tracked in SCID mice bearing human tumor xenografts by serial images obtained by scintigraphy or positron emission tomography (PET), respectively. These T cells selectively accumulate in EBV+ tumors expressing the T cells' restricting HLA allele but not in EBV- or HLA-mismatched tumors. The concentrations of transduced T cells detected in tumors and tissues are closely correlated with the concentrations of label retained at each site. Radiolabeled transduced T cells retain their capacity to eliminate targeted tumors selectively. This technique for imaging the migration of ex vivo-transduced antigen-specific T cells in vivo is informative, nontoxic, and potentially applicable to humans.     

Synthesis and physicochemical properties of 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl oligonucleotides

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.     

Biological activity of pentachlorophenol on the digestive gland cells of the freshwater mussel Unio tumidus

Many chlorinated phenols and their derivatives are used extensively as insecticides, fungicides and herbicides by industrial and agricultural users throughout the world. Among these substances, pentachlorophenol (PCP) is a broad-spectrum biocide, which is still used as a wood preservative. In this paper, the digestive gland cells were used to assess the effect of PCP in the range of concentrations 3.75-75 microM (0.01-0.2 ppm) on oxidative DNA damage, fluidity changes and peroxidation activity in the plasma membrane. The toxic property of PCP on DNA strand breakage was studied using the comet assay. The results showed that pentachlorophenol in the range of 37.5-75 microM contributed to these lesions. To demonstrate the changes in the fluidity of plasma membrane we used the spectrofluorimetric method using two fluorescence probes: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 12-(9-anthroyloxy) stearic acid (12-AS). It was shown that PC did not influence the surface of plasma membrane but contributed to the increase in the fluidity of the internal region of the lipid bilayer in the range of concentrations 18.75-75 microM (0.05-0.2 ppm). We also examined the effect of PCP on the lipid peroxidation. To imply its peroxidation properties the spectrophotometry method was used to measure the level of malondialdehyde (MDA), one of the endpoints of the peroxidation of polyunsaturated fatty acids. The obtained results showed that PCP in the used doses did not initiate the formation of lipid peroxides. Thus, our investigation indicates that PCP can behave as a prooxidant agent but its action depends on the used doses and parameters chosen for the research.