Isolation of High Quality DNA and RNA from Leaves of the Carnivorous Plant Drosera rotundifolia

Drosera rotundifolia belongs to the family of the sundews, a large group of carnivorous plants that carry stalked glands on the upper leaf surface to attract, trap and digest insects for food. Therefore, such plants can live in relatively poor ecosystems. They are frequently used as medicinal herbs and have various other interesting characteristics associated with them. In attempts to evaluate the gene pool of these plants, we experienced that many published protocols for nucleic acid isolation failed to yield DNA and RNA of sufficient quality for analysis. Therefore, we have developed CTAB (hexadecyltrimethylammoniumbromide)-based extraction protocols for the routine isolation of high-quality DNA and RNA from small amounts of in vitro-grown Drosera rotundifolia leaves. The methods developed are simple, fast and effective. The obtained DNA could be analyzed by PCR, restriction endonucleases and DNA gel blotting, and the obtained RNA was of sufficient quality for RT-PCR and RNA gel blotting.     

Role of yeast Rad5 and its human orthologs,HLTF and SHPRH in DNA damage tolerance

In the yeast Saccharomyces cerevisiae, the Rad6–Rad18 DNA damage tolerance pathway constitutes a major defense system against replication fork blocking DNA lesions. The Rad6–Rad18 ubiquitin-conjugating/ligase complex governs error-free and error-prone translesion synthesis by specialized DNA polymerases, as well as an error-free Rad5-dependent postreplicative repair pathway. For facilitating replication through DNA lesions, translesion synthesis polymerases copy directly from the damaged template, while the Rad5-dependent damage tolerance pathway obtains information from the newly synthesized strand of the undamaged sister duplex. Although genetic data demonstrate the importance of the Rad5-dependent pathway in tolerating DNA damages, there has been little understanding of its mechanism. Also, the conservation of the yeast Rad5-dependent pathway in higher order eukaryotic cells remained uncertain for a long time. Here we summarize findings published in recent years regarding the role of Rad5 in promoting error-free replication of damaged DNA, and we also discuss results obtained with its human orthologs, HLTF and SHPRH.     

Fibronectin matrix assembly suppresses dispersal of glioblastoma cells

Glioblastoma (GBM), the most aggressive and most common form of primary brain tumor, has a median survival of 12-15 months. Surgical excision, radiation and chemotherapy are rarely curative since tumor cells broadly disperse within the brain. Preventing dispersal could be of therapeutic benefit. Previous studies have reported that increased cell-cell cohesion can markedly reduce invasion by discouraging cell detachment from the tumor mass. We have previously reported that α5β1 integrin-fibronectin interaction is a powerful mediator of indirect cell-cell cohesion and that the process of fibronectin matrix assembly (FNMA) is crucial to establishing strong bonds between cells in 3D tumor-like spheroids. Here, we explore a potential role for FNMA in preventing dispersal of GBM cells from a tumor-like mass. Using a series of GBM-derived cell lines we developed an in vitro assay to measure the dispersal velocity of aggregates on a solid substrate. Despite their similar pathologic grade, aggregates from these lines spread at markedly different rates. Spreading velocity is inversely proportional to capacity for FNMA and restoring FNMA in GBM cells markedly reduces spreading velocity by keeping cells more connected. Blocking FNMA using the 70 KDa fibronectin fragment in FNMA-restored cells rescues spreading velocity, establishing a functional role for FNMA in mediating dispersal. Collectively, the data support a functional causation between restoration of FNMA and decreased dispersal velocity. This is a first demonstration that FNMA can play a suppressive role in GBM dispersal.     

γδ T cell homing to skin and migration to skin-draining lymph nodes is CCR7 independent

In most species, γδ T cells preferentially reside in epithelial tissues like the skin. Lymph duct cannulation experiments in cattle revealed that bovine dermal γδ T cells are able to migrate from the skin to the draining lymph nodes via the afferent lymph. For αβ T cells, it is generally accepted that epithelial and mucosal tissue egress is regulated by expression of the CCR7 chemokine receptor. In this study, we tracked the migratory route of bovine lymph-derived γδ T cells and examined their CCR7 cell surface expression in several compartments along this route. Total lymph cells from afferent and efferent origin were labeled with PKH fluorescent dyes and injected into the bloodstream. PKH(+) cells already reappeared in the afferent lymph after 4 h. The vast majority of the PKH(+) cells retrieved from the afferent lymph were of the WC1(+) γδ T cell phenotype, proving that this PKH(+) γδ T cell subset is able to home to and subsequently exit the skin. PKH(+) γδ T cells from afferent and efferent lymph lack CCR7 surface expression and display high levels of CD62L compared with CD4 T cells, which do express CCR7. Skin homing receptors CCR4 and CCR10 in contrast were transcribed by both CD4 and γδ T cells. Our findings suggest that γδ T cell skin egress and migration into the peripheral lymphatics is CCR7-independent and possibly mediated by CD62L expression.     

Dihydropyridine neuropeptide Y Y(1) receptor antagonists

Dihydropyridine 5a was found to be an inhibitor of neuropeptide Y(1) binding in a high throughput (125)I-PYY screening assay. Structure-activity studies around certain portions of the dihydropyridine chemotype identified BMS-193885 (6e) as a potent and selective Y(1) receptor antagonist. In a forskolin-stimulated c-AMP production assay using CHO cells expressing the human Y(1) receptor, 6e demonstrated full functional antagonism (K(b)=4.5 nM). Compound 6e inhibited NPY-induced feeding in satiated rats when dosed at 3.0 and 10.0 mg/kg (ip), and also decreased spontaneous overnight food consumption in rats at doses of 10 and 20 mg/kg (ip).     

Differentially functionalized diamines as novel ligands for the NPY2 receptor

The synthesis of novel ligands for the NPY(2) receptor using solid phase split pool methodology is described. One of the analogues, diamine 16, was found to be a potent NPY(2) binder.     

An apolipoprotein E-derived peptide mediates uptake of sterically stabilized liposomes into brain capillary endothelial cells

A promising strategy to solve the problems of insufficient membrane penetration of drugs and low target specificity is the localization of targeting and uptake-facilitating ligands on the surface of drug-carrier systems. This study investigated the role of a peptide derived from the LDL receptor (LDLr)-binding domain of apolipoprotein E (apoE) in initiating endocytosis in brain capillary endothelial cells. The highly cationic tandem dimer of apoE residues (141-150) was coupled covalently onto poly(ethylene glycol)-derivatized liposomes. Membrane binding and cellular uptake was monitored qualitatively by confocal-laser-scanning microscopy as well as quantitatively using a fluorescence assay. The peptide mediated an efficient, energy-dependent translocation of liposomes across the membrane of brain capillary endothelial cells. Liposomes without surface-located peptides displayed neither membrane accumulation nor cellular uptake. Low peptide affinity to LDLr and internalization of the complex into fibroblasts with up- and down-regulated receptor expression levels, as well as complex translocation into cells incubated with an antibody against the LDLr, pointed to a dominating role of an LDLr-independent transport route. Enzymatic digestion of heparan sulfate proteoglycan (HSPG) with heparinase I and addition of heparin and poly-l-lysin as competitors of HSPG and HSPG ligands, respectively, resulted in a significant loss in liposome internalization. The results suggested that HSPG played a major role in the apoE-peptide-mediated uptake of liposomes into endothelial cells of brain microvessels.     

Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis, showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use.     

Introgression of an intermediate <Emphasis Type="Italic">VRNH1</Emphasis> allele in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) leads to reduced vernalization requirement without affecting freezing tolerance

The process of vernalization is mainly controlled by two genes in winter barley (Hordeum vulgare L.), VRNH1 and VRNH2. A recessive allele at VRNH1 and a dominant allele at VRNH2 must be present to induce a vernalization requirement. In addition, this process is usually associated with greater low-temperature tolerance. Spanish barleys originated in areas with mild winters and display a reduced vernalization requirement compared with standard winter cultivars. The objective of this study was to investigate the genetic origin of this reduced vernalization requirement and its effect on frost tolerance. We introgressed the regions of a typical Spanish barley line that carry VRNH1 and VRNH2 into a winter cultivar, Plaisant, using marker-assisted backcrossing. We present the results of a set of 12 lines introgressed with all four possible combinations of VRNH1 and VRNH2, which were evaluated for vernalization requirement and frost tolerance. The reduced vernalization requirement of the Spanish parent was confirmed, and was found to be due completely to the effect of the VRNH1 region. The backcross lines showed no decline in frost tolerance compared with that of the recurrent parent unless they carried an extra segment of chromosome 5H. This extra segment, a carryover of the backcross process, apparently contained the well-known frost tolerance quantitative trait locus Fr-H2. We demonstrate that it is possible to manipulate the vernalization requirement with only minor effects on frost tolerance. This finding opens the path to creating new types of barley cultivars that are better suited to specific environments, especially in a climate-change scenario.