Membrane chemical stability and seed longevity

Here, we investigate the relationships between the chemical stability of the membrane surface and seed longevity. Dry embryos of long-lived tomato and short-lived onion seeds were labeled with 5-doxyl-stearic acid (5-DS). Temperature-induced loss of the electron spin resonance signal caused by chemical conversion of 5-DS to nonparamagnetic species was used to characterize the membrane surface chemical stability. No difference was found between temperature plots of 5-DS signal intensity in dry onion and tomato below 345 K. Above this temperature, the 5-DS signal remained unchanged in tomato embryos and irreversibly disappeared in onion seeds. The role of the physical state and chemical status of the membrane environment in the chemical stability of membrane surfaces was estimated for model systems containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) dried alone or in the presence of trehalose or glucose. Fourier transform infrared spectroscopy was used to follow temperature-induced structural changes in dry POPC. Spin-label technique was used to relate the chemical stability of 5-DS with the dynamic properties of the bilayer and 5-DS motion behavior. In all the models, the decrease in 5-DS signal intensity was always observed above T _m for the membrane surface. The 5-DS signal was irreversibly lost at high temperature when dry POPC was embedded in a glucose matrix. The loss of 5-DS signal was moderate when POPC was dried alone or in the presence of trehalose. Comparison of model and in vivo data shows that the differences in longevity between onion and tomato seeds are caused by differences in the chemical status of the membrane surface rather than the degree of its immobilization.     

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133⁺ cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.     

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

Angiotensin II (AngII) and its type receptor (AT1-R) play important roles in the development of cardiac hypertrophy. Low level of high density lipoprotein (HDL) is also an independent risk factor for cardiac hypertrophy. We therefore investigated in the present study whether HDL inhibits cardiac hypertrophy relatively to inhibition of AngII and AT1-R in both in vitro and in vivo experiments. Stimulation of cultured cardiomyocytes of neonatal rats with AngII for 24 h and infusion of AngII in mice for 2 weeks resulted in marked cardiac hypertrophic responses including increased protein synthesis, enlarged sizes of cardiomyocytes and hearts, upregulated phosphorylation levels of protein kinases and reprogrammed expression of specific genes, all of which were significantly attenuated by the treatment with HDL. Furthermore, AngII-treatment induced upregulation of AT-R expression either in cultured cardiomyocytes or in hearts of mice and HDL significantly suppressed the upregulation of AT1-R. Our results suggest that HDL may abrogate AngII-induced cardiac hypertrophy through downregulation of AT1-R expression.     

Isolation and characterization of eleven polymorphic microsatellite loci in Aegiphila sellowiana and their transferability

We isolated and characterized eleven polymorphic microsatellite loci for Aegiphila sellowiana an outcrossing pioneer tree species that is frequently used in reforestation programs of tropical riparian forests in Brazil. A total of 38 alleles were detected across a sample of 45 individuals of A. sellowiana, with an average number of 3.45 alleles per locus. The average polymorphic information content (PIC) was 0.430 and the observed (H_O) and expected (H_E) heterozygosity values varied from 0.156 to 1.000 and 0.145 to 0.730, respectively. Eight loci exhibited significant deviation from Hardy-Weinberg equilibrium (P ≤ 0.001) and 32 pair combinations of loci showed significant linkage disequilibrium (P ≤ 0.001). All 11 primers were tested for cross amplification in 12 species belonging to the family Lamiaceae and 5 species belonging to the related family Verbenaceae. The sequence and diversity information obtained using these microsatellites and their cross-transferability to other species of Lamiaceae as well as Verbenaceae will increase our understanding of genetic structures and species relationships within Aegiphyla and other genera of these families.     

Human papillomavirus infection in women with and without cervical cancer in Tbilisi, Georgia

Background: No accurate estimates of cervical cancer incidence or mortality currently exist in Georgia. Nor are there any data on the population-based prevalence of high-risk (HR) human papillomavirus (HPV) infection, which, in the absence of good-quality screening, is known to correlate with cervical cancer incidence. Methods: We obtained cervical cell specimens from 1309 women aged 18–59 years from the general population of Tbilisi, and also from 91 locally diagnosed invasive cervical cancers (ICC). DNA of 44 HPV types was tested for using a GP5+/6+-based PCR assay. Results: In the general population (of whom 2% reported a previous Pap smear) HPV prevalence was 13.5% (95% CI: 11.6–15.9), being highest in women aged 25–34 years (18.7%) and falling to between 8.6% and 9.5% for all age groups above 34 years. HR HPV prevalence was 8.6% overall, being 6.8% and 38.9% among women with normal and abnormal cytology, respectively. HPV45 (1.6%) was the most common type in women with normal cytology, whereas HPV16 predominated among women with cervical abnormalities (including 7 of 10 histologically confirmed cervical intraepithelial neoplasia 2/3) and among ICC (57.6%). The next most common types in ICC in Georgia were HPV45 and 18 (13.2 and 11.0%, respectively). Conclusions: We report a relatively high burden of HPV infection in Tbilisi, Georgia. Improving cervical cancer prevention, through screening and/or HPV vaccination, is an important public health issue in Georgia, where 70% of ICC are theoretically preventable by HPV16/18 vaccines.     

Karyotype and AFLP data reveal the phylogenetic position of the Brazilian endemic Hypochaeris catharinensis (Asteraceae)

The genus Hypochaeris offers an excellent model for studies of recent adaptive radiation in the South American continent. We used karyotype analysis with chromomycin?A₃ (CMA₃)/4??,6-diamidino-2-phenylindole (DAPI) banding and fluorescence in?situ hybridization (FISH), and amplified fragment length polymorphism (AFLP) fingerprinting to investigate for the first time the Brazilian endemic H.?catharinensis and define its position within the South American group of species. Strong CMA-positive signals were seen at the end of both arms of chromosome?3 and at the end of the long arm of chromosome?4. DAPI bands were only detected in subterminal position on short arm of chromosome?4. FISH with 5S and 35S ribosomal DNA (rDNA) probes revealed a single 5S rDNA locus on short arm of chromosome?2, typical for all other South American Hypochaeris taxa analyzed to date. The 35S rDNA locus was identified at subterminal position on the short arm of chromosome?3, as reported so far for only two of the known species (H.?lutea and H.?patagonica). The AFLP study included 55 individuals, comprising nine species of the South American Hypochaeris plus their putative ancestor H.?angustifolia. Eleven AFLP primer combinations generated a total of 401 fragments, of which 388 (96.7%) were polymorphic. High genetic similarities were observed among taxa, with all South American Hypochaeris species falling into one main cluster [100% bootstrap (BS)]. Hypochaeris catharinensis is closely related to H.?lutea (82% BS), forming a well-separated subcluster within the South American species. Taken together, the karyological and AFLP data contribute to the placement of H.?catharinensis within the phylogenetic framework of South American species of Hypochaeris and allow the definition of a novel and well-resolved phylogenetic group (the Lutea group).     

β-Arrestin Recruitment and G Protein Signaling by the Atypical Human Chemokine Decoy Receptor CCX-CKR

Chemokine receptors form a large subfamily of G protein-coupled receptors that predominantly activate heterotrimeric G_i proteins and are involved in immune cell migration. CCX-CKR is an atypical chemokine receptor with high affinity for CCL19, CCL21, and CCL25 chemokines, but is not known to activate intracellular signaling pathways. However, CCX-CKR acts as decoy receptor and efficiently internalizes these chemokines, thereby preventing their interaction with other chemokine receptors, like CCR7 and CCR9. Internalization of fluorescently labeled CCL19 correlated with β-arrestin2-GFP translocation. Moreover, recruitment of β-arrestins to CCX-CKR in response to CCL19, CCL21, and CCL25 was demonstrated using enzyme-fragment complementation and bioluminescence resonance energy transfer methods. To unravel why CCX-CKR is unable to activate G_i signaling, CCX-CKR chimeras were constructed by substituting its intracellular loops with the corresponding CCR7 or CCR9 domains. The signaling properties of chimeric CCX-CKR receptors were characterized using a cAMP-responsive element (CRE)-driven reporter gene assay. Unexpectedly, wild type CCX-CKR and a subset of the chimeras induced an increase in CRE activity in response to CCL19, CCL21, and CCL25 in the presence of the G_i inhibitor pertussis toxin. CCX-CKR signaling to CRE required an intact DRY motif. These data suggest that inactive G_i proteins impair CCX-CKR signaling most likely by hindering the interaction of this receptor with pertussis toxin-insensitive G proteins that transduce signaling to CRE. On the other hand, recruitment of the putative signaling scaffold β-arrestin to CCX-CKR in response to chemokines might allow activation of yet to be identified signal transduction pathways.     

Inhibitors of the bacterial cell wall biosynthesis enzyme MurC

A series of phosphinate transition-state analogues of the L-alanine adding enzyme (MurC) of bacterial peptidoglycan biosynthesis was prepared and tested as inhibitors of the Escherichia coli enzyme. Compound 4 was identified as a potent inhibitor of MurC from Escherichia coli with an IC(50) of 49nM.     

Optimization of a microarray sandwich-ELISA against hINF-gamma on a modified nitrocellulose membrane

The highly specific and highly sensitive ELISA (enzyme linked immunosorbent assay) technique is the most commonly used method for immunological diagnostics in general. In combination with protein microarrays and their ability to allow performing thousands of experiments in parallel, a promising tool for global analytical approaches with reduced consumption of time, analytes, and reagents is given. In this study a protein microarray-based sandwich-ELISA for human interferon-gamma (hINF-gamma) is established. In consideration of the immense importance of the surface chemistry, a new black nitrocellulose matrix that generates very high signal-to-noise ratios (SNR) and a very low autofluorescence was tested and optimized as microarray substrate. A validation of the applicability of the system was performed with a comparison to different commercially available systems. Experimental results show that the microarray-based ELISA is faster and easier to perform and shows a lower limit of detection (LOD) than a comparable system in a 96-well plate. The spotted slides with the capture antibody can be stored up to 1 month with no significant loss of signal intensity. A second model system with immobilized His-tagged restriction enzyme EcoRV and an anti-His antibody shows in coincidence the good applicability of the black nitrocellulose membrane and no cross-reactivity toward the ELISA.