Two-dimensional mapping as a tool for classification of green coffee bean species

Two species of the genus Coffea, Coffea arabica (Colombia) and Coffea canephora (Indiano Robusta) were analysed by two-dimensional (2-D) maps in order to obtain fingerprints of the expressed polypeptide chains and to determine which ones would characterize the two species. Green beans were milled under liquid nitrogen. A dry powder was produced by three different extraction protocols aimed at eliminating interfering substances (polyphenols). A reduced powder was produced by two successive extractions performed in acetone. Trichloroacetic acid (TCA; 10% w/v) and beta-mercaptoethanol (0.07% v/v) in acetone were used for the first extraction (a) and 10% w/v TCA in acetone was used for the second extraction (b). Proteins were then solubilized in a solution (40 microL per 1 mg powder) containing 7 M urea, 2 M thiourea, 3% w/v 3-(3-cholamidopropyldimethyl-amino)-1-propanesulfate, 1% v/v carrier ampholytes, 40 mM Tris, 5 mM tributylphosphine and 10 mM acrylamide as alkylating agent. Following incubation at room temperature for 1 hour and centrifugation (7000 rpm for 20 minutes), the supernatant was used for 2-D electrophoresis. The proteins were revealed by Sypro Ruby staining. Master maps of the five replicas of each species were compared by PDQuest analysis. The results of this differential proteome analysis were: sixteen proteins were expressed solely in C. canephora (var. Indiano Robusta) and five proteins were only found in C. arabica (var. Colombia). Another eight proteins were up-regulated in C. canephora (var. Indiano Robusta) in comparison to C. arabica (var. Colombia) and one was down-regulated in the same comparison. A number of these polypeptide chains were further characterized by mass spectrometry in the matrix-assisted laser desorption/ionisation-time of flight mode. Additionally, considering the low number of protein sequences of Coffea present in the databases we also investigated some spots with a more powerful tool, reversed phase-high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry, thus obtaining an internal peptide sequence. The general properties of the identified proteins are presented and discussed.     

Dynamic osmotic loading of chondrocytes using a novel microfluidic device

Many cells exhibit disparate responses to a mechanical stimulus depending on whether it is applied dynamically or statically. In this context, few studies have examined how cells respond to dynamic changes of the extracellular osmolality. In this study, we hypothesized that the cell size change response of cultured articular chondrocytes would be dependent on the frequency of applied osmotic loading. To test this hypothesis, we developed a novel microfluidic device, to apply hydrostatic pressure-driven dynamic osmotic loading by applying composition modulated flow, adapted from Tang and co-workers. This microfluidic device was used to study osmotic loads of +/-180 mOsm at a frequency up to 0.1 Hz with a constant minimal fluid-shear stress, and permit real-time monitoring of cell responses. Bovine articular chondrocytes were observed to exhibit increasing changes in cell volume with decreasing osmotic loading frequency. When the cell volume response was modeled by an exponential function, chondrocytes exhibited significantly different volume change responses to dynamic osmotic loading at 0.0125 Hz and static osmotic loading applied for a period of four minutes (Delta = +/-180 mOsm relative to the isotonic 360 mOsm). The intracellular calcium response at 0.0125 Hz was also monitored and compared with the response to static loading. Coupled with phenomenological or constitutive models, this novel approach could yield new information regarding cell material properties in response to dynamic loading that may contribute new insights into mechanisms of cellular homeostasis and mechanotransduction.     

Identification and phenotypic characterization of a subpopulation of T84 human colon cancer cells, after selection on activated endothelial cells

The extravasation of metastatic cells is regulated by molecular events involving the initial adhesion of tumor cells to the endothelium and subsequently the migration of the cells in the host connective tissue. The differences in metastatic ability could be attributed to properties intrinsic of the various primary tumor types. Thus, the clonal selection of neoplastic cells during cancer progression results in cells better equipped for survival and formation of colonies in secondary sites. A cell line (T84SF) exhibiting an altered phenotypic appearance was selected from a colon cancer cell line (T84) by repetitive plating on TNFalpha-activated human endothelial cells and subsequent selection for adherent cells. Cell growth, motility, chemoinvasive abilities, tyrosine phosphorylation signaling, and the metastasis formation in nude mice of the two cell lines was compared. T84SF cells displayed in vitro an higher proliferation rate and a more invasive behavior compared to the parental cells while formed in vivo a greater number of metastatic colonies in nude mice. As concerns the signaling underlying the phenotypes of the selected cells, we examined the general tyrosine phosphorylation levels in both cell lines. Our results indicate that T84SF have an increased basal tyrosine phosphorylation of several proteins among which src kinase was identified. Treatment of cells with a specific inhibitor of src activity caused a greater in vitro inhibition of proliferation and invasive properties of T84 parental cells with respect to T84SF cells and diminished metastasis formation in vivo. Altogether, these data provide evidences that this new cell line may be valuable for identifying molecular mechanisms involved in the metastatic progression of colon cancer.     

Lys300 plays a major role in the catalytic mechanism of maize polyamine oxidase

Maize polyamine oxidase (MPAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyses the oxidation of spermine and spermidine at the secondary amino groups. The structure of MPAO indicates a 30-A long U-shaped tunnel that forms the catalytic site, with residues Glu62 and Glu170 located close to the enzyme-bound FAD and residue Tyr298 in close proximity to Lys300, which in turn is hydrogen-bonded to the flavin N(5) atom via a water molecule (HOH309). To provide insight into the role of these residues in the catalytic mechanism of FAD reduction, we have performed steady-state and stopped-flow studies with wild-type, Glu62Gln, Glu170Gln, Tyr298Phe, and Lys300Met MPAO enzymes. We show that the steady-state enzyme activity is governed by an ionisable group with a macroscopic pK(a) of approximately 5.8. Kinetic analysis of the Glu62Gln, Glu170Gln, and Tyr298Phe MPAO enzymes have indicated (i) only small perturbations in catalytic activity as a result of mutation and (ii) steady-state pH profiles essentially unaltered when compared to the wild-type enzyme, suggesting that these residues do not play a critical role in the reaction mechanism. These kinetic observations are consistent with computational calculations that suggest that Glu62 and Glu170 are protonated over the pH range accessible to kinetic studies. Substitution of Lys300 with Met in MPAO resulted in a 1400-fold decrease in the rate of flavin reduction and a 160-fold decrease in the equilibrium dissociation constant for the Lys300Met-spermidine complex, consistent with a major role for this residue in the mechanism of substrate oxidation. A sizable solvent isotope effect (SIE = 5) accompanies FAD reduction in the wild-type enzyme and steady-state turnover (SIE = 2.3) of MPAO, consistent with the reductive half-reaction of MPAO making a major contribution to rate limitation in steady-state turnover. Studies using the enzyme-monitored turnover method indicate that oxidized FAD is the prominent form during steady-state turnover, consistent with the reductive half-reaction being rate-limiting. Our studies indicate the importance of Lys300 and probable importance of HOH309 to the mechanism of flavin reduction in MPAO. Possible roles for Lys300 and water in the mechanism of flavin reduction are discussed.     

Transcriptional profiling of gamma delta T cells identifies a role for vitamin D in the immunoregulation of the V gamma 9V delta 2 response to phosphate-containing ligands

Vitamin D is a steroid hormone that, in addition to its well-characterized role in calcium/phosphate metabolism, has been found to have regulatory properties for immune system function. The nuclear vitamin D receptor is widely expressed in tissues, but has also been shown to be regulated by hormones, growth factors, and cytokines. In this study we show that activation of human Vdelta2Vgamma9 T cells by nonpeptidic monoalkyl phosphates such as isopentenyl pyrophosphate leads to the up-regulation of the vitamin D receptor via a pathway that involves the classical isoforms of protein kinase C. We further show that this receptor is active by demonstrating that the ligand 1alpha,25-dihydroxyvitamin D3 (vitD3) significantly inhibits in a dose-dependent fashion phospholigand-induced gammadelta T cell expansion, IFN-gamma production, and CD25 expression. We also show that vitD3 negatively regulates signaling via Akt and ERK and, at high concentrations, potentiates Ag-induced cell death. As such, these data provide further support for the immunoregulatory properties of vitamin D, and suggest that the ability of vitD3 to negatively regulate the proinflammatory activity of gammadelta T cells may contribute to the protection this vitamin affords against inflammatory and autoimmune disorders dependent upon Th1-type responses.     

Immunohistochemical evaluation of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus

Control of the G1/S-phase transition as well as angiogenic switch are two of the most studied mechanisms in cancer. The current study examined the correlation between the immunohistochemical expression of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus (BE). Overall, p53 showed a much higher expression in BE patients (up to 50%) than in controls (1-10%) (P < 0.005). Also p21 showed a downregulation in BE when compared to normal esophagus (70% of cells vs. 65%), but the difference did not show any statistical significance (P = 0.45). pRb2/p130 was detected in 80% of cells in normal controls, but showed positive in only 20% of cells in BE biopsies. Additionally, Rb2/p130 expression was inversely correlated to that of VEGF, EZH2, and PCNA (P < 0.0001, P = 0.0032, P < 0.001, respectively). p27 stained more intensely and in a widespread manner (70%) cells in normal esophageal tissues but about only 30% in BE samples (P < 0.001). Lastly, in accordance with other reports, we also found p16 expressed by immunohistochemistry at high levels in normal controls and at low levels in BE (P < 0.001). In conclusion, p16, p21, p27, and p53 staining confirmed previously published data. Interestingly, pRb2/p130 expression was found significantly decreased in metaplastic epithelium compared to normal controls and showed significant inverse correlation with the expression of other markers, such as VEGF, EZH2, and PCNA. These data, taken together, indicate that these molecular events occurring in Barrett's metaplasia (BM) may represent one of the many steps taking place during esophageal malignant progression such as impairment of cell-cycle control, altered differentiation, and unbalanced angiogenesis.     

Development of SCAR Markers for Germplasm Characterisation in Olive Tree (Olea europea L.)

A set of 14 SCAR markers were developed starting from RAPD, AFLP and SAMPL analysis of several olive germplasm accessions. Eight RAPD, two AFLP and four SAMPL fragments were converted into dominant and codominant SCARs by cloning and sequencing the selected fragments. The markers obtained were evaluated on forty different olive cultivars from different Italian production areas (mainly from Liguria). The combined use of these SCARs made possible to univocally identify 26 cultivars while the remaining 14 will require the development of further markers since most of them are placed in a main group containing six genetically similar cultivars (among which Frantoio and Taggiasca) and four minor groups containing two cultivars each. A total of 31 different haplotypes were identified and the analysis of several individual plants indicated no intra-cultivar variability. Considering the SCAR polymorphism two alleles were scored for each markers with the only exception of markers IGPS3 and IGPS4 showing 4 alleles with 7 recognised groups and 5 alleles with 4 groups, respectively. Though less polymorphic in comparison with other markers like SSRs, the developed SCARs proved useful in genotype identification. In addition, they could potentially be used for breeding applications and forensic analysis. An erratum to this article is available at .     

Effects of a 50 Hz magnetic field on Dictyostelium discoideum (Protista)

Some studies have demonstrated that a few biological systems are affected by weak, extremely low frequency (ELF) electromagnetic fields (EMFs), lower than 10 mT. However, to date there is scanty evidence of this effect on Protists in the literature. Due to their peculiarity as single-cell eukaryotic organisms, Protists respond directly to environmental stimuli, thus appearing as very suitable experimental systems. Recently, we showed the presence of propionylcholinesterase (PrChE) activity in single-cell amoebae of Dictyostelium discoideum. This enzyme activity was assumed to be involved in cell-cell and cell-environment interactions, as its inhibition affects cell aggregation and differentiation. In this work, we have exposed single-cell amoebae of D. discoideum to an ELF-EMF of about 200 microT, 50 Hz, for 3 h or 24 h at 21 degrees C. A delay in the early phase of the differentiation was observed in 3 h exposed cells, and a significant decrease in the fission rate appeared in 24 h exposed cells. The PrChE activity was significantly lower in 3 h exposed cells than in the controls, whereas 24 h exposed cells exhibited an increase in this enzyme activity. However, such effects appeared to be transient, as the fission rate and PrChE activity values returned to the respective control values after a 24 h stay under standard conditions.     

Reproductive Consequences of Clonal Growth in Stenocereus eruca, a Rare Clonal Cactus of the Sonoran Desert

Stenocereus eruca is a prostrated, self-incompatible cactus endemic to the Sonoran Desert that regenerates primarily through clonal propagation. Clonal growth is expected to affect mate availability by influencing the number and spatial distribution of mating types. In this paper we examine the role of clonal growth on female fecundity through a series of pollination experiments in a population of S. eruca. We set up a pollen supplementation experiment using five distance treatments with pollen collected at 1, 10, 100, 1000 and 25000 m from receptor flowers during the years 2001 and 2002 and evaluated genetic sifmilarities between pairs of receptor-donor ramets through RAPD markers. Our data on fruit set, number of seeds/fruit, germination and overall fecundity revealed that S. eruca show a significant reduction in female fecundity when pollination occurs between ramets located at short distances (1 and 10 m), while genetic data showed high levels of similarity at those distances. The reduction in female fecundity is apparently a consequence of geitonogamy and inbreeding depression. Our data suggest that clonal growth and geitonogamy are likely to be partially responsible for the low levels of sexual reproduction and seedling recruitment observed in populations of S. eruca. Co-ordinating editor: H. Kudoh