Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

Identification of mutations that decrease the stability of a fragment of Saccharomyces cerevisiae chromosome III lacking efficient replicators

Eukaryotic chromosomes are duplicated during S phase and transmitted to progeny during mitosis with high fidelity. Chromosome duplication is controlled at the level of replication initiation, which occurs at cis-acting replicator sequences that are spaced at intervals of approximately 40 kb along the chromosomes of the budding yeast Saccharomyces cerevisiae. Surprisingly, we found that derivatives of yeast chromosome III that lack known replicators were replicated and segregated properly in at least 96% of cell divisions. To gain insight into the mechanisms that maintain these "originless" chromosome fragments, we screened for mutants defective in the maintenance of an "originless" chromosome fragment, but proficient in the maintenance of the same fragment that carries its normal complement of replicators (originless fragment maintenance mutants, or ofm). We show that three of these Ofm mutations appear to disrupt different processes involved in chromosome transmission. The OFM1-1 mutant seems to disrupt an alternative initiation mechanism, and the ofm6 mutant appears to be defective in replication fork progression. ofm14 is an allele of RAD9, which is required for the activation of the DNA damage checkpoint, suggesting that this checkpoint plays a key role in the maintenance of the "originless" fragment.     

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.     

Expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in mouse tissues and cell lines using an antibody against the enzyme amino-terminal domain

We have produced a polyclonal antibody that specifically recognizes cGMP-binding cGMP-specific phosphodiesterase (PDE5). The antibody was raised in rabbit using as immunogen a fusion protein, in which glutathione S-transferase was coupled to a 171 amino acid polypeptide of the N-terminal region of bovine PDE5. The antibody is able to immunoprecipitate PDE5 activity from mouse tissues and neuroblastoma extracts while it has no effect on all other PDE isoforms present in the extracts. PDE5 activity recovered in the immunoprecipitates retains its sensitivity to specific inhibitors such as zaprinast (IC(50)=0.6 microM) and sildenafil (IC(50)=3.5 nM). Bands of the expected molecular mass were revealed when solubilized immunoprecipitates were analysed in Western blots. The antibody selectively stained cerebellar Purkinje neurones, which are known to express high levels of PDE5 mRNA. Western blot analysis of mouse tissues revealed the highest expression signal in mouse lung, followed by heart and cerebellum, while a lower signal was evident in brain, kidney and a very low signal was present in the liver. In the hybrid neuroblastoma-glioma NG108-15 cells the antibody revealed a high PDE5 induction after dibutyryl-cAMP treatment.     

From cell cycle regulation to angiogenesis: dialogue between the basic and clinical sciences

Basic research in biological and medical disciplines has revealed fundamental aspects of the differentiation of single cells as well as the development of multicellular organisms. The combination of knowledge of intracellular and intercellular pathways controlling development and homeostasis in higher organisms is the key to understanding certain diseases that are associated with abnormalities in these pathways and developing strategies for fighting them. Today's high scientific output in a rapidly growing number of scientific journals requires great effort to keep up with the latest developments outside one's specialization. The tenth international conference of the International Society of Differentiation (ISD) therefore was a great opportunity for scientists of diverse fields of biological and medical research to learn about the latest developments in even remotely related branches of research and opening new perspectives. The authors have tried to conserve this spirit in reviewing main aspects of research presented at the conference.     

Prostaglandin E2 signalling pathway in human T lymphocytes from healthy and conjunctiva basal cell carcinoma-bearing subjects

Prostaglandin E-induced signal transduction pathways in human T cells from healthy and uveal melanoma-bearing subjects were studied. Transfection experiments showed that PGE2 was able to phosphorylate and activate the fusion trans-activator of the cAMP responsive element-binding protein (CREB). Phosphorylation was at least partially mediated by protein kinase A, as evidenced by the effects of specific kinase inhibitors. Western blotting experiments, which were performed to identify the CREB/ATF2 family members involved in the response to PGE2, revealed a modulation of proteins CREB1, CREB2 and ATF2 and phosphorylation of the 43 kDa form of CREB. Experiments of immunoprecipitation with CREB-binding protein (CBP) demonstrated that, after PGE2 treatment, all of the CREB/ATF isoforms studied, as well as the phosphorylated form of CREB (p-CREB), interacted with CBP. In basal conditions, T cells from patients with conjunctiva basal cell carcinoma showed the presence of p-CREB, which coimmunoprecipitated with CBP. CREB phosphorylation did not modify after PGE2 treatment whereas the p-CREB fraction bound to CBP increased in a delayed manner compared to normal subjects.     

Mechanisms controlling the integrity of replicating chromosomes in budding yeast

Cells are continually challenged by genomic insults that originate from chemical and physical agents diffused in the environment, but also normal cellular metabolism produces genotoxic effects. Moreover, DNA replication and recombination generate intermediates potentially dangerous for genome stability. Growing evidence show that many genetic disorders are characterized by high levels of chromosome alterations due to genomic instability, which is also a hallmark of cancer cells. Recent work shed some light on the molecular events that maintain the integrity of chromosomes during unperturbed S phase and in the face of odds.     

γ-Ray footprinting and fluorescence polarization anisotropy of a 30-mer synthetic DNA fragment with one 2′-deoxy-7-hydro-8- oxoguanosine lesion

The influence of the oxidative lesion 2'-deoxy-7-hydro-8-oxoguanosine (8-oxodG) on some conformational properties of DNA has been studied. Four 30-mer duplexes of the form [5'-GATCCTCTAGAGTC[G(*) or G]ACCTGCAGGCATGCA-3']:[3'-CTAGGAGATCTCAG[C or A]TGGACGTCCGTACGT-5'], in which G(*) is the 8-oxodG lesion, were synthesized in order to compare the effect of the GA mismatch and of the damaged G(*)C and G(*)A forms with the normal GC. Spectroscopic measurements performed by means of UV denaturation and circular dichroism experiments do not show gross changes of stability and overall structure in the damaged and mismatched samples. The control DNA and the samples containing GA mismatch show very similar gamma-rays cutting patterns, indicating that the introduction of the GA mismatch does not perturb the phosphate backbone geometry. In the samples containing the 8-oxodG there are some variations of the cleavage pattern near G(*) which are extended for almost one helical turn. Some differences are observed between G(*)C and G(*)A duplexes. In particular, in the G(*)C sample the reduced accessibility to OH radicals at the G15 site, observed in the control, spreads on the intrastrand adjacent bases and in the G(*)A sample a shift of the minimum is observed. The hydrodynamic radius R(h) derived by fluorescence polarization anisotropy decay exhibits a constant value of 11.4 +/- 0.2 A between 5 and 40 degrees C, in all the samples. The torsional constant alpha of each oligomer decreases when the temperature is raised and the alpha values of the damaged samples are higher than those of the normal ones.     

International conference on basic and clinical aspects of cell-cycle control

Scientists of numerous medical and life science disciplines met in Siena, Italy to discuss the latest proceedings in basic and clinical research. General models of interconnected linear and back-feeding cell-cycle control pathways provide a basis for applied molecular research. Cell-cycle determining factors essential for the control of cellular homeostasis either become markers to determine characteristics of a disease and/or become therapeutic targets. Apart from animal and tissue culture models, molecular theories finally have to stand proof in clinical application and evaluation. Therefore, the clinical feedback to the basic scientist's bench is essential for necessary adjustments of their models to improve future approaches to research challenges. A select group of speakers provided the audience with such an interdisciplinary dialogue at the first International Conference on Basic and Clinical Aspects of Cell-Cycle Control from May 29 to 31, 2000 in Siena, Italy.