Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics

Novel and powerful technologies such as DNA microarrays and proteomics have made possible the analysis of the expression levels of multiple genes simultaneously both in health and disease. In combination, these technologies promise to revolutionize biology, in particular in the area of molecular medicine as they are expected to reveal gene regulation events involved in disease progression as well as to pinpoint potential targets for drug discovery and diagnostics. Here, we review the current status of these technologies and highlight some studies in which they have been applied in concert to the analysis of biopsy specimens.     

DNA duplexes containing altered sugar residues as probes of EcoRII and MvaI endonuclease interactions with sugar-phosphate backbone

Oligonucleotides containing 1-(beta-D-2'-deoxy-threo-pentofuranosyl)cytosine (dCx) and/or 1-(beta-D-2'-deoxy-threo-pentofuranosyl)thymine (dTx) in place of dC and dT residues in the EcoRII and MvaI recognition site CC(A/T)GG were synthesized in order to investigate specific recognition of the DNA sugar-phosphate backbone by EcoRII and MvaI restriction endonucleases. In 2'-deoxyxylosyl moieties of dCx and dTx, 3'-hydroxyl groups were inverted, which perturbs the related individual phosphates. Introduction of a single 2'-deoxyxylosyl moiety into a dC x dG pair resulted in a minor destabilization of double-stranded DNA structure. In the case of a dA x dT pair the effect of a 2'-deoxyxylose incorporation was much more pronounced. Multiple dCx modifications and their combination with dTx did not enhance the destabilization effect. Hydrolysis of dCx-containing DNA duplexes by EcoRII endonuclease was blocked and binding affinity was strongly depended on the location of an altered sugar. A DNA duplex containing a dTx residue was cleaved by the enzyme, but kcat/K(M) was slightly reduced. In contrast, MvaI endonuclease efficiently cleaved both types of sugar-altered substrate analogs. However it did not cleave conformationally perturbed scissile bonds, when the corresponding unmodified bonds were perfectly hydrolyzed in the same DNA duplexes. Based on these data the possible contributions of individual phosphates in the recognition site to substrate recognition and catalysis by EcoRII were proposed. We observed strikingly non-equivalent inputs for different phosphates with respect to their effect on EcoRII-DNA complex formation.     

Spectrophotometric method of studying the cleavage of DNA-duplexes by restriction endonucleases]

A spectrophotometric method for continuous monitoring the cleavage of DNA duplexes by type II restriction endonucleases was proposed. The time course of cleavage of a 14-membered DNA duplex by MvaI endonuclease was obtained. The spectrophotometric method is characterized by rapidity and high precision in determining the kinetic parameters of the reaction. It can be recommended for testing the preparations for the presence of restriction endonucleases, rapid determination of the activity of any restriction endonucleases, highly precise quantitative analysis of the restriction enzyme catalysed reactions.     

Mechanism of CpG DNA methyltransferases M.SssI and Dnmt3a studied by DNA containing 2-aminopurine

Murine DNA methyltransferases Dnmt3a-CD and M.SssI from Spiroplasma methylate cytosines at CpG sites. The role of 6-oxo groups of guanines in DNA methylation by these enzymes has been studied using DNA substrates, which contained 2-aminopurine at different positions. Removal of the 6-oxo group of the guanine located adjacent to the target cytosine in the CpG site dramatically reduces the stability of the methyltransferase-DNA complexes and leads to a significant decrease in the methylation. Apparently, O6 of this guanine is involved in the recognition of CpG sites by the enzymes. Cooperative binding of Dnmt3a-CD to 2-aminopurine-containing DNA and the formation of nonproductive enzyme-substrate complexes were observed.     

Proteomic analysis of urinary fibrinogen degradation products in patients with urothelial carcinomas

Despite many years of research efforts and continued progress in the identification of urine markers for detection of bladder cancer, none of the markers described to date has been able to replace cystoscopy and urine cytology, the current gold standards for diagnosis. Here, we present a comprehensive gel-based proteomic study in which we have analyzed the presence and origin of fibrinogen (FG) and its degradation products (FDPs) in the urine of patients with and without urothelial carcinoma (UCs), with the aim of evaluating the potential of two-dimensional (2D) gel FDP profiling for detecting bladder cancer. A total of 151 urine samples collected from patients with Ucs of varying degrees of atypia and stages of invasion were compared with a control group consisting of 34 healthy volunteers with no clinical history of bladder cancer. The level and degree of degradation of FG in the urine were determined by 2D gel Western blotting in combination with enhanced chemilumenscence detection. Elevated levels of urine FG/FDPs were found in 99% of patients bearing superficial tumors, in 97% of the cases with early invasive disease, and in 96% of patients with highly invasive tumors. 2D gel profiling of urine FG/FDPs showed that the FG chains exhibited differential susceptibility to in situ proteolysis, with the α-chain being the most susceptible and the γ-chain the most resistant. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified peptide sequence regions in several of the most representative and common FDPs, which can be of value for producing novel specific antibodies to detect FG/FDPs in the urine. In addition, we present evidence indicating that FG is not produced by normal or malignant urothelium, although it is present both in the stroma of malignant tissue and tumor lesions. Taken together, the data indicate that increased levels of FG/FDPs amounts in the urine are a characteristic feature of bladder cancer, and emphasize the value of 2D gel profiling of urine FG/FDPs for detecting low-grade, noninvasive UCs.     

Details of Structure and Functioning of the Pharyngeal Jaw Apparatus of Ember Parrotfish Scarus rubroviolaceus (Scaridae)

Journal of Ichthyology - The structural details of some muscles, ligaments, aponeurotic structures, and osteological features of the pharyngeal jaw apparatus of ember parrotfish Scarus...     

Evidence for dynamics in proteins as a mechanism for ligand dissociation

Signal transduction, regulatory processes and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times (1/k(off)) should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into k(off) involving a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase whose binding affinities vary by six orders of magnitude. NMR relaxation-dispersion experiments revealed a common set of residues near the binding site that undergo a concerted millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with the binding affinity K(i) and k(off), suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although the forward rate of conformational exchange, k(conf,forward), is faster than k(off), the use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.     

Structural-functional analysis of diffusion in glucose absorption by rat small intestine enterocytes

To elucidate mechanisms providing transport of sugars across intestinal epithelium, on taking into account the current hypotheses (active transport, participation of paracellular transport and passive component of transcellular transport), it was important to reveal structural changes of tight junctions and distribution of the carriers of facilitated diffusion of GLUT2 and protein kinase C during absorption of glucose. On using confocal and electron microscopy, ultrastructural and immunocytochemical studies of enterocytes after perfusion of isolated rat small intestine fragment with 75 mM glucose (chronic experiment) have shown: 1) fluorescent labels of transporter GLUT2 and PKCbetaII are located in the apical area of enterocytes situated at the upper half of the villus. Antibodies against GLUT2, conjugated with gold, are revealed at the microvilli or apical membrane and in the area of terminal network; 2) no ultrastructural changes of the tight junction are detected on ultrathin sections and freeze--fracture replics. At the same time, fluorescent and gold labels against actin are concentrated in the vicinity of the lateral membrane in the tight junction area. The results obtained can serve a confirmation of a hypothesis that at high glucose concentrations GLUT2 participates in its transfer across the apical membrane.     

Morphofunctional features of visceral apparatus of silver arawana <Emphasis Type="Italic">Osteoglossum bicirrhosum</Emphasis> (Osteoglossidae)

The structure of some muscles—previously undescribed ligaments and aponeurotic structures of the visceral apparatus—of silver arawana Osteoglossum bicirrhosum has been studied in detail. A comparative analysis of the obtained results of anatomization and literature data is carried out. The peculiarities of the functioning of the visceral apparatus of silver arawana in the process of feeding are considered. New features of similarity in the structure of muscles of the visceral apparatus of Osteoglossidae and Salmonidae (by the example of salmon Salmo salar), which can provide a basis for confirming previously detected homologous features in the feeding stages of these two primitive groups of fish, are described.