Novel approaches to map protein interactions

Although we now have the sequence of the human genome at hand, we face the challenge of assigning function to the identified genes. Genes usually ascribe their function through proteins, and the role of proteins is to interact with other molecules. Therefore, if we could map the interactions of proteins we would be able to understand protein function. The challenge of mapping protein interactions is vast and many novel approaches have recently been developed for this task using molecular biology, mass spectrometry and chemiproteomic techniques.     

Novel approaches to energize microbial biocatalysts

An efficient and cheap energization of microbial biocatalysts is essential in current biotechnological processes. A promising alternative to the use of common organic or inorganic electron donors is the semiconductor nanoparticles (SNs) that absorb light and transfer electrons (photoelectrons) behaving as artificial photosynthetic systems (biohybrid systems). Excited photoelectrons generated by illuminated SNs are highly reductive and readily accepted by membrane-bound proteins and electron shuttles to drive specific cell reduction processes and energy generation in microbes. However, the operational mechanisms of these hybrid systems are still poorly understood, especially at the material–microbe interface, and therefore the design and production of efficient biohybrids are challenging. Some major limitations/challenges and future prospects of SNs as microbial energization systems are discussed.     

Novel approaches to molecular recognition using porphyrins

Studies of molecular recognition using designed and synthesised molecules provide valuable information on the principle and possible applications of artificial functional molecules. Porphyrin-based receptors have been used to elucidate haem-protein interactions and the basic mechanism of multi-point recognition.     

Novel approaches to the biosynthesis of vanillin

Microorganisms able to produce vanillin in excess of 6g/l from ferulic acid have now been isolated. In Pseudomonas strains, the metabolic pathway from eugenol via ferulic acid to vanillin has been characterised at the enzymic and molecular genetic levels. Attempts to introduce vanillin production into other organisms by genetic engineering have begun.     

Novel approaches to cancer therapy using oncolytic viruses

The goal of oncolytic therapy is to exploit the innate ability of viruses to infect tumor cells, replicate in tumor cells, and produce selective oncolysis while sparing normal cells. Although the concept that viruses can be oncolytic is not new, it is only in the last three decades that efforts have been directed at genetically mutating viruses to specifically target characteristics of cancer cells. Several viruses have the potential to infect, replicate and lyse tumor cells, each taking advantage of different host cancer cell biology. This review will focus on the major viruses under current investigation for oncolytic therapy, the mechanism by which they specifically eradicate tumors, and the clinical strategies currently under investigation.     

Novel approaches to the syntheses of N-substituted S-glycosyl-sulfenamides

Bis(tetra-O-acetyl-beta-D-glucopyranosyl)disulfide reacts, under silver ion activation, with primary and secondary aliphatic as well as aromatic amines to furnish the title compounds in moderate to good yields. The same derivatives could also be obtained from (tetra-O-acetyl)-beta-D-glucopyranosyl methanethiolsulfonate 1 by nucleophilic substitution with amines. It was shown that the polarization of the S-S-bond in 1 is enhanced by Ag+ so as to allow reaction with sterically hindered amines as well.     

Novel approaches to link apicobasal polarity to cell fate specification

Understanding the development of apicobasal polarity (ABP) is a long-standing problem in biology. The molecular components involved in the development and maintenance of APB have been largely identified and are known to have ubiquitous roles across organisms. Our knowledge of the functional consequences of ABP establishment and maintenance is far less comprehensive. Recent studies using novel experimental approaches and cellular models have revealed a growing link between ABP and the genetic program of cell lineage. This mini-review describes some of the most recent advances in this new field, highlighting examples from Caenorhabditis elegans and mouse embryos, human pluripotent stem cells, and epithelial cells. We also speculate on the most interesting and challenging avenues that can be explored.     

Novel approaches to tackling malarial drug resistance using yeast

Yeasts have a justified reputation as one of the world's most versatile organisms. Baker's yeast continues to live up to this recognition by joining the war against malaria. Yeast can now be used to study antifolate drug resistance patterns that depend on the dihydrofolate reductase enzyme (DHFR) from the malaria parasite.     

Novel approaches to the analysis of polysaccharide structures.

Recently, atomic force microscopy has been used to image a variety of polysaccharides and map their distribution on cell surfaces. The mechanical response of polysaccharides to tensile stress has been investigated in single-molecule force spectroscopy experiments. Small-angle X-ray scattering has provided a probe of polysaccharide structure operating in a size range (2-25 nm) that is intermediate between those accessible using nuclear magnetic resonance and light scattering.     

Novel approaches to the mode of action of colicins

According to the theory of Fredericq (1949) and Nomura (1964), colicins are attached by specific receptor sites in the cell walls of sensitive bacteria, which mediate their inhibitive effects. During last years, a great variety of experimental data have been accumulated, some of which cannot be easily interpreted in terms of this theory. There exist considerable discrepancies concerning the chemical nature and molecular weight of isolated receptors. The attachment of a colicin onto its receptor need not be irreversible. The inhibition of numerous membrane-associated functions in colicin-tolerant mutants suggests their pleiotropic deletion nature. The difference between colicin resistance and colicin tolerance does not seem to be clear-cut. Cells of stable L-forms of protoplast type, completely devoid of their walls, retain in most cases the same patterns of sensitivity to colioins as rods of the same strains. Experimental changes in the relationship between the cell wall and the cytoplasmic membrane decrease colicin sensitivity of the cells. Colicin E3 has been found to be a specific endoribonuclease, able to cleave a terminal fragment from the 16 S rRNA also in isolated ribosomesin vitro: not only in ribosomes from sensitivive bacteria, but also in those from resistant ones and from eukaryotic cells. A destabilization of the DNA helix was induced by colicin E2in vitro asin vivo. It seems that there exist two distinct types of colicin receptors with different functions: those in the cell wall, and those in the cytoplasmic membrane. Only the contact of colicins with the latter ones is biologically effective and starts both stages of their inhibitive effect: the reversible and the irreversible ones.     

Novel approaches and hurdles to somatic cloning in cattle

The overall efficiency of somatic cloning in cattle is still low. Many factors are necessary for successful birth of live offspring. Among them, the source of donor cells reveals the importance of the donor genotype but also the influence of the cell line itself. The cell cycle stage has been intensively investigated, and recent results indicate that, in cattle, the G0 stage of the donor nuclei is not a prerequisite for reprogramming, as highly proliferating cultured fibroblasts also result in live offspring after nuclear transfer. A technical approach using direct microinjection of fibroblast nuclei, instead of fusion of the whole cell, has proved to result in high in vitro development rates in cattle. However, full-term development of somatic cloned embryos is still limited by long-lasting effects and a high incidence of losses at periimplantation time (as well as in late gestation and around calving).     

Novel approaches to identify protein adducts produced by lipid peroxidation

Abstract

Lipid peroxidation is responsible for the generation of chemically reactive, diffusible lipid-derived electrophiles (LDEs) that covalently modify cellular protein targets. These protein modifications modulate protein activity and macromolecular interactions and induce adaptive and toxic cell signaling. Protein modifications induced by LDEs can be identified and quantified by affinity enrichment and liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based techniques. Tagged LDE analog probes with different electrophilic groups can be covalently captured by click chemistry for LC–MS/MS analyses, thereby enabling in-depth studies of proteome damage at the protein and peptide sequence levels. Conversely, click-reactive, thiol-directed probes can be used to evaluate thiol damage caused by LDE by difference. These analytical approaches permit systematic study of the dynamics of protein damage caused by LDE and mechanisms by which oxidative stress contribute to toxicity and diseases.     

Novel proteomic approaches for tissue analysis

Proteomics, the global study of protein expression and characteristics, has recently emerged as a key component in the field of molecular analysis. The dynamic nature of proteins, from ion channels to chaperones, presents a challenge, yet the understanding of these molecules provides a rich source of information. When applying proteomic analysis directly to human tissue samples, additional difficulties arise. The following article presents an overview of the current proteomic tools used in the analysis of tissues, beginning with conventional methods such as western blot analysis and 2D polyacrylamide gel electrophoresis. The most current high-throughput techniques being used today are also reviewed. These include protein arrays, reverse-phase protein lysate arrays, matrix-assisted laser desorption/ionization, surface-enhanced laser desorption/ionization and layered expression scanning. In addition, bioinformatics as well as issues regarding tissue preservation and microdissection to obtain pure cell populations are included. Finally, future directions of the tissue proteomics field are discussed.     

Novel proteomic approaches for tissue analysis

Proteomics, the global study of protein expression and characteristics, has recently emerged as a key component in the field of molecular analysis. The dynamic nature of proteins, from ion channels to chaperones, presents a challenge, yet the understanding of these molecules provides a rich source of information. When applying proteomic analysis directly to human tissue samples, additional difficulties arise. The following article presents an overview of the current proteomic tools used in the analysis of tissues, beginning with conventional methods such as western blot analysis and 2D polyacrylamide gel electrophoresis. The most current high-throughput techniques being used today are also reviewed. These include protein arrays, reverse-phase protein lysate arrays, matrix-assisted laser desorption/ionization, surface-enhanced laser desorption/ionization and layered expression scanning. In addition, bioinformatics as well as issues regarding tissue preservation and microdissection to obtain pure cell populations are included. Finally, future directions of the tissue proteomics field are discussed.     

Novel approaches to reversing anti-cancer drug resistance using gene-specific therapeutics.

One of the underlying mechanisms of multidrug resistance (MDR) is cellular overproduction of P-glycoprotein (P-gp), which acts as an efflux pump for various anti-cancer drugs. P-gp is encoded by a group of related genes termed MDR; only MDR1 is known to confer the drug resistance, and its overexpression in cancer cells has been a therapeutic target to circumvent the resistance. To overcome P-gp-mediated drug resistance, we have developed six anti-MDR1 hammerhead ribozymes and delivered them to P-gp-overproducing human leukemia cell line by a retroviral vector containing RNA polymerase III promoter. These ribozyme-transduced cells became vincristine-sensitive, concomitant with the decreases in MDR1 expression, P-gp amount and efflux pump function. Among the ribozymes tested, the anti-MDR1 ribozyme against the translation-initiation site exhibited the highest efficacy. The retrovirus-mediated transfer of this most potent anti-MDR1 ribozyme into a human lymphoma cell line, which was made resistant by infection of pHaMDR1/A retroviral vector and thus possessed a low degree of MDR due to P-gp expression relevant to clinical MDR, resulted in a complete reversal of MDR phenotype. In addition to retrovirus-mediated transfer of ribozymes, we evaluated the efficacy of cationic liposome-mediated transfer of ribozyme. Treatment of a P-gp-producing human breast cancer cell line with the liposome-ribozyme complex resulted in reversal of resistance, concomitant with the decreases in both MDR1 expression and P-gp amount. Confocal microscopic imaging of the cells after treatment with liposome/FITC-dextran showed cytoplasmic fluorescence that was abolished by cytochalasin B, indicating a high endocytotic activity in these cells. The endocytotic activity was well correlated with the success of cationic liposome-mediated transfer of MDR1 ribozyme. These distinct approaches using either retrovirus- or liposome-mediated transfer of anti-MDR1 ribozyme may be selectively applicable to the treatment of MDR cells with different properties such as endocytotic activity as a specific means to reverse resistance.