Structure and function analysis of the poliovirus cis-acting replication element (CRE)

The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5' noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CD(pro) complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.     

Oxidative damage to proteins in yeast cells exposed to adaptive levels of H(2)O(2)

When yeast cells are exposed to sublethal concentrations of oxidants, they adapt to tolerate subsequent lethal treatments. Here, we show that this adaptation involves tolerance of oxidative damage, rather than protection of cellular constituents. o- and m-tyrosine levels are used as a sensitive measure of protein oxidative damage and we show that such damage accumulates in yeast cells exposed to H(2)O(2) at low adaptive levels. Glutathione represents one of the main cellular protections against free radical attack and has a role in adaptation to oxidative stress. Yeast mutants defective in glutathione metabolism are shown to accumulate significant levels of o- and m-tyrosine during normal aerobic growth conditions.     

Specificity in the settlement -- modifying response of bacterial biofilms towards zoospores of the marine alga Enteromorpha

Previous studies have shown that the rate of settlement of zoospores of the green alga Enteromorpha is stimulated by mixed microbial biofilms and that the number of zoospores settling is positively correlated with the number of bacteria in the biofilm. In the present study the specificity of this relationship has been investigated. Ninety-nine strains of marine bacteria were isolated from natural biofilms on rocks and the surface of Enteromorpha plants. Isolates were screened by denaturing gradient gel electrophoresis (DGGE) to eliminate replicates and 16S rDNA sequencing identified a total of 37 unique strains. Phylogenetic analysis revealed that the isolated bacterial strains belonged to three groups gamma-Proteobacteria (28 strains), Cytophaga-Flavobacteria-Bacteroid (CFB) group (six strains) and alpha-Proteobacteria (one strain). Two strains were unassigned, showing < 93% sequence similarity with the CFB group. The main genera of gamma-Proteobacteria were Pseudoalteromonas (14 strains), Vibrio (five strains), Shewanella (five strains), Halomonas (three strains) and Pseudomonas (one strain). Spore settlement experiments were conducted on single-species biofilms, developed for different times on glass slides. The effect of correcting spore settlement values for biofilm density was evaluated. Results showed that the effect of bacterial strains on spore settlement was strain- but not taxon-specific and activity varied with the age of the biofilm. However, most of the strains belonging to genera Vibrio and Shewanella showed stimulation. Pseudoalteromonas strains showed a range of effects including settlement-inhibiting, paralysing and lysing activities. Spatial analysis of bacterial density in the presence and absence of spores revealed a range of different types of association between spores and bacteria. Overall, the spatial association between spores and bacteria appears to be independent of the overall quantitative influence of bacterial cells on spore settlement.     

Miniaturized analytical assays in biotechnology

Biotechnology today is a well-established paradigm in many areas of human endeavor, such as the pharmaceutical industry, agriculture, management of the environment and many others. Meanwhile, biology is undergoing a spectacular transition: whereas systematic biology was replaced gradually by molecular biology, the latter is rapidly being transformed into a new systematic era in which entire genomes are being charted by ever more sophisticated analytical techniques.In the wake of this onslaught of data, new fields are germinating, such as bioinformatics in an attempt to find answers to fundamental questions, answers that may be hidden in the massive amounts of data already available today.     

Inheritance of pyriproxyfen resistance in the whitefly, Bemisia tabaci (Q biotype)

The inheritance of resistance to pyriproxyfen, an insect growth regulator (a juvenoid, with ovicidal and larvicidal activities), was studied in the whitefly Bemisia tabaci (Gennadius). Two parental strains, both belonging to Q biotype, were assayed with pyriproxyfen; a susceptible strain (ALM-1) originating from Spain and a pyriproxyfen-resistant one (Pyri-R) from Israel. The resistance ratio between the two parental strains was approximately 7,000-fold. Concentration-mortality lines for F(1) heterozygous females from reciprocal crosses (SS female symbol X R male symbol and RR female symbol X S male symbol ) were derived by statistical modelling and proved intermediate to those of the parents. The pooled degree of dominance from both reciprocal crosses was +0.26, indicating that resistance was incompletely or partially dominant. Mortality curves for F(2) males produced by virgin F(1) heterozygous females displayed a broad plateau at 50% mortality, indicating that resistance to pyriproxyfen in B. tabaci is conferred primarily by a mutant allele at a single locus. The role of arrhenotoky in influencing the mode of inheritance of resistance, and its selection in field populations, is discussed.     

Computation of DNA backbone conformations

We present an algorithm for the computation of 2'-deoxyribose-phosphodiester backbone conformations that are stereochemically compatible with a given arrangement of nucleic acid bases in a DNA structure. The algorithm involves the sequential computation of 2'-deoxyribose and phosphodiester conformers (collectively referred to as a backbone 'segment'), beginning at the 5'-end of a DNA strand. Computation of the possible segment conformations is achieved by the initial creation of a fragment library, with each fragment representing a set of bond lengths, bond angles and torsion angles. Following exhaustive searching of sugar conformations, each segment conformation is reduced to a single vector, defined by a specific distance, angle and torsion angle, that allows calculation of the O(1)' position. A given 'allowed' conformation of a backbone segment is determined based on its compatibility with the base positions and with the position of the preceding backbone segment. Initial computation of allowable segment conformations of a strand is followed by the determination of continuous backbone solutions for the strand, beginning at the 3'-end. The algorithm is also able to detect repeating segment conformations that arise in structures containing geometrically repeating dinucleotide steps. To illustrate the utility and properties of the algorithm, we have applied it to a series of experimental DNA structures. Regardless of the conformational complexity of these structures, we are able to compute backbone conformations for each structure. Hence, the algorithm, which is currently implemented within a new computer program NASDAC (Nucleic Acids: Structure, Dynamics and Conformation), should have generally applicability to the computation of DNA structures.     

Aspects of cancer immunotherapy

Cancer immunotherapy has traditionally undergone a 'revolution' every decade, from the use of Bacille Calmette-Guérin by scarification in the 1970s, to interleukin-2 therapies in the 1980s, and monoclonal antibody treatments in the early 1990s. Usually the early reports on the use of such agents were encouraging, but when more patients were studied in multiple centres, the initial promising results could not be confirmed. Now in a new century, we have more reagents and methods available than ever before - indeed, with such a plethora of reagents it is difficult to envisage them being fully and appropriately tested within the next decade, by which time there will be even more reagents to test. However, there have been three major advances which should lead to substantial progress in cancer immunotherapy: (1) the widespread use of genetic engineering, enabling identification of candidate vaccine proteins and manipulation of their sequences; (2) the production of antigens, antibodies and cytokines in large amounts by recombinant technologies, and (3) an understanding of the mode of presentation of peptides by major histocompatibility complex Class I and Class II molecules and their recognition by T cells. Despite these advances, there are major problems facing cancer immunotherapy, such as the ability of tumours to mutate and evade the immune system and the difficulty of precisely defining the interactions of effector cells in mediating 'rejection' or destruction of a tumour. There are clearly immunological similarities with diseases such as malaria and schistosomiasis, where the invading foreign organisms can use a variety of strategies to resist an elicited immune response. The failure to find a suitable vaccine for these diseases must lead to some pessimism for the development of immunotherapy for an autologous tumour. However, there are promising studies now in progress which should give an indication of the most important directions to follow. This review provides a commentary on aspects of cancer immunotherapy and in particular will deal with: (1) the selection of antigens as vaccine components; (2) the modes of presentation of antigens, particularly by major histocompatibility complex Class I molecules; and (3) new modes of delivery of vaccine immunogens.     

Natively unfolded C-terminal domain of caldesmon remains substantially unstructured after the effective binding to calmodulin

The structure of C-terminal domain (CaD136, C-terminal residues 636-771) of chicken gizzard caldesmon has been analyzed by a variety of physico-chemical methods. We are showing here that CaD136 does not have globular structure, has low secondary structure content, is essentially noncompact, as it follows from high R(g) and R(S) values, and is characterized by the absence of distinct heat absorption peaks, i.e. it belongs to the family of natively unfolded (or intrinsically unstructured) proteins. Surprisingly, effective binding of single calmodulin molecule (K(d) = 1.4 +/- 0.2 microM) leads only to a very moderate folding of this protein and CaD136 remains substantially unfolded within its tight complex with calmodulin. The biological significance of these observations is discussed.