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Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The existence of approximately 90 antigenically distinct capsular serotypes has greatly complicated the development of an effective pneumococcal vaccine. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. PiuA and PiaA are the lipoprotein components of two pneumococcal iron ABC transporters and are required for full virulence in mouse models of infection. Here we describe a study of the distribution and genetic diversity of PiuA and PiaA within typical and atypical S. pneumoniae, Streptococcus oralis, and Streptococcus mitis strains. The genes encoding both PiuA and PiaA were present in all typical pneumococci tested, (covering 20 and 27 serotypes, respectively). The piuA gene was highly conserved within the typical pneumococci (0.3% nucleotide divergence), but was also present in 'atypical' pneumococci and the closely related species S. mitis and S. oralis, showing up to 10.4% nucleotide divergence and 7.5% amino acid divergence from the typical pneumococcai alleles. Conversely, the piaA gene was found to be specific to typical pueumococci, 100% conserved, and absent from the oral streptococci, including isolates of S. mitis known to possess pneumolysin and autolysin. These are desirable qualities for a vaccine candidate and as a diagnostic tool for S. pneumoniae.

This paper describes adoption of eukaryotic representational difference analysis of cDNA technique for prokayotic bacterial systems. Identification of five major gene products, involved in the iron-regulation in Neisseria meningitidis, using this technique validates its application to bacterial systems.

Multilocus enzyme electrophoresis was used in the analysis of 80 nonencapsulated Haemophilus influenzae isolates from patients with meningitis, otitis, chronic bronchitis and cystic fibrosis. Isolates were also recovered from healthy carriers. Genetic distribution of acute disease isolates was concluded as not random over the five electrophoretic types, although the genetic diversity within the groups was not different. The effect of bacterial persistence in the host on the genetic diversity of H. influenzae was also discussed.

Introduction of sites from the penA genes of Neisseria cinerea provides a mosaic structure for the penicillin-binding protein 2 genes (penA) of penicillin-resistant Neisseria meningitidis. An additional codon, Asp-345A, absent in the four N. cinerea isolates studied and which leads to a reduction in the affinity of PBP 2 in gonococci, is evident in the penA gene site obtained from N. cinerea in N. meningitidis K196.

Abstract Background A better understanding of the mechanisms involved in gas-phase fragmentation of peptides is essential for the development of more reliable algorithms for high-throughput protein identification using mass spectrometry (MS). Current methodologies depend predominantly on the use of derived m/z values of fragment ions, and, the knowledge provided by the intensity information present in MS/MS spectra has not been fully exploited. Indeed spectrum intensity information is very rarely utilized in the algorithms currently in use for high-throughput protein identification. Results In this work, a Bayesian neural network approach is employed to analyze ion intensity information present in 13878 different MS/MS spectra. The influence of a library of 35 features on peptide fragmentation is examined under different proton mobility conditions. Useful rules involved in peptide fragmentation are found and subsets of features which have significant influence on fragmentation pathway of peptides are characterised. An intensity model is built based on the selected features and the model can make an accurate prediction of the intensity patterns for given MS/MS spectra. The predictions include not only the mean values of spectra intensity but also the variances that can be used to tolerate noises and system biases within experimental MS/MS spectra. Conclusion The intensity patterns of fragmentation spectra are informative and can be used to analyze the influence of various characteristics of fragmented peptides on their fragmentation pathway. The features with significant influence can be used in turn to predict spectra intensities. Such information can help develop more reliable algorithms for peptide and protein identification.

Ageing is associated in many organisms with a reduction in motor movements. We have previously shown that the rate of feeding movements of the pond snail, Lymnaea, decreased with age but the underlying cause is not fully understood. Here, we show that dopamine in the cerebro‐buccal complex is an important signalling molecule regulating feeding frequency in Lymnaea and that ageing is associated with a decrease in CNS dopamine. A proteomic screen of young and old CNSs highlighted a group of proteins that regulate stress responses. One of the proteins identified was 14‐3‐3, which can enhance the synthesis of dopamine. We show that the Lymnaea 14‐3‐3 family exists as three distinct isoforms. The expression of the 29 kDa isoform (14‐3‐3Lym3) in the cerebro‐buccal complex decreased with age and correlated with feeding rate. Using a 14‐3‐3 antagonist (R18) we were able to reduce the synthesis of L‐DOPA and dopamine in ex vivo cerebro‐buccal complexes. Together these data suggest that an age‐related reduction in 14‐3‐3 can decrease CNS dopamine leading to a consequential reduction in feeding rate. [ABSTRACT FROM AUTHOR]

SUMO is a small post-translational modifier, that is attached to lysine residues in target proteins. It acts by altering protein-protein interactions, protein localisation and protein activity. SUMO chains can also act as substrates for ubiquitination, resulting in proteasome-mediated degradation of the target protein. SUMO is removed from target proteins by one of a number of specific proteases. The processes of sumoylation and desumoylation have well documented roles in DNA metabolism and in the maintenance of chromatin structure. To further analyse the role of this modification, we have purified protein complexes containing the S. pombe SUMO protease, Ulp2. These complexes contain proteins required for ribosome biogenesis, RNA stability and protein synthesis. Here we have focussed on two translation initiation factors that we identified as co-purifying with Ulp2, eIF4G and eIF3h. We demonstrate that eIF4G, but not eIF3h, is sumoylated. This modification is increased under conditions that produce cytoplasmic stress granules. Consistent with this we observe partial co-localisation of eIF4G and SUMO in stressed cells. Using HeLa cells, we demonstrate that human eIF4GI is also sumoylated; in vitro studies indicate that human eIF4GI is modified on K1368 and K1588, that are located in the C-terminal eIF4A- and Mnk-binding sites respectively. [ABSTRACT FROM AUTHOR]

Background: A better understanding of the mechanisms involved in gas-phase fragmentation of peptides is essential for the development of more reliable algorithms for high-throughput protein identification using mass spectrometry (MS). Current methodologies depend predominantly on the use of derived m/z values of fragment ions, and, the knowledge provided by the intensity information present in MS/MS spectra has not been fully exploited. Indeed spectrum intensity information is very rarely utilized in the algorithms currently in use for high-throughput protein identification. Results: In this work, a Bayesian neural network approach is employed to analyze ion intensity information present in 13878 different MS/MS spectra. The influence of a library of 35 features on peptide fragmentation is examined under different proton mobility conditions. Useful rules involved in peptide fragmentation are found and subsets of features which have significant influence on fragmentation pathway of peptides are characterised. An intensity model is built based on the selected features and the model can make an accurate prediction of the intensity patterns for given MS/MS spectra. The predictions include not only the mean values of spectra intensity but also the variances that can be used to tolerate noises and system biases within experimental MS/MS spectra. Conclusion: The intensity patterns of fragmentation spectra are informative and can be used to analyze the influence of various characteristics of fragmented peptides on their fragmentation pathway. The features with significant influence can be used in turn to predict spectra intensities. Such information can help develop more reliable algorithms for peptide and protein identification. [ABSTRACT FROM AUTHOR]

Abstract: We previously described a caspase-like activity, which we termed KIPase that is implicated in the turnover of the mammalian cell cycle regulator p27KIP1. KIPase cleaves a tetra-peptide substrate, Ac-DPSD-AMC, which mimics the target site in p27KIP1, and inhibitors based on this tetra-peptide are ineffective against other known caspases. Here we describe the purification and characterization of KIPase, and trace its activity to the β1 subunit of the 20S proteasome. Further analyses revealed that the activity of the β1 subunit is up-regulated as cells enter the cell cycle without concomitant change in the levels of the proteasome β1, β2 or β5 subunits. To our knowledge, this is the first description of cell cycle regulation of the caspase-like activity of the 20S proteasome. [Copyright &y& Elsevier]

Penicillin-resistant (penr) clinical isolates of Neisseria meningitidis, which do not produce β-lactamase, were first identified in Spain in 1985; the frequency of their recovery, which has been increasing in the past few years, reached 20% in 1989. Serogrouping, determination of serotypes and subtypes, and multilocus enzyme electrophoresis of the penr strains showed an extensive diversity. Resistance is due, at least in part, to a decreased affinity of penicillin-binding protein (PBP) 2 for penicillin. Similar low-affinity forms of PBP 2 are also found in penr isolates of Neisseria lactamica, Neisseria polysaccharea, and Neisseria gonorrhoeae. Genetic transformation of an N. meningitidis type strain to low-level penicillin resistance with DNA from resistant meningococci and other Neisseria species resulted in transformants that possessed low-affinity forms of PBP 2. These altered forms of PBP 2 have been shown to arise from recombinational events that replace parts of the PBP 2 gene with the corresponding regions from the PBP 2 genes of commensal Neisseria species. [ABSTRACT FROM PUBLISHER]

In this chapter 1 describe the PCR-coupled subtractive hybridization technique of representational difference analysis of cDNA (cDNA RDA). cDNA RDA is based on the representational difference analysis (RDA) method previously described by Lisitsyn et al., and can be used to identify genes whose expression is modified between two populations of cells. cDNA RDA is relatively inexpensive to perform and requires no prior knowledge of genome sequence data. The combining of PCR with a subtractive methodology results in a highly effective and extremely sensitive technique with application to very low amounts of starting material. The procedure can be divided into three main phases: PCR generation of amplicons representative of the starting populations of RNA molecules being compared; the two-step subtractive hybridization of these representations, leading to the enrichment of amplified fragments of differentially expressed genes and the sequential depletion of sequences common to both populations; and the purification, cloning, and sequencing of the resulting difference products.