Free-range layer chickens as a source of <Emphasis Type="Italic">Campylobacter</Emphasis> bacteriophage

Bacteriophage specific for Campylobacter were isolated from chicken excreta collected from established free-range layer breed stock. Bacteriophage were either propagated on a Campylobacter jejuni host with broad susceptibility to bacteriophage (NCTC 12662) or on Campylobacter isolates from the same samples. Campylobacters were confirmed as being C. jejuni and or C. coli, using a combination of standard biochemical tests and PCR analysis with genus and species specific primers. The bacteriophage displayed differential patterns of susceptibility against reference NCTC strains and contemporary C. jejuni /C. coli isolates from chicken excreta. Electron microscopy demonstrated that the phage possessed icosahedral heads and rigid contractile tails. Pulsed-field gel electrophoresis revealed the bacteriophage genomes to be double stranded DNA in the range of 140 kb in size and the restriction enzyme patterns of the DNAs indicate they are genetically related members of the Myoviridae family. This study showed that Campylobacter bacteriophage could easily be isolated from free-range chickens and form part of their normal microbiological biota of environmentally exposed birds.     

The importance of stress and genetic variation in human aggression

Both genetic and environmental factors have key roles in determining aggressive tendencies. In particular, reaction to stress appears to be an important factor in precipitating aggressive episodes and individuals may vary in their ability to cope with stressful environments depending on their genetic make up. Evidence from humans and primates indicates that adverse rearing conditions may interact with variants in stress and neurotransmitter pathway genes leading to antisocial and/or violent behaviour. Common alleles of some serotonin pathway genes, including those involved in its degradation (monoamine oxidase A, MAOA), or its re-uptake into pre-synaptic neurones (serotonin transporter, SERT) have been shown to confer functional variation. Examination of the interaction between the alleles of such polymorphisms (in particular those affecting MAOA) and environmental stressors suggest that they may provide protection against, or increase sensitivity to, abusive upbringing; an observation that may explain part of the variability in developmental outcomes associated with maltreatment.     

Allosteric control model of bone remodelling containing periodical modes

To help to understand the modelling process that occurs when a scaffold is implanted it is vital to understand the rather complex bone remodelling process prevalent in native bone. We have formulated a mathematical model that predicts osteoactivity both in scaffolds, as well as in bone in vivo and could set a basis for the more detailed allosteric models. The model is extended towards a bio-cybernetic vision of basic multicellular unit (BMU) action, when some of the regulation loops have been modified to reflect the allosteric control mechanisms, developed by Michaels-Menten, Hill, Koshland-Nemethy-Filmer, Monod-Wyman-Changeux. By implementation of this approach a four-dimensional system was obtained that shows steady cyclic behaviour using a wide range of constants with clear biological meaning. We have observed that a local steady state appears as a limiting cycle in multi-dimensional phase space and this is discussed in this paper. Physiological interpretation of this limiting four-dimension cycle possibly related to a conservative-like value has been proposed. Analysis and simulation of the model has shown an analogy between this conservative value, as a kind of substrate-energy regenerative potential of the bone remodelling system with a molecular nature, and to the classical physical value--energy. This dynamic recovery potential is directed against both mechanical and biomechanical damage to the bone. Furthermore, the current model has credibility when compared to the normal bone remodelling process. In the framework of widely recognised Hill mechanisms of allosteric regulation the cyclic attractor, described formerly for a pure cellular model, prevails for different forms of feedback control. This result indicates the viability of the proposed existence of a conservative value (analogous to energy) that characterises the recovery potential of the bone remodelling cycle. Linear stability analysis has been performed in order to determine the robustness of the basic state, however, additional work is required to study a wider range of constants.     

Sequence analysis and editing for bisulphite genomic sequencing projects

Bisulphite genomic sequencing is a widely used technique for detailed analysis of the methylation status of a region of DNA. It relies upon the selective deamination of unmethylated cytosine to uracil after treatment with sodium bisulphite, usually followed by PCR amplification of the chosen target region. Since this two-step procedure replaces all unmethylated cytosine bases with thymine, PCR products derived from unmethylated templates contain only three types of nucleotide, in unequal proportions. This can create a number of technical difficulties (e.g. for some base-calling methods) and impedes manual analysis of sequencing results (since the long runs of T or A residues are difficult to align visually with the parent sequence). To facilitate the detailed analysis of bisulphite PCR products (particularly using multiple cloned templates), we have developed a visually intuitive program that identifies the methylation status of CpG dinucleotides by analysis of raw sequence data files produced by MegaBace or ABI sequencers as well as Staden SCF trace files and plain text files. The program then also collates and presents data derived from independent templates (e.g. separate clones). This results in a considerable reduction in the time required for completion of a detailed genomic methylation project.     

Generalized substitution of isoencoding codons shortens the duration of papillomavirus L1 protein expression in transiently gene-transfected keratinocytes due to cell differentiation

Recently we reported that gene codon composition determines differentiation-dependent expression of the PV L1 genes in mouse primary keratinocytes (KCs) in vitro and in vivo (Zhao et al. 2005, Mol. Cell Biol. 25:8643–8655). Here, we investigated whether generalized substitution of isoencoding codons affects the duration of expression of PV L1 genes in mouse and human KCs in day 1 culture transiently transfected with native (Nat) and codon modified (Mod) L1 genes. Following transient transfection, KC continuously transcribed both Nat and Mod PV L1 genes for at least 12 days, with the levels of L1 mRNAs from the Mod L1 genes significantly higher than those from the Nat L1 genes. However, continuous L1 protein expression at day 9 post-transfection was observed for both mouse and human KCs transfected with the Nat L1 genes only. Further, aa-tRNAs prepared from D8 KC cultures enhanced translation of two PV Nat L1 DNAs in RRL lysate and PV Nat L1 mRNAs in D0 cell-free lysate, whereas aa-tRNAs from D0 KCs enhanced translation of PV Mod L1 mRNAs in D8 cell-free lysate. It appears that aa-tRNAs in less-differentiated and differentiated KCs differentially match the PV Nat and Mod L1 mRNAs to regulate their translations in vitro.     

Differential effect of cross-linking the CD98 heavy chain on fusion and amino acid transport in the human placental trophoblast (BeWo) cell line

CD98 (otherwise known as 4F2) is an integral membrane protein with multiple functions including amino acid transport, integrin activation, cell fusion and cell activation. The molecular mechanisms coordinating these multiple functions remain unclear. We have studied CD98 heavy chain (hc) function in a human placental trophoblast cell line (BeWo). We show that cross-linking of CD98hc by incubation of cells in the presence of functional monoclonal antibodies causes cellular re-distribution of the protein from the cytoplasm to the plasma membrane as measured by flow cytometry, western blotting and quantitative immuno-electron microscopy. The latter technique also indicated that CD98hc is trafficked between cell surface and cytoplasmic pools in vesicles. Increased cell surface CD98 correlates with increased cellular fusion in BeWo cells. In addition, we show reduced LAT 1 surface expression and neutral amino acid transport in the presence of the CD98 mabs. The results thus suggest that the function of CD98 in cell fusion is distinct from its role in cellular nutrient delivery.     

The role of tryptophan residues in the function and stability of the mechanosensitive channel MscS from Escherichia coli

Tryptophan (Trp) residues play important roles in many proteins. In particular they are enriched in protein surfaces involved in protein docking and are often found in membrane proteins close to the lipid head groups. However, they are usually absent from the membrane domains of mechanosensitive channels. Three Trp residues occur naturally in the Escherichia coli MscS (MscS-Ec) protein: W16 lies in the periplasm, immediately before the first transmembrane span (TM1), whereas W240 and W251 lie at the subunit interfaces that create the cytoplasmic vestibule portals. The role of these residues in MscS function and stability were investigated using site-directed mutagenesis. Functional channels with altered properties were created when any of the Trp residues were replaced by another amino acid, with the greatest retention of function associated with phenylalanine (Phe) substitutions. Analysis of the fluorescence properties of purified mutant MscS proteins containing single Trp residues revealed that W16 and W251 are relatively inaccessible, whereas W240 is accessible to quenching agents. The data point to a significant role for W16 in the gating of MscS, and an essential role for W240 in MscS oligomer stability.