Transcriptome analysis of peripheral blood mononuclear cells in human subjects following a 36 h fast provides evidence of effects on genes regulating inflammation, apoptosis and energy metabolism.

There is growing interest in the potential health benefits of diets that involve regular periods of fasting. While animal studies have provided compelling evidence that feeding patterns such as alternate-day fasting can increase longevity and reduce incidence of many chronic diseases, the evidence from human studies is much more limited and equivocal. Additionally, although several candidate processes have been proposed to contribute to the health benefits observed in animals, the precise molecular mechanisms responsible remain to be elucidated. The study described here examined the effects of an extended fast on gene transcript profiles in peripheral blood mononuclear cells from ten apparently healthy subjects, comparing transcript profiles after an overnight fast, sampled on four occasions at weekly intervals, with those observed on a single occasion after a further 24 h of fasting. Analysis of the overnight fasted data revealed marked inter-individual differences, some of which were associated with parameters such as gender and subject body mass. For example, a striking positive association between body mass index and the expression of genes regulated by type 1 interferon was observed. Relatively subtle changes were observed following the extended fast. Nonetheless, the pattern of changes was consistent with stimulation of fatty acid oxidation, alterations in cell cycling and apoptosis and decreased expression of key pro-inflammatory genes. Stimulation of fatty acid oxidation is an expected response, most likely in all tissues, to fasting. The other processes highlighted provide indications of potential mechanisms that could contribute to the putative beneficial effects of intermittent fasting in humans.

Metabolic shift of Escherichia coli under salt stress in the presence of glycine betaine.

An important area of food safety focuses on bacterial survival and growth in unfavorable environments. In order to understand how bacteria adapt to stresses other than nutrient limitation in batch cultures, we need to develop mechanistic models of intracellular regulation and metabolism under stress. We studied the growth of Escherichia coli in minimal medium with added salt and different osmoprotectants. To characterize the metabolic efficiency with a robust parameter, we identified the optical density (OD) values at the inflection points of measured "OD versus time" growth curves and described them as a function of glucose concentration. We found that the metabolic efficiency parameter did not necessarily follow the trend of decreasing specific growth rate as the salt concentration increased. In the absence of osmoprotectant, or in the presence of proline, the metabolic efficiency decreased with increasing NaCl concentration. However, in the presence of choline or glycine betaine, it increased between 2 and 4.5% NaCl before declining at 5% NaCl and above. Microarray analysis of the transcriptional network and proteomics analysis with glycine betaine in the medium indicated that between 4.5 and 5% NaCl, the metabolism switched from aerobic to fermentative pathways and that the response to osmotic stress is similar to that for oxidative stress. We conclude that, although the growth rate appeared to decrease smoothly with increasing NaCl, the metabolic strategy of cells changed abruptly at a threshold concentration of NaCl.

An appraisal of service users' structured activity requirements in an Irish forensic setting.

Participating in purposeful and structured daily activities is an important factor contributing to the health and well-being of forensic service users. A survey was carried out in an Irish forensic mental health setting to identify whether service users meet the standard of 25-h weekly activities, a standard set by the Quality Network for Forensic Mental Health Services, London. The findings indicate that 57 (61%) out of 93 service users fully meet the criteria. Furthermore, service users within the medium- and low-security environments appear to be engaging to an increased number of structured activities in comparison to those in acute units.

In vitro digestibility of beta-casein and beta-lactoglobulin under simulated human gastric and duodenal conditions: a multi-laboratory evaluation.

Initially the resistance to digestion of two cow's milk allergens, beta-casein, and beta-lactoglobulin (beta-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both beta-casein and beta-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of beta-casein digestion in the low-protease assay were slower, beta-Lg being pepsin resistant. During duodenal digestion, beta-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins.

Genomics of thermophilic campylobacter species.

The thermophilic Campylobacter species C. jejuni and C. coli are important human pathogens, which are major causes of bacterial gastroenteritis. The recent progress in genomics techniques has allowed for a rapid increase in our knowledge of the molecular biology of Campylobacter species, but needs to be matched by concurrent increases in our understanding of the unique biology of these organisms. Campylobacter species display significant levels of genomic variation via natural transformation, phase variation, plasmid transfer and infection with bacteriophages, and this poses a continuous challenge for studies on pathogenesis, physiology, epidemiology and evolution of Campylobacter. In this chapter we will review the current state of the art of the genomics of thermophilic Campylobacter species, and opportunities where genomics can further contribute to our understanding of the biology of these successful human pathogens.

Glycolytic enzymes associated with the cell surface of Streptococcus pneumoniae are antigenic in humans and elicit protective immune responses in the mouse.

Streptococcus pneumoniae is a leading cause of otitis media, sinusitis, pneumonia, bacteraemia and meningitis worldwide. The drawbacks associated with the limited number of various capsular polysaccharides that can be included in the polysaccharide-based vaccines focuses much attention on pneumococcal proteins as vaccine candidates. We extracted an enriched cell wall fraction from S. pneumoniae WU2. Approximately 150 soluble proteins could be identified by 2D gel electrophoresis. The proteins were screened by 2D-Western blotting using sera that were obtained longitudinally from children attending day-care centres at 18, 30 and 42 months of age and sera from healthy adult volunteers. The proteins were further identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Seventeen proteins were antigenic in children and adults, of which 13 showed an increasing antibody response with age in all eight children analysed. Two immunogenic proteins, fructose-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and a control protein with known low immunogenicity, heat shock protein 70 (DnaK), were expressed in Escherichia coli, purified and used to immunize mice. Mouse antibodies elicited to the recombinant (r) FBA and rGAPDH were cross-reactive with several genetically unrelated strains of different serotypes and conferred protection to respiratory challenge with virulent pneumococci. In addition, the FBA used in this study (NP_345117) does not have a human ortholog and warrants further investigation as a candidate for a pneumococcal vaccine. In conclusion, the immunoproteomics based approach utilized in the present study appears to be a suitable tool for identification of novel S. pneumoniae vaccine candidates.

Changes in the concentrations of free amino acids in milk during growth of Lactococcus lactis indicate biphasic nitrogen metabolism.

Analysis of the concentrations of free amino acids in milk during growth of Lactococcus lactis subsp, lactis revealed a biphasic pattern of change during the logarithmic phase. During the first period there was little overall change in the total concentration of amino acids in the medium. The second phase was characterized by increased net liberation of free amino acids. There were also qualitative differences in the amino acids that were taken up and utilized during each period. The concentrations of Val, Leu and Ile decreased only during the early phase, while those of Ser, Arg, Thr and Met decreased only during the second phase. Gly and Ala were utilized throughout logarithmic growth. Gly uptake appeared to be greater during the second period and accounted for the largest proportion of free amino acid utilization at this time. It is possible that the biphasic nature of amino acid nutrition was due to increased consumption in late log phase of peptides derived from milk proteins by proteolysis. Increased activity of the arginine deiminase pathway during late log phase was inferred from increased utilization of Arg and liberation of citrulline and ornithine.

Purification and Characterization of a Tripeptidase from Lactobacillus sake.

A tripeptidase was purified to homogeneity from the cell extract of Lactobacillus sake by ammonium sulfate precipitation, hydrophobic interaction chromatography, gel filtration chromatography, and two steps of anion exchange chromatography. After SDS-PAGE a single band of protein was detected of approximately 55 kDa. A similar molecular mass was estimated by gel filtration. The tripeptidase activity was optimal at pH 7.0 and at 40 degrees C. The enzyme was strongly inhibited by metal chelators, reducing agents, and bestatin while thiol group reagents, serine proteinase inhibitors, and aspartic proteinase inhibitors had no effect on the activity. The enzyme was activated by Mn(2+) and almost totally inhibited by Zn(2+) and to a lesser extent by Sn(2+). The enzyme only exhibited activity against tripeptides, and those hydrolyzed at higher rates were Ala-Ala-Ala, Ser-Ser-Ser, and Leu-Gly-Gly.

Cell membrane integrity and lysis in Lactococcus lactis: the detection of a population of permeable cells in post-logarithmic phase cultures.

A method was developed that enabled an analysis of the proportion of permeable cells in a culture of Lactococcus lactis. This used the fluorescence of propidium iodide (PI) when in contact with DNA and the impermeability of the intact cell membrane to this compound. A permeability index was suggested that expresses the PI-induced fluorescence of a cell suspension as a percentage of the value obtained from wholly permeabilized cells after treatment with cetyltrimethylammonium bromide. This method was applied to the determination of cell permeability in death phase cultures. A large proportion of unlysed cells was freely permeable to PI, a finding that may have some significance for the investigation of the role of cell lysis in cheese maturation. This method is suggested as a useful addition to the techniques available for the study of cell damage in a variety of fields, and for the screening of cheese starter bacteria.

A non-essential glutamyl aminopeptidase is required for optimal growth of Lactococcus lactis MG1363 in milk.

Degenerate PCR primers were designed from the N-terminal amino acid sequence of a glutamyl aminopeptidase (PepA) from Lactococcus lactis. These primers were used to screen a lambda library for clones containing the gene (pepA) encoding PepA. The DNA sequence of a 2.1 kb fragment containing pepA was determined. The sequence revealed the presence of one complete and two incomplete open reading frames (ORFs). The complete ORF encodes a putative protein of 353 amino acids with a predicted N-terminal sequence identical to that determined for purified PepA. The pepA gene was subcloned on an Escherichia coli plasmid vector and production of active PepA was confirmed by means of a zymogram. Mutants of L. lactis in which the pepA gene was inactivated grew to normal cell densities in milk but exhibited a reduced growth rate during the exponential phase. Thus whilst PepA is required for optimal growth it is not essential.

Monitoring tripeptidase activity using capillary electrophoresis. Comparison with the ninhydrin assay.

Capillary electrophoresis (CE) was used to assay the activity of a tripeptidase from a crude extract of Lactococcus lactis subsp. lactis NCDO 712 against the substrate, Gly-Gly-Phe and a comparison with a standard ninhydrin assay was made. Standard curves of the substrates and products showed a significantly variable colorimetric reaction to ninhydrin making accurate quantification of the tripeptidase problematic. The CE assay further demonstrated that the presence of contaminating enzymes in crude cell-free extracts can cause secondary reactions that are not apparent from the ninhydrin assay data. The CE assay was also able to generate enzyme kinetics data and monitor, during purification, the presence of co-eluting contaminating activities. The speed and sensitivity with CE allows routine analysis of the tripeptidase activity without any derivatization normally required for this enzyme.

Immunocytochemical localization of endopeptidase-24.11 in the nucleus tractus solitarius of the rat brain.

Recently, it has been hypothesized that the N-terminal portion of substance P (SP), SP(1-7), which results from the action of endopeptidase 24.11 (EC3.4.24.11), could be involved in mediating the depressor effects of baroreceptor afferent activation via its action on cells in the nucleus tractus solitarius (NTS). In this study, the binding of a monoclonal antibody to endopeptidase 24.11 was examined immunohistochemically at the level of the caudal medulla of the rat brain. By light microscopy, intense immunoreactivity was seen in the NTS, in fibers bordering the area postrema, and in the area postrema itself. After electron microscopy, endopeptidase 24.11-like immunoreactivity was seen to be associated with the cytoskeleton and plasma membrane in axons, dendrites and glial processes. Antigen was also associated with synaptic vesicles and plasma membranes in presynaptic terminals forming mainly axo-dendritic synapses typical of vagal afferent terminals involved in the baroreceptor reflex. Thus, endopeptidase 24.11 appears to be localized at sites where it could effectively process SP prior to its binding to postsynaptic receptors.

Initial observations of a kallikrein-like enzyme associated with the plasma membranes of rat adipocytes.

Homogenates of rat adipocytes and plasma membranes thereof were shown by radioimmunoassay to contain immunoreactive glandular kallikrein. On the basis of the hydrolysis of D-Val-Leu-Arg-p-nitroanilide, the kallikrein-like enzyme associated with the plasma membranes was found not to be stimulated by prior incubation with melittin or phospholipase A2. However, pre-incubation of the membrane preparation with trypsin did increase the activity of the enzyme. Furthermore, activation could also be achieved by incubating the plasma membranes with insulin at a dose that stimulated glucose uptake into intact adipocytes. On the other hand, incubation with insulin at a dose that did not increase glucose uptake into rat adipocytes was ineffective in activating the kallikrein-like enzyme.