The acetate recovery factor to correct tracer-derived dietary fat oxidation in humans

When using (13)C tracer to measure plasma fat oxidation, an acetate recovery factor should be determined in every subject to correct for label sequestration. Less is known regarding the acetate recovery factor for dietary fatty acid oxidation. We compiled data from six studies to investigate the determinants of the dietary acetate recovery factor (dARF) at rest and after physical activity interventions and compared the effects of different methods of dARF calculation on both the fat oxidation and its variability. In healthy lean subjects, dARF was 50.6 +/- 5.4% dose (n = 56) with an interindividual coefficient of variation of 10.6% at rest and 9.2% after physical activity modifications. The physical activity interventions did not impact dARF, and the intraindividual coefficient of variation was 4.6%. No major anthropological or physiological determinants were detected except for resting metabolic rate, which explains 7.4% of the dARF variability. Applying an individual or an average group dARF did not affect the mean and the variability of the derived dietary lipid oxidation at rest or after physical activity interventions. Using a mean dARF for a group leads to over- or underestimation of fat oxidation of less than 10% in individual subjects. Moreover, the use of a group or individual correction did not affect the significant relationship found between fasting respiratory exchange ratio and dietary fat oxidation. These data indicate that an average dARF can be applied for longitudinal and cross-sectional studies investigating dietary lipid metabolism.     

Vitamin D signaling in immune-mediated disorders: Evolving insights and therapeutic opportunities

1,25(OH)(2)D(3), the active form of vitamin D, is a central player in calcium and bone metabolism. More recently, important immunomodulatory effects have been attributed to this hormone. The widespread presence of the vitamin D receptor (VDR) in the immune system and the expression of the enzymes responsible for the synthesis of the active 1,25(OH)(2)D(3) regulated by specific immune signals, even suggest a paracrine immunomodulatory role for 1,25(OH)(2)D(3). Additionally, the different molecular mechanisms used by 1,25(OH)(2)D(3) to exert its immunomodulatory effects prove of a broad action radius for this compound. Both, the effects of vitamin D deficiency and/or absence of the VDR as well as intervention with pharmacological doses of 1,25(OH)(2)D(3) or one of its less-calcemic analogs, affects immune system behavior in different animal models of immune-mediated disorders, such as type 1 diabetes. This review aims to summarize the data as they stand at the present time on the role of vitamin D in the pathogenesis of immune-mediated disorders, with special focus on type 1 diabetes, and on the therapeutic opportunities for vitamin D in the prevention and treatment of this autoimmune disease in mouse models and humans.     

Unusual intracellular trafficking of Salmonella typhimurium in human melanoma cells

Salmonella spp. are enterobacteria capable of invading and replicating in both professional and non‐professional phagocytes. Here, we investigate the fate of S. typhimurium in human melanoma MelJuSo cells. The bacterium entered MelJuSo cells by a trigger mechanism and resided within a unique organelle, the Salmonella‐containing vacuole (SCV). The SCV acquired early endosomal markers transiently and then underwent a series of membrane modifications. In HeLa cells, vacuole maturation is characterized by the simultaneous acquisition of the lysosomal membrane glycoproteins (Lgps) Lamp1, CD63 and vacuolar (v)‐ATPase; in MelJuSo cells, however, acquisition of CD63 and v‐ATPase preceded that of Lamp1. A very striking event in MelJuSo cells was the arrest of bacterial septation starting from 8 h after infection. Bacteria nevertheless continued to elongate, remained morphologically intact and viable and were eventually exocytosed. This original feature was observed in several skin‐related cells including melanocytes, suggesting that it may provide the basis for an efficient host defence mechanism against Salmonella infection.     

Mode shift of an inhaled aerosol bolus is correlated with flow sequencing in the human lung.

We studied the effects on aerosol bolus inhalations of small changes in convective inhomogeneity induced by posture change from upright to supine in nine normal subjects. Vital capacity single-breath nitrogen washout tests were used to determine ventilatory inhomogeneity change between postures. Relative to upright, supine phase III slope was increased 33 +/- 11% (mean +/- SE, P < 0.05) and phase IV height increased 25 +/- 11% (P < 0.05), consistent with an increase in convective inhomogeneity likely due to increases in flow sequencing. Subjects also performed 0.5-microm-particle bolus inhalations to penetration volumes (V(p)) between 150 and 1,200 ml during a standardized inhalation from residual volume to 1 liter above upright functional residual capacity. Mode shift (MS) in supine posture was more mouthward than upright at all V(p), changing by 11.6 ml at V(p) = 150 ml (P < 0.05) and 38.4 ml at V(p) = 1,200 ml (P < 0.05). MS and phase III slope changes correlated positively at deeper V(p). Deposition did not change at any V(p), suggesting that deposition did not cause the MS change. We propose that the MS change results from increased sequencing in supine vs. upright posture.     

Effects of long-term spaceflight on mechanical properties of muscles in humans

The effects of long-term spaceflight(90-180 days) on the contractile and elastic characteristics ofthe human plantarflexor muscles were studied in 14 cosmonauts beforeand 2-3 days after landing. Despite countermeasures practicedaboard, spaceflight was found to induce a decrease in maximal isometrictorque (17%), whereas an index of maximal shortening velocity wasfound to increase (31%). In addition, maximal muscle activationevaluated during isokinetic tests decreased by 39%. Changes inmusculotendinous stiffness and whole joint stiffness were characterizedby means of quick-release movements and sinusoidal perturbations.Musculotendinous stiffness was found to be increased by 25%. Wholejoint stiffness decreased under passive conditions (21%), whereaswhole joint stiffness under active conditions remained unchanged afterspaceflight (1%). This invariance suggests an adaptive mechanism tocounterbalance the decrease in stiffness of passive structures by anincreased active stiffness. Changes in neural drive could participatein this equilibrium.

     

Synthesis and potential anti-inflammatory activity of some tetrahydrophthalazinones

A solid-phase route for the preparation of 4a,5,8,8a-tetrahydrophthalazinon-1-ones employing the Diels-Alder reaction has been developed. Some of the new compounds have been tested for inhibition of LPS-stimulated TNF-alpha production in human whole blood from patients with chronic obstructive pulmonary disease (COPD). This evaluation revealed two compounds 17 and 18 of interest, incorporating an arylpiperazine moiety, which were found to inhibit LPS-induced TNF-alpha release like the well known anti-inflammatory PDE4 inhibitors, rolipram and roflumilast.     

Vitamin D and 1,25-dihydroxyvitamin D3 as modulators in the immune system

Treatment from weaning until old age with 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)) prevents diabetes in NOD mice. It is mainly through its actions on dendritic cells (DCs), that 1,25(OH)(2)D(3) changes the function of potentially autoreactive T lymphocytes. In contrast, early life treatment (from 3 to 70 days of age) of NOD mice with vitamin D or 1,25(OH)(2)D(3) did not influence final diabetes incidence at 200 days of age. Also in spontaneous diabetic BB rats, diabetes could not be prevented by early life treatment (from 3 to 50 days of age) with vitamin D (1000 IU per day) or 1,25(OH)(2)D(3) (0.2 microg/kg per day or 1 microg/kg per 2 days). However, when NOD mice were made vitamin D deficient in early life (until 100 days of age), diabetes onset occurred earlier and final incidence was increased. These data further support a role for vitamin D and its metabolites in the pathogenesis of type 1 diabetes in NOD mice.     

Heme as an optical probe of a conformational transition of ovine recPrP

The prion protein occurs as a globular domain and a leading fragment whose structure is not well-defined. For the ovine species, all of the tryptophan residues are in the initial fragment, while the globular domain is rich in tyrosine residues. Using heme as a spectroscopic probe, we have studied the recombinant prion protein before and after a temperature-induced conformational change. As for most heme proteins, the absorption spectrum of heme-CO displays a red shift upon binding to the protein, and both the Y and W fluorescence are highly quenched. Flash photolysis kinetics of the PrP-heme-CO complex shows a low yield for the bimolecular phase, indicating a pocket around the hemes. By comparing the holoprotein and the truncated sequence corresponding to the globular domain, the stoichiometry was determined to be five hemes for the globular domain and two hemes for the leading fragment. At high temperature, the hemes are released; upon cooling, only two hemes bind, and only the tryptophan fluorescence is quenched; this would indicate that the globular domain has formed a more compact structure, which is inert with respect to the hydrophobic probe. The final state of polymerization is perturbed if the synthetic peptide "N3" (PrP residues 142-166, which include the first helix) is added to the prion protein solution; the temperature cycle no longer reduces the number of heme binding sites. This would indicate that the peptide may alter or inhibit the polymer formation.     

Specific recognition of rpsO mRNA and 16S rRNA by Escherichia coli ribosomal protein S15 relies on both mimicry and site differentiation

The ribosomal protein S15 binds to 16S rRNA, during ribosome assembly, and to its own mRNA (rpsO mRNA), affecting autocontrol of its expression. In both cases, the RNA binding site is bipartite with a common subsite consisting of a G*U/G-C motif. The second subsite is located in a three-way junction in 16S rRNA and in the distal part of a stem forming a pseudoknot in Escherichia coli rpsO mRNA. To determine the extent of mimicry between these two RNA targets, we determined which amino acids interact with rpsO mRNA. A plasmid carrying rpsO (the S15 gene) was mutagenized and introduced into a strain lacking S15 and harbouring an rpsO-lacZ translational fusion. Analysis of deregulated mutants shows that each subsite of rpsO mRNA is recognized by a set of amino acids known to interact with 16S rRNA. In addition to the G*U/G-C motif, which is recognized by the same amino acids in both targets, the other subsite interacts with amino acids also involved in contacts with helix H22 of 16S rRNA, in the region adjacent to the three-way junction. However, specific S15-rpsO mRNA interactions can also be found, probably with A(-46) in loop L1 of the pseudoknot, demonstrating that mimicry between the two targets is limited.