Influence of low dietary calcium during pregnancy and lactation on zinc levels in maternal blood and bone in rats.

The effect of low dietary calcium on maternal zinc nutritional status was studied. Two groups of 6 adult female Wistar rats were fed during pregnancy and lactation with experimental diets containing either 0.2 g (LCa) or 0.6 g (NCa) of calcium/100 g. Both diets contained/100 g: 20.0 g protein (potassium caseinate), 3.5 mg Zn, 0.6 g P. A third group (n = 6) was fed a "stock diet" (SG), containing/100 g: 24.8 g protein, 1.5 g Ca, 0.6 g P, 11.6 mg Zn. Maternal blood samples were drawn from the tail before mating (To), at delivery (D) and at weaning (W); dams were sacrificed at weaning and the right femur was excised. Determinations (atomic absorption spectrometry) were: Zinc in red blood cells (RBC), Zn and Ca in ashed femur. The results (mean +/- SD) were: RBCZn (microg/mL) at To: 8.65 +/- 1.80, which did not change in the SG or in the NCa groups, but increased significantly in the LCa group (p < 0.001) (D: 18.20 +/- 4.63; W: 26.70 +/- 6.02), regarding To. Femur Zn (microg/100 mg) showed an increase (p < 0.001) in the LCa group (30.2 +/- 2.1) regarding both SG (25.3 +/- 0.7) and NCa groups (24.1 +/- 0.7). Femur Ca (mg/100 mg) decreased (p < 0.05) in the LCa group (19.2 +/- 0.9) regarding both SG (24.0 +/- 0.5) and NCa groups (21.4 +/- 0.7) and leading to a significant increase in Zn/Ca ratio (p < 0.001) in the LCa group. Therefore, dietary calcium deficiency during pregnancy and lactation would produce an increase of Zn utilization, reflected in the increase of maternal blood Zn levels and in femur Zn content.     

Localization of an alkyl-acetyl-glycerol-CDP-choline: cholinephosphotransferase activity in submitochondrial fractions of Tetrahymena pyriformis

Tetrahymena pyriformis contains platelet-activating factor (PAF) as a minor lipid, which is biosynthesized de novo. A dithiothreitol-insensitive CDP-choline:cholinephosphotransferase (AAG-CPT), which utilizes alkyl-acetyl-glycerol as a substrate, had been detected in both the mitochondrial and microsomal fractions of the protozoan. In the present report, localization of this enzyme in submitochondrial fractions was studied. Cell fractionation was evaluated with enzyme and morphological markers. In this respect, succinate dehydrogenase, NADPH:cytochrome c reductase, glucose-6-phosphatase, alkaline phosphatase, monoaminoxidase, and cytochrome c oxidase activities were investigated. In the presence of antimycin A, mitochondrial activity of NADPH-cytochrome c reductase, was increased, while the microsomal one was reduced. Cardiolipin was distributed in the inner mitochondrial membrane. Alkaline phosphatase was found exclusively in the cytosol of the protozoan. The main portion of the dithiothreitol-insensitive AAG-CPT was localized in the inner mitochondrial membrane. Our data indicate that mitochondria are able to produce PAF, which might be associated with their function.     

Synovial histopathology of psoriatic arthritis,both oligo- and polyarticular,resembles spondyloarthropathy more than it does rheumatoid arthritis

At present only few biological data are available to indicate whether psoriatic arthritis (PsA) is part of the spondyloarthropathy (SpA) concept, whether it is a separate disease entity or a heterogeneous disease group with oligoarticular/axial forms belonging to SpA and polyarticular forms resembling rheumatoid arthritis (RA). To address this issue with regard to peripheral synovitis, we compared the synovial characteristics of PsA with those of ankylosing spondylitis (AS)/undifferentiated SpA (USpA) and RA, and compared the synovium of oligoarticular versus polyarticular PsA. Synovial biopsies were obtained from patients with RA, nonpsoriatic SpA (AS + USpA), and oligoarticular and polyarticular PsA. The histological analysis included examination(s) of the lining layer thickness, vascularity, cellular infiltration, lymphoid aggregates, plasma cells and neutrophils. Also, we performed immunohistochemical assessments of CD3, CD4, CD8, CD20, CD38, CD138, CD68, CD163, CD83, CD1a, CD146, α_Vβ₃, E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, S100A12, intracellular citrullinated proteins and major histocompatibility complex (MHC)–human cartilage (HC) gp39 peptide complexes. Comparing SpA (PsA + AS + USpA) with RA, vascularity, and neutrophil and CD163⁺ macrophage counts were greater in SpA (P < 0.05), whereas lining layer thickness and the number of CD83⁺ dendritic cells were greater in RA (P < 0.05). In RA, 44% of samples exhibited positive staining for intracellular citrullinated proteins and 46% for MHC–HC gp39 peptide complexes, whereas no staining for these markers was observed in SpA samples. We excluded influences of disease-modifying antirheumatic drug and/or corticosteroid treatment by conducting systematic analyses of treated and untreated subgroups. Focusing on PsA, no significant differences were observed between PsA and nonpsoriatic SpA. In contrast, vascularity (P < 0.001) and neutrophils were increased in PsA as compared with RA (P = 0.010), whereas staining for intracellular citrullinated proteins and MHC–HC gp39 peptide complexes was exclusively observed in RA (both P = 0.001), indicating that the same discriminating features are found in PsA and other SpA subtypes compared with RA. Exploring synovial histopathology between oligoarticular and polyarticular PsA, no significant differences were noted. Moreover, intracellular citrullinated proteins and MHC–HC gp39 peptide complexes, which are specific markers for RA, were observed in neither oligoarticular nor polyarticular PsA. Taken together, these data indicate that the synovial histopathology of PsA, either oligoarticular or polyarticular, resembles that of other SpA subtypes, whereas both groups can be differentiated from RA on the basis of these same synovial features, suggesting that peripheral synovitis in PsA belongs to the SpA concept.     

Infiltration of the synovial membrane with macrophage subsets and polymorphonuclear cells reflects global disease activity in spondyloarthropathy

Considering the relation between synovial inflammation and global disease activity in rheumatoid arthritis (RA) and the distinct but heterogeneous histology of spondyloarthropathy (SpA) synovitis, the present study analyzed whether histopathological features of synovium reflect specific phenotypes and/or global disease activity in SpA. Synovial biopsies obtained from 99 SpA and 86 RA patients with active knee synovitis were analyzed for 15 histological and immunohistochemical markers. Correlations with swollen joint count, serum C-reactive protein concentrations, and erythrocyte sedimentation rate were analyzed using classical and multiparameter statistics. SpA synovitis was characterized by higher vascularity and infiltration with CD163⁺ macrophages and polymorphonuclear leukocytes (PMNs) and by lower values for lining-layer hyperplasia, lymphoid aggregates, CD1a⁺ cells, intracellular citrullinated proteins, and MHC–HC gp39 complexes than RA synovitis. Unsupervised clustering of the SpA samples based on synovial features identified two separate clusters that both contained different SpA subtypes but were significantly differentiated by concentration of C-reactive protein and erythrocyte sedimentation rate. Global disease activity in SpA correlated significantly with lining-layer hyperplasia as well as with inflammatory infiltration with macrophages, especially the CD163⁺ subset, and with PMNs. Accordingly, supervised clustering using these synovial parameters identified a cluster of 20 SpA patients with significantly higher disease activity, and this finding was confirmed in an independent SpA cohort. However, multiparameter models based on synovial histopathology were relatively poor predictors of disease activity in individual patients. In conclusion, these data indicate that inflammatory infiltration of the synovium with CD163⁺ macrophages and PMNs as well as lining-layer hyperplasia reflect global disease activity in SpA, independently of the SpA subtype. These data support a prominent role for innate immune cells in SpA synovitis and warrant further evaluation of synovial histopathology as a surrogate marker in early-phase therapeutic trials in SpA.     

Beneficial effect of auto-aggregating Lactobacillus crispatus on experimentally induced colitis in mice

We tested the therapeutic relevance of auto aggregation in lactobacilli by comparing the effect on DSS induced colitis of viable Lactobacillus crispatus M247, isolated from healthy humans, to L. crispatus MU5, an isogenic spontaneous mutants of M247, the latter lacking the auto aggregation phenotype which allows the adhesion to human mucus. Aggregating L. crispatus M247, but not the non-aggregating MU5, was retrievable from mice feces and adherent to the colonic mucosa. Daily administration of L. crispatus M247, but not heat killed L. crispatus M247 or aggregation deficient L. crispatus MU5, dose-dependently reduced the severity of DSS colitis. Indeed, L. crispatus MU5 administered in a 30% sucrose solution, known to restore the aggregation phenotype, had a protective effect comparable to mice receiving L. crispatus M247. These results indicate that a surface-mediated property such as aggregation may play a pivotal role in the protective effects obtained by dietary supplementation with L. crispatus M247 during colitis.     

Iron requirement of Lactobacillus spp. in completely chemically defined growth media

A completely chemically-defined growth medium, containing guanine, thymine, cytidine, 2'-deoxyadenosine and 2'-deoxyuridine as DNA precursors, was developed for Lactobacillus johnsonii, on the basis of statistically designed techniques suitable for other lactobacilli. Particular focus was given to the nucleotide composition of different defined media, and to the specific nucleotide requirements of Lact. johnsonii. Most of the lactobacilli tested grew in a medium containing five free bases, four ribonucleosides or five deoxyribonucleosides. Adenine and guanine were replaceable by inosine. The requirement for thymine and cytosine was satisfied with uracil. The presence of inosine and uracil was identified as being essential for the growth of different Lactobacillus species, displaying their inability to synthesize purines and pyrimidines de novo. Defined recipes with different nucleotide composition were used to investigate iron requirements of lactobacilli. Only marginal differences in growth were observed in iron-depleted media supplemented with five free bases, four ribonucleosides or five deoxyribonucleosides; iron depletion had a greater effect on growth when inosine and uracil were supplied as the only nucleotide sources. The results suggest that iron plays a role in the pyrimidine and purine metabolism of lactobacilli. Lactobacillus spp., particularly Lact. johnsonii, require iron under particular environmental conditions with limited or specific nucleotide sources.     

Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA

In trans-translation transfer messenger RNA (tmRNA) and small protein B (SmpB) rescue ribosomes stalled on truncated or in other ways problematic mRNAs. SmpB promotes the binding of tmRNA to the ribosome but there is uncertainty about the number of participating SmpB molecules as well as their ribosomal location. Here, the interaction of SmpB with ribosomal subunits and ribosomes was studied by isolation of SmpB containing complexes followed by chemical modification of ribosomal RNA with dimethyl sulfate, kethoxal and hydroxyl radicals. The results show that SmpB binds 30S and 50S subunits with 1:1 molar ratios and the 70S ribosome with 2:1 molar ratio. SmpB-footprints are similar on subunits and the ribosome. In the 30S subunit, SmpB footprints nucleotides that are in the vicinity of the P-site facing the E-site, and in the 50S subunit SmpB footprints nucleotides that are located below the L7/L12 stalk in the 3D structure of the ribosome. Based on these results, we suggest a mechanism where two molecules of SmpB interact with tmRNA and the ribosome during trans-translation. The first SmpB molecule binds near the factor-binding site on the 50S subunit helping tmRNA accommodation on the ribosome, whereas the second SmpB molecule may functionally substitute for a missing anticodon stem–loop in tmRNA during later steps of trans-translation.     

Bacteremia in Children Hospitalized with Respiratory Syncytial Virus Infection

Background

The risk of bacteremia is considered low in children with acute bronchiolitis. However the rate of occult bacteremia in infants with RSV infection is not well established. The aim was to determine the actual rate and predictive factors of bacteremia in children admitted to hospital due to confirmed RSV acute respiratory illness (ARI), using both conventional culture and molecular techniques.

Methods

A prospective multicenter study (GENDRES-network) was conducted between 2011–2013 in children under the age of two admitted to hospital because of an ARI. Among those RSV-positive, bacterial presence in blood was assessed using PCR for Meningococcus, Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, in addition to conventional cultures.

Results

66 children with positive RSV respiratory illness were included. In 10.6% patients, bacterial presence was detected: H. influenzae (n = 4) and S. pneumoniae (n = 2). In those patients with bacteremia, there was a previous suspicion of bacterial superinfection and had received empirical antibiotic treatment 6 out of 7 (85.7%) patients. There were significant differences in terms of severity between children with positive bacterial PCR and those with negative results: PICU admission (100% vs. 50%, P-value = 0.015); respiratory support necessity (100% vs. 18.6%, P-value < 0.001); Wood-Downes score (mean = 8.7 vs. 4.8 points, P-value < 0.001); GENVIP scale (mean = 17 vs. 10.1, P-value < 0.001); and length of hospitalization (mean = 12.1 vs. 7.5 days, P-value = 0.007).

Conclusion

Bacteremia is not frequent in infants hospitalized with RSV respiratory infection, however, it should be considered in the most severe cases.     

Penetration and localization of furocoumarins in single living cells studied by microspectrofluorometry

The microspectrofluorometric technique has been used to study the penetration and the localization of psoralen, 4,5′,8-trimethylpsoralen and 4′-aminomethyltrioxsalen in single living L-cells. The concentration of the different compounds inside the cell reached a plateau in 2 min with psoralen and aminomethyltrioxsalen and in 20 min with trioxsalen. Washing of the cells with culture medium produced only a partial removal of the three furocoumarins, distributed apparently in equivalent amount in the nucleus and cytoplasm.     

Nucleotide and derived amino acid sequence of the cyanogenic β-glucosidase (linamarase) from white clover (Trifolium repens L.)

The nucleotide sequence and derived amino acid sequence of two different -glucosidase cDNA clones were determined. One clone (TRE104) was identified as the cyanogenic -glucosidase by homology with the N-terminal and internal peptide amino acid sequence of the purified enzyme. The biological function of the other -glycosidase (TRE361) is not known. Co-segregation of genomic restriction fragments uniquely identified by each cDNA clone shows that these two genes are linked in the white clover genome. Both TRE104 and TRE361 fragments co-segregate with cyanogenic -glucosidase activity. Extensive homology was found between the white clover -glucosidase sequences and a group of prokaryote and mammalian -glycosidases. This group of sequences has no homology with a separate set of -glucosidase genes isolated from fungi and the thermophilic bacterium Clostridium thermocellum.