The role of low-level lactate production in airway inflammation in asthma

Warburg and coworkers (Warburg O, Posener K, Negelein E. Z Biochem 152: 319, 1924) first reported that cancerous cells switch glucose metabolism from oxidative phosphorylation to aerobic glycolysis, and that this switch is important for their proliferation. Nothing is known about aerobic glycolysis in T cells from asthma. The objective was to study aerobic glycolysis in human asthma and the role of this metabolic pathway in airway hyperreactivity and inflammation in a mouse model of asthma. Human peripheral blood and mouse spleen CD4 T cells were isolated by negative selection. T cell proliferation was measured by thymidine incorporation. Cytokines and serum lactate were measured by ELISA. Mouse airway hyperreactivity to inhaled methacholine was measured by a FlexiVent apparatus. The serum lactate concentration was significantly elevated in clinically stable asthmatic subjects compared with healthy and chronic obstructive pulmonary disease controls, and negatively correlated with forced expiratory volume in 1 s. Proliferating CD4 T cells from human asthma and a mouse model of asthma produced higher amounts of lactate upon stimulation, suggesting a heightened glycolytic activity. Lactate stimulated and inhibited T cell proliferation at low and high concentrations, respectively. Dichloroacetate (DCA), an inhibitor of aerobic glycolysis, inhibited lactate production, proliferation of T cells, and production of IL-5, IL-17, and IFN-γ, but it stimulated production of IL-10 and induction of Foxp3. DCA also inhibited airway inflammation and hyperreactivity in a mouse model of asthma. We conclude that aerobic glycolysis is increased in asthma, which promotes T cell activation. Inhibition of aerobic glycolysis blocks T cell activation and development of asthma.     

Antifungal Activity of <Emphasis Type="Italic">Lactobacillus pentosus</Emphasis> ŁOCK 0979 in the Presence of Polyols and Galactosyl-Polyols

The antifungal activity of Lactobacillus pentosus ?OCK 0979 depends both on the culture medium and on the fungal species. In the control medium, the strain exhibited limited antagonistic activity against indicator food-borne molds and yeasts. However, the supplementation of the bacterial culture medium with polyols (erythritol, lactitol, maltitol, mannitol, sorbitol, xylitol) or their galactosyl derivatives (gal-erythritol, gal-sorbitol, gal-xylitol) enhanced the antifungal properties of Lactobacillus pentosus ?OCK 0979. Its metabolites were identified and quantified by enzymatic methods, HPLC, UHPLC-MS coupled with QuEChERS, and GC-MS. The presence of polyols and gal-polyols significantly affected the acid metabolite profile of the bacterial culture supernatant. In addition, lactitol and mannitol were used by bacteria as alternative carbon sources. A number of compounds with potential antifungal properties were identified, such as phenyllactic acid, hydroxyphenyllactic acid, and benzoic acid. Lactobacillus bacteria cultivated with mannitol synthesized hydroxy-fatty acids, including 2-hydroxy-4-methylpentanoic acid, a well-described antifungal agent. Scanning electron microscopy (SEM) and light microscopy confirmed a strong antifungal effect of L. pentosus ?OCK 0979.     

PC phosphorylation increases the ability of AFAP-110 to cross-link actin filaments

The actin filament-associated protein and Src-binding partner, AFAP-110, is an adaptor protein that links signaling molecules to actin filaments. AFAP-110 binds actin filaments directly and multimerizes through a leucine zipper motif. Cellular signals downstream of Src(527F) can regulate multimerization. Here, we determined recombinant AFAP-110 (rAFAP-110)-bound actin filaments cooperatively, through a lateral association. We demonstrate rAFAP-110 has the capability to cross-link actin filaments, and this ability is dependent on the integrity of the carboxy terminal actin binding domain. Deletion of the leucine zipper motif or PKC phosphorylation affected AFAP-110's conformation, which correlated with changes in multimerization and increased the capability of rAFAP-110 to cross-link actin filaments. AFAP-110 is both a substrate and binding partner of PKC. On PKC activation, stress filament organization is lost, motility structures form, and AFAP-110 colocalizes strongly with motility structures. Expression of a deletion mutant of AFAP-110 that is unable to bind PKC blocked the effect of PMA on actin filaments. We hypothesize that upon PKC activation, AFAP-110 can be cooperatively recruited to newly forming actin filaments, like those that exist in cell motility structures, and that PKC phosphorylation effects a conformational change that may enable AFAP-110 to promote actin filament cross-linking at the cell membrane.     

A Founder Mutation in the GK1 Gene Is Responsible for Galactokinase Deficiency in Roma (Gypsies)

Galactokinase deficiency is an inborn error in the first step of galactose metabolism. Its major clinical manifestation is the development of cataracts in the first weeks of life. It has also been suggested that carriers of the deficiency are predisposed to presenile cataracts developing at age 20-50 years. Newborn screening data suggest that the gene frequency is very low worldwide but is higher among the Roma in Europe. Since the cloning of the galactokinase gene (GK1) in 1995, only two disease-causing mutations, both confined to single families, have been identified. Here we present the results of a study of six affected Romani families from Bulgaria, where index patients with galactokinase deficiency have been detected by the mass screening. Genetic linkage mapping placed the disease locus on 17q, and haplotype analysis revealed a small conserved region of homozygosity. Using radiation hybrid mapping, we have shown that GK1 is located in this region. The founder Romani mutation identified in this study is a single nucleotide substitution in GK1 resulting in the replacement of the conserved proline residue at amino acid position 28 with threonine (P28T). The P28T carrier rate in this endogamous population is approximately 5%, suggesting that the mutation may be an important cause of early childhood blindness in countries with a sizeable Roma minority.     

Spatial-specific action of serotonin within the leech midbody ganglion

Serotonin is a conspicuous neuromodulator in the nervous system of many vertebrates and invertebrates. In previous experiments performed in the leech nervous system, we compared the effect of the amine released from endogenous sources [using selective serotonin reuptake inhibitors (SSRIs), e.g. fluoxetine] with that of bath-applied serotonin. The results suggested that the amine does not reach all its targets in a uniform way, but produces the activation of an interneuronal pathway that generated specific synaptic responses on different neurons. Taking into account that the release of the amine is often regulated at the presynaptic level, we have investigated whether autoreceptor antagonists mimic the SSRIs effect. We found that methiothepin (100 microM) produced similar effects than fluoxetine. To further test the hypothesis that endogenous serotonin produce its effect by acting locally at specific sites, we analyzed the effect of iontophoretic applications of serotonin. We found a site in the neuropil of the leech ganglia where serotonin application mimicked the effect of the SSRIs and the 5-HT antagonist. The results further support the view that the effect of serotonin exhibits a spatial specificity that can be relevant to understand its modulatory actions.     

Temporal Trends of HLA,CTLA-4 and PTPN22 Genotype Frequencies among Type 1 Diabetes in Continental Italy

The incidence of type 1 diabetes has, progressively, increased worldwide over the last decades and also in Continental Italian population. Previous studies performed in northern European countries, showed, alongside a general increase in the disease incidence, a decreasing frequency of the highest risk HLA genotype in type 1 diabetes populations, thus emphasizing the role of environmental factors. The aim of the study was to evaluate whether a decreasing trend of high risk HLA, CTLA-4 and PTPN22 genotypes would be present in type 1 diabetes subjects of Continental Italy, a country considered at low incidence of the disease compared to northern European populations. N = 765 type 1 diabetes patients diagnosed from 1980 to 2012 in Lazio region were included. For HLA, CTLA4 and PTPN22 temporal trend evaluation, subjects were subdivided into groups of years according to age at diagnosis. All subjects were typed for HLA-DRB1 and DQB1 by a reverse line blot. The CT60 polymorphism of the CTLA4 and C1858T of the PTPN22 gene were genotyped using ABI PRISM 7900HT (n = 419 and n = 364 respectively). HLA genotypes were divided in high, moderate and low risk categories. The proportion of the HLA risk categories was not statistically different over the three decades in subjects with age of onset <15 years and ≥15 years. The genotype distribution of CT60 polymorphism of CTLA4 gene did not show any change in the frequencies during time. The analysis of the PTPN22 C1858T variant revealed, instead, that the frequency of CT+TT susceptibility genotypes decreased during time (23.9% vs 13.6%, p = 0.017). We can hypothesize that the pressure of the diabetogenic environment could be milder and therefore not sufficient to reduce the need of a strong genetic background (HLA) “to precipitate” diabetes; the increased pressure of the environment could have, instead, some effects on minor susceptibility genes in our population.     

Metabolomics of Human Amniotic Fluid and Maternal Plasma during Normal Pregnancy

Metabolic profiles of amniotic fluid and maternal blood are sources of valuable information about fetus development and can be potentially useful in diagnosis of pregnancy disorders. In this study, we applied ¹H NMR-based metabolic profiling to track metabolic changes occurring in amniotic fluid (AF) and plasma (PL) of healthy mothers over the course of pregnancy. AF and PL samples were collected in the 2^nd (T2) and 3^rd (T3) trimester, prolonged pregnancy (PP) until time of delivery (TD). A multivariate data analysis of both biofluids reviled a metabolic switch-like transition between 2^nd and 3^rd trimester, which was followed by metabolic stabilization throughout the rest of pregnancy probably reflecting the stabilization of fetal maturation and development. The differences were further tested using univariate statistics at α = 0.001. In plasma the progression from T2 to T3 was related to increasing levels of glycerol, choline and ketone bodies (3-hydroxybutyrate and acetoacetate) while pyruvate concentration was significantly decreased. In amniotic fluid, T2 to T3 transition was associated with decreasing levels of glucose, carnitine, amino acids (valine, leucine, isoleucine, alanine, methionine, tyrosine, and phenylalanine) and increasing levels of creatinine, succinate, pyruvate, choline, N,N-dimethylglycine and urocanate. Lactate to pyruvate ratio was decreased in AF and conversely increased in PL. The results of our study, show that metabolomics profiling can be used to better understand physiological changes of the complex interdependencies of the mother, the placenta and the fetus during pregnancy. In the future, these results might be a useful reference point for analysis of complicated pregnancies.     

Kinetic studies of the reaction of heme-thiolate enzyme chloroperoxidase with peroxynitrite

The kinetics of the reaction of chloroperoxidase with peroxynitrite was studied under neutral and acidic pH by stopped-flow spectrophotometry. Chloroperoxidase catalyzed peroxynitrite decay with the rate constant, k_c, increasing with decreasing pH. The values of k_c obtained at pH 5.1, 6.1 and 7.1 were equal to: (1.96 ± 0.03) × 10⁶, (1.63 ± 0.04) × 10⁶ and (0.71 ± 0.01) × 10⁶ M⁻¹ s⁻¹, respectively. Chloroperoxidase was converted to compound II by peroxynitrite with pH-dependent rate constants: (12.3 ± 0.4) × 10⁶ and (3.8 ± 0.3) × 10⁶ M⁻¹ s⁻¹ at pH 5.1 and 7.1, respectively. After most of peroxynitrite had disappeared, the conversion of compound II into the ferric form of chloroperoxidase was observed. The recovery of the native enzyme was completed within 1 s and 5 s at pH 5.1 and 7.1, respectively. The possible reaction mechanisms of the catalytic decomposition of peroxynitrite by chloroperoxidase are discussed.     

Silica spicules and axial filaments of the marine sponge Stelletta grubii (Porifera,Demospongiae)

Summary In all cases an organic axial filament within the silica spicules of Stelletta grubii forms the core of the major axes of the glass. In the small, star-shaped silica spicules (asters) the filament is shown for the first time to be radial with an enlarged center; in the large four-rayed spicules (triaenes) it is four-rayed; and in the large single-rayed spicules (oxeas) the filament is single-rayed. In situ, the filament is not dissolved by boiling nitric acid and thus is apparently protected by encasement within the glass which can also be stratified. The small silica asters are formed by single cells which resemble the so-called spherulous cells of other sponges. The very large size of triaenes and oxeas suggests that they may possibly be formed by more than one cell. The diameter of the filament in the much smaller asters is much narrower than the filament in the larger spicules, indicating a possible relationship between filament diameter and spicule diameter. While the axial filament in larger spicules frequently has a triangular cross-section it can also be hexaognal. Some aster filaments also retain a close to hexagonal cross-section. Filaments freed from large spicules by hydrofluoric acid display a complex morphology; possibly there is an internal silicified core. Some reported aspects of filament morphology are, however, probably artefacts of desilicification with hydrofluoric acid. Offprint requests to: T.L. Simpson, Department of Biology, University of Hartford, West Harford, Connecticut 06117, USA (Permanent affiliation)