Distribution of glutathione transferases in Gram-positive bacteria and Archaea

Glutathione transferases (GSTs) have been widely studied in Gram-negative bacteria and the structure and function of several representatives have been elucidated. Conversely, limited information is available about the occurrence, classification and functional features of GSTs both in Gram-positive bacteria and in Archaea. An analysis of 305 fully-sequenced Gram-positive genomes highlights the presence of 49 putative GST genes in the genera of both Firmicutes and Actinobacteria phyla. We also performed an analysis on 81 complete genomes of the Archaea domain. Eleven hits were found in the Halobacteriaceae family of the Euryarchaeota phylum and only one in the Crenarchaeota phylum. A comparison of the identified sequences with well-characterized GSTs belonging to both Gram-negative and eukaryotic GSTs sheds light on their putative function and the evolutionary relationships within the large GST superfamily. This analysis suggests that the identified sequences mainly cluster in the new Xi class, while Beta class GSTs, widely distributed in Gram-negative bacteria, are under-represented in Gram-positive bacteria and absent in Archaea.     

Impaired myocardial metabolic reserve and substrate selection flexibility during stress in patients with idiopathic dilated cardiomyopathy

Under resting conditions, the failing heart shifts fuel use toward greater glucose and lower free fatty acid (FFA) oxidation. We hypothesized that chronic metabolic abnormalities in patients with dilated cardiomyopathy (DCM) are associated with the absence of the normal increase in myocardial glucose uptake and maintenance of cardiac mechanical efficiency in response to pacing stress. In 10 DCM patients and 6 control subjects, we measured coronary flow by intravascular ultrasonometry and sampled arterial and coronary sinus blood. Myocardial metabolism was determined at baseline, during atrial pacing at 130 beats/min, and at 15 min of recovery by infusion of [(3)H]oleate and [(13)C]lactate and measurement of transmyocardial arteriovenous differences of oxygen and metabolites. At baseline, DCM patients showed depressed coronary flow, reduced uptake and oxidation of FFA, and preferential utilization of carbohydrates. During pacing, glucose uptake increased by 106% in control subjects but did not change from baseline in DCM patients. Lactate release increased by 122% in DCM patients but not in control subjects. Cardiac mechanical efficiency in DCM patients was not different compared with control subjects at baseline but was 34% lower during stress. Fatty acid uptake and oxidation did not change with pacing in either group. Our results show that in DCM there is preferential utilization of carbohydrates, which is associated with reduced flow and oxygen consumption at rest and an impaired ability to increase glucose uptake during stress. These metabolic abnormalities might contribute to progressive cardiac deterioration and represent a target for therapeutic strategies aimed at modulating cardiac substrate utilization.     

The porcine <Emphasis Type="Italic">TBC1D1</Emphasis> gene: mapping,SNP identification,and association study with meat,carcass and production traits in Italian heavy pigs

TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1] is a Rab-GTPase-activating related protein implicated in regulating the trafficking of glucose transporter 4 (GLUT4 or SLC2A4) storage vesicles to the cell surface in response to insulin and AMPK-activating stimuli in skeletal muscle. Mutations in the human and mouse TBC1D1 genes confer risk of obesity or leanness. We identified five single nucleotide polymorphisms (SNPs) in the porcine TBC1D1 gene. One of them (FN677935:g.219G>A) was genotyped either by high resolution melting and PCR-RFLP analyses to study allele frequencies in a few pig breeds and evaluate association with meat production and carcass traits in five groups of sib-tested pigs of Italian Large White and Italian Duroc breeds. The g.219G>A SNP was associated (P < 0.05) with ham weight, back fat thickness and lean cuts content in Italian Large White and with visible intermuscular fat in Italian Duroc pigs.     

Structural differences in the hinge region of the glycoprotein hormone receptors: evidence from the sulfated tyrosine residues

Tyrosine sulfation is a late posttranslational modification of proteins that takes place in the Golgi network. In the past few years, this process has been identified as an important modulator of protein-protein interactions. Sulfated tyrosine residues have recently been identified in the C-terminal, so-called hinge region of the ectodomain of glycoprotein hormone receptors [TSH, LH/chorionic gonadotropin (CG), and FSH receptors] and were shown to play an important role in the interaction with their natural ligands. The position of two sulfated tyrosine residues in a Y-D/E-Y motif appears perfectly conserved in the alignment of TSH and LH receptors from different species, and site-directed mutagenesis experiments demonstrated that sulfation of the first residue of this motif was responsible for the functional effect on hormone binding. In contrast, the corresponding motif is not conserved in the FSH receptor, in which the first tyrosine residue is missing: the Y-D/E-Y motif is replaced by F(333)DY(335). We extend here our previous observation that, in this case, it is sulfation of the second sole tyrosine residue in the motif that is functionally important. An LH/CG receptor harboring an F(331)DY(333) motif (i.e. displaying decreased sensitivity to human CG) was used as a backbone in which short portions of the FSH receptor were substituted. Segments from the FSH receptor capable of restoring sensitivity to human CG were identified by transfection of the chimeras in COS-7 cells. These experiments identified key amino acid residues in the hinge region of the FSH receptor associated with the functional role of the second sulfated tyrosine residue in a Y-D/E-Y motif, allowing for efficient hormone binding. The experiments represent strong evidence that structural differences in the hinge regions of FSH and LH/CG receptors play a significant role in hormone-receptor-specific recognition.     

Gender-related effects on urine l-cystine metastability

Cystinuria is an autosomal recessive disease that causes l-cystine precipitation in urine and nephrolithiasis. Disease severity is highly variable; it is known, however, that cystinuria has a more severe course in males. The aim of this study was to compare l-cystine metastability in first-morning urine collected from 24 normal female and 24 normal male subjects. Samples were buffered at pH 5 and loaded with l-cystine (0.4 and 4 mM final concentration) to calculate the amount remaining in solution after overnight incubation at 4 °C; results were expressed as Z scores reflecting the l-cystine solubility in each sample. In addition, metabolomic analyses were performed to identify candidate compounds that influence l-cystine solubility. l-cystine solubility Z score was +0.44 ± 1.1 and ?0.44 ± 0.70 in female and male samples, respectively (p < 0.001). Further analyses showed that the l-cystine solubility was independent from urine concentration but was significantly associated with low urinary excretion of inosine (p = 0.010), vanillylmandelic acid (VMA) (p = 0.015), adenosine (p = 0.029), and guanosine (p = 0.032). In vitro l-cystine precipitation assays confirmed that these molecules induce higher rates of l-cystine precipitation in comparison with their corresponding dideoxy molecules, used as controls. In silico computational and modeling analyses confirmed higher binding energy of these compounds. These data indicate that urinary excretion of nucleosides and VMA may represent important factors that modulate l-cystine solubility and may represent new targets for therapy in cystinuria.     

Cytokine profile and proteome analysis in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis

The aim of this study was to analyze the type of immune response (Th1, Th2) and protein composition of bronchoalveolar lavage (BAL) of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Flow cytometry analysis of intracellular cytokines revealed different patterns: in IPF and SSc Th2 profiles were predominant, whereas in sarcoidosis Th1 prevailed. The proteomic analysis of BAL fluid (BALF) showed that there were quantitative differences between the three diseases. These were more evident between sarcoidosis and IPF, confirming our previous observations, whereas SSc had an intermediate profile between the two, however with some peculiarities. Comparison of BALF protein maps, constructed with the same quantity of total proteins, enabled us to identify the main profiles of the three diseases: an increase in plasma protein prevalent in sarcoidosis and also present in SSc, though for fewer proteins with respect to IPF and a greater abundance of low molecular weight proteins, mainly locally produced, in IPF. These findings are in line with the different pathogenesis of these diseases: IPF is considered a prevalently fibrotic disorder limited to the lung, with intense local production of functionally different proteins, whereas sarcoidosis and SSc are systemic immunoinflammatory diseases.     

Immune Regulatory Neural Stem/Precursor Cells Protect from Central Nervous System Autoimmunity by Restraining Dendritic Cell Function

Background

The systemic injection of neural stem/precursor cells (NPCs) provides remarkable amelioration of the clinico-pathological features of experimental autoimmune encephalomyelitis (EAE). This is dependent on the capacity of transplanted NPCs to engage concurrent mechanisms of action within specific microenvironments in vivo. Among a wide range of therapeutic actions alternative to cell replacement, neuroprotective and immune modulatory capacities of transplanted NPCs have been described. However, lacking is a detailed understanding of the mechanisms by which NPCs exert their therapeutic plasticity. This study was designed to identify the first candidate that exemplifies and sustains the immune modulatory capacity of transplanted NPCs.

Methodology/Principal Findings

To achieve the exclusive targeting of the peripheral immune system, SJL mice with PLP-induced EAE were injected subcutaneously with NPCs and the treatment commenced prior to disease onset. NPC-injected EAE mice showed significant clinical improvement, as compared to controls. Exogenous NPCs lacking the expression of major neural antigens were reliably (and for long-term) found at the level of draining lymph nodes, while establishing sophisticated anatomical interactions with lymph node cells. Importantly, injected NPCs were never found in organs other than lymph nodes, including the brain and the spinal cord. Draining lymph nodes from transplanted mice showed focal up-regulation of major developmental stem cell regulators, such as BMP-4, Noggin and Sonic hedgehog. In lymph nodes, injected NPCs hampered the activation of myeloid dendritic cells (DCs) and steadily restrained the expansion of antigen-specific encephalitogenic T cells. Both ex vivo and in vitro experiments identified a novel highly NPC-specific–BMP-4-dependent–mechanism hindering the DC maturation.

Conclusion/Significance

The study described herein, identifies the first member of the TGF β/BMP family of stem cell regulators as a novel tolerogenic factor released by NPCs. Full exploitation of this pathway as an efficient tool for vaccination therapy in autoimmune inflammatory conditions is underway.     

Stereospecific synthesis of "para-hydroxymexiletine" and sodium channel blocking activity evaluation

Both enantiomers of "para-hydroxymexiletine" (PHM), one of the main metabolites of mexiletine, were synthesized and fully characterized. Properties of (R)- and (S)-PHM, in terms of blocking potency and stereoselectivity on frog skeletal muscle Na(+) channels, were evaluated. The presence of a hydroxy group on the aryloxy moiety in the 4-position, as in PHM, reduced potency with respect to mexiletine in reducing I(Na max). However, PHM showed clear use-dependent behavior similar to that of mexiletine and, in contrast with what is observed with the parent compound, maintained its stereoselectivity during the use-dependent block. Chirality 16:72-78, 2004.