De novo expression of uncoupling protein 3 is associated to enhanced mitochondrial thioesterase-1 expression and fatty acid metabolism in liver of fenofibrate-treated rats

Clavaminate synthase (CAS), a 2-oxoglutarate (2OG) dependent dioxygenase, catalyses three steps in the biosynthesis of clavulanic acid. Crystals of CAS complexed with Fe(II), 2OG and deoxyguanidinoproclavaminate were exposed to nitric oxide (NO) acting as a dioxygen analogue. Prior to exposure with NO, the active site Fe(II) is octahedrally coordinated by a water molecule, the 2-oxo and 1-carboxylate groups of 2OG, and the side-chains of an aspartyl and two histidinyl residues. NO binds to the position previously occupied by the 2OG 1-carboxylate concomitant with rearrangement of the latter to the position previously occupied by the displaced water.     

Purification,cofactor analysis,and site-directed mutagenesis of Synechococcus ferredoxin-nitrate reductase

The narB gene of the cyanobacterium Synechococcus sp. strain PCC 7942 encodes an assimilatory nitrate reductase that uses photosynthetically reduced ferredoxin as the physiological electron donor. This gene was expressed in Escherichia coli and electrophoretically pure preparations of the enzyme were obtained using affinity chromatography with either reduced-ferredoxin or NarB antibodies. The electronic absorption spectrum of the oxidized enzyme showed a shoulder at around 320 nm and a broad absorption band between 350 and 500 nm. These features are indicative of the presence of an iron-sulfur centre(s) and accordingly metal analysis showed ca. 3 atoms of Fe per molecule of protein that could represent a [3Fe-4S] cluster. Further analysis indicated the presence of 1 atom of Mo and 2 molecules of ribonucleotide-conjugated molybdopterin per molecule of protein. This, together with the requirement of a mobA gene for production of an active enzyme, strongly suggests the presence of Mo in the form of the bis-MGD (bis-molybdopterin guanine dinucleotide) cofactor in Synechococcusnitrate reductase. A model for the coordination of the Mo atom to the enzyme is proposed. Four conserved Cys residues were replaced by site-directed mutagenesis. The effects of these changes on the enzyme activity and electronic absorption spectra support the participation of those residues in iron-sulfur cluster coordination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evaluation in rabbits of different anti-SHIV vaccine strategies based on DNA/fowlpox priming and virus-like particle boosting

Two different prime-boost immunization protocols were tested in rabbits and their immune response was evaluated and compared with the final aim of defining a vaccine strategy that might be able to protect non-human primates from infection with the pathogenic simian/human immunodeficiency virus, SHIV(89.6P). The two regimens were based on three priming immunizations with either an expression plasmid plus a fowlpox (FP) recombinant vector or with two FP recombinant vectors, each one expressing either the SIV(mac239) gag/pol or the HIV-1env(89.6P) genes. In both protocols, priming immunizations were followed by two boosts with SHIV-mimicking virus-like particles (VLP). A complete SHIV-specific response was observed in all animals. Interestingly, the DNA vaccine was three to 10 times more efficient than the FP recombinant in inducing an anti-gag humoral response. Real-time PCR confirmed the memory effect on T-cell subsets secreting interleukin-4 and interferon-gamma, as a consequence of stimulation of both arms of the immune system. Although both protocols were almost equally effective in eliciting homologous neutralizing antibodies and highlighted the efficacy of VLP administration for boosting, protocol A seemed to be more effective in promoting a balanced T-cell memory immune response and appears more promising for vaccine purposes.     

Conformationally-restricted analogues and partition coefficients of the 5-HT3 serotonin receptor ligands meta-chlorophenylbiguanide (mCPBG) and meta-chlorophenylguanidine (mCPG)

The present investigation examined two features of arylbiguanide and arylguanidine 5-HT(3) ligands: conformation and partition coefficients. Several conformationally-constrained analogues of mCPBG (2) and mCPG (11; K(i)=32 nM) were prepared and of these only 2-amino-5-chloro-3,4-dihydroquinazoline (14; K(i)=34 nM) retained high affinity. The partition coefficient of compound 11 (LogP(app)=-0.64) was less than that of its corresponding arylbiguanide 2 (LogP(app)=-0.38). The quinazoline structure may represent a pharmacologically-active conformation of these agents, and the arylbiguanides were found more lipid soluble than their arylguanidine counterparts at physiological pH.     

Phase Variation in Xenorhabdus nematophilus

Xenorhabdus nematophilus is a symbiotic bacterium that inhabits the intestine of entomopathogenic nematodes. The bacterium-nematode symbiotic pair is pathogenic for larval-stage insects. The phase I cell type is the form of the bacterium normally associated with the nematode. A variant cell type, referred to as phase II, can form spontaneously under stationary-phase conditions. Phase II cells do not elaborate products normally associated with the phase I cell type. To better define phase variation in X. nematophilus, several strains (19061, AN6, F1, N2-4) of this bacterium were analyzed for new phenotypic traits. An analysis of pathogenicity in Manduca sexta larvae revealed that the phase II form of AN6 (AN6/II) was significantly less virulent than the phase I form (AN6/I). The variant form of N2-4 was also avirulent. On the other hand, F1/II and 19061/II were as virulent as the respective phase I cells. Strain 19061/II was found to be motile, and AN6/II regained motility when the bacteria were grown in low-osmolarity medium. In contrast, F1/II remained nonmotile. The phase II cells did not produce the outer membrane protein, OpnB, that is normally induced during the stationary phase. Both phase I and phase II cells were able to support nematode growth and development. These findings indicate that while certain phenotypic traits are common to all phase II cells, other characteristics, such as virulence and motility, are variable and can be influenced by environmental conditions.     

The narA Locus of Synechococcus sp. Strain PCC 7942 Consists of a Cluster of Molybdopterin Biosynthesis Genes

The narA locus required for nitrate reduction in Synechococcus sp. strain PCC 7942 is shown to consist of a cluster of genes, namely, moeA, moaC, moaD, moaE, and moaA, involved in molybdenum cofactor biosynthesis. The product of the moaC gene of strain PCC 7942 shows homology in its N-terminal half to MoaC from Escherichia coli and in its C-terminal half to MoaB or Mog. Overexpression of the Synechococcus moaC gene in E. coli resulted in the synthesis of a polypeptide of 36 kDa, a size that would conform to a protein resembling a fusion of the MoaC and MoaB or Mog polypeptides of E. coli. Insertional inactivation of the moeA, moaC, moaE, and moaA genes showed that the moeA-moa gene cluster is required for growth on nitrate and expression of nitrate reductase activity in strain PCC 7942. The moaCDEA genes constitute an operon which is transcribed divergently from the moeA gene. Expression of the moeA gene and the moa operon was little affected by the nitrogen source present in the culture medium.     

Exploration of peptides bound to MHC class I molecules in melanoma

Advancements in high‐resolution HPLC and mass spectrometry have reinvigorated the application of this technology to identify peptides eluted from immunopurified MHC class I molecules. Three melanoma cell lines were assessed using w6/32 isolation, peptide elution and HPLC purification; peptides were identified by mass spectrometry. A total of 13 829 peptides were identified; 83–87% of these were 8–11 mers. Only approximately 15% have been described before. Subcellular locations of the source proteins showed even sampling; mRNA expression and total protein length were predictive of the number of peptides detected from a single protein. HLA‐type binding prediction for 10 078 9/10 mer peptides assigned 88–95% to a patient‐specific HLA subtype, revealing a disparity in strength of predicted binding. HLA‐B*27‐specific isolation successfully identified some peptides not found using w6/32. Sixty peptides were selected for immune screening, based on source protein and predicted HLA binding; no new peptides recognized by antimelanoma T cells were discovered. Additionally, mass spectrometry was unable to identify several epitopes targeted ex vivo by one patient's T cells.     

3,5-Diiodo-L-Thyronine Activates Brown Adipose Tissue Thermogenesis in Hypothyroid Rats

3,5-diiodo-l-thyronine (T2), a thyroid hormone derivative, is capable of increasing energy expenditure, as well as preventing high fat diet-induced overweight and related metabolic dysfunction. Most studies to date on T2 have been carried out on liver and skeletal muscle. Considering the role of brown adipose tissue (BAT) in energy and metabolic homeostasis, we explored whether T2 could activate BAT thermogenesis. Using euthyroid, hypothyroid, and T2-treated hypothyroid rats (all maintained at thermoneutrality) in morphological and functional studies, we found that hypothyroidism suppresses the maximal oxidative capacity of BAT and thermogenesis, as revealed by reduced mitochondrial content and respiration, enlarged cells and lipid droplets, and increased number of unilocular cells within the tissue. In vivo administration of T2 to hypothyroid rats activated BAT thermogenesis and increased the sympathetic innervation and vascularization of tissue. Likewise, T2 increased BAT oxidative capacity in vitro when added to BAT homogenates from hypothyroid rats. In vivo administration of T2 to hypothyroid rats enhanced mitochondrial respiration. Moreover, UCP1 seems to be a molecular determinant underlying the effect of T2 on mitochondrial thermogenesis. In fact, inhibition of mitochondrial respiration by GDP and its reactivation by fatty acids were greater in mitochondria from T2-treated hypothyroid rats than untreated hypothyroid rats. In vivo administration of T2 led to an increase in PGC-1α protein levels in nuclei (transient) and mitochondria (longer lasting), suggesting a coordinate effect of T2 in these organelles that ultimately promotes net activation of mitochondrial biogenesis and BAT thermogenesis. The effect of T2 on PGC-1α is similar to that elicited by triiodothyronine. As a whole, the data reported here indicate T2 is a thyroid hormone derivative able to activate BAT thermogenesis.     

Verbascoside down‐regulates some pro‐inflammatory signal transduction pathways by increasing the activity of tyrosine phosphatase SHP‐1 in the U937 cell line

Polyphenols are the major components of many traditional herbal remedies, which exhibit several beneficial effects including anti‐inflammation and antioxidant properties. Src homology region 2 domain‐containing phosphatase‐1 (SHP‐1) is a redox sensitive protein tyrosine phosphatase that negatively influences downstream signalling molecules, such as mitogen‐activated protein kinases, thereby inhibiting inflammatory signalling induced by lipopolysaccharide (LPS). Because a role of transforming growth factor β‐activated kinase‐1 (TAK1) in the upstream regulation of JNK molecule has been well demonstrated, we conjectured that SHP‐1 could mediate the anti‐inflammatory effect of verbascoside through the regulation of TAK‐1/JNK/AP‐1 signalling in the U937 cell line. Our results demonstrate that verbascoside increased the phosphorylation of SHP‐1, by attenuating the activation of TAK‐1/JNK/AP‐1 signalling. This leads to a reduction in the expression and activity of both COX and NOS. Moreover, SHP‐1 depletion deletes verbascoside inhibitory effects on pro‐inflammatory molecules induced by LPS. Our data confirm that SHP‐1 plays a critical role in restoring the physiological mechanisms of inducible proteins such as COX2 and iNOS, and that the down‐regulation of TAK‐1/JNK/AP‐1 signalling by targeting SHP‐1 should be considered as a new therapeutic strategy for the treatment of inflammatory diseases.     

Sprouty2 in the Dorsal Hippocampus Regulates Neurogenesis and Stress Responsiveness in Rats

Both the development and relief of stress-related psychiatric conditions such as major depression (MD) and post-traumatic stress disorder (PTSD) have been linked to neuroplastic changes in the brain. One such change involves the birth of new neurons (neurogenesis), which occurs throughout adulthood within discrete areas of the mammalian brain, including the dorsal hippocampus (HIP). Stress can trigger MD and PTSD in humans, and there is considerable evidence that it can decrease HIP neurogenesis in laboratory animals. In contrast, antidepressant treatments increase HIP neurogenesis, and their efficacy is eliminated by ablation of this process. These findings have led to the working hypothesis that HIP neurogenesis serves as a biomarker of neuroplasticity and stress resistance. Here we report that local alterations in the expression of Sprouty2 (SPRY2), an intracellular inhibitor of growth factor function, produces profound effects on both HIP neurogenesis and behaviors that reflect sensitivity to stressors. Viral vector-mediated disruption of endogenous Sprouty2 function (via a dominant negative construct) within the dorsal HIP of adult rats stimulates neurogenesis and produces signs of stress resilience including enhanced extinction of conditioned fear. Conversely, viral vector-mediated elevation of SPRY2 expression intensifies the behavioral consequences of stress. Studies of these manipulations in HIP primary cultures indicate that SPRY2 negatively regulates fibroblast growth factor-2 (FGF2), which has been previously shown to produce antidepressant- and anxiolytic-like effects via actions in the HIP. Our findings strengthen the relationship between HIP plasticity and stress responsiveness, and identify a specific intracellular pathway that could be targeted to study and treat stress-related disorders.