Genetic and biochemical characterization of MbeA,the relaxase involved in plasmid ColE1 conjugative mobilization

MbeA is a 60 kDa protein encoded by plasmid ColE1. It plays a key role in conjugative mobilization. MbeA*, a slightly truncated version of MbeA, was purified for in vitro analysis. MbeA* catalysed DNA cleavage and strand-transfer reactions using oligonucleotides embracing the ColE1 nic site, which was mapped to 5'-(1469)CTGG/CTTA(1462)-3'. Thus MbeA is the relaxase for ColE1 conjugal mobilization, in spite of the fact that it lacks a three histidine motif considered the invariant signature of conjugative relaxases. Amino acid sequence comparisons suggest MbeA is nevertheless related to the common relaxase protein family. For instance, MbeA residue Y19 could correspond to the invariant tyrosine in Motif I, whereas H97, E104 and N106 may constitute the equivalent residues to the histidine triad in Motif III. This hypothesis was tested by site-directed mutagenesis. MbeA amino acid residues Y19, H97, E104 and N106 were changed to alanine. MbeA mutant N106A showed reduced oligonucleotide cleavage and strand-transfer activities, whereas mutation in the other three residues resulted in proteins without detectable activity, suggesting they are directly implicated in catalysis of DNA-cleavage and strand-transfer reactions. A double substitution of E104 and N106 by histidines, therefore reconstituting the canonical histidine triad, restored relaxase activities to 1% of wild type. Thus, MbeA is a variant of the common relaxase theme with a HEN signature motif, which has to be added to the canonical three histidine motif of previously reported relaxases.     

The action of extracellular NAD+ on gluconeogenesis in the perfused rat liver

In the rat liver NAD⁺ infusion produces increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption. The aim of the present work was to investigate the possible action of this agent on gluconeogenesis using lactate as a gluconeogenic precursor. Hemoglobin-free rat liver perfusion in antegrade and retrograde modes was used with enzymatic determination of glucose production and polarographic assay of oxygen uptake. NAD⁺ infusion into the portal vein (antegrade perfusion) produced a concentration-dependent (25–100 μM) transient inhibition of oxygen uptake and gluconeogenesis. For both parameters inhibition was followed by stimulation. NAD⁺ infusion into the hepatic vein (retrograde perfusion) produced only transient stimulations. During Ca²⁺-free perfusion the action of NAD⁺ was restricted to small transient stimulations. Inhibitors of eicosanoid synthesis with different specificities (indo-methacin, nordihydroguaiaretic acid, bromophenacyl bromide) either inhibited or changed the action of NAD⁺. The action of NAD⁺ on gluconeogenesis is probably mediated by eicosanoids synthesized in non-parenchymal cells. As in the fed state, in the fasted condition extracellular NAD⁺ is also able to exert two opposite effects, inhibition and stimulation. Since inhibition did not manifest significantly in retrograde perfusion it is likely that the generating signal is located in pre-sinusoidal regions.     

Effect of the simultaneous administration of glucocorticoids and IL-15 on human NK cell phenotype,proliferation and function

We have previously reported a synergistic effect between hydrocortisone (HC) and IL-15 on promoting natural killer (NK) cell expansion and function. In the present study, we extend our findings to methylprednisolone (MeP) and dexamethasone (Dex), thus ascribing to glucocorticoids (GCs) a general feature as positive regulators of IL-15-mediated effects on NK cells. We demonstrate that each GC when combined with IL-15 in cultures of peripheral blood (PB)-derived CD56⁺ cells induces increased expansion of CD56⁺CD3⁻ cells displaying high cytolytic activity, IFN-γ production potential and activating receptor expression, including NKp30, NKp44, NKp46, 2B4, NKG2D and DNAM-1. Furthermore, GCs protected NK cells from IL-15-induced cell death. The combination of IL-15 with GCs favored the expansion of a relatively more immature CD16^low/neg NK cell population, with high expression of NKG2A and CD94, and significantly lower expression of KIR (CD158a and CD158b) and CD57, compared to IL-15 alone. IL-15-expanded NK cells, in the presence or absence of GCs, did not express CD62L, CXCR1 or CCR7. However, the presence of GCs significantly increased the density of CXCR3 and induced strong CXCR4 expression on the surface of NK cells. Our data indicate that IL-15/GC-expanded NK cells, apart from their increased proliferation rate, retain their functional integrity and exhibit a migratory potential rendering them useful for adoptive transfer in NK cell-based cancer immunotherapy.     

Angiotensin-(1-7) prevents diabetes-induced cardiovascular dysfunction

The aim of this study was to test the hypothesis that treatment with angiotensin-(1-7) [ANG-(1-7)] or ANG-(1-7) nonpeptide analog AVE-0991 can produce protection against diabetes-induced cardiovascular dysfunction. We examined the influence of chronic treatment (4 wk) with ANG-(1-7) (576 microg.kg(-1).day(-1) ip) or AVE-0991 (576 microg.kg(-1).day(-1) ip) on proteinuria, vascular responsiveness of isolated carotid and renal artery ring segments and mesenteric bed to vasoactive agonists, and cardiac recovery from ischemia-reperfusion in streptozotocin-treated rats (diabetes). Animals were killed 4 wk after induction of diabetes and/or treatment with ANG-(1-7) or AVE-0991. There was a significant increase in urine protein (231 +/- 2 mg/24 h) in diabetic animals compared with controls (88 +/- 6 mg/24 h). Treatment of diabetic animals with ANG-(1-7) or AVE-0991 resulted in a significant reduction in urine protein compared with vehicle-treated diabetic animals (183 +/- 16 and 149 +/- 15 mg/24 h, respectively). Treatment with ANG-(1-7) or AVE-0991 also prevented the diabetes-induced abnormal vascular responsiveness to norepinephrine, endothelin-1, angiotensin II, carbachol, and histamine in the perfused mesenteric bed and isolated carotid and renal arteries. In isolated perfused hearts, recovery of left ventricular function from 40 min of global ischemia was significantly better in ANG-(1-7)- or AVE-0991-treated animals. These results suggest that activation of ANG-(1-7)-mediated signal transduction could be an important therapeutic strategy to reduce cardiovascular events in diabetic patients.     

Nonsteroidal compounds designed to mimic potent steroid sulfatase inhibitors

Chemical synthesis and enzyme inhibition results are reported for a series of nonsteroidal sulfatase inhibitors, 1-(p-sulfamoyloxyphenyl)-5-(p-t-butylbenzyl)-5-alkanols and the lower active phenolic analogues. These compounds conserve some structural elements from the previously reported potent steroidal inhibitor 3-O-sulfamate-17alpha-(p-t-butylbenzyl)-17beta-hydroxy-estra-1,3,5(10)-triene, while the C18-methyl group and the hydrocarbon backbone represented by the steroid rings B, C, and D were replaced with a free conformational chain. Using estrone sulfate (100 microM) as substrate and homogenate of transfected HEK-293 cells as source of steroid sulfatase activity, the IC(50) values of the best inhibitors, the undecanol derivatives, were 0.4+/-0.1 and >300 nM, respectively, in the sulfamate and phenolic series. Although these sulfamoylated nonsteroidal inhibitors appear a bit less active than their steroidal analogues, they are however more potent than known inhibitors estrone-3-O-sulfamate and p-(O-sulfamoyl)-N-tetradecanoyl tyramine. The optimal side-chain length for the inhibition of steroid sulfatase activity was found to be six carbons, which corresponds to the number of carbons that mimic the B, C and D steroid rings, between C6 and C17. Furthermore, compounds with only the t-butylbenzyl group or the alkyl chain of six carbons are less potent inhibitors compared to the one that include both of these hydrophobic substituents. Such results suggest that compound from this later category better mimic the steroidal inhibitor.     

Improvement of calcium handling and changes in calcium-release properties after mini- or full-length dystrophin forced expression in cultured skeletal myotubes

Dystrophin is a cytoskeletal protein normally expressed underneath the sarcolemma of muscle fibers. The lack of dystrophin in Duchenne muscular Dystrophy (DMD) muscles results in fiber necrosis, which was proposed to be mediated by chronic calcium mishandling. The extensive comparison of dystrophic cells from human or mdx mice with normal muscles have suggested that the lack of dystrophin may alter the resting calcium permeability and steady-state levels of calcium, but this latter observation remains controversial. It is also not clear, whether calcium mishandling is resulting from the dystrophic process or if dystrophin can directly regulate calcium handling in muscle cells. This prompted us to determine if transfection of full-length dystrophin or Becker Muscular Dystrophy (BMD) minidystrophin, a candidate for viral-mediated gene therapy, could change calcium handling properties. We took advantage of specific properties of Sol8 cell line showing the absence of dystrophin expression together with a drastic calcium mishandling. Here, we show that full-length dystrophin allowed the recovery of a low resting intracellular-free calcium concentration together with lower calcium transients. We also show for the first time that stable expression of minidystrophin was able to restore normal calcium handling in Sol8 myotubes through a better control of steady-state levels, calcium transients, and subcellular calcium events. It suggests that dystrophin could play a regulatory role on calcium homeostasis apparatus and that functional links exist between calcium signaling and cytoskeleton.     

Genetic variations at the human growth hormone receptor (GHR) gene locus are associated with idiopathic short stature

GH plays an essential role in the growing child by binding to the growth hormone receptor (GHR) on target cells and regulating multiple growth promoting and metabolic effects. Mutations in the GHR gene coding regions result in GH insensitivity (dwarfism) due to a dysfunctional receptor protein. However, children with idiopathic short stature (ISS) show growth impairment without GH or GHR defects. We hypothesized that decreased expression of the GHR gene may be involved. To test this, we investigated whether common genetic variants (microsatellites, SNPs) in regulatory regions of the GHR gene region were associated with the ISS phenotype. Genotyping of a GT‐repeat microsatellite in the GHR 5′UTR in a Montreal ISS cohort (n = 37 ISS, n = 105 controls) revealed that the incidence of the long/short (L/S) genotype was 3.3× higher in ISS children than controls (P = 0.04, OR = 3.85). In an Italian replication cohort (n = 143 ISS, n = 282 controls), the medium/short (M/S) genotype was 1.9× more frequent in the male ISS than controls (P = 0.017, OR = 2.26). In both ISS cohorts, logistic regression analysis of 27 SNPs showed an association of ISS with rs4292454, while haplotype analysis revealed specific risk haplotypes in the 3′ haploblocks. In contrast, there were no differences in GT genotype frequencies in a cohort of short stature (SS) adults versus controls (CARTaGENE: n = 168 SS, n = 207 controls) and the risk haplotype in the SS cohort was located in the most 5′ haploblock. These data suggest that the variants identified are potentially genetic markers specifically associated with the ISS phenotype.     

Early locomotor behaviour in genetic stocks of chickens with different growth rates

Reduction in exercise increases the occurrence of lameness in meat-type chickens. Locomotor activity is dramatically reduced during the finishing period in chickens from fast-growing genetic types compared to slow-growing genetic types, but it is not known whether this difference is already present during the starting period and may be influenced by genetic factors. In order to define the effect of genetic origin on early locomotor behaviour, exercise was compared from 1 to 22 days of age in two meat-type chicken stocks differing in growth rate: male broilers (B) which grow fast and are often lame, and male "label rouge" chickens (L) which grow slowly and are rarely lame.Time budget (lying, standing, drinking, eating, walking) was measured by scanning in six repetitions of five birds (density=2.5 birds/m(2)) at 1, 8, 15 and 17 days of age. Standing bouts were analysed by focal sampling at 2-3, 6-7, 13-14 and 20-21 days of age.B chicks spent less time standing than L chicks at 15 days of age (B=13+/-2%, L=24+/-1%, P<0.01) and 17 days of age, and spent more time lying at 17 days of age (B=73+/-3%, L=60+/-4%, P<0.05).The major part (74%) of the total active time observed by focal sampling was linked to feeding activity. At 2 and 3 days, the activity of B chicks was half that of L chicks during standing bouts (duration of walking per bout: 19+/-4 s for B; 45+/-4 s for L, P<0.05). The activity observed by focal sampling during non-feeding bouts at 20-21 days was significantly correlated with the corresponding data recorded at 2-3 days in the same chicks in the B stock but not in the L stock.We concluded that (1) both B and L genetic stocks have the same overall activity during the first 3 days of age (scanning) but they exhibit different organisation and composition of standing bouts (focal sampling). (2) Genetic factors are probably involved in the expression of locomotor behaviour in very young chicks. (3) The correlations between the levels of activity at early and later ages suggest that selection of young mobile broiler chicks might increase activity at a later age and might therefore reduce the occurrence of leg abnormalities.     

Synthetic, spectroscopic and X-ray crystallographic structural studies on copper(II) complexes of the aminoguanizone of pyruvic acid

Copper(II) complexes of general empirical formula, CuX(Hagpa) · nH₂O and Cu(agpa) · 2H₂O (H₂agpa = aminoguanizone of pyruvic acid, X = Cl⁻, Br⁻, , CH₃COO⁻, , n = 0, 1, 1.5, 2), have been synthesized and characterized by IR, EPR spectroscopy and X-ray crystallography. The IR spectra of the complexes showed the ONN coordination of the ligand to copper(II) ion. The crystal structures of H₂agpa · H₂O and complexes [Cu(Hagpa)Br] and [Cu₂(Hagpa)₂(H₂O)₂(SO₄)] · DMSO showed an invariable conformation and coordination mode for the uninegatively charged tridentate ligand and revealed the formation of linear polymers in which bromide or sulfate anions bridge the copper(II) ions. The EPR spectra for complexes CuX(Hagpa) · nH₂O are described by spin Hamiltonian for S = 1/2, without hyperfine structure. The g-tensor is symmetrical for Cu(agpa) · 2H₂O, has tri-axial anisotropy for sulfate complexes, and exhibits axial symmetry for the other compounds investigated.