Effects of voltage-gated Na+ channel toxins from Tityus serrulatus venom on rat arterial blood pressure and plasma catecholamines

Scorpion toxins interact with ionic channels of excitable cells, leading to a massive release of neurotransmitters. Voltage-gated Na+ channel toxins are mainly responsible for the toxic effects of scorpion envenoming and can be classified into two classes: alpha- and beta-neurotoxins. TsTX-V and TsTX-I from Tityus serrulatus venom (TsV) are, respectively, examples of these toxins. In this work, we compared the effects of these toxins on mean arterial pressure (MAP) and catecholamines release in rats. Toxins were isolated by ion exchange chromatography (TsTX-I) followed by RP-HPLC (TsTX-V). All experiments were performed on conscious unrestrained rats previously catheterised. The toxins (15 and 30 microg/kg) and TsV (50 and 100 microg/kg) were injected intravenously. MAP was continuously monitored through femoral catheter. Epinephrine (E) and norepinephrine (NE) levels were determined by RP-HPLC with electrochemical detection, at 10 min before and 2.5, 30 and 90 min after treatments. Maximal pressor effects were observed at 2.5-3.5 min. TsV induced intense long lasting increase in MAP, as did TsTX-I. TsTX-V showed the lowest pressor effects. TsV showed the highest effects on catecholamines release, followed by TsTX-I and TsTX-V with maximal effect at 2.5 min, followed by a gradual reduction, however remaining higher than controls. Although both toxins act on Na+ channels, TsTX-I displayed significant and more intense effects on catecholamines release and blood pressure than TsTX-V. It seems that the toxicity of TsTX-V is not related only with its ability to release catecholamines, indicating that other neurotransmitters, may be involved in its toxicity.     

An isotope-edited FTIR investigation of the role of Ser-L223 in binding quinone (QB) and semiquinone (QB-) in the reaction center from Rhodobacter sphaeroides

In the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter sphaeroides, proton-coupled electron-transfer reactions occur at the secondary quinone (Q(B)) site. Several nearby residues are important for both binding and redox chemistry involved in the light-induced conversion from Q(B) to quinol Q(B)H(2). Ser-L223 is one of the functionally important residues located near Q(B). To obtain information on the interaction between Ser-L223 and Q(B) and Q(B)(-), isotope-edited Q(B)(-)/Q(B) FTIR difference spectra were measured in a mutant RC in which Ser-L223 is replaced with Ala and compared to the native RC. The isotope-edited IR fingerprint spectra for the C=O [see text] and C=C [see text] modes of Q(B) (Q(B)(-)) in the mutant are essentially the same as those of the native RC. These findings indicate that highly equivalent interactions of Q(B) and Q(B)(-) with the protein occur in both native and mutant RCs. The simplest explanation of these results is that Ser-L223 is not hydrogen bonded to Q(B) or Q(B)(-) but presumably forms a hydrogen bond to a nearby acid group, preferentially Asp-L213. The rotation of the Ser OH proton from Asp-L213 to Q(B)(-) is expected to be an important step in the proton transfer to the reduced quinone. In addition, the reduced quinone remains firmly bound, indicating that other distinct hydrogen bonds are more important for stabilizing Q(B)(-). Implications on the design features of the Q(B) binding site are discussed.     

Escobar syndrome is a prenatal myasthenia caused by disruption of the acetylcholine receptor fetal gamma subunit

Escobar syndrome is a form of arthrogryposis multiplex congenita and features joint contractures, pterygia, and respiratory distress. Similar findings occur in newborns exposed to nicotinergic acetylcholine receptor (AChR) antibodies from myasthenic mothers. We performed linkage studies in families with Escobar syndrome and identified eight mutations within the gamma -subunit gene (CHRNG) of the AChR. Our functional studies show that gamma -subunit mutations prevent the correct localization of the fetal AChR in human embryonic kidney-cell membranes and that the expression pattern in prenatal mice corresponds to the human clinical phenotype. AChRs have five subunits. Two alpha, one beta, and one delta subunit are always present. By switching gamma to epsilon subunits in late fetal development, fetal AChRs are gradually replaced by adult AChRs. Fetal and adult AChRs are essential for neuromuscular signal transduction. In addition, the fetal AChRs seem to be the guide for the primary encounter of axon and muscle. Because of this important function in organogenesis, human mutations in the gamma subunit were thought to be lethal, as they are in gamma -knockout mice. In contrast, many mutations in other subunits have been found to be viable but cause postnatally persisting or beginning myasthenic syndromes. We conclude that Escobar syndrome is an inherited fetal myasthenic disease that also affects neuromuscular organogenesis. Because gamma expression is restricted to early development, patients have no myasthenic symptoms later in life. This is the major difference from mutations in the other AChR subunits and the striking parallel to the symptoms found in neonates with arthrogryposis when maternal AChR auto-antibodies crossed the placenta and caused the transient inactivation of the AChR pathway.     

Fructose-fed rat hearts are protected against ischemia-reperfusion injury

High fructose-fed (HFF) rat model is known to develop the insulin-resistant syndrome with a very similar metabolic profile to the human X syndrome. Such metabolic modifications have been associated with a high incidence of cardiovascular disease. The role of free radical attack in diabetes mellitus and its cardiovascular complications have been abundantly documented. The present study examined the susceptibility to myocardial ischemic injury and the involvement of free radical attack and/or protection in the metabolic disorders of high FF rats. Rats were divided into two experimental groups that received diet for 4 weeks: a control group (C, n=28) receiving a standard diet and a HFF group (FF, n=28), in which 58% of the total carbohydrate was fructose. The euglycemic clamp technique was performed to assess insulin resistance. For the ischemia-reperfusion procedure, rat hearts were isolated and perfused at constant pressure before they were subjected to a 30-min occlusion of the left coronary artery followed by 120 mins of reperfusion. Hemodynamic parameters were measured throughout the protocol. Infarct-to-risk ratio (I/R) was assessed at the end of the protocol by 2,3,4-triphenyltetrazolium chloride staining and planimetric analysis. Lipid peroxidation, antioxidant enzyme activity, level of vitamin E, and trace element status were measured in blood samples from both groups. Rats of the FF group developed an insulin resistance indicated by the glucose infusion rate, which was decreased by 47%. Infarct size was significantly reduced in rats from the FF group (19.9% +/- 6.6%) compared to rats from the control group (34.6% +/- 4.9%), and cardiac functional recovery at reperfusion was improved in the FF group. Lipid peroxidation and oxidative stress were higher in the FF group, as indicated by higher malonedialdehyde level, whereas plasma vitamin E/triacylglycerol ratio was also enhanced in this group. This study indicates that fructose feeding affords protection against in vitro ischemia-reperfusion injury, potentially implicating vitamin E.     

New Insights in Trichoderma harzianum Antagonism of Fungal Plant Pathogens by Secreted Protein Analysis

Trichoderma harzianum ALL42 were capable of overgrowing and degrading Rhizoctonia solani and Macrophomina phaseolina mycelia, coiling around the hyphae with formation of apressoria and hook-like structures. Hyphae of T. harzianum ALL42 did not show any coiling around Fusarium sp. hyphae suggesting that mycoparasitism may be different among the plant pathogens. In this study, a secretome analysis was used to identify some extracellular proteins secreted by T. harzianum ALL42 after growth on cell wall of M. phaseolina, Fusarium sp., and R. solani. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. A total of 60 T. harzianum ALL42 secreted proteins excised from the gel were analyzed from the three growth conditions. While seven cell wall-induced proteins were identified, more than 53 proteins spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced. Endochitinase, β-glucosidase, α-mannosidase, acid phosphatase, α-1,3-glucanase, and proteases were identified in the gel and also detected in the supernatant of culture.     

Importance of the core structure of flavones in promoting inhibition of the mitochondrial respiratory chain

Flavonoids are a large group of polyphenolic compounds that have received considerable attention because of their biological and physiological importance. The flavone (2-phenyl-4H-1-benzopyran-4one) used in this work is found in some cereal grains and generates several biological activities, including: apoptosis induction, cell cycle arrest, caspase activation and inhibition of tumor cell proliferation. However, its effects on the hepatic mitochondrial metabolism are still unknown. We evaluated the effect of flavone on the metabolism of mitochondria isolated from rat liver. Polarographic experiments using 200 μmol L^?1 flavone and rat liver mitochondria oxidizing glutamate or succinate indicated that both substrates underwent: (i) reduction of state 3 respiration; (ii) stimulation of state 4 respiration; (iii) reduction of the respiratory control coefficient; and (iv) reduction of the ADP/O ratio. An analysis of the activity of enzymatic complexes in the respiratory chain showed that flavone acts between complexes I and III. Flavone reduced the membrane electric potential at doses of 100, 150 and 200 μmol L^?1. Flavone at certain doses (75–200 μmol L^?1) reduced mitochondrial swelling in the presence of valinomycin and KNO₃, suggesting that flavone could induce changes in mitochondrial membrane properties. These results demonstrate that the inhibition of mitochondrial enzymes in the respiratory chain coupled with the effects on membrane properties are promoted by the core structure of flavones, and these effects may be in part responsible for the cytotoxic effects of flavones.     

The N-terminus of PKR is responsible for the activation of the NF-kappaB signaling pathway by interacting with the IKK complex

The interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) has been shown to activate NF-kappaB independently of its kinase function after interaction with the IKK complex. In order to investigate the mechanism of NF-kappaB activation by PKR, we identified the domain of PKR responsible for stimulating the NF-kappaB pathway in PKR-deficient fibroblasts using an NF-kappaB dependent reporter assay. The N-terminal 1-265 AA of PKR activates NF-kappaB, whereas the 1-180 AA N-terminus restricted to the two dsRNA Binding Domains (DRBD), the third basic domain alone (AA 181-265), or the C-terminus of PKR (AA 266-550) were unable to stimulate the expression of the NF-kappaB dependent reporter gene. Using confocal microscopy, we confirmed that PKR full length as well as PKR N-terminus colocalized with IKKbeta. By GST-pulldown analysis, using different PKR domains, we then revealed the specific ability of the PKR N-terminus 1-265 to bind to and activate IKK and showed that this activation requires the integrity of the IKK complex. This activation is not only due to DRBDs since the DRBD fragment 1-180 failed to inhibit PKR 1-265 induced NF-kappaB activation. Our results therefore demonstrate that the ability of PKR to mediate NF-kappaB activation resides in its full N-terminus, and requires both DRBDs and the third basic domain.     

Pertussis toxin reduces the number of splenic Foxp3+ regulatory T cells

Pertussis toxin (PTx) is a bacterial toxin used to enhance the severity of experimental autoimmune diseases such as experimental autoimmune encephalomyelitis. It is known to promote permeabilization of the blood-brain barrier, maturation of APC, activation of autoreactive lymphocytes and alteration of lymphocyte migration. In this study, we show that i.v. injection of PTx in mice induces a decrease in the number of splenic CD4(+)CD25(+) regulatory T cells (Treg cells). Furthermore, PTx not only induces a depletion of the dominant CD4(+)CD25(+)Foxp3(+) subpopulation of splenic Treg cells, but also reduces to a similar extent the CD4(+)CD25(-)Foxp3(+) subpopulation. On a per cell basis, the suppressive properties of the remaining Treg cells are not modified by PTx treatment. The reduction in splenic Treg cells is associated with preferential migration of these cells to the liver. Additionally, Treg cells exhibit a high sensitivity to PTx-mediated apoptosis in vitro. Finally, in vivo depletion of Treg cells by injection of an anti-CD25 Ab, and PTx treatment, present synergistic experimental autoimmune encephalomyelitis exacerbating effects. Therefore, we identify a new effect of PTx and provide an additional illustration of the influence of microbial components on the immune system affecting the balance between tolerance, inflammation and autoimmunity.     

Hfq variant with altered RNA binding functions

The interaction between Hfq and RNA is central to multiple regulatory processes. Using site-directed mutagenesis, we have found a missense mutation in Hfq (V43R) which strongly affects2 the RNA binding capacity of the Hfq protein and its ability to stimulate poly(A) tail elongation by poly(A)-polymerase in vitro. In vivo, overexpression of this Hfq variant fails to stimulate rpoS–lacZ expression and does not restore a normal growth rate in hfq null mutant. Cells in which the wild-type gene has been replaced by the hfqV43R allele exhibit a phenotype intermediate between those of the wild-type and of the hfq minus or null strains. This missense mutation derepresses Hfq synthesis. However, not all Hfq functions are affected by this mutation. For example, HfqV43R represses OppA synthesis as strongly as the wild-type protein. The dominant negative effect of the V43R mutation over the wild-type allele suggests that hexamers containing variant and genuine subunits are presumably not functional. Finally, molecular dynamics studies indicate that the V43R substitution mainly changes the position of the K56 and Y55 side chains involved in the Hfq–RNA interaction but has probably no effect on the folding and the oligomerization of the protein.