Targeted Overexpression of Drosophila Transglutaminase-B Induced Characteristic Phenotypes in a Manner Similar to Transglutaminase-A

The Drosophila transglutaminase gene (CG7356) encodes two transglutaminases, dTG-A and dTG-B. To understand the roles of dTG-B during the development of the fly, we examined phenotypes induced through ectopic expression of dTG-B. Overexpression of dTG-B induced rough eye and extra wing crossvein phenotypes. These phenotypes were similar to those observed in the case of targeted overexpression of dTG-A.     

Distinct Subclasses of Small GTPases Interact with Guanine Nucleotide Exchange Factors in a Similar Manner

The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3–loop 7 [α3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and α3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and α3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.The small GTPases of the Ras superfamily are involved in regulating many intracellular processes, including cell growth and division, cell morphology and movement, vesicular transport, and nuclear events (4, 40, 41). These proteins, which act as molecular switches to control various functions in the cell, are in the active, or “on,” state when bound to GTP and the inactive, or “off,” state when bound to GDP. The immediate control of these GTPase-mediated events resides in the proteins which regulate their GTP- or GDP-binding status. Two classes of regulatory proteins have been identified: the guanine nucleotide exchange factors (GEFs), whose physiological function is to convert GTPases from a GDP-bound state to a GTP-bound state, and the GTPase-activating proteins (GAPs), which turn off the GTPases by activating an intrinsic GTPase activity (3, 42, 44). The GEFs stimulate guanine nucleotide release to yield a GEF–apo-GTPase reaction intermediate and, in part because the GTP concentration in cells is higher than that of GDP, the formation of active GTP-bound GTPase is favored (61).Most of our understanding of the physical interaction of these regulatory molecules with the small GTPases is based on studies of the Ras protein (3, 42–44). For example, it is known that Ras GAPs bind to the effector loop of Ras (3, 42–44). The Ras effector loop, comprising residues 30 to 45, also interacts with the known downstream targets of Ras (42–44, 79).Numerous groups have contributed to the effort to identify Ras residues which are involved in interactions with GEFs. Residues 62 to 75 in the switch II region of H-ras were found to be involved, as were residues 103 and 105 in the alpha-helix 3–loop 7 (α3-L7) region (16, 38, 49, 57, 59, 60, 68, 69, 73). The effector loop (switch I region) of Ras was also implicated in direct interactions with GEFs (5, 38, 47, 79). The switch I, switch II, and α3-L7 regions of H-ras are found adjacent to each other on the surface of the molecule, as would be expected for a surface domain involved in GEF binding (see Fig. ​Fig.7)7) (36). The recently described crystal structure of H-ras complexed with Sos demonstrates that each of these three regions is indeed at the interface of the Ras-Sos complex (5).Open in a separate windowFIG. 7Diagram showing the structure of H-ras bound to GDP. The effector loop (residues 35 to 42) (magenta), switch II region (residues 62 to 76) (cyan blue), and α3-L7 region (residues 101 to 109) (green) of Ras are on the surface of the molecule and are located next to each other. The remaining H-ras structure is shown in yellow. GDP is shown in red. Two orientations of the molecules are presented: A and B show one orientation, and C and D show the other orientation. The locations of several amino acid residues of H-ras in regions involved in binding GEFs are indicated. The switch II region and the α3-L7 region are presented as either stick models (A and C) or space-filling models (B and D).Ras GEFs exhibit a modest preference for binding GDP-bound forms of Ras, whereas Ras GAPs preferentially bind GTP-bound forms (28, 37, 45, 49, 74). Thus, the GEFs and GAPs which affect the nucleotide-binding status of Ras preferentially bind their respective substrates rather than their products. The high affinities for substrates likely reflect structural differences between the two nucleotide-bound forms of Ras. Significantly, the switch I and switch II regions of H-ras, known to have altered structures when bound to either GDP or GTP, fall within the regions implicated in interactions with GEFs and GAPs (66).Recently, the crystal structure of the Sec7 domain of human Arno, a GEF for the Arf GTPase, and an analysis of the interaction sites of these two proteins have been reported (48). The analysis revealed that Arf interacts with its exchange factor in a manner reminiscent of the Ras interaction with its GEFs. Arf appears to use three noncontiguous segments of its polypeptide to interact with Sec7. Importantly, these three regions of the Arf protein are analogous to those used by Ras to interact with its GEFs. The switch I region (effector loop) and switch II region of Arf and Ras interact with their GEFs (5, 38, 47, 48, 79). Also, Ras residues 103 to 105 in the α3-L7 region and the corresponding residues of Arf (residues 113 to 115) appear to bind GEFs (5, 24, 48, 68, 69). While the GEF-binding sequences of Arf and Ras are at analogous positions in the GTPases, GEF-binding sequences of Ras do not show homology with the Arf sequences. The finding that these two distantly related GTPases use analogous regions to interact with their GEFs raises several questions relating to other subclasses of GTPases. For example, do the Rho and Rab/YPT1 families of GTPases interact with their GEFs by using domains analogous to those used by Ras and Arf? Do the different families of GEF use a similar mechanism for catalyzing guanine nucleotide exchange on small GTPases?We undertook the present study to ask whether other small GTPases use the regions corresponding to the GEF-binding domain of H-ras to interact with their cognate GEFs. For this study, we chose the yeast YPT1 protein, which is a member of the Rab family of small GTPases (22, 29, 70). This family of proteins is involved in regulating vesicular transport (54, 55). Previously we used a yeast genetic screen to identify Ras residues which were involved in binding to Ras GEFs (49). This screen uses both a dominant interfering mutant and a constitutively active mutant of Ras. Here we created analogous YPT1 mutants and demonstrated that they could be used in a similar genetic screen. We demonstrated that the mechanism of dominant interference of YPT1 mutant N22 (YPT1-N22) is sequestration of an endogenous essential GEF for YPT1 such that a lethal phenotype occurs because endogenous YPT1 cannot be activated. Using both site-directed and random mutagenesis procedures, we identified a series of intragenic suppressors of YPT1-N22, among which we predicted would be mutants which fail to sequester essential GEFs for YPT1 due to the loss of a complete GEF-binding domain.Among the intragenic suppressor mutations, we identified 10 residues, at positions 42, 43, 49, 69, 71, 73, 75, 107, 109, and 115, which were involved in in vitro binding to DSS4, a GEF which can stimulate nucleotide exchange on YPT1 in vitro (10, 50). The positions of these residues correspond to the switch I, switch II, and α3-L7 regions of Ras, the same regions found to be important for Ras interaction with GEFs.Our findings suggest that the interaction of Ras with its specific GEFs may prove to be a useful model for analyzing the structural basis underlying the interaction of other small GTPases with their cognate GEFs. Further, our findings, together with an analysis of the interactions of Ras and Arf GTPases with their GEFs, indicate that small GTPases of the Ras superfamily use similar regions for interactions with GEFs, suggesting a similar catalytic mechanism of guanine nucleotide exchange for all small GTPases.     

Argon Inhalation Attenuates Retinal Apoptosis after Ischemia/Reperfusion Injury in a Time- and Dose-Dependent Manner in Rats

Purpose

Retinal ischemia and reperfusion injuries (IRI) permanently affect neuronal tissue and function by apoptosis and inflammation due to the limited regenerative potential of neurons. Recently, evidence emerged that the noble gas Argon exerts protective properties, while lacking any detrimental or adverse effects. We hypothesized that Argon inhalation after IRI would exert antiapoptotic effects in the retina, thereby protecting retinal ganglion cells (RGC) of the rat''s eye.

Methods

IRI was performed on the left eyes of rats (n = 8) with or without inhaled Argon postconditioning (25, 50 and 75 Vol%) for 1 hour immediately or delayed after ischemia (i.e. 1.5 and 3 hours). Retinal tissue was harvested after 24 hours to analyze mRNA and protein expression of Bcl-2, Bax and Caspase-3, NF-κB. Densities of fluorogold-prelabeled RGCs were analyzed 7 days after injury in whole-mounts. Histological tissue samples were prepared for immunohistochemistry and blood was analyzed regarding systemic effects of Argon or IRI. Statistics were performed using One-Way ANOVA.

Results

IRI induced RGC loss was reduced by Argon 75 Vol% inhalation and was dose-dependently attenuated by lower concentrations, or by delayed Argon inhalation (1504±300 vs. 2761±257; p<0.001). Moreover, Argon inhibited Bax and Bcl-2 mRNA expression significantly (Bax: 1.64±0.30 vs. 0.78±0.29 and Bcl-2: 2.07±0.29 vs. 0.99±0.22; both p<0.01), as well as caspase-3 cleavage (1.91±0.46 vs. 1.05±0.36; p<0.001). Expression of NF-κB was attenuated significantly. Immunohistochemistry revealed an affection of Müller cells and astrocytes. In addition, IRI induced leukocytosis was reduced significantly after Argon inhalation at 75 Vol%.

Conclusion

Immediate and delayed Argon postconditioning protects IRI induced apoptotic loss of RGC in a time- and dose-dependent manner, possibly mediated by the inhibition of NF-κB. Further studies need to evaluate Argon''s possible role as a therapeutic option.     

Prenatal Morphine Exposure Differentially Alters Addictive and Emotional Behavior in Adolescent and Adult Rats in a Sex-Specific Manner

The effects of prenatal opioid exposure in adult animals has been widely studied, but little is known about the effects of prenatal opioid on adolescents. Most of the risk behaviors associated with drug abuse are initiated during adolescence. The developmental state of the adolescent brain makes it vulnerable to initiate drug use and susceptible to drug-induced brain changes. In this study, pregnant rats were subcutaneously injected with an increasing dose of morphine (5 mg/kg, 7 mg/kg, 10 mg/kg) for 9 days since the gestation day 11. The effects of prenatal morphine (PNM) on learning and memory, anxiety- and depressive- like behavior, morphine induced conditioned place preference (CPP) as well as locomotor sensitization were tested in both adolescent and adult rats. The results showed that: (1) PNM decreased anxiety-like behavior in both adolescent and adult female rats, but not males; (2) PNM decreased depressive-like behavior in adolescent but increased depressive -like behavior in adult females; (3) PNM increased low dose morphine induced locomotor sensitization in females; (4) PNM decreased tyrosine hydroxylase (TH) expression in the prefrontal cortex but decreased dopamine D1 receptor expression in the nucleus-accumbens (NAc) in female rats. These results suggested that PNM altered the emotional and addictive behavior mainly in female rats, with female rats being less anxiety and depressive during adolescence, but more depressive in adult, and more sensitive to low dose morphine induced locomotor activity sensitization, which might be mediated in part by the differential expression of the TH, dopamine D1 receptors in the female brain.

     

Atomoxetine-Induced Increases in Monoamine Release in the Prefrontal Cortex are Similar in Spontaneously Hypertensive Rats and Wistar-Kyoto Rats

Spontaneously hypertensive rats (SHRs) are used as a model for attention-deficit/hyperactivity disorder (ADHD), since SHRs are hyperactive and show defective sustained attention in behavioral tasks. The psychostimulants amphetamine and methylphenidate and the selective norepinephrine reuptake inhibitor atomoxetine are used as ADHD medications. The effects of high K⁺ stimulation or psychostimulants on brain norepinephrine or dopamine release in SHRs have been previously studied both in vitro and in vivo, but the effects of atomoxetine on these neurotransmitters have not. The present study examined the effects of administration of atomoxetine on extracellular norepinephrine, dopamine, and serotonin levels in the prefrontal cortex of juvenile SHRs and Wistar-Kyoto (WKY) rats. Baseline levels of prefrontal norepinephrine, dopamine, and serotonin were similar in SHRs and WKY rats. Systemic administration of atomoxetine (3 mg/kg) induced similar increases in prefrontal norepinephrine and dopamine, but not serotonin, levels in both strains. Furthermore, there was no difference in high K⁺-induced increases in extracellular norepinephrine, dopamine, and serotonin levels in the prefrontal cortex between SHRs and WKY rats. These findings indicate that monoamine systems in the prefrontal cortex are similar between SHRs and WKY rats.     

Anthrax Lethal Toxin Kills Macrophages in a Strain-Specific Manner by Apoptosis or Caspase-1-Mediated Necrosis

Murine macrophages have been classified as either susceptible or nonsusceptible to killing by anthrax lethal toxin (LT) depending upon genetic background. While considered resistant to LT killing, we found that bone marrow-derived macrophages (BMMs) from DBA/2, AKR, and C57BL/6 mice were slowly killed by apoptosis following LT exposure. LT killing was not restricted to in vitro assays, as splenic macrophages were also depleted in LT-injected C57BL/6 mice. Human macrophages, also considered LT resistant, similarly underwent slow apoptosis in response to LT challenge. In contrast, LT triggered rapid necrosis and a broad protein release in BMMs derived from BALB/c and C3H/HeJ, but not C57BL/6 mice. Released proteins included processed interleukin-18, confirming reports of inflammasome and caspase-1 activation in LT-mediated necrosis in macrophages. Complete inhibition of caspase-1 activity was required to block LT-mediated necrosis. Strikingly, minimal residual caspase-1 activity was sufficient to trigger significant necrosis in LT-treated macrophages, indicating the toxicity of caspase-1 in this process. IL-18 release does not trigger cytolysis, as IL-18 is released late and only from LT-treated macrophages undergoing membrane perturbation. We propose that caspase-1-mediated macrophage necrosis is the source of the cytokine storm and rapid disease progression reported in LT-treated BALB/c mice.     

Mesp1 Patterns Mesoderm into Cardiac,Hematopoietic, or Skeletal Myogenic Progenitors in a Context-Dependent Manner

1. Download : Download high-res image (212KB)
2. Download : Download full-size image

     

Low Concentrations of Hydrogen Peroxide or Nitrite Induced of Paracoccidioides brasiliensis Cell Proliferation in a Ras-Dependent Manner

Paracoccidioides brasiliensis, a causative agent of paracoccidioidomycosis (PCM), should be able to adapt to dramatic environmental changes inside the infected host after inhalation of air-borne conidia and transition to pathogenic yeasts. Proteins with antioxidant functions may protect fungal cells against reactive oxygen (ROS) and nitrogen (RNS) species generated by phagocytic cells, thus acting as potential virulence factors. Ras GTPases are involved in stress responses, cell morphology, and differentiation in a range of organisms. Ras, in its activated form, interacts with effector proteins and can initiate a kinase cascade. In lower eukaryotes, Byr2 kinase represents a Ras target. The present study investigated the role of Ras in P. brasiliensis after in vitro stimulus with ROS or RNS. We have demonstrated that low concentrations of H₂O₂ (0.1 mM) or NO₂ (0.1–0.25 µM) stimulated P. brasiliensis yeast cell proliferation and that was not observed when yeast cells were pre-incubated with farnesyltransferase inhibitor. We constructed an expression plasmid containing the Byr2 Ras-binding domain (RBD) fused with GST (RBD-Byr2-GST) to detect the Ras active form. After stimulation with low concentrations of H₂O₂ or NO₂, the Ras active form was observed in fungal extracts. Besides, NO₂ induced a rapid increase in S-nitrosylated Ras levels. This alternative posttranslational modification of Ras, probably in residue Cys123, would lead to an exchange of GDP for GTP and consequent GTPase activation in P. brasiliensis. In conclusion, low concentrations of H₂O₂ or NO₂ stimulated P. brasiliensis proliferation through Ras activation.     

白介素1β时间依赖性地抑制大鼠皮层神经元钾电流

白细胞介素1β（Interleukin-1β,IL-1β）是一种重要的促炎细胞因子,在中枢神经系统中发挥着广泛的生物学功能。大量研究表明,IL-1β的作用非常复杂,在不同的模型和条件下作用不同,包括神经损伤或者神经毒性作用。电压门控钾通道调节神经元电学性质,也参与多种中枢神经系统的病理学过程。虽然IL-1β和钾通道都在脑损伤和脑疾病过程中发挥重要作用,但目前还很少有它们之间关系的研究报导。文章作者以原代培养的大鼠皮层神经元为材料,使用全细胞膜片钳技术,研究了10 ng/mL的IL-1β在不同处理时间下对皮层神经元电压依赖性钾电流的影响。根据电流的性质,可以将记录到的钾电流分为瞬时外向电流（IA）为主的IA样电流和延迟整流电流（IK）为主的IK样电流两部分,结果显示：IL-1β处理8 h对二者没有作用;处理24 h可使IA样和IK样电流的幅度降低20%左右。以上结果提示IL-1β对大鼠皮层神经元电压门控钾电流具有抑制作用,并且这种抑制可能具有时间依赖性。     

The TSC-mTOR Pathway Mediates Translational Activation of TOP mRNAs by Insulin Largely in a Raptor- or Rictor-Independent Manner

The stimulatory effect of insulin on protein synthesis is due to its ability to activate various translation factors. We now show that insulin can increase protein synthesis capacity also by translational activation of TOP mRNAs encoding various components of the translation machinery. This translational activation involves the tuberous sclerosis complex (TSC), as the knockout of TSC1 or TSC2 rescues TOP mRNAs from translational repression in mitotically arrested cells. Similar results were obtained upon overexpression of Rheb, an immediate TSC1-TSC2 target. The role of mTOR, a downstream effector of Rheb, in translational control of TOP mRNAs has been extensively studied, albeit with conflicting results. Even though rapamycin fully blocks mTOR complex 1 (mTORC1) kinase activity, the response of TOP mRNAs to this drug varies from complete resistance to high sensitivity. Here we show that mTOR knockdown blunts the translation efficiency of TOP mRNAs in insulin-treated cells, thus unequivocally establishing a role for mTOR in this mode of regulation. However, knockout of the raptor or rictor gene has only a slight effect on the translation efficiency of these mRNAs, implying that mTOR exerts its effect on TOP mRNAs through a novel pathway with a minor, if any, contribution of the canonical mTOR complexes mTORC1 and mTORC2. This conclusion is further supported by the observation that raptor knockout renders the translation of TOP mRNAs rapamycin hypersensitive.     

Endothelin A Receptor Antagonist,Atrasentan, Attenuates Renal and Cardiac Dysfunction in Dahl Salt-Hypertensive Rats in a Blood Pressure Independent Manner

Proteinuria is a hallmark of chronic kidney disease (CKD) and cardiovascular disease (CVD), and a good predictor of clinical outcome. Selective endothelin A (ET_A) receptor antagonist used with renin-angiotensin system (RAS) inhibitors prevents development of proteinuria in CKD. However, whether the improvement in proteinuria would have beneficial effects on CVD, independent of RAS inhibition, is not well understood. In this study, we investigated whether atrasentan, an ET_A receptor antagonist, has renal and cardiovascular effects independent of RAS inhibition. Male Dahl salt sensitive (DSS) rats, at six weeks of age, received water with or without different doses of atrasentan and/or enalapril under high salt (HS) diet or normal diet (ND) for 6 weeks. At the end of 12th week, atrasentan at a moderate dose significantly attenuated proteinuria and serum creatinine without reducing mean arterial pressure (MAP), thereby preventing cardiac hypertrophy and improving cardiac function. ACE inhibitor enalapril at a dose that did not significantly lowered BP, attenuated cardiac hypertrophy while moderately improving cardiac function without reducing proteinuria and serum creatinine level. Nonetheless, combined therapy of atrasentan and enalapril that does not altering BP exerted additional cardioprotective effect. Based on these findings, we conclude that BP independent monotherapy of ET_A receptor antagonist attenuates the progression of CKD and significantly mitigates CVD independent of RAS inhibition.     

Displacement Behaviour Solving a Silent Contradiction

Displacement acts, once a hot topic in ethology, but wrapped in silence for two decades since, have recently been suggested to indicate and relax social tension (Maestripieri et al., 1992; Wiepkema, 1987). The first of these contentions seems to be in contradiction with some of the classical ethological studies of displacement behaviour, in particular those supporting the disinhibition hypothesis, since the latter would not predict any positive correlation between amount of tension (i.c. intensity of the conflict) and the occurrence of displacement acts.A critical examination of these studies reveals that a positive correlation of the sort has been found, but that proponents of the disinhibition hypothesis tried to explain it in terms of their own model, rather than taking it at its face value. (The reason for this is that they viewed it as pleading for the surplus hypothesis which they rejected). It can be shown that at least some of their explanations are grounded on assumptions which are arbitrary. Also, the disinhibition hypothesis does not account for the occurrence of displacement behaviour in contexts with tension but without conflict.This discussion leads to a new interpretation of displacement behaviour, in which tension is the direct cause, whether generated by conflict or otherwise, and relaxation is brought about by a breaking of the cognitive symmetry between actors. It is indicated how this interpretation may be tested with experiments.     

Regional Cerebral Glucose Metabolism and Blood Flow During the Silent Phase of Methylmercury Neurotoxicity in Rats

Methylmercuric chloride was given to rats in a neurotoxic dose regimen (six daily doses of 8 mg kg-1 p.o.). During the silent (asymptomatic) phase of intoxication, the rates of cerebral glucose influx and cerebral glucose phosphorylation were measured simultaneously using 2-deoxyglucose. Regional cerebral blood flow was also measured using iodoantipyrine. The unidirectional flux of glucose into brain was not affected by methylmercury, and differences in the rates of glucose phosphorylation from region to region remained coupled to the regional cerebral blood flow. However, the blood flow was reduced throughout the brain, an observation suggesting that the operational level of metabolically regulated blood flow had been reset. Thus, in spite of a generalised reduction in blood flow, there was no indication of impaired cerebral glucose supply or utilization during the silent phase of methylmercury intoxication.