Electroacupuncture inhibits dendritic spine remodeling through the srGAP3-Rac1 signaling pathway in rats with SNL

Previous studies have shown that peripheral nerve injury can lead to abnormal dendritic spine remodeling in spinal dorsal horn neurons. Inhibition of abnormal dendritic spine remodeling can relieve neuropathic pain. Electroacupuncture (EA) has a beneficial effect on the treatment of neuropathic pain, but the specific mechanism remains unclear. Evidence has shown that slit-robo GTPase activating protein 3 (srGAP3) and Rho GTPase (Rac1) play very important roles in dendritic spine remodeling. Here, we used srGAP3 siRNA and Rac1 activator CN04 to confirm the relationship between SrGAP3 and Rac1 and their roles in improving neuropathic pain with EA. Spinal nerve ligation (SNL) was used as the experimental model, and thermal withdrawal latency (TWL), mechanical withdrawal threshold (MWT), Western blotting, immunohistochemistry and Golgi-Cox staining were used to examine changes in behavioral performance, protein expression and dendritic spines. More dendritic spines and higher expression levels of srGAP3 were found in the initial phase of neuropathic pain. During the maintenance phase, dendritic spines were more mature, which was consistent with lower expression levels of srGAP3 and higher expression levels of Rac1-GTP. EA during the maintenance phase reduced the density and maturity of dendritic spines of rats with SNL, increased the levels of srGAP3 and reduced the levels of Rac1-GTP, while srGAP3 siRNA and CN04 reversed the therapeutic effects of EA. These results suggest that dendritic spines have different manifestations in different stages of neuropathic pain and that EA may inhibit the abnormal dendritic spine remodeling by regulating the srGAP3/Rac1 signaling pathway to alleviate neuropathic pain.

Complete screening of exons 2, 3, and 4 of kras and nras genes reveals a higher number of clinically relevant mutations than food and drug administration quantitative polymerase chain reaction-based commercial kits

Abstract Background: The presence of clinically relevant mutations in KRAS and NRAS genes determines the response of anti-epidermal growth factor receptor antibody therapy for metastatic colorectal cancer (mCRC). The only quantitative polymerase chain reaction (qPCR)-based diagnostic tests approved by the Food and Drug Administration (FDA) screen merely for mutations in codons 12 and 13 of KRAS. Objective: The objective of the study was to study the frequency of clinically relevant mutations in KRAS and NRAS genes that are not included in FDA-approved qPCR tests. Methods: Formalin-fixed paraffin-embedded tumor specimens from 1113 mCRC Mexican patients from different health institutions across the country were analyzed by Sanger sequencing for KRAS mutations in exons 2, 3, and 4. Furthermore, 83 were analyzed in exons 2, 3, and 4 of NRAS. Results: From the specimens tested for KRAS, 33.69% harbored a mutation. From these, 71.77% were in codon 12 and 27.69% in codon 13 (both located in exon 2). Codons 59 (exon 3) and 146 (exon 4) accounted for the remaining 0.54%. From the 83 specimens, in which NRAS was analyzed, three mutations were found in codon 12 (3.61%). Approximately 6% of RAS mutated specimens would have been falsely reported as RAS wild type if an FDA-approved qPCR diagnostic test had been used. Conclusions: While these kits based on qPCR can be very practical and highly sensitive, their mutation coverage ignores mutations from poorly genetically characterized populations.

Association between MFN2 gene polymorphisms and the risk and prognosis of acute liver failure: a case-control study in a Chinese population

This study aimed to determine the role of mitofusin 2 (MFN2) gene polymorphisms in the risk and prognosis of acute liver failure (ALF). A total of 298 blood samples were collected from 138 ALF patients (case group) and 160 healthy participants (control group). Coagulation function, glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), total bilirubin (TB), blood ammonia and lactic acid (LA) were measured. The predictive evaluation of MFN2 gene polymorphisms in the risk and prognosis of ALF patients was estimated using Kaplan-Meier survival analysis, haplotype analysis, binary logistic regression analysis and Cox regression analysis. Higher levels of GPT, GOT, TB, blood ammonia and LA were observed in ALF patients with the GG genotype of rs873457 or the TT genotype of rs4846085 than in those with the CC genotype of these two SNPs. The GTACAGC and GTGTGGC haplotypes were a protective factor and a risk factor for ALF, respectively. Blood ammonia and LA levels were independent risk factors and the CC genotype of rs873457 and the CC genotype of rs4846085 were protective factors for ALF. ALF patients with the GG genotype of rs873457 or the TT genotype of rs4846085 had a lower survival rate than those with other genotypes of these two SNPs. The rs4846085 and rs873457 polymorphisms were both independent factors affecting the prognosis of ALF patients. MFN2 gene polymorphisms (rs873457, rs2336384, rs1474868, rs4846085 and rs2236055) may be associated with ALF and the rs873457 and rs4846085 polymorphisms are correlated with the risk and prognosis of ALF.

O papel de RhoA e Rac1 GTPases nas respostas celulares após danos no DNA induzidos por radiação ionizante gama / The role of RhoA and Rac1 GTPases in cellular responses after DNA damage induced by ionizing gamma radiation

O mecanismo pelo qual uma célula responde a algum dano no seu material genético é extremamente importante. Isto ocorre pela rápida ativação da maquinaria de reparo de danos no DNA, a qual é composta por uma rede intrincada de sinalização proteica, culminando no reparo do DNA; porém se o dano for irreparável ocorre ativação de mecanismos de morte celular. RhoA,e Rac1 pertencem a família das pequenas proteínas sinalizadoras Rho GTPases, as quais atuam como interruptores moleculares ciclando entre estado ativo (ligada a GTP) e inativo (ligada a GDP). Os componentes desta família estão relacionados ao controle dos mais diversos processos celulares como, por exemplo, remodelamento do citoesqueleto, migração, adesão, endocitose, progressão do ciclo celular e oncogênese. No entanto, apesar das proteínas Rho GTPases estarem envolvidas em um amplo espectro de atividades biológicas, há poucas informações sobre seu papel na manutenção da integridade genômica quando células são submetidas a algum agente genotóxico. Para investigar o envolvimento das GTPases RhoA e Rac1 nas respostas de células submetidas a radiação gama, foram gerados, a partir de células de carcinoma de cervix humano - HeLa, sublinhagens clonais mutantes de RhoA e Rac1 expressando exogenamente RhoA constitutivamente ativa (HeLa-RhoA V14), RhoA dominante negativa (HeLa-RhoA N19), Rac1 constitutivamente ativa (HeLa-Rac1 V12) e Rac1 dominante negativa (HeLa-Rac N17). Após estas linhagens celulares serem expostas a diferentes doses de radiação gama, observamos que ambas GTPases, RhoA e Rac1, são ativadas em resposta aos efeitos da radiação. Além disso, a modulação da atividade destas enzimas, através das mutações, levou a uma alteração das respostas celulares frente aos danos no DNA, como uma redução da capacidade de reparar quebras simples e duplas nas fitas do DNA. Por outro lado, a deficiência de RhoA ou Rac1 GTPase levou a uma redução da ativação de Chk1 e Chk2 ou da fosforilação da histona H2AX, respectivamente, prejudicando os mecanismos de detecção de danos no DNA e levando as células a permanecerem mais tempo nos pontos de checagem G1/S e/ou G2/M do ciclo celular. Esses fatores contribuíram de modo expressivo para a redução da proliferação e sobrevivência celular levando as células à morte. Por fim, ensaios celulares de reparo de danos de um DNA exógeno através de mecanismos de Recombinação Homóloga (HR) e Recombinação Não-Homóloga de extremidades (NHEJ), demonstraram que a inibição da atividade de RhoA reduz significativamente a eficiência de ambas vias de reparo. Desta maneira, este trabalho demonstra e reforça a existência de mais um viés de atuação das pequenas GTPases RhoA e Rac1, agora em células HeLa, nas respostas celulares aos danos induzidos por exposição a radiação gama, modulando a sobrevivência, proliferação e indiretamente modulando resposta ao reparo do DNA através da via de Recombinação Homóloga e Não-Homóloga

The mechanism by which a cell responds to DNA damage is extremely important. This occurs by a quick activation of the DNA damage repair machinery, which consists of an intricate protein signaling network culminating in DNA repair. But if the damages are irreparable occurs there is activation of cell death mechanisms. RhoA and Rac1 belong to family of small Rho GTPases, signaling proteins that act as molecular switches cycling between the active state (GTP-bound) and inactive state (GDP-bound). Members of this family are implicated in the control of diverse cellular process such as cytoskeletal remodeling, migration, adhesion, endocytosis, cell cycle progression, and oncogenesis. However, despite Rho proteins are involved in a broad spectrum of biological activities, there is just a few information about their roles in the maintenance of genomic integrity, that is, when the cells are subjected to some kinf of genotoxic agent. To investigate the involvement of the GTPases RhoA and Rac1 in cellular responses to gamma radiation, we generated from human cervix carcinoma cells - HeLa, clonal sublines of RhoA and Rac1 mutants, exogenous and stably expressing the constitutively active RhoA (HeLa-RhoA V14), the dominant negative RhoA (HeLa-RhoA N19), the constitutively active Rac1 (HeLa-Rac1 V12) and the dominant negative Rac1 (HeLa-Rac1 N17). After all these cell lines have been exposed to different doses of gamma radiation, we found that both GTPases, RhoA and Rac1, are activated in response to the radiation effects. Furthermore, the modulation of two enzymes activity, by using the mutant clones, led to a change in cellular responses to the DNA damage, as the reduction in the capacity of repairing DNA single and double strand breaksr. On the other hand, the deficiency of RhoA or Rac1 GTPase led to a reduction of Chk1 and Chk2 activation, or on the phosphorylation of histone H2AX, respectively, hindering the mechanisms of DNA damage detection and arresting cells in the G1/S and/or G2/M checkpoints of cell cycle. These factors significantly contributed to the reduction of cell proliferation and survival, leading cells to death. Finally, cellular assays of DNA damage repair of exogenous DNA by Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ), demonstrated that RhoA inhibition significantly reduced the repair efficiency of both pathways. Thus, this work demonstrates and reinforces the existence of other biological functions of small GTPases RhoA and Rac1 in HeLa cells, by regulating cellular responses to DNA damage induced by exposure to gamma radiation, modulating the survival, proliferation and indirectly modulating the response to DNA damage repair pathway through the Homologous Recombination and Non-Homologous Recombination

Toxoplasma gondii and the Immunity-Related GTPase (IRG) resistance system in mice: a review

The Immunity Related GTPases (IRG proteins) constitute a large family of interferon-inducible proteins that mediate early resistance to Toxoplasma gondii infection in mice. At least six members of this family are required for resistance of mice to virulent T. gondii strains. Recent results have shown that the complexity of the resistance arises from complex regulatory interactions between different family members. The mode of action against T. gondii depends on the ability of IRG proteins to accumulate on the parasitophorous vacuole of invading tachyzoites and to induce local damage to the vacuole resulting in disruption of the vacuolar membrane. Virulent strains of T. gondiiovercome the IRG resistance system, probably by interfering with the loading of IRG proteins onto the parasitophorous vacuole membrane. It may be assumed that T. gondii strains highly virulent for mice will be disadvantaged in the wild due to the rapid extinction of the infected host, while it is self-evident that susceptibility to virulent strains is disadvantageous to the mouse host. We consider the possibility that this double disadvantage is compensated in wild populations by segregating alleles with different resistance and susceptibility properties in the IRG system.

Identificacao e caracterizacao de transcritos humanos: novas famílias de pequenas GTPases e novos Longos RNAs intrônicos não-codificantes / Identification and caracterization of human transcripts: novel small GTPase gene families and novel Long Intronic non-coding RNAs

Terminado o sequenciamento do genoma humano, as atenções se voltaram para a determinação do conjunto completo de transcritos humanos. Diversos trabalhos sugerem que enquanto apenas uma pequena fração de mRNAs codificantes para proteína não é conhecida, existe um grande número de RNAs não-codificantes (ncRNAs) ainda não caracterizados. Nesse contexto, o presente trabalho visou explorar as informações de expressão gênica contidas em ESTs para identificar e caracterizar novos transcritos humanos. A busca genômica por membros de famílias gênicas relacionadas com câncer levou a identificação de novas pequenas GTPases, destacando uma subfamília que deve apresentar função supressora tumoral em próstata. Uma classe de ncRNAs longos, sem splicing, expressos antisenso a partir de regiões intrônicas foi descrita utilizando plataformas de microarrays, construídas pelo grupo, enriquecidas com seqüências sem anotação. O perfil de expressão de 23 ncRNAs intrônicos estava significativamente correlacionado com o grau de diferenciação de tumores de próstata (Gleason Score), e pode ser utilizado como candidato a marcador molecular de prognóstico. Um total de 39 ncRNAs intrônicos responderam à estimulação por andrógeno, apontando para um mecanismo regulatório da expressão intrônica por sinais fisiológicos hormonais. A biogênese da expressão intrônica parece ser complexa, pois uma fração não é transcrita pela RNA Polimerase II. A transcrição intrônica estava correlacionada com uso de exons em células tratadas com andrógeno. Assinaturas de expressão intrônica conservadas em tecidos humanos e de camundongos, e interações de transcritos intrônicos com proteínas regulatórias foram observadas. Este trabalho contribui com novas e originais evidências que dão apoio ao papel postulado para esses ncRNAs no controle fino do programa transcricional humano

Effect of detergents and lipids on transducin photoactivation by rhodopsin

Rhodopsin samples, isolated using four different extraction procedures, were used to investigate the photodependent activation of the GTPase activity of transducin. A complete inhibition of transducin light-dependent GTP hydrolytic activity was observed when rhodopsin purified in the presence of 1 per cent digitonin, following rod outer segment (ROS) solubilization with 1 per cent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate (CHAPS), WAS used for its activation [0 pmol of inorganic phosphate (Pi) released/min/pmol of rhodopsin]. Rhodopsin, isolated in the presence of 1 per cent digitonin following ROS solubilization with 1 per cent digitonin, was capable of stimulating slightly transducin GTPase activity, with an initial rate of 1 pmol of GTP hydrolyzed/min/pmol of rhodopsin. However, rhodopsin purified in the presence of 0.2 per cent n-dodecyl-beta-D-maltoside (DM), following ROS solubilization with either 1 per cent CHAPS or 1 per cent DM, stimulated the enzymatic activity of transducin in a light-dependent manner, with an initial rate of 5 pmol of Pi released/min/pmol of rhodopsin. Addition of 0.075 per cent egg phosphatidylcholine (PC) to the four different solubilized rhodopsin samples significantly enhanced light-stimulated GTP hydrolysis by transducin, with initial rates increasing from 0 to 1, 1 to 2, and 5 to 30 pmol of Pi released/min/pmol of rhodopsin, respectively. Furthermore, DM-solubilized rhodopsin induced the hydrolysis of the maximun amount of GTP by transducin at 0.0075 per cent PC, while digitonin-solubilized rhodopsin only stimulated the GTPase activity of transducin to a similar value, when the amount of the photoreceptor protein was increased 4-fold and 0.15 per cent PC was added to the assay mixture. These results suggest that the effective photoactivation of transducin by rhodopsin requires phospholipids, which seem to be differentially eliminated with the detergent extraction procedure utilized during ROS membranes solubilization and photopigment isolation.

Identification of GTPase genes in the protozoa parasites Trypanosoma cruzi and Leishmania amazonensis

A PCR based assay was designed in order to amplify putative ras gene sequences of the GTPase superfamily eventually present in Leishmania amazonensis and Trypanosoma cruzi. A set of primers corresponding to the conserved motifs G1 and G3 of the GTP binding proteins was synthesized. Sequencing of six PCR products (three from Leishmania and three from Trypanosoma) identified, however, two other different GTPases. The 270 bp L. amazonensis clone, pLef-11, shared an amino acid identity of around 80 percent with an eukaryotic elongation factor 1a of protein synthesis. On the other hand, the 168 bp T. cruzi clone, pTCr1, demonstrated over 60 percent amino acid identity to ras-related proteins of the rab-YPT-SEC4 family involved in control of vesicular traffic. To our knowledge, this is the first report of GTP binding protein genes in trypanosomatids