GTP Activity Research Articles

The pro-oncogenic noncanonical activity of a RAS•GTP:RanGAP1 complex facilitates nuclear protein export.

Canonical RAS signaling, including PI3K/AKT- and RAF/MEK-dependent activities, results mainly from RAS•GTP interaction with its effectors at the plasma membrane. Here, we identified a fundamental, oncogenic, noncanonical RAS•GTP activity that increases XPO1-dependent export of nuclear protein cargo into the cytoplasm and is independent of PI3K/AKT and RAF/MEK signaling. This RAS-dependent step acts downstream from XPO1 binding to nuclear protein cargo and is mediated by a perinuclear protein complex between RAS•GTP and RanGAP1 that facilitates hydrolysis of Ran•GTP to Ran•GDP, which promotes release of nuclear protein cargo into the cytoplasm. The export of nuclear EZH2, which promotes cytoplasmic degradation of the DLC1 tumor suppressor protein, is a biologically important component of this pro-oncogenic activity. Conversely, preventing nuclear protein export contributes to the antitumor activity of KRAS inhibition, which can be further augmented by reactivating the tumor suppressor activity of DLC1 or potentially combining RAS inhibitors with other cancer treatments.

Structural Basis of Nucleotide Selectivity in Pyruvate Kinase

Nucleoside triphosphates are indispensable in numerous biological processes, with enzymes involved in their biogenesis playing pivotal roles in cell proliferation. Pyruvate kinase (PYK), commonly regarded as the terminal glycolytic enzyme that generates ATP in tandem with pyruvate, is also capable of synthesizing a wide range of nucleoside triphosphates from their diphosphate precursors. Despite their substrate promiscuity, some PYKs show preference towards specific nucleotides, suggesting an underlying mechanism for differentiating nucleotide bases. However, the thorough characterization of this mechanism has been hindered by the paucity of nucleotide-bound PYK structures. Here, we present crystal structures of Streptococcus pneumoniae PYK in complex with four different nucleotides. These structures facilitate direct comparison of the protein-nucleotide interactions and offer structural insights into its pronounced selectivity for GTP synthesis. Notably, this selectivity is dependent on a sequence motif in the nucleotide recognition site that is widely present among prokaryotic PYKs, particularly in Firmicutes species. We show that pneumococcal cell growth is significantly impaired when expressing a PYK variant with compromised GTP and UTP synthesis activity, underscoring the importance of PYK in maintaining nucleotide homeostasis. Our findings collectively advance our understanding of PYK biochemistry and prokaryotic metabolism.

ELMO interacts with DOCK180 to enhance coelomocyte phagocytosis in Vibrio splendidus challenged Apostichopus japonicus

Engulfment and cell motility protein (ELMO) was identified as a novel, evolutionarily conserved upstream regulator of the RAC signaling pathway in mammals, which was essential for diverse physiological and pathological processes of cells, such as phagocytosis and cell migration. However, the functions and the underlying mechanisms of ELMO1 are generally poorly understood in invertebrates. In this study, we found that the endocytosis and GTP activity were significantly enriched in AjELMO1-interfered coelomocytes of Apostichopus japonicus by RNA-seq. Moreover, overexpression or interference of AjELMO1 significantly altered the coelomocyte phagocytosis levels induced by Vibrio splendidus AJ01, leading to an increase and decrease in the intracellular AJ01 burden by flow cytometry and bacteriophage plate counting in vivo and in vitro. Further functional analysis with different endocytosis inhibitors treatment revealed that AjELMO1 mediated AJ01 internalization via the actin-dependent endocytic pathways through the changes of F-actin/G-actin, but not the clathrin-, macropinocytosisi-, and dynamin-dependent endocytic pathways. The dedicator of cytokinesis protein 1 (AjDOCK180) was identified as an interacting protein of AjELMO1 using co-IP and GST-Pull down followed by mass spectrometry identification. Further function analysis also revealed that AjDOCK180 promotes coelomocyte phagocytosis through the actin endocytosis pathway via the changes in the phagocytosis levels, the intracellular AJ01 burden, and the ratio of F-actin/G-actin in coelomocytes. Additionally, we found that the phagocytic activity in sea cucumber coelomocytes promoted by AjELMO1 can be blocked by interference with AjDOCK180. Altogether, our data demonstrate that AjELMO1-AjDOCK180 is involved in resisting AJ01 infection via medicating coelomocyte phagocytosis through the actin-dependent pathway.

Distinctive in vitro ATP Hydrolysis Activity of AtVIPP1, a Chloroplastic ESCRT-III Superfamily Protein in Arabidopsis.

Vesicle-inducing protein in plastid 1 (VIPP1), characteristic to oxygenic photosynthetic organisms, is a membrane-remodeling factor that forms homo-oligomers and functions in thylakoid membrane formation and maintenance. The cyanobacterial VIPP1 structure revealed a monomeric folding pattern similar to that of endosomal sorting complex required for transport (ESCRT) III. Characteristic to VIPP1, however, is its own GTP and ATP hydrolytic activity without canonical domains. In this study, we found that histidine-tagged Arabidopsis VIPP1 (AtVIPP1) hydrolyzed GTP and ATP to produce GDP and ADP in vitro, respectively. Unexpectedly, the observed GTPase and ATPase activities were biochemically distinguishable, because the ATPase was optimized for alkaline conditions and dependent on Ca2+ as well as Mg2+, with a higher affinity for ATP than GTP. We found that a version of AtVIPP1 protein with a mutation in its nucleotide-binding site, as deduced from the cyanobacterial structure, retained its hydrolytic activity, suggesting that Arabidopsis and cyanobacterial VIPP1s have different properties. Negative staining particle analysis showed that AtVIPP1 formed particle or rod structures that differed from those of cyanobacteria and Chlamydomonas. These results suggested that the nucleotide hydrolytic activity and oligomer formation of VIPP1 are common in photosynthetic organisms, whereas their properties differ among species.

PRMT5 Interacting Partners and Substrates in Oligodendrocyte Lineage Cells.

The protein arginine methyl transferase PRMT5 is an enzyme expressed in oligodendrocyte lineage cells and responsible for the symmetric methylation of arginine residues on histone tails. Previous work from our laboratory identified PRMT5 as critical for myelination, due to its transcriptional regulation of genes involved in survival and early stages of differentiation. However, besides its nuclear localization, PRMT5 is found at high levels in the cytoplasm of several cell types, including oligodendrocyte progenitor cells (OPCs) and yet, its interacting partners in this lineage, remain elusive. By using mass spectrometry on protein eluates from extracts generated from primary oligodendrocyte lineage cells and immunoprecipitated with PRMT5 antibodies, we identified 1196 proteins as PRMT5 interacting partners. These proteins were related to molecular functions such as RNA binding, ribosomal structure, cadherin and actin binding, nucleotide and protein binding, and GTP and GTPase activity. We then investigated PRMT5 substrates using iTRAQ-based proteomics on cytosolic and nuclear protein extracts from CRISPR-PRMT5 knockdown immortalized oligodendrocyte progenitors compared to CRISPR-EGFP controls. This analysis identified a similar number of peptides in the two subcellular fractions and a total number of 57 proteins with statistically decreased symmetric methylation of arginine residues in the CRISPR-PRMT5 knockdown compared to control. Several PRMT5 substrates were in common with cancer cell lines and related to RNA processing, splicing and transcription. In addition, we detected ten oligodendrocyte lineage specific substrates, corresponding to proteins with high expression levels in neural tissue. They included: PRC2C, a proline-rich protein involved in methyl-RNA binding, HNRPD an RNA binding protein involved in regulation of RNA stability, nuclear proteins involved in transcription and other proteins related to migration and actin cytoskeleton. Together, these results highlight a cell-specific role of PRMT5 in OPC in regulating several other cellular processes, besides RNA splicing and metabolism.

ΔNp63α suppresses cell invasion through downregulating Rac1 activity

ΔNp63α, a member of the p53 family of transcription factors, is overexpressed in a number of cancers and plays a role in proliferation, differentiation, migration and invasion. We showed previously that ΔNp63α suppresses cell invasion by positively regulating miR-320a, resulting in the downregulation of its direct target protein kinase C gamma (PKCg) and a corresponding reduction in phosphorylated Ras-related C3 botulinum toxin substrate 1 (Rac1). Here, we further show that ΔNp63α inhibits Rac1 GTPase activity. Using a Rac1 pulldown assay to measure Rac1-GTP levels, we showed that silencing ΔNp63α in A431 cells led to a significant upregulation of Rac1-GTP. Silencing ΔNp63α in A431 cells also led to an increase in p21-activated kinase (PAK1) phosphorylation, confirming the negative regulation of ΔNp63α on Rac1. Inhibiting Rac1 GTP activity using EHop-016 led to a significant reduction in both pRac1 and pPAK1 levels. Treatment with Ehop-016 reversed the increase in cell invasion observed upon silencing ΔNp63α. RT-PCR analysis performed on an array of 43 Rac guanine exchange factors (Rac-GEFs) showed that 6 GEFs are significantly upregulated by silencing ΔNp63α in A431 cells. Taken together, our data suggest that ΔNp63α negatively regulates Rac1 GTP activity via downregulation of a Rac-GEF, thereby reducing Rac1-GTP levels and inhibiting cancer cell invasion.

Growth and metabolic responses of grouper juveniles (Epinephelus coioides) fed diets containing varying levels of leucine

We conducted an 8-week feeding trial to evaluate the effects of dietary leucine on growth performance, body and muscle composition, hepatic and plasma components, gut morphology, and relative expression levels of genes in the mTOR pathway. Six isonitrogenous and isolipidic diets with increasing levels of Leu (1.88%, 2.84%, 3.51%, 4.45%, 5.27%, and 6.06%, respectively) were formulated. Each diet was fed to 4-replicate groups of 20 groupers (average initial body weight of 35.0 g/fish) twice daily, to apparent satiety. Weight gain and specific growth rates; feed intake; body protein, lipid, and muscle protein content; and liver GOT and branched chain α-keto acid dehydrogenase kinase activities showed positive linear and/or quadratic responses to dietary Leu levels. In contrast, body moisture and ash content, liver triglyceride and total-cholesterol content, and plasma GOT and GTP activity had negative linear and/or quadratic responses to different dietary Leu levels. There were no differences in the feed conversion ratio and hepatosomatic index, condition factor, plasma GOT activity, and plasma triglyceride and total-cholesterol content among treatments. The mucosal fold number in three gut segments (proximal intestine (PI), middle intestine (MI), and distal intestine (DI)) and mucosal fold length and muscle layer thickness in the DI showed positive quadratic responses to dietary Leu levels. In addition, mucosal fold length and number, and muscle layer thickness in the PI were greater that those observed for the MI and DI. The mRNA level of SLC3A2, SLC7A5, RagC/D, Ragulator, Raptor, mTOR, mLST8, and Tel2 in the muscle and/or gut showed positive quadratic responses to dietary Leu levels, whereas negative quadratic responses were observed for mRNA levels of RagA/B and V-ATPase in the muscle and/or gut, and SLC7A5 in the liver. The optimal amount of Leu required for this fish was estimated to be 8.88% of the dietary protein content, based on the regression analysis of percent weight gains against dietary Leu levels. The results indicate that appropriate dietary Leu levels (range 3.51%–4.45%) could help to improve growth performance and nutrient deposition, maintain gut structural integrity and positively regulate genes in the mTOR signaling pathway in the fish.

Dephosphorylation of cGAS by PPP6C impairs its substrate binding activity and innate antiviral response

The cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in host defense by sensing cytosolic DNA derived from microbial pathogens or mis-located cellular DNA. Upon DNA binding, cGAS utilizes GTP and ATP as substrates to synthesize cGAMP, leading to MITA-mediated innate immune response. In this study, we identified the phosphatase PPP6C as a negative regulator of cGAS-mediated innate immune response. PPP6C is constitutively associated with cGAS in un-stimulated cells. DNA virus infection causes rapid disassociation of PPP6C from cGAS, resulting in phosphorylation of human cGAS S435 or mouse cGAS S420 in its catalytic pocket. Mutation of this serine residue of cGAS impairs its ability to synthesize cGAMP upon DNA virus infection. In vitro experiments indicate that S420-phosphorylated mcGAS has higher affinity to GTP and enzymatic activity. PPP6C-deficiency promotes innate immune response to DNA virus in various cells. Our findings suggest that PPP6C-mediated dephosphorylation of a catalytic pocket serine residue of cGAS impairs its substrate binding activity and innate immune response, which provides a mechanism for keeping the DNA sensor cGAS inactive in the absence of infection to avoid autoimmune response.

ΔNp63α suppresses cell invasion through downregulating Rac1 activity

ΔNp63α, a member of the p53 family of transcription factors, is overexpressed in a number of cancers and plays a role in proliferation, differentiation, migration and invasion. We showed previously that ΔNp63α suppresses cell invasion by positively regulating miR‐320a, resulting in the downregulation of its direct target protein kinase C gamma (PKCg) and a corresponding reduction in phosphorylated Ras‐related C3 botulinum toxin substrate 1 (Rac1). Here, we further show that ΔNp63α inhibits Rac1 GTP activity. Using a Rac1 pulldown assay to measure Rac1‐GTP levels, we showed that silencing ΔNp63α in A431 cells led to a significant upregulation of Rac1‐GTP. Silencing ΔNp63α in A431 cells also led to an increase in p21‐activated kinase (PAK1) phosphorylation, confirming the negative regulation of ΔNp63α on Rac1. Inhibiting Rac1 GTP activity using Ehop‐016 led to a significant reduction in both pRac1 and pPAK1 levels. Treatment with Ehop‐016 reversed the increase in cell invasion observed upon silencing ΔNp63α. RT‐PCR analysis performed on an array of 43 Rac guanine exchange factors (Rac‐GEFs) showed that 6 GEFs are significantly upregulated by silencing ΔNp63α in A431 cells. Taken together, our data suggest that ΔNp63α negatively regulates Rac1 GTP activity via downregulation of a Rac‐GEF, thereby reducing Rac1‐GTP levels and inhibiting cancer cell invasion.Support or Funding InformationThis work was supported by a grant from the National Cancer Institute [1R01CA154715] to M.P.K.

TUFT1 promotes metastasis and chemoresistance in triple negative breast cancer through the TUFT1/Rab5/Rac1 pathway

BackgroundTriple negative breast cancer (TNBC) is a breast cancer (BC) subtype that is characterized by its strong invasion and a high risk of metastasis. However, the specific mechanisms underlying these phenotypes are unclear. TUFT1 plays an important role in BC and impacts the proliferation and survival of BC cells. Recent studies have shown that TUFT1 mediates intracellular lysosome localization and vesicle transport by regulating Rab GTPase, but the relevance of this activity in TNBC is unknown. Therefore, our aim was to systematically study the role of TUFT1 in the metastasis and chemoresistance of TNBC.MethodsWe measured TUFT1, Rab5-GTP, and Rac1-GTP expression levels in samples of human TNBC by immunohistochemistry (IHC) and conducted univariate and multivariate analyses. shRNA-mediated knockdown and overexpression, combined with transwell assays, co-immunoprecipitation, a nude mouse xenograft tumor model, and GTP activity assays were used for further mechanistic studies.ResultsTUFT1 expression was positively correlated with Rab5-GTP and Rac1-GTP in the TNBC samples, and co-expression of TUFT1 and Rab5-GTP predicted poor prognosis in TNBC patients who were treated with chemotherapy. Mechanism studies showed that TUFT1 could activate Rab5 by binding to p85α, leading to activation of Rac1 through recruitment of Tiam1, and concurrent down-regulation of the NF-κB pathway and proapoptotic factors, ultimately promoting metastasis and chemoresistance in TNBC cells.ConclusionsOur findings suggest that the TUFT1/Rab5/Rac1 pathway may be a potential target for the effective treatment of TNBC.

TUFT1 Promotes Triple Negative Breast Cancer Metastasis, Stemness, and Chemoresistance by Up-Regulating the Rac1/β-Catenin Pathway.

Objectives: Triple negative breast cancer (TNBC) is a subtype of breast cancer with stronger invasion and metastasis, but its specific mechanism of action is still unclear. Tuft1 plays an important regulatory role in the survival of breast cancer cells; however, its role in regulating TNBC metastatic potential has not been well-characterized. Our aim was therefore to systematically study the mechanism of TUFT1 in the metastasis, stemness, and chemoresistance of TNBC and provide new predictors and targets for BC treatment.Methods: We used western blotting and IHC to measure TUFT1and Rac1-GTP expression levels in both human BC samples and cell lines. A combination of shRNA, migration/invasion assays, sphere formation assay, apoptosis assays, nude mouse xenograft tumor model, and GTP activity assays was used for further mechanistic studies.Results: We demonstrated that silencing TUFT1 in TNBC cells significantly inhibited cell metastasis and stemness in vitro. A nude mouse xenograft tumor model revealed that TUFT1 knockdown greatly decreased spontaneous lung metastasis of TNBC tumors. Mechanism studies showed that TUFT1 promoted tumor cell metastasis and stemness by up-regulating the Rac1/β-catenin pathway. Moreover, mechanistic studies indicated that the lack of TUFT1 expression in TNBC cells conferred more sensitive to chemotherapy and increased cell apoptosis via down-regulating the Rac1/β-catenin signaling pathway. Further, TUFT1 expression positively correlated with Rac1-GTP in TNBC samples, and co-expression of TUFT1 and Rac1-GTP predicted poor prognosis in TNBC patients who treated with chemotherapy.Conclusion: Our findings suggest that TUFT1/Rac1/β-catenin pathway may provide a potential target for more effective treatment of TNBC.

Evaluation of drug dependent persons’ health on the basis of routine laboratory test results

IntroductionThe aim of this study was to evaluate the frequency of reference value deviations in blood chemistry in patients treated for substance abuse and to demonstrate the relationship between the type of dependence and abnormalities in biochemistry studies. Material and methodThe study involved 93 subjects hospitalised in the brief dependence and codependence therapy department. Patients were divided into three groups: alcohol, opiate and amphetamine dependent. The study was conducted in the first two weeks of stay and concerned demographic data and blood chemistry results. ResultsOver 40% of all subjects had a higher than normal GTP activity, more than 20% higher than normal levels of cholesterol and ALT, and more than 16% higher AST. Persons addicted to amphetamine were shown to have the highest level of LDL, while the lowest was in those addicted to alcohol. The highest average GTP, AST, ALT liver injury markers occurred in alcohol and opiate dependent persons and the lowest in amphetamine addicts. DiscussionThis study confirmed the results of other studies that persons addicted to amphetamine had better biochemical parameters of liver functioning than opiate or alcohol addicts. ConclusionThe most commonly assessed biochemical changes of reference values observed above concerned total cholesterol, AST, ALT, GTP. Alcohol or opiate dependent persons had more often increased activity of GTP, AST and ALT than those addicted to amphetamine.

The type III TGFβ receptor is required for hyaluronan‐mediated epicardial invasive cell motility (542.5)

The epicardium originates from the proepicardial organ (PEO). Both the PEO and epicardium require directed cell‐motility to cover the heart and instruct epicardially derived cells for coronary vasculature formation. The type III transforming growth factor‐beta receptor (Tgfbr3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Utilizing a primary epicardial cell line derived from Tgfbr3(+/+) and Tgfbr3(‐/‐) e13.5 mouse embryos , we show that Tgfbr3(‐/‐) epicardial cells are 34.4% deficient in 2D migration relative to wild type. Furthermore, we show that high‐molecular weight Hyaluronan (HMWHA) drives 3D cellular invasion in wild type epicardial cells, but not Tgfbr3(‐/‐) epicardial cells. We show Src kinase is required for HMWHA stimulated filamentous actin (f‐actin) polymerization in wild‐epicardial cells. Furthermore, HMWHA stimulates Y416Src phosphorylation in wild type, but Src is not activated in Tgfbr3(‐/‐) epicardial cells. RhoA and Rac1 GTPase are downstream of Src kinase, and directly upstream of f‐actin polymerization; HMWHA robustly induces RhoA and Rac1 GTP binding and activity in wild type, but not Tgfbr3(‐/‐) epicardial cells. This deficit in RhoA and Rac1 activation results in reduced formation of f‐actin stress fibers in HMWHA stimulated Tgfbr3(‐/‐) epicardial cells. Taken together, we demonstrate that the type III TGFβ receptor is required for activation of basic cell motility pathways stimulated by Hyaluronan that are required for proper coronary vasculature formation.Grant Funding Source: NIH ES 04940; ES06694; SWEHSC P30ES006694

반응표면분석을 이용한 청국장 제조시 마늘의 첨가조건 최적화

마늘을 이용한 청국장의 제조조건을 최적화 하고자 중심합성계획에 따라 마늘의 첨가량(<TEX>$X_1$</TEX>), 마늘 열처리시간(<TEX>$X_2$</TEX>) 및 청국장 발효시간(<TEX>$X_3$</TEX>)을 독립변수로 하고, 점질물 생성량(<TEX>$Y_1$</TEX>), 산도(<TEX>$Y_2$</TEX>), 아미노태 질소량(<TEX>$Y_3$</TEX>), <TEX>${\gamma}$</TEX>-GTP(<TEX>$Y_4$</TEX>) 및 ABTS 라디칼 소거능(<TEX>$Y_5$</TEX>)을 종속변수로 하여 반응표면분석을 실시하였다. 점질물 생성량의 정상점은 최대점으로 실제변수인 마늘의 첨가량이 6.53%, 열처리 시간 6.81분, 청국장 발효시간 55.18시간에서 13.02%의 최적 값을 보였다. 산도는 청국장의 발효시간이 길어질수록 증가하였으며, 최소값인 0.50%를 나타내는 마늘 첨가량의 실제변수는 7.75%, 열처리 시간은 3.42분, 청국장 발효는 58.60시간이었다. 아미노태 질소 함량은 80.58~158.82 mg% 범위였는데 정상점은 안장점으로 능선분석을 통해 얻어진 아미노태 질소의 최고값은 156.97 mg%였다. 이 때 마늘 첨가량 및 열처리 시간은 각각 6.21% 및 14.85분, 청국장 발효시간은 58.04시간이었다. <TEX>${\gamma}$</TEX>-GTP 활성은 353.66 mU/mL의 최대값을 가질 때, 마늘 첨가량, 마늘 열처리 시간 및 청국장 발효시간이 각각 5.73%, 6.99분, 57.96시간 이었다. ABTS 라디칼 소거능은 마늘 첨가량과 청국장 발효시간을 낮추고 마늘의 열처리 시간을 높이는 경우 76.43%의 최고값 얻을 수 있었는데, 이에 따른 실제변수의 마늘의 첨가량과 열처리시간은 각각 3.78% 및 14.28분과 청국장 발효시간은 57.99시간이었다. 이상의 결과를 종합하여 볼 때 마늘이 첨가된 청국장 제조를 위해 마늘은 불린 콩 무게 대비 3.78~7.75%로 첨가하되 3.42~14.85분의 범위에서 스팀가열한 후 사용하며, 55~59시간 정도 발효시키는 것이 적합하였다. This study was performed to determine the optimal composition of Cheonggukjang added with garlic. The experiment utilized a central composite design (CCD). The evaluation was carried out by means of response surface methodology (RSM), which included 18 experimental points with three independent variables : the content of the garlic (1.3~9.7%, <TEX>$X_1$</TEX>), the steaming time of garlic (0~15.1 min, <TEX>$X_2$</TEX>), and the fermentation time of Cheonggukjang (48.2~71.8 h, <TEX>$X_3$</TEX>). The viscous substance (<TEX>$Y_1$</TEX>), acidity (<TEX>$Y_2$</TEX>), amino-type nitrogen (<TEX>$Y_3$</TEX>), <TEX>${\gamma}$</TEX>-GTP activity (<TEX>$Y_4$</TEX>) and ABTS radical scavenging activity (<TEX>$Y_5$</TEX>). were assessed in four replicates with five dependent variables. The maximum content of the viscous substance was 13.02% at 6.53% (<TEX>$X_1$</TEX>), 6.81 min (<TEX>$X_2$</TEX>) and 55.18 h (<TEX>$X_3$</TEX>). The acidity was increased when the fermentation time was longer, and the minimum acidity point was 0.50% at 7.75% (<TEX>$X_1$</TEX>), 3.42 min (<TEX>$X_2$</TEX>) and 58.60 h (<TEX>$X_3$</TEX>), respectively. The content of the amino-type nitrogen at the experimental range studied was was 80.58~158.82 mg%, and the stationary point was at saddle point. Using ridge analysis, the maximum point was 156.97 mg% at 6.21% (<TEX>$X_1$</TEX>), 14.85 min (<TEX>$X_2$</TEX>) and 58.04 h(<TEX>$X_3$</TEX>). The optimum conditions of <TEX>${\gamma}$</TEX>-GTP activity was 5.73% (<TEX>$X_1$</TEX>), 6.99 min (<TEX>$X_2$</TEX>) and 57.96 h(<TEX>$X_3$</TEX>), respectively, at the maximum point was 353.66 mU/mL. The maximum point of ABTS radical scavenging activity was 76.43% at 3.78% (<TEX>$X_1$</TEX>), 14.28 min (<TEX>$X_2$</TEX>) and 57.99 h(<TEX>$X_3$</TEX>) at the saddle point, when the garlic steaming time was longer.

Serum ^|^gamma;-GTP Activity Is Closely Associated with Serum CRP Levels in Non-Overweight and Overweight Middle-Aged Japanese Men

The relationship between γ-glutamyltransferase (γ-GTP) and C-reactive protein (CRP) has not been established, particularly in the lean or non-overweight population. In the present study, we examined the associations between γ-GTP and CRP in non-overweight and overweight middle-aged Japanese men. We conducted a cross-sectional study of 4,271 apparently healthy men aged 40 to 64 y (mean±SD, 50.5±6.6 y) who participated in health checkups. Associations between serum CRP levels, other clinical parameters, and lifestyle factors were analyzed using Spearman's rank correlation coefficient analysis and multiple linear regression analysis in the non-overweight (body mass index [BMI]<25 kg/m(2)) and overweight (BMI≥25 kg/m(2)) men. Associations between serum γ-GTP activity and serum CRP levels were analyzed using analysis of covariance by comparisons of serum CRP levels of four subgroups according to γ-GTP status. In non-overweight men, BMI, high-density lipoprotein cholesterol, triacylglycerols, fasting blood glucose, aspartate aminotransferase, γ-GTP, and smoking habit were positively associated with serum CRP levels. In overweight men, BMI, diastolic blood pressure, triacylglycerols, and γ-GTP were positively associated with serum CRP levels. After adjustment for age, BMI, smoking status, and alcohol intake, dose-response relationships were observed between γ-GTP and CRP levels in both overweight and non-overweight men. The results of this study indicate that an increase in serum γ-GTP activity is closely associated with elevated CRP levels in both non-overweight and overweight middle-aged Japanese men.

구속스트레스를 가한 흰쥐의 지질 대사에 대한 흑마늘 추출물의 효과

흑마늘의 구속 스트레스에 대한 유효농도를 확인하고자 2, 7 및 14 brix로 조절한 흑마늘 추출물을 급이한 흰쥐의 체내 지질대사를 중심으로 분석하였다. 흑마늘 추출물 14 brix 급이군의 식이섭취량은 <TEX>$547.64{\pm}13.99$</TEX> g/4 weeks로 대조군에 비해 유의적으로 높았다. 모든 장기에서 흑마늘 추출물의 첨가 급이에 따른 무게의 변화는 통계적인 유의차는 없었다. 흑마늘 추출물 첨가 급이군의 혈당은 흑마늘 추출물의 농도가 높아질수록 낮아지는 경향을 보여 2 brix 추출물 급이군의 혈당은 <TEX>$142.97{\pm}6.51$</TEX> mg/dl 였으나 14 brix 급이군에서는 <TEX>$121.32{\pm}3.50$</TEX> mg/dl로 정상군 보다도 더 낮았다. 총 콜레스테롤 함량은 <TEX>$54.54{\pm}1.63{\sim}65.84{\pm}2.61$</TEX> mg/dl의 범위였는데, 14 brix의 흑마늘 추출물 첨가 급이군의 혈청 총 콜레스테롤 농도는 <TEX>$54.54{\pm}1.63$</TEX> mg/dl로 가장 낮은 함량이었으며, 정상군에 비해서도 유의적으로 낮은 함량이었다. 중성지질의 함량도 동일한 경향이었다. HDL-콜레스테롤 농도는 구속대조군에 비해 흑마늘 추출물 7 및 14 brix 급이군에서 유의적으로 높았으며, LDL VLDL-콜레스테롤은 이와 상반되는 결과를 나타내었다. 혈청 GOT 및 GTP 활성도는 흑마늘 추출물의 농도가 높아질수록 낮아져 흑마늘 추출물의 급이는 간보호 활성을 나타낼 것으로 기대된다. 간장내 총 지질 및 중성지질의 함량은 구속대조군에 비해 흑마늘 추출물 급이군에서 유의적으로 낮았으나 총콜레스테롤은 서로간에 유의차가 없었다. 간장내 지질 과산화물인 TBARS 함량은 2 brix의 흑마늘 추출물 급이군은 구속대조군과 유의적인 차이가 없었으나 7 및 14 brix 급이군은 정상군보다 더 낮은 함량이었다. 간장내 글리코겐 함량은 구속대조군에 비해 14 brix 흑마늘 추출물 급이군에서만 유의적으로 낮았다. 이상의 결과들을 종합하여 볼 때 7 brix 이상의 흑마늘 추출물 급이는 흰쥐의 지질대사를 개선하고, 간보호 활성을 나타냄으로써 구속 스트레스로 해소에 기여할 것으로 추정된다. In order to determine the effective concentration, 2, 7, and 14 brix black garlic extracts (BGE) were fed to restraint stressed rats and the lipid metabolism was analyzed. The dietary intake of the 14 brix BGE-fed group was <TEX>$547.64{\pm}13.99$</TEX> g/4 weeks and it was significantly higher than that of the control group. The weight of all organs from the all BGE-fed groups showed no significant differences. While BGE concentration increased, BGE-fed groups tend to have low content of blood glucose. It was the lowest in the 14 brix BGE-fed group (<TEX>$121.32{\pm}3.50$</TEX> mg/dl). Total cholesterol content was <TEX>$54.54{\pm}1.63{\sim}65.84{\pm}2.61$</TEX> mg/dl. Total cholesterol content of the 14 brix BGE-fed group was the lowest and, in addition, lower than the normal group. Triglyceride content of serum showed the same tendency. HDL-cholesterol concentrations of the restraint stressed control (RSC) group were significantly higher than those of 7 and 14 brix BGE-fed groups. LDL and VLDL-cholesterol contents showed contrary results. GOT, GTP, and ALP activity of serum were lower when BGE concentration was higher. It is expected that BGE has a liver protective activity. In the liver, total lipids and triglyceride contents were significantly lower in BGE-fed groups than in the ISC group. However, total cholesterol content showed no significant difference between groups. TBARS content in liver of the 2 brix BGE-fed group was not significantly different than control, while 7 and 14 brix BGE-fed groups were lower than control. Glycogen content in the liver was only significantly lower in the 14 brix BGE-fed group than in the RSC group. Findings indicate that feed of BGE over 7 brix contributes to resolving the restraint-stressed rats by improving the lipid metabolism and liver protective activity.

GTP cyclohydrolase 1 gene haplotypes as predictors of SSRI response in Japanese patients with major depressive disorder

Tetrahydrobiopterin (BH4) plays an important role in the biosynthesis of serotonin, melatonin and catecholamines, all of which are implicated in the pathophysiology of mood disorders (MDs), including major depressive disorder (MDD) and bipolar disorder (BP). Production of BH4 is regulated by GTP cyclohydrolase transcription and activity. Thus, we considered the GTP cyclohydrolase gene (GCH1) to be a good candidate gene in the pathophysiology of MDs and of the serotonin selective reuptake inhibitors (SSRIs) response in MDD, and conducted a case-control study utilizing three SNPs (rs8007267, rs3783641 and rs841) and moderate sample sizes (405 MDD patients, including 262 patients treated by SSRIs, 1022 BP patients and 1805 controls). A multiple logistic regression analysis was carried out to compare the frequencies of each SNP genotype for the target phenotype across patients and controls in several genetic models, while adjusting for possible confounding factors. A clinical response was defined as a decrease of more than 50% from the baseline score on the Structured Interview Guide for Hamilton Rating Scale for Depression (SIGH-D) within 8 weeks, and clinical remission as a SIGH-D score of less than 7 at 8 weeks. No associations between three SNPs in GCH1 and MDD or BP were observed; however, GCH1 was associated with SSRI therapeutic response in MDD in all the marker's haplotype analysis (Global P value=0.0379). Results suggest that GCH1 may predict response to SSRI in MDD in the Japanese population. Nevertheless, a replication study using larger samples may be required for conclusive results, since our sample size was small.

NADPH oxidase-mediated Rac1 GTP activity is necessary for nongenomic actions of the mineralocorticoid receptor in the CA1 region of the rat hippocampus

Mineralocorticoid receptors (MRs) in the central nervous system play important roles in spatial memory, fear memory, salt sensitivity, and hypertension. Corticosterone binds to MRs to induce presynaptic vesicle release and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor aggregation, which are necessary for induction of long-term potentiation under psychological stress. On the other hand, cognitive dysfunction is an important problem clinically in patients with hypertension, diabetes, and cerebral infarction, and all of these conditions are associated with an increase in reactive oxygen species (ROS) generation. Oxidative stress has been shown to modify the genomic actions of MRs in the peripheral organs; however, there have been no reports until now about the relation between the nongenomic actions of MRs and ROS in the central nervous system. In this study, we investigated the relationship between ROS and the nongenomic actions of MR. We examined the nongenomic actions of MR by measuring the slope of the field excitatory postsynaptic potentials and found that ROS induced an additive increase of these potentials, which was accompanied by Rac1 GTP activation and ERK1/2 phosphorylation. An NADPH oxidase inhibitor, apocynin, blocked the nongenomic actions of MRs. A Rac1 inhibitor, NSC23766, was also found to block synaptic enhancement and ERK1/2 phosphorylation induced by NADPH and corticosterone. We concluded that NADPH oxidase activity and Rac1 GTP activity are indispensable for the nongenomic actions of MRs and that Rac1 GTP activation induces ERK1/2 phosphorylation in the brain.

Dtorsin, the Drosophila Ortholog of the Early-Onset Dystonia TOR1A (DYT1), Plays a Novel Role in Dopamine Metabolism

Dystonia represents the third most common movement disorder in humans. At least 15 genetic loci (DYT1-15) have been identified and some of these genes have been cloned. TOR1A (formally DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. However, the function of torsinA has yet to be fully understood. Here, we have generated and characterized a complete loss-of-function mutant for dtorsin, the only Drosophila ortholog of TOR1A. Null mutation of the X-linked dtorsin was semi-lethal with most male flies dying by the pre-pupal stage and the few surviving adults being sterile and slow moving, with reduced cuticle pigmentation and thin, short bristles. Third instar male larvae exhibited locomotion defects that were rescued by feeding dopamine. Moreover, biochemical analysis revealed that the brains of third instar larvae and adults heterozygous for the loss-of-function dtorsin mutation had significantly reduced dopamine levels. The dtorsin mutant showed a very strong genetic interaction with Pu (Punch: GTP cyclohydrolase), the ortholog of the human gene underlying DYT14 dystonia. Biochemical analyses revealed a severe reduction of GTP cyclohydrolase protein and activity, suggesting that dtorsin plays a novel role in dopamine metabolism as a positive-regulator of GTP cyclohydrolase protein. This dtorsin mutant line will be valuable for understanding this relationship and potentially other novel torsin functions that could play a role in human dystonia.

WNT-5A stimulates the GDP/GTP exchange at pertussis toxin-sensitive heterotrimeric G proteins

The lipoglycoproteins of the WNT family act on seven transmembrane-spanning Class Frizzled receptors. Here, we show that WNT-5A evokes a proliferative response in a mouse microglia-like cell line (N13), which is sensitive to pertussis toxin, thus implicating the involvement of heterotrimeric G proteins of the Gi/o family. We continue to show that WNT-5A stimulation of N13 membranes and permeabilized cells evokes the exchange of GDP for GTP of pertussis toxin-sensitive G proteins employing [γ-35S]GTP assay and activity state-specific antibodies to GTP-bound Gi proteins. Our functional analysis of the PTX-sensitivity of WNT-induced G protein activation and PCR analysis of G protein and FZD expression patterns suggest that WNT-5A stimulation leads to the activation of Gi2/3 proteins in N13 cells possibly mediated by FZD5, the predominant FZD expressed. In summary, we provide for the first time molecular proof that WNT-5A stimulation results in the activation of heterotrimeric Gi2/3 proteins in mammalian cells with physiological protein stochiometry.