Intracellular localization of CK2α as a prognostic factor in invasive breast carcinomas.

Overexpression of the ubiquitous protein kinase, CK2α, has been reported in various human cancers. Here, we demonstrate that nuclear and nucleolar CK2α localization in invasive ductal carcinomas of the breast is a reliable predictor of poor prognosis. Cellular localization of CK2α in nuclei and nucleoli was analyzed immunohistochemically using surgical tissue blocks from 112 patients, who had undergone surgery without neoadjuvant chemotherapy. Clinical data collection and median follow-up period were for more than 5 y. In total, 93.8% of patients demonstrated elevated CK2α expression in nuclei and 36.6% of them displayed elevated expression predominantly in nucleoli. Clinicopathological malignancy was strongly correlated with elevated nuclear and nucleolar CK2α expression. Recurrence-free survival was significantly worse (P = .0002) in patients with positive nucleolar CK2α staining. The 5-y survival rate decreased to a roughly 50% in nucleolar CK2α-positive patients of triple-negative (P = .0069) and p Stage 3 (P = .0073) groups. In contrast, no patients relapsed or died in the triple-negative group who exhibited a lack of nucleolar CK2α staining. Evaluation of nucleolar CK2α staining showed a high secondary index with a hazard ratio of 6.629 (P = .001), following lymph node metastasis with a hazard ratio of 14.30 (P = .0008). Multivariate analysis demonstrated that nucleolar CK2α is an independent factor for recurrence-free survival. Therefore, we propose that histochemical evaluation of nucleolar CK2α-positive staining may be a new and robust prognostic indicator for patients who need further treatment. Functional consequences of nucleolar CK2 dysfunction may be a starting point to facilitate development of novel treatments for invasive breast carcinoma.

A novel complement inhibitor sMAP-FH targeting both the lectin and alternative complement pathways.

Inhibition of the complement activation has emerged as an option for treatment of a range of diseases. Activation of the lectin and alternative pathways (LP and AP, respectively) contribute to the deterioration of conditions in certain diseases such as ischemia-reperfusion injuries and age-related macular degeneration (AMD). In the current study, we generated dual complement inhibitors of the pathways MAp44-FH and sMAP-FH by fusing full-length MAp44 or small mannose-binding lectin-associated protein (sMAP), LP regulators, with the N-terminal five short consensus repeat (SCR) domains of complement factor H (SCR1/5-FH), an AP regulator. The murine forms of both fusion proteins formed a complex with endogenous mannose-binding lectin (MBL) or ficolin A in the circulation when administered in mice intraperitoneally. Multiple complement activation assays revealed that sMAP-FH had significantly higher inhibitory effects on activation of the LP and AP in vivo as well as in vitro compared to MAp44-FH. Human form of sMAP-FH also showed dual inhibitory effects on LP and AP activation in human sera. Our results indicate that the novel fusion protein sMAP-FH inhibits both the LP and AP activation in mice and in human sera, and could be an effective therapeutic agent for diseases in which both the LP and AP activation are significantly involved.

Cutting Edge: Role of MASP-3 in the Physiological Activation of Factor D of the Alternative Complement Pathway.

The complement system, a part of the innate immune system, can be activated via three different pathways. In the alternative pathway, a factor D (FD) plays essential roles in both the initiation and the amplification loop and circulates as an active form. Mannose-binding lectin-associated serine proteases (MASPs) are key enzymes of the lectin pathway, and MASP-1 and/or MASP-3 are reported to be involved in the activation of FD. In the current study, we generated mice monospecifically deficient for MASP-1 or MASP-3 and found that the sera of the MASP-1-deficient mice lacked lectin pathway activity, but those of the MASP-3-deficient mice lacked alternative pathway activity with a zymogen FD. Furthermore, the results indicate that MASP-3 but not MASP-1 activates the zymogen FD under physiological conditions and MASP-3 circulates predominantly as an active form. Therefore, our study illustrates that, in mice, MASP-3 orchestrates the overall complement reaction through the activation of FD.

Dictyostelium Differentiation-Inducing Factor-1 Promotes Glucose Uptake, at Least in Part, via an AMPK-Dependent Pathway in Mouse 3T3-L1 Cells.

Differentiation-inducing factor-1 (DIF-1) is a chlorinated alkylphenone (a polyketide) found in the cellular slime mold Dictyostelium discoideum. DIF-1 and its derivative, DIF-1(3M) promote glucose consumption in vitro in mammalian cells and in vivo in diabetic rats; they are expected to be the leading antiobesity and antidiabetes compounds. In this study, we investigated the mechanisms underlying the actions of DIF-1 and DIF-1(3M). In isolated mouse liver mitochondria, these compounds at 2-20 µM promoted oxygen consumption in a dose-dependent manner, suggesting that they act as mitochondrial uncouplers, whereas CP-DIF-1 (another derivative of DIF-1) at 10-20 µM had no effect. In confluent mouse 3T3-L1 fibroblasts, DIF-1 and DIF-1(3M) but not CP-DIF-1 induced phosphorylation (and therefore activation) of AMP kinase (AMPK) and promoted glucose consumption and metabolism. The DIF-induced glucose consumption was reduced by compound C (an AMPK inhibitor) or AMPK knock down. These data suggest that DIF-1 and DIF-1(3M) promote glucose uptake, at least in part, via an AMPK-dependent pathway in 3T3-L1 cells, whereas cellular metabolome analysis revealed that DIF-1 and DIF-1(3M) may act differently at least in part.

Astrocyte-Derived Exosomal microRNA miR-200a-3p Prevents MPP+-Induced Apoptotic Cell Death Through Down-Regulation of MKK4.

Astrocytes release exosomes that regulate neuronal cell function. 1-methyl-4-phenylpyridinium (MPP+) is a well-known neurotoxin used to induce cell death in in vitro Parkinson's disease models, and microRNA (miRNA) transferred by released exosomes can regulate its mechanisms. Here, we demonstrated that exosomes released from normal astrocytes (ADEXs), but not exosomes derived from MPP+-stimulated astrocytes (MPP+-ADEXs), significantly attenuate MPP+-induced cell death in SH-SY5Y cells and primary mesencephalic dopaminergic neuron cultures, and reduce expression of mitogen-activated protein kinase kinase 4 (MKK4), an important upstream kinase in the c-Jun N-terminal kinase cell death pathway. Similar neuroprotective results were obtained from primary hippocampal neuron cultures, an in vitro glutamate excitotoxicity model. Through small-RNA sequencing of exosomal miRNA, we identified miR-200a-3p as the most down-regulated miRNA expressed in MPP+-ADEXs. miRNA target analysis and reporter assay confirmed that miR-200a-3p targets MKK4 through binding to two independent sites on the 3'-UTR of Map2k4/MKK4 mRNA. Treatment with miR-200a-3p mimic suppressed both MKK4 mRNA and protein expressions, and attenuated cell death in MPP+-treated SH-SY5Y cells and glutamate-treated hippocampal neuron cultures. Our results suggest that normal astrocytes release miR-200a-3p which exhibits a neuroprotective effect through down-regulation of MKK4.

Mitochondrial reactive oxygen species suppress humoral immune response through reduction of CD19 expression in B cells in mice.

Reactive oxygen species (ROS) are implicated in the modulation of diverse processes including immune responses. To evaluate the effects of metabolic ROS produced by mitochondria on B-cell function and development, we created transgenic (Tg) mice expressing a phosphorylation-defective mutant of succinate dehydrogenase A in B cells (bSDHAY215F ). Splenic B cells in male, but not female, bSDHAY215F mice produced three times more ROS than those in the control mice, and had decreased production of IgM, IgG1 , and IgG3 , and affinity maturation of IgG1 against T-cell-dependent antigens. Following immunization, the male bSDHAY215F mice further displayed suppressed germinal center (GC) formation, and proliferation of GC B cells. Signaling analysis revealed defects in the intrinsic BCR responses, such as activation of Lyn, Btk, and PLCγ2, thus resulting in reduced intracellular Ca2+ mobilization. Notably, the expression levels of B-cell co-receptor CD19 and its interaction with Lyn after BCR ligation were significantly reduced in B cells from male bSDHAY215F mice. These results suggest that mitochondrial ROS suppress humoral immune responses through reduction of CD19 expression and resultant BCR signaling in B cells. Therefore, B-cell immunity may be more labile to oxidative stress in male mice than in female mice.

Derivatives of Dictyostelium differentiation-inducing factors inhibit lysophosphatidic acid-stimulated migration of murine osteosarcoma LM8 cells.

Osteosarcoma is a common metastatic bone cancer that predominantly develops in children and adolescents. Metastatic osteosarcoma remains associated with a poor prognosis; therefore, more effective anti-metastatic drugs are needed. Differentiation-inducing factor-1 (DIF-1), -2, and -3 are novel lead anti-tumor agents that were originally isolated from the cellular slime mold Dictyostelium discoideum. Here we investigated the effects of a panel of DIF derivatives on lysophosphatidic acid (LPA)-induced migration of mouse osteosarcoma LM8 cells by using a Boyden chamber assay. Some DIF derivatives such as Br-DIF-1, DIF-3(+2), and Bu-DIF-3 (5-20 µM) dose-dependently suppressed LPA-induced cell migration with associated IC50 values of 5.5, 4.6, and 4.2 µM, respectively. On the other hand, the IC50 values of Br-DIF-1, DIF-3(+2), and Bu-DIF-3 versus cell proliferation were 18.5, 7.2, and 2.0 µM, respectively, in LM8 cells, and >20, 14.8, and 4.3 µM, respectively, in mouse 3T3-L1 fibroblasts (non-transformed). Together, our results demonstrate that Br-DIF-1 in particular may be a valuable tool for the analysis of cancer cell migration, and that DIF derivatives such as DIF-3(+2) and Bu-DIF-3 are promising lead anti-tumor agents for the development of therapies that suppress osteosarcoma cell proliferation, migration, and metastasis.

Synthesis of prenylated quinolinecarboxylic acid derivatives and their anti-obesity activities.

Mitochondrial uncoupling is one of the therapeutic strategies used to control energy metabolism in various metabolic diseases and in obesity. Ppc-1 (1), a prenylated quinolinecarboxylic acid isolated from cellular slime molds, shows uncoupling activity in vitro and anti-obesity activity in vivo. In this study, we synthesized Ppc-1 (1) and its derivatives, and revealed the structure-activity relationship of uncoupling activities. The triprenylated compound 18 showed mitochondrial uncoupling activity that was more potent than that of Ppc-1 (1). Compound 18 also suppressed weight gain in mice without undesired effects such as lesions on tissues. These results indicate that compound 18 could be used as a seed compound for new anti-obesity drugs.

Cell cycle-dependent gene networks for cell proliferation activated by nuclear CK2α complexes.

Nuclear expression of protein kinase CK2α is reportedly elevated in human carcinomas, but mechanisms underlying its variable localization in cells are poorly understood. This study demonstrates a functional connection between nuclear CK2 and gene expression in relation to cell proliferation. Growth stimulation of quiescent human normal fibroblasts and phospho-proteomic analysis identified a pool of CK2α that is highly phosphorylated at serine 7. Phosphorylated CK2α translocates into the nucleus, and this phosphorylation appears essential for nuclear localization and catalytic activity. Protein signatures associated with nuclear CK2 complexes reveal enrichment of apparently unique transcription factors and chromatin remodelers during progression through the G1 phase of the cell cycle. Chromatin immunoprecipitation-sequencing profiling demonstrated recruitment of CK2α to active gene loci, more abundantly in late G1 phase than in early G1, notably at transcriptional start sites of core histone genes, growth stimulus-associated genes, and ribosomal RNAs. Our findings reveal that nuclear CK2α complexes may be essential to facilitate progression of the cell cycle, by activating histone genes and triggering ribosomal biogenesis, specified in association with nuclear and nucleolar transcriptional regulators.

Coronary vasospasm induced in transgenic mouse with increased phospholipase C-δ1 activity.

BACKGROUND: We reported that phospholipase C (PLC)-δ1 activity was enhanced 3-fold in patients with coronary spastic angina. We detected variant PLC-δ1 with replacement of arginine 257 by histidine (R257H) showing increased enzymatic activity. We tested the hypothesis that increased PLC-δ1 activity causes enhanced coronary vasomotility. METHODS AND RESULTS: We generated transgenic (TG) mice with human R257H variant PLC-δ1 in vascular smooth muscle cells. PLC enzymatic activity in the coronary artery was increased by 2.57 and 1.89 times, respectively, in homozygous and heterozygous TG compared with wild-type (WT) mice. ST elevation after ergometrine occurred in 17 of 18 homozygous TG, 6 of 20 heterozygous TG, and 3 of 22 WT mice (P<0.01, homozygous TG versus WT; P<0.05, homozygous TG versus heterozygous TG; P=NS, heterozygous TG versus WT). ST elevation was associated with bradyarrhythmias in homozygous TG mice. Focal coronary artery narrowing was documented with the microvascular filling technique in 3 of 5 homozygous TG mice after ergometrine but not in any of 7 WT mice (P<0.05). In the isolated Langendorff hearts, coronary perfusion pressure was increased after ergometrine in homozygous TG mice (P<0.01) but not in heterozygous TG or WT mice. Coronary perfusion pressure increase after prostaglandin F2α was similar among homozygous TG, heterozygous TG, and WT mice. Cultured rat aortic smooth muscle cells transfected with variant PLC-δ1 showed a higher PLC activity than those with WT PLC-δ1 (P<0.05) and furthermore showed greater intracellular Ca2+ response to acetylcholine in variant than in WT PLC-δ1 (P<0.05). CONCLUSIONS: Increased PLC-δ1 activity enhances coronary vasomotility such as that seen in patients with coronary spastic angina.

Mitochondrial c-Src regulates cell survival through phosphorylation of respiratory chain components.

Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr(193) of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr(215) of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.

CpG methylation prevents YY1-mediated transcriptional activation of the vimentin promoter.

Vimentin exhibits a complex pattern of tissue-specific and developmentally regulated expression, but the mechanisms underlying the complex transcriptional regulation remain poorly understood. Here we examined whether vimentin expression can be regulated by CpG methylation of the vimentin promoter. Two subclones of the rat C6 glioma cells were established with (C6vim+) and without (C6vim-) vimentin. Bisulfite genomic sequencing revealed that the vicinity of the transcription start site within the vimentin promoter is highly methylated in C6vim- cells but not in C6vim+ cells. Treatment of C6vim- cells with a demethylating agent, 5-aza-2'-deoxycytidine, restored vimentin expression, indicating that hypermethylation of the promoter region correlates with transcriptional silencing of the vimentin gene. Electrophoretic mobility shift assay (EMSA) and transient transfection experiments demonstrated that YY1 is a key transcriptional activator regulating vimentin expression and that CpG methylation is sufficient to prevent the binding of YY1 to the vimentin promoter. These data suggest that the inability of YY1 to access the hypermethylated promoter may be one of the mechanisms that mediate vimentin downregulation.

p122 protein enhances intracellular calcium increase to acetylcholine: its possible role in the pathogenesis of coronary spastic angina.

OBJECTIVE: Phospholipase C-δ1 activity is enhanced in patients with coronary artery spasm, and a p122 protein was recently cloned to potentiate phospholipase C-δ1 activity. To investigate the role of p122 in enhanced vasomotility, we examined p122 expression in the cultured skin fibroblasts obtained from patients with and without coronary spasm, intracellular Ca(2+) concentration ([Ca(2+)]i) [corrected] at baseline and after stimulation with acetylcholine in the cells transfected with p122, and promoter in genomic DNA. METHODS AND RESULTS: [corrected] p122 protein and gene expression levels in patients with coronary spasm (n=11) were enhanced compared with levels in control subjects (n=9) (P<0.01 for both). [Ca(2+)](i) at baseline and the peak increase in [Ca(2+)](i) in response to acetylcholine were both 2 times higher in cells transfected with p122 than in those without p122. Conversely, knockdown of p122 resulted in diminished [Ca(2+)](i) response. In the p122 promoter analysis, the -228G/A and -1466C/T variants revealed the increase in luciferase activity. Although the -1466C/T variant was similar between 144 patients with coronary spasm and 148 controls, the -228G/A variant was more frequent in male patients than in male controls (P<0.05). CONCLUSIONS: The p122 protein is upregulated in patients with coronary spasm, causing increased [Ca(2+)](i) to acetylcholine, and thereby seems to be related to enhanced coronary vasomotility.

Prenylated quinolinecarboxylic acid compound-18 prevents sensory nerve fiber outgrowth through inhibition of the interleukin-31 pathway.

Interleukin-31 (IL-31) is involved in excessive development of cutaneous sensory nerves in atopic dermatitis (AD), leading to severe pruritus. We previously reported that PQA-18, a prenylated quinolinecarboxylic acid (PQA) derivative, is an immunosuppressant with inhibition of p21-activated kinase 2 (PAK2) and improves skin lesions in Nc/Nga mice as an AD model. In the present study, we investigate the effect of PQA-18 on sensory nerves in lesional skin. PQA-18 alleviates cutaneous nerve fiber density in the skin of Nc/Nga mice. PQA-18 also inhibits IL-31-induced sensory nerve fiber outgrowth in dorsal root ganglion cultures. Signaling analysis reveals that PQA-18 suppresses phosphorylation of PAK2, Janus kinase 2, and signal transducer and activator of transcription 3 (STAT3), activated by IL-31 receptor (IL-31R), resulting in inhibition of neurite outgrowth in Neuro2A cells. Gene silencing analysis for PAK2 confirms the requirement for STAT3 phosphorylation and neurite outgrowth elicited by IL-31R activation. LC/MS/MS analysis reveals that PQA-18 prevents the formation of PAK2 activation complexes induced by IL-31R activation. These results suggest that PQA-18 inhibits the IL-31 pathway through suppressing PAK2 activity, which suppresses sensory nerve outgrowth. PQA-18 may be a valuable lead for the development of a novel drug for pruritus of AD.

Dietary syringic acid reduces fat mass in an ovariectomy-induced mouse model of obesity.

OBJECTIVE: Postmenopausal women are at increased risk of metabolic diseases such as obesity and diabetes. Therefore, the chemoprevention of postmenopausal changes in health via dietary supplements is important. Syringic acid (SA) is a phenolic compound present in the fruit of the assai palm, Euterpe oleracea, and in the mycelium of the shiitake mushroom, Lentinula edodes. This compound shows no affinity for estrogen receptors and may exert disease-preventive effects. Reportedly, dietary SA ameliorates high-fat diet-induced obesity in mice; however, its effects on estrogen deficiency-induced obesity are still unclear. Therefore, in this study, we investigated whether and how dietary SA affects these factors in ovariectomized (OVX) mice. METHODS: Ten-week-old OVX mice were fed SA-containing diets (100 mg/kg body weight/d) for 12 weeks. Their body weights, food intake, and uterus weights as well as other parameters were measured and comparisons were made with mice in the control group. RESULTS: Dietary SA did not affect the body weight, food intake, or uterus weight of OVX mice over the study period; however, the SA-fed group showed lower fat mass (ie, visceral, subcutaneous, and total fat) than the OVX-control group (11.1 ±â€Š3.3 vs. 8.3 ±â€Š2.4, P < 0.05; 7.9 ±â€Š1.1 vs. 5.9 ±â€Š1.6, P < 0.05; 19.0 ±â€Š4.2 vs. 14.1 ±â€Š3.8, P < 0.05, respectively). Furthermore, blood analysis revealed that SA-treatment resulted in a dose-dependent decrease and increase in serum triglyceride (59.2 ±â€Š8.3 vs. 43.9 ±â€Š12.2 mg/dL P < 0.05) and adiponectin (7.7 ±â€Š0.3 vs. 9.5 ±â€Š0.6 µg/mL, P < 0.05) levels, respectively. CONCLUSIONS: These results suggest that the SA diet improves lipid metabolism without affecting the uterus in OVX mice. Therefore, dietary SA has potential applicability for the prevention of postmenopausal obesity and type 2 diabetes.

Dysregulation of very long chain acyl-CoA dehydrogenase coupled with lipid peroxidation.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease of unknown etiology. We previously revealed increased oxidative stress and high expression of antioxidant proteins in culture cell lines established from lesional lung tissues with IPF (Kabuyama Y, Oshima K, Kitamura T, Homma M, Yamaki J, Munakata M, Homma Y. Genes Cells 12: 1235-1244, 2007). In this study, we show that IPF cells contain high levels of free cholesterol and its peroxidized form as compared with normal TIG7 lung fibroblasts, suggesting that radical oxygen species (ROS) are generated within specific organelles. To understand the molecular basis underlying the generation of ROS in IPF cells, we performed proteomic analysis of mitochondrial proteins from TIG and IPF cells. This analysis shows that the phosphorylation of Ser586 of very long chain acyl-CoA dehydrogenase (VLCAD) is significantly reduced in IPF cells. Similar results are obtained from immunoblotting with anti-pS586 antibody. Kinase activity toward a peptide containing Ser586 from IPF cells is significantly lower than that from TIG cells. Furthermore, a phosphorylation-negative mutant (S586A) VLCAD shows reduced electron transfer activity and a strong dominant-negative effect on fatty acid beta-oxidation. The ectopic expression of the S586A mutant induced human embryonic kidney (HEK) 293 cells to produce significantly high amounts of oxidized lipids and hydrogen peroxide. HEK293 cells expressing the S586A mutant exhibit a reduction in cell growth and an enhancement in apoptosis. These results suggest a novel regulatory mechanism for homeostatic VLCAD activity, whose dysregulation might be involved in the production of oxidative stress and in the pathogenesis of IPF.

Focal adhesion-localization of START-GAP1/DLC1 is essential for cell motility and morphology.

There is a class of GTPase activating proteins for the Rho family GTPases (RhoGAPs) that contain the steroidogenic acute regulatory protein (STAR)-related lipid transfer (START) domain. In mammals three genes encode such proteins and they are designated START-GAP1-3 or deleted in liver cancer 1-3 (DLC1-3). In this study, we examined the intracellular localization and roles of START-GAP1/DLC1 in cell motility. Immunofluorescence microscopic analysis of NRK cells and HeLa cells revealed that START-GAP1 was localized in focal adhesions. Amino acid residues 265-459 of START-GAP1 were found to be necessary for focal adhesion targeting and we name the region "the focal adhesion-targeting (FAT) domain." It was previously known that ectopic expression of START-GAP1 induced cell rounding. We demonstrated that the FAT domain of START-GAP1 was partially required for this morphological change. Furthermore, expression of this domain in HeLa cells resulted in dissociation of endogenous START-GAP1 from focal adhesions as a dominant negative modulator, reducing cell migration and spreading. Taken together, START-GAP1 is targeted to focal adhesions via the FAT domain and regulates actin rearrangement through down-regulation of active RhoA and Cdc42. Its absence from focal adhesions could, therefore, cause abnormal cell motility and spreading.

Comparison of neuropathic pain and neuronal apoptosis following nerve root or spinal nerve compression.

Altered dorsal root ganglion (DRG) function is associated with neuropathic pain following spinal nerve injury. However, compression of the cauda equina and dorsal rhizotomy proximal to the DRG do not induce significant pain, whereas in the spinal nerve and peripheral nerve, injury distal to the DRG does induce neuropathic pain. Caspase signaling induces apoptosis, and caspase inhibitors prevent pain-related behavior. The degree of DRG neuronal apoptosis is thought to play a role in pain behavior. We suggest that differences in pain behavior according to the injury sites within the DRG may be related to imbalances in apoptotic injuries. The aim of this study was to determine which compression injury was more painful and to compare behavior with expression of tumor necrosis factor (TNF)-alpha in DRG and apoptosis in the DRG following crush injury to the L5 nerve root or L5 spinal nerve. Sprague-Dawley rats received a crush injury to the L5 spinal nerve (distal to the DRG), crush injury to the L5 nerve root (proximal to the DRG), or no crush injury (sham). Mechanical allodynia was determined by the von Frey test. Expression of TNF-alpha was compared among three groups using immunoblot findings. Furthermore, we compared the percentage of neurons injured in the DRG using immunostaining for apoptotic cells and localization of activated caspase 3. Mechanical allodynia was observed in both crush injury groups. The duration of mechanical allodynia in the distal crush group was significantly longer than in the proximal crush group (P < 0.05). TNF-alpha expression was increased in DRG neurons following injury. DRG apoptosis in the distal crush group was significantly higher than in the proximal group at each time point (P < 0.05). This study suggests that spinal nerve crush injuries produce a greater degree of DRG apoptosis than do corresponding nerve root crush injuries, and that the former injuries are associated with longer lasting mechanical allodynia. Thus, differences in the time course of mechanical allodynia might be associated with an imbalance in DRG apoptosis.

IQGAP1 activates PLC-δ1 by direct binding and moving along microtubule with DLC-1 to cell surface.

Phospholipase C (PLC)-δ1, activated by p122RhoGTPase-activating protein (GAP)/deleted in liver cancer-1 (p122RhoGAP/DLC-1), contributes to the coronary spastic angina (CSA) pathogenesis. The present study aims to further investigate the p122RhoGAP/DLC-1 protein. We examined molecules assisting this protein and identified a scaffold protein-IQ motif-containing GTPase-activating protein 1 (IQGAP1). IQGAP1-C binds to the steroidogenic acute regulatory-related lipid transfer (START) domain of p122RhoGAP/DLC-1, and PLC-δ1 binds to IQGAP1-N, forming a complex. In fluorescence microscopy, small dots of PLC-δ1 created fine linear arrays like microtubules, and IQGAP1 and p122RhoGAP/DLC-1 were colocated in the cytoplasm with PLC-δ1. Ionomycin induced the raft recruitment of the PLC-δ1, IQGAP1, and p122RhoGAP/DLC-1 complex by translocation to the plasma membrane (PM), indicating the movement of this complex is along microtubules with the motor protein kinesin. Moreover, the IQGAP1 protein was elevated in skin fibroblasts obtained from patients with CSA, and it enhanced the PLC activity and peak intracellular calcium concentration in response to acetylcholine. IQGAP1, a novel stimulating protein, forms a complex with p122RhoGAP/DLC-1 and PLC-δ1 that moves along microtubules and enhances the PLC activity.

Prenylated quinolinecarboxylic acid derivative prevents neuronal cell death through inhibition of MKK4.

The development of neuroprotective agents is necessary for the treatment of neurodegenerative diseases. Here, we report PQA-11, a prenylated quinolinecarboxylic acid (PQA) derivative, as a potent neuroprotectant. PQA-11 inhibits glutamate-induced cell death and caspase-3 activation in hippocampal cultures, as well as inhibits N-Methyl-4-phenylpyridinium iodide- and amyloid ß1-42-induced cell death in SH-SY5Y cells. PQA-11 also suppresses mitogen-activated protein kinase kinase 4 (MKK4) and c-jun N-terminal kinase (JNK) signaling activated by these neurotoxins. Quartz crystal microbalance analysis and in vitro kinase assay reveal that PQA-11 interacts with MKK4, and inhibits its sphingosine-induced activation. The administration of PQA-11 by intraperitoneal injection alleviates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced degeneration of nigrostriatal dopaminergic neurons in mice. These results suggest that PQA-11 is a unique MKK4 inhibitor with potent neuroprotective effects in vitro and in vivo. PQA-11 may be a valuable lead for the development of novel neuroprotectants.