Cu(II)-Catalyzed [3 + 1 + 1 + 1] Cyclization of 1,3-Diketones and 2-Naphthols Using N,N-Dimethylethanolamine as a Dual Carbon Synthon for the Synthesis of 2H-Chromenes.

Reactions with diverse C1 synthons to realize homologation were well explored. However, homologations occurring twice with one C1 synthon in a reaction were less reported. We disclose herein a Cu(II)-catalyzed novel and efficient synthesis of 2H-chromenes from 2-naphthols, 1,3-diketones, and N,N-dimethylethanolamine (DMEA) as a dual carbon synthon. Various 2H-chromenes with different functional groups are constructed in moderate to good yields. This is the first report that DMEA acts as a dual C1 synthon.

Application of N,N-Dimethylethanolamine as a One-Carbon Synthon for the Synthesis of Pyrrolo[1,2-a]quinoxalines, Quinazolin-4-ones, and Benzo[4,5]imidazoquinazolines via [5 + 1] Annulation.

The synthesis of N-heterocycles composes a significant part of synthetic chemistry. In this report, a Cu(II)-catalyzed green and efficient synthesis of pyrrolo[1,2-a]quinoxaline, quinazolin-4-one, and benzo[4,5]imidazoquinazoline derivatives was developed, employing N,N-dimethylethanolamine (DMEA) as a C1 synthon. Green oxidant O2 is critical in these transformations, facilitating the formation of a key intermediate─a reactive iminium ion. The method conducted under mild conditions is compatible with a diversity of functional groups, providing an appealing alternative to the previously developed protocols.

Rottlerin suppresses growth of human pancreatic tumors in nude mice, and pancreatic cancer cells isolated from Kras(G12D) mice.

The purpose of the study was to examine the molecular mechanisms by which rottlerin inhibited growth of human pancreatic tumors in Balb C nude mice, and pancreatic cancer cells isolated from Kras(G12D) mice. AsPC-1 cells were injected subcutaneously into Balb c nude mice, and tumor-bearing mice were treated with rottlerin. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. The expression of components of Akt, Notch, and Sonic Hedgehog (Shh) pathways were measured by the immunohistochemistry, Western blot analysis, and/or q-RT-PCR. The effects of rottlerin on pancreatic cancer cells isolated from Kras(G12D) mice were also examined. Rottlerin-treated mice showed a significant inhibition in tumor growth which was associated with suppression of cell proliferation, activation of capase-3 and cleavage of PARP. Rottlerin inhibited the expression of Bcl-2, cyclin D1, CDK2 and CDK6, and induced the expression of Bax in tumor tissues compared to untreated control. Rottlerin inhibited the markers of angiogenesis (Cox-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9), thus blocking production of tumorigenic mediators in tumor microenvironment. Rottlerin also inhibited epithelial-mesenchymal transition by up-regulating E-cadherin and inhibiting the expression of Slug and Snail. Furthermore, rottlerin treatment of xenografted tumors or pancreatic cancer cells isolated from Kras(G12D) mice showed a significant inhibition in Akt, Shh and Notch pathways compared to control groups. These data suggest that rottlerin can inhibit pancreatic cancer growth by suppressing multiple signaling pathways which are constitutively active in pancreatic cancer. Taken together, our data show that the rottlerin induces apoptosis and inhibits pancreatic cancer growth by targeting Akt, Notch and Shh signaling pathways, and provide a new therapeutic approach with translational potential for humans.

Embelin suppresses growth of human pancreatic cancer xenografts, and pancreatic cancer cells isolated from KrasG12D mice by inhibiting Akt and Sonic hedgehog pathways.

Pancreatic cancer is a deadly disease, and therefore effective treatment and/or prevention strategies are urgently needed. The objectives of this study were to examine the molecular mechanisms by which embelin inhibited human pancreatic cancer cell growth in vitro, and xenografts in Balb C nude mice, and pancreatic cancer cell growth isolated from KrasG12D transgenic mice. XTT assays were performed to measure cell viability. AsPC-1 cells were injected subcutaneously into Balb c nude mice and treated with embelin. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. The expression of Akt, and Sonic Hedgehog (Shh) and their target gene products were measured by the immunohistochemistry, and Western blot analysis. The effects of embelin on pancreatic cancer cells isolated from 10-months old KrasG12D mice were also examined. Embelin inhibited cell viability in pancreatic cancer AsPC-1, PANC-1, MIA PaCa-2 and Hs 766T cell lines, and these inhibitory effects were blocked either by constitutively active Akt or Shh protein. Embelin-treated mice showed significant inhibition in tumor growth which was associated with reduced expression of markers of cell proliferation (Ki67, PCNA and Bcl-2) and cell cycle (cyclin D1, CDK2, and CDK6), and induction of apoptosis (activation of caspase-3 and cleavage of PARP, and increased expression of Bax). In addition, embelin inhibited the expression of markers of angiogenesis (COX-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9) in tumor tissues. Antitumor activity of embelin was associated with inhibition of Akt and Shh pathways in xenografts, and pancreatic cancer cells isolated from KrasG12D mice. Furthermore, embelin also inhibited epithelial-to-mesenchymal transition (EMT) by up-regulating E-cadherin and inhibiting the expression of Snail, Slug, and ZEB1. These data suggest that embelin can inhibit pancreatic cancer growth, angiogenesis and metastasis by suppressing Akt and Shh pathways, and can be developed for the treatment and/or prevention of pancreatic cancer.

NleB, a bacterial effector with glycosyltransferase activity, targets GAPDH function to inhibit NF-κB activation.

Modulation of NF-κB-dependent responses is critical to the success of attaching/effacing (A/E) human pathogenic E. coli (EPEC and EHEC) and the natural mouse pathogen Citrobacter rodentium. NleB, a highly conserved type III secretion system effector of A/E pathogens, suppresses NF-κB activation, but the underlying mechanisms are unknown. We identified the mammalian glycolysis enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an NleB-interacting protein. Further, we discovered that GAPDH interacts with the TNF receptor-associated factor 2 (TRAF2), a protein required for TNF-α-mediated NF-κB activation, and regulates TRAF2 polyubiquitination. During infection, NleB functions as a translocated N-acetyl-D-glucosamine (O-GlcNAc) transferase that modifies GAPDH. NleB-mediated GAPDH O-GlcNAcylation disrupts the TRAF2-GAPDH interaction to suppress TRAF2 polyubiquitination and NF-κB activation. Eliminating NleB O-GlcNAcylation activity attenuates C. rodentium colonization of mice. These data identify GAPDH as a TRAF2 signaling cofactor and reveal a virulence strategy employed by A/E pathogens to inhibit NF-κB-dependent host innate immune responses.

Impaired osteoblast function in GPRC6A null mice.

GPRC6A is a widely expressed orphan G protein-coupled receptor that senses extracellular amino acids, osteocalcin, and divalent cations in vitro. GPRC6A null (GPRC6A(-/-)) mice exhibit multiple metabolic abnormalities including osteopenia. To investigate whether the osseous abnormalities are a direct function of GPRC6A in osteoblasts, we examined the function of primary osteoblasts and bone marrow stromal cell cultures (BMSCs) in GPRC6A(-/-) mice. We confirmed that GPRC6A(-/-) mice exhibited a decrease in bone mineral density (BMD) associated with reduced expression of osteocalcin, ALP, osteoprotegerin, and Runx2-II transcripts in bone. Osteoblasts and BMSCs derived from GPRC6A(-/-) mice exhibited an attenuated response to extracellular calcium-stimulated extracellular signal-related kinase (ERK) activation, diminished alkaline phosphatase (ALP) expression, and impaired mineralization ex vivo. In addition, siRNA-mediated knockdown of GPRC6A in MC3T3 osteoblasts also resulted in a reduction in extracellular calcium-stimulated ERK activity. To explore the potential relevance of GPRC6A function in humans, we looked for an association between GPRC6A gene polymorphisms and BMD in a sample of 1000 unrelated American Caucasians. We found that GPRC6A gene polymorphisms were significantly associated with human spine BMD. These data indicate that GRPC6A directly participates in the regulation of osteoblast-mediated bone mineralization and may mediate the anabolic effects of extracellular amino acids, osteocalcin, and divalent cations in bone.

GPRC6A null mice exhibit osteopenia, feminization and metabolic syndrome.

BACKGROUND: GPRC6A is a widely expressed orphan G-protein coupled receptor that senses extracellular amino acids, osteocalcin and divalent cations in vitro. The physiological functions of GPRC6A are unknown. METHODS/PRINCIPAL FINDINGS: In this study, we created and characterized the phenotype of GPRC6A(-/-) mice. We observed complex metabolic abnormalities in GPRC6A(-/-) mice involving multiple organ systems that express GPRC6A, including bone, kidney, testes, and liver. GPRC6A(-/-) mice exhibited hepatic steatosis, hyperglycemia, glucose intolerance, and insulin resistance. In addition, we observed high expression of GPRC6A in Leydig cells in the testis. Ablation of GPRC6A resulted in feminization of male GPRC6A(-/-) mice in association with decreased lean body mass, increased fat mass, increased circulating levels of estradiol, and reduced levels of testosterone. GPRC6A was also highly expressed in kidney proximal and distal tubules, and GPRC6A(-/-) mice exhibited increments in urine Ca/Cr and PO(4)/Cr ratios as well as low molecular weight proteinuria. Finally, GPRC6A(-/-) mice exhibited a decrease in bone mineral density (BMD) in association with impaired mineralization of bone. CONCLUSIONS/SIGNIFICANCE: GPRC6A(-/-) mice have a metabolic syndrome characterized by defective osteoblast-mediated bone mineralization, abnormal renal handling of calcium and phosphorus, fatty liver, glucose intolerance and disordered steroidogenesis. These findings suggest the overall function of GPRC6A may be to coordinate the anabolic responses of multiple tissues through the sensing of extracellular amino acids, osteocalcin and divalent cations.

Parathyroid-specific interaction of the calcium-sensing receptor and G alpha q.

The calcium-sensing receptor regulates various parathyroid gland functions, including hormone secretion, gene transcription, and chief cell hyperplasia through G alpha q- and G alpha i-dependent signaling pathways. To determine the specific function of G alpha q in these processes, we generated transgenic mice using the human parathyroid hormone promoter to drive overexpression of a dominant negative G alpha q loop minigene to selectively disrupt G alpha q function in the parathyroid gland. The G alpha q loop mRNA was highly expressed in the parathyroid gland but not in other tissues of these transgenic mice. Gross appearance, body weight, bone mineral density, and survival of the transgenic mice were indistinguishable from those of their wild-type littermates. Adult transgenic mice, however, exhibited an increase in parathyroid hormone mRNA and in its basal serum level as well as in gland size. The response of the parathyroid gland to hypocalcemia was found to be reduced in sensitivity in the transgenic mice when compared to their wild-type controls. Abnormalities of the parathyroid gland function in these transgenic mice were similar to those of heterozygous G alpha q(+/-) and calcium sensing receptor(+/-) mice. These studies demonstrate the feasibility of selectively targeting the parathyroid gland to investigate signaling mechanisms downstream of the calcium receptor.

Role of interleukin-4 and monocyte chemoattractant protein-1 in the neuropathogenesis of X4 simian human immunodeficiency virus infection in macaques.

Recent studies on the coreceptor usage of human immunodeficiency virus (HIV) strains associated with acquired immunodeficiency syndrome (AIDS) dementia have shown that both X4 and R5 viruses are involved in the process. The disease is associated with enhanced virus replication and monocyte chemoattractant protein (MCP)-1 production in macrophages in the brain. Using the macaque model of the disease, the authors show here that X4, macrophage-tropic simian human immunodeficiency virus (SHIV) required the enhancing effect of interleukin (IL)-4 to achieve equivalent concentrations of virus and MCP-1 that are produced in macrophages infected with R5 viruses alone. Confocal microscopy showed that macrophages in the encephalitic brains were the major producers of MCP-1. The authors surmise, therefore, that whereas R5 viruses maybe capable of causing the disease as a primary pathogen, X4 viruses may require IL-4, induced by opportunistic pathogens, for induction of the neuropathological syndrome.

A randomized trial of docosahexaenoic acid supplementation during the third trimester of pregnancy.

OBJECTIVE: To hypothesize that higher intake of docosahexaenoic acid, an n-3 long chain polyunsaturated fatty acid, would increase duration of gestation and birth weight in US women. METHODS: This was a randomized, double-blind, controlled, clinical trial. Subjects were enrolled in an ambulatory clinic where they received prenatal care. This was a population-based sample. Most subjects received government assistance for medical care and most were black (73%). Subjects were enrolled between the 24th and 28th week of pregnancy and consumed docosahexaenoic acid (33 or 133 mg) from eggs until parturition. Gestational age and birth weight were the main study outcomes. Infant length and head circumference, preterm birth, and low birth weight were secondary outcomes. RESULTS: Eighty-three percent of subjects completed the study (291 of 350 enrolled). No subject was discontinued for an adverse event. After controlling for important predefined risk factors and confounding variables, gestation increased by 6.0 +/- 2.3 days (P =.009) in the higher docosahexaenoic acid group. Birth weight, length, and head circumference increased, but did not reach statistical significance (P =.06-.18), although the increases could be clinically important indications of enhanced intrauterine growth. No safety concerns were raised by the study. CONCLUSION: Duration of gestation increased significantly when docosahexaenoic acid intake was increased during the last trimester of pregnancy. The increase in gestation was similar to that reported for interventions with much larger amounts of n-3 long chain polyunsaturated fatty acids.