Generation of monoclonal antibody against 6-Keto PGF and development of ELISA for its quantification in culture medium.

Prostacyclin or prostaglandin I (PGI), a metabolite of arachidonic cyclooxygenase pathway, has been demonstrated as an effector of adipocyte differentiation. However, due to its instability in biological fluid, it is difficult to evaluate the role of PGI in regulating adipocyte differentiation in different stages in culture. Therefore, this study aimed to establish a simple and rapid method for the production of monoclonal antibody against 6-Keto PGFα, a stable PGI metabolite, and its quantification to determine the role of PGI in culture medium.

Eicosapentaenoic Acid Induces the Inhibition of Adipogenesis by Reducing the Effect of PPARγ Activator and Mediating PKA Activation and Increased COX-2 Expression in 3T3-L1 Cells at the Differentiation Stage.

Obesity has received increasing attention in recent years because it is a factor in the development of non-communicable diseases. The current study aimed to analyze how representative fatty acids (FAs) such as palmitic acid, stearic acid, oleic acid, α-linolenic acid (ALA), and eicosapentaenoic acid (EPA) affected adipogenesis when/if introduced at the differentiation stage of 3T3-L1 cell culture. These FAs are assumed to be potentially relevant to the progression or prevention of obesity.

Prostaglandin D Added during the Differentiation of 3T3-L1 Cells Suppresses Adipogenesis via Dysfunction of D-Prostanoid Receptor P1 and P2.

We previously reported that the addition of prostaglandin, (PG)D, and its chemically stable analog, 11-deoxy-11-methylene-PGD (11d-11m-PGD), during the maturation phase of 3T3-L1 cells promotes adipogenesis. In the present study, we aimed to elucidate the effects of the addition of PGD or 11d-11m-PGD to 3T3-L1 cells during the differentiation phase on adipogenesis. We found that both PGD and 11d-11m-PGD suppressed adipogenesis through the downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) expression.

Arachidonic Acid Added during the Differentiation Phase of 3T3-L1 Cells Exerts Anti-Adipogenic Effect by Reducing the Effects of Pro-Adipogenic Prostaglandins.

A linoleic acid (LA) metabolite arachidonic acid (AA) added to 3T3-L1 cells is reported to suppress adipogenesis. The purpose of the present study aimed to clarify the effects of AA added during the differentiation phase, including adipogenesis, the types of prostaglandins (PG)s produced, and the crosstalk between AA and the PGs produced. Adipogenesis was inhibited by AA added, while LA did not.

Prostaglandin D2 and its analog, 11d-11m-PGD2, added during the differentiation phase contribute to adipogenic program inhibition in 3T3-L1 cells.

We previously reported that prostaglandin (PG)D2 and its isosteric analog, 11-deoxy-11-methylene-PGD2 (11d-11m-PGD2), promote adipogenesis in 3T3-L1 cells during the maturation phase. Focusing on the differentiation phase, although both PGs inhibited adipogenesis, this effect was canceled out by PGI2 and PGJ2 derivatives. Thus, PGD2 and 11d-11m-PGD2 play different roles during the phases, but do not affect PGI2- and PGJ2-derivative-induced adipogenesis.

Anti-inflammatory and bioavailability studies on dietary 3,5,4'-trihydroxy-6,7-methylenedioxyflavone-O-glycosides and their aglycone from indigo leaves in a murine model of inflammatory bowel disease.

Persicaria tinctoria (Aiton) Spach, also called Polygonum tinctorium Lour., (family Polygonaceae) for indigo plant has been traditionally useful as a medicinal or edible plant with a variety of biological activities. Of these, much attention has been paid to their anti-inflammatory activities.

Constituents of flavonol -glycosides and antioxidant activities of extracts from seeds, sprouts, and aerial parts of Lour.

Lour. (family Polygonaceae), known as indigo plant, has been useful as a medicinal or edible plant abundant in polyphenolic compounds. We have recently shown that flavonol -glycosides with 3,5,4'-trihydroxy-6,7-methylenedioxyflavone (TMF) are predominant flavonoids in indigo leaves.

Skatole regulates intestinal epithelial cellular functions through activating aryl hydrocarbon receptors and p38.

Intestinal bacteria produce skatole (3-methylindole) from tryptophan in dietary proteins and ingesting large quantities of animal protein is associated with increased fecal skatole concentrations. Although possibly associated with disrupted intestinal homeostasis, the influence of skatole on intestinal epithelial cellular function has not been characterized in detail. The present study aimed to determine whether skatole induces intestinal epithelial cell (IEC) dysfunction.

Comparison of pro-adipogenic effects between prostaglandin (PG) D and its stable, isosteric analogue, 11-deoxy-11-methylene-PGD, during the maturation phase of cultured adipocytes.

Prostaglandin (PG) D is relatively unstable and dehydrated non-enzymatically into PGJ derivatives, which are known to serve as pro-adipogenic factors by activating peroxisome proliferator-activated receptor (PPAR) γ, a master regulator of adipogenesis. 11-Deoxy-11-methylene-PGD (11d-11m-PGD) is a novel, chemically stable, isosteric analogue of PGD in which the 11-keto group is replaced by an exocyclic methylene. Here we attempted to investigate pro-adipogenic effects of PGD and 11d-11m-PGD and to compare the difference in their ways during the maturation phase of cultured adipocytes.

In Vitro Anti-inflammatory and Antioxidant Activities of 3,5,4'-Trihydroxy-6,7-Methylenedioxyflavone-O-Glycosides and Their Aglycone from Leaves of Polygonum tinctorium Lour.

Polygonum tinctorium Lour (indigo plant) has been regarded as a useful medicinal plant for traditional herbal medicine. The polyphenolic fraction of indigo leaves exhibited anti-inflammatory activities as determined by the suppressed synthesis of nitric oxide (NO) in cultured RAW264 macrophage cells. The acid hydrolysate of the fraction showed much more potent effect than the unhydrolyzed one.

Antioxidant activities and structural characterization of flavonol O-glycosides from seeds of Japanese horse chestnut (Aesculus turbinata BLUME).

We attempted to evaluate the contents and distribution of antioxidants in the whole seeds, seed shells, and peeled seeds of the Japanese horse chestnut. The seed shells exhibited the highest antioxidant activities due to the presence of highly polymeric proanthocyanidins as we have reported recently. On the other hand, the peeled seeds predominantly contained flavonols such as quercetin and kaempferol at a high level of 66.

Elevated serum MDA and depleted non-enzymatic antioxidants, macro-minerals and trace elements are associated with bipolar disorder.

Genetic and neurobiological factors are considered to be the major causes of mood and mental disorders. However, over the past few years, increased levels of serum malondialdehyde and altered levels of various non-enzymatic antioxidants and essential minerals involved in abnormal functional activity have been identified as major contributing factors to the pathogenesis of several neurological disorders. The aim of this study was to determine the levels of the serum lipid peroxidation product malondialdehyde (MDA), antioxidants (vitamin A, E and C), macro-minerals (calcium, potassium and sodium) and trace elements (zinc, iron and selenium) in patients with bipolar disorder and to explore their role in disease progression.

Stimulation of fat storage by prostacyclin and selective agonists of prostanoid IP receptor during the maturation phase of cultured adipocytes.

We have previously shown that cultured adipocytes have the ability to biosynthesize prostaglandin (PG) I called alternatively as prostacyclin during the maturation phase by the positive regulation of gene expression of PGI synthase and the prostanoid IP receptor. To clarify how prostacyclin regulates adipogenesis, we investigated the effects of prostacyclin and the specific agonists or antagonists for the IP receptor on the storage of fats during the maturation phase of cultured adipocytes. Exogenous PGI and the related selective agonists for the IP receptor including MRE-269 and treprostinil rescued the storage of fats attenuated by aspirin, a cyclooxygenase inhibitor.

Pretreatment of cultured preadipocytes with arachidonic acid during the differentiation phase without a cAMP-elevating agent enhances fat storage after the maturation phase.

Arachidonic acid (AA) and the related prostanoids exert complex effects on the adipocyte differentiation depending on the culture conditions and life stages. Here, we investigated the effect of the pretreatment of cultured 3T3-L1 preadipocytes with exogenous AA during the differentiation phase without 3-isobutyl-1-methylxanthine (IBMX), a cAMP-elevating agent, on the storage of fats after the maturation phase. This pretreatment with AA stimulated appreciably adipogenesis after the maturation phase as evident with the up-regulated gene expression of adipogenic markers.

The tyrosine-sorting motif of the vacuolar sorting receptor VSR4 from Arabidopsis thaliana, which is involved in the interaction between VSR4 and AP1M2, μ1-adaptin type 2 of clathrin adaptor complex 1 subunits, participates in the post-Golgi sorting of VSR4.

μ1-Adaptin of adaptor protein (AP) 1 complex, AP1M, is generally accepted to load cargo proteins into clathrin-coated vesicles (CCVs) at the trans-Golgi network through its binding to cargo-recognition sequences (CRSs). Plant vacuolar-sorting receptors (VSRs) function in sorting vacuolar proteins, which are reportedly mediated by CCV. We herein investigated the involvement of CRSs of Arabidopsis thaliana VSR4 in the sorting of VSR4.

New Gateway-compatible vectors for a high-throughput protein-protein interaction analysis by a bimolecular fluorescence complementation (BiFC) assay in plants and their application to a plant clathrin structure analysis.

Protein-protein interactions (PPI) play key roles in various biological processes. The bimolecular fluorescence complementation (BiFC) assay is an excellent tool for routine PPI analyses in living cells. We developed new Gateway vectors for a high-throughput BiFC analysis of plants, adopting a monomeric Venus split just after the tenth β-strand, and analyzed the interaction between Arabidopsis thaliana coated vesicle coatmers, the clathrin heavy chain (CHC), and the clathrin light chain (CLC).

Identification of new flavonol O-glycosides from indigo (Polygonum tinctorium Lour) leaves and their inhibitory activity against 3-hydroxy-3-methylglutaryl-CoA reductase.

Indigo plant (Polygonum tinctorium Lour) has been utilized as a medicinal plant with a variety of biological activities. We have recently detected higher levels of flavonoids in indigo leaves. This study was undertaken to conduct the simultaneous analysis of those flavonoids using total extracts from indigo leaves by ultra-performance liquid chromatography-electrospray ionization-time-of-flight/mass spectrometry(E) (UPLC-ESI-TOF/MS(E)).

Identification and quantitative analysis of polyphenolic compounds from the indigo plant (Polygonum tinctorium Lour).

The indigo plant (Polygonum tinctorium Lour) has been used traditionally as a medicinal plant with a variety of biological effects. Of these, polyphenolic ingredients are postulated to contribute to these activities. However, the identification and quantification of polyphenolic compounds in indigo plants have not been conducted comprehensively until now.

Endogenous synthesis of prostacyclin was positively regulated during the maturation phase of cultured adipocytes.

Prostacyclin alternatively called prostaglandin (PG) I2 is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI2 has not been determined comprehensively at different life stages of adipocytes.

Cultured preadipocytes undergoing stable transfection with cyclooxygenase-1 in the antisense direction accelerate adipogenesis during the maturation phase of adipocytes.

The arachidonate cyclooxygenase (COX) pathway is involved in the generation of several types of endogenous prostaglandins (PGs) with opposite effects on adipogenesis at different life stages of adipocytes. However, the specific role of COX isoforms, the rate-limiting enzymes for the pathway, remains elusive in the regulation of the endogenous synthesis of PGs. This study was aimed at the selective suppression of the constitutive COX-1 in cultured preadipocytes by the isolation of cloned preadipocytes transfected stably with a mammalian expression vector harboring cDNA encoding mouse COX-1 in the antisense direction.

A monoclonal antibody specific for Δ12-prostaglandin J2 and its utilization in the immunological assay in cell culture system of adipocytes.

Prostaglandin (PG) D(2) can be produced in adipocytes and dehydrated to PGs of J(2) series, including Δ(12)-PGJ(2) and 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)), which serve as pro-adipogenic prostanoids through the activation of peroxisome proliferator-activated receptor γ. To accomplish the quantification of Δ(12)-PGJ(2) in the cell culture system of adipocytes, the present study aimed to develop a sensitive and specific immunological assay for Δ(12)-PGJ(2). Here, we established a cloned hybridoma cell line secreting a monoclonal antibody specifically recognizing Δ(12)-PGJ(2) and utilized for the development of its solid-phase enzyme-linked immunosorbent assay (ELISA).

Prostaglandin J2 series induces the gene expression of monocyte chemoattractant protein-1 during the maturation phase of cultured adipocytes.

Prostaglandin (PG) J(2) series including Δ(12)-PGJ(2) and 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) is the dehydration products of PGD(2) that are biosynthesized through the cyclooxygenase (COX) pathway. These prostanoids are active ligands for peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis in adipocytes. Here we investigated whether PGJ(2) derivatives can modulate the gene expression of monocyte chemoattractant protein-1 (MCP-1), a pro-inflammatory chemokine, during the maturation phase of adipocytes.

Generation of monoclonal antibody for 15-deoxy-Δ¹²,¹⁴-prostaglandin J₂ and development of enzyme-linked immunosorbent assay for its quantification in culture medium of adipocytes.

15-deoxy-Δ¹²,¹⁴-prostaglandin J₂ (15d-PGJ₂) is a biologically active molecule serving as a pro-adipogenic factor or an anti-inflammatory regulator. This compound is one of naturally occurring derivatives formed by the non-enzymatic dehydration of PGD₂. To determine the endogenous synthesis of 15d-PGJ₂, a convenient immunological approach is useful.

Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids.

Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD(2) and its non-enzymatic dehydration products, PGJ(2) series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD(2) and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction.