Lipocalin-type prostaglandin D2 synthase protein regulates glial cell migration and morphology through myristoylated alanine-rich C-kinase substrate: prostaglandin D2-independent effects

Prostaglandin D synthase (PGDS) is responsible for the conversion of PGH(2) to PGD(2). Two distinct types of PGDS have been identified: hematopoietic-type PGDS (H-PGDS) and lipocalin-type PGDS (L-PGDS). L-PGDS acts as both a PGD(2)-synthesizing enzyme and as an extracellular transporter of various lipophilic small molecules. Although L-PGDS is one of the most abundant proteins in the cerebrospinal fluid, little is known about the function of L-PGDS in the central nervous system (CNS). To better understand the role of L-PGDS in the CNS, effects of L-PGDS on the migration and morphology of glial cells were investigated. The L-PGDS protein accelerated the migration of cultured glial cells. Expression of the L-pgds gene was detected in glial cells and neurons. L-PGDS protein also induced morphological changes in glia similar to the characteristic phenotypic changes in reactive gliosis. L-PGDS-induced cell migration was associated with augmented formation of actin filaments and focal adhesion, which was accompanied by activation of AKT, RhoA, and JNK pathways. L-PGDS protein injected into the mouse brain promoted migration and accumulation of astrocytes in vivo. Furthermore, the cell migration-promoting effect of L-PGDS on glial cells was independent of the PGD(2) products. The L-PGDS protein interacted with myristoylated alanine-rich protein kinase C substrate (MARCKS) to promote cell migration. These results demonstrate the critical role of L-PGDS as a secreted lipocalin in the regulation of glial cell migration and morphology. The results also indicate that L-PGDS may participate in reactive gliosis in an autocrine or paracrine manner, and may have pathological implications in neuroinflammatory diseases.     

Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase

Prostaglandin D₂ synthase (PGDS) catalyzes the isomerization of prostaglandin H₂ (PGH₂) to prostaglandin D₂ (PGD₂). PGD₂ produced by hematopoietic prostaglandin D₂ synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH₂, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC₅₀s ranging from 6 to 807 nM in SPA and 82 nM to 10 μM in the LAD2 cell assay.     

Texture analysis of cartilage T<Subscript>2</Subscript> maps: individuals with risk factors for OA have higher and more heterogeneous knee cartilage MR T<Subscript>2</Subscript> compared to normal controls - data from the osteoarthritis initiative

Introduction  

The goals of this study were (i) to compare the prevalence of focal knee abnormalities, the mean cartilage T₂ relaxation time, and the spatial distribution of cartilage magnetic resonance (MR) T₂ relaxation times between subjects with and without risk factors for Osteoarthritis (OA), (ii) to determine the relationship between MR cartilage T₂ parameters, age and cartilage morphology as determined with whole-organ magnetic resonance imaging scores (WORMS) and (iii) to assess the reproducibility of WORMS scoring and T₂ relaxation time measurements including the mean and grey level co-occurrence matrix (GLCM) texture parameters.     

Increased expression of lipocalin-type-prostaglandin D synthase in ulcerative colitis and exacerbating role in murine colitis

The pathogenesis of ulcerative colitis (UC) is unclear, but enhancement of disease activity by usage of nonsteroidal anti-inflammatory drugs suggests involvement of prostanoid in its pathophysiology. However, biological effect of prostaglandin (PG) D(2) on intestinal inflammation remains unknown. We investigated the expression of enzymes for PGD(2) synthesis, prostaglandin D synthase (PGDS), and its relation to the activity of colitis in UC patients. The role of lipocalin-type PGDS (L-PGDS) using a murine colitis model was also assessed. Tissue samples were obtained by colonic biopsies from patients with UC. Expression levels of mRNAs for L-PGDS and hematopoietic-type PGDS were investigated by quantitative RT-PCR. COX-2 and L-PGDS expression was investigated by immunohistochemistry. Localization of L-PGDS expression was also determined by in situ hybridization. In experimental study, mice were treated with dextran sodium sulfate in the drinking water to induce colitis. The degree of colonic inflammation was compared with L-PGDS(-/-) mice and control mice. The level of L-PGDS mRNA expression was increased in UC patients in parallel with disease activity. Colocalization of L-PGDS and cyclooxygenase (COX) 2 was observed in lamina proprial infiltrating cells and muscularis mucosa in UC patients. The level of hematopoietic PGDS mRNA expression did not differ from control mucosa. Dextran sodium sulfate treatment to L-PGDS(-/-) mice showed lower disease activity than control mice. We reported for the first time the presence of L-PGDS in the COX-2-expressing cells in the mucosa of active UC patients and that only L-PGDS increased with disease activity. An animal model study suggests that PGD(2) derived from L-PGDS-expressing cells plays proinflammatory roles in colitis.     

Expression of human α<Subscript>1</Subscript>-proteinase inhibitor in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Background  

Human α₁-proteinase inhibitor (α₁-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, α₁-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant α₁-PI (r-α₁-PI) could provide an attractive alternative. Although r-α₁-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation.     

Reactive oxygen species are involved in regulating α<Subscript>1</Subscript>-adrenoceptor-activated vascular smooth muscle contraction

Background  

Reactive oxygen species (ROS) were shown to mediate aberrant contractility in hypertension, yet the physiological roles of ROS in vascular smooth muscle contraction have remained elusive. This study aimed to examine whether ROS regulate α₁-adrenoceptor-activated contraction by altering myosin phosphatase activities.     

Structural Basis of the Catalytic Mechanism Operating in Open-Closed Conformers of Lipocalin Type Prostaglandin D Synthase

Lipocalin type prostaglandin D synthase (L-PGDS) is a multifunctional protein acting as a somnogen (PGD₂)-producing enzyme, an extracellular transporter of various lipophilic ligands, and an amyloid-β chaperone in human cerebrospinal fluid. In this study, we determined the crystal structures of two different conformers of mouse L-PGDS, one with an open cavity of the β-barrel and the other with a closed cavity due to the movement of the flexible E-F loop. The upper compartment of the central large cavity contains the catalytically essential Cys⁶⁵ residue and its network of hydrogen bonds with the polar residues Ser⁴⁵, Thr⁶⁷, and Ser⁸¹, whereas the lower compartment is composed of hydrophobic amino acid residues that are highly conserved among other lipocalins. SH titration analysis combined with site-directed mutagenesis revealed that the Cys⁶⁵ residue is activated by its interaction with Ser⁴⁵ and Thr⁶⁷ and that the S45A/T67A/S81A mutant showed less than 10% of the L-PGDS activity. The conformational change between the open and closed states of the cavity indicates that the mobile calyx contributes to the multiligand binding ability of L-PGDS.Prostaglandin (PG)⁶ D synthase (PGDS; PGH₂ d-isomerase (EC 5.3.99.2)) (1, 2) produces PGD₂, having 9α-hydroxy and 11-keto groups, from PGH₂, which bears the chemically labile 9,11-endoperoxide group and is produced as a common intermediate of all prostanoids by the action of cyclooxygenase (PGH₂ synthase). Two distinct types of PGDS have evolved from phylogenetically distinct protein families (2, 3). One is hematopoietic PGDS (H-PGDS), which belongs to the σ class of GSH S-transferases (4, 5), and the other is lipocalin type PGDS (L-PGDS), a member of the lipocalin family (6, 7). L-PGDS is the only enzyme in the lipocalin family and is identical to β-trace, a major protein in human cerebrospinal fluid (8, 9). Although H-PGDS and L-PGDS catalyze the same reaction, their amino acid sequences and tertiary structures are quite different from each other, indicating that these enzymes are a new example of functional convergence (2, 3).L-PGDS is expressed in the heart, central nervous system, and male genital organs of various mammals and is involved in various physiological and pathological functions (reviewed in Refs. 6 and 7). In the brain, L-PGDS produces PGD₂, which is involved in the regulation of pain and non-rapid eye movement sleep, as was shown in studies using gene knock-out mice (10, 11) and human enzyme transgenic mice (12). L-PGDS is regulated by SOX9 and is involved in the differentiation of male genital organs (13–15). This enzyme is also expressed in adipocytes (16), vascular smooth muscle cells (17), and myocardial cells (18, 19) and is involved in adipocyte differentiation, the progression of arteriosclerosis (20), and the protection against hypoxemia (18) or ischemia/reperfusion injury (19). L-PGDS binds various lipophilic compounds, such as retinoids (21), bilirubin, biliverdin (22), gangliosides (23), and amyloid-β peptides (24, 25), with high affinity, acting as an extracellular transporter of these compounds and serving as an endogenous amyloid-β chaperone to prevent amyloid deposition in vivo (24).Although many biochemical and physiological studies suggest important roles of PGD₂ and L-PGDS/β-trace in the regulation of sleep and other biological functions, the crystal structure of L-PGDS has not been resolved. In this study, we determined the crystal structures of two different forms of the Δ^1–24-C65A mutant of mouse L-PGDS in both open and closed conformations. L-PGDS was shown to possess a typical lipocalin fold, the β-barrel, which is a unique structural component specific to L-PGDS and comprises a mobile E-F loop and a large central cavity with two compartments. By performing site-directed mutagenesis of Δ^1–24-L-PGDS and the Δ^1–24-C65A mutant, we found that the Cys⁶⁵ surrounded by the hydroxyl side chains of Ser⁴⁵, Thr⁶⁷, and Ser⁸¹ was activated to contribute to the catalysis by L-PGDS.     

Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR<Subscript>1</Subscript>

Background  

Smoke released from burning vegetation functions as an important environmental signal promoting the germination of many plant species following a fire. It not only promotes the germination of species from fire-prone habitats, but several species from non-fire-prone areas also respond, including some crops. The germination stimulatory activity can largely be attributed to the presence of a highly active butenolide compound, 3-methyl-2H-furo[2,3-c]pyran-2-one (referred to as karrikin 1 or KAR₁), that has previously been isolated from plant-derived smoke. Several hypotheses have arisen regarding the molecular background of smoke and KAR₁ action.     

Endophytic infection modifies organic acid and mineral element accumulation by rice under Na<Subscript>2</Subscript>CO<Subscript>3</Subscript> stress

Background and aims

Saline and alkali soils severely impact plant growth. Endophyte and plant associations are known to significantly modify plant metabolism. This study reports the effects of a type of endophyte on organic acid (OA) accumulation and ionic balance in rice under Na₂CO₃ stress.

Methods

Rice seedlings with (E+) and without (E-) endophytic infection were subjected to different levels of Na₂CO₃ stress (0, 5, 10, 15, and 20 mM) for two weeks. Organic acids and mineral elements in the leaves and roots were determined.

Results

Seedlings with endophytic infection accumulated mainly citrate and fumarate, with some malate and succinate in the leaves. In the roots, accumulation of malate and fumarate was enhanced significantly by endophytic infection, while less citrate and succinate was accumulated under Na₂CO₃ stress, which suggested that leaves and roots use different mechanisms to control OA metabolism. Endophytes reduced the total Na and Na:K ratios, but increased ST values, the percent changes of other measured nutrients, Chl content, and dry weight per plant under Na₂CO₃ stress.

Conclusions

Endophytic infection plays a key role in maintaining plant growth by improving nutrient uptake and adjusting OA accumulation under Na₂CO₃ stress. The application of endophytes can enhance the resistance of rice to salinity.

     

An effective approach for generating a three-Cys<Subscript>2</Subscript>His<Subscript>2</Subscript> zinc-finger-DNA complex model by docking

Background  

Determination of protein-DNA complex structures with both NMR and X-ray crystallography remains challenging in many cases. High Ambiguity-Driven DOCKing (HADDOCK) is an information-driven docking program that has been used to successfully model many protein-DNA complexes. However, a protein-DNA complex model whereby the protein wraps around DNA has not been reported. Defining the ambiguous interaction restraints for the classical three-Cys₂His₂ zinc-finger proteins that wrap around DNA is critical because of the complicated binding geometry. In this study, we generated a Zif268-DNA complex model using three different sets of ambiguous interaction restraints (AIRs) to study the effect of the geometric distribution on the docking and used this approach to generate a newly reported Sp1-DNA complex model.     

Spatial temperature distribution in human hairy and glabrous skin after infrared CO<Subscript>2</Subscript> laser radiation

Background  

CO₂ lasers have been used for several decades as an experimental non-touching pain stimulator. The laser energy is absorbed by the water content in the most superficial layers of the skin. The deeper located nociceptors are activated by passive conduction of heat from superficial to deeper skin layers.     

Application of <Emphasis Type="Italic">in vivo</Emphasis> micro-computed tomography in the temporal characterisation of subchondral bone architecture in a rat model of low-dose monosodium iodoacetate-induced osteoarthritis

Introduction  

Osteoarthritis (OA) is a complex, multifactorial joint disease affecting both the cartilage and the subchondral bone. Animal models of OA aid in the understanding of the pathogenesis of OA and testing suitable drugs for OA treatment. In this study we characterized the temporal changes in the tibial subchondral bone architecture in a rat model of low-dose monosodium iodoacetate (MIA)-induced OA using in vivo micro-computed tomography (CT).     

Dietary fatty acid intake affects the risk of developing bone marrow lesions in healthy middle-aged adults without clinical knee osteoarthritis: a prospective cohort study

Introduction  

Fatty acids have been implicated in osteoarthritis (OA), yet the mechanism by which fatty acids affect knee structure and consequently the risk of knee OA has not been fully elucidated. Higher intakes of fatty acids have been shown to be associated with the risk of bone marrow lesions (BMLs) in a healthy population. The aim of this study was to examine the association between fatty acid consumption and the incidence of BMLs in healthy middle-aged adults without clinical knee OA.     

Chondrogenic differentiation potential of osteoarthritic chondrocytes and their possible use in matrix-associated autologous chondrocyte transplantation

Introduction  

Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA.     

The association between leptin,interleukin-6, and hip radiographic osteoarthritis in older people: a cross-sectional study

Introduction  

The associations between leptin, interleukin (IL)-6, and hip radiographic osteoarthritis (OA) have not been reported, and their roles in obesity-related hip OA are unclear. The aim of this study was to describe the associations between leptin, IL-6, and hip radiographic osteoarthritis (ROA) in older adults.     

A subgroup of plant aquaporins facilitate the bi-directional diffusion of As(OH)<Subscript>3</Subscript> and Sb(OH)<Subscript>3</Subscript>across membranes

Background  

Arsenic is a toxic and highly abundant metalloid that endangers human health through drinking water and the food chain. The most common forms of arsenic in the environment are arsenate (As(V)) and arsenite (As(III)). As(V) is a non-functional phosphate analog that enters the food chain via plant phosphate transporters. Inside cells, As(V) becomes reduced to As(III) for subsequent extrusion or compartmentation. Although much is known about As(III) transport and handling in microbes and mammals, the transport systems for As(III) have not yet been characterized in plants.     

Gum arabic modified Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> nanoparticles cross linked with collagen for isolation of bacteria

Background  

Multifunctional magnetic nanoparticles are important class of materials in the field of nanobiotechnology, as it is an emerging area of research for material science and molecular biology researchers. One of the various methods to obtain multifunctional nanomaterials, molecular functionalization by attaching organic functional groups to nanomagnetic materials is an important technique. Recently, functionalized magnetic nanoparticles have been demonstrated to be useful in isolation/detection of dangerous pathogens (bacteria/viruses) for human life. Iron (Fe) based material especially FePt is used in the isolation of ultralow concentrations (< 10² cfu/ml) of bacteria in less time and it has been demonstrated that van-FePt may be used as an alternative fast detection technique with respect to conventional polymerase chain reaction (PCR) method. However, still further improved demonstrations are necessary with interest to biocompatibility and green chemistry. Herein, we report the synthesis of Fe₃O₄ nanoparticles by template medication and its application for the detection/isolation of S. aureus bacteria.     

Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay

Background  

X-linked ocular albinism type 1 (OA1) is caused by mutations in OA1 gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment.     

Optical biosensor differentiates signaling of endogenous PAR<Subscript>1</Subscript> and PAR<Subscript>2</Subscript> in A431 cells

Background  

Protease activated receptors (PARs) consist of a family of four G protein-coupled receptors. Many types of cells express several PARs, whose physiological significance is mostly unknown.