ROCKing cytokine secretion balance in human T cells

Balanced regulation of cytokine secretion in T cells is critical for maintenance of immune homeostasis and prevention of autoimmunity. The Rho-associated kinase (ROCK) 2 signaling pathway was previously shown to be involved in controlling of cellular movement and shape. However, recent work from our group and others has demonstrated a new and important role of ROCK2 in regulating cytokine secretion in T cells. We found that ROCK2 promotes pro-inflammatory cytokines such as IL-17 and IL-21, whereas IL-2 and IL-10 secretion are negatively regulated by ROCK2 under Th17-skewing activation. Also, in disease, but not in steady state conditions, ROCK2 contributes to regulation of IFN-γ secretion in T cells from rheumatoid arthritis patients. Thus, ROCK2 signaling is a key pathway in modulation of T-cell mediated immune responses underscoring the therapeutic potential of targeted inhibition of ROCK2 in autoimmunity.     

Activation of the pro-migratory bone morphogenetic protein receptor 1B gene in human MDA-MB-468 triple-negative breast cancer cells that over-express CYP2J2

Secondary metastases are the leading cause of mortality in patients with breast cancer. Cytochrome P450 (CYP) 2J2 (CYP2J2) is upregulated in many human tumors and generates epoxyeicosanoids from arachidonic acid that promote tumorigenesis and metastasis, but at present there is little information on the genes that mediate these actions. In this study MDA-MB-468 breast cancer cells were stably transfected with CYP2J2 (MDA-2J2 cells) and Affymetrix microarray profiling was undertaken. We identified 182 genes that were differentially expressed in MDA-2J2 cells relative to control (MDA-CTL) cells (log[fold of control] ≥2). From gene ontology pathway analysis bone morphogenetic protein (BMP) receptor 1B (BMPR1B) emerged as an important upregulated gene in MDA-2J2 cells. Addition of the BMPR1B ligand BMP2 stimulated the migration of MDA-2J2 cells, but not MDA-CTL cells, from 3D-matrigel droplets. Migration of MDA-2J2 cells was prevented by the BMPR antagonist dorsomorphin. These findings indicate that over-expression of CYP2J2 in MDA-MB-468-derived breast cancer cells activates BMPR1B expression that may contribute to increased migration. Targeting BMPR1B may be a novel approach to inhibit the metastatic activity of breast cancers that contain high levels of CYP2J2.     

Two fibrinogen-like proteins,FGL1 and FGL2 are disulfide-linked subunits of oligomers that specifically bind nonviable spermatozoa

Nevertheless, a nonviable sperm population is present in the cauda epididymidis of many species. Degenerating spermatozoa release enzymes that could have detrimental effects on the viability of neighboring cells, and they are source of autoantigens that induce an autoimmune response if they escape the blood-epididymis barrier. Does the epididymis have specialized protective mechanism(s) to segregate the viable sperm population from defective spermatozoa? Previously, we identified a fibrinogen-like protein-2 (fgl2) that specifically binds to and polymerizes into a cocoon-like complex coating defective spermatozoa and sperm fragments. The objective of the present study is to identify the subunit composition of the fgl2-containing oligomers both in the soluble and cocoon-like complex. Our proteomic studies indicate that the 260/280 kDa oligomers (termed eFGL) contain two distinct disulfide-linked subunits; 64 kDa fgl2 and 33 kDa fgl1. Utilizing a PCR-based cloning strategy, the 33 kDa polypeptide has been identified as fibrinogen-like protein-1 (fgl1). Immunocytochemical studies revealed that fgl1 selectively binds to defective spermatozoa in the cauda epididymidis. Northern blot analysis and in situ hybridization demonstrated the high expression of fgl1 in the principal cells of the proximal cauda epididymidis. Co-immunoprecipitation analyses of cauda epididymal fluid, using anti-fgl2, demonstrate that both fgl1 and fgl2 are present in the soluble eFGL. Our study is the first to show an association of fgl1 and fgl2 both in the soluble and in the sperm-associated eFGL. We conclude that our results provide new insights into the mechanisms by which the potentially unique epididymal protein functions in the recognition and elimination of defective spermatozoa.     

WFS1 and non-syndromic low-frequency sensorineural hearing loss: A novel mutation in a Portuguese case

Low-frequency sensorineural hearing loss (LFSNHL) is an unusual type of HL in which frequencies at 2000 Hz and below are predominantly affected. Most of the families with LFSNHL carry missense mutations in WFS1 gene, coding for wolframin.     

Chemical constituents isolated from Disporum viridescens leaves and their inhibitory effect on nitric oxide production in BV2 microglial cells

Excessive NO (nitric oxide) has been associated with the pathogenesis of various neurodegenerative diseases including Alzheimer’s disease (AD). In our screening system using LPS-activated BV2 microglial cells, the methanolic extract of Disporum viridescens leaves was found to have significant NO inhibitory activity. Bioactivity-guided isolation yielded a new phenylpropanoid characterized as 4-ally-2,6-dimethoxyphenyl 1-O-β-d-apiofuranosyl (1 → 6)-β-d-glucopyranoside (12) with 21 known compounds from the leaves of D. viridescens. Among them, compounds 2 and 4 significantly inhibited NO production. Thus, we further elucidated the anti-inflammatory mechanism of these lignans. Especially, compound 4 inhibited the expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) through the suppression of the MAPK signaling pathway. Taken together, the anti-inflammatory activities of the active constituents isolated from D. viridescens leaves could have therapeutic potential against neurodegenerative diseases.     

Effect of externally added carnitine on the synthesis of acetylcholine in rat cerebral cortex cells

Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 μM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of β-oxidation might be correlated with the lower level of acetylcholine synthesis.     

The neutralization of acidic coal mine lakes by additions of natural organic matter: a mesocosm test

Cylindrical polyethylene enclosures 3 m in length and 1 m in diameter reaching from the surface to the bottom were constructed in an acid (pH=3.1) lake on a coal surface mine in southern Illinois. Wheat straw was added to the enclosures to test the effects of dissimilatory sulfate reduction on water chemistry. Added straw increased sulfide concentrations, raised pH to 6.5, reduced O₂ and increased acid neutralizing capacity of the enclosed water columns when compared with a control enclosure and with the open lake. Generation of acid neutralizing capacity exceeded the standing stock of sulfide indicating that sulfide was removed either by precipitation of FeS or outgassing of H₂S. The pH and acid neutralizing capacity within the enclosures eventually returned to the level of the surrounding lake because of water exchange around the enclosure walls. Our results show that additions of organic matter to acid surface mine lakes result in the generation of acid neutralizing capacity.     

Synthesis of stromal glycosaminoglycans in response to injury

Our goal is to examine the synthesis and deposition of corneal glycosaminoglycans (GAGs) in response to a wound created by the insertion of porous discs into stromal interlamellar pockets. The disc and the surrounding stromal tissue were assayed and compared to contralateral control stroma and to sham operated corneas at 14,42, and 84 days. The tissue and/or discs were removed and labeled with ³⁵S-sulfate for 18 h; GAGs were extracted with 4 M guanidine–HCl. Extracts were chromatographed on Q-Sepharose columns, bound proteoglycans were eluted with a linear salt gradient, and radioactive fractions were analyzed. Total GAG content was determined colorimetrically, using dimethylmethylene blue. Specific GAGs were determined using enzymatic digestion with selective polysaccharide lyases and protein cores were examined using SDS–PAGE. The nonbound fractions from the chromatography were assayed for TGF-β using Western blot analysis and for hyaluronic acid using an ¹²⁵I-radiometric assay. Specific GAGs were localized 42 days after the disc had been implanted in the stroma. The placement of the discs into the stroma resulted in a decrease in the total amount of GAG. However, the ratio of dermatan-chondroitin sulfate and heparan sulfate to keratan sulfate increased in the surrounding tissue and disc. Hyaluronic acid was elevated at day 14 in the surrounding tissue, and not until day 84 in the disc. Western blot analysis of surrounding tissue extracts revealed forms of TGF-β that migrated with an apparent molecular mass of 63 and 43 kDa. The results indicate that the insertion of discs into interlamellar pockets causes changes in the sulfation and proportion of the glycosaminoglycans in the surrounding tissue and the disc. These changes are coincident with the appearance of TGF-β. After 84 days, the population of glycosaminoglycans in the disc begins to resemble the surrounding stroma. This model will allow us to examine further the synthesis and deposition of proteins following an extensive wound in which cells must migrate to the wound site and then undergo extensive remodeling. © 1995 Wiley-Liss, Inc.