We previously demonstrated a differential activation of the endothelin-1 (ET-1) pathway in male and female deoxycorticosterone (DOCA)-salt hypertensive rats, with the male rats exhibiting marked alterations in vascular and pressor responses to ET-1 and Suc-[Glu,(9)Ala(11,15)]-ET-1(8-21) (IRL-1620), an ET(B) agonist. Mechanisms underlying these gender differences are unclear, and we hypothesized that the ovarian hormones attenuate vascular ET(B) responses in female DOCA-salt rats. Female Wistar rats were randomized in 3 groups: sham-operated, ovariectomized (OVX), and OVX plus hormone replacement with estradiol (E) or estradiol/progesterone (EP). Two weeks later, rats were uninephrectomized and further randomized in DOCA-salt (subcutaneous injections of desoxycorticosterone and drinking water containing NaCl/KCl) and control normotensive (subcutaneous injections of vehicle and tap water). Blood pressure was evaluated both by direct and standard tail-cuff methods. Responses to IRL-1620 were evaluated in vivo/in situ in the mesenteric microcirculation. mRNA expression of ET-1 and ET(A/B) receptors was evaluated in mesenteric arteries by reverse transcription-polymerase chain reaction and expressed relative to GAPDH. OVX-DOCA rats developed a more severe form of hypertension than did DOCA rats. Treatment with E or EP restored blood pressure to levels observed in DOCA rats. In the mesentery, IRL-1620 induced vasodilatation in control rats, a mild vasoconstriction in DOCA rats, and marked vasoconstriction in OVX-DOCA rats. Both E and EP decreased IRL-1620-induced vasoconstriction in the DOCA group. In the normotensive group, OVX did not change blood pressure or IRL-1620-induced vasodilation. Removal of the ovaries increased ET-1 mRNA in arteries from DOCA and control rats, although treatment with E or EP reversed these changes. Vascular ET(B) receptor mRNA levels were greatly enhanced in OVX-DOCA but not OVX-control rats. Hormone replacement with E or EP restored ET(B) receptor expression in the DOCA group. A greater blood pressure-lowering effect of bosentan (ET(A)/ET(B) blocker) was observed in OVX-DOCA rats. The observation that OVX worsens hypertension as well as the altered ET(B) receptor-mediated responses and the effects of bosentan in female DOCA rats supports our suggestion that the ovarian hormones modulate ET-1/ET(B) receptor vascular responses/expression in DOCA-salt hypertension.

We previously established that COX-2 overexpression promotes breast cancer progression and metastasis. As long-term use of COX-2 inhibitors (COX-2i) can promote thrombo-embolic events, we tested an alternative target, prostaglandin E2 receptor <em>EP</em>4 subtype (<em>EP</em>4), downstream of COX-2. Here we used the highly metastatic syngeneic murine C3L5 breast cancer model to test the role of <em>EP</em>4-expressing macrophages in vascular endothelial growth factor (VEGF)-C/D production, angiogenesis, and lymphangiogenesis in situ, the role of <em>EP</em>4 in stem-like cell (SLC) functions of tumor cells, and therapeutic effects of an <em>EP</em>4 antagonist RQ-15986 (<em>EP</em>4A). C3L5 cells expressed all <em>EP</em> receptors, produced VEGF-C/D, and showed high clonogenic tumorsphere forming ability in vitro, functions inhibited with COX-2i or <em>EP</em>4A. Treating murine macrophage RAW 264.7 cell line with COX-2i celecoxib and <em>EP</em>4A significantly reduced VEGF-A/C/D production in vitro, measured with quantitative PCR and Western blots. Orthotopic implants of C3L5 cells in C3H/HeJ mice showed rapid tumor growth, angiogenesis, lymphangiogenesis (CD31/LYVE-1 and CD31/PROX1 immunostaining), and metastasis to lymph nodes and lungs. Tumors revealed high incidence of <em>EP</em>4-expressing, VEGF-C/D producing macrophages identified with dual immunostaining of F4/80 and <em>EP</em>4 or VEGF-C/D. Celecoxib or <em>EP</em>4A therapy at non-toxic doses abrogated tumor growth, lymphangiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in treated mice revealed markedly reduced VEGF-A/C/D and phosphorylated Akt/ERK proteins, VEGF-C/D positive macrophage infiltration, and proliferative/apoptotic cell ratios. Knocking down COX-2 or <em>EP</em>4 in C3L5 cells or treating cells in vitro with celecoxib or <em>EP</em>4A and treating tumor-bearing mice in vivo with the same drug reduced SLC properties of tumor cells including preferential co-expression of COX-2 and SLC markers ALDH1A, CD44, OCT-3/4, <em>β</em>-catenin, and SOX-2. Thus, <em>EP</em>4 is an excellent therapeutic target to block stem-like properties, angiogenesis, and lymphangiogenesis induced by VEGF-A/C/D secreted by cancer cells and tumor infiltrating macrophages.

Predicting invasion depth of superficial esophageal squamous cell carcinoma is crucial in determining the precise indication for endoscopic resection because the rate of lymph node metastasis increases in proportion to the invasion depth of the carcinoma. Previous studies have shown a close relationship between microvascular patterns observed by Narrow Band Imaging magnifying endoscopy and invasion depth of the superficial carcinoma. Thus, the Japan Esophageal Society (JES) developed a simplified magnifying endoscopic classification for estimating invasion depth of superficial esophageal squamous cell carcinomas. We conducted a prospective study to evaluate the diagnostic values of type B vessels in the pretreatment estimation of invasion depth of superficial esophageal squamous cell carcinomas utilizing JES classification, the criteria of which are based on the degree of irregularity in the microvascular morphology. Type A microvessels corresponded to noncancerous lesions and lack severe irregularity; type B, to cancerous lesions, and exhibit severe irregularity. Type B vessels were subclassified into BBBEP or T1a-LPM, T1a-MM or T1b-SM1, and T1b-SM2 tumors, respectively. We enrolled 211 patients with superficial esophageal squamous cell carcinoma. The overall accuracy of type B microvessels in estimating tumor invasion depth was 90.5 %. We propose that the newly developed JES magnifying endoscopic classification is useful in estimating the invasion depth of superficial esophageal squamous cell carcinoma.

In this study changes in the diameter and compliance of the distal abdominal aorta (76 healthy Caucasian males, 5-71 years old) were determined non-invasively and related to age by means of an ultrasound phase-locked echo-tracking system. The diameter of the aorta increased not only in the period between 5 and 25 years of age, but also by about 30% between the ages of 25 and 71. The pressure diameter curves at 25, 51 and 70 years were non-linear with flattening between 90 and 110 mmHg, and the slope of the curves declined with age. Thus, the pressure strain elastic modulus (Ep) and stiffness (beta) increased (i.e. compliance decreased) in an exponential manner according to age. A group of 37 males with aneurysmal widening of the distal abdominal aorta had a significant increase in Ep when compared to an age-matched control group. Furthermore, the ranges for both Ep and beta were much larger in the aneurysm group than in the control group, indicating diversity in the pathogenesis of the disease. The non-invasive ultrasonic method of phase-locked echo-tracking is an appropriate method for studying compliance in major arteries under a variety of pathophysiological conditions.

Twelve college-age men exercised on a bicycle ergometer to VO2max and at 60, 70, and 80% VO2max for 30 min to determine the effects of exercise intensity on plasma beta-endorphin (B-EP). The time course for alterations in B-EP and the relationship to lactate were also examined. Following the VO2max test, the three submaximal intensities were completed on separate days using a counter-balanced design. Blood was sampled from an indwelling venous catheter at rest during exercise and recovery to assess the time course response. B-EP content was determined by radioimmunoassay (Immunonuclear) with less than 5% cross-reactivity to B-LPH. At rest, B-EP content was similar across visits, 4.34 +/- 0.36 pmol.l-1. The 60% intensity did not elevate B-EP at any time measured. B-EP content increased by 15 min at 70% VO2max with a further increase at 30 min. B-EP remained elevated during the 20 min recovery. At 80% VO2max B-EP content increased by 5 min. B-EP continued to increase during the exercise and peaked at 21.91 +/- 2.03 pmol.l-1 5 min into the recovery. Lactate showed a mild correlation with B-EP (r = 0.43) at 80% VO2max. A significant correlation (r = 0.78) between lactate and B-EP did occur with the VO2max test. It is concluded that an exercise intensity of at least 70% VO2max for 15 min is needed to increase plasma B-EP. Furthermore, the higher the exercise intensity the more rapid the onset for increases in plasma B-EP.

Nucleotide sequencing of a 4.15 kb DNA fragment from megaplasmid 2 of Rhizobium meliloti 2011 revealed the location of the genes exoH, exoK and exoL. The putative proteins encoded by these genes have molecular weights of 41, 30, and 44 kDa, respectively. The hydrophobicity profile of the ExoH amino acid sequence resembles that of transmembrane proteins. The predicted exoL gene product does not contain hydrophobic regions, indicating a cytoplasmic localization. The exoK gene product is characterized by a putative signal peptide and exhibits significant homology to endo-beta-1,3-1,4-glucanases of bacilli and Clostridium thermocellum. R. meliloti exoK mutants induced pink nodules and synthesized a reduced amount of exopolysaccharide (EPS). Colonies of this mutant showed a delay in the appearance of the Calcofluor white fluorescence. In addition, the formation of the characteristic halo was strongly delayed. R. meliloti exoL and exoH mutants induced pseudonodules. The exoH, but not the exoL mutant, synthesized an EPS that could be precipitated by cetyl pyridinium chloride (CPC) and also by ethanol. Plasmid integration mutagenesis revealed promoter regions preceding exoH, exoK and exoL.

Inoculation of the right hind paw with Mycobacterium butyricum rapidly led to swelling and inflammation. The afflicted limb showed an enhanced sensitivity to noxious pressure (hyperalgesia) and a reduced sensitivity to noxious heat 24 h following treatment. Both naloxone and MR 2266 (which has greater activity at kappa-opioid receptors) further increased the sensitivity to pressure (that is, potentiated the hyperalgesia) but did not affect the response to heat. They did not affect the response of the uninflamed paw. At 1 week, only MR 2266 was effective. At both 24 h and 1 week, the inflamed paw showed pronounced supersensitivity to the antinociceptive action of morphine against noxious pressure. At both 24 h and (to a greater extent) 1 week, a rise in levels of immunoreactive (ir)-dynorphin (DYN) was seen in the ipsilateral dorsal horn of the lumbar spinal cord. There was no alteration in the contralateral dorsal horn or in either ventral horn. Furthermore, levels of ir-met-enkephalin (ME) and ir-leu-enkephalin (LE) were unaffected. There was no difference in the density of mu-, delta- or kappa-binding sites in any part of the lumbar cord, at either 24 h or 1 week, between ipsilateral and contralateral tissue. By 3 and 5 weeks postinoculation, the symptoms had spread to the contralateral hind limb and ir-DYN was elevated in the contralateral dorsal horn and the ipsilateral ventral horn. At 5 weeks, levels of ir-ME and ir-LE also were increased in the ipsilateral and contralateral dorsal horns, but not in the contralateral ventral horn. Furthermore, levels of ir-DYN were increased in the cervico-thoracic spinal cord, and rats displayed adrenal hypertrophy and a rise in plasma levels of ir-beta-endorphin (beta-EP). These data indicate: (1) Peripheral inflammation localized to a single limb selectively modifies levels of ir-DYN in ipsilateral dorsal horn. The effect is specific to DYN as compared to ME and LE. The density of mu-, delta-, or kappa-receptors in the lumbar spinal cord is unmodified. (2) The altered response to opioid agonists and antagonists shown by rats with an inflamed limb may be selective to the injured tissue. (3) Alterations in opioid systems associated with unilateral hind limb inflammation may not be exclusively chronic in nature: they appear very rapidly (within 24 h) of the induction of pain. With time, the contralateral limb becomes affected and, eventually, the effects resemble those seen with generalized polyarthritis.

We recently identified two isoforms of mouse prostaglandin (PG) E receptor EPEPEP 3 beta, which are produced by alternative splicing and different only in the carboxyl-terminal domain (Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, Y., T., Honda, A., Watabe, A., Hirata, M., Narumiya, S., and Ichikawa, A. (1993) J. Biol. Chem. 268, 2712-2718). We examined here agonist-induced desensitization of the two isoforms using Chinese hamster ovary cells stably expressing these isoforms. Exposure of the EPEPEPbeta isoform to PGE2 did not affect its dose-response curve for PGE2, and no sequestration or decrease in the receptor number was observed in this isoform. Thus, alternative splicing produced the two isoforms with different carboxyl-terminal domains, which are different in sensitivity to agonist-induced desensitization.

Escherichia coli and related Gram-negative bacteria contain an energy-coupled transport system through the outer membrane which consists of the proteins TonB, ExbB, ExbD anchored in the cytoplasmic membrane and receptors in the outer membrane. Differences in the activities of the Escherichia coli and the Serratia marcescens TonB proteins were used to identify TonB functional domains. In E.coli TonB segments were replaced by equivalent fragments of S. marcescens TonB and the activities of the resulting chimaeric proteins were determined. In addition, E. coli TonB was truncated at the C-terminal end, and point mutants were generated using bisulphite. From the results obtained we draw the following conclusions: an important site of interaction between TonB and ExbB is located in the N-terminal region of TonB within or close to the cytoplasmic membrane since an N-terminal 44-residue fragment of TonB was stabilized by ExbB and interfered with wild-type TonB activity. In addition, the activity of a TonB derivative in which histidine residue 20 was replaced by arginine was strongly reduced, and a double mutant containing arginine-7 to histidine and alanine-22 to threonine substitutions displayed an impaired uptake of ferrichrome. Furthermore, the domain around residue 160 is involved in TonB activity. S. marcescens TonB segments of this region in E. coli TonB conferred S. marcescens TonB activities, and E. coli TonB point mutants displayed strongly impaired activities for the uptake of colicin B and M and ferric siderophores. Plasmid-encoded tonB mutants of this region showed negative complementation of chromosomal wild-type tonB, and certain tonB mutants suppressed colicin B TonB-box mutants. Uptake of colicins required different domains in TonB, for colicin B and M around residue 160 and for colicin Ia, a domain closer to the C-terminal end. Tandem duplication of the E. coli (EP)X(KP) region by insertion of the S. marcescens (EP)X(KP) region (38 residues) and replacement of lysine residue 91 by glutamate did not alter TonB activity so that no evidence was obtained for this region to be implicated in receptor binding. The aberrant electrophoretic mobility of TonB was caused by the proline-rich sequence since its removal resulted in a normal mobility.

Many Gram-negative bacteria use the multi-protein type II secretion system (T2SS) to selectively translocate virulence factors from the periplasmic space into the extracellular environment. In Vibrio cholerae the T2SS is called the extracellular protein secretion (Eps) system,which translocates cholera toxin and several enzymes in their folded state across the outer membrane. Five proteins of the T2SS, the pseudopilins, are thought to assemble into a pseudopilus, which may control the outer membrane pore EpsD, and participate in the active export of proteins in a "piston-like" manner. We report here the 2.0 A resolution crystal structure of an N-terminally truncated variant of EpsH, a minor pseudopilin from Vibrio cholerae. While EpsH maintains an N-terminal alpha-helix and C-terminal beta-sheet consistent with the type 4a pilin fold, structural comparisons reveal major differences between the minor pseudopilin EpsH and the major pseudopilin GspG from Klebsiella oxytoca: EpsH contains a large beta-sheet in the variable domain, where GspG contains an alpha-helix. Most importantly, EpsH contains at its surface a hydrophobic crevice between its variable and conserved beta-sheets, wherein a majority of the conserved residues within the EpsH family are clustered. In a tentative model of a T2SS pseudopilus with EpsH at its tip, the conserved crevice faces away from the helix axis. This conserved surface region may be critical for interacting with other proteins from the T2SS machinery.

One of the systems proposed to mediate the reinforcing effects of ethanol is the endogenous opioid system. The objective of the present studies was to investigate the effects of various concentrations of ethanol on the release of beta-endorphin (beta-EP) by the hypothalami of mice showing either high (C57BL/6) or low (DBA/2) voluntary ethanol consumption. Results indicated that the release of beta-EP, either under basal conditions or in the presence of ethanol, was higher from the hypothalami of the C57BL/6 than of the DBA/2 mice. After exposure to various concentrations of ethanol, it was observed that for both strains of mice, low concentrations of ethanol (10, 20 and 25 mM) induced a more pronounced increase in the release of hypothalamic beta-EP than high concentrations of ethanol (30 and 60 mM) leading to an inverse U-shaped dose-response curve. Maximum release for both strains of mice was obtained at 20 mM ethanol. High-performance liquid chromatography analysis indicated that beta-EP 1-31 in the nonacetyl, opiate active form was the major form of the beta-EP-sized peptides released by the hypothalami of both strains of mice. The tissue content of beta-EP-like peptides was similar in the hypothalami of both strains of mice; however, the content of pro-opiomelanocortin mRNA was significantly higher in the hypothalami of the C57BL/6 mice. These genetically determined differences in the basal activity and in the response of the hypothalamic beta-EP system to ethanol may be partially responsible for the different ethanol consumption exhibited by these two strains of mice.

The mechanisms by which leptin influences energy homeostasis are not entirely understood. Several observations indicate that proopiomelanocortin (POMC) is involved in the regulation of food intake and may be a mediator of leptin action. To further study this interaction, a sensitive solution hybridization assay was used to compare the levels of POMC mRNA in the medial basal hypothalamus (MBH) of lean (+/+, +/fa(f)) and obese leptin receptor-deficient (fa(f)/fa(f)) rats. POMC peptide products were also measured by RIA in the same animals. Cytoplasmic POMC RNA levels were significantly reduced by 53% in obese rats as compared with lean controls: 0.30 +/- 0.04 vs. 0.64 +/- 0.07 pg/microgram total RNA (p < 0.02). Significant reductions in mean concentrations of hypothalamic POMC-derived peptides from the same dissections were detected in the obese rats vs. lean controls: alpha-MSH 1.77 +/- 0.07 vs. 2.34 +/- 0.10; beta-EP 4.06 +/- 0.24 vs. 5.86 +/- 0.36; gamma(3)-MSH 5.32 +/- 0. 20 vs. 6.52 +/- 0.12 ng/mg protein (p < 0.001). To determine whether leptin stimulates POMC gene transcription, the acute effect of an intracerebroventricular (i.c.v.) injection of leptin (5 microgram) on POMC primary transcript was quantified in the MBH of lean rats after a 16-hour fast. There was a significant 167% increase in mean POMC hnRNA levels 3 h after i.c.v. leptin injection (1.15 +/- 0.22 pg/MBH; p < 0.02), but not after 1 h (0.76 +/- 0.08 pg/MBH), compared to saline controls (0.69 +/- 0.08 pg/MBH). 4 h after the injection of leptin, POMC hnRNA was still increased, but to a lesser extent (140%), as compared with control animals (p = 0.006). These studies demonstrate for the first time in the leptin receptor-deficient rat that there is an associated decrease in POMC gene expression and peptide levels in the MBH. Furthermore, the acute increase in the levels of POMC primary transcript in non-obese rats after a single i.c.v. injection of leptin supports a role for leptin in the regulation of POMC gene transcription. Taken together, these studies provide further evidence that POMC is an important mediator of the effects of leptin on food intake and energy expenditure.

Until recently, it was generally accepted that the vascularization of solid tumors occurred exclusively through the sprouting and co-option from pre-existing blood vessels. Growing evidence now suggests that bone marrow-derived endothelial progenitor cells (EP) circulate in the blood and may play an important role in the formation of new blood vessels in certain tumors. Whether endothelial progenitors participate in the vascularization of brain tumors has not yet been evaluated. In this study, we examined the contribution of EP to tumor angiogenesis in a murine glioma tumor model. Donor bone marrow cells obtained from transgenic mice constitutively expressing beta-galactosidase or GFP either ubiquitously or transcriptionally regulated by an endothelial specific promotor Tie-2 were injected into lethally irradiated adult mice. After bone marrow reconstitution by donor cells, mice were implanted with syngeneic GL261 murine glioma cells. Morphological and confocal 3-dimensional analysis showed that the majority of the engrafted donor marrow cells were expressing hematopoietic and/or microglia markers, but did not appreciably contribute to the tumor vasculature. Implantation of glioma cells genetically engineered to overexpress VEGF produced highly vascularized tumors. However, the number of endothelial progenitors incorporated in the tumor vasculature did not increase. These data strongly suggest that neovascularization in the brain might fundamentally be regulated by the sprouting of pre-existing vessels and implicate that circulating endothelial progenitors do not play a significant role in this process.

The terminal branch of the general secretion pathway (Gsp or type II secretion system) is used by several pathogenic bacteria for the secretion of their virulence factors across the outer membrane. In these secretion systems, a complex of 12-15 Gsp proteins spans from the pore in the outer membrane via several associated signal or energy-transducing proteins in the inner membrane to a regulating ATPase in the cytosol. The human pathogen Vibrio cholerae uses such a system, called the Eps system, for the export of the cholera toxin and other virulence factors from its periplasm into the lumen of the gastrointestinal tract of the host. Here, we report the atomic structure of the periplasmic domain of the EpsM protein from V.cholerae, which is a part of the interface between the regulating part and the rest of the Eps system. The crystal structure was determined by Se-Met MAD phasing and the model was refined to 1.7A resolution. The monomer consists of two alphabetabeta-subdomains forming a sandwich of two alpha-helices and a four-stranded antiparallel beta-sheet. In the dimer, a deep cleft with a polar rim and a hydrophobic bottom made by conserved residues is located between the monomers. This cleft contains an extra electron density suggesting that this region might serve as a binding site of an unknown ligand or part of a protein partner. Unexpectedly, the fold of the periplasmic domain of EpsM is an undescribed circular permutation of the ferredoxin fold.

BACKGROUND

Proteomic investigations have revealed alterations in cytoskeletal proteins expressed in human acute lymphoblastic leukemia cells that are resistant to microtubule-disrupting agents. We characterized gamma-actin expression in antimicrotubule drug-resistant leukemia and examined the effect of altered gamma-actin in resistance of acute lymphoblastic leukemia to antimicrotubule agents.

METHODS

Two-dimensional polyacrylamide gel electrophoresis and mass spectrometry were used to identify actin proteins in human acute lymphoblastic leukemia cell lines resistant to vinblastine (CCRF-CEM/VLBB (CCRF-CEM/dEpoBEP cells. Expression of gamma-actin (normalized to that of beta2-microglobulin [beta2M]) in primary leukemia cells obtained from patients at diagnosis (n = 44) and relapse (n = 25) was examined using semiquantitative reverse transcription-polymerase chain reaction. Statistical significance of changes in the ratio of gamma-actin to beta2M expression between diagnosis and relapse samples was determined by two-sided unpaired Student's t tests.

RESULTS

We identified novel mutant forms of gamma-actin and the concomitant loss of wild-type gamma-actin in CCRF-CEM/VLBBEP cells transfected with gamma-actin siRNA displayed higher relative resistance to paclitaxel (P<.001), vinblastine (P = .04), and epothilone B (P = .045) than mock-transfected cells. No gamma-actin gene mutations were identified in 37 samples of primary leukemia cells (eight from patients at diagnosis, 29 from patients at relapse). Gamma-actin gene expression was lower in acute lymphoblastic leukemia samples collected at clinical relapse (n = 25; mean gamma-actin/beta2M = 0.53) than in samples collected at diagnosis (n = 44; mean gamma-actin/beta2M = 0.68; difference = 0.15, 95% confidence interval [CI] = 0.04 to 0.27, P = .01).

CONCLUSIONS

These data provide functional and associative clinical evidence of a novel form of drug resistance that involves interactions between gamma-actin and microtubules.

BACKGROUND

The involvement of the cyclooxygenases (COX), in particular COX-2, is well documented for many tumours, e.g. colon, breast and prostate cancer, by both experimental and clinical studies. There are epidemiological data from subjects using NSAIDs, and experimental evidence supporting the hypothesis of prostaglandins (PGs) as regulators of tumourigenesis in the ovary. One of the end products of PG-synthesis, PGE2, regulates several key-processes, which are characteristic for tumour growth, e.g. angiogenesis, proliferation and apoptosisis. The present study investigated the pathway for PGE2-synthesis and signalling in ovarian tumourigenesis by analysing specimen from normal ovaries (n = 18), benign (B) (n = 8), borderline type (BL) (n = 6) and malignant tumours (AC) (n = 22). The expression and cell-specific localization of COX-1, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1) and two of the receptors for PGE2, EP1 and EP2, were examined by immunoblotting (IB) and immunohistochemistry (IHC).

RESULTS

The results are in line with earlier studies demonstrating an increase of COX-2 in AC compared to the normal ovary, B and BL tumours. Increased expressions were also observed for COX-1, mPGES-1 and EP-1 which all were significantly (p < 0.05) augmented in less differentiated AC (grades: moderately-, poorly- and undifferentiated). The increase of COX-2 was also correlated to stage (FIGO classification) with significant elevations in stages II and III. EP1 was increased in stage III while no significant alterations were demonstrated for COX-1, mPGES-1 or EP2 for stage. IHC revealed staining of the tumour cells, but also increase of COX-1, COX-2, mPGES-1 and EP1-2 in the stromal compartment of AC (grades: moderately-, poorly- and undifferentiated). This observation suggests interactions between tumour cells and stromal cells (fibroblasts, immune cells), e.g. paracrine signalling mediated by growth factors, cytokines and possibly PGs.

CONCLUSIONS

The increases of COX-1, COX-2, mPGES-1 and EP1-2 in epithelial ovarian cancer, supports the hypothesis that PGE2-synthesis and signalling are of importance for malignant transformation and progression. The observed augmentations of COX-1, COX-2 and mPGES-1 have implications for future therapeutic strategies.

Current antipsychotic drugs primarily target dopamine D2 receptors (D2Rs), in conjunction with other receptors such as those for serotonin. However, these drugs have serious side effects such as extrapyramidal symptoms (EPS) and diabetes. Identifying a specific D2R signaling pathway that could be targeted for antipsychotic effects, without inducing EPS, would be a significant improvement in the treatment of schizophrenia. We report here that the D2R forms a protein complex with Disrupted in Schizophrenia 1 (DISC1) that facilitates D2R-mediated glycogen synthase kinase (GSK)-3 signaling and inhibits agonist-induced D2R internalization. D2R-DISC1 complex levels are increased in conjunction with decreased GSK-3α/β (Ser21/9) phosphorylation in both postmortem brain tissue from schizophrenia patients and in Disc1-L100P mutant mice, an animal model with behavioral abnormalities related to schizophrenia. Administration of an interfering peptide that disrupts the D2R-DISC1 complex successfully reverses behaviors relevant to schizophrenia but does not induce catalepsy, a strong predictor of EPS in humans.

Classically, the prostaglandin E(2) (PGE(2)) receptor EP(4) has been classified as coupling to the Galpha(s) subunit, leading to intracellular cAMP increases. However, EP(4) signaling has been revealed to be more complex and also involves coupling to pertussis toxin-sensitive Galpha(i) proteins and beta-arrestin-mediated effects. There are now many examples of selective activation of independent pathways by G protein-coupled receptor (GPCR) ligands, a concept referred to as functional selectivity. Because most EP(4) ligands had thus far only been functionally characterized by their ability to stimulate cAMP production, we systematically determined the potencies and efficacies of a panel of EP(4) ligands for activation of Galpha(s), Galpha(i), and beta-arrestin relative to the endogenous ligand PGE(2). For this purpose, we adapted three bioluminescence resonance energy transfer (BRET) assays to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists. PGE(2) was the most selective in activating Galpha(s), whereas PGF(2alpha) and PGE(1) alcohol were the most biased for activating Galpha(i1) and beta-arrestin, respectively. We observed reversal in order of potencies between beta-arrestin 2 and Galpha(i1) functional assays comparing PGE(1) alcohol and either PGF(2alpha), PGD(2), or 7-[(1R,2R)-2-[(E,3R)-3-hydroxy-4-(phenoxy)but-1-enyl]-5-oxocyclopentyl]heptanoic acid (M&B28767). Most ligands were full agonists for the three pathways tested. Our results have implications for the use of PGE(2) analogs in experimental and possibly clinical settings, because their activity spectra on EP(4) differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP(4) agonists should be applicable for the study of other GPCRs.

Maternal stress is a key risk factor in neurodevelopmental disorders, which often have a sex bias in severity and prevalence. We previously identified O-GlcNAc transferase (OGT) as a placental biomarker in our mouse model of early prenatal stress (EPS), where OGT levels were lower in male compared with female tissue and were further decreased following maternal stress. However, the function of placental OGT in programming the developing brain has not been determined. Therefore, we generated a transgenic mouse with targeted placental disruption of Ogt (Pl-OGT) and examined offspring for recapitulation of the adult EPS phenotype. Pl-OGT hemizygous and EPS male placentas showed similar robust changes in gene expression patterns suggestive of an altered ability to respond to endocrine and inflammatory signals, supporting placental OGT as an important mediator of EPS effects. ChIP-Seq for the O-GlcNAc mark identified the 17 beta hydroxysteroid dehydrogenase-3 (Hsd17b3) locus in male EPS placentas, which correlated with a reduction in Hsd17b3 expression and concordant reduced testosterone conversion. Remarkably, Pl-OGT adult offspring had reduced body weights and elevated hypothalamic-pituitary-adrenal stress axis responsivity, recapitulating phenotypes previously reported for EPS males. Further, hypothalamic microarray gene-set enrichment analyses identified reduced mitochondrial function in both Pl-OGT and EPS males. Cytochrome c oxidase activity assays verified this finding, linking reduced placental OGT with critical brain programming. Together, these studies confirm OGT as in important placental biomarker of maternal stress and demonstrate the profound impact a single placental gene has on long-term metabolic and neurodevelopmental programming that may be related to an increased risk for neurodevelopmental disorders.

A Lactobacillus plantarum strain producing exopolysaccharides (EPSs) was isolated from corn silage. When this strain, named L. plantarum EP56, was grown on a chemically defined medium, two EPS fractions were isolated. The cell-bound EPS fraction (EPS-b) was composed of a single high-molecular-mass polymer of 8.5x10(5) Da containing glucose, galactose and N-acetylgalactosamine in a molar ratio of approximately 3:1:1 and traces of glycerol and phosphoglycerol. The released EPS fraction (EPS-r) was composed of the high-molecular-mass bound polysaccharide and a second polymer of 4x10(4) Da containing glucose, galactose and rhamnose in a molar ratio of 3:1:1 and traces of glycerol and phosphoglycerol. EPS-b and EPS-r contained phosphate which contributes to their negative net charge. Studies on polysaccharide production and location showed that both polymers were synthesized during the exponential growth phase and that the EPS-b polymer was progressively released into the culture medium during the stationary growth phase. Carbon source and temperature influenced EPS synthesis when L. plantarum EP56 was grown in a chemically defined medium. Lactose was the most efficient carbon source among the five tested (glucose, galactose, fructose, lactose and sucrose). EPS production was also increased when the incubation temperature is lowered.

Three methods were used to prepare the cell-bound exopolysaccharides (c-EPS) of Lactobacillus plantarum 70810, and the maximum yield (64.17 mg/mL) was obtained by ultrasonic extraction. After anion exchange and gel filtration chromatography, the c-EPS was fractionated as a single peak with a molecular weight of 169.6 kD. Its structural characteristics were investigated by gas chromatography (GC), methylation, Fourier-transform infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR) analysis. Based on obtained data, the novel c-EPS was found to be a galactan containing a backbone of α-D-(1→6)-linked galactcosyl, β-D-(1→4)-linked galactcosyl, β-D-(1→2,3)-linked galactcosyl residues and a tail end of β-D-(1→)-linked galactcosyl residues. Preliminary in vitro tests revealed that c-EPS significantly inhibited the proliferation of HepG-2, BGC-823, especially HT-29 tumor cells. Our results suggested that the c-EPS produced by L. plantarum 70810 might be suitable for use as functional foods and natural antitumor drugs.

The lipopolysaccharide (LPS) constituents of the gram-negative bacterial wall are among the most potent activators of inflammation. In the current study, we examined the effect of subcutaneous injection of Escherichia coli LPS on leukocyte influx into the normal and injured brain using endogenous peroxidase (EP). Normal brain parenchyma does not contain granulocytes and this does not change after indirect trauma, in facial axotomy. However, systemic injection of 1 mg LPS led to a gradual appearance of EP-positive parenchymal granulocytes within 12 h, with a maximum at 1-4 days after injection. Facial axotomy (day 14) led to a further 50-300% increase in granulocyte number. Of the five mouse strains tested in the current study, four--Balb/C, FVB, C57Bl/6, and C3H/N--showed vigorous granulocyte influx (60-90 cells per 20-microm section in axotomized facial nucleus, 20-40 cells per section on the contralateral side). The influx was an order of magnitude lower in the SJL mice. The peroxidase-positive cells were immunoreactive for neutrophil antigen 7/4 and alpha M beta 2 integrin, were negative for IBA1 (monocytes) and CD3 (T cells), and could be prelabeled by subcutaneous injection with rhodamine B isothiocyanate (RITC), confirming their origin as blood-borne granulocytes. All RITC-positive cells were IBA1 negative. This influx of granulocytes was accompanied by a disruption of the blood-brain barrier to albumin and induction of the cell adhesion molecule ICAM-1 on affected blood vessels. Transgenic deletion of ICAM-1 led to a more than 50% reduction in the number of infiltrating granulocytes compared to litter-matched wild-type controls, in normal brain as well as in axotomized facial motor nucleus. In summary, systemic injection of LPS leads to invasion of granulocytes into the mouse brain and a breakdown of the blood-brain barrier to blood-borne cells and to soluble molecules. Moreover, this mechanism may play a pathogenic role in the etiology of meningitis and in severe bacterial sepsis.

Aerobic, alkaliphilic bacteria were isolated and characterized from water and sediment samples collected in the winter season, January 2002 from alkaline Lonar lake, India, having pH 10.5. The total number of microorganisms in the sediment and water samples was found to be 10(2)-10(6) cfu g(-1) and 10(2)-10(4) cfu ml(-1), respectively. One hundred and ninety-six strains were isolated using different enrichment media. To study the bacterial diversity of Lonar lake and to select the bacterial strains for further characterization, screening was done on the basis of pH and salt tolerance of the isolates. Sixty-four isolates were subjected to phenotypic, biochemical characterization and 16S rRNA sequencing. Out of 64, 31 bacterial isolates were selected on the basis of their enzyme profile and further subjected to phylogenetic analysis. Phylogenetic analysis indicated that most of the Lonar lake isolates were related to the phylum Firmicutes, containing Low G+C, Gram-positive bacteria, with different genera: Bacillus, Paenibacillus, Alkalibacillus, Exiguobacterium, Planococcus, Enterococcus and Vagococcus. Seven strains constituted a Gram-negative bacterial group, with different genera: Halomonas, Stenotrophomonas and Providencia affiliated to gamma-Proteobacteria, Alcaligenes to beta-Proteobacteria and Paracoccus to alpha-Proteobacteria. Only five isolates were High G+C, Gram-positive bacteria associated with phylum Actinobacteria, with various genera: Cellulosimicrobium, Dietzia, Arthrobacter and Micrococcus. Despite the alkaline pH of the Lonar lake, most of the strains were alkalitolerant and only two strains were obligate alkaliphilic. Most of the isolates produced biotechnologically important enzymes at alkaline pH, while only two isolates (ARI 351 and ARI 341) showed the presence of polyhydroxyalkcanoate (PHA) and exopolysaccharide (EPS), respectively.

Catenins (alpha-, beta- and gamma- or plakoglobin) are cytoplasmic cadherin-associated proteins. Studies on cultured cells have suggested that both alpha-catenin and plakoglobin are important for the adhesive function of cadherins. alpha-catenin binds to both beta-catenin and plakoglobin and may link the cadherin/catenin complex to actin filaments. Separate domains of plakoglobin bind to cadherin and alpha-catenin, suggesting it may act as a bridge between these molecules. However, plakoglobin may have other activities: it is expressed in both desmosomal junctions in association with desmogleins and the cytoplasm in conjunction with APC, and previous work suggests it may act in a dorsal signalling pathway when overexpressed in Xenopus embryos. Here, we have studied the roles of alpha-catenin and plakoglobin directly, by depleting the maternal mRNAs coding for each of them in developing Xenopus embryos. We find that depletion of maternal alpha-catenin causes the loss of intercellular adhesion at the blastula stage, similar to that reported previously for EP cadherin. Depletion of plakoglobin results in a partial loss of adhesion, and a loss of embryonic shape, but does not affect dorsal signalling.