To define the cellular targets for c-kit ligand (KL) and to study their functional properties and composite antigenic profile, we isolated cells expressing c-kit receptor (KR) from bone marrow (BM), peripheral blood, and fetal liver (FL) using immunoadherence to a recently obtained antibody (SR-1) against the human KR. Cells isolated by this approach (designated SR-1Ad) have the morphology of blasts and represent 1% to 4% of the original BM or FL populations. SR-1Ad cells from either source are highly enriched in progenitors (12% to 73%) and respond to KL in distinct patterns. In SR-1Ad cells from BM, the greatest impact of KL stimulation is on burst-forming units-erythroid (BFU-E), whereas in SR1-Ad cells from FL, the most significant KL effect is on a mixed erythroid/nonerythroid progenitor (erythroid/macrophage, colony-forming unit-mix [CFU-Mix]). When antibody SR-1 is continually present in culture, it neutralizes the effects of added KL. Furthermore, in the absence of added KL, it greatly diminishes the erythropoietin- and interleukin-3-dependent BFU-E growth in BM; whereas in FL, a wider spectrum of inhibition is observed, with CFU-Mix most severely curtailed. SR-1Ad cells coexpress other progenitor-associated antigens in a combination reflecting the dominant presence of erythroid progenitors (high expression of CD34, DR, CD38, and Ep-1; low expression of CD33). Several cytoadhesion molecules, ie, alpha L/beta 2 and alpha 4/beta 1 integrins, and intercellular adhesion molecule 1 and homing cell adhesion molecule 1, are also coexpressed. Our data provide new information on the isolation and characterization of KR expressing cells from normal, adult, and fetal hematopoietic tissues. On these biologically relevant target cells, the impact of ligand-induced stimulation or antibody-mediated ablation of KR function has been gauged.

Despite the characterization of some Burkholderia cepacia complex exopolysaccharides (EPSs), little is known about the role of EPSs in the pathogenicity of B. cepacia complex organisms. We describe 2 Burkholderia cenocepacia (genomovar III) isolates obtained from a patient with cystic fibrosis (CF): the nonmucoid isolate C8963 and the mucoid isolate C9343. Both isolates had identical random amplified polymorphic DNA patterns. C9343 produced a capsule composed of the EPSs PS-I and PS-II, as well as alpha -1,6-glucan. These isolates exhibited several phenotypic differences: C8963 synthesized octanoyl-homoserine lactone and produced biofilms, but C9343 did not; in a mouse model of pulmonary infection, C8963 was cleared more rapidly than was C9343; and C9343 interacted poorly with macrophages and neutrophils, compared with C8963, suggesting that the C9343 capsule interfered with cell-surface interactions. Overproduction of EPS by C9343 resulted in a mucoid appearance and interfered with cell-surface interactions and clearance in an animal model. This mucoid colonial appearance could enhance the persistence and virulence of this important CF-related pathogen.

Ectopic pregnancy (EP) and normal intrauterine pregnancy (IUP) serum proteomes were quantitatively compared to systematically identify candidate biomarkers. A 3-D biomarker discovery strategy consisting of abundant protein immunodepletion, SDS gels, LC-MS/MS, and label-free quantitation of MS signal intensities identified 70 candidate biomarkers with differences between groups greater than 2.5-fold. Further statistical analyses of peptide quantities were used to select the most promising 12 biomarkers for further study, which included known EP biomarkers, novel EP biomarkers (ADAM12 and ISM2), and five specific isoforms of the pregnancy specific beta-1-glycoprotein family. Technical replicates showed good reproducibility and protein intensities from the label-free discovery analysis compared favorably with reported abundance levels of several known reference serum proteins over at least 3 orders of magnitude. Similarly, relative abundances of candidate biomarkers from the label-free discovery analysis were consistent with relative abundances from pilot validation assays performed for five of the 12 most promising biomarkers using label-free multiple reaction monitoring of both the patient serum pools used for discovery and the individual samples that constituted these pools. These results demonstrate robust, reproducible, in-depth 3-D serum proteome discovery, and subsequent pilot-scale validation studies can be achieved readily using label-free quantitation strategies.

The inhibitory effects of three prostanoid analogues, EP 045, EP 092 and pinane thromboxane A2 (PTA2), on the aggregation of human platelets in vitro have been investigated. In diluted platelet-rich plasma (PRP), EP 045 (20 microM) and EP 092 (1 microM) completely inhibited irreversible aggregation responses to thromboxane A2 (TXA2), prostaglandin H2 (PGH2) and five chemically stable thromboxane mimetics, including 11,9-epoxymethano-PGH2 and 9,11-azo-PGH2. Reversible aggregation produced by the prostanoid analogue, CTA2, was also inhibited. The block of the stable agonist action was surmountable. In plasma-free platelet suspensions EP 045 and EP 092 were more potent antagonists. Schild analysis indicated a competitive type of antagonism for EP 045 (affinity constant of 1.1 X 10(7) M-1); the nature of the EP 092 block is not clear. Primary aggregation waves induced by ADP, platelet activating factor (Paf) and adrenaline were unaffected by EP 045 and EP 092, whereas the corresponding second phases of aggregation were suppressed. Aggregation and 5-hydroxytryptamine (5-HT) release induced by either PGH2 or 11,9-epoxymethano-PGH2 were inhibited in a parallel manner by EP 045. Inhibition of thromboxane biosynthesis is not involved in these effects. EP 045 and EP 092 did not raise adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in the platelet suspensions. In plasma-free platelet suspensions PTA2 produced a shape change response which could be blocked by EP 045. PTA2, therefore, has a thromboxane-like agonist action. The block of the aggregatory action of 11,9-epoxymethano-PGH2 by PTA2 appears to be mainly due to competition at the thromboxane receptor. However, PTA2 produced a slight rise in cyclic AMP levels; this could be due to a very weak stimulant action on either PGI2 or PGD2 receptors present in the human platelet. Functional antagonism by PTA2 may therefore augment its thromboxane receptor blocking activity. The results are discussed in terms of (a) the specificity of antagonism produced by EP 045, EP 092 and PTA2, (b) the validity of affinity constant determinations for receptor antagonists when aggregation is the biological response, and (c) the characteristics of the human platelet thromboxane receptor in comparison with those of thromboxane receptors in smooth muscle.

Fibronectin (Fn) is involved in the early stages of bone formation, and prostaglandin E (PGE) is an important factor regulating osteogenesis. Here we found that PGE(2) enhanced extracellular Fn assembly in rat primary osteoblasts, as shown by immunofluorescence staining and enzyme-linked immunosorbent assay. PGE(2) also increased the protein levels of Fn by using Western blotting analysis. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptor, antisense oligonucleotides revealed that the EP(1) receptor but not other PGE receptors is involved in PGE(2)-mediated up-regulation of Fn. At the mechanistic level, Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)), phosphatidylinositol-phospholipase C inhibitor (U73122), or Src inhibitor (PP2) attenuated the PGE(2)-induced Fn expression. Protein kinase C (PKC) inhibitor (GF109203X) also inhibited the potentiating action of PGE(2). Furthermore, treatment with antisense oligonucleotides of various PKC isoforms, including alpha, beta, epsilon, and delta, demonstrated that alpha isozyme plays an important role in the enhancement action of PGE(2) on Fn assembly. Flow cytometry and reverse transcription-PCR showed that PGE(2) and 17-phenyl trinor PGE(2) (EP(1)/EP(3) agonist) increased the surface expression and mRNA level of alpha5 or betabetaEP(1)/phospholipase C/PKCalpha/c-Src signaling pathway.

OBJECTIVE

We discuss pathology-based characterization and classification of acquired immune and inflammatory myopathies (IIMs).

RESULTS

Several types of IIMs do not fit well into the typical IIM subclassifications: dermatomyositis, polymyositis and inclusion body myositis (IBM). Myopathologic features that can provide additional diagnostic clarification in IIM are types of muscle fiber pathology; immune changes (cellular and humoral); and tissues with distinctive involvement (connective tissue, vessels and muscle fibers). Pathologic classification categories include immune myopathies with perimysial pathology (IMPP), a group that can be associated with antisynthetase antibodies; myovasculopathies, including childhood dermatomyositis; immune polymyopathies, active myopathies with little inflammation such as the myopathy with signal recognition particle antibodies; immune myopathies with endomysial pathology (IM-EP), illustrated by brachio-cervical inflammatory myopathy (BCIM); histiocytic inflammatory myopathies, like sarcoid myopathy; and inflammatory myopathies with vacuoles, aggregates and mitochondrial pathology (IM-VAMP), which have inclusion body myositis as a pathologic subtype and are poorly treatable. Some myopathologic features, like B-cell foci and alkaline phosphatase staining of capillaries or perimysium, are more likely to be present in treatable categories of IIM.

CONCLUSIONS

Myopathology can be used to classify IIM. Identification of distinctive myopathologic changes in IIM can improve diagnostic and prognostic accuracy and focus treatment, therapeutic trials and studies of pathogenic factors.

Ethanol has been shown to enhance the in vitro release of hypothalamic beta-endorphin (beta-EP). In the present study, the pattern of beta-EP release by the hypothalamus of two strains of mice, bred selectively for their preference (C57BL/6) or aversion (DBA/2) to ethanol, was investigated using a tissue perifusion system. The tissues were perifused with 20 mM ethanol for 30 min and the immunoreactive beta-EP content was estimated in perifusates collected every 2 min. Ethanol induced an enhanced release of hypothalamic beta-EP characterized by an initial spike followed by a gradual decrease toward baseline levels in both strains of mice. The ethanol-induced increase in beta-EP release by the hypothalamus of the C57BL/6 mice was more pronounced and longer lasting than that by the hypothalamus of the DBA/2 mice. Similar to beta-EP, an immediate sharp increase of corticotropin-releasing hormone (CRH) release was induced by ethanol which, however, did not present a spike but was maintained significantly higher than spontaneous release for the duration of ethanol exposure. Both ethanol-induced beta-EP and CRH release returned to basal levels within 10 min following removal of ethanol. That beta-EP levels did not remain elevated for the duration of ethanol exposure was not due to tissue depletion of releasable beta-EP pool, since exposure of the hypothalami to 10(-8) M CRH for 10 min, immediately after the perifusion with 20 mM ethanol, resulted in a large increase of beta-EP release. A second ethanol exposure 30 min after the first one did not induce an increase in beta-EP release. However, when the recovery period from the first ethanol exposure was extended to 60 min, a significant increase in the release of hypothalamic beta-EP was observed from the hypothalamus of the C57BL/6 but not of the DBA/2 mice. It is concluded that hypothalamic endorphinergic neurons present a fast, transient increase of beta-EP release in the presence of 20 mM ethanol, and become insensitive to subsequent ethanol exposures for a period of about 60 min. In addition, genetically determined differences exist with regards to the magnitude and duration of the ethanol-stimulated release of beta-EP, as well as on the length of the ethanol nonresponsive period. These differences may explain in part the differences in the voluntary ethanol consumption exhibited by these strains of mice.

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with beta-1,3, beta-1,4, beta-1,6, alpha-1,3, and alpha-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGROmegaexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGROmegaexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGROmegaexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, approximately 50 microg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-beta-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.

Prostaglandin (PG) E(2) is an important modulator of the actions of angiotensin (Ang) II. In the present study, we investigated the renal microvascular actions of PGE(2) and the EP receptor subtypes involved. Ibuprofen potentiated Ang II-induced vasoconstriction in in vitro perfused normal rat kidneys and augmented afferent arteriolar, but not efferent arteriolar, responses in the hydronephrotic rat kidney model. This preglomerular effect of endogenous prostanoids was mimicked by exogenous PGE(2), which reversed Ang II-induced afferent arteriolar vasoconstriction at concentrations of 0.1 to 10 nmol/L without affecting the efferent arteriole. The PGE(2)-induced vasodilation was potentiated by the phosphodiesterase inhibitor Ro 20-1724 and was mimicked by 11-deoxy-PGE(1) (0.01 to 1 nmol/L). Butaprost, which acts preferentially at EP(2) receptors, was relatively ineffective. Whereas 0.1 to 10 nmol/L PGE(2) elicited vasodilation, higher concentrations (1 to 10 micromol/L) restored Ang II-induced afferent arteriolar vasoconstriction. This response was blocked by pertussis toxin (200 microg/mL) and was mimicked by the EP(1)/EP(3) agonist sulprostone (1 to 300 nmol/L). Reverse transcription-polymerase chain reaction of individually isolated afferent arterioles revealed the presence of message for EP(4) and all 3 EP(3) splice variants (alpha, beta, and gamma) but not EP(1) or EP(2). Our findings thus indicate that PGE(2) elicits both vasodilatory and vasoconstrictor actions on the afferent arteriole. The vasodilation is mediated by EP(4) receptors coupled to cAMP, presumably via G(alphas). The vasoconstriction is mediated by an EP(3) receptor coupled to G(alphai) and appears to reflect a functional antagonism of the EP(4)-induced vasodilation.

Inhibitors of erythropoiesis have been found in the blood of uremic patients but their nature has not been identified. These patients have excess blood levels of parathyroid hormone (PTH) and it is possible that PTH inhibits erythropoiesis. The present study was undertaken to examine the effect of intact PTH molecules and some of its fragments on human peripheral blood and mouse bone marrow burst-forming units-erythroid (BFU-E), on mouse bone marrow erythroid colony-forming unit (CFU-E), and granulocyte macrophage progenitors (CFU-GM), and evaluate the interaction between PTH and erythropoietin (Ep) on human BFU-E. Intact PTH (1-84 bPTH) in concentrations (7.5-30 U/ml;) comparable to those found in blood of uremic patients produced marked and significant (P less than 0.01) inhibition of BFU-E and mouse marrow GFU-GM, but not of mouse marrow CFU-E. Inactivation of 1-84 bPTH abolished its action on erythropoiesis. Increasing the concentration of Ep in the media from 0.67 to 1.9 U/ml overcame the inhibitory effect of 1-84 bPTH on BFU-E. The N-terminal fragment of PTH (1-34 bPTH) and 53-84 hPTH had no effect on BFU-E. The results demonstrate that (a) either the intact PTH molecule or a C-terminal fragment(s) bigger than 53-84 moiety exerts the inhibitory effect on erythropoiesis, and (b) adequate amounts of Ep can overcome this action of PTH. The data provide one possible pathway for the participation of excess PTH in the genesis of the anemia of uremia.

The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant (M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation. Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production of the protease ( approximately 130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type. Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels CodY represses production of an unknown protease and is involved in biofilm formation.

Levels of prostaglandin E(2) (PGE(2)) in human and rodent breast cancers are higher than surrounding normal tissues. PGE(2) exhibits biological activity through binding to membrane receptors, EP(1-4). The present study was designed to investigate the effects of ONO-8711, a newly synthesized selective PGE receptor EP(1) antagonist, on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced breast cancer development. Starting at 7 weeks of age, female Sprague-Dawley (SD) rats were given PhIP (85 mg/kg body weight) by gavage four times weekly for two weeks. Dietary administration of ONO-8711 at 400 or 800 p.p.m. delayed occurrence of breast tumors for 2 or 4 weeks, respectively. At 20 weeks after the last dosing of PhIP, all animals were killed and complete autopsy was made. All breast tumors were diagnosed as invasive ductal adenocarcinomas histopathologically. Administration of ONO-8711 at 800 p.p.m. significantly decreased PhIP-induced breast cancer incidence, multiplicity and volume compared with those of rats fed the control diet (56% versus 79%, P < 0.05, 1.2 versus 2.5, P < 0.05, 0.7 versus 1.4 cm(3), P < 0.01, respectively). Apoptosis was significantly increased in breast cancer cells by feeding of ONO-8711 at 800 p.p.m. of 158% (P < 0.05). EP(1) receptor was detected by reverse transcription-polymerase chain reaction (RT-PCR) in breast cancers, not in normal tissues. These results suggest that EP(1) receptor is associated with breast cancer development and selective PGE receptor EP(1) antagonists may possess chemopreventive effects through the induction of apoptosis without any side effects.

Ethyl pyruvate (EP) was recently identified as a stable lipophilic derivative of pyruvic acid with significant antineoplastic activities. The high mobility group box-BBB (Akt) pathways play a crucial role in tumorigenesis and development of many malignant tumors. We tried to observe the effects of ethyl pyruvate on liver cancer growth and explored its effects in hepatocellular carcinoma model. In this study, three hepatocellular carcinoma cell lines were treated with ethyl pyruvate. An MTT colorimetric assay was used to assess the effects of EP on cell proliferation. Flow cytometry and TUNEL assays were used to analyze apoptosis. Real-time PCR, Western blotting and immunofluorescence demonstrated ethyl pyruvate reduced the HMGBEP could induce apoptosis and slow the growth of liver cancer. Moreover, EP decreased the expression of HMGBBax/Bcl-2 ratio. In conclusion, this study demonstrates that ethyl pyruvate induces apoptosis and cell-cycle arrest in G phase in hepatocellular carcinoma cells, plays a critical role in the treatment of cancer.

Significant growth phase-dependent differences were noted in the transcriptome of the hyperthermophilic bacterium Thermotoga maritima when it was cocultured with the hyperthermophilic archaeon Methanococcus jannaschii. For the mid-log-to-early-stationary-phase transition of a T. maritima monoculture, 24 genes (1.3% of the genome) were differentially expressed twofold or more. In contrast, methanogenic coculture gave rise to 292 genes differentially expressed in T. maritima at this level (15.5% of the genome) for the same growth phase transition. Interspecies H2 transfer resulted in three- to fivefold-higher T. maritima cell densities than in the monoculture, with concomitant formation of exopolysaccharide (EPS)-based cell aggregates. Differential expression of specific sigma factors and genes related to the ppGpp-dependent stringent response suggests involvement in the transition into stationary phase and aggregate formation. Cell aggregation was growth phase dependent, such that it was most prominent during mid-log phase and decayed as cells entered stationary phase. The reduction in cell aggregation was coincidental with down-regulation of genes encoding EPS-forming glycosyltranferases and up-regulation of genes encoding beta-specific glycosyl hydrolases; the latter were presumably involved in hydrolysis of beta-linked EPS to release cells from aggregates. Detachment of aggregates may facilitate colonization of new locations in natural environments where T. maritima coexists with other organisms. Taken together, these results demonstrate that syntrophic interactions can impact the transcriptome of heterotrophs in methanogenic coculture, and this factor should be considered in examining the microbial ecology in anaerobic environments.

Although the liver is the major site of erythropoietin (Ep) production in the fetus, this function is assumed by kidneys in the adult. The mechanisms underlying the liver to kidney switch of Ep formation are not understood. We studied the natural progression of this transition in sheep by measuring Ep production in response to anemia in normal and bilaterally nephrectomized fetal and newborn sheep beginning at about 80 d gestation (normal gestation: 140 d). Removal of both kidneys before induction of anemia did not affect Ep formation up to about 120 d of gestation. A significant reduction (29%, P < 0.02) in Ep synthesis was first noted at about 130 d of gestation (initiation of switch). This level of nephrectomy-induced reduction of Ep formation persisted until about 15 d after birth. Thereafter, bilateral nephrectomy caused further significant decreases (P < 0.05) in Ep production, gradually resulting in near total absence of Ep production at about day 40 postpartum (completion of switch). Chronic administration of testosterone (12 mg/wk) or estradiole benzoate (1.5 mg/d, 5 d/wk) to the fetus/newborn beginning at 85-90 d of gestation enhanced or suppressed erythropoiesis, respectively, but failed to affect the time at which the liver to kidney switch was initiated and/or completed. By contrast, a significant delay (P < 0.001) in the onset, but not completion of the switch occurred in animals that were either thyroidectomized or rendered chronically anemic beginning in the second third of the gestation period. Administration of thyroxin (1.2 mg/d, 5 d/wk) to thyroidectomized fetus/newborns not only prevented the delay in the initiation of the switch, but also accelerated the rate at which the switch was completed. These results demonstrate that in sheep (a) the liver to kidney switch of Ep production is initiated in utero during the last third of the gestation period, but is completed after birth, (b) this transition occurs gradually; the assumption of Ep producing capacity by the kidney is not preceded by an abrupt loss of hepatic Ep formation; and (c) the switch is not affected by changes in sex hormone levels during the prenatal-postnatal growth periods, but is profoundly influenced by alterations in thyroid hormone and oxygen supply-demand levels.

OBJECTIVE

This review will highlight recent studies in the role of prostaglandins in regulating the epithelial response to injury in the gastrointestinal tract.

RESULTS

Prostaglandins, particularly PGE2, regulate intestinal epithelial apoptosis and proliferation in the face of injury. In the dextran sodium sulphate colitis model, PGE2, produced through cyclooxygenase-2, supports epithelial proliferation. Two studies demonstrated that PGE2 is an important mediator of the protective effects of toll-like receptor signaling in the dextran sulphate sodium model. One study suggested that toll-like receptor signaling induced cyclooxygenase-2 expression whereas the other suggested that toll-like receptor signaling induces the repositioning of cyclooxygenase-2 expressing stromal cells. PGE2 is also protective of small intestinal epithelial cells in the radiation injury model. In this model PGE2 decreases radiation-induced apoptosis and increases crypt survival. PGE2 binds to EP receptors; EPbeta-catenin pathway.

CONCLUSIONS

Endogenous PGE2 has pro-proliferative and antiapoptotic effects on epithelial cells in gastrointestinal injury.

The surface of Trypanosoma brucei brucei insect forms is covered by an invariant protein coat consisting of procyclins. There are six or seven procyclin genes that encode unusual proteins with extensive tandem repeat units of glutamic acid (E) and proline (P) (referred to as EP repeats), and two genes that encode proteins with internal pentapeptide (GPEET) repeats. Although the EP forms of procyclins have been isolated and characterized by several laboratories, evidence for GPEET procyclin has largely been confined to the expression of its mRNA. To characterize GPEET procyclin further, we isolated the protein from T. b. brucei strain 427. We found that label from [3H]myristic acid and [3H]ethanolamine was incorporated into GPEET procyclin and we demonstrated the protein's covalent modification with a glycosylphosphatidylinositol anchor. The major form of GPEET procyclin showed an apparent molecular mass of 22-32 kDa, was susceptible to proteolytic treatment and was found to be phosphorylated. Surprisingly, our results show that GPEET procyclin represents the major form of procyclin in T. b. brucei 427 culture forms and that the ratio of EP to GPEET procyclin can vary considerably between different cell lines.

Many different receptors can stimulate cAMP synthesis in the heart, but not all elicit the same functional responses. For example, it has been recognized for some time that prostaglandins such as PGE1 increase cAMP production and activate PKA, but they do not elicit responses like those produced by beta-adrenergic receptor (betaAR) agonists such as isoproterenol (isoprenaline), even though both stimulate the same signalling pathway. In the present study, we confirm that isoproterenol, but not PGE1, is able to produce cAMP-dependent stimulation of the L-type Ca(2+) current in guinea pig ventricular myocytes. This is despite finding evidence that these cells express EP(4) prostaglandin receptors, which are known to activate G(s)-dependent signalling pathways. Using fluorescence resonance energy transfer-based biosensors that are either freely diffusible or bound to A kinase anchoring proteins, we demonstrate that the difference is due to the ability of isoproterenol to stimulate cAMP production in cytosolic and caveolar compartments of intact cardiac myocytes, while PGE1 only stimulates cAMP production in the cytosolic compartment. Unlike other receptor-mediated responses, compartmentation of PGE1 responses was not due to concurrent activation of a G(i)-dependent signalling pathway or phosphodiesterase activity. Instead, compartmentation of the PGE1 response in cardiac myocytes appears to be due to transient stimulation of cAMP in a microdomain that can communicate directly with the bulk cytosolic compartment but not the caveolar compartment associated with betaAR regulation of L-type Ca(2+) channel function.

The etiology of chronic pelvic pain syndrome (CPPS)/chronic prostatitis category III remains unknown. Whereas a subset of men respond to antimicrobial therapy, gram positive bacteria isolated from expressed prostatic secretions (EPS) are often considered to be commensal rather than pathogenic. We wished to study oxidative stress as a marker of tissue injury and response in EPS of men with CPPS to determine whether infection with gram positive bacteria is associated with increased oxidative stress. A total of 300 EPS specimens from 100 men with CPPS were collected for microscopy, culture, and biochemical and molecular assays. Oxidant injury was measured by 8-isoprostane F2alpha (IsoP) levels and total antioxidant capacity as Trolox equivalents. Total RNA from EPS was used for gene expression of heme oxygenase-1 (HO-1) and granzyme B. The only bacteria found in EPS were gram positive. For our analysis, these men were classified as having chronic bacterial prostatitis (category II). IsoP levels (pg/mL) were highest in men with category II prostatitis (7315 +/- 1428) followed by nonbacterial prostatitis (category IIIa, 2043 +/- 561), prostatodynia (category IIIb, 319 +/- 81), and asymptomatic controls (298 +/- 99). IsoP levels decreased significantly after successful treatment with antibiotics or an antioxidant supplement (Prosta-Q). Antioxidant capacity was detected in 11 out of 18, 4 out of 16, and 1 out of 16 men tested with category II, IIIa, and IIIb prostatitis, respectively. No correlation was observed between IsoP levels and the number of white blood cells in EPS. HO-1 and granzyme B expression was highest in men with category II prostatitis than in men with either category III prostatitis or asymptomatic controls. On the basis of elevated oxidative stress, clinical response to antibiotics, and post-treatment reduction in oxidative stress, we conclude that gram positive bacteria in some men with CPPS may be pathogens. It is speculated that oxidative stress may be a key pathway in some men with CPPS that can be targeted with antioxidant therapy.

In an effort to understand the reaction mechanism of a B2 metallo-beta-lactamase, steady-state and pre-steady-state kinetic and rapid freeze quench electron paramagnetic resonance (EPR) studies were conducted on ImiS and its reaction with imipenem and meropenem. pH dependence studies revealed no inflection points in the pH range of 5.0-8.5, while proton inventories demonstrated at least 1 rate-limiting proton transfer. Site-directed mutagenesis studies revealed that Lys224 plays a catalytic role in ImiS, while the side chain of Asn233 does not play a role in binding or catalysis. Stopped-flow fluorescence studies on ImiS, which monitor changes in tryptophan fluorescence on the enzyme, and its reaction with imipenem and meropenem revealed biphasic fluorescence time courses with a rate of fluorescence loss of 160 s(-)(1) and a slower rate of fluorescence regain of 98 s(-)(1). Stopped-flow UV-vis studies, which monitor the concentration of substrate, revealed a rapid loss in absorbance during catalysis with a rate of 97 s(-)(1). These results suggest that the rate-limiting step in the reaction catalyzed by ImiS is C-N bond cleavage. Rapid freeze quench EPR studies on Co(II)-substituted ImiS demonstrated the appearance of a rhombic signal after 10 ms that is assigned to a reaction intermediate that has a five-coordinate metal center. A distinct product (EP) complex was also observed and began to appear in 18-19 ms. When these results are taken together, they allow for a reaction mechanism to be offered for the B2 metallo-beta-lactamases and demonstrate that the mono- and dinuclear Zn(II)-containing enzymes share a common rate-limiting step, which is C-N bond cleavage.

OBJECTIVE

This study determined the ultimate tensile strength (UTS) of enamel (E), dentin (D) and dentin-enamel junction (DEJ) using the microtensile technique. It was hypothesized that the UTS of dental structures varies according to location and nature.

METHODS

Intact occlusal enamel surfaces from extracted human third molars were etched with 37% phosphoric acid and bonded with a one-bottle adhesive system. The bonded occlusal surfaces received a resin composite build-up and teeth were serially, vertically sectioned into several 0.7 mm thick slabs. Each slab was then trimmed to a dumbbell-shaped specimen with irrigated diamond burs to reduce the cross-sectional area to approximately 0.5 mm2 at E, D or DEJ. E was tested according to its prismatic orientation (parallel, EP; and transversally, ET) and D as function of depth (superficial, DS; middle, DM and deep, DD). Specimens were tested in tension in an Instron testing machine at 0.5 mm/min. Results were analyzed by one-way ANOVA and Duncan's Multiple Range test.

RESULTS

UTS mean values (N = 20) were, MPa (SD): DEJ, 46.9 (13.7)b; EP, 42.1 (11.9)b; ET, 11.5 (4.7)d; DS, 61.6 (16.2)a; DM, 48.7 (16.6)b and DD, 33.9 (7.9)c. Enamel stressed transversally to its prismatic orientation was significantly weaker (p < 0.05) Dentin depth significantly affected its UTS (p < 0.05) DEJ presented UTS that was similar to EP and DM (p>> 0.05)

CONCLUSIONS

The UTS of dental structures varies according to its nature and location.

OBJECTIVE

Females develop cardiovascular diseases and abdominal aortic aneurysms later in life than males. In this study we investigated the diameter and compliance of the distal abdominal aorta in healthy females of varying ages. The results were compared with those obtained previously from healthy males in order to assess potential sex-related differences in the aging process of the abdominal aorta.

METHODS

An ultrasound phase-locked echo-tracking system was used to determine differences in diameter and pulsatile diameter change of the distal abdominal aorta in 69 Caucasian females from 4 to 74 years. Pressure strain elastic modulus (Ep) and stiffness (beta) were calculated from diameter, pulsatile diameter change and blood pressure obtained by the auscultatory method. Compliance was defined as the inverse of Ep and beta. The pressure dependence of Ep and beta was evaluated in 10 females with intraarterial blood pressure measurement at rest and during isometric exercise.

RESULTS

The diameter of the distal abdominal aorta increased not only in the period between the ages of 5 and 25 years, but also by about 24% between 25 and 70 years (p < 0.0001). From about the age of 25 years the diameter was smaller in females than in males (p < 0.01). Ep and beta increased nearly linearly with advancing age in females from 0.18 x 10(5) to 1.17 x 10(5) N/m2 (Ep) and from 1.85 to 8.51 (beta). In males the increase in Ep and beta was greater and exponential in nature (p < 0.001). Ep but not beta increased significantly during blood pressure increase (p < 0.05). Stiffness (beta) may therefore be a more useful index of arterial compliance than Ep.

CONCLUSIONS

This investigation demonstrates age and sex-related differences in diameter and compliance in the normal human abdominal aorta and implies that degenerative changes appear later in females than in males.

The immunomodulatory effects of probiotics were assessed following exposure of normal peripheral blood mononuclear cells (PBMC), cord blood cells and the spleen-derived monocyte/macrophage cell line CRL-9850 to Lactobacillus acidophilus LAVRI-A1, Lb. rhamnosus GG, exopolysaccharides (EPS)-producing Streptococcus thermophilus St1275, Bifidobacteriun longum BL536, B. lactis BBMC following bacterial stimulation was measured, using live, heat-killed or mock gastrointestinal tract (GIT)-exposed bacteria, and results show that (i) all bacterial strains investigated induced significant secretion of pro- and anti-inflammatory cytokines from PBMC-derived monocytes/macrophages; and (ii) cytokine levels increased relative to the expansion of bacterial cell numbers over time for cells exposed to live cultures. Bifidobacteria and S. thermophilus stimulated significant concentrations of transforming growth factor (TGF)-β, an interleukin necessary for the differentiation of regulatory T cells (T(reg) )/T helper type 17 (Th17) cells and, as such, the study further examined the induction of Th17 and T(reg) cells after PBMC exposure to selected bacteria for 96 h. Data show a significant increase in the numbers of both cell types in the exposed populations, measured by cell surface marker expression and by cytokine production. Probiotics have been shown to induce cytokines from a range of immune cells following ingestion of these organisms. These studies suggest that probiotics' interaction with immune-competent cells produces a cytokine milieu, exerting immunomodulatory effects on local effector cells, as well as potently inducing differentiation of Th17 and T(reg) cells.

Fatal cases of acute shock complicating Clostridium sordellii endometritis following medical abortion with mifepristone (also known as RU-486) used with misoprostol were reported. The pathogenesis of this unexpected complication remains enigmatic. Misoprostol is a pharmacomimetic of PGE(2), an endogenous suppressor of innate immunity. Clinical C. sordellii infections were associated with intravaginal misoprostol administration, suggesting that high misoprostol concentrations within the uterus impair immune responses against C. sordellii. We modeled C. sordellii endometritis in rats to test this hypothesis. The intrauterine but not the intragastric delivery of misoprostol significantly worsened mortality from C. sordellii uterine infection, and impaired bacterial clearance in vivo. Misoprostol also reduced TNF-alpha production within the uterus during infection. The intrauterine injection of misoprostol did not enhance mortality from infection by the vaginal commensal bacterium Lactobacillus crispatus. In vitro, misoprostol suppressed macrophage TNF-alpha and chemokine generation following C. sordellii or peptidoglycan challenge, impaired leukocyte phagocytosis of C. sordellii, and inhibited uterine epithelial cell human <em>beta</em>-defensin expression. These immunosuppressive effects of misoprostol, which were not shared by mifepristone, correlated with the activation of the G(s) protein-coupled E prostanoid (<em>EP</em>) receptors <em>EP</em>2 and <em>EP</em>4 (macrophages) or <em>EP</em>4 alone (uterine epithelial cells). Our data provide a novel explanation for postabortion sepsis leading to death and also suggest that PGE(2), in which production is exaggerated within the reproductive tract during pregnancy, might be an important causal determinant in the pathogenesis of more common infections of the gravid uterus.