ING-1(heMAb), a Human Engineered monoclonal antibody to epithelial cell adhesion molecule (Ep-CAM), was evaluated for its in vitro and in vivo activity. The dissociation constant of ING-1(heMAb) for binding to Ep-CAM on HT-29 human colon tumor cells was 2 to 5 nM, similar to chimeric ING-1. In antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays, ING-1(heMAb) caused a concentration-dependent lysis of BT-20 breast, MCF-7 breast, HT-29 colon, and CACO-2 colon tumor cells, with maximum cytolysis at approximately 1 microg/ml. After an intravenous injection in rats, plasma ING-1(heMAb) levels declined with an alpha half-life of 8 to 11 hours, and a beta half-life of 20 days, typical of an IgG in a species without the target for ING-1. In nude mice with human HT-29 colon tumors, plasma ING-1(heMAb) levels declined more rapidly than in non-tumor-bearing mice, suggesting an enhanced clearance via the tumor-associated human Ep-CAM. In nude mice, intravenous treatments with ING-1(heMAb) twice a week for 3 weeks significantly suppressed the growth of human HT-29 colon and PC-3 prostate tumors in a dose-dependent manner, with 1.0 mg/kg providing the greatest benefit. These results indicate that Human Engineered ING-1(heMAb) is a high-affinity antibody with potent in vitro activity that targets and suppresses the growth of human tumors in vivo.

To study the role of opioid peptides in human obesity, plasma beta-endorphin (beta EP), beta-lipotropin (beta LPH), and cortisol resting values, circadian rhythms, and responses to hypoglycemia were studied in 6 prepubertal and 6 pubertal obese adolescents (at least 40% above ideal body weight) and in 10 normal subjects matched for age, sex, and pubertal development. Baseline plasma beta LPH and beta EP concentrations in both obese children and adolescents were twice as high as those in normal controls, while cortisol levels were not different. Cortisol, beta EP, and beta LPH levels had a clear circadian rhythmicity in all subjects, with the exception of obese pubertal boys whose plasma beta EP concentrations were constant throughout the day. After insulin administration, the fall in blood sugar was similar in all groups. Plasma cortisol and beta EP responses were similar in both obese and normal prepubertal subjects. In obese pubertal adolescents, beta EP did not increase significantly after hypoglycemia, although it did increase in normal weight pubertal subjects. In normal prepubertal subjects, the circadian rhythms of beta EP and beta LPH secretion and release induced by hypoglycemia suggest the presence of a well developed neuroendocrine control of proopiomelanocortin-related peptide secretion. In prepubertal obese children, the increased plasma beta EP and beta LPH levels with the maintenance of their circadian rhythm and responsivity to hypoglycemia suggest overactivity of anterior pituitary secretion. In obese adolescents, in spite of the normal rhythm of beta LPH and cortisol, beta EP levels did not change throughout the day, thus suggesting beta EP secretion from nonpituitary sources in these subjects. The present study indicates a possible direct role for hyperendorphinemia in the induction of overeating in obese children and adolescents.

BACKGROUND

Electroporation (EP) is a widely used non-viral gene transfer method. We have attempted to develop an exact protocol to maximize DNA expression while minimizing tissue damage following EP of skeletal muscle in vivo. Specifically, we investigated the effects of varying injection techniques, electrode surface geometry, and plasmid mediums.

RESULTS

We found that as the amount of damage increased in skeletal muscle in response to EP, the level of beta-galactosidase (beta-gal) expression drastically decreased and that there was no evidence of beta-gal expression in damaged fibers. Two specific types of electrodes yielded the greatest amount of expression. We also discovered that DNA uptake in skeletal muscle following intra-arterial injection of DNA was significantly enhanced by EP. Finally, we found that DMSO and LipoFECTAMINE, common enhancers of DNA electroporation in vitro, had no positive effect on DNA electroporation in vivo.

CONCLUSIONS

When injecting DNA intramuscularly, a flat plate electrode without any plasmid enhancers is the best method to achieve high levels of gene expression.

The potential of Lactobacillus rhamnosus R for producing exopolysaccharide (EPS) when grown on basal minimum medium supplemented with glucose or lactose was investigated. EPS production by L. rhamnosus R is partially growth associated and about 500 mg of EPS per liter was synthesized with both sugars. The product yield coefficient (Y(EPS/S)) was 3.15 (0.0315 g of EPS [g of lactose](-1)) and 2.88 (0.0288 g of EPS [g of glucose](-1)). It was clearly shown that the amount of EPS produced declined upon prolonged fermentation. Degradation of EPS in fermentation processes was also assessed by measuring its molecular weights and viscosities. As these reductions might have a negative effect on the yield and viscosifying properties of EPS, it was essential to examine possible causes related to this breakdown. The decrease in viscosities and molecular weights of EPS withdrawn at different cultivation times permitted us to suspect the presence of a depolymerizing enzyme in the fermentation medium. Our study on enzymatic production profiles showed a large spectrum of glycohydrolases (alpha-D-glucosidase, beta-D-glucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucuronidase, and some traces of alpha-L-rhamnosidase). These enzymes were localized, two of them (alpha-D-glucosidase and beta-D-glucuronidase) were partially purified and characterized. When incubated with EPS, these enzymes were capable of lowering the viscosity of the polymer as well as liberating some reducing sugars. Upon prolonged incubation (27 h), the loss of viscosity was increased up to 33%.

The effects of adenosine (ADE) and ADE agonists on erythropoietin (Ep) production were determined using percent (%) 59Fe incorporation in red cells of exhypoxic polycythemic mice. The hemisulfate salt of ADE produced a significant increase in % 59Fe incorporation in response to hypoxia in concentrations of 400 to 1600 nmol/kg/day (i.v.). 5'-N-ethyl-carboxamideadenosine (NECA), a selective A2 receptor agonist, increased radioiron incorporation in a dose-dependent manner (10-100 nmol/kg/day, i.v.). In contrast, N6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, did not affect radioiron incorporation in concentrations up to 1600 nmol/kg/day (i.v.). Albuterol, a beta 2-adrenergic agonist, enhanced % 59Fe incorporation in polycythemic mice and low doses of CHA (50 and 100 nmol/kg/day), which were not effective alone on % 59Fe incorporation in polycythemic mice exposed to hypoxia, inhibited the enhancement in radioiron induced by albuterol (25 and 100 micrograms/kg/day, i.p.) plus hypoxia. Theophylline (20 and 80 mg/kg/day, i.p.), a well-known antagonist of ADE receptors, blocked the ADE and NECA enhancement in radioiron incorporation at a dose of theophylline alone which produced only a slight enhancement of % 59Fe incorporation. These results suggest that ADE may both inhibit through A1 receptor activation and increase via A2 receptor stimulation the production of Ep.

The surface polysaccharides of Rhizobium leguminosarum 128C53 sm(r)rif(r) (parent) and its exo(-1) mutant were isolated and characterized. The parent carries out normal symbiosis with its host, pea, while the exo(-1) mutant does not nodulate the pea. The following observations were made. (a) The parent produces lipopolysaccharide (LPS), typical acidic extracellular polysaccharide (EPS), and three additional polysaccharides, PS1, PS2, and PS3. The PS1 and PS2 fractions are likely to be the capsular polysaccharide (CPS) and are identical in composition to the EPS. The PS3 fraction is a small-molecular-weight glucan. (b) The exo(-1) mutant produces LPS, EPS, and a PS3 fraction, but does not produce significant amounts of either PS1 or PS2. The LPS from the exo(-1) mutant appears to be identical to the parental LPS. Analysis of the EPS from exo(-1) shows that it consists of two polysaccharides. One polysaccharide is identical to the LPS and comprises 70% of the exo(-1) EPS. The second polysaccharide is identical to the exo(-1) PS3 and comprises 30% of the exo(-1) EPS. This result shows that the exo(-1) mutant does not produce any of the typical acidic parental EPS and that the major polysaccharide released into the media by the exo(-1) mutant is intact LPS. The exo(-1) mutant PS3 fraction was found to contain two polysaccharides, PS3-1 and PS3-2. The PS3-2 polysaccharide is identical to the parental PS3 described above. The PS3-1 polysaccharide has a composition similar to the polysaccharide portion of the LPS. This result suggests that the exo(-1) mutant produces LPS polysaccharide fragments. These LPS polysaccharide fragments are not produced by the parent strain.

The purpose of the present study was to investigate the effect of time of beta-endorphin (beta-EP) administration on lordosis in ovariectomized female rats injected subcutaneously (sc) with estradiol benzoate (EB) and progesterone (Prog). Intracerebroventricular (icv) injections of beta-EP and naloxone (NLX), an opioid receptor antagonist, were administered at the various stages of sc steroid hormone priming. Facilitation of lordosis induced by 10 microg beta-EP was observed exclusively within the initial 6 h of estrogen action, after which inhibition of lordosis occurred. At 12 h after EB priming, at the time of sc Prog treatment (or 43 h after EB priming), icv injection of 10 microg beta-EP significantly inhibited lordosis. Lordosis was significantly facilitated by icv injections of 1 and 10 microg beta-EP at the time of sc EB priming, but not by 0.1 microg beta-EP. A dose-response relationship was identified for lordosis in experimental animals receiving icv injection of beta-EP. Lordosis was inhibited by icv injections of 1 and 10 microg beta-EP at 1 h before the test (or 47 h after EB priming). Lordosis was significantly inhibited by icv injection of NLX at all stages. From the present results, it seems that two different mechanisms are involved in endorphinergic modulation of rats' sexual receptivity: (a) the endorphinergic system at the initial stages of estrogen action facilitates the estrogen activation of lordosis; (b) the endorphinergic system at the final stages of steroid action inhibits lordosis. Moreover, there exists a critical time between 6 and 12 h after estrogen priming for endorphinergic mediation to modulate estrogen action.

In humans, proopiomelanocortin (POMC) and the peptides derived from it have been individually identified in plasma under differing conditions. However, direct quantitative comparison has proved difficult because of the limitations of RIAs. Using a panel of monoclonal antibodies recognizing different regions of POMC, we have developed specific two-site immunoradiometric assays (IRMAs) for the ACTH precursors (POMC and pro-ACTH), ACTH, beta-lipotropin (beta LPH), beta-endorphin (beta EP), and the N-terminal POMC fragment (N-POC). We have quantified these peptides directly in plasma from normal subjects under basal conditions and in response to different regulatory factors. Basal levels of ACTH precursors, 5-40 pmol/L, were greater than or equal to ACTH, less than 0.9-11.3 pmol/L; N-POC, 5.6-16.8 pmol/L; beta LPH, 2.5-6.7 pmol/L; and beta EP less than or equal to 1.7 pmol/L. ACTH, N-POC, beta LPH, and beta EP levels increased in parallel in response to metyrapone (n = 8) and decreased in response to dexamethasone (n = 8), whereas ACTH precursor concentrations did not respond. After human CRH administration, peripheral concentrations of ACTH, N-POC, and beta LPH showed similar increments (median increment, 163%, 145%, and 172%, respectively; n = 6). POMC peptide responses to human CRH were also assessed in inferior petrosal sinuses draining the pituitary in 20 patients with pituitary-dependent Cushing's disease. In these patients, the increment in ACTH after CRH exceeded that in ACTH precursors by 4-fold (median, 459% and 96%). An increase in the ratios of ACTH/N-POC and ACTH/beta LPH was also apparent after CRH stimulation. The increment in beta EP after CRH always exceeded the increments in POMC and beta LPH. In summary, these data suggest that significant concentrations of ACTH precursors are present in the circulation of normal subjects, that ACTH precursors are not regulated in the same way as the processed POMC peptides, and that ACTH and beta EP are preferentially released from the pituitary in response to CRH.

The central nervous system (CNS) is one of the main target tissues for sex steroid hormones, which act both through genomic mechanisms, modulating synthesis, release, and metabolism of many neuropeptides and neurotransmitters, and through nongenomic mechanisms, influencing electrical excitability, synaptic function, and morphological features. The identification of the brain as a de novo source of neurosteroids modulating cerebral function, suggests that the modifications in mood and cognitive performances occurring in postmenopausal women could also be related to a modification in the levels of neurosteroids, particularly allopregnanolone and DHEA, GABA-A agonist, and antagonist, respectively. The selective estrogen receptor modulators (SERMs) are compounds that activate the estrogen receptors with different estrogenic and antiestrogenic tissue-specific effects. In addition to the effects of the classic steroid hormones on the CNS, the study of selective estrogen receptor modulators impact on the neuroendocrine system has recently provided encouraging results, indicating that raloxifene analog LY 117018 and the new generation SERM EM-652 have an estrogen-like action on beta-endorphin and on allopregnanolone in ovariectomized rats, while they exert an anti-estrogenic effect in fertile rats and in ovariectomized rats treated with estrogens. In addition, raloxifene administration in postmenopausal women plays an estrogen-like effect on circulating beta-EP and allopregnanolone levels, and it restores the response of beta-EP and allopregnanolone to neuroendocrine tests. In conclusion, the positive effects of HRT on mood and cognition in postmenopausal women occur via the modulation of neuroendocrine pathways and probably also of neurosteroidogenesis. The effects of raloxifene on mood and cognition encourage the efforts in the research of an ideal estrogen replacement therapy, showing all the positive effects of estrogens and fewer side effects.

The aim of the present study was to evaluate the relationship between central and peripheral concentrations of proopiocortin-related peptides in different periods of life. One hundred and eighty-nine plasma samples from normal subjects (18-87 years) obtained in basal conditions, and 20 cerebrospinal fluid (CSF) samples obtained by lumbar puncture from healthy volunteers (18-75 years) were studied. beta Lipotropin (beta LPH), beta endorphin (beta EP) and ACTH were measured by specific RIA after silicic acid plasma extraction and gel chromatography (beta LPH and beta EP). No sex differences were found in the patterns of the three peptides either in the plasma or in CSF. In the plasma samples, both beta LPH and beta EP concentrations showed a pattern throughout life which was expressed by a paraboloid function with the lowest values found in young and old subjects and with peaks at 51.3 and 48.2 years, respectively. On the contrary, ACTH values failed to be represented by a significant linear or curvilinear regression and presented only a slight decrease in subjects over 75 years of age. CSF levels of beta LPH were significantly lower in 45-76 year old subjects (18.8 +/- 12.6 fmol/ml, M +/- SD) than in 18-44 year old subjects (34.5 +/- 15.8; p less than 0.05), as were those of beta EP (elderly: 41.2 +/- 19.7; young: 94.2 +/- 36.7; p less than 0.05), which showed a significantly linear inverse correlation with age (r = 0.6062, p less than 0.01). These CSF samples did not show any ACTH variations connected with age.(ABSTRACT TRUNCATED AT 250 WORDS)

The E-series prostaglandins (PGEs) are complex lipid regulators of B lymphocyte function. They inhibit the growth of certain B lymphoma lines. We report that heterogeneity with respect to PGE-induced growth inhibition correlates with the maturation state of the B cell lines. Specifically, the pre-B cell line 70Z/3 and the immature lymphoma CH31 are extremely sensitive to PGE2. To a lesser degree, other immature lymphomas (CH33, ECH408.1 and WEHI-231) are sensitive to PGE2. More mature lymphomas (BAL-17, CH12 and CH27) and fully differentiated myelomas (J558 and MOPC-315) are insensitive to PGE2. It is unknown what subtype of PGE receptor(s) (EPs) are expressed by B lymphocytes. It is also unknown if modulation of EP receptor expression could account for the differences in the sensitivity of these B cell lines to PGE2. To investigate these issues, reverse transcriptase polymerase chain reaction, Northern blot and DNA sequencing analyses were employed to obtain a definitive EP receptor subtype profile for these B cell lines, and for normal splenic B lymphocytes. Both normal and transformed B lymphocytes express mRNA encoding EPEPEPB lineage cells do not express EPEPB cell lines are clonal, indicating that EPEPEPEPEPEPB cell lines despite their differing susceptibilities to PGE-induced growth inhibition. Conversely, the polyclonal activator LPS selectively upregulates EPB cell line CH12, but not in the LPS-sensitive pre-B cell line, 70Z/3. The activator LPS does not affect EPEPEPEPEPEPEPEPB lineage cells.

BACKGROUND

We have previously shown that alcohol suppresses the natural killer (NK) cell activity of splenic lymphocytes partly by reducing the secretion of opioid peptide beta-endorphin (beta-EP) and its positive influence on NK-cell cytolytic activity in rats. The inhibition of NK-cell cytolytic activity was also associated with a reduced number of NK cells after chronic ethanol administration. Hence, the possibility arises that chronic ethanol may alter NK cell proliferation, survival, or both. In this study, we investigated whether ethanol treatment for 1 to 4 weeks reduces the proliferation of other lymphocyte subsets and whether beta-EP regulates ethanol's effect on lymphocyte proliferation.

METHODS

Male rats were ad libitum-fed rat chow, pair-fed with isocaloric liquid diet, or fed with ethanol-containing liquid diet for 1, 2, 3, or 4 weeks. Groups of these rats were infused with beta-EP with or without the delta-receptor antagonist naltrindol into the paraventricular nucleus of the hypothalamus. Splenocytes were isolated and used for flow cytometric analysis of the changes in the number of various lymphocyte subsets. Lymphocyte proliferation was determined by mitogen stimulation assays.

RESULTS

Ethanol consumption resulted in a reduction of the number of CD161+ NK cells, CD3+ T lymphocytes, CD4+ T-helper cells, and CD8a+ cytotoxic T cells in a time-dependent fashion. Alcohol consumption also suppressed the proliferative response of lymphocyte subsets to concanavalin A, phytohemagglutinin, and lipopolysaccharide. Beta-EP promoted the lymphocytes' proliferative response to mitogens, whereas naltrindol blocked the effects of the opioid. Chronic alcohol consumption reduced the proliferative response of lymphocytes to beta-EP.

CONCLUSIONS

These results suggest that chronic alcohol administration reduces immune function partly by decreasing the opioid-regulated mitogen-stimulated proliferation of lymphocyte subsets.

Variations in the essential oil composition of Achillea millefolium L. growing in Estonia and in other European countries, were determined. The oils were obtained in yields of 0.9-9.5 mL kg-1. A total of 102 components were identified. The quantitatively most important components of yarrow were sabinene, beta-pinene, 1,8-cineole, artemisia ketone, linalool, alpha-thujone, beta-thujone, camphor, borneol, fenchyl acetate, bornyl acetate, (E)-beta-caryophyllene, germacrene D, caryophyllene oxide, beta-bisabolol, delta-cadinol, chamazulene etc. Samples from Estonia contained high amounts of monoterpenes and chamazulene. High amounts of monoterpenes and chamazulene were also found in samples from Hungary, Greek, Moldavia, Latvia, Lithuania and Germany. The oils from France, Belgium, Russia, Armenia, Spain and Italy were rich in oxygenated monoterpenes and contained a little amount of chamazulene. The drugs from Greece, Estonia, Moldavia and Scotland were rich in sesquiterpenes. The Millefolii herba grown in Estonia conforms to the European Pharmacopoeia (EP) standards in the aspect of the essential oil contents.

Pulse-induced permeabilization of cellular membranes, generally referred to as electroporation (EP), has been used for years as a tool to increase macromolecule uptake in tissues, including nucleic acids, for gene therapeutic applications, and this technique has been shown to result in improved immunogenicity. In this study, we assessed the utility of EP as a tool to improve the efficacy of HB-110, a novel therapeutic DNA vaccine against chronic hepatitis B, now in phase 1 of clinical study in South Korea. The potency of HB-110 in mice was shown to be improved by EP. The rapid onset of antigen expression and higher magnitude of humoral and cellular responses in electric pulse-treated mice revealed that EP may enable a substantial reduction in the dosage of DNA vaccine required to elicit a response similar in magnitude to that achievable via conventional administration. This study also showed that EP-based vaccination at 4-week-intervals elicited a cellular immune response which was about two-fold higher than the response elicited by conventional vaccination at 2-week intervals. These results may provide a rationale to reduce the clinical dose and increase the interval between the doses in the multidose vaccination schedule. Electric pulsing also elicited a more balanced immune response against four antigens expressed by HB-110: S, preS, Core, and Pol.

The reconstruction of epicardial potentials (EPs) from body surface potentials (BSPs) can be characterized as an ill-posed inverse problem which generally requires a regularized numerical solution. Two kinds of errors/noise: geometric errors and measurement errors exist in the ECG inverse problem and make the solution of such problem more difficulty. In particular, geometric errors will directly affect the calculation of transfer matrix A in the linear system equation AX = B. In this paper, we have applied the truncated total least squares (TTLS) method to reconstruct EPs from BSPs. This method accounts for the noise/errors on both sides of the system equation and treats geometric errors in a new fashion. The algorithm is tested using a realistically shaped heart-lung-torso model with inhomogeneous conductivities. The h-adaptive boundary element method [h-BEM, a BEM mesh adaptation scheme which starts from preset meshes and then refines (adds/removes) grid with fixed order of interpolation function and prescribed numerical accuracy] is used for the forward modeling and the TTLS is applied for inverse solutions and its performance is also compared with conventional regularization approaches such as Tikhonov and truncated single value decomposition (TSVD) with zeroth-, first-, and second-order. The simulation results demonstrate that TTLS can obtain similar results in the situation of measurement noise only but performs better than Tikhonov and TSVD methods where geometric errors are involved, and that the zeroth-order regularization is the optimal choice for the ECG inverse problem. This investigation suggests that TTLS is able to robustly reconstruct EPs from BSPs and is a promising alternative method for the solution of ECG inverse problems.

The Italian Society of Hematology (SIE) and two affiliate societies (SIES and GITMO) commissioned a project to develop clinical practice guidelines for the treatment of nodal diffuse large B-cell non Hodgkin lymphomas (DLBCL). Key questions clinically relevant to the management of patients with nodal DLBCL were formulated by an Advisory Committee (AC) and approved by an Expert Panel (EP) composed of eight senior hematologists. After a comprehensive and systematic literature review, the EP formulated therapy recommendations and graded them according to the supporting evidence. An explicit approach to consensus methodologies was used for evidence interpretation and for producing recommendations in the absence of strong evidence. The EP formulated recommendations on which first-line therapy to choose in patients with nodal DLBCL. Patients of all ages, with stage I-II disease and no adverse prognostic factors should receive abbreviated chemotherapy with an anthracycline-containing regimen plus involved field radiotherapy (35-40 Gy). Patients with stage I-II disease and at least one adverse prognostic factor, or with stage III-IV disease, should receive frontline chemoimmunotherapy with CHOP, CHOP-like or third-generation chemotherapy plus rituximab. Recommendations on stem cell transplantation and on which therapy to adopt for refractory or relapsed patients were also formulated.

Migration of vascular smooth cells from the media to the intima essentially contributes to neointima formation after percutaneous transluminal angioplasty and stent implantation. The stable prostacyclin mimetic iloprost has been shown to inhibit neointima formation in experimental restenosis, but it is currently unknown whether this may be caused by an antimigratory effect. Hence, the present study analyses (i) the influence of G(s)-coupled prostacyclin (IP) receptors on cell migration and (ii) verifies whether EP(3) receptors with opposite (i.e., G(i)) coupling may conversely stimulate cell migration. In a modified Boyden chamber model, it was shown that iloprost dose-dependently inhibits the migration of primary human arterial smooth muscle cells, which constitutively express the IP receptor. On the other hand, human arterial smooth muscle cell migration was stimulated by the EP(3) receptor agonist M&B 28.767. To independently study the effects of these receptors, IP or EP(3) receptors were stably overexpressed in chinese hamster ovary cells (CHO-IP and CHO-EP(3)). Chemotaxis of CHO cells transfected with G(s)-coupled IP receptors was concentration-dependently inhibited by iloprost (2-100 nM), while there was no effect of iloprost on mock-transfected CHO. By contrast, CHO-cells that overexpressed EP(3) receptors showed a significant, concentration dependent (1-100 nM) increase of cell migration in presence of the selective EP(3) agonist M&B 28.767. It is concluded that the prostacyclin mimetic iloprost inhibits vascular cell migration, which probably depends on a G(s)-mediated increase of intracellular cAMP. EP(3) receptors conversely stimulate CHO migration.

Wild-type (WT) Rat-1 fibroblasts express undetectable quantities of the prostaglandin E(2) (PGE(2)) EPEPEPEPbeta-stimulated increases in CTGF mRNA. A similar pattern of CTGF expression in response to PGE(2) and forskolin is observed in neonatal rat primary smooth muscle cell cultures. When WT Rat-1 cells are stably transfected with the EPbeta-stimulated increase in CTGF mRNA. PGE(2) does not have this effect on cells expressing the EPEPEPEPbeta-stimulated CTGF production and suggest a role for PGE(2) in increasing CTGF mRNA levels in the absence of the EPEP transfectants nor the WT Rat-1 cells responded to PGE(2) with inositol phosphate production.

The prostaglandin E-prostanoid (<em>EP</em>)3 receptor signals primarily through the inhibitory G protein Gi, thereby decreasing intracellular cAMP levels. To study the signal transduction properties of the rabbit <em>EP</em>3 receptor, five splice variants were expressed in HEK293tsA201 cells: 72A, 74A, 77A, 80A and the novel splice variant NT, which lacks the C-terminal sequence. The ability of the <em>EP</em>3 receptor splice variants to modulate expression of a <em>beta</em>-galactosidase reporter gene under the control of a promoter containing cAMP response elements (CRE) was assessed. Each splice variant induced sulprostone-mediated increase in <em>beta</em>-galactosidase enzymatic activity with EC50 ranging from 0.8 nM for the NT splice variant to 3.1 nM for the 77A splice variant. Substitution of either Asp338 with Ala, or Arg329 with Ala or Glu in the 77A splice variant resulted in a loss of receptor-evoked increases in <em>beta</em>-galactosidase activity, whereas substitution of Lys300 with alanine had no effect on signal transduction. These phenotypes correlate with the inhibition of cAMP generation by direct cAMP measurement. Signal transduction was insensitive to pretreatment of cells with pertussis toxin, suggesting that a nonGi/Go pathway is activated by the <em>EP</em>3 receptor. Direct measurement of second messenger levels confirmed that there was no increase in cAMP levels mediated by the 77A splice variant, however, there was a modest increase in intracellular Ca2+. Partial blockade of the reporter activity with kinase inhibitors demonstrates that CRE activation is mediated in part by a Ca2+-dependent kinase pathway. These data suggest that the <em>EP</em>3 receptor signals through a novel cAMP response element binding protein/CRE pathway.

The binding of tritiated beta-endorphin (3H-beta-EP) to brain homogenates is described. This has been difficult to achieve due to the lack of availability of 3H-beta-EP and to technical difficulties associated with high non-specific binding of beta-EP. We now report that 3H-beta-EP binding is saturable, stereospecific, has high affinity and is inhibited by sodium. Its dissociation rate is ten-fold longer than that of naloxone. Its regional distribution exhibits interesting differences from naloxone and enkephalin binding. ACTH1-24 appears to displace it more effectively than it displaces 3H-naloxone. The results are discussed in terms of multiple transmitter systems and the multiple opiate receptor hypothesis.

In the present investigation reconstitution of Na+,K(+)-ATPase increases the number of phosphorylation sites (EP) of solubilized enzyme from 4.2 +/- 0.3 nmol/mg to 6.9 +/- 0.6 nmol/mg. The latter figure corresponds to one phosphorylation site per alpha beta-promoter. A cholesterol content>> 10 mol% in the liposome bilayer and a high extracellular [Na+] are necessary to obtain this high value. Spontaneous dephosphorylation after maximum phosphorylation in Na+ is biphasic both in solubilized enzyme and after reconstitution. The rate of dephosphorylation compares with the specific hydrolytic Na(+)-ATPase activity measured at exactly identical conditions for all three preparations assuming parallel dephosphorylation of at least two phosphointermediates. The distribution of EP-species is found to vary among the three enzyme preparation used, i.e., membrane bound, solubilized, and reconstituted Na+,K(+)-ATPase, however in all the equilibrium is strongly poised away from the E1P-form.

The opportunistic pathogen Burkholderia cenocepacia contains three soluble carbohydrate-binding proteins, related to the fucose-binding lectin PA-IIL from Pseudomonas aeruginosa. All contain a PA-IIL-like domain and two of them have an additional N-terminal domain that displays no sequence similarities with known proteins. Printed arrays screening performed on the shortest one, B. cenocepacia lectin A (BC2L-A), demonstrated the strict specificity for oligomannose-type N-glycan structures (Lameignere E, Malinovská L, Sláviková M, Duchaud E, Mitchell EP, Varrot A, Sedo O, Imberty A, Wimmerová M. 2008. Structural basis for mannose recognition by a lectin from opportunistic bacteria Burkholderia cenocepacia. Biochem J. 411:307-318.). The disaccharides alphaMan1-2Man, alphaMan1-3Man, and alphaMan1-6Man and the trisaccharide alphaMan1-3(alphaMan1-6)Man were tested by titration microcalorimetry in order to evaluate their affinity for BC2L-A in solution and to characterize the thermodynamics of the binding. Oligomannose analogs presenting two mannoside residues separated by either flexible or rigid spacer were also tested. Only the rigid one yields to high affinity binding with a fast kinetics of clustering, while the flexible analog and the trimannoside display moderate affinities and no clustering effect on short time scale. The crystal structures of BC2L-A have been obtained in complex with alphaMan1-3Man disaccharide and alphaMan1(alphaMan1-6)-3Man trisaccharide. The lengthy time required for the co-crystallization with the trisaccharide allowed for the formation of cluster since in the BC2L-A-trimannose complex solved at 1.1 A resolution, the sugar creates a bridge between two adjacent dimers, yielding to molecular strings. AFM experiments were performed in order to visualize the filaments formed in solution by this type of interaction.

Retroviral gene transfer of the murine erythropoietin receptor (EpR) cDNA into the pro-B-cell line, Ba/F3, was used to generate cells expressing high EpR levels. One of the resulting clones, Ba/F3 clone C5, contained 5 integrated copies of the gene and expressed, at the cell surface, a single affinity class of EpRs at 10 to 15 times the level present on spleen cells from phenylhydrazine-treated mice. Cross-linking studies with clone C5, using 125I-Ep, yielded the same two 105- and 88-Kd major species as that seen with typical erythroid cells. This was distinct from that obtained with EpR-transfected COS cells or L cells, which gave species of 88 and 65 Kd. This suggests that the biologically active EpR complex generated in this Ba/F3 cell line may closely resemble that present in native Ep-responsive erythroid progenitor cells. Tyrosine phosphorylation experiments showed that several proteins in clone C5 cells were rapidly phosphorylated on tyrosine residues in response to Ep, one being the EpR itself. The proportion of cell surface EpRs tyrosine phosphorylated in response to Ep was substantial, reaching a maximum of approximately 10% within 30 minutes of incubation at 37 degrees C. A comparison of Ep- and murine interleukin-3 (mIL-3)-induced tyrosine phosphorylation patterns in clone C5 cells showed that both growth factors stimulated the tyrosine phosphorylation of proteins with molecular weights of 135, 93, 70, and 55 Kd. This could suggest that the Ep and mIL-3 receptors are capable of using the same tyrosine kinase in these cells.

Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10(7) cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of beta-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined.