We have previously identified prostaglandin EP(3)receptors in left ventricular myocardium. To assess the potential contribution of this receptor subtype to the anti-ischemic properties of E-type prostaglandins (i.e. PGE(1)), two groups of anesthetized open-chest minipigs were subjected to LAD occlusion (1 h) and reperfusion (3 h). In one group, the selective EP(3)receptor agonist M&B 28.767 (2 pmol/kgxmin) was infused into the LAD from 20 min before ischemia until the end of reperfusion. The other group received vehicle. M&B 28.767 did not alter the systemic hemodynamics, but significantly reduced infarct size (tetrazolium staining) and creatine kinase release by 53% and 48%, respectively. Ischemia-induced ventricular arrhythmias were mostly reduced. Further experiments analysed the effects of EP(3)receptor stimulation on normoxic myocardium. PGE(1), an unselective agonist to all EP receptor subtypes, as well as M&B 28.767 (2 pmol/kgxmin of each into the LAD) reduced the action potential duration (epicardial monophasic electrodes) and almost prevented the inotropic response to intravenous isoprenaline. This dual response is consistent with the EP(3)receptor coupling to an inhibitory G protein. This was confirmed in separate experiments with stable Chinese hamster ovary cell transfectants expressing the porcine EP(3)receptor, where M&B 28.767 inhibited the forskolin-induced increase in cAMP in a concentration-dependent manner. It is concluded that the protection of reperfused ischemic myocardium by E-type prostaglandins is mediated by EP(3)receptors, which seems to involve a combined activation of repolarizing membrane currents and an inhibition of deleterious effects caused by ischemia-induced catecholamine release.

Microbial biofilms contribute to biofouling in a wide range of processes from medical implants to processed food. The extracellular polymeric substances (EPS) are implicated in imparting biofilms with structural stability and resistance to cleaning products. Still, very little is known about the structural role of the EPS in Gram-positive systems. Here, we have compared the cell surface and EPS of surface-attached (biofilm) and free-floating (planktonic) cells of Bacillus cereus, an organism routinely isolated from within biofilms on different surfaces. Our results indicate that the surface properties of cells change during biofilm formation and that the EPS proteins function as non-specific adhesions during biofilm formation. The physicochemical traits of the cell surface and the EPS proteins give us an insight into the forces that drive biofilm formation and maintenance in B. cereus.

This study was performed in order to characterize the relationship between adhesion and biofilm formation abilities of drinking water-isolated bacteria (Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp.). Adhesion was assessed by two distinct methods: thermodynamic prediction of adhesion potential by quantifying hydrophobicity and the free energy of adhesion; and by microtiter plate assays. Biofilms were developed in microtiter plates for 24, 48 and 72 h. Polystyrene (PS) was used as adhesion substratum. The tested bacteria had negative surface charge and were hydrophilic. PS had negative surface charge and was hydrophobic. The free energy of adhesion between the bacteria and PS was>> 0 mJ/m(2) (thermodynamic unfavorable adhesion). The thermodynamic approach was inappropriate for modelling adhesion of the tested drinking water bacteria, underestimating adhesion to PS. Only three (B. cepacia, Sph. capsulata and Staphylococcus sp.) of the six bacteria were non-adherent to PS. A. calcoaceticus, Methylobacterium sp. and M. mucogenicum were weakly adherent. This adhesion ability was correlated with the biofilm formation ability when comparing with the results of 24 h aged biofilms. Methylobacterium sp. and M. mucogenicum formed large biofilm amounts, regardless the biofilm age. Given time, all the bacteria formed biofilms; even those non-adherents produced large amounts of matured (72 h aged) biofilms. The overall results indicate that initial adhesion did not predict the ability of the tested drinking water-isolated bacteria to form a mature biofilm, suggesting that other events such as phenotypic and genetic switching during biofilm development and the production of extracellular polymeric substances (EPS), may play a significant role on biofilm formation and differentiation. This understanding of the relationship between adhesion and biofilm formation is important for the development of control strategies efficient in the early stages of biofilm development.

Extracellular polysaccharides play an important role in aggregation and surface colonization of plant-associated bacteria. In this work, we report the time course production and monomer composition of the exopolysaccharide (EPS) produced by wild type strain and several mutants of the plant growth promoting rhizobacterium (PGPR) Azospirillum brasilense. In a fructose synthetic medium, wild type strain Sp7 produced a glucose-rich EPS during exponential phase growth and an arabinose-rich EPS during stationary and death phase growth. D-glucose or L-arabinose did not support cell growth as sole carbon sources. However, glucose and arabinose-rich EPSs, when used as carbon source, supported bacterial growth. Cell aggregation of Sp7 correlated with the synthesis of arabinose-rich EPS. exoB (UDP-glucose 4'-epimerase), exoC (phosphomannomutase) and phbC (poly-beta-hydroxyburyrate synthase) mutant strains, under tested conditions, produced arabinose-rich EPS and exhibited highly cell aggregation capability. A mutant defective in LPS production (dTDP 4-rhamnose reductase; rmlD) produced glucose-rich EPS and did not aggregate. These results support that arabinose content of EPS plays an important role in cell aggregation. Cell aggregation appears to be a time course phenomenon that takes place during reduced metabolic cell activity. Thus, aggregation could constitute a protected model of growth that allows survival in a hostile environment. The occurrence of exoC and rmlD was detected in several species of Azospirillum.

Celiac Sprue is an inflammatory disease of the small intestine triggered by ingestion of dietary gluten, a family of glutamine and proline rich proteins found in common foodgrains such as wheat, rye, and barley. One potential therapy for this lifelong disease anticipates using an oral protease to detoxify gluten in vivo. Recent studies have shown that <em>EP</em>-<em>B</em>2 (endoprotease <em>B</em>, isoform 2) from barley is a promising example of such a glutenase, thus warranting its large-scale production for animal safety and human clinical studies. Here we describe a scaleable fermentation, refolding and purification process for the production of gram to kilogram quantities of pro-<em>EP</em>-<em>B</em>2 (zymogen form of <em>EP</em>-<em>B</em>2) in a lyophilized form. A fed-batch E. coli fermentation system was developed that yields 0.3-0.5 g purified recombinant protein per liter culture volume. Intracellular degradation of pro-<em>EP</em>-<em>B</em>2 during the fermentation was overcome by manipulating the fermentation temperature and duration of protein expression. A simple refolding protocol was developed using a fast dilution method to refold the enzyme at concentrations greater than 0.5 mg/mL. Kinetic analysis showed that pro-<em>EP</em>-<em>B</em>2 refolding is a first-order reaction with an estimated rate constant of 0.15 h(-1). A lyophilization procedure was developed that yielded protein with 85% recoverable activity after 7 weeks of storage at room temperature. The process was successfully scaled up to 100 L with comparable purity and recovery.

In order to characterize the epidemiological and clinical picture of post-streptococcal acute glomerulonephritis (PSAGN), a prospective study was designed to investigate all admissions to a general hospital of a local health service in Chile. The protocol included the investigation of previous streptococcal infections (SI), clinical symptoms and signs, socioeconomic situation (SES), throat and skin swabs for the isolation of group A beta-hemolytic streptococci, sequential determination of serum antistreptolysin O (ASO) titer, anti-DNAase B antibodies, and C3. During the 20 years studied, 926 cases were admitted (56% males). Incidence showed an endemic period (EP) 1980-1983, an epidemic outbreak (EO) 1984-1989, and a late period (LP) 1990-1999, with a rate per 100,000 inhabitants of 6.2, 13.2, and 1.7, respectively. The clinical picture was similar in the three periods. SES was homogeneous, with 80% of the population in low and middle-low categories. The average size of the family was 6.9 compared with 4.8 in the general population. Pyoderma was more frequent than pharyngeal infection, and more so during the EO. The isolation rate of group A beta-hemolytic streptococci from the pharynx was 20% compared with 60% from skin swabs. During EP, the most prevalent serotypes were T14-M0 and T1-M1 from the pharynx and TImp19-M0 from the skin. During EO, T14-M0 was more prevalent (30%). M or T classification was possible in EP and EO in 80%-85% of all strains isolated from the two locations. Significant titers for ASO and anti-DNAase B were found on admission: 55% and 75%, respectively. Both tests allowed identification of 100% of previous SI. In conclusion, the incidence of PSAGN had an uneven trend during the observed period. EO was mainly due to skin infection and a predominance of one serotype, T14-MO, was observed. After the EO, the yearly rate gradually decreased from 13.2 in 1988 to 0.0 in 1999, a rate similar to that of industrialized nations.

Chimeric antigen receptor (CAR) T-cell therapy has emerged as an option for relapsed/refractory aggressive B-cell lymphomas that have failed 2 lines of therapy. Failures usually occur early after infusion. The purpose of our study was to identify factors that may predict failure, particularly early progression (EP), within the first month after infusion. Characteristics of 116 patients were analyzed at the time of decision (TD) to use commercial CAR (axicabtagene ciloleucel, n = 49; tisagenlecleucel n = 67) and at the time of treatment (TT), together with total metabolic tumor volume (TMTV) at TT. With a median follow-up of 8.2 months, 55 patients failed treatment; 27 (49%) were early progressors. The estimated 12-month progression-free survival (PFS) and overall survival (OS) were 47.2% (95% confidence interval [CI], 38.0-58.6) and 67.0% (95% CI, 57-79), respectively. Univariate analyses for PFS and OS identified Eastern Cooperative Oncology Group Performance Status (ECOG PS) ≥2, stage III/IV disease, extranodal (EN) sites ≥2, elevated lactate dehydrogenase (LDH), increased C-reactive protein (CRP), high International Prognostic Index at TD and at TT, as well as increased CRP, bulky mass, and high TMTV at TT, as risk factors. Multivariate analyses for PFS, EP, and OS identified elevated LDH and EN sites ≥2 at TD and the same predictors at TT (ie, increased CRP, EN sites ≥2, and TMTV >80 mL). In summary, risk factors identified for early progression at TD and at TT were EN involvement (≥2 sites) and lymphoma burden (LDH, TMTV).

Gestational trophoblastic diseases (GTD) are a group of pregnancy-related disorders representing rare human tumours. They encompass premalignant disorders including complete (CHM), partial hydatidiform mole (PHM), exaggerated placental site (EPS), and placental-site nodule (PSN) as well as malignant disorders (also known as "gestational trophoblastic neoplasia [GTN]") including invasive mole, choriocarcinoma (CC), placenta-site trophoblastic tumour (PSTT), and epitheloid trophoblastic tumours (ETT) (Fig. 1). Originally, GTD develop from abnormal proliferation of trophoblastic tissue and form botryoid arranged vesicles. Premalignant moles are usually treated by suction curettage while persistent and recurrent moles and malignant forms require systemic therapy with methotrexate or combination chemotherapy consisting of etoposide, actimomycin D, methotrexate, vincristine, and cyclophosphamide (EMA-CO). β-human chorion gonadotropin (β-hCG) plays a crucial role in diagnosis and monitoring therapeutic effects. Since the definitive diagnosis cannot be obtained by histology in most cases, persistent or recurrent disease is diagnosed by elevated or persistent serum levels of β-hCG. While curing rates are described to be as high as 98 %, GTD may initially present, recur, or end up as a metastasising systemic disease. This underlines the importance of a regular and consistent follow-up after treatment.

In this study, we examined the endothelin (ET) receptor subtype involved in mitogenic signaling in human primary and metastatic melanoma cell lines. In a reverse transcriptase-polymerase chain reaction (RT-PCR) study, ET(B) mRNA expression in metastatic melanoma cells was decreased from that of primary melanoma. Only RPM-EP, a primary recurrent melanoma cell line, showed strong ET(A) mRNA expression. ET-1 and ET-3 stimulated DNA synthesis of primary and recurrent cutaneous melanoma cells in serum-deprived cultures. The growth response to ET-1 in metastatic melanoma cells was decreased from that in primary melanoma cells. [125I]-IRL-1620 binding to PM-WK, a primary melanoma cell line, was significantly blocked by excessive amounts of unlabeled BQ-788. [125I]-IRL-1620 binding to metastatic melanoma cells was significantly decreased from that of primary melanoma cells. From these results, we conclude that the mitogenic effects of ET in human primary melanoma are mainly mediated through ET(B) receptors and that down-regulation of ET(B) receptors causes the decreased growth response of ET-1 in metastatic melanoma cells.

An increase in initial chemotherapy intensity was evaluated in 29 patients with high risk metastatic non-seminomatous germ cell tumours (NSGCT) of the testis, defined by the presence of multiple large lung metastases, liver, bone or brain metastases, or the combination of large abdominal mass with high serum concentration of the tumour markers alpha-foetoprotein (AFP) or beta subunit of human chorionic gonadotrophin (HCG) (AFP greater than 500 ku/l or HCG greater than 1000 iu/l). Four courses of bleomycin, vincristine and cisplatin (BOP) were given at 7 day intervals, followed by three courses of etoposide, cisplatin with or without bleomycin (BEP or EP) at 21 day intervals for a total of 13 weeks of chemotherapy. Twenty-three (85%) of 27 evaluable patients have remained continuously free from disease progression at a median of 24 months (range 14-38 months) from chemotherapy and the actuarial 2 year freedom from progression rate is 86% (95% CI = 73-99%). Three patients died from non-malignant causes, two of bleomycin pneumonitis and one from complications of cystic fibrosis. Thus cause specific overall survival in the total population of treated patients is 79%. With appropriate limitation of bleomycin dosage, this approach is well tolerated and results compare favourably with less intensive induction schedules based on initial 21-28 day cycles.

BACKGROUND

In the frame of a research line dedicated to better clarify the role of exopolysaccharides (EPS) in bacterial virulence, EPS produced by species of the Burkholderia cepacia complex (Bcc), namely Burkholderia multivorans, Burkholderia cenocepacia, and a Bcc member of undetermined genomovar, all isolated at the Cystic Fibrosis Regional Centre of Florence (Italy), were investigated for they structural properties.

METHODS

Three strains of B. multivorans, three of B. cenocepacia and one of a Bcc member of undetermined genomovar were isolated from CF patients. The reference strains C1576 and J2315, for genomovar II and III, respectively, were included in the study. The bacteria were grown on solid media, the exopolysaccharides produced were purified, and their structures were determined. In addition, sugar analysis of sputum samples was accomplished to search for EPS produced in vivo.

RESULTS

Six strains out of seven produced the exopolysaccharide cepacian, while one strain of B. multivorans produced a completely different polymer, previously known in the literature as PS1. Two strains synthesised very small amounts of EPS. No definitive evidence for the presence of cepacian in sputum samples was found.

CONCLUSIONS

Most strains examined produced abundant amounts of polysaccharides. Cepacian was the most common EPS isolated and its production was not associated to a particular genomovar.

Sex steroids may modulate the secretion of beta-endorphin (beta-EP). Naloxone (Nal), an opioid antagonist, has been used as a probe of central opioid activity. Nal-evoked responses of PRL and LH were evaluated in the midluteal (ML) and late follicular (LF) phases of ovulatory women (Pre) and compared to responses of oophorectomized women before and after the administration of conjugated estrogens (CE) and again after CE and progestin administration. In the ML and LF phases, serum LH increased significantly (P less than 0.05 and P less than 0.01, respectively) during Nal infusion for 4 h, while PRL did not change. In oophorectomized women, there were no significant changes in LH or PRL during Nal infusion. After 3 weeks of CE treatment (1.25 mg daily), LH increased during Nal infusion (P less than 0.05), as did PRL (P less than 0.01). After treatment with CE and medroxyprogesterone acetate (MPA), LH and PRL both increased (P less than 0.05 and P less than 0.01, respectively). The area under the LH curve during Nal infusion after CE and MPA treatment was greater than that after CE alone. Both of these responses were comparable to those of the LF and ML phases of Pre women. During Nal infusion, LH pulse frequency increased in the ML compared to the LF phase of the cycle and, in oophorectomized women, was greater after CE and CE with MPA treatment compared to pretreatment values (P less than 0.05). LH amplitudes during Nal infusion were highest in the ML phase and after CE and MPA treatment in oophorectomized women, and these LH amplitudes were similar. No correlation was found between peripheral plasma beta-EP and Nal-evoked LH responses. No differences were evident in plasma beta-EP levels between Pre and oophorectomized women. In conclusion, 1) endogenous opioid activity is low in oophorectomized women; 2) treatment with estrogen increases opioid activity, and the addition of a progestin increases this activity further; and 3) these data support the contention that sex steroids exert a profound influence on endogenous opioid activity.

14C-labeled extracellular products of a natural microphytobenthic community and two species of benthic diatoms (Nitzschia hybridaeformis and Amphora coffeaeformis) were fractionated into extracellular dissolved organic carbon (14C-EDOC), organic carbon extracted with EDTA (14C-EDTA-extractable OC) and extracellular polymeric substances (14C-EPS). The biodegradation of this labeled extracellular organic carbon by bacteria in sediments was examined to determine the processes of enzymatic degradation of photosynthetically-produced extracellular organic carbon from microphytobenthos in an intertidal flat ecosystem. In addition, primary production as well as extracellular enzyme activities (beta- and alpha-glucosidase) were measured to evaluate the possible relationship between organic carbon production and microbiological degradation at the Isshiki intertidal flat in Mikawa Bay, Japan. With all three 14C-fractions extracted from a natural microphytobenthic assemblage and two species of benthic diatoms, more than 50% of the added substrates were mineralized within 24 h by the bacterial community in sediments. At that time, the percentage of high-molecular-weight compounds (>5 K MW) to total MW compounds of 14C-EDTA-extractable OC and 14C-EPS fractions decreased within 24 h from 50.9 to 6.6% and 74.5 to 11.1%, respectively. In situ, beta- and alpha-glucosidase activity in sediment was higher than in the seawater column (at a depth of 1 m), though the photosynthetic production of microphytobenthos was equal to that of phytoplankton. Based on our previous studies that microphytobenthos produced much more extracellular products than phytoplankton, it is assumed from these results that carbon flowing into the microbial loop through the mediation of enzymatic degradation of extracellular products in a benthic system exceeds that in the overlying water column.

Recent work by our group has shown that an exopolysaccharide (EPS)-producing starter pair, Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii subsp. bulgaricus MR-1R, can significantly increase moisture retention in low-fat mozzarella (D. B. Perry, D. J. McMahon, and C. J. Oberg, J. Dairy Sci. 80:799-805, 1997). The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. Analysis of low-fat mozzarella made with different combinations of MR-1C, MR-1R, and the non-EPS-producing starter culture strains S. thermophilus TA061 and Lactobacillus helveticus LH100 showed that S. thermophilus MR-1C was responsible for the increased cheese moisture level. To investigate the role of the S. thermophilus MR-1C EPS in cheese moisture retention, the epsE gene in this bacterium was inactivated by gene replacement. Low-fat mozzarella made with L. helveticus LH100 plus the non-EPS-producing mutant S. thermophilus DM10 had a significantly lower moisture content than did cheese made with strains LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the S. thermophilus MR-1C EPS indicated that the polymer has a novel basic repeating unit composed of D-galactose, L-rhamnose, and L-fucose in a ratio of 5:2:1.

The plasma beta-endorphin (beta-EP) and beta-lipotropin (beta-LPH) response of men, eumenorrheic women, and amenorrheic women (n = 6) to 1 h of rest or to a bicycle ergometer test [20 min at 30% maximum O2 uptake (VO2max), 20 min at 60% VO2max, and at 90% VO2max to exhaustion] was studied in both normal (22 degrees C) and cold (5 degrees C) environments. beta-EP and beta-LPH was measured by radioimmunoassay in venous samples collected every 20 min during rest or after each exercise bout. Exhaustive exercise at ambient temperature (Ta) 22 degrees C induced significant increases in plasma beta-EP and beta-LPH in all subjects as did work at 60% VO2max in amenorrheic and eumenorrheic women. During work at Ta 5 degrees C, the relative increase in beta-EP and beta-LPH was suppressed in eumenorrheic women and completely prevented in amenorrheic women. Although significant lowering of beta-EP and beta-LPH was observed in men and eumenorrheic women during rest at 5 degrees C, amenorrheic women maintained precold exposure levels. These findings suggest that plasma beta-EP and beta-LPH may reflect a thermoregulatory response to heat load. There appears to be a sexual dimorphism in exercise- and cold-induced release of beta-EP and beta-LPH and amenorrhea may be accompanied by alterations in these responses.

OBJECTIVE

Intracranial recordings were taken from the basal ganglia (BG) in order to explore the possible role of the BG in the cognitive processing of sensory information.

METHODS

Ten patients with intractable temporal lobe epilepsy, who were candidates for epilepsy surgery, underwent intracranial recordings with depth electrodes. A frontal approach was used for the insertion of diagonal depth electrodes into the amygdalo-hippocampal complex (AH complex). These electrodes passed through the BG. The putamen was explored in 8 patients; the nucleus caudatus and pallidum were explored in two patients. The contingent negative variation (CNV) paradigm was tested using auditory warning stimuli and visual imperative stimuli followed by a hand flexion. The auditory and visual middle and late latency potentials evoked by the warning and imperative stimuli were analyzed.

RESULTS

(1) Auditory evoked potentials (EPs): the amplitude potential gradient was observed with latencies of (a) 150-195ms (9 patients); (b) 215-290ms (9 patients); and (c) 350-600ms (10 patients). Negative potentials, with latencies of 100 and 110ms were observed in two patients. (2) Visual EPs: (a) 160-195ms (9 patients); (b) 210-295ms (9 patients); and (c) 330-550ms (7 patients). Negative potentials with latencies between 100 and 120ms were observed in 4 patients. CNV was obtained from the BG in 8 patients; a phase reversal was observed twice.

CONCLUSIONS

(1) The BG generate middle and late latency EPs in a cognitive paradigm linked to the motor task. (2) The BG generate CNV. (3) The BG could play an integrative role in the processing of sensory, cognitive, and motor information.

1. The prostanoid receptor(s) on human airways smooth muscle (HASM) cells that mediates the inhibitory effect of PGE(2) on interleukin (IL)-1 beta-induced granulocyte/macrophage colony-stimulating factor (GM-CSF) release has been classified. 2. IL-1 beta evoked the release of GM-CSF from HASM cells, which was suppressed by PGE(2), 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3)-selective), ONO-AE1-259 and butaprost (both EP(2)-selective) with pIC(50) values of 8.61, 7.13, 5.64, 8.79 and 5.43, respectively. EP-receptor agonists that have selectivity for the EP(1)-(17-phenyl-omega-trinor PGE(2)) and EP(3)-receptor (sulprostone) subtypes as well as cicaprost (IP-selective), PGD(2), PGF(2 alpha) and U-46619 (TP-selective) were poorly active or inactive at concentrations up to 10 microM. 3. AH 6809, a drug that can be used to selectively block EP(2)-receptors in HASM cells, antagonised the inhibitory effect of PGE(2), 16,16-dimethyl PGE(2) and ONO-AE1-259 with apparent pA(2) values of 5.85, 6.09 and 6.1 respectively. In contrast, the EP(4)-receptor antagonists, AH 23848B and L-161,982, failed to displace to the right the concentration-response curves that described the inhibition of GM-CSF release evoked by PGE(2) and ONO-AE1-259. 4. Inhibition of GM-CSF release by PGE(2) and 8-Br-cAMP was abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA) but not by H-89, a purported small molecule inhibitor of PKA. 5. We conclude that prostanoid receptors of the EP(2)-subtype mediate the inhibitory effect of PGE(2) on GM-CSF release from HASM cells by recruiting a PKA-dependent pathway. In addition, the data illustrate that caution should be exercised when using H-89 in studies designed to assess the role of PKA in biological processes.

An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.

In our companion paper, the physiological functions of pancreatic β cells were analyzed with a new β-cell model by time-based integration of a set of differential equations that describe individual reaction steps or functional components based on experimental studies. In this study, we calculate steady-state solutions of these differential equations to obtain the limit cycles (LCs) as well as the equilibrium points (EPs) to make all of the time derivatives equal to zero. The sequential transitions from quiescence to burst-interburst oscillations and then to continuous firing with an increasing glucose concentration were defined objectively by the EPs or LCs for the whole set of equations. We also demonstrated that membrane excitability changed between the extremes of a single action potential mode and a stable firing mode during one cycle of bursting rhythm. Membrane excitability was determined by the EPs or LCs of the membrane subsystem, with the slow variables fixed at each time point. Details of the mode changes were expressed as functions of slowly changing variables, such as intracellular [ATP], [Ca(2+)], and [Na(+)]. In conclusion, using our model, we could suggest quantitatively the mutual interactions among multiple membrane and cytosolic factors occurring in pancreatic β cells.

OBJECTIVE

To determine the stiffness of large arteries in patients with Takayasu's arteritis (TA).

METHODS

A noninvasive ultrasonic method was used to measure Peterson's elastic modulus (Ep), Young's elastic modulus (YM). and the stiffness constant beta of the common carotid arteries in 13 and of the common femoral arteries in 16 patients with TA and in healthy control subjects.

RESULTS

The median carotid artery Ep, YM, and stiffness index beta were statistically significantly higher in patients with TA than in controls (p=0.019, 0.013, and 0.004, respectively). The median femoral artery Ep and YM were also higher in patients with TA than in controls (p=0.005 and 0.039, respectively), and the stiffness constant beta (p=0.127) also tended to be higher in patients with TA relative to healthy persons.

CONCLUSIONS

Carotid artery compliance is strongly diminished in patients with TA. A comparable but somewhat less conspicuous change is seen in the femoral arteries.

Reaction of cytochrome P450 enzymes with arylacetylenes results in heme N-alkylation [e.g., Komives, E. A., and Ortiz de Montellano, P. R., (1985) J. Biol. Chem. 260, 3330-3336] and/or protein modification [e.g., Gan, L.-S. L., Acebo, A. L. and Alworth, W. L. (1984) Biochemistry 23, 3827-3836]. To clarify the factors that determine whether heme or protein alkylation occurs, we have investigated the cytochrome P450 1A1-catalyzed oxidation of 1-ethynylpyrene (1-EP) and phenylacetylene (PA). Cytochrome P450 1A1 in microsomes from beta-naphthoflavone-induced rats is inactivated in a time- and NADPH-dependent manner by 1-EP and PA. Parallel loss of the heme chromophore is observed with PA but not with 1-EP, although partial heme chromophore loss is observed when the purified, reconstituted enzyme is inactivated by either agent. Product analysis shows that 1-EP and PA are oxidized to, respectively, (1'-pyrenyl)-acetic and phenylacetic acids. In contrast to the inactivation of cytochrome P450 2B1 by PA, no isotope effect is observed on enzyme inactivation or metabolite formation when the acetylenic hydrogen is replaced by deuterium in either 1-EP or PA. Inactivation of cytochrome P450 1A1 by 1-EP results in covalent binding of 0.8-0.9 equiv (relative to total cytochrome P450 content) of the inhibitor to the microsomal protein. The results demonstrate that a single isozyme can be inactivated, depending on the structure of the arylacetylene, by heme or protein alkylation. Spectroscopic binding constants (Ks) show that 1-EP binds to the enzyme with>> 2000 times greater affinity that PA.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

To explore the mechanism of electroacupuncture (EA)-induced cumulative analgesic effects on chronic pain in rats with or without ovariectomy (OVX).

METHODS

A total of 110 female Wistar rats were randomized into normal control (n=10), chronic constrictive injury (CCI, n=10), CCI+EA (n=30), OVX+CCI (n=30), and OVX+CCI+EA (n=30) groups. Each of the latter 3 groups was further divided into 2 days (2 d), 2 weeks (2 W) and 3 weeks (3 W) subgroups, respectively (n=10 in each subgroup). The CCI pain model was established by ligature of the right sciatic nerve, and the memory impairment model duplicated by OVX. The paw withdrawal latency (PWL, pain threshold) of the bilateral footplates was detected by radiant heat irradiation, and the bilateral difference in PWL (PWLD) was used to evaluate changes in the pain reaction. Morris water maze test was conducted for evaluating the rats' learning-memory ability. EA was applied to bilateral Zusanli (ST36) and Yanglingquan (GB34) for 2 d, 2 W and 3 W, respectively. Pituitary and hypothalamic beta-endorphin (EP) and adrenocorticotrophic hormone (ACTH) contents were detected by immunoradioassay.

RESULTS

Compared with the CCI group, PWLD of the CCI+EA-3 W group decreased significantly (P<0.05). Compared with the OVX+CCI group, PWLD of the OVX+CCI+EA-3 W group was lowered considerably (P<0.05), but the value was markedly higher than its basal value and those of the normal control and CCI+EA groups (P<0.05). In comparison with the sham-OVX group, the escape latency, swimming distance (SD) in the target quadrant and total SD were increased remarkably in the OVX group (P<0.05), while the number of target platform crossings was decreased significantly (P<0.05), suggesting an impairment of the OVX rats' learning-memory ability. In simple CCI rats, both beta-EP and ACTH contents of the pituitary increased markedly (P<0.05), and those of the hypothalamus decreased obviously compared to the normal control group (P<0.05). After EA, pituitary and hypothalamic ACTH levels were significantly lowered at 2 d and hypothalamic ACTH and beta-EP contents increased obviously at 3 W in comparison with the CCI group (P<0.05). In OVX+CCI rats, following EA, pituitary beta-EP contents at 2 d, 2 W and 3 W, and hypothalamic beta-EP and ACTH contents at 2 W and hypothalamic ACTH levels at 3 W increased significantly (P<0.05), but hypothalamic beta-EP level at 3W decreased markedly (P<0.05). The effects of repeated EA in lowering pituitary ACTH and raising hypothalamic beta-EP and ACTH levels disappeared after OVX+CCI.

CONCLUSIONS

Repeated EA has a cumulative analgesic effect, which is closely associated with its effects in regulating pituitary and hypothalamic beta-EP and ACTH levels. OVX may weaken the analgesic effect of EA by affecting hypothalamic-pituitary axis activity.

Human 5-hydroxytryptamine(7) (5-HT(7)) receptors display characteristics shared with receptors believed to form a tight physical coupling with G protein in the absence of ligand. Some receptors apparently preassociated with G(i/o) and G(q/11) are reported to inhibit the signaling of other similarly coupled G protein-coupled receptors by limiting their access to activate a common G protein pool. Therefore, we determined whether 5-HT(7) receptor expression was sufficient to limit signaling of endogenously expressed G(s)-coupled receptors in human embryonic kidney (HEK) 293 cells. Using the ecdysone-inducible expression system, which allows for the titration of increasing receptor density in the same clonal cell line, we compared the effects of 5-HT(4(b)) and 5-HT(7(a,b,d)) receptor expression on adenylyl cyclase (AC) stimulation by the endogenous G(s)-coupled beta-adrenergic (betaAR) and prostanoid EP (EPR) receptors. betaAR- and EPR-stimulated AC activity was attenuated by 5-HT(7) receptor expression in both membrane preparations and intact HEK293 cells. betaAR- and EPR-stimulated AC activity was unaffected by expression of the G(s)-coupled 5-HT(4) receptor. The mechanism of this heterologous desensitization seems independent of protein kinase A activation, nor does it occur at the level of G protein activation because 1) betaAR- and EPR-stimulated AC activity was not restored to control values when Galpha(s) was overexpressed; and 2) beta(1)AR and beta(2)AR activation of Galpha(s) was unaffected by the expression of 5-HT(7) receptors. In addition, overexpression of AC isoforms was unable to rescue betaAR- and EPR-stimulated AC activity. Therefore, 5-HT(7) receptors probably limit access and/or impede activation of AC by betaAR and EP receptors. Although the 5-HT(7) receptor may preassociate with G protein and/or AC, the mechanism of this heterologous desensitization remains elusive.

Treatment with 13-cis retinoic acid (13-cis RA) has been shown to significantly improve the clinical outcome of children with high-risk neuroblastoma. Despite the large number of studies investigating the cellular effects of retinoids in neuroblastoma cells, the influence of RA isomerisation and the factors that determine the extent of RA isomerisation and uptake are unknown. The aim of this study was to establish the extent of extra- and intracellular isomerisation of 13-cis RA and all-trans retinoic acid (ATRA) in neuroblastoma cell lines, and to investigate the influence of isomerisation on their growth inhibitory effects and on the regulation of expression of cellular retinoic acid binding protein II (CRABP II) and RAR-beta. Limited extracellular isomerisation was observed up to 72 hr after incubation of four neuroblastoma cell lines with 10 microM 13-cis RA or ATRA. The retinoic acid isomer present initially in the medium accounted for >75% of extracellular retinoid exposure. By contrast, incubation with 13-cis RA resulted in intracellular levels of ATRA comparable to those of 13-cis RA. This degree of intracellular isomerisation was not observed after ATRA incubations, with 13-cis RA accounting for <10% of total intracellular retinoids. No differences were observed in the sensitivity of three N-type neuroblastoma cell lines to either 13-cis RA (IC(50): 11.2-13.9 microM) or ATRA (IC(50): 12.9-14.4 microM), despite 10-fold differences in intracellular retinoid levels. A decrease in sensitivity to 13-cis RA (IC(50)=137 microM), as compared to ATRA (IC(50)=41 microM), was observed in the S-type cell line SH S EP. RAR-beta was induced in a dose-dependent manner in SH SY 5Y cells following incubation with ATRA, whereas a weaker and delayed induction was observed with 13-cis RA. Similarly, incubation with ATRA resulted in a greater induction of CRABP II in these cells. In summary, these results indicate either an intracellular conversion of 13-cis RA to ATRA or a selective uptake of ATRA and suggest that this may mediate the differential activity of 13-cis RA in neuroblastoma cell subtypes.