To get high yield of ethanol-soluble proteins (EP) and the antioxidant peptides from Sphyrna lewini muscle, orthogonal experiments (L(9)(3)(4)) were applied to optimize the best extraction conditions and enzyme hydrolysis conditions. The yield of EP reached 5.903±0.053% under the optimum conditions of ethanol concentration 90%, solvent to material ratio 20:1, extraction temperature of 40°C and extraction time of 80min. The antioxidant SEPH (EP hydrolysate of S. lewini muscle) was prepared by using papain under the optimum conditions of enzymolysis time 2h, total enzyme dose 1.2%, enzymolysis temperature 50°C and pH 6, and its DPPH radical scavenging activity reached 21.76±0.42% at the concentration of 10mg/ml. Two peptides (F42-3 and F42-5) were isolated from SEPH by using ultrafiltration, anion-exchange chromatography, gel filtration chromatography and RP-HPLC. The structures of F42-3 and F42-5 were identified as Trp-Asp-Arg and Pro-Tyr-Phe-Asn-Lys with molecular weights of 475.50Da and 667.77Da, respectively. F42-3 and F42-5 exhibited good scavenging activity on hydroxyl radical (EC(50) 0.15mg/ml and 0.24mg/ml), ABTS radical (EC(50) 0.34mg/ml and 0.12mg/ml), and superoxide anion radical (EC(50) 0.09mg/ml and 0.11mg/ml), but moderate DPPH radical (EC(50) 3.63mg/ml and 4.11mg/ml). F42-3 and F42-5 were also effectively against lipid peroxidation in the model system and peroxyl free radical scavenging in β-carotene linoleic acid assay. Their high activities were due to the smaller size and the presence of antioxidative amino acids within the peptide sequences.

In this study, age and sex differences in diameter and compliance of the common carotid artery (CCA) were evaluated in 119 healthy subjects with a phase-locked echo-tracking system. The diameter and pulsatile diameter changes were measured, and pressure strain elastic modulus (Ep) and stiffness (beta) were calculated and used as the inverse estimate of compliance. The carotid diameter increased more rapidly in males and was larger than in females from 25 years of age. The relative diameter change was equal in both sexes, and decreased from 12% to 14% in younger subjects to approximately 5% in elderly subjects. Compliance decreased almost linearly and in parallel in males and females up to 45 years of age. Between 45 and 60 years the decrease was more marked in females than in males, whereas it was by far more marked in males between 60 and 70 years of age.

OBJECTIVE

The objective was three-fold; (1) to test the erosive potential (EP) of various soft drinks, (2) to determine properties related to the soft drinks that were important for EP, and (3) to test possibilities of reducing the EP of soft drinks by modification.

METHODS

Sixteen soft drinks from the Icelandic market including three modified soft drinks were used. The pH, calcium, phosphorus, and titratable acid (TA) to pH 5.5, 7.0, and 10.0 were determined in each drink. From these results the buffer capacity (beta) at pH 4.5, 6.3, and 8.5, degree of saturation with respect to hydroxyapatite (DS(HAP)), and critical pH (DS(HAP)=1) were calculated. One orange juice was modified by addition of various concentrations of calcium and phosphate. EP was determined as weight loss from tooth pieces after immersion into the soft drinks for 24 and 72 h as well as calcium increase in the soft drink upon immersion.

RESULTS

EP of the drinks varied from 0-10% weight loss and 0-31 mmol calcium increase. The pH in carbonated and sport drinks was lower than in fruit juices, whereas TA and beta was considerably higher in fruit juices. Significant correlations were obtained between EP and TA, beta, pH, and DS(HAP) (r(s)=0.69-0.90). Addition of calcium and phosphate to the experimental drinks considerably decreased their EP.

CONCLUSIONS

We conclude that several properties related to soft drinks have an impact on their EP upon long exposure time to teeth and that moderate modification could be a helpful measure to reduce the EP of soft drinks.

Previous studies provide evidence that fetal ethanol exposure induces hypothalamic-pituitary-adrenal (HPA) and pituitary beta-endorphin (beta-EP) hyperresponsiveness to acute stressors. The present study demonstrates significant effects of in utero ethanol exposure on the parallel response patterns of the HPA axis and the pituitary beta-EP system to repeated exposures to a stressor, restraint stress, and indicates sex differences in response. Together, data from the two experiments indicate that, after repeated restraint exposures, fetal ethanol-exposed (E) males and females both show significantly increased plasma levels of adrenocorticotropin (ACTH), and E males also show significantly increased plasma levels of beta-endorphin-like immunoreactivity (beta-EPLIR), compared with their respective pair-fed and control counterparts. Marginal increases in the corticosterone response of E males and the beta-EPLIR response of E females, compared with their controls, were also observed. In addition, delayed or deficient habituation to restraint stress was observed in the beta-EPLIR response of E males and the ACTH response of E females. These data demonstrate that fetal E-exposed males and females both exhibit hormonal hyperresponsiveness and/or deficits in recovery after repeated exposures to restraint stress, but that the patterns of response may differ depending on the number and duration of restraint exposures, the time course measured, and whether the endpoint measured is corticosterone, ACTH, or beta-EPLIR. In addition, the finding that E and pair-fed animals both differed from their respective controls in certain developmental and hormonal measures suggests that prenatal nutritional factors may play a role in mediating some of the changes that are observed.

BACKGROUND

Nicotine improves the deficiencies of sensory gating function in schizophrenic patients and in dilute brown non-Agouti (DBA/2) mice. This effect of nicotine has been attributed to activation of the alpha7 nicotinic acetylcholine receptor (nAChR) subtype.

OBJECTIVE

The aim of this study was to determine whether the activation of another nAChR subtype, the central nervous system (CNS) prominent alpha4<em>beta</em>2 receptor, also contributes to the effects of nicotine on sensory gating in DBA/2 mice.

METHODS

Unanesthetized DBA/2 mice were treated either with nicotine, the alpha4betabeta-erythroidine, the noncompetitive nAChR antagonist mecamylamine, or a combination of an antagonist and nicotine. Thereafter, gating was assessed by recording hippocampal evoked potentials (EP), which were elicited by pairs of auditory clicks. The EP response to the second click, or test amplitude (TAMP), was divided by the EP response to the first click, or condition amplitude (CAMP), to derive gating T:C ratios.

RESULTS

Nicotine significantly (p<0.05) lowered T:C ratios by 42%, while significantly increasing CAMP by 55%. After a pretreatment with dihydro-beta-erythroidine, nicotine still significantly lowered T:C ratios by 28%; however, the nicotine-induced increase of CAMP was blocked. Mecamylamine blocked the effect of nicotine on both T:C ratios and CAMP.

CONCLUSIONS

Activation of alpha4betabetaEPs may be indicative of a profile that would improve information processing in schizophrenia and other CNS diseases.

An in vitro clonal assay for a class of human hemopoietic progenitors (CFU-GEMM) with several characteristics of pluripotential stem cells has been previously described. In the presence of medium conditioned by leukocytes stimulated with phytohemagglutinin (PHA-LCM) and erythropoietin (Ep), CFU-GEMM give rise to mixed hemopoietic colonies containing granulocytic, erythroid, monocyte-macrophage, and megakaryocytic elements. In initial studies we found that CFU-GEMM were present in equal but low frequencies in blood (B) and bone marrow (M) mononuclear cell populations. However, when the culture system was modified by the substitution of Iscove's modified Dulbecco's medium for alpha-MEM and the addition of mercaptoethanol, a significant enhancement of mixed colony formation occurred, and an approximately 3-4-fold difference in the frequency of CFU-GEMM between B and M emerged. Replating studies showed the formation of secondary differentiated hemopoietic colonies and at least a limited capacity for self-renewal of CFU-GEMM. The in vitro growth of normal CFU-GEMM was highly dependent on hemopoietin(s) present in PHA-LCM. In vitro detection of CFU-GEMM, however, requires only relatively low permissive concentrations of Ep, in contrast to the high Ep requirement for optimal BFU-E growth in vitro. These and other data described demonstrate CFU-GEMM to be a distinct multipotential stem cell class whose assay may prove useful in the study of human blood dyscrasias.

BACKGROUND

Pseudomonas aeruginosa is a gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A-L) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the "scaffolding" polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A-L biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated.

RESULTS

In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanýi type O:2a,c (Lanýi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure: -4)-α-L-GalNAcA-(1-3)-α-D-QuiNAc-(1-3)- α-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic β-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically in minimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least ∼20% of dry weight) of LPS-like material.

CONCLUSIONS

We characterized the chemical structure of the LPS O-antigen and showed that the O-antigen polysaccharide is an abundant extracellular carbohydrate of PA14. We present evidence that LPS-like material is found as a component of a biofilm matrix of P. aeruginosa.

Erythropoietin (EP) is required by late stage erythroid progenitor cells to prevent apoptosis. In a previous study (Gregoli and Bondurant, 1997, Blood 90:630-640), it was shown that rapid proteolytic conversion of procaspase 3 to the fully activated enzyme occurred when erythroblasts were deprived of EP for as little as 2 h. In the present study, protein and mRNA analyses of erythroblasts indicated the presence of the proenzyme precursors of caspases 1, 2, 3, 5, 6, 7, 8, and 9. The effects of various caspase inhibitors on caspase 3 processing and on apoptosis were examined. These inhibitors were benzyloxycarbonyl (z-) and fluoromethyl-ketone (FMK) derivatives of peptides that serve as substrates for selected caspases. z-VAD-FMK, t-butoxycarbonyl-aspartate-FMK (Boc-D-FMK), and z-IETD-FMK blocked the initial cleavage of procaspase 3, while z-DEVD-FMK, z-VEID-FMK, and z-VDVAD-FMK did not block the initial cleavage but had some effect on blocking apoptosis. The peptide inhibitor z-FA-FMK, which inhibits cathepsins B and L but is not known to inhibit caspases, altered caspase 3 processing to a final 19 kDa large subunit that appeared to retain enzymatic activity. The action of z-FA-FMK in preventing the usual conversion to a 1 7 kDa subunit suggests the possibility that a noncaspase protease may be involved in caspase 3 processing. Studies with the peptide inhibitors and EP were done to determine the short- and long-term effectiveness of the caspase inhibitors in protecting EP-deprived cells from apoptosis. Although several of the inhibitors were effective, z-IETD-FMK was studied most extensively because of its specificity for enzymes which cleave procaspase 3 at aspartate 175 (IETD175). Large percentages of EP-deprived erythroblasts treated with z-IETD-FMK appeared morphologically normal and negative by a DNA strand breakage (TUNEL) assay at 24 h (75%) compared to EP-deprived controls (10%) which were not treated with inhibitor. However, inhibitor-treated erythroid progenitors deprived of EP for 24 h and then resupplied with EP showed only a modest improvement in long-term survival compared to cells which did not receive the caspase inhibitor during the 24 h EP deprivation. Thus, while the manifestations of apoptosis were delayed in most cells by inhibiting caspase activity, the processes initiating the loss of cell viability due to EP deprivation were irreparablein the majority of the cells and eventually led to their deaths.

Because of increased interest in surface carbohydrates of Rhizobium in relation to host specificity, phenol-water extractions were carried out of whole cells of Rhizobium strains of the species R. leguminosarum, R. phaseoli, R. trifolii and R. meliloti. Fractionation of the crude extracts with cetavlon afforded polysaccharide mixtures, which were essentially free of RNA and acidic exopolysaccharide (EPS). They could be separated into a high molecular weight heteropolysaccharide fraction of lipopolysaccharide (LPS) nature and a low molecular weight glucan fraction. Glucan turned out to be the principal polysaccharide component of the cells (up to 10% of the dry cell weight), when cultivated in carbohydrate-rich media, and to be present as firmly attached capsular material. Glucan (mol wt 3000) structure was elucidated by methylation and periodate oxidation techniques. Methylation yielded 3, 4, 6-tri-O-methyl-D-glucose, characterized by GLC-MS, as the only product of hydrolysis of the fully methylated glucan. The glucan consumed 1 mole of periodate per mole anhydroglucose unit and gave sophorose on partial hydrolysis. From these data a linear beta-1,2-linked glucan structure was deduced. The occurrence of beta-1,2-glucan and the implications for the specific binding properties of Rhizobium cells are discussed.

Adenylyl cyclases (AC) are important regulators of airway smooth muscle function, because β-adrenergic receptor (AR) agonists stimulate AC activity and increase airway diameter. We assessed expression of AC isoforms in human bronchial smooth muscle cells (hBSMC). Reverse transcriptase-polymerase chain reaction and immunoblot analyses detected expression of AC2, AC4, and AC6. Forskolin-stimulated AC activity in membranes from hBSMC displayed Ca(2+)-inhibited and G(βγ)-stimulated AC activity, consistent with expression of AC6, AC2, and AC4. Isoproterenol-stimulated AC activity was inhibited by Ca(2+) but unaltered by G(βγ), whereas butaprost-stimulated AC activity was stimulated by G(βγ) but unaffected by Ca(2+) addition. Using sucrose density centrifugation to isolate lipid raft fractions, we found that only AC6 localized in lipid raft fractions, whereas AC2 and AC4 localized in nonraft fractions. Immunoisolation of caveolae using caveolin-1 antibodies yielded Ca(2+)-inhibited AC activity (consistent with AC6 expression), whereas the nonprecipitated material displayed G(βγ)-stimulated AC activity (consistent with expression of AC2 and/or AC4). Overexpression of AC6 enhanced cAMP production in response to isoproterenol and beraprost but did not increase responses to prostaglandin E(2) or butaprost. β(2)AR, but not prostanoid EP(2) or EP(4) receptors, colocalized with AC5/6 in lipid raft fractions. Thus, particular G protein-coupled receptors couple to discreet AC isoforms based, in part, on their colocalization in membrane microdomains. These different cAMP signaling compartments in airway smooth muscle cells are responsive to different hormones and neurotransmitters and can be regulated by different coincident signals such as Ca(2+) and G(βγ).

The present study investigates the effects of gastrin-17 on human colon cancer HT-29 cells to examine whether gastrin receptor (CCK-2), cyclooxygenase (COX-1, COX-2) isoforms and prostaglandin receptor pathways interact to control cell growth. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis demonstrated that HT-29 cells are endowed with the naive expression of CCK-2 receptor (short splice variant), COX-1, COX-2 and prostaglandin EP(4) receptor, but not gastrin. Gastrin-17 significantly promoted cell growth and DNA synthesis. Both these stimulating effects were abolished by L-365,260 or GV150013 (CCK-2 receptor antagonists), but were unaffected by SC-560 (COX-1 inhibitor). L-745,337 (COX-2 inhibitor) or AH-23848B (EP(4) receptor antagonist) partly reversed gastrin-17-induced cell growth, while they fully antagonized the enhancing action on DNA synthesis. HT-29 cells responded to gastrin-17 with a significant increase in prostaglandin E(2) release. This enhancing effect was completely counteracted by L-365,260, GV150013 or L-745,337, while it was insensitive to cell incubation with SC-560. Exposure of HT-29 cells to gastrin-17 was followed by an increased phosphorylation of both extracellular regulated kinases (ERK-1/ERK-2) and Akt. Moreover, gastrin-17 enhanced the transcriptional activity of COX-2 gene promoter and stimulated COX-2 expression. These latter effects were antagonized by L-365,260 or GV150013, and could be blocked also by PD98059 (inhibitor of ERK-1/ERK-2 phosphorylation) or wortmannin (inhibitor of phosphatidylinositol 3-kinase). Analogously, gastrin-17-induced prostaglandin E(2) release was prevented by PD98059 or wortmannin. The present results suggest that (a) in human colon cancer cells endowed with CCK-2 receptors, gastrin-17 is able to enhance the transcriptional activity of COX-2 gene through the activation of ERK-1/ERK-2- and phosphatidylinositol 3-kinase/Akt-dependent pathways; (b) these stimulant actions lead to downstream increments of COX-2 expression, followed by prostaglandin E(2) production and EP(4) receptor activation; (c) the recruitment of COX-2/prostaglandin pathways contributes to the growth-promoting actions exerted by gastrin-17.

BACKGROUND

Several studies have reported the presence of electroencephalography (EEG) abnormalities or altered evoked potentials (EPs) during sepsis. However, the role of these tests in the diagnosis and prognostic assessment of sepsis-associated encephalopathy remains unclear.

METHODS

We performed a systematic search for studies evaluating EEG and/or EPs in adult (≥ 18 years) patients with sepsis-associated encephalopathy. The following outcomes were extracted: a) incidence of EEG/EP abnormalities; b) diagnosis of sepsis-associated delirium or encephalopathy with EEG/EP; c) outcome.

RESULTS

Among 1976 citations, 17 articles met the inclusion criteria. The incidence of EEG abnormalities during sepsis ranged from 12% to 100% for background abnormality and 6% to 12% for presence of triphasic waves. Two studies found that epileptiform discharges and electrographic seizures were more common in critically ill patients with than without sepsis. In one study, EEG background abnormalities were related to the presence and the severity of encephalopathy. Background slowing or suppression and the presence of triphasic waves were also associated with higher mortality. A few studies demonstrated that quantitative EEG analysis and EP could show significant differences in patients with sepsis compared to controls but their association with encephalopathy and outcome was not evaluated.

CONCLUSIONS

Abnormalities in EEG and EPs are present in the majority of septic patients. There is some evidence to support EEG use in the detection and prognostication of sepsis-associated encephalopathy, but further clinical investigation is needed to confirm this suggestion.

The population of group B streptococci (GBS) associated with invasive infections in nonpregnant adults from 2001 to 2008 was analyzed in isolates submitted from 24 hospital laboratories in Portugal (n = 225). The isolates were characterized by antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and surface protein gene profiling. GBS invasive cases were found more frequently among men in all age groups. In addition, serotype Ia was the most frequent in our collection, whereas serotype V is dominant elsewhere. Serotype Ia was represented mainly by a single PFGE cluster defined by sequence type 23 (ST23) and surface protein gene eps and by ST24 and bca, similarly to neonatal invasive infections in Portugal, indicating that the same genetic lineages can be responsible for both vaginal colonization and invasive disease in all age groups. In contrast, the hypervirulent serotype III/ST17 neonatal lineage was responsible for a minority of infections. Serotype V isolates were distributed into two genetic lineages, one defined by ST1 and surface protein gene alp3 and macrolide resistant, and another presenting with ST2 and eps and fully susceptible to all antimicrobials tested. The erm(TR) gene was the most frequently found among erythromycin-resistant isolates, while the bovine-associated tet(O) gene was found in a minority of tetracycline-resistant isolates. Our data emphasize the importance of local identification of the genetic lineages responsible for GBS invasive infections in nonpregnant adults. The dominance of serotype Ia in invasive disease in Portugal highlights the importance of this serotype in GBS pathogenesis.

The purpose of this study was to examine the effects of three different run training programs on plasma responses of beta-endorphin (beta-EP), adrenocorticotropin (ACTH), and cortisol to maximal treadmill exercise. Subjects were randomly assigned to one of three training groups: sprint intervals (SI) (N = 8), endurance (E) (N = 10), or combination (C) (N = 7). Training was monitored for 10 wk, and maximal treadmill exercise tests were administered pre-training and after 2, 4, 6, 8, and 10 wk of training. Blood samples were obtained (pre-training and after 10 wk) before, immediately after, and 5 and 15 min following the maximal exercise tests. All groups significantly (P less than 0.05) increased maximal oxygen consumption values at 8 and 10 wk of the training period. Significant exercise-induced increase in plasma beta-EP, ACTH, cortisol, and blood lactate were observed for both pre- and post-training tests in all training groups. The SI group demonstrated significant post-training increases in beta-EP, ACTH, cortisol, and 5 min post-exercise blood lactate concentrations in response to maximal exercise. No training-induced hormonal changes were observed for the E group. While exercise-induced increases were observed, the C group exhibited significant post-training reductions in plasma responses of beta-EP, ACTH, and blood lactate concentrations in response to maximal exercise. Still, resting and post-exercise increases in plasma cortisol concentrations were significantly higher in magnitude in the post-training test. Lactate was significantly correlated with beta-EP (r = 0.72), ACTH (r = 0.70), and cortisol (r = 0.64).(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation to different nutritional environments is essential for life cycle completion by all Trypanosoma brucei sub-species. In the tsetse fly vector, L-proline is among the most abundant amino acids and is mainly used by the fly for lactation and to fuel flight muscle. The procyclic (insect) stage of T. b. brucei uses L-proline as its main carbon source, relying on an efficient catabolic pathway to convert it to glutamate, and then to succinate, acetate and alanine as the main secreted end products. Here we investigated the essentiality of an undisrupted proline catabolic pathway in T. b. brucei by studying mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase (TbP5CDH), which catalyzes the irreversible conversion of gamma-glutamate semialdehyde (γGS) into L-glutamate and NADH. In addition, we provided evidence for the absence of a functional proline biosynthetic pathway. TbP5CDH expression is developmentally regulated in the insect stages of the parasite, but absent in bloodstream forms grown in vitro. RNAi down-regulation of TbP5CDH severely affected the growth of procyclic trypanosomes in vitro in the absence of glucose, and altered the metabolic flux when proline was the sole carbon source. Furthermore, TbP5CDH knocked-down cells exhibited alterations in the mitochondrial inner membrane potential (ΔΨm), respiratory control ratio and ATP production. Also, changes in the proline-glutamate oxidative capacity slightly affected the surface expression of the major surface glycoprotein EP-procyclin. In the tsetse, TbP5CDH knocked-down cells were impaired and thus unable to colonize the fly's midgut, probably due to the lack of glucose between bloodmeals. Altogether, our data show that the regulated expression of the proline metabolism pathway in T. b. brucei allows this parasite to adapt to the nutritional environment of the tsetse midgut.

The barley cysteine proteinase B (EPB) is the main protease responsible for the degradation of endosperm storage proteins providing nitrogenous nutrients to support the growth of young seedlings. The expression of this enzyme is induced in the germinating seeds by the phytohormone, gibberellin, and suppressed by another phytohormone, abscisic acid. In situ hybridization experiments indicate that EPB is expressed in the scutellar epithelium within 24 h of seed germination, but the aleurone tissue surrounding the starchy endosperm eventually becomes the main tissue expressing this enzyme. The EPB gene family of barley consists of two very similar genes, EPBEPBEPBEPBEPB family can be classified as cathepsin L-like endopeptidases and is most closely related to two legume cysteine proteinases (Phaseolus vulgaris EP-C1 and Vigna mungo SHEP) which are also involved in seed storage protein degradation. The promoters of EPBEPBbeta-glucuronidase, and introduced into barley aleurone cells using the particle bombardment method. Transient expression studies indicate that EPB promoters are sufficient to confer the hormonal regulation of these genes.

The entire coding sequences for five possible human cadherins, named cadherin-4, -8, -11, -12 and -13, were determined. The deduced amino acid sequences of cadherin-4 and cadherin-13 showed high homology with those of chicken R-cadherin or chicken T-cadherin, suggesting that cadherin-4 and cadherin-13 are mammalian homologues of the chicken R-cadherin or T-cadherin. Comparison of the extracellular domain of these proteins with those of other cadherins and cadherin-related proteins clarifies characteristic structural features of this domain. The domain is subdivided into five subdomains, each of which contains a cadherin-specific motif characterized by well-conserved amino acid residues and short amino acid sequences. Moreover, each subdomain has unique features of its own. The comparison also provides additional evidence for two structurally different types of cadherins: the first type includes B-, E-, EP-, M, N-, P- and R-cadherins and cadherin-4; the second type includes cadherin-5 through cadherin-12. Cadherin-13 lacks the sequence corresponding to the cytoplasmic domain of typical cadherins, but the extracellular domain shares most of the features common to the extracellular domain of cadherins, especially those of the first type of cadherins, suggesting that cadherin-13 is a special type of cadherin. These results, and those of other recent cloning studies, indicate that many cadherins with different properties are expressed in various tissues of different organisms.

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use with a wide variety of diseases. Alone, this technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. In this study we evaluated the MEA for its use in DNA vaccination using Hepatitis B virus as the infectious model. We utilized the guinea pig model because their skin is similar in thickness and morphology to humans. The plasmid encoding Hepatitis B surface antigen (HBsAg) was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to injection only groups. While this electrode requires further study, our results suggest that the MEA has potential for use in electrically mediated intradermal DNA vaccination.

To study the efficacy of electro-acupuncture for the relief of labor pain, and to build a better understanding of how electro-acupuncture might influence the neuroendocrine system, 36 primiparas were randomly divided into an electro-acupuncture group and a control group. Assessments of pain intensity and degree of relaxation during labor were analyzed. The differences between the electro-acupuncture group and the control group on the concentration of beta-endorphin (beta-EP) and 5-hydroxytryptamine (5-HT) in the peripheral blood were compared. The electro-acupuncture group was found to exhibit a lower pain intensity and a better degree of relaxation than the control group (p = 0.018; p = 0.031). There existed a significant difference in the concentration of beta-EP and 5-HT in the peripheral blood between the two groups at the end of the first stage (p = 0.037; p = 0.030). Electro-acupuncture was found to be an effective alternative or complementary therapy in the relief of pain during labor. The benefit of electro-acupuncture for relieving labor pain may be based on the mechanism of producing a synergism of the central nervous system (CNS) with a direct impact on the uterus through increasing the release of beta-EP and 5-HT into the peripheral blood.

Serum beta-endorphin levels during a graded exercise test to exhaustion. Med. Sci. Sports Exerc., Vol. 19, No. 2, pp. 78-82, 1987. Nine untrained college age males completed a graded exercise protocol to maximal capacity on a bicycle ergometer to determine if there was a relationship between intensity of exercise and serum beta-endorphin (beta-EP) levels. Subjects fasted for 12 h and abstained from physical activity at least 24 h prior to testing. Subjects completed the Multiple Affect Adjective Check List prior to and following exercise to ascertain if psychological state would be associated with beta-EP levels. The initial workload was 150 kilopond meters and was increased 150 kilopond meters every 3 min until VO2max or leg fatigue occurred. Expired gases were continuously analyzed, and a venous blood sample was drawn from an indwelling catheter during the final 30 s of each stage and 5-min post-exercise. beta-EP levels were determined from serum using a radioimmunoassay technique and corrected for cross-reactivity with beta-lipotropin using affinity chromatography. Resting beta-EP levels were 25.3 +/- 4.1 pg X ml-1 and did not demonstrate significant changes during any stage of exercise. A correlation analysis (r = 0.30) revealed no significant relationship between exercise intensity and beta-EP levels. Following exercise, beta-EP levels were significantly increased compared to resting values (38.8 +/- 4.8 pg X ml-1). In addition, psychological state was unaffected by exercise despite significant increases in recovery beta-EP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoglobulin (Ig) genes expressed in mature B lymphocytes can undergo somatic hypermutation upon cell interaction with antigen and T cells. The mutation mechanism had previously been shown to depend upon transcription initiation, suggesting that a mutator factor was loaded on an RNA polymerase initiating at the promoter and causing mutations during elongation (Peters, A., and U. Storb. 1996. Immunity. 4:57-65). To further elucidate this process we have created an artificial substrate consisting of alternating EcoRV and PvuII restriction enzyme sites (EPS) located within the variable (V) region of an Ig transgene. This substrate can easily be assayed for the presence of mutations in DNA from transgenic lymphocytes by amplifying the EPS insert and determining by restriction enzyme digestion whether any of the restriction sites have been altered. Surprisingly, the EPS insert was mutated many times more frequently than the flanking Ig sequences. In addition there were striking differences in mutability of the different nucleotides within the restriction sites. The data favor a model of somatic hypermutation where the fine specificity of the mutations is determined by nucleotide sequence preferences of a mutator factor, and where the general site of mutagenesis is determined by the pausing of the RNA polymerase due to secondary structures within the nascent RNA.

OBJECTIVE

To investigate the expression of beta-adrenoceptor (AR) subtypes responsible for detrusor smooth muscle relaxation.

METHODS

Isolated rat detrusor smooth muscle was examined by tension measurement and reverse transcription polymerase chain reaction (RT-PCR).

RESULTS

Norepinephrine (NE), epinephrine (EP) and isoproterenol (ISO) were found to relax the detrusor muscle pre-contracted by 6 x 10(-7) M carbachol in the presence of 10(-6) M phentolamine. NE relaxed the detrusor muscle as potently as EP. This potency order (NE=EP) thus indicated the <em>beta</em>-ARs of the rat detrusor muscle to possibly be a <em>beta</em>1 subtype. However, in the presence of 10(-6) M propranolol, <em>beta</em>1- and <em>beta</em>2- but not <em>beta</em>3-AR antagonist, NE showed a more potent relaxation than EP. This observation indicated that the rat detrusor muscle also possesses <em>beta</em>3-AR. RT-PCR revealed all three subtypes of <em>beta</em>-AR mRNA, namely <em>beta</em>1-, <em>beta</em>2- and <em>beta</em>3-AR mRNA, to be expressed in rat detrusor smooth muscle cells.

CONCLUSIONS

We concluded that betabeta-ARs of rat detrusor smooth muscle are considered to be mixed populations consisting of three subtypes which play an important role in relaxing smooth muscle in response to catecholamines.

Diazepam binding inhibitor (DBI)/endozepine (EP)/acyl-CoA-binding protein (ACBP) is a small, highly conserved protein which has been independently isolated and characterized from different species using several different biological systems. To further investigate the structural and functional properties of this protein, we have cloned the homologous gene for DBI/EP/ACBP from the budding yeast Saccharomyces cerevisiae. The yeast gene contains no introns and encodes a polypeptide of 87 amino acids (including the initiating methionine), identical in length to the human gene product with 48% conservation of amino acid residues. The most highly conserved domain consists of 7 contiguous residues which are identical in all known protein species from yeast, birds, and mammals. This domain has previously been shown to constitute the hydrophobic binding site on DBI/EP/ACBP for acyl-CoA esters and is located within the second helical region of the molecule. Major and minor mRNA species of approximately 520 and 740 nucleotides, respectively, were detected in exponentially growing yeast. Sequences similar to those implicated in the regulation of fatty acid synthesis and beta-oxidation in yeast were detected in the promoter region of the gene. The presence of a highly conserved DBI/EP/ACBP gene in a primitive organism such as yeast provides support for the basic biological role of DBI/EP/ACBP as an acyl-CoA-binding protein and suggests that many of the biological functions attributed to it in higher organisms may result from its ability to interact with acyl-CoA. Hence, we have designated the yeast gene as ACB, for acyl-CoA-binding protein.

In the bivalve mollusc Mytilus edulis shell thickening occurs from the extrapallial (EP) fluid wherein secreted shell matrix macromolecules are thought to self-assemble into a framework that regulates the growth of CaCO(3) crystals, which eventually constitute approximately 95% of the mature shell. Herein is the initial report on the purification and characterization of a novel EP fluid glycoprotein, which is likely a building block of the shell-soluble organic matrix. This primary EP fluid protein comprises 56% of the total protein in the fluid and is shown to be a dimer of 28,340 Da monomers estimated to be 14.3% by weight carbohydrate. The protein is acidic (pI = 4.43) and rich in histidine content (11.14%) as well as in Asx and Glx residues (25.15% total). The N terminus exhibits an unusual repeat sequence of histidine and aspartate residues that occur in pairs: NPVDDHHDDHHDAPIVEHHD approximately. Ultracentrifugation and polyacrylamide gel electrophoresis demonstrate that the protein binds calcium and in so doing assembles into a series of higher order protomers, which appear to have extended structures. Circular dichroism shows that the protein-calcium binding/protomer formation is coupled to a significant rearrangement in the protein's secondary structure in which there is a major reduction in beta-sheet with an associated increase in alpha-helical content of the protein. A model for shell organic matrix self-assembly is proposed.