Eribis peptide 94 (EP 94) is a novel enkephalin analog, thought to interact with the μ- and δ-opioid receptors. The purpose of the present study was to examine the cardioprotective potential of EP 94 in two clinically relevant porcine models of myocardial ischaemia and reperfusion, and to investigate if such an effect is associated with an increased expression of endothelial nitric oxide synthase (eNOS). Forty-one anesthetized pigs underwent 40min of coronary occlusion followed by 4h of reperfusion. In Protocol I, balloon occlusion of the left anterior descending artery was performed with concurrent intravenous administration of (A) vehicle (n=7), (B) EP 94 (1ug/kg) after 5, 12, 19 and 26min of ischaemia (n=4) or (C) EP 94 (1ug/kg) after 26, 33, 40min of ischaemia (n=6). In Protocol II, open-chest pigs were administered (D) vehicle (n=6) or (E) 0.2ug/kg/min of EP 94 (n=6) through an intracoronary infusion into the jeopardized myocardium, started after 30min of ischaemia and maintained for 15min. The hearts were stained and the protein content of eNOS measured. EP 94 reduces infarct size when administered both early and late during ischaemia compared with vehicle (infarct size group A 61.6±2%, group B 50.2±3% and group C 49.2±2%, respectively, P<0.05), as well as when infused intracoronary (infarct size group D 82.2±3.9% and group E 61.2±2.5% respectively, P<0.01). Phosphorylated eNOS Ser(1177) in relation to total eNOS was significantly increased in the group administered EP 94, indicating activation of nitric oxide production.

Hepatocyte growth factor (HGF) is a multifunctional protein that signals through the MET receptor. HGF stimulates cell proliferation, cell dispersion, neuronal survival and wound healing. In the inner ear, levels of HGF must <em>b</em>e fine-tuned for normal hearing. In mice, a deficiency of HGF expression limited to the auditory system, or an over-expression of HGF, cause neurosensory deafness. In humans, noncoding variants in <i>HGF</i> are associated with nonsyndromic deafness <i>DFNB39</i> However, the mechanism <em>b</em>y which these noncoding variants causes deafness was unknown. Here, we reveal the cause of this deafness using a mouse model engineered with a noncoding intronic 10<em>b</em>p deletion (del10) in <i>Hgf</i> Male and female mice homozygous for del10 exhi<em>b</em>it moderate-to-profound hearing loss at four weeks of age as measured <em>b</em>y tone <em>b</em>urst auditory <em>b</em>rainstem responses (ABRs). The wild type +80 millivolt endocochlear potential (<em>EP</em>) was significantly reduced in homozygous del10 mice compared to wild type littermates. In normal cochlea, <em>EPs</em> are dependent on ion homeostasis mediated <em>b</em>y the stria vascularis (SV). Previous studies showed that developmental incorporation of neural crest cells into the SV depends on signaling from HGF/MET. We show <em>b</em>y immunohistochemistry that in del10 homozygotes, neural crest cells fail to infiltrate the developing SV intermediate layer. Phenotyping and RNAseq analyses reveal no other significant a<em>b</em>normalities in other tissues. We conclude that, in the inner ear, the noncoding del10 mutation in <i>Hgf</i> leads to developmental defects of the SV and consequently dysfunctional ion homeostasis and, a reduction in the <em>EP</em>, recapitulating human DFNB39 nonsyndromic deafness.(<em>b</em>)Significance Statement:</<em>b</em>) Hereditary deafness is a common, clinically and genetically heterogeneous neurosensory disorder. Previously we reported that human deafness DFNB39 is associated with noncoding variants in the 3'UTR of a short isoform of <i>HGF</i> encoding hepatocyte growth factor. For normal hearing, HGF levels must <em>b</em>e fine-tuned as an excess or deficiency of HGF cause deafness in mouse. Using a <i>Hgf</i> mutant mouse with a small 10 <em>b</em>ase pair deletion recapitulating a human <i>DFNB39</i> noncoding variant, we demonstrate that neural crest cells fail to migrate into the stria vascularis intermediate layer, resulting in a significantly reduced endocochlear potential, the driving force for sound transduction <em>b</em>y inner ear hair cells. HGF-associated deafness is a neurocristopathy <em>b</em>ut, unlike many other neurocristopathies, it is not syndromic.

This report examines the role of pre- and post-migration trauma in explaining differences in refugee and immigrant mental health. Data were derived from mother-youth refugee and immigrant dyads from six countries of origin who were living in Canada at the time of the study. Youth reports of emotional problems (EP) and aggressive behavior (AB) were the mental health outcomes. EP and AB were regressed on predictor blocks: a) status (refugee versus immigrant), visible minority, and gender; b) premigration trauma and postmigration discrimination; c) parent and youth human and social capital; d) poverty, neighborhood, and schools. Refugees suffered higher levels of EP and AB, premigration traumas, and discrimination. Postmigration perception of discrimination predicted both EP and AB and explained immigrant versus refugee differences in EP. Antirefugee discrimination net of discrimination based on immigrant or visible minority status has deleterious mental health consequences.

Equine Piroplasmosis (EP) is a tick-borne disease caused by apicomplexan protozoan parasites, Babesia caballi and Theileria equi. The disease is responsible for serious economic losses to the equine industry. It principally affects donkeys, horses, mules, and zebra but DNA of the parasites has also been detected in dogs and camels raising doubt about their host specificity. The disease is endemic in tropical and temperate regions of the world where the competent tick vectors are prevalent. Infected equids remain carrier for life with T. equi infection, whilst, infection with B. caballi is cleared within a few years. This review focuses on all aspects of the disease from the historical overview, biology of the parasite, epidemiology of the disease (specifically highlighting other non-equine hosts, such as dogs and camels), vector, clinical manifestations, risk factors, immunology, genetic diversity, diagnosis, treatment, and prevention.

Despite previous evidence for a potent inflammatory response after a traumatic brain injury (TBI), it is unknown whether exercise preconditioning (EP) improves outcomes after a TBI by modulating inflammatory responses.

We performed quantitative real-time PCR (qPCR) to quantify the genes encoding 84 cytokines and chemokines in the peripheral blood and used ELISA to determine both the cerebral and blood levels of interleukin-6 (IL-6). We also performed the chromatin immunoprecipitation (ChIP) assay to evaluate the extent of nuclear factor kappa-B (NF-κB) binding to the DNA elements in the IL-6 promoter regions. Also, we adopted the Western blotting assay to measure the cerebral levels of heat shock protein (HSP) 70, synapsin I, and β-actin. Finally, we performed both histoimmunological and behavioral assessment to measure brain injury and neurological deficits, respectively.

We first demonstrated that TBI upregulated nine pro-inflammatory and/or neurodegenerative messenger RNAs (mRNAs) in the peripheral blood such as CXCL10, IL-18, IL-16, Cd-70, Mif, Ppbp, Ltd, Tnfrsf 11b, and Faslg. In addition to causing neurological injuries, TBI also upregulated the following 14 anti-inflammatory and/or neuroregenerative mRNAs in the peripheral blood such as Ccl19, Ccl3, Cxcl19, IL-10, IL-22, IL-6, Bmp6, Ccl22, IL-7, Bmp7, Ccl2, Ccl17, IL-1rn, and Gpi. Second, we observed that EP inhibited both neurological injuries and six pro-inflammatory and/or neurodegenerative genes (Cxcl10, IL-18, IL-16, Cd70, Mif, and Faslg) but potentiated four anti-inflammatory and/or neuroregenerative genes (Bmp6, IL-10, IL-22, and IL-6). Prior depletion of cerebral HSP70 with gene silence significantly reversed the beneficial effects of EP in reducing neurological injuries and altered gene profiles after a TBI. A positive Pearson correlation exists between IL-6 and HSP70 in the peripheral blood or in the cerebral levels. In addition, gene silence of cerebral HSP70 significantly reduced the overexpression of NF-κB, IL-6, and synapsin I in the ipsilateral brain regions after an EP in rats.

TBI causes neurological deficits associated with stimulating several pro-inflammatory gene profiles but inhibiting several anti-inflammatory gene profiles of cytokines and chemokines. Exercise protects against neurological injuries via stimulating an anti-inflammatory HSP70/NF-κB/IL-6/synapsin I axis in the injured brains.

<A<em>b</em>stractText>Targeting cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) is difficult <em>b</em>ecause of their similarities with normal stem cells (NSCs). <em>Ep</em>CAM can identify CSCs from <em>Ep</em>CAM+AFP+HCC cases, <em>b</em>ut is also expressed on NSCs. We aimed to distinguish the two using integrated protein, mRNA and miRNA profiling.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>iTRAQ <em>b</em>ased protein profiling and Next Generation Sequencing (NGS) was performed on <em>Ep</em>CAM+/<em>Ep</em>CAM^- cells isolated from HCC (<em>Ep</em>+CSC, <em>Ep</em>^- HCC) and <em>Ep</em>CAM+ cells from non-cancerous/non-cirrhotic control liver tissues (<em>Ep</em>+NSC). Validations were done using qRT-PCR, flowcytometry and western <em>b</em>lotting followed <em>b</em>y in vitro and in vivo functional studies.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>11 proteins were overexpressed (>3 fold) in <em>Ep</em>+CSCs compared to <em>Ep</em>^- HCC and <em>Ep</em>+NSC cells. However, RNA-sequencing confirmed the <em>Ep</em>+CSC specific up-regulation of only HSPA8, HNRNPC, MPST and GAPDH mRNAs among these. Data<em>b</em>ase search com<em>b</em>ined with miRNA profiling revealed <em>Ep</em>⁺ CSC specific down-regulation of 29 miRNAs targeting these four genes. Of these, only miR-26<em>b</em>-5p was found to target <em>b</em>oth HSPA8 and <em>Ep</em>CAM. Validation of HSPA8 overexpression and miR-26<em>b</em>-5p down-regulation followed <em>b</em>y linear regression analysis esta<em>b</em>lished a negative correlation <em>b</em>etween the two. Functional studies demonstrated that reduced miR-26<em>b</em>-5p expression increased the spheroid formation, migration, invasion and tumourigenicity of <em>Ep</em>⁺ CSCs. Furthermore, anti-miR-26<em>b</em>-5p increased the num<em>b</em>er of <em>Ep</em>⁺ CSCs with a concomitant overexpression of stemness genes and reduction of proapoptotic protein BBC3, which is a known su<em>b</em>strate of HSPA8.</p><p><div>(<em>b</em>)CONCLUSION</<em>b</em>)</div>miR-26<em>b</em>-5p imparts metastatic properties and helps in maintenance of <em>Ep</em>⁺ CSCs via HSPA8. Thus, miR-26<em>b</em>-5p and HSPA8 could serve as molecular targets for selectively eliminating the <em>Ep</em>⁺ CSC population in human HCCs.</p>

High-frequency electrical stimulation of specific brain structures, known as deep brain stimulation (DBS), is an effective treatment for movement disorders, but mechanisms of action remain unclear. We examined the time-dependent effects of DBS applied to the entopeduncular nucleus (EP), the rat homolog of the internal globus pallidus, a target used for treatment of both dystonia and Parkinson's disease (PD). We performed simultaneous multi-site local field potential (LFP) recordings in urethane-anesthetized rats to assess the effects of high-frequency (HF, 130 Hz; clinically effective), low-frequency (LF, 15 Hz; ineffective) and sham DBS delivered to EP. LFP activity was recorded from dorsal striatum (STR), ventroanterior thalamus (VA), primary motor cortex (M1), and the stimulation site in EP. Spontaneous and acute stimulation-induced LFP oscillation power and functional connectivity were assessed at baseline, and after 30, 60, and 90 minutes of stimulation. HF EP DBS produced widespread alterations in spontaneous and stimulus-induced LFP oscillations, with some effects similar across regions and others occurring in a region- and frequency band-specific manner. Many of these changes evolved over time. HF EP DBS produced an initial transient reduction in power in the low beta band in M1 and STR; however, phase synchronization between these regions in the low beta band was markedly suppressed at all time points. DBS also enhanced low gamma synchronization throughout the circuit. With sustained stimulation, there were significant reductions in low beta synchronization between M1-VA and STR-VA, and increases in power within regions in the faster frequency bands. HF DBS also suppressed the ability of acute EP stimulation to induce beta oscillations in all regions along the circuit. This dynamic pattern of synchronizing and desynchronizing effects of EP DBS suggests a complex modulation of activity along cortico-BG-thalamic circuits underlying the therapeutic effects of GPi DBS for conditions such as PD and dystonia.

With an increase in antibiotic-resistant strains, the nosocomial pathogen Acinetobacter baumannii has become a serious threat to global health. Glycoconjugate vaccines containing fragments of bacterial exopolysaccharide (EPS) are an emerging therapeutic to combat bacterial infection. Herein, we characterize the bacteriophage ΦAB6 tailspike protein (TSP), which specifically hydrolyzed the EPS of A. baumannii strain 54149 (Ab-54149). Ab-54149 EPS exhibited the same chemical structure as two antibiotic-resistant A. baumannii strains. The ΦAB6 TSP-digested products comprised oligosaccharides of two repeat units, typically with stoichiometric pseudaminic acid (Pse). The 1.48-1.89-Å resolution crystal structures of an N-terminally-truncated ΦAB6 TSP and its complexes with the semi-hydrolyzed products revealed a trimeric β-helix architecture that bears intersubunit carbohydrate-binding grooves, with some features unusual to the TSP family. The structures suggest that Pse in the substrate is an important recognition site for ΦAB6 TSP. A region in the carbohydrate-binding groove is identified as the determinant of product specificity. The structures also elucidated a retaining mechanism, for which the catalytic residues were verified by site-directed mutagenesis. Our findings provide a structural basis for engineering the enzyme to produce desired oligosaccharides, which is useful for the development of glycoconjugate vaccines against A. baumannii infection.

BACKGROUND

Whether subgroups of functional dyspepsia (FD) should be treated with different approaches is controversially discussed in research. As our previous study has demonstrated the effect of acupuncture in FD treatment, we now further analyze the therapeutic effect of acupuncture in the treatment of postprandial distress syndrome (PDS) and epigastric pain syndrome (EPS).

METHODS

A retrospective analysis was conducted in 465 eligible PDS patients and 241 EPS patients. 4 acupuncture groups (group A: specific acupoints along the stomach meridian; group B: non-specific acupoints along the stomach meridian; group C: alarm and transport acupoints; group D: specific acupoints along the gallbladder meridian) were compared with a non-acupoint sham acupuncture group and an itopride group. The patients were treated in 5 consecutive sessions per week for 4 weeks and were followed-up for 12 weeks afterwards. Primary outcome of the study was defined as response rate and symptom improvement as measured by the Symptom Index of Dyspepsia, while secondary outcome was designated as improvement in quality of life (QoL) as determined by the Nepean Dyspepsia Index.

RESULTS

Symptoms of dyspepsia and QoL were improved from baseline in all groups. In EPS patients, no statistically significant differences could be observed in response rate (p = 0.239) and symptoms improvement (p = 0.344 for epigastric pain; p = 0.465 for epigastric burning). In contrast, PDS patients of the acupuncture group A showed higher response rate (53.2% vs. 19.7%, p<0.001; 53.2% vs. 35.1%, p = 0.025) and score change in postprandial fullness (1.01 vs. 0.27, p<0.001; 1.01 vs. 0.57, p<0.001), early satiation (0.81 vs. 0.21, p<0.001; 0.81 vs. 0.39, p=0.001), and QoL (14.5 vs. 4.33, p<0.001; 14.5 vs. 8.5, p<0.001) compared to the sham acupuncture and itopride group.

CONCLUSIONS

FD patients with PDS responded better to the acupuncture therapies, especially at the specific acupoints along the stomach meridian. The positive therapeutic effect of acupuncture on PDS was correlated with the improvement in postprandial fullness.

BACKGROUND

ClinicalTrial.gov NCT00599677.

OBJECTIVE

To utilize dynamic magnetic resonance angiography (MRA) to characterize aortic stiffness (beta) and elastic modulus (Ep) as indexes of wall compliance during the cardiac cycle and determine any influence of different endograft designs or the presence of endoleaks on these indexes.

METHODS

Eleven consecutive patients (11 men; median age 74 years, range 63-78) with abdominal aortic aneurysm (AAA) selected for endovascular repair were scanned pre- and postoperatively. Aortic area and diameter changes during the cardiac cycle were determined using dynamic MRA at 4 levels: 3 cm above the renal arteries, between the renal arteries, 1 cm below the renal arteries, and at the level of maximum aneurysm sac diameter. Ep and beta were calculated. Data are presented as median (range); p<0.05 was considered significant.

RESULTS

Preoperatively, Ep and beta were significantly higher at the level of the aneurysm sac compared to all other levels (p<0.05). Following EVAR, stiffness increased at this level (p<0.05). After implantation, patients with an Excluder endograft demonstrated Ep and beta measurements at the aneurysm neck that were 94% and 60% higher, respectively, compared to those with a Talent (p<0.05) endograft. The presence of an endoleak had no effect on Ep or beta.

CONCLUSIONS

This study introduces the feasibility of dynamic MRA imaging-based calculations of aortic elastic modulus and stiffness. AAA patients demonstrate increased Ep and beta at the level of the aneurysm sac. EVAR results in increased aneurysm sac Ep and beta. Stent-graft design seems to alter Ep and beta within the aneurysm neck, which may have consequences for endograft durability. The presence of an endoleak does not seem to have an effect on Ep or beta.

BACKGROUND

Nkx2.5 is a conserved homeodomain (HD) containing transcription factor essential for early cardiac development. We generated a DNA nonbinding missense mutation, I183P in the HD, similar to the missense HD mutation found in patients. Transgenic mice expressing this mutation under beta-MHC promoter [beta-MHC(I183P)] showed a postnatal lethal phenotype with heart failure. In contrast, mice expressing the mutation under alpha-MHC promoter [alpha-MHC(I183P)] survive, with later onset heart failure. The aim of this study was to investigate the interrelationship between lethal cardiac failure and the electrophysiologic (EP) phenotypes using cardiac-specific promoters with mutant gene expression at different stages of development and maturation.

RESULTS

In beta-MHC(I183P) and wild-type littermates, six-lead ECG and in vivo endocardial EP studies were performed at 2.5, 3, 4, and 5 weeks of age. In alpha-MHC(I183P) and their wild-type controls, ECGs were acquired at 3, 19, 31, and 64 weeks and in vivo EP studies assessed at 19 +/- 4 weeks of age. Beta-MHC(I183P) mice display AV nodal, atrial, and ventricular EP dysfunction by 3 weeks of age. Bradycardia and PR prolongation were evident on telemetered ambulatory ECG of beta-MHC(I183P) mice. In contrast, alpha-MHC(I183P) mice had no abnormalities on serial ECG through 31 weeks or EP findings at 19 weeks, except increased myocardial tissue refractoriness. However, by 64 weeks, PR intervals lengthened in alpha-MHC(I183P) mice.

CONCLUSIONS

Both prenatal and postnatal overexpression of DNA nonbinding mutant Nkx2.5 are associated with AV conduction malfunction and heart failure; however, more profound progressive EP defects are seen when this mutation expresses during fetal and neonatal periods. These conduction abnormalities may contribute to the lethal heart failure and early mortality evident in DNA nonbinding mutant Nkx2.5 mice.

Earlier studies showed that PGD2 produced both increases and decreases in short-circuit current across the canine proximal colon. PGD2 metabolites had opposing effects: 11 beta-PGF2 alpha elicited only increases in Isc, and 13,14-dihydro-15-keto-PGD2 elicited only decreases. The stimulant effects involved FP receptors, but the receptors involved in mediating the inhibitory effects remained undefined. Here we show that the tissue, although it is capable of producing both PGD2 and PGE2, did not produce 11 beta-PGF2 alpha in measurable amounts. The selective DP receptor agonist BW 245C did not mimic the inhibitory effects of PGD2, producing only dose-dependent increases in short-circuit current. Further, these responses were not significantly inhibited by BW A868C. Cross-desensitization experiments suggested that the stimulant effects of BW 245C involved the EP receptor. However, on a standard preparation (rabbit platelets), both PGD2 and BW 245C inhibited ADP-induced aggregation and were antagonized by BW A868C. 11 beta-PGF2 alpha had no effects. The decreases in short-circuit current across the canine colonic epithelium elicited by PGD2 and 13,14-dihydro-15-keto PGD2 are not mimicked by other prostanoids, nor by the selective agonist BW 245C, and thus appear to involve receptors other than the classical DP receptor.

A number of recent studies suggest that interleukin 1 beta (IL-1 beta) is a major mediator of hypothalamo-pituitary-adrenal (HPA) responses following infectious aggression. We investigated whether IL-1 beta mediates long-term changes in HPA activity and studied the cellular regulation of the anterior pituitary. To mimic chronically elevated IL-1 beta production thought to occur during infectious diseases, osmotic pumps (Alzet type) were implanted in the peritoneal cavity of male rats and hIL-1 beta was infused continuously at rates of 1 or 3 micrograms/day. Effects of hIL-1 beta action on plasma ACTH, beta-endorphin (beta-EP) and corticosterone (CORT) secretion and on anterior pituitary (AP), ACTH and beta-EP content were followed. In addition, hypothalamic (HT) CRH mRNA and in AP, CRH receptor (CRH-Rc) mRNA, POMC nuclear primary transcript RNA, POMC nuclear intermediate processing RNA and POMC nuclear and cytoplasmic mRNA were quantified using a highly sensitive solution hybridization nuclease protection assay. Continuous infusion of hIL-1 beta stimulated the HPA axis at varying degrees. Increased HT CRH gene expression, AP POMC gene transcription, ACTH and beta-EP release occurred only during the first 3 days of the treatment. A long-lasting enhancement of ACTH and beta-EP synthesis and of POMC gene expression resulted from activated POMC gene transcription followed by an increased POMC mRNA stability and decreased POMC mRNA turnover. In the AP, stimulation of ACTH and beta-EP secretion and POMC gene transcription disappeared after continuous IL-1 beta treatment, possibly in part due to a refractory process mediated by decreased CRH-Rc gene expression in corticotropes.

We investigated in this study the response of the fetal hypothalamo-pituitary-adrenal (HPA) system during an acute maternal stress in rats, in order to find out a possible role of developing fetal hypothalamus and to correlate its function to the androgen unbalance during the critical period of sex-specific brain differentiation. Pregnant rats of days 18-22 of gestation were subjected to an acute forced immobilization, and plasma levels of corticosterone (B) and ACTH were measured in mothers and fetuses. Hypothalamic contents of CRH and beta-endorphin (EP), pituitary content of ACTH, and plasma levels of B and ACTH were measured in mothers and fetuses under the maternal stress on day 20 of gestation. By an acute exposure to the 20 minutes' stress, plasma levels of B and ACTH elevated significantly in mothers on each day of gestation. A significant increase of fetal plasma ACTH was detected from day 18 in males, and from day 20 in females. During the maternal stress on day 20 of gestation, hypothalamic contents of CRH and EP decreased significantly in male and female fetuses, when plasma levels of B and ACTH elevated significantly. These results indicate that fetal HPA axis seems to actually respond to the maternal stress during the late gestational period. Further, a release of CRH under the stress together with an activation of EP system in the fetal hypothalamus suggests a possible mechanism regulating the androgen secretion by the fetal hypothalamus via changes of the LH levels.

Common migraine (CM) is an evolutive disease characterized by a progressive increase in the number of attacks and a consequent reduction in the free periods, eventually reaching a state of continuous migraine with interparoxysmal headache (MIH). To evaluate the role of central pro-opiocortin-related peptides in the pathogenesis of the disease, cerebrospinal fluid (CSF) levels of beta-lipotropin (beta-LPH), beta-endorphin (beta-EP) and ACTH were measured in two groups of migraine sufferers with increasing severity of the disease (CM and MIH), and in healthy controls. ACTH values were similar in the 3 groups, while beta-LPH levels were significantly lower (P less than 0.005) in patients affected by MIH (10.4 +/- 8.6 fmol/ml) than in patients with CM (35.7 +/- 8.3) and in controls (32.9 +/- 15.33). beta-EP levels were closely correlated with the severity of the disease: they decreased significantly from those found in healthy controls (86.1 +/- 37 fmol/ml) to those of CM sufferers (38.5 +/- 3.5; P less than 0.005) and showed a further significant fall (P less than 0.01) to the lowest levels which were found in MIH patients (14.8 +/- 9.8). These data showing that the progressive evolution of migraine is concomitant with a progressive impairment in the CSF levels of beta-EP, sustain the concept that non-organic central pain is related to a reduced activity of the neurons responsible for the CSF content of beta-EP.

The relationship between polyclonal B cell activation and immunosuppressor effects induced by F5'EP-Sm, a non-cytotoxic protein secreted by Streptococcus mutans, was studied in C57BL/6 mice. Mice treated with F5'EP-Sm exhibited a considerable increase in splenic nonspecific Ig plaque-forming cells (PFC) compared with untreated mice. The isotypic pattern of non-specific PFC responses favours IgG2a approximately equal to IgG2b greater than IgG3 greater than IgG1 approximately equal to IgM, when taken as a ratio between treated and untreated animals. When F5'EP-Sm was administered 2 days before immunization with sheep red blood cells (SRBC), the non-specific PFC production was accompanied by an ephemeral increase in specific PFC against SRBC 1 day after immunization, which was quickly replaced by a strong immunosuppression. In contrast, when F5'EP-Sm was injected after priming, there was little or no demonstrable suppression of specific PFC, and the increase of non-specific PFC was much less evident. The kinetic curves representing increase or decrease in relation to controls of specific and non-specific PFC are almost mirror images in each of the isotypes. The in vivo suppressor effect was abrogated in thymectomized mice, although the involvement of the T cell compartment is probably secondary to the B cell mitogen effect, since T-depleted spleen cells proliferate and synthesize non-specific Ig when stimulated in vitro with F5'EP-Sm.

An ultrasound phase-locked, echo-tracking system was used to determine the dynamic properties of the distal abdominal aorta in 10 Caucasian male subjects (mean age, 25 years). Recordings were made at rest and during the blood pressure increase resulting from isometric exercise. The pressure diameter curve was nonlinear with an inflection at about 90-110 mmHg. Above this pressure range, the vessel was stiffer (less compliant), but the pressure diameter relationship was roughly linear above as well as below the inflection. Individual pressure diameter curves showed hysteresis, i.e., the aorta had a smaller diameter during expansion than during retraction at corresponding pressures. The pressure strain elastic modulus (Ep) and stiffness (beta) were at rest [Mean Arterial Pressure (MAP), 81 mmHg] 0.70 10(5) N/m2 and 6.0, respectively. During isometric exercise (MAP, 122 mmHg), Ep increased significantly by 91% and stiffness (beta) nonsignificantly by 27%. The variability of the compliance determinations was 5% when the ultrasonic system was combined with intra-arterial blood pressure measurements and less than 7% when combined with auscultatory blood pressure measurements. It is concluded that the phase-locked, echo-tracking system fulfills clinical requirements for routine measurements of vascular compliance.

The effects of recombinant human erythropoietin (rEp) on murine hematopoietic progenitors were studied using a serum-free culture. A high concentration of rEp stimulated the formation of mixed erythroid-megakaryocyte colonies (EM colonies) and blast cell colonies, as well as erythroid colonies, erythroid bursts, and megakaryocyte colonies from normal mouse bone marrow cells. Direct effects of rEp on EM colony, megakaryocyte colony, and erythroid burst formation were confirmed by depletion of accessory cells such as T cells, B cells, and macrophages from crude bone marrow cells, and inhibition of the colonies by the addition of rabbit anti-rEp antibody to the culture in a dose-dependent fashion. Replating experiments were performed to confirm the differentiating ability of blast cell colonies grown in the presence of rEp. Most of the blast cell colonies yielded not only secondary erythroid colonies but also megakaryocyte colonies in the presence of 2 IU/mL rEp. Some of the blast cell colonies produced secondary EM colonies in the presence of 16 IU/ml rEp of 2 IU/mL rEp plus interleukin-3, although no granulocyte-macrophage colonies were found in the secondary culture. These results suggest that Ep acts not only as a late-acting factor that is specific for erythroid progenitors, but also as a bipotential EM-stimulating factor for murine hematopoietic cells.

The black yeast Aureobasidium pullulans (de Bary) Arnaud is known to synthesize the exopolysaccharide pullulan, a poly-alpha-1,6-maltotriose. Nine strains were found to produce additional aubasidan-like EPS, i.e. glucans with alpha-1,4-D-, beta-1,6-D- and beta-1,3-D-glycosidic bonds. These strains had previously been found to deviate in genotypic characters. Additional physiological differences were found: the optimal nitrogen source for exopolysaccharide production in liquid medium was NaNO3 for aubasidan-producing strains, and (NH4)2SO4 for the remaining strains. A new variety, A. pullulans var. aubasidani Yurlova, is described for the strains producing aubasidan-like components. The new variety can be distinguished from A. pullulans var. pullulans by the absence of assimilation of methyl-alpha-D-glucoside and lactose.

Evacuolated protoplasts (EP) retain transcriptional activity responding, after UV-light treatment, to the expression of chalcone synthase (CHS); this behaviour is similar to the parsley cell culture and protoplasts from which the EP were derived. Chemical lysis of the EP with low concentrations of a detergent in a minimal volume had no negative effect on the inducibility of CHS by UV-containing white light. Based on these observations a new method is presented here for establishing a light-responsive in vitro transcription system from evacuolated protoplasts of a parsley (Petroselinum crispum) cell culture. A 615 bp promoter fragment of the CHS gene, fused to the beta-Glucuronidase (GUS) reporter gene, was accurately transcribed as in transiently transformed protoplasts and the reaction was highly sensitive to alpha-amanitin. A 226 bp CHS promoter/GUS reporter construct with mutated cis-acting elements was unable to stimulate GUS transcription, whilst a 341 bp 35S-promoter from the cauliflower mosaic virus (CaMV) was constitutively expressed in dark- or light-treated lysates. The expression of the CHS full-length promoter/GUS construct in the cell-free system was three-fold increased by white or red light compared with the dark level. These results demonstrate that within this in vitro system there must exist a largely intact signal transduction chain between photoreceptor(s) and the CHS promoter. As such it will be a valuable tool for elucidating signalling mechanisms and functional assays of trans-acting factors acting at the end of the pathway.

Sporadic colorectal cancer (CRC) insurgence and progression depend on the activation of Wnt/β-catenin signaling. Dickkopf (DKK)-1 is an extracellular inhibitor of Wnt/β-catenin signaling that also has undefined β-catenin-independent actions. Here we report for the first time that a proportion of DKK-1 locates within the nucleus of healthy small intestine and colon mucosa, and of CRC cells at specific chromatin sites of active transcription. Moreover, we show that DKK-1 regulates several cancer-related genes including the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), which are involved in detoxification of chemotherapeutic agents. Nuclear DKK-1 expression is lost along CRC progression; however, it remains high in a subset (15%) of CRC patients (n = 699) and associates with decreased progression-free survival (PFS) after chemotherapy administration and overall survival (OS) [adjusted HR, 1.65; 95% confidence interval (CI), 1.23-2.21; P = 0.002)]. Overexpression of ALDH1A1 and REPS2 associates with nuclear DKK-1 expression in tumors and correlates with decreased OS (P = 0.001 and 0.014) and PFS. In summary, our findings demonstrate a novel location of DKK-1 within the cell nucleus and support a role of nuclear DKK-1 as a predictive biomarker of chemoresistance in colorectal cancer.

We have characterized a novel substrate for somatic hypermutation, confirming that non-Ig sequences can be targeted for mutation and demonstrating that this substrate allows for the rapid assay for mutations. An artificial sequence containing alternating EcoRV and PvuII sites (EPS) was inserted into the Vkappa167 transgene, which is known to be a target for mutation. To assay for somatic hypermutation, the EPS is amplified using flanking transgene primers, and the PCR product is subsequently digested with either EcoRV or PvuII. A mutation is seen as the appearance of a larger fragment, indicating a base change in a restriction enzyme site. The original transgene, Vkappa167/EPS, showed evidence of a low level of mutation in both splenic hybridomas and Peyer's patch-derived or immunized splenic BEPS were made, Vkappa167/POX and Vkappa167/PEPS. While none of the Vkappa167/POX transgenic lines demonstrated mutation, the Vkappa167/PEPS transgene was highly mutated in BB cells with high peanut agglutinin levels at a frequency similar to that of endogenous Ig genes. An analysis of splenic RNA from the unimmunized transgenic mice indicated that the levels of stable message in splenic B cells could not be correlated with the mutation seen in GC B cells. The mutable Vkappa167/PEPS transgenic line is a unique tool to study somatic hypermutation in vivo.

Genetic and biochemical analysis of exopolysaccharide (EPS) production in lactic acid bacteria has been a growing field of interest in the food industry. We previously identified and characterized a gene cluster composed of 13 genes (epsA to epsM) responsible for EPS production in Streptococcus thermophilus Sfi6. Here we report one further gene, pbp2b, that is connected to EPS production. Mutants with a gene disruption in pbp2b were no longer able to produce EPS, exhibited a reduced growth-rate, and their cell morphology was altered. The predicted gene product showed significant homology to the class B penicillin-binding proteins 2b of Streptococcus pneumoniae, Streptococcus sanguis and Streptococcus mitis involved in peptidoglycan synthesis. Upstream of pbp2b, we further identified two genes which showed significant homology to the E. coli folD and urfl, which is an unidentified open reading frame presumed to be involved in DNA repair. Downstream of pbp2b, we identified a gene that showed homology to the Bacillus subtilis and the Escherichia coli recM or recR which, respectively, are involved in the methyl-dependent DNA mismatch repair. In S. thermophilus, pbp2b and recM were transcribed from their own promoters as monocistronic mRNAs and are therefore organized as independent transcriptional units.

OBJECTIVE

To report the results of a randomized phase II trial of docetaxel with and without estramustine phosphate (EP) in patients with hormone-refractory prostate cancer (HRPC).

METHODS

Patients with progressive HRPC were randomized to receive docetaxel 70 mg/m(2) on day 1 (arm A), or docetaxel 70 mg/m(2) on day 2 plus oral EP three times daily, at a total daily dose of 840 mg, on days 1-5 (arm B). The primary objective of the trial was to evaluate the activity of the treatments in terms of the response in prostate-specific antigen (PSA) level.

RESULTS

Forty-five of the 49 patients centrally randomized to arm A and 44 of the 46 in arm B were evaluable for activity. The PSA level decreased by>> or =50% in 40% of the patients in arm A and in 75% of those in arm B. The median time to PSA progression was 20 weeks in arm A and 30 weeks in arm B. The patients in arm B had an improvement in pain over time.

CONCLUSIONS

These data support the existence of a possible advantage in combining docetaxel and EP, which should be verified in a specific randomized phase III study.