Endogenous opioid peptides have a role in the regulation of the hypothalamic-pituitary-adrenal axis. Recently, beta-endorphin (EP) has been thought to inhibit CRF release in vivo and in vitro. In the present study we examined the effects of central administration of EP on ACTH secretion and gene expression of both CRF in the hypothalamus and POMC in the anterior pituitary gland (AP) during basal and insulin-induced hypoglycemia in pentobarbital-anesthetized rats. Administration of EP in the lateral ventricle decreased basal CRF levels in the median eminence and inhibited basal and hypoglycemia-induced ACTH secretion in a dose-dependent manner. Hypoglycemia-induced POMC mRNA levels in the AP and CRF mRNA levels in the hypothalamus were also dose-dependently inhibited by the administration of EP. The inhibitory effect of EP was reversed by naloxone. These results suggest that 1) central administration of EP acts through the opioid receptor to inhibit hypoglycemia-induced CRF gene expression in the hypothalamus and CRF release, which results in a decrease in ACTH secretion and POMC mRNA levels in the AP; and 2) the active site of EP is the CRF neuron in the paraventricular nucleus.

We explored in the duck hepatitis B virus (DHBV) model the impact of electroporation (EP)-mediated DNA vaccine delivery on the neutralizing humoral response to viral preS/S large envelope protein. EP enhanced the kinetics and magnitude of anti-preS response compared to the standard needle DNA injection (SI). Importantly, EP dramatically enhanced the neutralizing potency of the humoral response, since antibodies induced by low DNA dose (10 μg) were able to highly neutralize DHBV and to recognize ten antigenic regions, including four neutralization epitopes. Whereas, SI-induced antibodies by the same low DNA dose were not neutralizing and the epitope pattern was extremely narrow, since it was limited to only one epitope. Thus, EP-based delivery was able to improve the dose efficiency of DNA vaccine and to maintain a highly neutralizing, multi-specific B-cell response, suggesting that it may be an effective approach for chronic hepatitis B therapy at clinically feasible DNA dose.

We previously demonstrated that MIBP1 and RFX1 polypeptides associate in vivo to form a complex that binds to the MIF-1 element in the c-myc gene and the major histocompatibility complex class II X-box recognition sequence. We now show that the EP element, a key regulatory sequence within hepatitis B virus enhancer I, also associates with MIBP1 and RFX1. Using polyclonal antisera directed against either oligonucleotide-purified MIBP1 or a peptide derived from the major histocompatibility complex class II promoter-binding protein RFX1, we showed that MIBP1 and RFX1 are both present in the DNA-protein complexes at the EP site. In addition, while the EP element can act cooperatively with several adjacent elements to transactivate hepatitis B virus expression, we demonstrated that the EP site alone can repress transcription of simian virus 40 promoter in a position- and orientation-independent manner, suggesting a silencer function in hepatocarcinoma cells.

CRF, a hypothalamic peptide, is a potent stimulator of POMC synthesis and secretion in the pituitary. POMC biosynthesis has been documented in the testis, specifically in Leydig cells, and recent studies suggest that CRF is synthesized locally in the testis. A reverse hemolytic plaque assay and immunocytochemistry with Leydig cell-specific antibodies were used to study the effect of CRF on secretion of the POMC peptide beta-endorphin (beta EP) from normal rat primary Leydig cell cultures. In enriched Leydig cell preparations incubated with beta EP antiserum (diluted 1:50) then with complement (diluted 1:25), approximately 15% of immunocytochemically identified Leydig cells formed plaques. Preabsorption of the antiserum with beta EP (2 micrograms/microliters antiserum) overnight at 4 C abolished the formation of plaques. Increasing concentrations of CRF (from 10(-1) to 10(-7) M) resulted in an approximately 80% increase in both the percentage of plaque-forming cells and the mean plaque size. When the CRF antagonist CRF-(9-41) (10(-6) M) was added in the presence of CRF, the increases in plaque number and average size did not occur. These results demonstrate that Leydig cells have functional CRF receptors and that beta EP secretion from these cells is stimulated by CRF.

We observe an enormous spontaneous exchange bias (~300-600 Oe)--measured in an unmagnetized state following zero-field cooling--in a nanocomposite of BiFeO(3) (~94%)-Bi(2)Fe(4)O(9) (~6%) over a temperature range 5-300 K. Depending on the path followed in tracing the hysteresis loop--positive (p) or negative (n)--as well as the maximum field applied, the exchange bias (H(E)) varies significantly with | - H(Ep) |>> | H(En) |. The temperature dependence of H(E) is nonmonotonic. It increases, initially, till ~150 K and then decreases as the blocking temperature T(B) is approached. All these rich features appear to be originating from the spontaneous symmetry breaking and consequent onset of unidirectional anisotropy driven by "superinteraction bias coupling" between the ferromagnetic core of Bi(2)Fe(4)O(9) (of average size ~19 nm) and the canted antiferromagnetic structure of BiFeO(3) (of average size ~112 nm) via superspin glass moments at the shell.

Activin A as a predictor of pregnancy failure has been the focus of heated debate, but the value of a combined activin A and follistatin (FS) measurement in serum to predict pregnancy failure has not been reported yet. We assessed whether a single serum measurement of the two physiological antagonists at 6-8 weeks gestation could differentiate ectopic pregnancies (EP) or missed abortions (MA) from healthy intrauterine pregnancies (IUP). activin A concentrations were significantly lower in women with EP (n = 30, median value of 264 pg/mL) and women with MA (n = 30, median value of 350 pg/mL) compared to IUP (n = 33, median value of 788 pg/mL); P < 0.001. At a threshold value of 505 pg/mL, activin A had 87.9% sensitivity and 100% specificity and negative predictive value of 0.974 for discriminating an ectopic pregnancy from viable pregnancies. FS was able to discriminate IUP from EP (ROC curve P < 0.001) as was their ratio (ROC curve P = 0.008), but was unable to discriminate a MA from an EP. In EP, activin A did not correlate with beta HCG levels. The present findings support the thesis that activin A or FS could be considered promising biomarkers for the discrimination between an IUP and a failed pregnancy (MA or EP).

OBJECTIVE

We investigated genotoxic effects of occupational exposure to lead acetate in pottery-glaze ceramic workers.

METHODS

The study was carried out in 30 exposed workers and 30 matched controls, to whom several biochemical parameters-the blood lead (B-Pb; range: exposed, 41.68-404.77; controls, 12-52) and cadmium (B-Cd) level, the activity of delta-aminolevulinic acid dehydratase (ALAD), erythrocyte protoporphyrin (EP), the level of vitamin B(12) and folate in serum-were measured. The genotoxic effects were evaluated by the alkaline comet assay, the DNA diffusion assay and micronucleus test in peripheral blood lymphocytes.

RESULTS

Subjects exposed to lead had significantly higher B-Pb level and, consequently, increased values of tail intensity (TI), frequency of apoptotic and necrotic cells, and frequency of micronuclei (MN). In contrast, their activity of ALAD, the level of vitamin B(12) and folate in serum were significantly lower compared to controls. Poisson regression analysis showed a significant correlation of profession, duration of exposure, smoking, level of cadmium in blood, ALAD and EP with primary DNA damage. A majority of primary damage repairs in a short period after exposure to a genotoxic agent. In addition, the influence of gender and level of vitamin B(12) and folate in serum MN frequency in exposed group was observed.

CONCLUSIONS

In this study, DNA diffusion and micronucleus test showed higher influence of tested parameters to DNA damage. The results indicate a need for concomitant use of at least two different biomarkers of exposure when estimating a genetic risk of lead exposure.

The reason for the particularly increased risk for cardiovascular complications in diabetic women is still unclear. We have previously found decreased distensibility of elastic arteries in type I diabetic women, indicating increased cardiac load, not seen in type I diabetic men, which might be one contributing factor. Whether the effect of gender is different in muscular arteries in type I diabetic patients has not been assessed. As estimates of arterial distensibility we measured stiffness (beta) and pressure strain elastic modulus (Ep) in the muscular common femoral artery using echo-tracking sonography in 30 women (mean age 34 years, range 20-61) and 26 men (mean age 38 years, range 22-56) with type I diabetes. The results were compared with those of 89 healthy individuals of corresponding age and gender and with previously published results from elastic arteries in these patients obtained at the same occasion. The internal common femoral diameter was significantly decreased in both diabetic men and women. In sharp contrast to the highly significant decreased distensibility of the elastic abdominal aorta and common carotid artery in the type I diabetic women, the distensibility of the common femoral artery did not clearly differ between patients and controls, neither for women nor for men. Thus, the gender difference in changes of arterial distensibility found in elastic arteries was absent or far less obvious in the femoral artery. In conclusion, female gender seems to affect the mechanical properties of elastic, but not of large muscular arteries in type I diabetic patients. Thus, putative gender differences in arterial changes in type I diabetes are to be sought in elastic rather than muscular arteries.

Brain beta-endorphin (beta-EP) was measured in the rat during pregnancy, parturition, and the postpartum period. beta-EP increased in the hypothalamus, midbrain, and amygdala during gestation and remained elevated through delivery until 1-2 days postpartum. The concentration of beta-EP increased in the hypothalamus from 31.8 +/- 1.4 (+/- SE) ng/mg protein in nonpregnant controls to 41.4 +/- 1.8 and 39.2 +/- 1.9 during early (8-10 days) and late (18-20 days) pregnancy, respectively, and in the midbrain from 3.20 +/- 0.17 to 5.21 +/- 0.30 and 5.25 +/- 0.64 ng/mg protein (P less than 0.01). In another experiment, the brain content of beta-EP expressed as nanograms per region, increased from 12.6 +/- 0.29 to 14.7 +/- 0.33 in the hypothalamus, from 4.09 +/- 0.44 to 6.03 +/- 0.34 in the midbrain, and from 0.93 +/- 0.11 to 1.32 +/- 0.06 ng in the amygdala at 16-17 days of gestation compared with that in nonpregnant controls (P less than 0.01). When hypothalamic beta-EP was measured 1 week postpartum in lactating and nonlactating rats, a significant decline in the beta-EP concentration of both groups was noted compared with that measured during pregnancy; beta-EP levels were similar in the lactating and nonlactating rats. We conclude that pregnancy and parturition are associated with significant changes in brain beta-EP and suggest that beta-EP of central rather than peripheral origin may mediate changes in pain perception and maternal behavior during pregnancy.

In the rat, body temperature (bt) is highest, and plasma iron (Fe) and zinc (Zn) concentrations are lowest at night while the rat is most active; the inverse is true during the day. Based on data implicating endogenous pyrogen (EP) as a mediator of the rise in body temperature and fall in plasma trace metal levels during infection we hypothesized that the circadian rise in body temperature and fall in plasma Fe and Zn levels may be attributed to a cyclic release of EP. To test this hypothesis: (1) Rats were injected ip with an antipyretic dose of sodium salicylate (300 mg/kg). The result was a reduction (P less than 0.05) in bt at night. (2) Rats were injected during the day with 1 ml each of plasma collected from rats during the night. As a control, rat plasma collected during the day was injected at this same time point. A rise (P less than 0.05) in bt was observed only in animals who had received plasma collected at night. These results support the hypothesis that a pyrogen, perhaps EP, is present in the plasma of rats at night. The release of EP during periods of greatest activity may have an adaptive role since rats are more likely to come into contact with pathogens during these times. If EP were released during periods of activity, the likelihood of severe infection occurring would be diminished. To test this hypothesis, two groups of rats were injected with Salmonella typhimurium, one group at midnight (A) and one group at noon (B). The mortality rate was 25% in group A and 60% in group B (P less than 0.025). These data support the hypothesis that the immune/host defense of rats to S. typhimurium is more effective at night, possibly due to an increased level of circulating pyrogen.

The chick model has been instrumental in illuminating genes that regulate early vertebrate development and pattern formation. Targeted ectopic gene expression is critical to dissect further the complicated gene interactions that are involved. In an effort to develop a consistent method to ectopically introduce and focally express genes in chick mesoderm, we evaluated and optimized several gene delivery methods, including implantation of 293 cells laden with viral vectors, direct adenoviral injection, and electroporation (EP). We targeted the mesoderm of chick wing buds between stages 19 and 21 (Hamburger and Hamilton stages) and used beta-galactosidase and green fluorescent protein (GFP) to document gene transfer. Expression constructs using the cytomegalovirus (CMV) promoter, the beta-actin promoter, and vectors with an internal ribosomal entry sequence linked to GFP (IRES-GFP) were also compared. After gene transfer, we monitored expression for up to 3 days. The functionality of ectopic expression was demonstrated with constructs containing the coding sequences for Shh, a secreted signaling protein, or Hoxb-8, a transcription factor, both of which can induce digit duplication when ectopically expressed in anterior limb mesoderm. We identified several factors that enhance mesodermal gene transfer. First, the use of a vector with the beta-actin promoter coupled to the 69% fragment of the bovine papilloma virus yielded superior mesodermal expression both by markers and functional results when compared with several CMV-driven vectors. Second, we found the use of mineral oil to be an important adjuvant for EP and direct viral injection to localize and contain vector within the mesoderm at the injection site. Lastly, although ectopic expression could be achieved with all three methods, we favored EP confined to the mesoderm with insulated microelectrodes (confined microelectroporation- CMEP), because vector construction is rapid, the method is efficient, and results were consistent and reproducible.

Eight obese patients (exceeding ideal body weight by 50% or more) with no endocrinological or metabolic disorders and 8 healthy, age-matched, normal-weight volunteers were submitted to an overnight short dexamethasone (DXM) suppression test and to a psychological assessment through various psychometric scales. Plasma B-Endorphin (B-EP), B-Lipotropin (B-LPH), ACTH and cortisol concentrations were evaluated in basal conditions, as well as 9 and 17 hours after late night administration of 1 mg DXM in both groups. All hormones were measured by radioimmunoassay, either directly in the plasma (ACTH and cortisol) or after silicic acid extraction and Sephadex G-75 column chromatography (B-LPH and B-EP). In obese patients, plasma B-EP levels in basal conditions were three times higher than in normal weight controls and remained unaltered by DXM suppression. ACTH and B-LPH, in contrast, were within the normal range and were significantly reduced by DXM. In 3 of the 8 patients, plasma cortisol concentrations at 17 hours post-DXM were greater than 50 ng/ml indicating an early escape from the suppression. Psychometric evaluations revealed a prevalence of depressive personality in obese patients. These data indicate an hypersecretion of B-EP in obese patients, which is only partially dependent on hypothalamic control.

In the present study we have examined the response of endogenous opiates (beta-EP and Met-enk) and ACTH to a particular type of thermal stimulus such as sauna in 8 young healthy subjects. Sauna-induced hyperthermia resulted in an increase of plasma beta-EP and ACTH, but appeared to have no significant effect on circulating Met-enk. The different responses of ACTH, beta-EP and Met-enk to heat exposure indicate that hyperthermia represents a form of stress, which can trigger off a well-defined neuroendocrine reaction.

BACKGROUND

Unknown primary tumors are highly malignant diseases which portend a dire prognosis. We designed a prospective high dose-intensity policy with the aim of improving the results obtained with conventional chemotherapy.

METHODS

Chemotherapy regimens were determined according to clinical features. In patients younger than 61 years with an ECOG performance status of 0 or 1, poorly differentiated adenocarcinoma or poorly differentiated carcinoma, and no evidence of brain or bone marrow involvement (group A), the treatment plan included four sequential high-dose courses with hematopoietic progenitor cell and growth factor support. Peripheral blood progenitor cells were collected by apheresis as the leukocyte counts recovered from the nadir induced by the first cycle of chemotherapy (doxorubicin 75 mg/m2, cyclophosphamide 6000 mg/m2). Patients then received two cycles of etoposide (800 mg/m2) and carboplatin (900 mg/m2) separated by one cycle of doxorubicin (75 mg/m2) and cyclophosphamide (3000 mg/m2). G-CSF (5 micrograms/kg/d) was given until engraftment. It was planned that cycles would be delivered every three weeks. The remaining patients (group B) received alternative cycles of AC (doxorubicin 50 mg/m2, cyclophosphamide 1000 mg/m2) and EP (etoposide 300 mg/m2, cisplatin 100 mg/m2). Cycles were given at two-week intervals with GM-CSF support (5 micrograms/kg/d) from day 4 to day 10. Patients without measurable lesions were included, since the major endpoint was survival.

RESULTS

Sixty patients entered the study. Twenty patients were assigned to group A and 40 patients to group B. In group A, 5 of 12 patients with measurable lesions (42%; 95% confidence interval (95% CI): 22%-62%) achieved major responses to chemotherapy, including one complete response. The duration of the overall median survival was 11 months. In group B, a major response was observed in 12 (39%; 95% CI: 28%-50%) of 31 patients with measurable lesions, including three complete responses. The overall median survival was 8 months. Hematological toxicities were noteworthy in both groups. Two toxic deaths occurred in group B.

CONCLUSIONS

Using these doses and schedules of chemotherapy, a high-dose intensity policy does not appear to improve the outcome of patients with carcinoma of unknown primary site. Alternative studies dealing with new drugs are required.

BACKGROUND

Much research has confirmed the favorable effect of irinotecan/cisplatin (IP) and etoposide/cisplatin (EP) on extensive-stage small cell lung cancer (E-SCLC). This study investigated two sequential orders of IP and EP in the treatment of E-SCLC. We also compared the efficacy and safety of IP and EP in first-line chemotherapy in E-SCLC.

METHODS

Ninety-three untreated patients with E-SCLC were randomly allocated to two groups. Group A received IP as first-line therapy until progression and then changed to EP; group B received EP as first-line therapy until tumor progression followed by IP. The primary endpoints were overall survival and time to second tumor progression. The secondary endpoints were first progression-free survival (PFS), ie, time from randomization to first occurrence of tumor progression after first-line treatment with IP or EP, tumor response, and safety of the different sequential treatment orders of IP and EP.

RESULTS

Median overall survival was 15.4 months in group A (IP followed by EP) versus 15.7 months in group B (EP followed by IP; P=0.483). The median time to second tumor progression was 9.5 months in group A versus 9.9 months in group B (P=0.361). As first-line and second-line therapy, IP achieved a 95.9% and 60% disease control rate, respectively, and EP achieved 95.6% and 59% disease control rate. The median first PFS was not significantly different between group A and group B (6.5 months and 6.3 months, respectively; P=0.256). Grade 3/4 diarrhea appeared to be significantly more frequent with IP than with EP. The probability of anemia and thrombocytopenia was not significantly different between the two groups. However, significantly more patients who received the IP regimen as second-line treatment developed grade 3/4 neutropenia than those who received the IP regimen as first-line therapy.

CONCLUSIONS

There were no statistically significant differences in between the two sequences of IP and EP in the treatment of E-SCLC. Except EP regimen, IP may be another reserved regimen in the first-line treatment of E-SCLC.

We demonstrated the genotype-dependent removal of human norovirus particles with a microfiltration (MF) membrane in the presence of bacteria bearing histo-blood group antigens (HBGAs). Three genotypes (GII.3, GII.4, and GII.6) of norovirus-like particles (NoVLPs) were mixed with three bacterial strains (Enterobacter sp. SENG-6, Escherichia coli O86:K61:BBGAs as extracellular polymeric substances (EPS). This MF membrane removal of NoVLPs was not significant when EPS was removed from cells of Enterobacter sp. SENG-6. GII.6 NoVLP was not rejected with the MF membrane in the presence of E. coli O86:K61:BEPS of E. coli O86:K61:BB-antigen in lipopolysaccharide (LPS) of E. coli O86:K61:BBGA-negative strain, was mixed with NoVLPs. These results demonstrate that the location of HBGAs on bacterial cells is an important factor in determining the genotype-dependent removal efficiency of norovirus particles with the MF membrane. The presence of HBGAs in mixed liquor suspended solids from a membrane bioreactor (MBR) pilot plant was confirmed by immune-transmission electron microscopy, which implies that bacterial HBGAs can contribute to the genotype-dependent removal of human noroviruses with MBR using MF membrane.

Emphysema is the major component of chronic obstructive pulmonary disease (COPD). During emphysema, elastin breakdown in the lung tissue originates from the release of large amounts of elastase by inflammatory cells. Elevated levels of elastin-derived peptides (EP) reflect massive pulmonary elastin breakdown in COPD patients. Only the EP containing the GXXPG conformational motif with a type VIII β-turn are elastin receptor ligands inducing biological activities. In addition, the COOH-terminal glycine residue of the GXXPG motif seems a prerequisite to the biological activity. In this study, we endotracheally instilled C57BL/6J mice with GXXPG EP and/or COOH-terminal glycine deleted-EP whose sequences were designed by molecular dynamics and docking simulations. We investigated their effect on all criteria associated with the progression of murine emphysema. Bronchoalveolar lavages were recovered to analyze cell profiles by flow cytometry and lungs were prepared to allow morphological and histological analysis by immunostaining and confocal microscopy. We observed that exposure of mice to EP elicited hallmark features of emphysema with inflammatory cell accumulation associated with increased matrix metalloproteinases and desmosine expression and of remodeling of parenchymal tissue. We also identified an inactive COOH-terminal glycine deleted-EP that retains its binding-activity to EBP and that is able to inhibit the in vitro and in vivo activities of emphysema-inducing EP. This study demonstrates that EP are key actors in the development of emphysema and that they represent pharmacological targets for an alternative treatment of emphysema based on the identification of EP analogous antagonists by molecular modeling studies.

BACKGROUND

Echinacea purpurea L. (EP) is a popular herbal antioxidant and immunomodulator. The present study was conducted to evaluate the effects of EP on meat quality and oxidative status in broilers. Two hundred and fifty (1-day-old) male broilers (Arbor Acres) were randomly allocated to five groups including the control (corn-soybean meal diet) and 0.1, 0.5, 1.0 and 2.0% EP powder groups, with two replicates per treatment group.

RESULTS

The results indicated that the addition of 0.5% and 1.0% EP significantly increased water-holding capacity and decreased storage loss of breast and thigh fillets at 35 days old. For fillet colour, L* (lightness) values were lower, and a* (redness) and b* (yellowness) values were higher with EP supplementation. Lower crude fat contents were observed in EP groups in comparison with control at 35 days of age in breast and thigh fillets, respectively. Production of malondialdehyde was slightly reduced in serum of EP supplemented birds compared to the control group. Results for Trolox equivalent antioxidant capacity, catalase and superoxide dismutase were significantly higher for the 0.5, 1.0 and 2.0% EP supplemental groups than control group in serum. Liver and spleen tissues results showed that the antioxidative enzymes activities were higher with EP powder at 35 days of age.

CONCLUSIONS

Dried EP can be used as a feed additive to improve the meat quality and oxidative status in Arbor Acres broilers.

We investigated the anti-tumor effect of human interferon-beta (HuIFN-beta) against malignant melanoma. In vitro study revealed that HuIFN-beta not only inhibited proliferation of melanoma cells (seven cell lines: MM-AN, MM-BP, MM-LH, MM-RU, PM-WK, RPM-EP, RPM-MC) but also induced apoptosis in a dose dependent fashion, though the sensitivity to HuIFN-beta was different among cell lines. In addition, we administered HuIFN-beta into cutaneous metastatic lesions of melanoma and evaluated clinical and histopathological effects. Although the size of the metastatic cutaneous lesion did not change by the intralesional injection of HuIFN-beta, histopathological examination revealed apoptotic changes of melanoma cells along with dense lymphohistiocytic infiltration. The present study confirmed direct and indirect inhibitory effects of HuIFN-beta on human melanoma cells and suggests that local higher concentration of HuIFN-beta is needed to eradicate melanoma lesions.

EPS-1 was an exopolysaccharide produced by the medicinal fungus Cordyceps sinensis (Cs-HK1). In the present study, EPS-1 was sulfated with chlorosulfonic acid (CSA)-pyridine (Pyr) at different volume ratios, yielding four sulfated derivatives, SEPS-1A, B, C and D, with different degrees of substitution (DS: 0.25-1.38) and molecular weights (17.1-4.1 kDa). The sulfation of EPS-1 occurred most frequently at the C-6 hydroxyl groups due to their higher reactivity. In aqueous solution, the native EPS-1 formed random coils or aggregated networks, but the sulfated derivatives formed single helices. The antioxidant activities of the sulfated EPS-1 derivatives for scavenging hydroxyl radicals (•OH) and 2,2-azinobis-3-ehtylbenzothiazolin-6-sulfonic acid radicals (ABTS•+) were significantly increased with increasing DS and decreasing molecular weight (MW). Sulfation has thus been shown to be an effective and favorable strategy for improving the physico-chemical properties and bioactivities of fungal polysaccharides.

Vascular changes are common in acromegaly (ACM). Current therapies can normalise the levels of both growth hormone (GH) and insulin-like growth factor (IGF1).

OBJECTIVE

To establish whether the ACM vascular changes in patients with effectively managed disease are different from those in patients with an active condition.

METHODS

64 ACM patients were tested for serum GH (random and during an oral glucose tolerance test) and IGF1. Ultrasonography of the right common carotid (RCC) explored structural (the carotid diameter and intima-media thickness index (IMT)) and functional (the augmentation index (AIx), elastic modulus (Ep), and local pulse wave velocity (PWV)) arterial parameters in the ACM patients (groups A and B) and an age- and sex-matched control group of 21 patients without acromegaly (group C).

RESULTS

The ACM patients were divided into 2 subgroups that had similar cardiovascular risk factor profiles: A (n=10, with controlled ACM), and B (n=54, with active ACM). The AIx was higher in groups A (27.7% [2.2-54.3]) and B (20.0% [ - 38.2-97.1]) than in group C (3.5% [ - 11.3-31.1]), p=0.01 and 0.002, respectively. The group B patients presented with poorer functional carotid wall parameters than the control subjects: Ep-95.5 [33-280] KPa vs. 77.5 [39-146] KPa, p=0.01; and PWV-6 [3.6-10.4] m/s vs. 5.4 [3.9-7.2] m/s, p=0.03.The ACM patients had greater RCC diameters (6.4 ± 0.6 mm vs. 5.7 ± 0.6 mm, p<0.001) and IMT values (0.72 ± 0.13 mm vs. 0.58 ± 0.08 mm, p<0.001) than the subjects in group C.

CONCLUSIONS

Both the controlled and active ACM patients showed structural arterial changes. After 1 year of disease control, the patients with controlled ACM showed improvements in the functional, but not the structural, arterial parameters compared with the patients with an active condition.

Adenylyl cyclases (ACs) are important regulators of airway smooth muscle function, because β-adrenergic receptor (βAR) agonists stimulate AC activity and cAMP production. We have previously shown in a number of cell types that AC6 selectively couples to βAR and these proteins are coexpressed in lipid rafts. We overexpressed AC2, AC3, and AC6 in mouse bronchial smooth muscle cells (mBSMCs) and human embryonic kidney (HEK)-293 cells by using recombinant adenoviruses and assessed their localization and regulation by various G protein-coupled receptors (GPCRs). AC3 and AC6 were expressed primarily in caveolin-rich fractions, whereas AC2 expression was excluded from these domains. AC6 expression enhanced cAMP production in response to isoproterenol but did not increase responses to butaprost, reflecting the colocalization of AC6 with β(2)AR but not E prostanoid type 2 receptor (EP(2)R) in lipid raft fractions. AC2 expression enhanced butaprost-stimulated cAMP production but had no effect on the β(2)AR-mediated response. AC3 did not couple to any GPCR tested. Forskolin-induced arborization of mBSMCs was assessed as a functional readout of cAMP signaling. Arborization was enhanced by overexpression of AC6 and AC3, but AC2 had no effect. GPCR-stimulated arborization mirrored the selective coupling observed for cAMP production. With the addition of the phosphodiesterase 4 (PDE4) inhibitor rolipram AC2 accelerated forskolin-stimulated arborization. Thus, AC2 selectively couples to EP(2)R, but signals from this complex are limited by PDE4 activity. AC3 does not seem to couple to GPCR in either mBSMCs or HEK-293 cells, so it probably exists in a distinct signaling domain in these cells.

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme beta-glucuronidase. The sequences encoding C28 and human enzyme beta-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGkappa signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-beta-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme beta-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug.

In oviparous vertebrates estrogens induce hepatic synthesis of vitellogenin (VG), a blood protein sequestered in vitellogenic oocytes and from which lipovitellin (LV) and phosvitin are derived. Our objective was to identify VG in the channel catfish, Ictalurus punctatus. An intraperitoneal injection of estradiol-17 beta into adult male fish induced a dose-dependent accumulation of a 150 kDa protein (EP) in the plasma. EP was detectable in Coomassie blue-stained polyacrylamide gels within 24 hr after injection of 2 mg hormone/100 g body weight. During the next 4 days, EP increased from 5 to about 25% of the total plasma protein. Electrophoretic mobility, peptide mapping, and immunological crossreactivity showed EP to be indistinguishable from a plasma protein in adult females with vitellogenic ovaries. Two major yolk polypeptides, YP1 (120 kDa) and YP2 (29.6 kDa), were precipitated by (NH4)2SO4 from a yolk protein extract. YP1 but not YP2 reacted with an anti-EP polyclonal antiserum in Western blots. Peptide mapping after proteolysis with trypsin showed YPs 1 and 2 to be unique and revealed structural homologies between YP1 and EP. Liver but not pancreatic explants from an estradiol-treated male synthesized and secreted a [35S]methionine-labeled, 150 kDa protein beginning about 2 hr after initial exposure to the label. We tentatively conclude that EP and YP1 represent VG and LV, respectively. YP2 remains unidentified.