Ecdysteroid metabolism in mammals: The fate of ingested 20-hydroxyecdysone in mice and rats.

Phytoecdysteroids are molecules derived from sterol metabolism and found in many plants. They display a wide array of pharmacological effects on mammals (e.g. anabolic, anti-diabetic). Although these effects have been long established, the molecular targets involved remain to be identified. Like endogenous steroid hormones and bile acids, which are biochemically related, ingested or injected phytoecdysteroids undergo a set of reactions in mammals leading to the formation of numerous metabolites, only some of which have been so far identified, and it is presently unknown whether they represent active metabolites or inactivation products. In the large intestine, ecdysteroids undergo efficient 14-dehydroxylation. Other changes (reductions, epimerization, side-chain cleavage) are also observed, but whether these occur in the liver and/or large intestine is not known. The purpose of this study was to investigate the pharmacokinetics of 20-hydroxyecdysone (20E), the most common phytoecdysteroid, when administered to mice and rats, using, when required, tritium-labelled molecules to permit metabolic tracking. Bioavailability, the distribution of radioactivity and the kinetics of formation of metabolites were followed for 24-48 hours after ingestion and qualitative and quantitative analyses of circulating and excreted compounds were performed. In mice, the digestive tract always contains the majority of the ingested 20E. Within 30 min after ingestion, 20E reaches the large intestine, where microorganisms firstly remove the 14-hydroxyl group and reduce the 6-one. Then a very complex set of metabolites (not all of which have yet been identified) appears, which correspond to poststerone derivatives formed in the liver. We have observed that these compounds (like bile acids) undergo an entero-hepatic cycle, involving glucuronide conjugation in the liver and subsequent deconjugation in the intestine. Despite the very short half-life of ecdysteroids in mammals, this entero-hepatic cycle helps to maintain their plasma levels at values which, albeit low (≤0.2 µM), would be sufficient to evoke several pharmacological effects. Similar 20E metabolites were observed in mice and rats; they include in particular 14-deoxy-20E, poststerone and 14-deoxypoststerone and their diverse reduction products; the major products of this metabolism have been unambiguously identified. The major sites of metabolism of exogenous ecdysteroids in mammals are the large intestine and the liver. The entero-hepatic cycle contributes to the metabolism and to maintaining a low, but pharmacologically significant, concentration of ecdysteroids in the blood for ca. 24 h after ingestion. These data, together with parallel in vitro experiments provide a basis for the identification of 20E metabolite(s) possibly involved in the physiological effects associated with ecdysteroids in mammals.

Peripheral inflammation exacerbates α-synuclein toxicity and neuropathology in Parkinson's models.

AIMS: Parkinson's disease and related disorders are devastating neurodegenerative pathologies. Since α-synuclein was identified as a main component of Lewy bodies and neurites, efforts have been made to clarify the pathogenic mechanisms of α-synuclein's detrimental effects. α-synuclein oligomers are the most harmful species and may recruit and activate glial cells. Inflammation is emerging as a bridge between genetic susceptibility and environmental factors co-fostering Parkinson's disease. However, direct evidence linking inflammation to the harmful activities of α-synuclein oligomers or to the Parkinson's disease behavioural phenotype is lacking. METHODS: To clarify whether neuroinflammation influences Parkinson's disease pathogenesis, we developed: (i) a 'double-hit' approach in C57BL/6 naive mice where peripherally administered lipopolysaccharides were followed by intracerebroventricular injection of an inactive oligomer dose; (ii) a transgenic 'double-hit' model where lipopolysaccharides were given to A53T α-synuclein transgenic Parkinson's disease mice. RESULTS: Lipopolysaccharides induced a long-lasting neuroinflammatory response which facilitated the detrimental cognitive activities of oligomers. LPS-activated microglia and astrocytes responded differently to the oligomers with microglia activating further and acquiring a pro-inflammatory M1 phenotype, while astrocytes atrophied. In the transgenic 'double-hit' A53T mouse model, lipopolysaccharides aggravated cognitive deficits and increased microgliosis. Again, astrocytes responded differently to the double challenge. These findings indicate that peripherally induced neuroinflammation potentiates the α-synuclein oligomer's actions and aggravates cognitive deficits in A53T mice. CONCLUSIONS: The fine management of both peripheral and central inflammation may offer a promising therapeutic approach to prevent or slow down some behavioural aspects in α-synucleinopathies.

The role of ozonized oil and a combination of tobramycin/dexamethasone eye drops in the treatment of viral conjunctivitis: a randomized clinical trial.

PURPOSE: To determine whether topical tobramycin 0.3%/dexamethasone 0.1% plus ozonized oil eye drops reduces clinical signs and infectious viral titers of presumed viral conjunctivitis more than tobramycin/dexamethasone eye drops alone. METHODS: Prospective, single-blind, randomized, parallel-groups trial. Eighty patients with a clinical diagnosis of presumed viral conjunctivitis were randomizedly divided into two treatment groups: a study group and a control group, 40 for each group. Patients in the study group received topical tobramycin 0.3%/dexamethasone 0.1% eye drops, plus ozonized oil eye drops, both four times daily; patients in the control group received only topical tobramycin 0.3%/dexamethasone eye drops four times daily. The treatment was for seven days in both groups. Swabs were taken from the conjunctival fornix for adenovirus PCR analysis on the day of recruitment and at seven days follow-up. Clinical signs were also recorded on the day of recruitment and at follow-up examination: the main outcomes were conjunctival injection and conjunctival chemosis, graded on a 4-point clinical scale, presence or absence of superficial punctate keratitis and subepithelial corneal infiltrates. RESULTS: No statistically significant difference was reached in adenoviral infection negativization between the two groups, although the study group showed a higher number of PCR negative results at seven days follow-up. PCR real time detected adenoviral infection in 17 of 24 patients on the day of recruitment and it was positive in 4 patients on the seventh day (viral positivity reduction of 76%). In the control group PCR was positive for adenovirus in 18 of 24 patients on the day of recruitment and in 7 patients at seven days follow-up (reduction of 61%). There was statistically significant difference on conjunctival clinical signs between the study and control groups. Significant difference was also found on superficial punctate keratitis resolution between the study and the control group. In the former superficial punctate keratitis was detected in 14 eyes on the first day and in 5 eyes after seven days while in the latter superficial punctate keratitis was found in 124 eyes on the first day and in 6 eyes on the seventh day. No difference was found in subepithelial corneal infiltrates appearance between the two groups. CONCLUSIONS: The use of ozonized-oil containing eye drops in combination with topical tobramycin 0.3%/dexamethasone 0.1% eye drops four times daily seems to reduce the signs of conjunctivitis, and the duration of viral infection, although it does not affect the subepithelial corneal infiltrates appearance.

Evaluation of the agreement between TNM 7th and 8th in a population-based series of cutaneous melanoma.

BACKGROUND: The 8th edition of TNM has introduced new rules for staging cutaneous melanoma. OBJECTIVE: To compare TNM 7th and 8th editions in defining pathological stages of melanoma. METHODS: A population-based series of 1847 skin melanoma from Romagna cancer registry (Italy) incident during 2003-2012 has been used to measure the agreement (with Cohen's kappa) between TNM 8th and 7th editions in defining melanoma stage. Disease-specific survival has been computed for each stage according to TNM 7th and 8th. RESULTS: The agreement between the two TNM editions was quite good when considered on average (kappa = 70.7%), moderate for stage I (61.5%), nearly perfect for stage II (95.0%), but extremely poor for stage III (8.1%). The overall melanoma-specific observed survival was 90.8% at 5 year and 88.9% at 10 year with a strong prognostic effect of stage. CONCLUSION: TNM 8th edition introduces several changes which do not seem really helpful in addressing the care of stage I melanoma and may complicate the definition and comparability of stage III.

Indoor air quality at life and work environments in Rome, Italy.

The air quality of three different microenvironments (school, dwelling, and coffee bar) located in the city of Rome, Italy, was assessed. Indoor and outdoor concentrations of polycyclic aromatic hydrocarbons (PAHs) associated with PM2.5 particles were determined during an intensive 3-week sampling campaign conducted in March 2013. In interiors, total particulate PAHs ranged from 1.53 to 4.96 ng/m(3) while outdoor air contained from 2.75 to 3.48 ng/m(3). In addition, gaseous toxicants, i.e., NO2, NOx , SO2, O3, and BTEX (benzene, toluene, ethyl-benzene, and xylene isomers), were determined both in internal and external air. To solve the origin of indoor and outdoor PAHs, several source apportionment methods were applied. Multivariate analysis revealed that emissions from motor vehicles, biomass burning for heating purposes, and soil resuspension were the major sources of PAHs in the city. No linear correlation was established between indoor and outdoor values for PM2.5 and BTEX; the respective indoor/outdoor concentration ratios exceed unity except for PM2.5 in the no smoking home and benzene in all school floors. This suggests that important internal sources such as tobacco smoking, cleaning products, and resuspension dust contributed to indoor pollution. Using the monitoring stations of ARPA Lazio regional network as reference, the percentage within PAH group of benzo[a]pyrene, which is the WHO marker for the carcinogenic risk estimates, was ca. 50% higher in all locations investigated.

Emotional demands as a risk factor for mental distress among nurses.

BACKGROUND: Although it is widely acknowledged that in certain occupations emotional demands may be a critical phenomenon for workers' health, this has been traditionally taken for granted and their role in the stress process has not often been directly assessed. OBJECTIVES: To examine the relationship between emotional demands and mental distress, adjusting for the potential effect of common psychosocial factors (workload, job control, social support, role stressors, and poor relationships) and personal psychological factors (i.e. having been diagnosed with anxiety or depressive disorder). METHODS: A cross-sectional study on a sample of nurses of the National Healthcare Service was carried out (N = 256, 81.3% women). The psychosocial factors considered were assessed by means of widely known and validated scales. The examined health outcome (i.e. mental distress) was operationalized by means of the General Health Questionnaire (12-item version). Covariates: gender, age, tenure and shiftwork. ANALYSES: a series of logistic regressions. RESULTS: Exposure to emotional demands was a risk factor for mental distress. The resulting risk was not altered when adjusting for other psychosocial and personal factors. In the final model emotional demands, workload and role stressors, in addition to having been diagnosed with anxiety or depressive disorder, were significant risk factors for nurses' mental distress. CONCLUSIONS: Emotional demands may substantially impact on nurses mental distress. These results give rise to concern in relation to work-stress prevention in certain professions, given that emotional demands are not included in the most common psychosocial risk assessment tools currently available, which may then miss identifying an important precondition of work stress.

Particle size distribution of n-alkanes and polycyclic aromatic hydrocarbons (PAHS) in urban and industrial aerosol of Algiers, Algeria.

The distribution of ambient air n-alkanes and polycyclic aromatic hydrocarbons (PAHs) associated to particles with aerodynamic diameters lesser than 10 µm (PM(10)) into six fractions (five stages and a backup filter) was studied for the first time in Algeria. Investigation took place during September of 2007 at an urban and industrial site of Algiers. Size-resolved samples (<0.49, 0.49-0.95, 0.95-1.5, 1.5-3.0, 3.0-7.2, and 7.2-10 µm) were concurrently collected at the two sampling sites using five-stage high-volume cascade impactors. Most of n-alkanes (~72 %) and PAHs (~90 %) were associated with fine particles ≤ 1.5 µm in both urban and industrial atmosphere. In both cases, the n-alkane contents exhibited bimodal or weakly bimodal distribution peaking at the 0.95-1.5-µm size range within the fine mode and at 7.3-10 µm in the coarse mode. Low molecular weight PAHs displayed bimodal patterns peaking at 0.49-0.95 and 7.3-10 µm, while high molecular weight PAHs exhibited mono-modal distribution with maximum in the <0.49-µm fraction. While the mass mean diameter of total n-alkanes in the urban and industrial sites was 0.70 and 0.84 µm, respectively, it did not exceed 0.49 µm for PAHs. Carbon preference index (~1.1), wax% (10.1-12.8), and the diagnostic ratios for PAHs all revealed that vehicular emission was the major source of these organic compounds in PM(10) during the study periods and that the contribution of epicuticular waxes emitted by terrestrial plants was minor. According to benzo[a]pyrene-equivalent carcinogenic power rates, ca. 90 % of overall PAH toxicity across PM(10) was found in particles ≤ 0.95 µm in diameter which could induce adverse health effects to the population living in these areas.

[Comparison between the risk of mobbing at work and conditions of job strain and effort-reward imbalance in relation to stress-related disorders: a study in the public administration]. / Confronto tra rischio mobbing e condizioni di job strain ed effort-reward imbalance in relazione a disturbi stress-correlati: studio nella pubblica amministrazione.

BACKGROUND: A limitation of previous research on mobbing at work was that the individual consequences of the phenomenon were often assessed on 'clinical' samples of victims. Studies in organizations are less numerous and those available rarely evaluated the potential effect of mobbing over and above that of other psychosocial factors which provided robust evidence of adverse health effects. OBJECTIVES: The relationship between exposure to mobbing and stress-related psychophysical conditions was investigated taking in consideration the possible concomitant exposure to job strain and effort-reward imbalance. METHODS: Cross-sectional study on public administration administrative employees (No. = 538, 48% women). The psychosocial factors considered were assessed by means of well known and validated scales. The four health outcomes considered were psychological caseness (as operationalized by means of the general health questionnaire-12-item version), depression, gastritis and colitis/irritable colon syndrome as indicated by having reported a medical diagnosis of such conditions. Covariates: gender, age, body mass index, smoking habits and experience of a traumatic event in the last year. Analysis consisted of a series of logistic regressions. RESULTS: Exposure to mobbing was significantly associated with all the outcomes considered over and above the covariates. The inclusion in the models of job strain and effort-reward imbalance did not substantially alter the results. CONCLUSIONS: The relationship between mobbing at work and health outcomes is not confounded by concomitant exposure to job strain and effort-reward imbalance. These results offer further confirmation of the uniqueness of mobbing as a psychosocial risk factor.

Attention deficit induced by blockade of N-methyl D-aspartate receptors in the prefrontal cortex is associated with enhanced glutamate release and cAMP response element binding protein phosphorylation: role of metabotropic glutamate receptors 2/3.

The hypothesis that attention deficits induced by the hypofunction of N-methyl d-aspartate (NMDA) receptors in the prefrontal cortex (PFC) might be associated with increased glutamate release and changes in the phosphorylation of the cyclic adenosine monophosphate response element-binding protein on serine 133 (p-S(133)CREB) was investigated in this study. Infusion of 50 ng/side 3-(R)-2-carboxypiperazin-4-propyl-1-phosphonic acid ((R)-CPP), a competitive glutamate NMDA receptor antagonist, into the medial prefrontal cortex (mPFC) of rats performing the five-choice serial reaction time (5-CSRT) task, reduced accuracy of visual discrimination (measured by % correct responses) and enhanced impulsivity (measured by the number of premature responses) and compulsivity (measured by the number of perseverative responses). The mGluR2/3 receptor agonist, LY379268, injected s.c. at 0.1 mg/kg, reduced (R)-CPP-induced impairment in attentional functioning (accuracy) and impulsivity but not compulsive perseveration. In parallel studies using microdialysis technique and Western blot analysis we found that (R)-CPP (100 µM) infused in the medial prefrontal cortex increased glutamate efflux whereas injected in the medial prefrontal cortex at a dose causing impairments in attentional performance (50 ng/side) increased p-S(133)CREB in the frontal cortex (FC), decreased it in the caudate-putamen (CPu) and was without effect in the nucleus accumbens (NAC). LY379268 at the dose effective in reducing (R)-CPP-induced behavioral deficit reduced both the (R)-CPP-induced rise in glutamate efflux in the prefrontal cortex and the increase in p-S(133)CREB in the frontal cortex but was without effect on the decrease in p-S(133)CREB in the caudate-putamen. The data provide evidence that enhanced glutamate release and phosphorylation of cAMP response element binding protein (CREB) on serine 133 may be associated to attention deficit and loss of impulse control. Furthermore they suggest that mGluR2/3 agonists have a therapeutic potential for cognitive deficits.

Anticonvulsant effects and behavioural outcomes of rAAV serotype 1 vector-mediated neuropeptide Y overexpression in rat hippocampus.

Neuropeptide Y (NPY) is an endogenous peptide with powerful anticonvulsant properties. Its overexpression in the rat hippocampus, mediated by the local application of recombinant adeno-associated viral (rAAV) vectors carrying the human NPY gene, results in significant reduction of seizures in acute and chronic seizure models. In this study, we characterized a more efficient rAAV-NPY vector to improve cell transfection in the injected area. The changes included pseudotyping with the AAV vector serotype 1 (rAAV1), and using the strong constitutive hybrid CBA promoter, which contains a cytomegalovirus enhancer and chicken beta-actin promoter sequences. We compared NPY expression and the associated anticonvulsant effects of this new vector, with those mediated by the former rAAV vector with chimeric serotype 1/2 (rAAV1/2). In addition, we investigated whether rAAV serotype 1 vector-mediated chronic NPY overexpression causes behavioural deficits that may detract from the clinical utility of this therapeutic approach. We report that rAAV-NPY serotype 1 vector has significantly improved anticonvulsant activity when compared with serotype 1/2 vector, as assessed by measuring EEG seizure activity in kainic acid treated rats. rAAV1-mediated NPY overexpression in naive rats did not result in alterations of physiological functions such as learning and memory, anxiety and locomotor activity. In addition, we did not observe glia activation, or humoral immune responses against serotype 1 vector, which could inactivate gene expression. Our findings show that rAAV1-NPY vector with the CBA promoter mediates powerful anticonvulsant effects and seems to be safe in rodents, thus it may be considered a vector of choice for possible clinical applications.

Evaluation of principal cannabinoids in airborne particulates.

The determination of delta(9)-tetrahydrocannabinol (Delta9-THC), cannabidiol (CND) and cannabinol (CNB), primary active components in cannabis preparation, was carried out on airborne particulates by applying a specific procedure consisting of soot extraction by ultrasonic bath, purification by solvent partitioning, derivatization with N-(t-butyldimethylsilyl)-N-methyl-trifluoroacetamide, and separation/detection through gas chromatography coupled with tandem mass spectrometry. The optimized procedure was found suitable for measuring the three psychotropic substances at concentrations ranging from ca. 0.001 to ca. 5.0 ng cm(-3) of air, with recoveries always higher than 82%, accuracy >7.3% and precision >90%. Application of the procedure performed on field in Rome and Bari, Italy, demonstrated that all three compounds contaminate the air in Italian cities whereas in Algiers, Algeria, only cannabinol, the most stable in the atmosphere, exceeded the limit of quantification of the method. The relative percentages of the three cannabinoids in general reproduced those typical of the Cannabis sativa plant and were very different from those found in human blood, urine and sweat.

Cerebrospinal fluid beta-glucocerebrosidase activity is reduced in Dementia with Lewy Bodies.

The autophagy-lysosomal degradation pathway plays a role in the onset and progression of neurodegenerative diseases. Clinical and genetic studies indicate that mutations of beta-glucocerebrosidase represent genetic risk factors for synucleinopathies, including Parkinson's Disease (PD) and Dementia with Lewy Bodies (DLB). We recently found a decreased activity of lysosomal hydrolases, namely beta-glucocerebrosidase, in cerebrospinal fluid of PD patients. We have thus measured the activity of these enzymes - alpha-mannosidase (EC 3.2.1.24), beta-mannosidase (EC 3.2.1.25), beta-glucocerebrosidase (EC 3.2.1.45), beta-galactosidase (EC 3.2.1.23) and beta-hexosaminidase (EC 3.2.1.52) - in cerebrospinal fluid of patients suffering from DLB, Alzheimer's Disease (AD), Fronto-Temporal Dementia (FTD) and controls. Alpha-mannosidase activity showed a marked decrease across all the pathological groups as compared to controls. Conversely, beta-glucocerebrosidase activity was selectively reduced in DLB, further suggesting that this enzyme might specifically be impaired in synucleinopathies.

Funtional characterization of four novel MAN2B1 mutations causing juvenile onset alpha-mannosidosis.

Alpha-mannosidosis is a recessively inherited disorder due to the deficiency of the lysosomal alpha-mannosidase. We report the molecular analysis performed in two patients with the late onset form of alpha-mannosidosis. Four new alleles were identified: three missense mutations involving highly conserved residues, c.597 C>A (p.H200N), c.1553 T>C (p.L518P) and c.2746 C>A (p.R916S) and a single nucleotide deletion, c.2660delC. In vitro expression studies in COS-1 cells demonstrated that pH200N, p.L518P and p.R916S proteins are expressed but retained no residual enzyme activity. These data are supported by structural 3D analysis which predicted that both p.L518P and p.R916S could affect the interaction of the small E-domain with the active site domain or the main body of the structure while the pH200N might alter substrate binding or other catalytic properties. Finally, the c.2660delC causes a frameshift introducing a premature stop codon (p.T887SfsX45), presuming to be a severe mutation.

[Reference values of urinary mercury in the Italian population]. / Valori di riferimento del mercurio urinario nella popolazione Italiana.

This paper shows the results of a polycentric study performed to assess the reference values of urinary mercury (U-Hg) in Italian population. 374 subjects from four Italian cities (Bari, Brescia, Genova e Siena) have been examined. A questionnaire on life style, dietary habits, occupational or environmental exposure to Hg and clinical history has been administered to every participant and number and surface of dental amalgams have been verified for all subjects. The determination of U-Hg has been performed on urinary extemporary samples by hydride generation atomic absorption method (HG-AAS); urinary creatinine has been determinated to reduce the intraindividual variability. U-Hg reference values were: 0.21-3.20 micrograms/g creat (5 degrees and 95 degrees percentile) and 0.12-6.04 micrograms/g creat (range). Moreover study results have shown that number and surface of dental amalgams, dietary fish intake and body mass index (BMI) influenced significatively U-Hg excretion. U-Hg reference values from this polycentric study resulted comparable to those assessed in other European countries, whereas the mean U-Hg observed in the referent Italian population was lower.

Reversal of visual attention dysfunction after AMPA lesions of the nucleus basalis magnocellularis (NBM) by the cholinesterase inhibitor donepezil and by a 5-HT1A receptor antagonist WAY 100635.

RATIONALE: Degeneration of the cholinergic magnocellular neurons in the basal forebrain and their cortical projections is a major feature of the neuropathology of Alzheimer's disease (AD). In addition to memory dysfunction, attentional functions are also impaired in AD. OBJECTIVE: We investigated the extent to which the cholinesterase inhibitor donepezil reversed the attentional performance deficit in nucleus basalis magnocellularis (NBM) lesioned rats. We also examined the effects of a selective and potent 5-HT(1A) receptor antagonist, WAY 100635, on the attentional deficit of NBM lesioned rats. METHODS: We injected alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) into the NBM to selectively destroy cholinergic neurons projecting to the neocortex. Attentional functions were examined using the 5-CSRT task, in which hungry rats were required to locate brief visual targets presented randomly in one of five locations in a specially designed chamber. RESULTS. AMPA lesions of the NBM caused marked reductions in choline acetyltransferase activity (ChAT) ranging from 30 to 46% in medial areas of the cortex (medial-frontal and cingulate) and from 58 to 72% in more lateral areas (anterior-dorso-lateral and parietal). AMPA lesioned rats made fewer correct responses (choice accuracy), longer latency to correct response and an increase in the number of premature and perseverative responses. These impairments showed some recovery over the next 12 weeks. Reducing the duration of the visual stimulus reinstated the impairments in choice accuracy. The anticholinesterase inhibitor donepezil at 1.0 mg/kg but not 0.5 mg/kg reversed the impairments in choice accuracy and correct response latency. The premature and perseverative over-responding of AMPA lesioned rats remained unchanged. A dose of 0.1 mg/kg WAY 100635 to AMPA-lesioned rats improved their choice accuracy but did not shorten correct response latencies. The number of premature responses was reduced by WAY 100635 but perseverative over-responding was not affected. CONCLUSIONS: The attentional impairments induced due to cortical cholinergic dysfunction may be ameliorated by cholinergic treatments such as cholinesterase inhibitors. In addition, 5-HT(1A) receptors and the cortical cholinergic system exert balanced opposition in regulating attentional performance in the rat. Blockade of 5-HT(1A) receptors may be useful to treat some aspects of attentional dysfunction in AD.

Biomembranes enriched with TGFbeta1 favor bone matrix protein expression by human osteoblasts in vitro.

The use of growth factors in oral tissue regeneration is currently under investigation. When growth factors are combined with commercial materials, the in vitro mechanisms of action still remain unclear. The present study first evaluated the capacity of barrier membranes, used in oral surgery, to sequester TGFbeta(1). Resorbable HYAFF, paroguide, poly DL-lactide and nonresorbable PTFE membranes were immersed in MEM containing 0.2 ng (125)I-TGFbeta(1) for different periods of time. It was found that HYAFF membrane and paroguide sequestered the most TGFbeta(1), which was then released in its active form (as shown by the CCL64 cell line bioassay). Untreated membranes and membranes enriched with TGFbeta(1) were then used as substrate for human bone cells to evaluate the synthesis of the osteoblast phenotype, as indicated by specific parameters. Results showed that membranes enriched with TGFbeta(1) increased alkaline phosphatase activity, collagen, and osteocalcin production more than untreated membranes. HYAFF and paroguide membranes, which sequestered the most of TGFbeta(1), were the most suitable for stimulating bone matrix proteins.

Time-dependent induction of anxiogenic-like effects after central infusion of urocortin or corticotropin-releasing factor in the rat.

RATIONALE: Corticotropin-releasing factor (CRF) and urocortin (Ucn) belong to the CRF-related family, share a high degree of structural homology and bind to CRF receptors. However, compared with CRF, Ucn was shown to display either weaker or similar anxiogenic-like effects in vivo. OBJECTIVE: To compare the anxiogenic-like responses of rats injected intracerebroventricularly (ICV) with different doses of either rat/human CRF (r/hCRF) or rat Ucn (rUcn) at different intervals after injection. METHODS: Rats were tested on three validated paradigms of emotional behavior [i.e. elevated plus-maze (EPM), defensive withdrawal (DW) and conflict test (CT)] 5 and 30 min after treatment. RESULTS: In the EPM test only r/hCRF, but not rUcn, produced anxiogenic-like effects at the dose of 1.0 microg, when the peptides were injected 5 min before testing. At 30 min after injection, both peptides caused a significant reduction of open arms exploration, rUcn being effective at 0.01 microg. In the DW test both peptides were equally potent in decreasing the exploratory behavior and increasing the time spent in the chamber at the dose of 1.0 microg when tested 30 min after injection. In the CT both rUcn (0.25-1.0 microg) and r/hCRF (0.75-1.0 microg) decreased significantly the responding in the punished component. However, rUcn reduced food responding also in the unpunished component possibly due to its powerful anorectic activity. CONCLUSIONS: Comparison of anxiogenic-like activities of r/hCRF and rUcn at doses up to 1.0 microg revealed striking differential effects that depended on the time of testing after ICV peptide injection, and on the paradigm of anxiety used. These results suggest that the onset of r/hCRF and rUcn actions related to behavioral responses to anxiety is likely to depend on brain peptide-specific mechanisms including binding properties to CRF-receptors, differential distribution to specific functional brain sites and the distribution and effectiveness of binding-protein interactions.

Synthesis and secretion of transforming growth factor-beta1 by human desmoid fibroblast cell line and its modulation by toremifene.

The present study provides evidence that the in vitro cultured fibroblast cell line from desmoid tumors differs from normal fibrobasts in its extracellular matrix (ECM) macromolecule composition and is modulated by treatment with toremifene, an antiestrogen that reduces tumor mass by an unknown mechanism. The results showed increased transforming growth factor-beta 1 (TGF-beta1) production, TGF-beta1 mRNA expression, and TGF-beta1 receptor number in desmoid fibroblasts compared with normal cells. As desmoid fibroblasts did not produce tumor necrosis factor-alpha (TNF-alpha) but were sensitive to it, which enhanced glycosaminoglycans (GAG) accumulation, we assessed the TGF-beta1 effects on TNF-alpha production by human monocytes. Our results showed TGF-beta1 significantly increased TNF-alpha secretion by monocytes. Toremifene mediated its effects in desmoid fibroblasts via an estrogen receptor-independent pathway. It inhibited GAG accumulation and the secretion of both latent and active forms of TGF-beta1 and had an inhibitory effect on TNF-alpha production by monocytes. Our results suggest that in reducing TGF-beta1 production by desmoid fibroblasts and TNF-alpha production by monocytes, toremifene may restore the balance between the two growth factors.

Stimulation of 5-HT(1A) receptors in the dorsal raphe ameliorates the impairment of spatial learning caused by intrahippocampal 7-chloro-kynurenic acid in naive and pretrained rats.

OBJECTIVE: The present study investigated the effect of stimulating 5-HT(1A) receptors in the dorsal raphe on the impairment of spatial learning caused by intrahippocampal 7-chloro-kynurenic acid (7-Cl-Kyn) in naive rats and in rats familiar with the general requirements of the task. METHODS: A week after implantation of cannulae to give access to the dorsal raphe (DR) and the CA1 region of the dorsal hippocampus, rats started their 5 days acquisition training on a two-platform spatial discrimination task in a water maze. On each acquisition day, WAY 100635 and 8-OH-DPAT alone or in combination were injected into the dorsal raphe (DR) 5 min before intrahippocampal injections of 7-Cl-Kyn which was given 10 min before the training session. Similar experiments were conducted in rats that had been familiarized with the general requirements of the task by pretraining them in the water maze in the absence of distal cues. RESULTS: 7-Cl-Kyn (3 microg/microl), injected bilaterally in the CA1 region of the dorsal hippocampus, impaired choice accuracy with no significant effect on choice latency. Rats treated with 7-Cl-Kyn tended to spend more time swimming close to the pool walls and made more errors of omission than controls in the first two sessions. Administered into the DR, the 5-HT1A receptor agonist 8-OH-DPAT (1 microg/0.5 microl) had no effect on any parameter of rats' performance but antagonized the impairment of choice accuracy caused by intrahippocampal 7-Cl-Kyn. Injected into the DR, 1 microg/0.5 microl WAY 100635, a 5-HT(1A) receptor antagonist, had no effect on rats' performance or on the impairment caused by intrahippocampal 7-Cl-Kyn, but antagonized the effect of 8-OH-DPAT on the 7-Cl-Kyn-induced deficit. The non-mnemonic behavioral disturbances shown by naive rats treated with 7-Cl-Kyn were greatly reduced in pretrained rats which, nevertheless, showed a marked impairment of choice accuracy similar to that of naive rats. As in previous experiments, administration of 1 microg/0.5 microl 8-OH-DPAT in the dorsal raphe antagonized the impairment of choice accuracy caused by intrahippocampal 7-Cl-Kyn without any effect on other parameters of rats' performance. CONCLUSIONS: The results show that stimulation of presynaptic 5-HT(1A) receptors in the dorsal raphe counteracts the deficit in spatial learning caused by a reduced NMDA-mediated excitatory input on pyramidal cells in the hippocampus. The possible mechanisms and the importance of these findings for the symptomatic treatment of memory disorders in man are discussed.

Promoter characterization and expression of the gene coding for the human GM2 activator protein.

Genomic clones of the human GM2 activator protein have been isolated and analyzed. The 5' region of the gene demonstrated promoter activity as ascertained by its ability to drive luciferase gene expression in transfected COS cells. This sequence contains GC rich region and several putative promoter elements were present, including Sp1, AP2, cAMP-responsive element, and B-cell-specific activating protein. Analysis of tissue distribution of the GM2 activator protein gene revealed tissue-specific variations in transcript levels. Placenta, bone marrow, mammary gland, bladder, lymph node, and spleen had the highest mRNA levels.