AMPA/kainate and NMDA-like glutamate receptors at the chromatophore neuromuscular junction of the squid: role in synaptic transmission and skin patterning

Glutamate receptor types were examined at the chromatophore synapses of the squids Alloteuthis subulata and Loligo vulgaris, where nerve-induced muscle contraction causes chromatophore expansion. Immunoblotting with antibody raised against a squid AMPA receptor (sGluR) demonstrated that AMPA/kainate receptors are present in squid skin. Application of l-glutamate evoked chromatophore muscle contractions in both ventral and dorsal skins, while NMDA was only active on a subpopulation of dorsal chromatophores. In dorsal skin, neurotransmission was partly blocked by either AMPA/kainate receptor antagonists (CNQX and DNQX) or NMDA receptor antagonists (AP-5 and MK-801) or completely blocked by simultaneous application of both classes of antagonists. In isolated muscle fibres, ionophoretic application of l-glutamate evoked fast inward CNQX- and DNQX-sensitive currents with reversal potentials around +14 mV and a high conductance to Na+. In fibres from dorsal skin only, a slower outward glutamate-sensitive current appeared at positive holding potentials. At negative potentials, currents were potentiated by glycine or by removing external Mg2+ and were blocked by AP-5 and MK-801. Glutamate caused a fast, followed by a slow, transient increase in cytoplasmic Ca2+. The slow component was increased in amplitude and duration by glycine or by lowering external Mg2+ and decreased by AP-5 and MK-801. In cells from ventral skin, no 'NMDA-like responses' were detected. Thus, while AMPA/kainate receptors mediated fast excitatory synaptic transmission and rapid colour change over the whole skin, activation of both AMPA/kainate and NMDA-like receptors in a subpopulation of dorsal chromatophores prolonged the postsynaptically evoked Ca2+ elevation causing temporally extended colour displays with behavioural significance.     

Differential scanning calorimetry study on drug release from an inulin-based hydrogel and its interaction with a biomembrane model: pH and loading effect

Inulin has been derivatized with methacrylic anhydride (MA) and succinic anhydride (SA) to obtain a methacrylated/succinilated derivative (INU-MA-SA) able to produce a pH sensitive hydrogel after UV irradiation. The hydrogel was characterized and loaded with diflunisal (10.4, 17 and 24%, w/w) chosen as a model drug. The drug release from INU-MA-SA-based hydrogel to a biomembrane model made by unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) was investigated at pH 4.0 and 7.4 by differential scanning calorimetry (DSC) that appears to be a suitable technique to follow the transfer kinetics of a drug from a controlled release system to a biomembrane model. The drug release from the hydrogel was compared with the dissolution of drug solid form by examining the effects exerted on the thermotropic behaviour of the DMPC unilamellar vesicles. The transferred drug and the release rate were affected by the drug loading as well as by the pH of the external medium. In particular the release was not linearly related to the drug loading but an intermediate loading allowed a better release at both investigated pHs, with a faster and more complete release observed at pH 7.4.     

Combination of DNA repair gene single nucleotide polymorphisms and increased levels of DNA adducts in a population-based study.

Inherited single nucleotide polymorphisms (SNPs) of DNA repair genes may contribute to variations in DNA repair capacity and susceptibility to cancer. We investigated the role of SNPs in three DNA repair genes (X-ray repair cross-complementing group 1-Arg399Gln, exon 10; X-ray repair cross-complementing group 3-Thr241Met, exon 7; and xeroderma pigmentosum-D-Lys751Gln, exon 23) and their combination, in modulating the levels of "bulky" DNA adducts in a population sample of 628 Italian healthy individuals belonging to the prospective European project "European Prospective Investigation into Cancer and Nutrition." DNA-adduct levels were measured as relative adduct level per 10(9) nucleotides by (32)P-post DNA labeling assay in WBCs from peripheral blood. Genotyping was performed by PCR-RFLP analysis or primer extension/denaturing high-performance liquid chromatography technique. We found a dose-response relationship between the number of at-risk alleles and levels of adducts (P = 0.0046). Individuals with at least three variant alleles had a statistically significant odds ratio (OR) for being in the highest tertile of adducts compared with those with undetectable adducts [three alleles, adjusted OR = 5.07, 95% confidence interval (CI) = 1.29-19.9; four alleles, adjusted OR = 5.03, 95% CI = 1.18-21.45; five alleles, adjusted OR = 7.65, 95% CI = 0.94-62.2]. Our study suggests that the combined effect of multiple variant alleles may be more important than the investigation of single SNP in modulating DNA repair capacity.     

Induction of chromosomal instability in colonic cells by the human polyomavirus JC virus

Most colorectal cancers display chromosomal instability, which is characterized by gross chromosomal rearrangements, loss of heterozygosity and aneuploidy. We have previously demonstrated a link between JC virus strains Mad-1 and Delta98 and colorectal cancer. Others have also associated the virus to the induction of colon cancer and aneuploid brain tumors by producing a highly tumorigenic protein named T antigen (TAg), which binds to beta-catenin and inactivates key proteins such as p53. The aim is to demonstrate that JC virus is capable of inducing chromosomal instability in colonic cells. We used the human colon cancer cell line RKO as a model. The cell line has wild-type p53, wild-type beta-catenin and APC and is diploid. Neuroblastoma JCI cells, which are infected with the virus, VA13 fibroblasts, which are transformed by the SV40 TAg, were used as positive controls. HCT116, which has mutated beta-catenin, and SW480, which is a model of CIN, were also used as controls. The genomes of the Mad-1 and Delta98 strains were transfected into cells. As negative controls we used pUC or no plasmids. Cells were collected at 0, 7, 14, and 21 days after transfection. PCR was used for the detection of TAg and the regulatory region DNA sequences at different time frames and Southern blot of whole genomic extracts for viral DNA integration into the host genome. Immunofluorescence and Western blot were performed for TAg, viral capsid proteins, and nuclear beta-catenin expressions, whereas coimmunoprecipitation was used to detect protein interactions. Karyotype analysis and electron microscopy were performed to seek chromosomal instability and cell abnormalities, respectively. Retention of viral sequences was observed for Mad-1- and Delta98-transfected RKO cells at all time frames with PCR only, whereas Southern blot analysis showed nonintegrated sequences at T7 alone. TAg and capsid protein expressions, as well as increased p53 and nuclear beta-catenin, were observed between T0 and T7 for Mad-1 and Delta98 alone. Also, interaction between TAg and both p53 and beta-catenin was also observed between T0 and T7. Chromosomal instability, characterized by chromosomal breakage, dicentric chromosomes, and increasing ploidy, was observed at all time frames for Mad-1 and Delta98, as well as cell abnormalities. In conclusion, we demonstrate that JC virus Mad-1 and Delta98 are able to induce chromosomal instability in colonic cells with a hit and run mechanism that involves an early interaction with beta-catenin and p53.     

Malaria clusters among illegal Chinese immigrants to Europe through Africa

Between November 2002 and March 2003, 17 cases of malaria (1 fatal) were observed in illegal Chinese immigrants who traveled to Italy through Africa. A further cluster of 12 was reported in August, 2002. Several immigrants traveled by air, making the risk of introducing sudden acute respiratory syndrome a possibility should such illegal immigrations continue.     

Prenatal ultrasound diagnosis of Toriello-Carey syndrome

Toriello-Carey syndrome is a rare malformative complex, described for the first time in 1988, characterized by agenesis of the corpus callosum, facial anomalies, cardiac defects and hypotonia. Relatively few neonatal cases have been reported. We describe here the first prenatal ultrasound diagnosis of the syndrome based on the detection of agenesis of the corpus callosum and spongious cardiomyopathy in a 22-week-old fetus of a couple with positive family history. The first sib of the couple was diagnosed with Toriello-Carey syndrome at 1 year of age, and had, in addition to the typical facial anomalies not detectable by ultrasound, agenesis of the corpus callosum and the same heart lesion (spongious cardiomyopathy). This report demonstrates that prenatal diagnosis of Toriello-Carey syndrome is feasible in the second trimester of pregnancy.     

Effect of different doses of progestin on uterine leiomyomas in postmenopausal women

OBJECTIVE(S): To evaluate the effects of two doses of medroxyprogesterone acetate (MPA) on uterine leiomyoma sizes and on uterine bleeding pattern in postmenopausal women treated with oral hormone replacement therapy (HRT). STUDY DESIGN: Thirty natural postmenopausal women affected by uterine leiomyomas were enrolled and treated with oral micronized estradiol (E(2)) at dose of 2 mg per day, and randomized to receive in association MPA at dose of 2.5 mg daily (group A) or of 5 mg daily (group B). At the beginning of the study and after 1 year of treatment, uterine leiomyomata dimensions were measured using transvaginal ultrasonography (TV-USG). The subjects were instructed to note on a daily dairy the number and severity of abnormal uterine bleeding (AUB) episodes. RESULTS: After 1 year of therapy, a significant changes in mean uterine leiomyomas size was observed in the group treated with higher dose of MPA. No significant differences in uterine bleeding were detected between the two groups. CONCLUSION(S): In postmenopausal women with uterine leiomyomas, it is necessary to use the minimal efficacious dose of progestin during HRT because of a higher risk to increase the tumors dimensions.     

The stress of birth enhances in vitro spontaneous and IL-8-induced neutrophil chemotaxis in the human newborn

The birth process induces fetal stress. Stress has profound effects on the immune system, also by acting on the trafficking of leukocytes, a process in which adhesion and chemotaxis are primordial and critical events for the development of effective antimicrobial defenses. The newborn is rapidly challenged by a microflora at the epithelia linings and therefore depending on early, innate immunity onset. The objective of the study was to investigate the immune response in cord blood from newborns in relation to different degrees of fetal stress, with focus on neutrophil chemotaxis. We analyzed in vitro transmigration ability of neutrophils and their CD11b expression, measured total white blood count (WBC) and the major leukocyte populations, interleukin (IL)-8, interferon (IFN)-gamma, and soluble E-Selectin, as well as relevant immuno-modulating hormones in infants born at term after Cesarean section prior to the start of labor (n = 55), normal vaginal delivery (n = 87), and assisted delivery (n = 26). Arterial pH and lactate were used as stress markers. We found that spontaneous and IL-8-induced transmigration ability of neutrophils from newborns after normal delivery was significantly higher compared with that of neutrophils from Cesarean section or from adults. With a progressive increase in fetal stress, there were significant elevations in total WBC, in particular neutrophils and monocytes, as well as an enhanced IL-8 and soluble E-Selectin level. Assisted delivery, associated with the highest degree of fetal stress in addition had an enhanced lymphocyte and monocytes count as well as an increased IFN-gamma level. There were significant direct correlations between neutrophils and monocytes, respectively, with cortisol, beta-endorphin, and prolactin. Interferon-gamma was directly related to dopamine, as well as to the lymphocyte and monocyte count. The setting of the HPA-axis at birth is a promoter of an alarm response and a surge of neuroendocrine immuno-modulating factors that enhances antimicrobial defenses of the newborn. We speculate that IL-8 induced by normal labor may be a priming factor for an increased neutrophil chemotaxis through the pre-activated endothelium of the fetus. Assisted delivery may trigger excessive recruitment of additional inflammatory cells and IFN-gamma release.     

Study of formulation variables influencing the drug release rate from matrix tablets by experimental design.

Experimental design was utilized to simultaneously investigate the effect of varying the type of diluent (insoluble Calcium phosphate or water-soluble arabic gum) and the diluent/matrix ratio on the drug release behaviour from both lipophilic (glyceryl behenate, Compritol) or hydrophilic (hydroxypropylmethylcellulose) matrix tablets. Ketoprofen, theophylline and sodium sulphadiazine were selected as model drugs on the basis of their respectively very low, medium and high water-solubility, in order to evaluate the influence of this parameter as well. The selected response variables were the dissolution efficiency (i.e. the area under the dissolution curve) after one and six hours and the time necessary to dissolve 10% drug. Tablets obtained by direct compression of drug-diluent-matrix ternary mixtures prepared according to the experimental plan provided for by an asymmetric screening matrix, were tested for drug release properties using a USP paddle apparatus. Graphic analysis of the effects allowed identification, for each examined drug, of the formulation factors active on the selected responses and determination of the proper level of the variables to be selected for the response improvement. The different results obtained with the three examined drugs pointed out the role of the drug solubility in determining the influence of formulation parameters on drug release rate from matrix tablets.