Influence of the cannabinoid agonist HU 210 on cocaine- and CQP 201-403-induced behavioural effects in rat.

Acute injection of the cannabinoid agonist HU 210 (6.25-100 microg/kg, i.p.) dose-dependently inhibited rat locomotor activity and rearing, while subchronic treatment with the drug (once daily for 7 days) at the same doses only diminished locomotion. Acute but not subchronic administration of HU 210 (12.5-50 microg/kg, i.p.) potently counteracted acute and subchronic cocaine (15 mg/kg, i.p.)-induced hyperlocomotion and enhanced rearing. The acute cannabinoid (6.25-100 microg/kg, i.p.) also inhibited locomotor activity, stereotyped behaviour and shaking elicited by the D1/D2 agonist CQP 201-403 (500 microg/kg, i.p.). On the contrary, subchronic treatments with HU 210 enhanced CQP 201-403-induced locomotor activity and potently stimulated escape attempts. Discussion centers on the influence of cannabinoids on experimental models of psychosis.     

Airborne microorganisms in a subterranean archaeological area of the basilica of San Lorenzo in Lucina (Rome)

The basilica of San Lorenzo in Lucina in Rome was built in the fifth century on the foundations of a Roman insula used for dwellings and commercial activities, where the first Christian communities had held their meetings. This archaeological area is still rather isolated since it can only be visited by groups for one/two hours once a month. Such a situation is ideal for studying both the quantity of microorganisms in the air under undisturbed conditions and/or conveyed by visitors. The biodeterioration of subterranean remains is determined by the development of bacteria, cyanobacteria, actinomycetes, algae and fungi. On the walls of this underground archaeological area the main form of alteration is the efflorescence. In order to compare the stone and airborne microflora, the microorganisms living on stone and the airborne ones have been detected. Air samples were taken both during the visits and under non-turbulent conditions utilizing three sampling methods: gravitational, intake by multi-stage impact and intake by single-stage impact. The results have made it possible to determine the microbial content of the air, as well as the sampling schedule required for rarely visited underground environments. Furthermore, a comparison of the three methods used showed that the adoption of one or the other sampling method gave different complementary information on airborne microflora. This revised version was published online in June 2006 with corrections to the Cover Date.     

The P2 purinergic receptors of human dendritic cells: identification and coupling to cytokine release.

We investigated the expression of purinoceptors in human dendritic cells, providing functional, pharmacological, and biochemical evidence that immature and mature cells express P2Y and P2X subtypes, coupled to increase in the intracellular Ca(2+), membrane depolarization, and secretion of inflammatory cytokines. The ATP-activated Ca(2+) change was biphasic, with a fast release from intracellular stores and a delayed influx across the plasma membrane. A prolonged exposure to ATP was toxic to dendritic cells that swelled, lost typical dendrites, became phase lucent, detached from the substrate, and eventually died. These changes were highly suggestive of expression of the cytotoxic receptor P2X(7), as confirmed by ability of dendritic cells to become permeant to membrane impermeant dyes such as Lucifer yellow or ethidium bromide. The P2X(7) receptor ligand 2',3'-(4-benzoylbenzoyl)-ATP was a better agonist then ATP for Ca(2+) increase and plasma membrane depolarization. Oxidized ATP, a covalent blocker of P2X receptors, and the selective P2X(7) antagonist KN-62 inhibited both permeabilization and Ca(2+) changes induced by ATP. The following purinoceptors were expressed by immature and mature dendritic cells: P2Y(1), P2Y(2), P2Y(5), P2Y(11) and P2X(1), P2X(4), P2X(7). Finally, stimulation of LPS-matured cells with ATP triggered release of IL-1 beta and TNF-alpha. Purinoceptors may provide a new avenue to modulation of dendritic cells function.     

Chinese hamster ovary cells with constitutively expressed sialidase antisense RNA produce recombinant DNase in batch culture with increased sialic acid

Under some cell culture conditions, recombinant glycoprotein therapeutics expressed in Chinese hamster ovary (CHO) cells lose sialic acid during the course of the culture (Sliwkowski et al., 1992; Munzert et al., 1996). A soluble sialidase of CHO cell origin degrades the expressed recombinant protein and has been shown to be released into the culture fluid as the viability of the cells decreases. To reduce the levels of the sialidase and to prevent desialylation of recombinant protein, a CHO cell line has been developed that constitutively expresses sialidase antisense RNA. Several antisense expression vectors were prepared using different regions of the sialidase gene. Co-transfection of the antisense constructs with a vector conferring puromycin resistance gave rise to over 40 puromycin resistant clones that were screened for sialidase activity. A 5' 474 bp coding segment of the sialidase cDNA, in the inverted orientation in an SV 40-based expression vector, gave maximal reduction of the sialidase activity to about 40% wild-type values. To test if this level of sialidase would lead to increased sialic acid content of an expressed recombinant protein, the 474 antisense clone was employed as a host for expression of human DNase as a model glycoprotein. The sialic acid content of the DNase produced in the antisense cultures was compared with material made in the wild-type parental cell line. About 20-37% increase in sialic acid content, or 0.6-1.1 mole of additional sialic acid out of a total of 3.0 mole on the product, was found on the DNase made in the antisense cell lines.     

Use of hidden Markov capture–recapture models to estimate abundance in the presence of uncertainty: Application to the estimation of prevalence of hybrids in animal populations

Estimating the relative abundance (prevalence) of different population segments is a key step in addressing fundamental research questions in ecology, evolution, and conservation. The raw percentage of individuals in the sample (naive prevalence) is generally used for this purpose, but it is likely to be subject to two main sources of bias. First, the detectability of individuals is ignored; second, classification errors may occur due to some inherent limits of the diagnostic methods. We developed a hidden Markov (also known as multievent) capture–recapture model to estimate prevalence in free‐ranging populations accounting for imperfect detectability and uncertainty in individual's classification. We carried out a simulation study to compare naive and model‐based estimates of prevalence and assess the performance of our model under different sampling scenarios. We then illustrate our method with a real‐world case study of estimating the prevalence of wolf (Canis lupus) and dog (Canis lupus familiaris) hybrids in a wolf population in northern Italy. We showed that the prevalence of hybrids could be estimated while accounting for both detectability and classification uncertainty. Model‐based prevalence consistently had better performance than naive prevalence in the presence of differential detectability and assignment probability and was unbiased for sampling scenarios with high detectability. We also showed that ignoring detectability and uncertainty in the wolf case study would lead to underestimating the prevalence of hybrids. Our results underline the importance of a model‐based approach to obtain unbiased estimates of prevalence of different population segments. Our model can be adapted to any taxa, and it can be used to estimate absolute abundance and prevalence in a variety of cases involving imperfect detection and uncertainty in classification of individuals (e.g., sex ratio, proportion of breeders, and prevalence of infected individuals).     

Mesoporous silica chips for selective enrichment and stabilization of low molecular weight proteome

The advanced properties of mesoporous silica have been demonstrated in applications, which include chemical sensing, filtration, catalysis, drug delivery and selective biomolecular uptake. These properties depend on the architectural, physical and chemical properties of the material, which in turn are determined by the processing parameters in evaporation‐induced self‐assembly. In this study, we introduce a combinatorial approach for the removal of the high molecular weight proteins and for the specific isolation and enrichment of low molecular weight species. This approach is based on mesoporous silica chips able to fractionate, selectively harvest and protect from enzymatic degradation, peptides and proteins present in complex human biological fluids. We present the characterization of the harvesting properties of a wide range of mesoporous chips using a library of peptides and proteins standard and their selectivity on the recovery of serum peptidome. Using MALDI‐TOF‐MS, we established the correlation between the harvesting specificity and the physicochemical properties of mesoporous silica surfaces. The introduction of this mesoporous material with fine controlled properties will provide a powerful platform for proteomics application offering a rapid and efficient methodology for low molecular weight biomarker discovery.     

Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection

The proliferative response of PBMC to hepatitis B virus (HBV) envelope, core, and e Ag was analyzed prospectively in 21 patients with acute self-limited HBV infection and compared with the response of patients with chronic HBV infection and different levels of HBV replication (i.e., hepatitis e Ag (HBeAg)- or anti-HBe-positive) and liver damage (i.e., chronic active hepatitis or chronic asymptomatic carriers). Our results indicate that: 1) HBV-infected subjects who develop a self-limited acute hepatitis show a vigorous PBMC response to hepatitis B core Ag and HBeAg, as expression of T cell activation; 2) appearance of a detectable lymphocyte response to HBV nucleocapsid Ag is temporally associated with the clearance of HBV envelope Ag; 3) in patients with chronic HBV infection the level of T cell responsiveness to hepatitis B core Ag and to HBeAg is significantly lower than that observed during acute infection; 4) T cell sensitization to HBV envelope Ag in acute and chronic HBV infection is usually undetectable and when measurable is expressed transiently and at low levels. These results may reflect immune events of pathogenetic relevance with respect to evolution of disease and viral clearance.