Effect of (-)-cathinone, a khat leaf constituent, on dopaminergic firing and dopamine metabolism in the rat brain

The effect of (-)-cathinone (CAT), an alkaloid from khat leaves, on brain dopamine (DA) metabolism and on the firing rate of nigral DA neurons was studied in rats, in comparison with that of d-amphetamine. Like d-amphetamine, CAT (8-40 mg/kg i.p.) decreased DOPAC levels in the caudate nucleus, nucleus accumbens and frontal cortex, without modifying DA concentrations. CAT showed approximately one fifth of the potency of d-amphetamine in this effect. CAT, injected i.v. to unanesthetized, paralyzed rats, inhibited the firing rate of DA neurons in the substantia nigra, pars compacta, showing a similar potency to that of d-amphetamine in this respect. CAT-induced inhibition of dopaminergic firing was reversed by haloperidol.     

Low molecular weight protein tyrosine phosphatase and caveolin-1: interaction and isoenzyme-dependent regulation

Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are small enzymes that are ubiquitous in many organisms. They are important in biological processes such as cell proliferation, adhesion, migration, and invasiveness. LMW-PTP is expressed in mammalian cells as two isoforms (IF1 and IF2) originating through alternative splicing. We have previously shown that IF2 targets lipid rafts called caveolae and interacts with caveolin-1, their major structural protein. Caveolae are cholesterol- and sphingolipid-rich membrane microdomains that have been implicated in a variety of cellular functions, including signal transduction events. Caveolin-1 contains a scaffolding region that contributes to the binding of the protein to the plasma membrane and mediates protein omo- and etero-oligomerization. Interaction of many signaling molecules with the scaffolding domain sequesters them into caveolae and inhibits or suppresses their activities. Caveolin-interacting proteins usually have a typical sequence motif, also present in all the LMW-PTPs, which is characterized by aromatic or large hydrophobic residues in specific positions. We have examined here the interaction of the LMW-PTP isoforms with caveolin-1 and its molecular mechanism, together with the consequences for their tyrosine phosphatase activities. We found that IF1 and IF2 are both capable of interacting with defined regions of caveolin-1 and that their putative caveolin binding sequence motif is not responsible for the association. The formation of LMW-PTP/caveolin-1 complexes is accompanied by modulation of the enzyme activities, and the inhibitory effect elicited against IF1 is stronger than that against IF2. The caveolin scaffolding domain is directly involved in the observed phenomena.     

Biocompatibility of denture base acrylic resins evaluated in culture of L929 cells. Effect of polymerisation cycle and post-polymerisation treatments

Objective: The purpose of this study was to evaluate the effect of two post‐polymerisation treatments and different cycles of polymerisation on the cytotoxicity of two denture base resins. Materials and methods: The resins tested were Lucitone 550 and QC 20. Discs of resins were fabricated following the manufacturer's instructions. Lucitone 550 was processed by long cycle or short cycle. The resin QC 20 was processed by reverse cycle or normal cycle. The specimens were divided into groups: (i) post‐polymerised in microwave for 3 min at 500 W; (ii) post‐polymerised in water‐bath at 55°C for 60 min and (iii) without post‐polymerisation. Eluates were prepared by placing three discs into a sterile glass vial with 9 ml of Eagle's medium and incubated at 37°C for 24 hours. L929 cells were seeded into 96 well culture plates and DNA synthesis was assessed by ³H‐thymidine incorporation assay. Results: The results were submitted to two‐way anova and Tukey HSD test. QC 20 specimens polymerised by the normal cycle and submitted to microwave post‐polymerisation were graded as moderately cytotoxic. Similar results were observed for Lucitone 550 processed by long cycle without post‐polymerisation. The other experimental groups were graded as not cytotoxic. After water‐bath post‐polymerisation, specimens of Lucitone 550 processed by long cycle produced significantly lower inhibition of DNA synthesis than the other groups. Conclusion: The long cycle increased the cytotoxicity of Lucitone 550 and water‐bath post‐polymerisation reduced the cytotoxicity of Lucitone 550 processed by long cycle.     

Factors Influencing the Seasonal Phenology and Composition of Zooplankton Communities in Mountain Temporary Pools

In 2001 nine temporary pools of the northern Apennines (Italy) were visited on 13 occasions during the ice‐free season (May to October). The aims of this research were to define the relationships between hydroperiod and other environmental variables and the zooplankton. In total, 49 zooplankton taxa were identified: 36 rotifers, 5 cladocerans, 6 copepods and 2 anostracans. Our results indicate that hydroperiod is a major determinant affecting zooplankton species richness. The highest number of taxa was found in the pond having the longest duration. Distinctive species assemblages were observed in different habitat types: pools with the shortest hydroperiod were characterised by organisms with brief life cycles (e.g. rotifers) and/or typical of temporary habitat (e.g. anostracans). Of the physical and chemical characteristics, pH and chlorophyll‐a appeared to have the largest influence on zooplankton distribution in the studied pools. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The interaction of native calf thymus DNA with Fe(III)-dipyrido[3,2-a:2',3'-c]phenazine

The mono and bis dipyrido[3,2-a:2′,3′-c]phenazine (dppz) adducts of iron(III) chloride, i.e. [Fe(dppz)]Cl₃ and [Fe(dppz)₂]Cl₃, have been synthesized and characterized. The interaction of the Fe^IIIdppz hydrolyzed aquo complex with native calf thymus DNA has been monitored as a function of the metal complex-DNA molar ratio, by variable temperature UV absorption spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. The results obtained in solution at various ionic strength values give support for a tight intercalative binding of the Fe^IIIdppz cation with DNA. In particular, the appearance of induced CD bands, caused by the addition of Fe^IIIdppz, indicate the existence of a rigid metal complex-DNA-binding leading to dominating chiral organization of Fe^IIIdppz species within the DNA double helix. The trend of selected CD bands with the molar concentration of Fe^IIIdppz emphasizes that the presence of high amounts of metal complex induces also the formation of DNA-Fe^IIIdppz supramolecular aggregates in solution. The analysis of fluorescence measurements allowed us to calculate a value of the intercalative binding constant comparable to that obtained by UV spectrophotometric titration. Finally, the temperature dependence of the absorbance at 258 nm shows that the metal complex strongly increases the DNA melting temperature already at metal complex-DNA molar ratio equal to 0.25 suggesting that metal complex intercalation effectively hinders DNA denaturation. Overall, the results of the present study point out that the Fe^IIIdppz aquo complex has DNA-binding properties analogous to those previously reported for the tris-chelate Fe^II(phen)₂dppz complex (phen = 1,10-phenantroline).     

Resistance of Human Erythrocyte Membranes to Triton X-100 and C<Subscript>12</Subscript>E<Subscript>8</Subscript>

Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4°C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C₁₂E₈. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C₁₂E₈, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C₁₂E₈ in comparison to intact cells, while the difference in the S values between Triton X-100 and C₁₂E₈ DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C₁₂E₈ detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs.     

Prognostic Value of Beta-Tubulin-3 and c-Myc in Muscle Invasive Urothelial Carcinoma of the Bladder

Background

To date, putative prognostic biomarkers have shown limited utility from the clinical perspective for bladder urothelial carcinoma. Herein, the expression of beta-tubulin-3 and c-Myc was evaluated to determine their prognostic potential.

Methods

In formalin fixed-paraffin embedded blocks, immunohistochemical expression of c-Myc and beta-tubulin-3 was evaluated. H score ranging from 0 to 300 was obtained by multiplying the percentage of positive cells by intensity (0–3); c-Myc and beta-tubulin-3 expression was defined: 0: negative, 1: weakly positive, 2: strongly positive.

Results

beta-tubulin-3 and c-Myc immunoexpression was available for 46 cases. At the univariate analysis, node-involvement, beta-tubulin-3 and c-Myc overexpression discriminate shorter DFS (HR 2.19, p = 0.043; HR 3.10, p = 0.24 and HR 3.05, p = 0.011, respectively); 2-yrs DFS log-rank analysis according to low versus high level of immunoexpression were statistically significant; beta-tubulin-3, 53% low vs 12.7% high (p = value 0.02) and c-Myc 28 low vs 8 high (p-value 0.007). Patients displaying negative beta-tubulin-3/c-Myc had statistically significant better 2-yrs DFS than those with mixed expression or double positivity (54.5% versus 18.7% versus 0%, log-rank p = 0.006).

Conclusions

c-Myc and beta-tubulin-3 show improvement for prognostic risk stratification in patients with muscle invasive bladder urothelial carcinoma. These molecular pathways may also be candidate to improve predictiveness to targeted therapies.     

A height-wood-seed axis which is preserved across climatic regions explains tree dominance in European forest communities

Plant Ecology - Environmental constraints on both functional traits and trait–trait correlation patterns are extremely relevant research questions to understand assembling mechanisms and...     

The intrachain disulfide bridge is responsible of the unusual stability properties of novel acylphosphatase from Escherichia coli

Acylphosphatase (AcP) activity in prokaryotes was classically attributed to some aspecific acid phosphatases. We identified an open reading frame for a putative AcP in the b0968 Escherichia coli gene and purified the recombinant enzyme after checking by RT-PCR that it was indeed expressed. EcoAcP has a predicted typical fold of the AcP family but displays a very low specific activity and a high structural stability differently from its mesophilic and similarly to its hyperthermophilic counterparts. Site directed mutagenesis suggests that, together with other structural features, the intrachain S–S bridge in EcoAcP is involved in a remarkable thermal and chemical stabilization of the protein without affecting its catalytic activity.