99mTc-labeling of colchicine using [99mTc(CO)3(H2O)3]+ and [99mTc triple bond N]2+ core for the preparation of potential tumor-targeting agents

Multidrug resistance (MDR) mediated by over-expression of P-glycoprotein (Pgp) is one of the major causes of failure of chemotherapy in cancer treatment. Colchicine, a naturally occurring alkaloid, is a Pgp substrate and acts as an antimitotic agent by binding to microtubules. Hence, Colchicine and its analogues radiolabeled with 99mTc may have potential for visualization of MDR in tumors. Here we report 99mTc-labeling of colchicine derivatives using [99mTc(CO)3(H2O)3]+ and [99mTc triple bond N]2+ cores. Trimethylcolchicinic acid synthesized from colchicine was used as the precursor to prepare iminodiacetic acid and dithiocarbamate derivatives which were then radiolabeled with [99mTc(CO)3(H2O)3]+ and [99mTc triple bond N]2+ cores, respectively. Radiolabeling yield for both the complexes was > 98% as observed by HPLC and TLC patterns. In vitro studies in tumor cell lines showed significant uptake for 99mTc-carbonyl as well as for 99mTc-nitrido colchicine complexes. Biodistribution studies in Swiss mice bearing fibrosarcoma tumor showed 4.1 +/- 1.2% ID/g of uptake at 30 min pi for 99mTc(CO)3-complex as against 0.42 +/- 0.24% ID/g for the 99mTcN-complex. 99mTc(CO)3-colchicine complex exhibited better pharmacokinetics with lower liver accumulation as compared to the 99mTcN-complex. Thus, colchicine radiolabeled with [99mTc(CO)3(H2O)3]+ core is more promising with respect to in vivo distribution characteristics in tumor model.     

Ixodid ticks of road-killed wildlife species in southern Italy: new tick-host associations and locality records

In this study 1,868 questing Ixodes ricinus ticks (nymphs and adults), collected in six sites from three counties—Giurgiu, Sibiu, and Tulcea—in Romania, were examined by polymerase chain reaction (PCR) followed by reverse line blot (RLB) for detection of Borrelia burgdorferi sensu lato presence. The bacteria were found in 18% of the investigated ticks. The prevalence of infection did not differ significantly between nymphs (19.1%) and adults (15.4%). Three B. burgdorferi s.l. genospecies were detected: B. afzelii (61.1%), B. garinii (31.2%), and B. valaisiana (7.7%). No mixed infections were detected. The highest infection prevalence in nymphs was detected at Cristian (Sibiu County)—22.0%, whereas in adults it was at Comana (Giurgiu County)—19.8%. This preliminary study provides evidence that Lyme disease spirochetes are present in various regions of Romania, and at a relatively high prevalence in their vectors, thus posing a risk of infection to human subjects in the areas infested by ticks.     

Autochthonous and migratory birds as a dispersion source for Ixodes ricinus in southern Italy

The present study was carried out in a protected wooded area, which is part of the Parco Regionale Gallipoli Cognato Piccole Dolomiti Lucane, one of the most important ecological reserves in southern Italy. From April 2010 to April 2011, 212 birds, comprising 22 species from 12 families, were captured and examined for ticks. A total of 75 (35.4?%) birds were found infested by ticks, with 451 ticks being collected. All ticks were identified as Ixodes ricinus, of which 241 (53.4?%) were larvae and 210 nymphs (46.6?%). The highest intensity of infestation was found in April 2010, when 117 ticks were retrieved on 25 birds. No ticks were found on birds (n?=?5) netted in December 2010. High infestation rates were recorded on blackbirds (Turdus merula) (90 %; 29 birds examined) and on mistle thrushes (Turdus viscivorus) (100?%; 2 birds examined). The highest intensity of infestation by larvae was found on wrens (5.6 larvae/bird) and by nymphs on mistle thrushes (11.5 nymphs/bird). Temperature and number of hours of light showed to influence the activity of larvae and nymphs. These data support the notion that birds may be responsible for the heterogeneous distribution of I. ricinus in Europe, thus playing a role in the epidemiology of certain tick-borne pathogens.     

Stacking small segments of the 1D chromosome of bread wheat containing major gluten quality genes into durum wheat: transfer strategy and breeding prospects

Using a chromosome engineering strategy, we previously developed two durum wheat recombinant lines, each containing on chromosome 1A a short segment of bread wheat chromosome 1D with either the Glu-D1 (PL line) or the Gli-D1/Glu-D3 (PS line) genes. Since PL and PS transfers produced substantial but different effects on durum gluten properties, in the present work stacking of their 1DS and 1DL segments into the same chromosome 1A was undertaken to investigate their combined effect in durum wheat and to potentially widen the spectrum of the crop end-uses. Development of genetic and genomic in situ hybridization (GISH)-based physical maps of PS and PL recombinant arms facilitated selection of carriers of the double-recombinant (PS?+?PL) 1A chromosome among F₂ progeny from the PS?×?PL cross. With the 1DS transfer spanning the terminal 17% of recombinant 1AS, and the interstitial 1DL segment occupying 16% of recombinant 1AL, PS?+?PL chromosomes were generated by pairing events occurred with a 68.4% frequency in 1A regions shared by parental chromosomes. Homozygous double-recombinant F₃ plants exhibited no significant differences for relevant agronomic traits compared to sib lines possessing either one or no 1D segment. Among quality parameters preliminarily assessed, SDS sedimentation values increased by 12% in PS and by over 32 and 38% in PL and PS?+?PL lines, respectively, compared to null controls. As a whole, the novel recombinant genotype offers good prospects for direct exploitation in breeding, and hence for an effective contribution to the enhancement of the crop value.     

Candida albicans cell wall comprises a branched beta-D-(1-->6)-glucan with beta-D-(1-->3)-side chains

The structure of immunogenic and immunomodulatory cell wall glucans of Candida albicans is commonly interpreted in terms of a basic polysaccharide consisting of a beta-D-(1-->3)-linked glucopyranosyl backbone possessing beta-D-(1-->6)-linked side chains of varying distribution and length. This proposed molecular architecture has been re-evaluated by the present study on the products of selective enzymolysis of insoluble C. albicans glucan particles (GG). High resolution 1H (400 and 700 MHz) and 13C (100 and 175 MHz) NMR analyses were performed on a soluble beta-glucan preparation (GG-Zym) obtained by GG digestion with endo-beta-D-(1-->3)-glucanase and on its high- (Pool 1) and low-molecular weight (Pool 2) sub-fractions. The resonances typical of uniformly beta-D-(1-->6)- and beta-D-(1-->3)-linked linear glucans, together with additional multiplets assigned to short-chain oligoglucosides, were detected in GG-Zym. Pool 1 (46.3+/-6.4% of GG-Zym content) consisted of beta-D-(1-->6)-linked glucopyranosyl polymers, with short beta-D-(1-->3)-branched side chains of 2.20+/-0.02 units (branching degree (DB)=0.14+/-0.03). Pool 2 was a mixture of glucose and linear short-chain beta-D-(1-->3)-oligoglucosides. Further digestion of Pool 1 by beta-D-(1-->6)-glucanase yielded a mixture of glucose and short beta-D-(1-->6)-linked, either linear or beta-D-(1-->3,6) branched, oligomers. These endoglucanase digestion patterns were consistent with the presence in C. albicans cell wall glucans of beta-D-(1-->6)-linked glucopyranosyl backbones possessing beta-D-(1-->3)-linked side chains, a structure very close to that of beta-D-(1-->6)-glucan from Saccharomyces cerevisiae yeast. This finding may provide the grounds for further elucidation of the cell wall structure and a better understanding of the biological properties of C. albicans beta-glucans.     

Hypoxia-induced modifications in plasma membranes and lipid microdomains in A549 cells and primary human alveolar cells

We evaluated the response to mild hypoxia exposure of A549 alveolar human cells and of a continuous alveolar cell line from human excised lungs (A30) exposed to 5% O(2) for 5 and 24 h. No signs of increased peroxidation and of early apoptosis were detected. After 24 h of hypoxia total cell proteins/DNA ratio decreased significantly by about 20%. Similarly, we found a decrease in membrane phospholipid and cholesterol content. The membrane fluidity assessed by fluorescence anisotropy measurements was unchanged. We also prepared the detergent resistant membrane fraction (DRM) to analyze the distribution of the two types of lipid microdomains, caveolae and lipid rafts. The DRM content of Cav-1, marker of caveolae, was decreased, while CD55, marker of lipid rafts, increased in both cell lines. Total content of these markers in the membranes was unchanged indicating remodelling of their distribution between detergent-resistant and detergent-soluble fraction of the cellular membrane. The changes in protein markers distribution did not imply changes in the corresponding mRNA, except in the case of Cav-1 for A30 line. In the latter case we found a parallel decrease in Cav-1 and in the corresponding mRNA. We conclude that an exposure to a mild degree of hypoxia triggers a significant remodelling of the lipid microdomains expression, confirming that they are highly dynamic structures providing a prompt signalling platform to changes of the pericellular microenvironment.