Metal-ion-dependent biological properties of a chelator-derived somatostatin analogue for tumour targeting

Somatostatin-based radioligands have been shown to have sensitive imaging properties for neuroendocrine tumours and their metastases. The potential of [(55)Co(dotatoc)] (dotatoc =4,7,10-tricarboxymethyl-1,4,7,10-tetraazacyclododecane-1-ylacetyl-D-Phe-(Cys-Tyr-D-Trp-Lys-Thr-Cys)-threoninol (disulfide bond)) as a new radiopharmaceutical agent for PET has been evaluated. (57)Co was used as a surrogate of the positron emitter (55)Co and the pharmacokinetics of [(57)Co(dotatoc)] were investigated by using two nude mouse models. The somatostatin receptor subtype (sst1-sst5) affinity profile of [(nat)Co(dotatoc)] on membranes transfected with human somatostatin receptor subtypes was assessed by using autoradiographic methods. These studies revealed that [(57)Co(dotatoc)] is an sst2-specific radiopeptide which presents the highest affinity ever found for the sst2 receptor subtype. The rate of internalisation into the AR4-2J cell line also was the highest found for any somatostatin-based radiopeptide. Biodistribution studies, performed in nude mice bearing an AR4-2J tumour or a transfected HEK-sst2 cell-based tumour, showed high and specific uptake in the tumour and in other sst-receptor-expressing tissues, which reflects the high receptor binding affinity and the high rate of internalisation. The pharmacologic differences between [(57)Co(dotatoc)] and [(67)Ga(dotatoc)] are discussed in terms of the structural parameters found for the chelate models [Co(II)(dota)](2-) and [Ga(III)(dota)](-) whose X-ray structures have been determined. Both chelates show six-fold coordination in pseudo-octahedral arrangements.

New copper(II) complexes with isoconazole: Synthesis, structures and biological properties

There is an increasing demand for novel metal-based complexes with biologically relevant molecules in technology and medicine. Three new Cu(II) coordination compounds with antifungal agent isoconazole (L), namely mononuclear complexes CuCl2(L)(2) (1), and Cu(O2CMe)(2)(L)(2)center dot 2H(2)O (2) and coordination polymer Cu(pht)(L)(2)(n) (3) (where H(2)pht - o-phthalic acid) were synthesized and characterized by IR spectroscopy, thermogravimetric analysis and X-ray crystallography. X-ray analysis showed that in all complexes, the isoconazole is coordinated to Cu(II) centres by a N atom of the imidazole fragment. In complex I, the square-planar environment of Cu(II) atoms is completed by two N atoms of isoconazole and two chloride ligands, whereas the Cu(II) atoms are coordinated by two N atoms from two isoconazole ligands and two O atoms from the different carboxylate residues: acetate in 2 and phthalate in 3. The formation of an infinite chain through the bridging phthalate ligand is observed in 3. The biosynthetic ability of micromycetes Aspergillus niger CNMN FD 10 in the presence of the prepared complexes 1-3 as well as the antifungal drug isoconazole were studied. Complexes 2 and 3 accelerate the biosynthesis of enzymes (beta-glucosidase, xylanase and endoglucanase) by this fungus. Moreover, a simplified and improved method for the preparation of isoconazole nitrate was developed.

Sugar modification as a means to increase the biological performance of oligonucleotides

Modification of the ribose unit in DNA and RNA profoundly influences the self-rcognition and the biological properties of the nucleic acids. Conformational restriction of the ribose units, as in LNA and tricyclo-DNA, has been identified as a powerful tool to increase DNA and RNA affinity as well as biological stability and antisense properties. Apart from that sugar modified DNA analogues, as homo-DNA, have shown to be orthogonal base-pairing systems which by virtue of non-crosscommunicating with the natural nucleic acids open novel applications in biotechnology.

Synthesis and properties of isobicyclo-DNA

We present the synthesis of the isobicyclo-DNA building blocks with the nucleobases A, C, G and T, as well as biophysical and biological properties of oligonucleotides derived thereof. The synthesis of the sugar part was achieved in 5 steps starting from a known intermediate of the tricyclo-DNA synthesis. Dodecamers containing single isobicyclo-thymidine incorporations, fully modified A- and T-containing sequences, and fully modified oligonucleotides containing all four bases were synthesized and characterized. Isobicyclo-DNA forms stable duplexes with natural nucleic acids with a pronounced preference for DNA over RNA as complements. The most stable duplexes, however, arise by self-pairing. Isobicyclo-DNA forms preferentially B-DNA-like duplexes with DNA and A-like duplexes with complementary RNA as determined by circular dichroism (CD) spectroscopy. Self-paired duplexes show a yet unknown structure, as judged from CD spectroscopy. Biochemical tests revealed that isobicyclo-DNA is stable in fetal bovine serum and does not elicit RNaseH activity.

An investigation into the mechanical and aesthetic properties of new generation coated nickel-titanium wires in the as-received state and after clinical use.

BACKGROUND/OBJECTIVES The purpose of this study was to compare the mechanical, structural, and aesthetic properties of two types of aesthetic coated nickel-titanium (NiTi) wires compared with comparable regular NiTi wires in the as-received state and after clinical use. MATERIALS/METHODS Sixty one subjects were randomly assigned to four groups (N = 61), two groups of coated wires and two groups of comparable, non-coated controls (n = 15/group). The period in the mouth ranged from 4 to 12 weeks after insertion. In total, 121 wires (61 retrieved and 60 as-received) were used in the study. The percentages of coating retention and loss were extrapolated from scans. A brief survey of five questions with three choices was given to all patients. Differential scanning calorimetry (DSC) and three-point bending tests were done on as-received and used wires. RESULTS The surface characterization by the percentage of resin remaining indicated that most wires in both test groups lost a significant amount of coating. A patient survey indicated that this was a noticeable feature for patients. DSC analysis of the wires indicated that the metallurgical properties of the coated wires were not similar to the uncoated wires in the as-received condition. Three-point bending results indicate a wide variation in test results with large standard deviations among all the groups. LIMITATIONS The extent of coating loss requires investigating, as do the biological properties of the detached coating. CONCLUSIONS Both wires lost a significant amount of aesthetic coating after varying periods in the mouth. The metallurgical testing of these findings may indicate that these wires perform differently in the mouth.

Quassinoids: From traditional drugs to new cancer therapeutics

Quassinoids are a group of compounds extracted from plants of the Simaroubaceae family, which have been used for many years in folk medicine. These molecules gained notoriety after the initial discovery of the anti-leukemic activity of one member, bruceantin, in 1975. Currently over 150 quassinoids have been isolated and classified based on their chemical structures and biological properties investigated in vitro and in vivo. Many molecules display a wide range of inhibitory effects, including anti-inflammatory, anti-viral, anti-malarial and anti-proliferative effects on various tumor cell types. Although often the exact mechanism of action of the single agents remains unclear, some agents have been shown to affect protein synthesis in general, or specifically HIF-1α and MYC, membrane polarization and the apoptotic machinery. Considering that future research into chemical modifications is likely to generate more active and less toxic derivatives of natural quassinoids, this family represents a powerful source of promising small molecules targeting key prosurvival signaling pathways relevant for diverse pathologies. Here, we review available knowledge of functionality and possible applications of quassinoids and quassinoid derivatives, spanning traditional use to the potential impact on modern medicine as cancer therapeutics.

Tritrichomonas foetus isolates from cats and cattle show minor genetic differences in unrelated loci ITS-2 and EF-1alpha

The protozoan parasite Tritrichomonas foetus is well known as an important causative agent of infertility and abortion in cattle (bovine trichomonosis). This World Organisation for Animal Health (O.I.E.) notifiable disease is thought to be under control in many countries including Switzerland. In recent studies, however, T. foetus has also been identified as an intestinal parasite that causes chronic large-bowel diarrhoea in cats. Since the feline isolates were considered indistinguishable from bovine isolates, the possibility and risk of parasite transmission from cats to cattle and vice versa has been intensively discussed in current literature. Therefore, we investigated if cat and cattle isolates are genetically distinct from each other or in fact represent identical genotypes. For this purpose, two independent genetic loci were selected that turned out to be well-suited for a PCR sequencing-based genotyping of trichomonad isolates: (i) previously published internal transcribed spacer region 2 (ITS-2) and (ii) a semi-conserved sequence stretch of the elongation factor-1 alpha (EF-1alpha) gene used for the first time in the present study. Respective comparative analyses revealed that both loci were sufficiently variable to allow unambiguous genetic discrimination between different trichomonad species. Comparison of both genetic loci confirmed that T. suis and T. mobilensis are phylogenetically very close to T. foetus. Moreover, these two genetic markers were suited to define host-specific genotypes of T. foetus. Both loci showed single base differences between cat and cattle isolates but showed full sequence identity within strains from either cat or cattle isolates. Furthermore, an additional PCR with a forward primer designed to specifically amplify the bovine sequence of EF-1alpha was able to discriminate bovine isolates of T. foetus from feline isolates and also from other trichomonads. The implications these minor genetic differences may have on the biological properties of the distinct isolates remain to be investigated.

Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset neurological disease resulting from mutations in the SACS gene encoding sacsin, a 4,579-aa protein of unknown function. Originally identified as a founder disease in Québec, ARSACS is now recognized worldwide. Prominent features include pyramidal spasticity and cerebellar ataxia, but the underlying pathology and pathophysiological mechanisms are unknown. We have generated an animal model for ARSACS, sacsin knockout mice, that display age-dependent neurodegeneration of cerebellar Purkinje cells. To explore the pathophysiological basis for this observation, we examined the cell biological properties of sacsin. We show that sacsin localizes to mitochondria in non-neuronal cells and primary neurons and that it interacts with dynamin-related protein 1, which participates in mitochondrial fission. Fibroblasts from ARSACS patients show a hyperfused mitochondrial network, consistent with defects in mitochondrial fission. Sacsin knockdown leads to an overly interconnected and functionally impaired mitochondrial network, and mitochondria accumulate in the soma and proximal dendrites of sacsin knockdown neurons. Disruption of mitochondrial transport into dendrites has been shown to lead to abnormal dendritic morphology, and we observe striking alterations in the organization of dendritic fields in the cerebellum of knockout mice that precedes Purkinje cell death. Our data identifies mitochondrial dysfunction/mislocalization as the likely cellular basis for ARSACS and indicates a role for sacsin in regulation of mitochondrial dynamics.

Xanthohumol ameliorates atherosclerotic plaque formation, hypercholesterolemia, and hepatic steatosis in ApoE-deficient mice

SCOPE: Xanthohumol (XN), a prenylated antioxidative and anti-inflammatory chalcone from hops, exhibits positive effects on lipid and glucose metabolism. Based on its favorable biological properties, we investigated whether XN attenuates atherosclerosis in western-type diet-fed apolipoprotein-E-deficient (ApoE(-/-) ) mice. METHODS AND RESULTS: XN supplementation markedly reduced plasma cholesterol concentrations, decreased atherosclerotic lesion area, and attenuated plasma concentrations of the proinflammatory cytokine monocyte chemoattractant protein 1. Decreased hepatic triglyceride and cholesterol content, activation of AMP-activated protein kinase, phosphorylation and inactivation of acetyl-CoA carboxylase, and reduced expression levels of mature sterol regulatory element-binding protein (SREBP)-2 and SREBP-1c mRNA indicate reduced lipogenesis in the liver of XN-fed ApoE(-/-) mice. Concomitant induction of hepatic mRNA expression of carnitine palmitoyltransferase-1a in ApoE(-/-) mice-administered XN suggests increased fatty acid beta-oxidation. Fecal cholesterol concentrations were also markedly increased in XN-fed ApoE(-/-) mice compared with mice fed western-type diet alone. CONCLUSION: The atheroprotective effects of XN might be attributed to combined beneficial effects on plasma cholesterol and monocyte chemoattractant protein 1 concentrations and hepatic lipid metabolism via activation of AMP-activated protein kinase.

Different vascular smooth muscle cell apoptosis in the human internal mammary artery and the saphenous vein. Implications for bypass graft disease

BACKGROUND: The remarkable patency of internal mammary artery (MA) grafts compared to saphenous vein (SV) grafts has been related to different biological properties of the two blood vessels. We examined whether proliferation and apoptosis of vascular smooth muscle cells (VSMC) from human coronary artery bypass vessels differ according to patency rates. METHODS AND RESULTS: Proliferation rates to serum or platelet-derived growth factor (PDGF)-BB were lower in VSMC from MA than SV. Surface expression of PDGF beta-receptor was slightly lower, while that of alpha-receptor was slightly higher in MA than SV. Cell cycle distribution, expression of cyclin E, cdk2, p21, p27, p57, and cdk2 kinase activity were identical in PDGF-BB-stimulated cells from MA and SV. However, apoptosis rates were higher in MA than SV determined by lactate dehydrogenase release, DNA fragmentation, and Hoechst 33258 staining. Moreover, caspase inhibitors (Z-VAD-fmk, Boc-D-fmk) abrogated the different proliferation rates of VSMC from MA versus SV. Western blotting and GSK3-beta kinase assay revealed lower Akt activity in VSMC from MA versus SV, while total Akt expression was identical. Adenoviral transduction of a constitutively active Akt mutant abrogated the different proliferation rates of VSMC from MA versus SV. CONCLUSIONS: Higher apoptosis rates due to lower Akt activity rather than different cell cycle regulation account for the lower proliferation of VSMC from MA as compared to SV. VSMC apoptosis may protect MA from bypass graft disease.

Distinct subcellular expression patterns of neutral endopeptidase (CD10) in prostate cancer predict diverging clinical courses in surgically treated patients

PURPOSE: Neutral endopeptidase (CD10), an ectopeptidase bound to the cell surface, is thought to be a potential prognostic marker for prostate cancer. EXPERIMENTAL DESIGN: Prostate cancer patients (N = 3,261) treated by radical prostatectomy at a single institution were evaluated by using tissue microarray. Follow-up data were available for 2,385 patients. The cellular domain (membranous, membranous-cytoplasmatic, and cytoplasmatic only) of CD10 expression was analyzed immunohistochemically and correlated with various clinical and histopathologic features of the tumors. RESULTS: CD10 expression was detected in 62.2% of cancer samples and occurred preferentially in higher Gleason pattern (P < 0.0001). CD10 expression positively correlated with adverse tumor features such as elevated preoperative prostate-specific antigen (PSA), higher Gleason score, and advanced stage (P < 0.0001 each). Survival analyses showed that PSA recurrence was significantly associated with the staining pattern of CD10 expression. Outcome significantly declined from negative over membranous, membranous-cytoplasmatic, to exclusively cytoplasmatic CD10 expression (P < 0.0001). In multivariate analysis, CD10 expression was an independent predictor for PSA failure (P = 0.0343). CONCLUSIONS: CD10 expression is an unfavorable independent risk factor in prostate cancer. The subcellular location of CD10 protein is associated with specific clinical courses, suggesting an effect on different important biological properties of prostate cancer cells. The frequent expression of CD10 in prostate cancer and the strong association of CD10 with unfavorable tumor features may qualify this biomarker for targeted therapies.

Zebrafish (Danio rerio) as a model organism for investigating endocrine disruption

Endocrine-disrupting compounds (EDCs) are widespread in the aquatic environment and can cause alterations in development, physiological homeostasis and health of vertebrates. Zebrafish, Danio rerio, has been suggested as a model species to identify targets as well as modes of EDC action. In fact, zebrafish has been found useful in EDC screening, in EDC effects assessment and in studying targets and mechanisms of EDC action. Since many of the environmental EDCs interfere with the sex steroid system of vertebrates, most EDC studies with zebrafish addressed disruption of sexual differentiation and reproduction. However, other targets of EDCs action must not be overlooked. For using a species as a toxicological model, a good knowledge of the biological traits of this species is a pre-requisite for the rational design of test protocols and endpoints as well as for the interpretation and extrapolation of the toxicological findings. Due to the genomic resources available for zebrafish and the long experience with zebrafish in toxicity testing, it is easily possible to establish molecular endpoints for EDC effects assessment. Additionally, the zebrafish model offers a number of technical advantages including ease and cost of maintenance, rapid development, high fecundity, optical transparency of embryos supporting phenotypic screening, existence of many mutant strains, or amenability for both forward and reverse genetics. To date, the zebrafish has been mainly used to identify molecular targets of EDC action and to determine effect thresholds, while the potential of this model species to study immediate and delayed physiological consequences of molecular interactions has been instrumentalized only partly. One factor that may limit the exploitation of this potential is the still rather fragmentary knowledge of basic biological and endocrine traits of zebrafish. Information on species-specific features in endocrine processes and biological properties, however, need to be considered in establishing EDC test protocols using zebrafish, in extrapolating findings from zebrafish to other vertebrate species, and in understanding how EDC-induced gene expression changes translate into disease.