Alpha(v)beta3 integrin expression up-regulates cdc2, which modulates cell migration

The alphavbeta3 integrin has been shown to promote cell migration through activation of intracellular signaling pathways. We describe here a novel pathway that modulates cell migration and that is activated by alphavbeta3 and, as downstream effector, by cdc2 (cdk1). We report that alphavbeta3 expression in LNCaP (beta3-LNCaP) prostate cancer cells causes increased cdc2 mRNA levels as evaluated by gene expression analysis, and increased cdc2 protein and kinase activity levels. We provide three lines of evidence that increased levels of cdc2 contribute to a motile phenotype on integrin ligands in different cell types. First, increased levels of cdc2 correlate with more motile phenotypes of cancer cells. Second, ectopic expression of cdc2 increases cell migration, whereas expression of dominant-negative cdc2 inhibits migration. Third, cdc2 inhibitors reduce cell migration without affecting cell adhesion. We also show that cdc2 increases cell migration via specific association with cyclin B2, and we unravel a novel pathway of cell motility that involves, downstream of cdc2, caldesmon. cdc2 and caldesmon are shown here to localize in membrane ruffles in motile cells. These results show that cdc2 is a downstream effector of the alphavbeta3 integrin, and that it promotes cell migration.     

Determination of cocaine, benzoylecgonine and cocaethylene in human hair by solid-phase microextraction and gas chromatography-mass spectrometry

The present work describes a highly precise and sensitive method developed to detect cocaine (COC), benzoylecgonine (BE, its main metabolite) and cocaethylene (CE, transesterification product of the coingestion of COC with ethanol) in human head hair samples. The method was based on an alkylchloroformate derivatization of benzoylecgonine and the extraction of the analytes by solid-phase microextraction (SPME). Gas chromatography-mass spectrometry (GC-MS) was used to identify and quantify the analytes in selected ion monitoring mode (SIM). The limits of quantification and detection (LOQ and LOD) were: 0.1 ng/mg for COC and CE, and 0.5 ng/mg for BE. Good inter- and intra-assay precision was observed. The dynamic range of the assay was 0.1-50 ng/mg. The method is not time consuming and was shown to be easy to perform.     

Simultaneous liquid chromatography-tandem mass spectrometric determination of albendazole sulfoxide and albendazole sulfone in plasma

This paper describes a simple, fast, sensitive and reliable method for the simultaneous determination of albendazole sulfoxide (ASOX) and albendazole sulfone (ASON), the two most important metabolites of the drug albendazole (ABZ), in plasma samples using liquid chromatography and tandem mass spectrometry. After liquid-liquid extraction with dichloromethane, the two albendazole metabolites and the internal standard phenacetin were resolved in a CN column using the mobile phase methanol-water (4:6, v/v) acidified with 1% acetic acid. Detection by electrospray mass spectrometry was carried out in the positive ion mode. The method was linear up to 2500 and 250 ng/ml for ASOX and ASON, respectively, with mean recoveries of more than 85%. The precision and accuracy data, based on within- and between-day variations over 5 days, were lower than 15%. The quantitation limits of 0.5 and 5.0 ng/ml for ASON and ASOX are low enough for the method to be suitable for pharmacokinetic studies. Pharmacokinetic data obtained with the proposed method following oral administration of ABZ to a patient with neurocysticercosis are also reported.     

1H-NMR study of the quadruplex [d(TGGGT)]4 containing a modified thymine

A NMR structural study of quadruplex [d(TGGGT)]4 containing a modified thymine is reported. The three dimensional structure of the complex is very similar to those of other parallel stranded quadruplexes. The modified thymines (T*) are able, at least in the minimised structures, to form a tetrad containing extra H-bonds through the hydroxyl groups. Nevertheless, in this new tetrad the modified thymines are slightly open towards the solvent respect to the unmodified T-tetrad.     

Malondialdehyde in benign prostate hypertrophy: a useful marker?

Benign prostate hypertrophy (BPH) is the most common benign tumor in men due to obstruction of the urethra and, finally, uremia. Malondialdehyde (MDA) is a product derived from peroxidation of polyunsaturated fatty acids and related esters. Evaluation of MDA in serum represents a non-invasive biomarker of oxidative stress. Prostate-specific antigen (PSA) is a sensitive marker for prostatic hypertrophy and cancer. We analyzed MDA serum levels to evaluate the oxidative stress in BPH. To this end, 22 BPH patients and 22 healthy donors were enrolled. Data show an increase of MDA level in BPH patients and a positive correlation between PSA and MDA levels. In conclusion, we describe a previously unknown relationship between PSA and MDA as an index of inflammation and oxidative stress in BPH.     

Synthesis and opioid activity of N,N-dimethyl-Dmt-Tic-NH-CH(R)-R' analogues: acquisition of potent delta antagonism

N,N-Dimethylation of the H-Dmt-Tic-NH-CH(R)-R′ series of compounds produced no significant affect on the high δ-opioid receptor affinity (K_i=0.035–0.454 nM), but dramatically decreased that for the μ-opioid receptor. The effect of N-methylation was independent of the length of the linker (R); however, the bioactivities were affected by the chemical composition of the third aromatic group (R′): phenyl (Ph) (5′–8′) elicited a greater reduction in μ-affinity (40–70-fold) compared to analogues containing 1H-benzimidazole-2-yl (Bid) (9-fold). The major consequences of N,N-dimethylation on in vitro bioactivity were: (i) a loss of δ-agonism coupled with the appearance of potent δ antagonism (4′–7′) (pA₂=8.14–9.47), while 1 exhibited only a 160-fold decreased δ agonism (1′) and the δ antagonism of 8 enhanced >10-fold (pA₂=10.62, 8′); and (ii) a consistent loss of μ-affinity resulted in enhanced δ-opioid receptor selectivity. With the exception of compound 1′, the change in the hydrophobic environment at the N-terminus and formation of a tertiary amine by N,N-dimethylation in analogues of the Dmt-Tic pharmacophore produced potent δ-selective antagonists.     

Eradication of Helicobacter pylori infection improves blood pressure values in patients affected by hypertension

Background. Arterial hypertension is a risk factor for atherosclerosis of whose pathogenesis is unknown. Growing evidence underscores the causative role of endothelial dysfunction. A possible association between Helicobacter pylori infection and cardiovascular and autoimmune disorders has been found. The release of cytotoxic substances either of bacterial origin or produced by the host may represent mediators of these systemic sequelae. The aim of our study was to determine the prevalence of H. pylori infection in hypertensive patients and the effects of H. pylori eradication on blood pressure and on digestive symptoms. Materials and Methods. Seventy‐two hypertensive patients (34 male and 38 female; mean age 53 ± 12 years) and 70 normotensive controls (35 male and 35 female; mean age 52 ± 10 years) were enrolled. All patients were subjected to a first ambulatory blood pressure monitoring (ABPM) at enrollment, a ¹³C urea breath test and a test for IgG‐CagA antibodies, and completed the validated dyspepsia questionnaire. H. pylori‐positive patients were treated with triple therapy (amoxicillin, clarithromycin and ranitidine bismute citrate) for 7 days. Control of eradication was assessed by ¹³C urea breath test, and all patients underwent a second ABPM 6 months after enrollment. Results. H. pylori infection was 55% in hypertensive patients, with 90% CagA positivity, and 50% in controls, with 60% CagA positivity. At the first ABPM, blood pressure values were similar in H. pylori‐positive and ‐negative individuals; positive patients showed a significant increase in pyrosis and epigastric pain compared to negative patients. H. pylori was eradicated in 80% of patients and in 85% of controls. At the second ABPM, we found a statistically significant decrease in 24‐hour mean blood pressure values when compared to the first ABPM only in the eradicated hypertensive group. Conclusions. Our study demonstrated a significant decrease in blood pressure values, in particular in diastolic blood pressure values, after H. pylori eradication in hypertensive patients. A high prevalence of CagA positivity was found. The association between cardiovascular disease and H. pylori infection seems pronounced only in CagA‐positive patients. The possible links between hypertensive disease and H. pylori infection may involve the activation of the cytokine cascade with the release of vasoactive substances from the primary site of infection, or molecular mimicry between the CagA antigens of H. pylori and some peptides expressed by endothelial cells and smooth muscle cells.     

Dog keeping in Taiwan: its contribution to the problem of free-roaming dogs

This study conducted a quantitative ethnographic analysis of the influence of demographic factors and early experience (childhood exposure to dogs) on Taiwanese dog-keeping practices and behavior. A telephone survey of a randomly selected sample of 2,001 Taiwan residents determined their dog ownership histories, current patterns of dog ownership and disposal, and other dog-related activities. The results suggest that low rates of neutering, easy availability of low- or no-cost puppies, a tendency to allow owned dogs free access to the outdoors, unrealistic expectations of dog ownership, canine behavioral problems, and religious and cultural taboos against euthanasia and shelter relinquishment have contributed to the recent increase in the numbers of free-roaming dogs in Taiwan. Logistic regression analyses determined that a relatively small number of demographic and experiential variables predicted dog ownership and disposal patterns. The most important of these was childhood experience of living with household dogs. In light of these findings, future efforts to reduce the stray dog problem should focus on enforcing registration fees, particularly for unsterilized animals; low-cost neutering schemes; and educational programs designed to promote neutering, improve knowledge of canine behavior and behavior problems, and develop more realistic expectations and attitudes toward dog ownership. Marked Taiwanese resistance to canine euthanasia and shelter relinquishment suggests a need for alternative methods of managing the existing free-roaming dog population.     

Dual inhibition of focal adhesion kinase and epidermal growth factor receptor pathways cooperatively induces death receptor-mediated apoptosis in human breast cancer cells

The focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) are protein-tyrosine kinases that are overexpressed and activated in human breast cancer. To determine the role of EGFR and FAK survival signaling in breast cancer, EGFR was stably overexpressed in BT474 breast cancer cells, and each signaling pathway was specifically targeted for inhibition. FAK and EGFR constitutively co-immunoprecipitated in EGFR-overexpressing BT474 cells. In low EGFR-expressing BT474-pcDNA3 vector control cells, inhibition of FAK by the FAK C-terminal domain caused detachment and apoptosis via pathways involving activation of caspase-3 and -8, cleavage of poly(ADP-ribose) polymerase, and caspase-3-dependent degradation of AKT. This apoptosis could be rescued by the dominant-negative Fas-associated death domain, indicating involvement of the death receptor pathway. EGFR overexpression did not inhibit detachment induced by the FAK C-terminal domain, but did suppress apoptosis, activating AKT and ERK1/2 survival pathways and inhibiting cleavage of FAK, caspase-3 and -8, and poly(ADP-ribose) polymerase. Furthermore, this protective effect of EGFR signaling was reversed by EGFR kinase inhibition with AG1478. In addition, inhibition of FAK and EGFR in another breast cancer cell line (BT20) endogenously overexpressing these kinases also induced apoptosis via the same mechanism as in the EGFR-overexpressing BT474 cells. The results of this study indicate that dual inhibition of FAK and EGFR signaling pathways can cooperatively enhance apoptosis in breast cancers.