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Radiocontrast medium induced nephrotoxicity is a major clinical problem. There is considerable interest in reducing the incidence of acute renal failure due to the use of radiocontrast media (RCM). Reduction of renal blood flow and direct toxic effect on renal tubular epithelial cells have been postulated as major causes of RCM nephropathy. Understanding the molecular mechanisms by which RCM cause cell damage may allow the development of pharmacological therapy to prevent their nephrotoxicity. In this work we have investigated the signaling pathways that may be affected by RCM. The incubation of human renal tubular proximal cells with sodium diatrizoate, iopromide and iomeprol caused a marked dephosphorylation of the kinase Akt on Ser473 within 5min of incubation. RCM also caused a decrease in cell viability, which was substantially alleviated by transfecting the cells with a constitutively active form of Akt. Further downstream targets of Akt, including the Forkhead family of transcription factors FKHR and FKHRL1, were also dephosphorylated by RCM at Thr24 and Thr32, respectively. The P70S6 kinase was also dephosphorylated at Thr389 and Ser371 by RCM. However there was a more dramatic decrease in phosphorylation of the phosphorylated form of mammalian target of rapamycin (mTOR) and of the extracellular-signal regulated kinases (ERK) 1/2 caused by sodium diatrizoate than by iopromide. These results demonstrate the effect of RCM on some intracellular signaling pathways that may allow understanding of the mechanism of their toxicity and may allow the development of strategies to overcome their adverse effects.

An analytical HPLC method was developed to determine monomeric anthocyans as catechin (CA), flavonols as rutin (RU) and phenol acids as gallic acid (GA) adsorbed on the cell wall of 23 Saccharomyces cerevisiae strains grown on two media containing high levels of phenolic compounds, from grape seeds and grape skins, and on one control medium. Microbial biomass purification by liquid-liquid extraction of the phenolics is followed by reversed-phase chromatographic separation and CA, RU and GA detection by ultraviolet detector. The method was linear over the studied range of concentrations: GA at 0.12-0.96 microg/ml, CA at 0.25-20.00 microg/ml and RU at 0.02-0.20 microg/ml. The correlation coefficient for each analyte was greater than 0.9983. The recovery was greater than 85% for both GA and RU, and greater than 94% for CA. The detection limits for GA, CA and RU were determined to be 0.015, 0.025 and 0.029 microg/mg of biomass, respectively. The proposed method is highly responsive for the determination of different phenolics, and seems to be useful to evaluate their adsorption profile on yeasts.

Ptprj is a ubiquitously expressed murine gene encoding a receptor-type protein tyrosine phosphatase, which has recently been proposed as a candidate gene on the locus Scc1 for colon cancer susceptibility. It has been demonstrated that PTPRJ, the human homologue of Ptprj, is involved in the control of cell growth and adhesion, being furthermore altered in several types of cancer including mammary, thyroid, lung, colon, and pancreatic cancers. To investigate the biological functions of Ptprj, we have generated mice deficient in this receptor protein tyrosine phosphatase. Ptprj-deficient mice are viable, fertile, and show no gross anatomical alterations. Furthermore, neither changes in life span nor spontaneous tumor appearance were observed in Ptprj-null mice. Our results indicate that Ptprj is dispensable for normal growth and development in mice.

Context: It is unknown whether genetic factors that play an important role in body weight homeostasis influence the response to laparoscopic adjustable gastric banding (LAGB)., Objective: We investigated the impact of common polymorphisms in four candidate genes for insulin resistance on weight loss after LAGB., Design: The design was a 6-month follow-up study., Setting: The study setting was hospitalized care., Patients: A total of 167 unrelated morbidly obese subjects were recruited according to the following criteria: age, 18-66 yr inclusive; and body mass index greater than 40 kg/m2 or greater than 35.0 kg/m2 in the presence of comorbidities., Intervention: LAGB was used as an intervention., Main Outcome Measure: Measure of correlation between weight loss and common polymorphisms in candidate genes for insulin resistance and obesity was the main outcome measure., Results: The following single nucleotide polymorphisms were detected by digestion of PCR products with appropriate restriction enzymes: Gly972Arg of the insulin receptor substrate-1 gene, Pro12Ala of the proliferator-activated receptor-gamma gene, C-174G in the promoter of IL-6 gene, and G-866A in the promoter of uncoupling protein 2 gene. Baseline characteristics including body mass index did not differ between the genotypes. At the 6-month follow-up after LAGB, carriers of G-174G IL-6 genotype had lost more weight than G-174C or C-174C genotype (P = 0.037), and carriers of A-866A uncoupling protein 2 genotype had lost more weight as compared with G-866G (P = 0.018) and G-866A (P = 0.035) genotype, respectively. Weight loss was lower in carriers of Gly972Arg insulin receptor substrate-1 genotype than Gly972Gly carriers, but not statistically significant (P = 0.06). No difference between carriers of Pro12Ala and Pro12Pro proliferator-activated receptor-gamma genotype was observed., Conclusions: These data demonstrate that genetic factors, which play an important role in the regulation of body weight, may account for differences in the therapeutic response to LAGB.

Recently a novel class of non-competitive AMPA receptor (AMPAR) antagonists, such as, N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (PS3Ac) have been developed using molecular modeling studies. In this study we present a validated method for detecting PS3Ac in biological matrices by high performance liquid chromatography with ultraviolet detection. In this study PS3Ac was administered to Wistar rats. After intraperitoneal administration, the plasma concentrations of PS3Ac and its potential metabolic products, i.e., PS3OH, PS3 and PS3OHAc were determined. Serum samples (0.5 ml) were purified by solid-phase extraction of analytes using Oasis cartridges. The chromatographic separation was performed on a LiChrosorb RP-1 at 30 degrees C. The eluent was made of potassium dihydrogen phosphate/acetonitrile in ratio of 50:50 (v/v); the flow rate was 1 ml/min. The detection was performed at 220 nm. The method exhibited a large linear range from 0.05 to 5 microg/ml for all studied compounds. The intra-assay accuracy ranged from 92% determined at 0.1 microg/ml of PS3OH, to 108% determined at 0.05 microg/ml of PS3OHAc. The average coefficient of variation of inter-assay was 6.27%. The average recovery from plasma was 78.5%. The limits of quantification for all the tetrahydroisoquinoline derivatives was 20 ng. The method proved to be highly sensitive and specific for the determination of the studied compounds in rat plasma and has been successfully applied to the evaluation of the pharmacokinetic profile of the inoculated compound.

In this work, we present molecular modeling studies carried out using six DNA sequences and six azinomycin analogues, including the naturally occurring compound azinomycin B, selected on the basis of known cell cytotoxicity and structural analogies (epoxide and aziridine alkylating moieties). Among several computational methods the Stochastic Dynamics with Energy Minimization (SDEM) approach yielded results superior to the others with the natural compound (r(2) > 0.9) and was adopted for studying other DNA adducts, obtaining good correlation between the average theoretical cross-linking properties and the antitumor activity scale. As a result, some interesting SAR considerations have been highlighted and a cross-linking conformation different from that of the azinomycin was identified in a less potent, simplified analogue.

Interferon alpha (IFN-alpha) has been widely used in the treatment of human solid and haematologic malignancies. Although the antitumour activity of IFN-alpha is well recognised at present, no major advances have been achieved in the last few years. Recent findings have provided new information on the molecular mechanisms of the antitumour activity of the cytokine. In fact, IFN-alpha appears to block cell proliferation, at least in part, through the induction of apoptotic effects. This cytokine can also regulate the progression of tumour cells through the different phases of the cell cycle inducing an increase of the expression of the cyclin-dependent kinase inhibitors p21 and p27. However, it must be considered that IFN-alpha is a physiologic molecule with ubiquitously expressed receptors that is likely to activate survival mechanisms in the cell. We have recently identified an epidermal growth factor (EGF) Ras-dependent protective response to the apoptosis induced by IFN-alpha in epidermoid cancer cells. The identification of tissue- and/or tumour-specific survival pathways and their selective targeting might provide a new approach to improve the efficacy of IFN-alpha-based treatment of human cancer. Moreover, new pegylated species of IFN-alpha are now available with a more favourable pharmacokinetic profile. We will review these achievements, and we will specifically address the topic of IFN-alpha-based molecularly targeted combinatory antitumour approaches.

Objective: The aim of this study was to investigate whether diabetic patients carrying the Arg(972) insulin receptor substrate-1 (IRS-1) variant are at increased risk for secondary failure to sulfonylurea., Research Design and Methods: A total of 477 unrelated Caucasian type 2 diabetic patients were recruited according to the following criteria: onset of diabetes after age 35 years, absence of ketonuria at diagnosis, and anti-GAD(-) antibody. Type 2 diabetes was diagnosed according to the American Diabetes Association criteria. Patients with secondary sulfonylurea failure were defined as those requiring insulin due to uncontrolled hyperglycemia (fasting plasma glucose >300 mg/dl) despite sulfonylurea-metformin combined therapy, appropriate diet, and absence of any conditions causing hyperglycemia., Results: Of the total patients, 53 (11.1%) were heterozygous for the Arg(972) IRS-1 variant, 1 (0.2%) was homozygous, and the remainder (88.7%) were homozygous for the wild-type allele. The genotype frequency of the Arg(972) IRS-1 variant was 8.7% among diabetic patients well controlled with oral therapy and 16.7% among patients with secondary failure to sulfonylurea (odds ratio 2.1 [95% CI 1.18-3.70], P = 0.01). Adjustment for age, sex, BMI, metabolic control, age at diagnosis, duration of diabetes, and Pro12Ala polymorphism of peroxisome proliferator-activated receptor-gamma2 gene in a logistic regression analysis with secondary failure to sulfonylurea as a dependent variable did not change this association (2.0 [1.38-3.86], P = 0.038)., Conclusions: These data demonstrate that the Arg(972) IRS-1 variant is associated with increased risk for secondary failure to sulfonylurea, thus representing a potential example of pharmacogenetics in type 2 diabetes.

Objectives: The systematic parasagittal sextant biopsy technique under transrectal ultrasound guidance, routinely performed to diagnose and stage prostate cancer, has been shown to outperform directed or random biopsies, revolutioning our ability to detect carcinoma of the prostate. Different biopsy schemes have been proposed with similar positive percentage of cancer detection. The present study evaluate from the patient's perspective the complications with the use of two different systematic biopsy protocols with 14 and 8 cores., Material and Methods: Between January 1999 and February 2000, 177 consecutive patients, mean age 64.1 +/- 7.7 years, referred for normal screening digital rectal examination (DRE) and prostate specific antigen (PSA) level 4-10 ng/ml, were submitted to a transrectal US examination followed by lesion directed and 14 scheme systematic biopsies to detect prostate cancer. Biopsies were obtained from conventional sextant biopsies (6 core) and 3 alternate sites which included: the right and left extreme lateral peripheral zone between anterior tissue and posterior gland base (2 core); the right and left transition zone, immediately adjacent to the urethra anterior and posterior (4 core) and the right and left central gland in the mid zone typical of benign prostatic hyperplasia (BPH) (2 core). All specimens were separated for specific location identification. Cancer was identified in 61 patients (34.46%). Traditional sextant biopsies showed 23 patients (37.7%) with positive core to detect cancer, while a sextant regimen incorporating lateral peripheral zone biopsies and transitional zone detected 19 cancer (31.1%). The combination of lateral peripheral and transitional zone alone detected cancer in 59 patients. No cancer was detected in central gland. The lateral peripheral zone was the most frequently positive site biopsy followed by the transitional zone. According the results of our study from April 2000 we started to consider a novel scheme to reduce number of biopsies maintaining the same sensitivity. A subsequent group of 121 patients, mean age 61 +/- 4.6 years, enrolled from April 2000 to May 2001, underwent a transrectal US examination followed by lesion directed and 8 scheme systematic biopsies. None of the patients had previously undergone prostate biopsy. In all patients a visual analog score (VAS) questionnaire about pain and complications was obtained 7 days after the procedure., Results: Of the 149 patients who completed the questionnaire 9.9% found the procedure moderately to extremely painful afterwards, with a VAS > 5, the commonest of these complications being pain and voiding difficulties with a mean value of 15.8%, systemic symptoms as fever or sweats with a mean value of 7.65% of cases. Between the two groups submitted to 14 or 8 scheme biopsies, we detect a statistically significant difference for urethral bleeding (7.3% vs 4.9% p value 0.05) and rectal bleeding (10.3% vs 3.7% p value 0.04), systemic symptoms 10.3% vs 5.0% p value 0.05) and painful voiding afterwards (5.8% vs 2.4% p value 0.02)., Conclusions: Ultrasound guided transrectal biopsy of the prostate is a well tolerated and effective method for obtaining multiple biopsy specimens from the prostate with low incidence of serious complications. The absolute value of referred complications with VAS < 5 results high, but the rate of major complications results low. The 8 biopsy scheme, including sampling in peripherial zone at midgland and transition zone periuretrally toward the base, should be considered in a initial biopsy scheme to reduce number of biopsy and enhancing sensitivity, with a significant less degree of complication rate compared to a extensive 14 biopsy scheme.

Crohn' s disease (CD) is a chronic intestinal inflammatory disorder characterized by aberrant mucosal Th1 cell activation and production of IL-12, the major Th1-driving factor. The T cell response to IL-12 is dependent on the expression of a specific receptor composed of two subunits, termed IL-12Rbeta1 and IL-12Rbeta2. The content of IL-12Rbeta2, as measured at the mRNA level, is crucial in regulating Th1 differentiation. In this study we therefore investigated IL-12Rbeta2 RNA transcripts in CD. IL-12Rbeta2 expression was increased in active CD as well as Helicobacter pylori (HP)-associated gastritis and Salmonella colitis compared with that in inactive CD, ulcerative colitis, noninflammatory controls, and celiac disease. In contrast, IL-12Rbeta1 transcripts were expressed at comparable levels in all samples. In CD, IL-12Rbeta2 expression strictly correlated with tyrosine phosphorylation of STAT4, a key component of the IL-12-dependent Th1 polarization. This was associated with a pronounced expression of IFN-gamma. Transcripts for IL-12/p40 were detected in CD, HP-positive, and Salmonella colitis patients, but not in celiac disease, indicating that IL-12Rbeta2 up-regulation occurs only in IL-12-associated Th1 gastrointestinal diseases. Finally, we showed that stimulation of lamina propria mononuclear cells with IL-12 enhanced IL-12Rbeta2, suggesting that IL-12 regulates IL-12Rbeta2 expression in human gastrointestinal mucosa. The data show that the signaling pathway used by IL-12 to induce Th1 differentiation is increased at the site of disease in CD, further supporting the view that IL-12/IL-12R signals contribute to the inflammatory response in this condition.