Knowledge of sexual consent as a protective factor against sexual violence perpetration among first-year college men: a moderation analysis.

This study examined whether knowledge of sexual consent buffers the relationship between risk factors for sexual violence (SV) and SV perpetration among first-year college men. The study data were drawn from a longitudinal study with 1144 first-year college men. A series of generalized linear models were conducted to examine whether knowledge of sexual consent moderated the relationship between SV risk factors and SV perpetration. Knowledge of sexual consent moderated the effect of hypermasculinity (P < 0.001), binge drinking (P < 0.001), rape-supportive social norms (P = 0.007) and peer support for SV (P < 0.001) such that there was a positive association between risk factors and SV perpetration among those with lower, but not higher, knowledge of sexual consent. Knowledge of sexual consent did not significantly moderate the relationship between SV perpetration and outcome expectancies for non-consensual sex (P = 0.387) and pornography use (P = 0.494). Knowledge of sexual consent may counteract risk factors for SV perpetration among young college men. The findings highlight the need for consent education to be incorporated in youth comprehensive sexual education to increase knowledge of sexual consent prior to college and campus-based SV prevention programming delivered to college students.

Pilot study on the effects of high molecular weight sodium hyaluronate in the treatment of chronic pharyngitis.

Although several therapeutic approaches are available at present for the treatment of chronic pharyngitis, new therapeutic strategies acting on pharyngeal mucous function should be investigated in order to improve symptoms and quality of life. High-molecular weight hyaluronate performs important functions on mucociliary clearance, tissues hydration, defense against micro-organisms, and on tissue repair as well, but at present there is no clinical evidence of its exogenous use in patients with chronic pharyngitis. Our open, randomized controlled study was carried out to investigate efficacy, and tolerability of exogenous high molecular weight sodium hyaluronate (SH) at the dosage of 9 mg three times a day for a period of 30 days, in patients with chronic pharyngitis. Results show significant improvements of symptoms and cytology in active group (A, n = 10) vs. control group (B, n = 10). Good compliance and no adverse events were reported in group A. In conclusion, SH was effective and safe in patients with chronic pharyngitis.

Future research and collaboration: the "SINERGIE" project on HCV (South Italian Network for Rational Guidelines and International Epidemiology).

The SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology) project is intended to set up a collaborative network comprising virologists, clinicians and public health officials dealing with patients affected by HCV disease in the Calabria Region. A prospective observational data-base of HCV infection will be developed and used for studies on HCV natural history, response to treatment, pharmaco-economics, disease complications, and HCV epidemiology (including phylogenetic analysis). With this approach, we aim at improving the identification and care of patients, focusing on upcoming research questions. The final objective is to assist in improving care delivery and inform Public Health Authorities on how to optimize resource allocation in this area.

Impact of SARS-CoV-2 on dentistry: a review of literature.

OBJECTIVE: SARS-CoV-2 is a new Coronavirus identified as the cause of Coronavirus disease in 2019 (COVID-19). The epidemic spread in China and beyond its borders, involving 114 countries with more than 5 million dead. On March 11, the WHO declared the spread of SARS-CoV-2 to be a pandemic and encouraged nations to adopt harsh restrictive measures. Therefore, patients more and more often turn to dental offices only for emergencies. Healthcare professionals, including dentists, are at high infectious risk. In fact, the closeness to the oral cavity and nasopharynx and the use of drills or ultrasonic devices that cause aerosol release, make dental professions at high risk of bacterial and viral infections. The way patients are treated has changed. In fact, it should be mandatory to carry out a pre-treatment telephone triage and the use of mouthwashes to reduce bacterial load. In the current pandemic, it is necessary to adopt specific safety protocols that can protect dental operators as well as limit the spread of the virus. The purpose of this review is to present an overview on ways to reduce the risk of SARS-CoV-2 contagion in dentistry by focusing on the immediate situation as well as by looking towards the future. MATERIALS AND METHODS: To reach the review purpose, we selected a series of studies using keywords "COVID-19" OR "SARS-CoV-2" in association with "dentistry" AND "safety protocols" AND "healthcare procedures" AND "individual protection dispositive" AND "air transmission" AND "droplet". We selected papers exclusively in English language, up to 1st January 2022. RESULTS: During future phases of the pandemic, everywhere in the World, it is necessary to impose all dentistry team both a serological screening and the vaccination, as already established for all health staff in Italy. CONCLUSIONS: For own safety, it is an important for the whole dentistry category constantly update the devices and the protocols adopted, as well as monitoring the real infectious threats, which may occur.

Quantitative muscle mass biomarkers are independent prognosis factors in primary central nervous system lymphoma: The role of L3-skeletal muscle index and temporal muscle thickness.

OBJECTIVE: To investigate the role of quantitative muscle biomarkers assessed with skeletal muscle index at the third lumbar vertebra (L3-SMI) and temporal muscle thickness (TMT) in predicting progression-free and overall survival in patients with primary central nervous system lymphoma (PCNSL) undergoing first-line high-dose methotrexate-based chemotherapy. METHODS: L3-SMI and TMT were calculated on abdominal CT and brain high-resolution 3D-T1-weighted MR images, respectively, using predefined validated methods. Standardized sex-specific cut-off values were used to divide patients in different risk categories. Kaplan-Meier plots were calculated, and survival analysis was performed using log-rank tests, univariate, and multivariable Cox-regression models, calculating hazard ratios (HR) and 95% confidence intervals (CI), also adjusting for potential confounders (age, sex, and performance status). RESULTS: Forty-three patients were included in this study. Median follow-up was 23 months (interquartile range 12-40); at median follow-up, rates of progression-free and overall survival for the cohort were 46% and 57%, respectively. Thirteen (30%) and 11 (26%) patients showed L3-SMI or TMT values below the predefined cut-offs. In Cox-regression multivariable analysis patients with low L3-SMI or TMT showed significantly worse progression-free (HR 4.40, 95% CI 1.66-11.61, p = 0.003; HR 4.40, 95% CI 1.68-11.49, p = 0.003, respectively) and overall survival (HR 3.16, 95% CI 1.09-9.11, p = 0.034; HR 4.93, 95% CI 1.78-13.65, p = 0.002, respectively) compared to patients with high L3-SMI or TMT. CONCLUSIONS: Quantitative muscle mass evaluation assessed by both L3-SMI and TMT is a promising tool to identify PCNSL patients at high risk of negative outcome. Confirmatory studies on larger independent series are warranted.

Aging resistance, mechanical properties and translucency of different yttria-stabilized zirconia ceramics for monolithic dental crown applications.

OBJECTIVES: The dental market moves towards high-translucency monolithic zirconia dental crowns, which are usually placed either with - or without - a thin glaze layer. The microstructural features and the mechanical performances of these materials are still controversial, as well as their susceptibility to aging. This paper aims at studying these aspects in the current generation of zirconia dental crowns showing different degrees of translucency. METHODS: Four different commercial zirconia materials were investigated, including one standard 'full-strength' 3Y-TZP and three grades with improved translucency. The microstructural features (phase composition and assemblage, grain size) were carefully studied, as well as mechanical properties (biaxial bending strength and indentation toughness), translucency and aging behavior (in autoclave at 134°C). Aging was conducted on crowns with and without glaze to better represent clinical uses. RESULTS: Important differences are found in terms of microstructures among the materials in terms of cubic phase content and yttria in the tetragonal phase, leading to different optical, mechanical and aging resistance properties. We show that higher cubic phase content leads to better translucency and stability in water steam, but at the expense of strength and toughness. A compromise is always inevitable between translucency and aging resistance on one side and mechanical properties on the other side.

Intermittent hypoxia as a means to improve aerobic capacity in type 2 diabetes.

Physical inactivity and a low maximal aerobic capacity (VO2max) strongly predict morbidity and mortality in patients with type 2 diabetes (T2D). Patients with T2D have a reduced VO2max when compared with healthy individuals of similar age, weight, and physical activity levels, and this lower aerobic capacity is usually attributed to a reduced oxygen delivery to the working muscles. The oxygen carrying capacity of the blood, as well as increases in cardiac output and blood flow, contribute to the delivery of oxygen to the active muscles during exercise. Hemoglobin mass (Hb mass), a key determinant of oxygen carrying capacity, is suggested to be reduced in patients with T2D following the observation of a lower blood volume (BV) in combination with normal hematocrit levels in this population. Therefore, a lower Hb mass, in addition to a reported lower BV and impaired cardiovascular response to exercise, likely contributes to the reduced oxygen delivery and VO2max in patients with T2D. While exercise training increases Hb mass, BV, and consequently VO2max, the majority of patients with T2D are not physically active, highlighting the need for alternative methods to improve VO2max in this population. Exposure to hypoxia triggers the release of erythropoietin, the hormone regulating red blood cell production, which increases Hb mass and consequently BV. Exposure to mild intermittent hypoxia (IH), characterized by few and short episodes of hypoxia at a fraction of inspired oxygen ranging between 10 and 14% interspersed with cycles of normoxia, increased red blood cell volume, Hb mass, and plasma volume in patients with coronary artery disease or chronic obstructive pulmonary disease, which resulted in an improved VO2max in both populations. We hypothesize that 12 exposures to mild IH over a period of 4weeks will increase Hb mass, BV, cardiac function, and VO2max in patients with T2D. Therefore, exposures to mild IH may increase oxygen delivery and VO2max without the need to perform exercise in patients with T2D.

Optimizing a canine survival model of orthotopic lung transplantation.

INTRODUCTION: While acute models of orthotopic lung transplantation have been described in dogs, the technical considerations of developing a survival model in this species have not been elaborated. Herein, we describe optimization of a canine survival model of orthotopic lung transplantation. METHODS: Protocols of orthotopic left lung transplantation and single lung ventilation were established in acute experiments (n=9). Four dogs, serving as controls, received autologous, orthotopic lung transplants. Allogeneic transplants were performed in 16 DLA-identical and 16 DLA-mismatched unrelated recipient dogs. Selective right lung ventilation was utilized in all animals. A Malecot tube was left in the pleural space connected to a Heimlich valve for up to 24 hours. To date, animals have been followed up to 24 months by chest radiography, pulmonary function tests, bronchoscopy with lavage, and open biopsies. RESULTS: Long-term survival was achieved in 34/36 animals. Two recipients died intraoperatively secondary to cardiac arrest. All animals were extubated on the operating table, and in all cases the chest tube was removed within 24 hours. Major complications included thrombosis of the pulmonary artery and subcritical stenosis of bronchial anastamosis. One recipient underwent successful treatment of a small bowel intussusception. CONCLUSIONS: We report our experience in developing a survival canine model of orthotopic single lung transplantation. While short-term survival following canine lung transplantation is achievable, we report particular considerations that facilitate animal comfort, early extubation, and lung reexpansion in the immediate postoperative period, further optimizing use of this species for experimental modeling of long-term complications after lung transplantation.

Cellular sensitivity to beta-diketonato complexes of ruthenium(III), chromium(III) and rhodium(III).

The aim of this study was to investigate cellular response to several ruthenium(III), chromium(III) and rhodium(III) compounds carrying bidentate beta-diketonato ligands: [(acac)--acetylacetonate ligand, (tfac)--trifluoroacetylacetonate ligand]. Cell sensitivity studies were performed on several cell lines (A2780, cisplatin-sensitive and -resistant U2-OS and U2-OS/Pt, HeLa, B16) using growth-inhibition assay. Effect of intracellular GSH depletion on cell sensitivity to the agents was analyzed in A2780 cells. Flow cytometry was used to assess apoptosis by Annexin-V-FITC/PI staining, and to analyze induction of caspase-3 activity. Possible DNA binding/damaging affinity was investigated, by inductively coupled mass spectrometry, and by 14C-thymidine / 3H-uridine incorporation assay. Cell sensitivity studies showed that the pattern of sensitivity to Ru(tfac)3 complex of the two cisplatin-sensitive/-resistant osteosarcoma cell lines, U2-OS and U2-OS/Pt, was similar to that of A2780 cells (72 h exposure), with the IC50 being around 40 microM. The growth-inhibitory effect of Ru(acac)3 ranged over 100 microM, while Cr(III) and Rh(III) complexes were completely devoid of antitumor action in vitro. Ru(tfac)3 exhibited strong potential for apoptosis induction on A2780 cells (up to 40%) and caused cell cycle arrest in the S phase as well as decrease of the percent of G1 and G2 cells. Ru(acac)3-induced apoptosis was slightly higher than 10%, whereas activation of caspase-3 in HeLa cells was moderate. DNA binding study revealed that only Cr(acac)3 was capable of binding DNA, while Cr(III) and Ru(III) compounds possess potential to inhibit DNA/RNA synthesis. In conclusion, only Ru(III) complexes showed potential for antitumor action.

Adverse drug reactions associated with off-label use of ketorolac, with particular focus on elderly patients. An analysis of the Italian pharmacovigilance database and a population based study.

OBJECTIVE: This study aims to evaluate the frequency of off-label use of ketorolac in Italy and the related suspected adverse drug reactions (ADRs) reported. METHODS: All the suspected cases associated with ketorolac recorded in the Italian Pharmacovigilance database were retrieved. Case evaluations were carried out in order to identify the off-label use of ketorolac. Moreover, an analysis of the inappropriate use of ketorolac was conducted using the 'Arianna' database of Caserta local health unit. RESULTS: Up to December 2014, 822 reports of suspected ADRs related to ketorolac were retrieved in the database. The use of ketorolac was classified as off-label for 553 reports and on-label for 269. Among the off-label cases, 58.6% were serious compared to 39.0% of on-label cases. Gastrointestinal events were more frequently reported with off-label use. The analysis of Arianna database showed that 37,729 out of 61,910 patients, were treated off-label. CONCLUSIONS: The off-label use of ketorolac is widespread in Italy. This use increases the risk of serious ADR, especially in in case of prolonged duration of treatment and in elderly patients. The Italian Medicine Agency has decided to accurately monitor the appropriate use of the drug in Italy and, if necessary, take measures in order to minimize the risks.

bcl-2 but not p53 expression is associated with resistance to chemotherapy in advanced breast cancer.

Programmed cell death is an important determinant of the response to chemotherapy. Among the factors controlling this process, a significant role is played by bcl-2 and p53, the expression of which, together with estrogen receptor content and tumor proliferative activity, was investigated by means of immunohistochemistry in 55 advanced breast cancer patients (median age, 60 years; range, 25-71 years). Analysis of bcl-2 expression identified two groups of patients with a significant difference in response rate. A total of 17 patients (31%) responded to chemotherapy (5 had a complete response and 12 had a partial response): 14 of 32 (44%) bcl-2-negative patients (< 40% stained cells) and only 3 of 23 (13%) bcl-2-positive patients (> or = 40% of stained cells; P = 0.019 by Fisher's exact test). The two groups were well balanced in terms of age, performance status, disease-free survival, menopausal status, and type of chemotherapy. bcl-2-negative tumors showed a tendency toward a higher p53 expression and proliferation rate, whereas an excess of bone as the dominant disease site was evident among the bcl-2-positive ones. However, the only variable to result significantly different between the two groups was estrogen receptor expression (P = 0.004). A multivariate logistic regression model showed that bcl-2 maintained its power of discriminating two groups with a different probability of responding to chemotherapy, although the greatest contribution was given by dominant disease site and type of chemotherapy. In conclusion, the results of this study suggest a possible role for bcl-2 in predicting resistance to chemotherapy.

Inductively coupled plasma mass spectroscopy quantitation of platinum-DNA adducts in peripheral blood leukocytes of patients receiving cisplatin- or carboplatin-based chemotherapy.

Platinum-DNA adducts can be assayed in peripheral blood leukocytes by means of atomic absorption spectroscopy and ELISA, and high adduct levels have been correlated previously with favorable clinical response to platinum-based chemotherapy. Our purpose was to study adduct formation in peripheral blood leukocytes by means of a new method, inductively coupled plasma mass spectroscopy (ICP-MS), and to correlate adduct formation with clinical response and toxicity. Platinum (Pt)-DNA adducts were measured by means of ICP-MS in leukocytes of 66 patients receiving a cisplatin- or carboplatin-based chemotherapy, collected either before the beginning of treatment and incubated in vitro with cisplatin or 1 and 24 h after the administration of drug to the patient. The Pt-DNA adduct level in leukocytes from patients exposed to drug in vitro was 14.33 +/- 14.71 fmol/microgram DNA (mean +/- SD), which was not significantly different from the value of 23.4 +/- 19.53 fmol/microgram DNA observed in leukocytes from nine healthy volunteers. In samples collected after the administration of chemotherapy, Pt-DNA adducts ranged from 1.91 +/- 3.59 fmol/microgram DNA (mean +/- SD) at the 1-h time point to 2.61 +/- 3.35 fmol/microgram DNA at 24 h (P > 0.05). Adduct levels in leukocytes exposed in vitro did not correlate with adduct levels from patients treated with cisplatin-based chemotherapy (r = 0.085 and 0.011 at 1 and 24 h, respectively). At 24 h, adduct levels in patients receiving cisplatin (3.15 +/- 3.64 fmol/microgram DNA, mean +/- SD) were significantly higher (P = 0.02) than those observed in patients treated with standard dose carboplatin (0.57 +/- 0.73 fmol/microgram DNA) and also higher than those in patients receiving high-dose carboplatin (1.18 +/- 1.06 fmol/microgram DNA), although the latter difference did not reach statistical significance (P = 0.071). No differences in adduct levels (mean +/- SD) were evident between patients responsive (3.23 +/- 3.51 fmol/microgram DNA) and nonresponsive (2.34 +/- 3.01 fmol/microgram DNA) to chemotherapy. In the homogeneous group of patients treated with combination of cisplatin and 5FU, received dose intensity, hemoglobin decrease, and posttreatment creatinine could not be linked with the extent of leukocyte adduct formation. The data presented here demonstrate that ICP-MS allows the detection of adducts in patients treated with cisplatin or carboplatin and suggest that adduct formation in leukocytes is not a major determinant of response or toxicity.

Potential risk factors for the development of acute renal failure in preterm newborn infants: a case-control study.

AIMS: To determine in a case-control study possible associations between the development of acute renal failure in preterm newborns and therapeutic interventions, particularly drug treatments. METHODS: The study population was 172 preterm infants of <38 weeks gestation; 71 had acute renal failure and 101 were controls closely matched for gestational age and birth weight. Maternal and neonatal information was collected for both groups through questionnaires and interviews. Routine data on renal variables were also collected. Univariate and multivariate logistic regression analyses were performed. RESULTS: Very low birthweight infants were at high risk of acute renal failure (79% of cases were <1500 g). However, the acute renal failure was transient. Mothers of infants with acute renal failure received more drugs during pregnancy and delivery (mainly antibiotics and non-steroidal anti-inflammatory drugs). Of the possible therapeutic interventions, intubation, catheterisation, and phototherapy were mainly applied to case subjects. A low Apgar score and patent ductus arteriosus were diagnosed in a greater percentage of neonates with acute renal failure. Moreover, in the first few days of life and before diagnosis of acute renal failure, case subjects received more drugs (antibiotics, non-steroidal anti-inflammatory drugs, and diuretics) and for a longer time. In the multivariate logistic analysis, medullary hyperechogenicity (odds ratio (OR) 4.491; 95% confidence interval (CI) 1.879 to 10.731) and ceftazidime administration (OR 5.082; 95% CI 1.493 to 17.297) were associated with a greater risk of acute renal failure. CONCLUSIONS: The results suggest the need for careful monitoring of very low birthweight infants and attention to drug treatments, as it is difficult to differentiate between normality and renal failure in the first few days of life.

Melatonin can be differentially metabolized in the rat to produce N-acetylserotonin in addition to 6-hydroxy-melatonin.

It has been generally agreed that the metabolism of the pineal hormone melatonin (aMT) consists of 6-hydroxylation followed by sulfate or glucuronide conjugation. The urinary assay of 6-hydroxy-melatonin (6-HaMT) is valued as a means of providing integrated information on aMT production. However, we show, in this study, that aMT has two principal urinary metabolites, N-acetylserotonin (NAS) as well as 6-HaMT. Rats were administered varying doses of aMT and their urines were collected and analyzed by thin layer chromatography and gas chromatography-mass spectrometry (GCMS). Thin layer chromatography of the urinary metabolites showed the expected pattern, a major spot at Rf 46%, the position of 6-sulfatoxy-melatonin, a less intense spot at Rf 32%, the position of 6-glucuronide-melatonin and a weak spot at Rf 78%, the unconjugated metabolite. However, after deconjugation and derivitization, GCMS analysis of the urines, or of the spot at Rf 46%, showed two products, one of which had the same GC retention time and mass spectrum as 6-HaMT, whereas the other had the GC retention time and mass spectrum of NAS. When deuterated aMT was administered, GCMS analysis showed the presence of deuterated 6-HaMT and deuterated NAS, proving that NAS was metabolized directly from aMT and not produced somewhere else in the body in response to aMT. Finally, GCMS analysis of urines after the administration of 6-HaMT or of NAS showed only one metabolic product in each case, i.e. 6-HaMT and NAS, respectively. This suggested that the conversion of aMT to 6-HaMT and NAS resulted from two independent metabolic pathways. It is understandable that research workers who relied entirely on chromatography should have failed to distinguish NAS and its conjugates from 6-HaMT and its conjugates since the chromatographic and staining properties of the two indoles are almost indistinguishable.

Secretion of a bone resorbing factor by chick thyroid glands in organ culture.

The ability of the thyroid gland to secrete a bone resorbing factor in vitro was studied using glands obtained from 20-day-old chick embryos. The glands were incubated in a modified BGJ medium containing 1 mg/ml bovine serum albumin under 5% CO2-40% O2 at 37 C. The culture media were assayed in vitro by measuring the stimulation of the release of previously incorporated 45Ca from cultured 19-day fetal rat bone shafts over a 48 h period. The glands secreted a stimulator of bone resorption which did not appear to be parathyroid hormone (PTH). The dose-response curve for the thyroid gland factor was not parallel to that obtained using PTH and secretion was not under calcium control. Neither thyroxine (T4) nor triiodothyronine (T3) produced a marked stimulation of bone resorption over a wide range of doses. Bone resorption stimulated by the thyroid gland factor was inhibited by calcitonin (CT). Concentrations of TH and thyroid gland factor which were minimally effective when tested separately, produced a marked synergistic response when added together. This synergism was not seen when T4, T3, PGE1, or PGE2 were tested with PTH. Media obtained by culturing explants of embryonic chick liver, heart and muscle did not have bone resorbing activity. Secretion of the bone resorbing factor by thyroid glands was blocked by Indomethacin (10(-5)M) but the effects of the factor on bone were not blocked by this agent. These results suggest that the thyroid gland is capable of secreting a stimulator of bone resorption, possibly a prostaglandin, which is capable of synergizing with PTH, and which may represent a tissue factor which under certain circumstances may exert an influence on bone.

Melatonin is metabolized to N-acetyl serotonin and 6-hydroxymelatonin in man.

To investigate whether melatonin (aMT) can be metabolized to N-acetyl serotonin (NAS), a low dose of deuterated aMT was administered to four normal subjects, and their urine samples were analyzed for the presence of deuterated NAS and deuterated 6-hydroxymelatonin (6-HaMT). In one set of experiments, the urine samples were subjected to column chromatography to separate the glucuronide and sulfate conjugates for independent analysis. In another, an internal standard (NAS-sulfate) was used for quantification and total conjugate analysis. Measurement was by gas chromatography-mass spectrometry, and the molecular ions of deuterated and nondeuterated NAS and 6-HaMT were monitored. Deuterated aMT was metabolized to deuterated NAS and deuterated 6-HaMT. The proportion of NAS was less in the sulfate than in the glucuronide conjugates and, overall, represented 15% of the total. Since demethylation is not a pathway that occurs with other pineal methoxyindoles, even at a much larger dose, it seems to be a significant finding with regard to aMT. Thus, it may be important to elucidate the differential metabolism of aMT at different time points and in different age groups.

gamma-Glutamyl transpeptidase catalyses the extracellular detoxification of cisplatin in a human cell line derived from the proximal convoluted tubule of the kidney.

Nephrotoxicity is a side-effect and the main factor limiting the clinical use of cisplatin. In vivo, the administration of the cysteine-containing tripeptide glutathione (GSH) has been found to reduce nephrotoxicity, but the biochemical mechanism of this protective action is not fully understood. The present study was designed to gain insights into the mechanism by which GSH prevents cisplatin nephrotoxicity. We also wanted to verify the hypothesis of whether the protective action of GSH is mediated by products of the extracellular breakdown of GSH catalysed by gamma-glutamyl transpeptidase (GGT), an enzyme that is highly expressed in kidney tubular cells. The study was performed in HK-2 cells, derived from the immortalisation of human kidney proximal tubule cells. We investigated the influence of modulators of GGT activity and/or thiols on the antiproliferative activity of cisplatin and on the intracellular GSH content. We determined the antiproliferative activity of cisplatin, platinum cellular accumulation and DNA platination following precomplexing of the drug with thiols. The antiproliferative effect of cisplatin was minimally affected by the addition of GSH. However, when the antiproliferative assay was performed in the presence of glycyl-glycine (GlyGly), to serve as a transpeptidation acceptor and thus to stimulate GGT-mediated GSH catabolism, cisplatin-induced growth inhibition was largely prevented. This effect was not mediated through an increase of intracellular GSH levels, which were not affected by the GlyGly supplementation. The thiol dipeptide cysteinyl-glycine, i.e. the GSH catabolite generated by GGT activity, showed a higher reactivity against cisplatin in vitro than GSH, as was shown by the more rapid oxidation of its -SH groups. The cisplatin/GSH or cisplatin/cysteinyl-glycine adducts did not display an antiproliferative effect. However, 2 h precomplexing with GSH in the presence of GGT, or directly with the GSH catabolite cysteinyl-glycine, decreased the antiproliferative effect of cisplatin and drug-induced DNA platination to a greater extent than precomplexing with GSH alone. The results of the present study show that, in HK-2 cells, extracellular GSH decreases the antiproliferative effects of cisplatin only upon its hydrolysis by GGT, thereby supporting the hypothesis that the extracellular metabolism of GSH by GGT plays a role in modulating cisplatin nephrotoxicity. A primary role in the protection of HK-2 cells appears to be played by cysteinyl-glycine, the proximal product of the GGT-mediated hydrolysis of GSH, which shows a high reactivity against CDDP resulting in the rapid inactivation of the drug.

Introduction of a novel HRP substrate-Nanogold probe for signal amplification in immunocytochemistry.

Amplification of immunological signals with catalyzed reporter deposition (CARD) allows improved detection of scarce tissue antigens in light and electron microscopy. The technique takes advantage of the oxidation ability of horseradish peroxidase (HRP), in the presence of hydrogen peroxide, to yield the accumulation of one of its specific reporter-tagged substrates. This immunocytochemical approach continues to be improved by the introduction of new reporter molecules tagged to tyramine or to other HRP substrates. In this study we introduced a novel HRP substrate tagged to Nanogold particles. The amplification protocol is based on the application of a specific primary antibody, a biotinylated secondary antibody, streptavidin-HRP, and an HRP substrate coupled to Nanogold, followed by silver intensification. In addition to amplification of immunological signals of high resolution, direct accumulation of Nanogold particles at target sites by enzymatic activity of HRP improves the efficiency of the technique compared to other amplification protocols. Moreover, this approach combines the CARD amplification potentials with the ultrasmall gold probe and the silver intensification method. Immunolabeling obtained by light and electron microscopy, as well as immunodot assay using this new amplification strategy, appear to be highly sensitive, specific, and of enhanced intensity.

Immediate response of 5-methoxytryptophol in the circulation to hypoglycaemic stress induced by insulin.

Pineal indoles have been shown to affect the release of anterior pituitary hormones but details of the interrelationships are lacking. Using a new gas chromatography-mass spectrometry (g.c.-m.s.) assay the concentration of 5-methoxytryptophol (ML) was measured in plasma samples obtained from 16 children undergoing investigation of pituitary function for delayed growth. All the children received an insulin tolerance test (ITT) to study their endocrine response to stress. Some children received luteinizing hormone releasing hormone (LH-RH) and/or thyrotrophin releasing hormone (TRH). The change in concentration of ML during an ITT was similar to the change in concentration of blood sugar; a drop at 20 min followed by a rise at 30 min. This was not significantly altered by the administration of LH-RH or TRH, nor was there a different pattern of response in children who were deficient in growth hormone as opposed to those with idiopathic delayed growth. The fall in concentration of ML with stress may mediate the increased secretion of pituitary hormones. Alternatively, the pineal gland may respond directly to insulin.