RESUMEN
The reported method aims to be a powerful aid for the simultaneous determination of tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and tetrahydrocannabivarin (THCV) in oily based preparations. The chromatographic separation was carried out using an Hypersil Gold PFP (50â¯×â¯2.1â¯mm, 1.9⯵m) column, using H2Oâ¯+â¯2â¯mM ammonium formate + 0.2 % formic acid (M1) and Methanol + 2â¯mM ammonium formate + 0.2 % formic acid (M2) as mobile phases. The flow rate was set 0.4â¯mL/min. Specifically, this method was validated in terms of linearity, limit of detections and quantifications (LODs and LOQs), accuracy (precision and trueness, both intra and interday), selectivity, and matrix effects. This procedure allowed quantifying seven phytocannabinoids in less than 10â¯min. The validated method shows a good linearity within the range 0.25-1000â¯ng/mL, while precision and trueness (intra- and inter-day) were below <13.25 % and 7.59 %, respectively. Regarding the matrix effect, the method satisfies all the requirements, except for the THC and THCV, where it reaches about 120 %. This element does not affect the method performances as it has been observed that this value is constant and reproducible and therefore does not involve errors in the quantitative analysis. The method was tested and applied on more 70 different oily based preparations. Furthermore, starting from four different cannabis cultivar (FM2, Bedrolite, Bedrocan, and Bediol), it allowed to evaluate the reproducibility of the magistrali preparations. The real samples, in fact, derive from different local pharmacies, and were analyzed by the accredited UNI CEI EN ISO/IEC 17025:2018, Pharmatoxicology Laboratory (ACCREDIA, lab n. 2274 ASLPE, accreditation number 1822â¯L), accordingly to the current regulations.
Asunto(s)
Cannabis , Espectrometría de Masas en Tándem , Cannabinol , Cromatografía Liquida , Dronabinol/análisis , Reproducibilidad de los ResultadosRESUMEN
A fast off-line FPSE-HPLC-PDA method has been reported that allows simultaneous clean up and determination of six non-steroidal anti-inflammatory drugs (NSAIDs) in saliva samples from healthy volunteers. Particularly, furprofen, indoprofen, ketoprofen, fenbufen, flurbiprofen, and ibuprofen were chromatographically resolved. Benzyl paraben was chosen as the internal standard (BzPB, IS). These target compounds were successfully extracted from human saliva using fabric phase sorptive extraction (FPSE) and then analysed in the liquid chromatographic system by means of a short analytical column (Symmetry C18, 75 × 4.6 mm, 3.5 µm) using acetonitrile (AcN) and phosphate buffer (PBS, 30 mM; pH = 2.5) as the mobile phases. The method, validated through the calculation of all analytical parameters in accordance of International Guidelines, was applied to real saliva sample analysis collected from informed volunteers. The proposed approach that included the use of sol-gel polytetrahydrofuran (sol-gel PTHF) sorbent immobilized on cellulose support and C18 stationary phase used in HPLC, showed high potential as a fast tool for future clinical and forensic applications. The herein reported results encourage potential future application of FPSE in the forensic field. Furthermore, the FPSE membrane was tested in dried saliva spot mode (DSS) in order to check its potential use as a sampling device, also for forensic applications.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Flurbiprofeno/química , Fenilpropionatos/química , Saliva/química , Antiinflamatorios no Esteroideos/farmacocinética , Celulosa/química , Cromatografía Líquida de Alta Presión , Femenino , Flurbiprofeno/farmacocinética , Humanos , Límite de Detección , Masculino , Estructura Molecular , Parabenos/normas , Fenilpropionatos/farmacocinética , Microextracción en Fase SólidaRESUMEN
Antimicrobial peptides (AMPs) selectively kill bacteria by disrupting their cell membranes, and are promising compounds to fight drug-resistant microbes. Biophysical studies on model membranes have characterized AMP/membrane interactions and the mechanism of bilayer perturbation, showing that accumulation of cationic peptide molecules in the external leaflet leads to the formation of pores ("carpet" mechanism). However, similar quantitative studies on real cells are extremely limited. Here, we investigated the interaction of the dansylated PMAP23 peptide (DNS-PMAP23) with a Gram-positive bacterium, showing that 107 bound peptide molecules per cell are needed to kill it. This result is consistent with our previous finding for Gram-negative strains, where a similar high threshold for killing was determined, demonstrating the general relevance of the carpet model for real bacteria. However, in the case of the Gram-positive strain, this number of molecules even exceeds the total surface available on the bacterial membrane. The high affinity of DNS-PMAP23 for the anionic teichoic acids of the Gram-positive cell wall, but not for the lipopolysaccharides of Gram-negative bacteria, provides a rationale for this finding. To better define the role of anionic lipids in peptide/cell association, we studied DNS-PMAP23 interaction with E. coli mutant strains lacking phosphatidylglycerol and/or cardiolipin. Surprisingly, these strains showed a peptide affinity similar to that of the wild type. This finding was rationalized by observing that these bacteria have an increased content of other anionic lipids, thus maintaining the total membrane charge essentially constant. Finally, studies of DNS-PMAP23 association to dead bacteria showed an affinity an order of magnitude higher compared to that of live cells, suggesting strong peptide binding to intracellular components that become accessible after membrane perturbation. This effect could play a role in population resistance to AMP action, since dead bacteria could protect the surviving cells by sequestering significant amounts of peptide molecules. Overall, our data indicate that quantitative studies of peptide association to bacteria can lead to a better understanding of the mechanism of action of AMPs.
Asunto(s)
Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Pared Celular/efectos de los fármacos , Relación Estructura-Actividad , Secuencia de Aminoácidos/genética , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Pared Celular/química , Pared Celular/ultraestructura , Bacterias Gramnegativas/química , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/patogenicidad , Bacterias Grampositivas/química , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/patogenicidad , Humanos , Lipopolisacáridos/química , Pruebas de Sensibilidad MicrobianaAsunto(s)
Avalanchas , Epinefrina/orina , Norepinefrina/orina , Adulto , Asfixia , Síndrome de Aplastamiento , Desastres , Femenino , Humanos , Hipotermia/orina , Italia , Masculino , Traumatismo Múltiple , Factores de TiempoRESUMEN
Limited information about the distribution of different bovine papillomavirus (BPV) types in Italy is available; therefore, this study aimed to investigate the presence of BPVs in bovine lesions in the Emilia Romagna region. Sixty-four proliferative lesions were collected between december 2011 and december 2014, and subsequently analysed by qualitative PCR with genus- and type-specific primer pairs, as well as rolling circle amplification (RCA). The results demonstrated, for the first time in Italy, the presence of BPV 6, 7, 8, 10 and 12 and also types previously described elsewhere. In addition, the high prevalence of viral co-infections in this sample set provides new information about viral tropism.
Asunto(s)
Enfermedades de los Bovinos/virología , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/veterinaria , Animales , Bovinos , Femenino , Italia , Papillomaviridae/clasificación , Infecciones por Papillomavirus/virologíaRESUMEN
The genus Micipsella comprises three species of filariae to date identified in lagomorphs only, whereas the other genera belonging to the subfamily Splendidofilariinae are described as parasites of birds, reptiles and mammals. In the present study seven specimens of Micipsella numidica (Seurat, 1917), collected from the hare Lepus europaeus in Italy, were characterized genetically by molecular amplification of the mitochondrial genes (12S rDNA; cox1) and the 5S rDNA gene spacer region. Phylogenetic trees inferred using available sequences from filariae and those identified in this study evidenced a close relationship between M. numidica and Splendidofilariinae of other mammals and reptiles (Rumenfilaria andersoni and Madathamugadia hiepei). The present findings, apart from adding new data about the hosts in Italy, support the taxonomic position of M. numidica and highlight the substantial biological and molecular differences existing between Splendidofilariinae and other Onchocercidae. The study also contributes to our knowledge of the molecular/genetic diagnosis of filarial parasites of veterinary and medical concern in any vertebrate or invertebrate host.
Asunto(s)
Filariasis/veterinaria , Filarioidea/clasificación , Filarioidea/aislamiento & purificación , Liebres/parasitología , Animales , Análisis por Conglomerados , ADN de Helmintos/química , ADN de Helmintos/genética , ADN Mitocondrial/química , ADN Mitocondrial/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Filariasis/parasitología , Filarioidea/genética , Italia , Filogenia , ARN Ribosómico/genética , ARN Ribosómico 5S/genética , Análisis de Secuencia de ADNRESUMEN
We identified a novel papillomavirus (CePV1) in a fibropapilloma of a 1.5 year old male red deer (Cervus elaphus) shot in the Italian Alps in Brescia province. PV particles were first observed by electron microscopy and PV DNA was then identified by PCR using degenerate primers. Subsequently we cloned the entire genome and determined its complete sequence. CePV1 genome is 8009 bp long and contains all 9 ORFs and the long untranslated regulatory region characteristic for Delta-papillomaviruses. Pairwise nucleotide alignments and phylogenetic analyses based on concatenated E1-E2-L1 ORFs allowed to determine the highest similarity with the Capreolus caprelus papillomavirus CcaPV1. The analysis of the host-parasite phylogenetic tree interactions suggest the co-divergence of CePV1 and C. elaphus while the identified topological incongruences leading us to speculate that CcaPV1 could eventually be the result of an earlier host switch event.
Asunto(s)
Ciervos/virología , Papillomaviridae/clasificación , Infecciones por Papillomavirus/veterinaria , Filogenia , Animales , Masculino , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , Reacción en Cadena de la PolimerasaRESUMEN
Hyperthermia produces regression of human cancer. Because hyperthermia has produced only limited results, attention has focused on searching for substances able to sensitize tumour cells to the effects of hyperthermia. The flavonoid quercetin has been reported to be a hyperthermic sensitizer in ovarian and uterine cervical tumours and in leukaemia. Quercetin and tamoxifen inhibit melanoma cell growth. We therefore investigated whether quercetin and tamoxifen can sensitize M10, M14 and MNT1 human melanoma cells to hyperthermia. We observed that both quercetin and tamoxifen synergize with hyperthermia (42.5 degrees C) in reducing the clonogenic activity of M14 and MNT1 and in inducing apoptotic cell death in all three cell lines. As revealed by flow cytometric and Northern blot analyses, quercetin and tamoxifen reduced heat shock protein-70 expression at both protein and mRNA levels. Our results suggest that quercetin and tamoxifen can be usefully combined with hyperthermia in the therapy of recurrent and/or metastatic melanoma.
Asunto(s)
Apoptosis/efectos de los fármacos , Hipertermia Inducida , Melanoma/patología , Quercetina/farmacología , Tamoxifeno/farmacología , Northern Blotting , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/genética , Calor , Humanos , Etiquetado Corte-Fin in Situ , Melanocitos/efectos de los fármacos , Melanocitos/patología , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/terapia , Quercetina/uso terapéutico , ARN/genética , ARN/metabolismo , Tamoxifeno/uso terapéutico , Temperatura , Células Tumorales CultivadasRESUMEN
Platelet activation has been suggested to play an important role in the pathogenesis of prethrombotic states and thus may be responsible for decompression illness during compressed air (scuba) diving. To investigate the effect of physical, mental, and environmental stress on platelet activation during immersion in ice-cold water, we examined 10 male breath-hold divers (BHD), 10 elite BHD (eBHD), and 10 scuba divers during immersion in an ice-covered lake at moderate altitude. Platelet activation was examined by surface expression of activation-dependent glycoproteins CD62p, CD63, and CD42a with flow cytometry 10 min before and 1 min and again 24 h after diving. Plasma epinephrine level was also measured. In addition, the relationship between the activated platelets and the epinephrine level was evaluated. The percentage of platelet activation increased from 2.1 +/- 0.4 to 5.7 +/- 0.3, 1.8 +/- 0.3 to 12.9 +/- 0.8, and 3.7 +/- 0.9 to 31.2 +/- 0.8 in BHD, eBHD, and scuba divers, respectively. The percentage of platelet activation returned to pre-immersion levels in BHD and eBHD divers 24 h after diving, but was still higher in scuba divers. A positive relationship exists between the plasma epinephrine level and the percentage of the platelet activation. This study suggests that physical and mental stress enhance platelet activation during diving in ice-cold water.
Asunto(s)
Frío , Buceo/fisiología , Epinefrina/sangre , Activación Plaquetaria/fisiología , Estrés Fisiológico/fisiopatología , Adulto , Análisis de Varianza , Biomarcadores/sangre , Citometría de Flujo , Humanos , Masculino , Estrés Fisiológico/sangreAsunto(s)
Apoptosis/efectos de los fármacos , Ácido Micofenólico/toxicidad , Neuroblastoma/patología , Antibióticos Antineoplásicos/toxicidad , Western Blotting , División Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Guanosina/farmacología , Humanos , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
In the period 1987-1997 6 patients with Hürthle cell carcinomas and 4 patients with Hürthle cell adenomas underwent primary surgical treatment (8.1% of all thyroid carcinomas). The diagnosis of Hürthle cell tumor was based on the presence of more then 75% Hürthle cells and the malignity on capsular or/and vascular invasion. All the patients with Hürthle cell cancer underwent total thyroidectomy, in three cases with Hürthle cell adenoma thyroid lobectomy was performed and in one case total thyroidectomy. Follow-up time ranged from 1 to 8 years after surgery (mean 4.5 years). There was no death and no recurrence. The Authors have studied the nuclear DNA content in Hürthle cell tumors: 3 adenomas were euploid and 1 was aneuploid, 4 carcinomas were aneuploid and 2 were euploid. The results in Authors' study of the DNA content and nuclear DNA ploidy are not uniformly consistent enough to allow a distinction between benign and malignant neoplasms and to evaluate the prognosis, but the number of patients and the follow up are still too limited.
Asunto(s)
Adenoma Oxifílico , Neoplasias de la Tiroides , Adenoma Oxifílico/diagnóstico , Adenoma Oxifílico/patología , Adenoma Oxifílico/cirugía , Adulto , Anciano , ADN de Neoplasias , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ploidias , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/cirugía , Tiroidectomía , Resultado del TratamientoRESUMEN
In a series of in vitro experiments we characterised the relationship between DNA distribution in the G1, S and G2/M phases of cell cycle and PDE and GST activity in CaCo-2 cells. The DNA distribution in CaCo-2 cells, was assessed by flow cytometry, with fluorescent dyes at different time points of culture. The exponential increase in cell number continued until day 10 when there was cell saturation. The effect of medium replacement on PDE activity was assayed in the first 10 h after medium replacement. The 6th hour is the time at which PDE activity was found to be highest. We have assayed the PDE enzyme with cGMP and cAMP as substrates. Only cAMP was consumed from this enzyme. We found a very close correlation between the DNA distribution in the various phases of the cell cycle and the PDE activity. PDE activity was very high during the active replication phase, whereas GST activity was high after confluency.
Asunto(s)
Carcinoma/enzimología , Neoplasias del Colon/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Carcinoma/patología , Ciclo Celular , Diferenciación Celular , División Celular , Neoplasias del Colon/patología , ADN de Neoplasias/biosíntesis , Citometría de Flujo , Glutatión Transferasa/metabolismo , Humanos , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
In seven rabbits subjected to suprarenal aortic coarctation hypertension, the segments above and below the coarctation were tested for the antioxidant defences (i.e. acid-soluble thiol compounds, selenium-dependent and selenium-independent glutathione peroxidase, glutathione reductase, glutathione transferase) and thiobarbituric acid-reactive substances. Seven sham-operated rabbits served as controls. Systolic blood pressure proximal to the ligature increased significantly with respect to pre-operative values after 16 days (117 +/- 8.3 vs 71.7 +/- 5.2 mmHg, P less than 0.05), while pressure distal to the ligature remained normotensive. Higher values of acid-soluble thiol compounds, thiobarbituric acid-reactive substances and increased activities of selenium-dependent glutathione peroxidase, glutathione reductase and glutathione transferase were assayed in the suprarenal with respect to the subrenal segment in both groups. However, the values of the upper segments were more elevated in the experimental group than in controls, but no differences were observed in the lower segments. Glutathione peroxidase activity assayed with cumene hydroperoxide was higher than the activity assayed with hydrogen peroxide in the hypertensive segments, but no differences were detected in the substenotic and control segments. Furthermore, an isoenzymatic form of glutathione transferase, analogous to rat 8-8 glutathione transferase isoenzyme, was detected by immunodiffusion in the hypertensive aorta. The following conclusions may be drawn: (1) a biochemical gradient in glutathione-related enzymes, acid-soluble thiol compounds and thiobarbituric acid-reactive substances between the proximal and distal aorta seems to exist in control rabbits; (2) suprarenal aortic coarctation induces a significant increase in glutathione-related antioxidant defences and thiobarbituric acid-reactive substances of the hypertensive aortic wall.
Asunto(s)
Coartación Aórtica/metabolismo , Glutatión/metabolismo , Hipertensión Renovascular/metabolismo , Animales , Antioxidantes/metabolismo , Aorta/metabolismo , Coartación Aórtica/complicaciones , Radicales Libres , Glutatión Peroxidasa/metabolismo , Glutatión Transferasa/metabolismo , Hipertensión Renovascular/etiología , Masculino , ConejosRESUMEN
In 24 rabbits fed a hyperlipidic diet (0.5% cholesterol, 5% lard and 5% peanut oil) for 10 (group A1), 30 group B1) and 60 days, (Group C1), compared to 24 control rabbits fed a standard diet for the same periods, antioxidant defence system (total superoxide dismutase, catalase, total thiol compounds selenium-dependent and selenium-independent glutathione peroxidase, glutathione reductase, glutathione transferase) and lipid peroxidation (thiobarbituric acid-reactive substances) in the aortic wall were tested. The percent of intima with grossly apparent atherosclerosis, is assessed by staining with the lipophilic dye Sudan IV, was negligible in group A1, but increased progressively in groups B1 (22.7-6.7%) and C1 (56.8-8.8%). Compared to the controls, a significant rise in superoxide dismutase activity was observed after 30 days of hyperlipidic diet, with a further marked increase at 60 days. Total thiol compounds and selenium-dependent glutathione peroxidase activity rose progressively from 10 to 30 and 60 days in cholesterol-fed rabbits. On the contrary, catalase, glutathione reductase and glutathione transferase activities significantly decreased in all experimental groups. Selenium-independent glutathione peroxidase activity was not detectable. Thiobarbituric acid-reactive substances increased about 3 times in hyperlipidemic rabbits. In conclusion, the changes in aortic antioxidant defence mechanisms and lipid peroxidation precede the massive vascular lipid infiltration in cholesterol-fed rabbits; some antioxidant mechanisms are stressed (superoxide, dismutase, glutathione peroxidase, total thiol compounds), whereas others are depressed (catalase, glutathione reductase, and glutathione transferase), thus potentially reducing or increasing vascular susceptibility to oxidative injury.