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Background: Coronavirus disease 2019 (COVID-19) is a pandemic disease that is causing a public health emergency. Characteristics and clinical significance of myocardial injury remain unclear., Methods: This retrospective single-center study analyzed 189 patients who received a COVID-19 diagnosis out of all 758 subjects with a high sensitive troponin I (Hs-TnI) measurement within the first 24 h of admission at the Policlinico A.Gemelli (Rome, Italy) between February 20th 2020 to April 09th 2020., Results: The prevalence of myocardial injury in our COVID-19 population is of 16%. The patients with cardiac injury were older, had a greater number of cardiovascular comorbidities and higher values of acute phase and inflammatory markers and leucocytes. They required more frequently hospitalization in Intensive Care Unit (10 [32.3%] vs 18 [11.4%]; p = .003) and the mortality rate was significantly higher (17 [54.8%] vs. 15 [9.5%], p < .001). Among patients in ICU, the subjects with myocardial injury showed an increase need of endotracheal intubation (8 out of 9 [88%] vs 7 out of 19[37%], p = .042). Multivariate analyses showed that hs-TnI can significantly predict the degree of COVID-19 disease, the intubation need and in-hospital mortality., Conclusions: In this study we demonstrate that hs-Tn can significantly predict disease severity, intubation need and in-hospital death. Therefore, it may be reasonable to use Hs-Tn as a clinical tool in COVID-19 patients in order to triage them into different risk groups and can play a pivotal role in the detection of subjects at high risk of cardiac impairment during both the early and recovery stage., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aim: To evaluate whether changes in bowel perfusion parameters measured by dynamic-CEUS (D-CEUS) can be used for monitoring response to therapy in active Crohn disease (CD)., Methods: Fifty-four CD patients were evaluated with d-CEUS before (T0) and after 2 (T1), 6 (T2) and 12 weeks (T3) of anti-TNFα therapy. Variations from baseline were calculated for: peak intensity, PI; area under the curve, AUC; slope of wash in, P w ; time to peak, TP; mean transit time, MTT (median percentage values) and were correlated with combined endoscopic/clinical response after 12 weeks and clinical relapse within 6 months., Results: 70% of patients achieved combined endoscopic/clinical response (responders). The reduction in PI, AUC, P w and MTT between T1 and T0 was higher in responders. Relapsers (21%) showed significantly lower reduction in delta PI and P w at T1 and T2. At T3 they showed a new increase in PI and lower reduction in delta P w. In relapsers, AUC showed a significantly lower decrease at T2 and T3, TP showed a significant reduction at T3 and MTT showed a progressive increase at the different time-points, reaching the statistical significance at T3., Conclusions: d-CEUS might become a reliable predictor of combined endoscopic/clinical response and clinical relapse in CD., Competing Interests: Declaration of Competing Interest No potential conflicts of interest. No financial support., (Copyright © 2020 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: The clinical picture of obsessive-compulsive disorder encompasses a broad range of symptoms that are related to multiple psychological domains, including perception, cognition, emotion, and social relatedness. As obsessive-compulsive symptoms (OCS) frequently have an early onset, there are limited data about OCS in older populations (≥65 years) and, in particular, in elderly subjects with Parkinson disease (PD)., Objective: This study aimed to estimate the prevalence of OCS using a self-report measure (Obsessive-Compulsive Inventory-Revised) and to identify associated sociodemographic and clinical factors in a sample of elderly PD patients compared to a comparison group of similarly aged healthy volunteers., Results: The mean age was 74 ± 6 years in the PD patients and 73 ± 7 years in the comparison group. The mean disease duration was 9.6 ± 5.8 years. Among the PD patients, 30.7% reported at least one OCS or a related disorder compared to 21.1% in the comparison group. Hoarding was significantly more common in PD patients than in the comparison group., Conclusions: Subclinical OCS were present at a high percentage in both PD patients and comparison group. The OCS phenotype in PD may present differently, as hoarding was more common in PD patients., (Copyright © 2019 American Association for Geriatric Psychiatry. Published by Elsevier Inc. All rights reserved.)

Background: In liver cirrhosis, the renin-angiotensin-aldosterone system is involved in the pathogenesis of portal hypertension. Its effector, angiotensin II, is generated by angiotensin-converting enzyme (ACE). Serum ACE levels are affected by I/D polymorphism of its gene, with alleles I and D being associated, respectively, with lesser and greater activity of the enzyme. In cirrhotic patients carrying the ACE I allele, an increased risk for gastro-oesophageal varices was observed., Aim: The aim of our study was to evaluate whether ACE I/D polymorphism influenced portal pressure., Methods: Fifty-one consecutive cirrhotic patients were divided based on ACE genotype (DD, ID, and II). Kidney and liver function tests, upper endoscopy, and hepatic venous pressure gradient measurement (HVPG) were performed in all patients., Results: The presence of the ACE I allele was associated with a higher HVPG value (18.7±6.4 vs 10.3±6.3mmHg; P<.001), higher frequency of large gastrooesophageal varices (59.3% vs 25.0%; P<.05), and higher frequency of variceal bleeding (63.0% vs 29.2%; P<.05). No significant differences were found between patients with and those without the ACE I allele regarding Child-Pugh score, MELD score, ascites, and hepatic encephalopathy., Conclusion: ACE I/D polymorphism seems to influence the severity of portal hypertension and the risk of variceal bleeding in liver cirrhosis, regardless of the severity of liver disease., (Copyright © 2018 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: There is uncertainty about the optimal method for measuring the decompressive craniectomy (DC) surface area and how large the DC should be., Methods: A radiological technique for measuring the surface area of removed bone flaps in a series of 73 DCs was developed. Preoperative and early postoperative computed tomography scans of each patient were evaluated. Midline shift (MLS) was considered the key factor for successful DC and was assigned to either normal (0-4 mm) or pathological (≥5 mm) ranges. The association between postoperative MLS and patient survival at 12 months was assessed., Results: Measurements of all removed bone flaps yielded a mean surface area of 7759 mm 2 . The surface area of the removed bone flap did not influence survival (surviving 7643 mm 2 vs. deceased 7372 mm 2 ). The only factor associated with survival was reduced postoperative MLS (P < 0.034). Risk of death was 14.4 (3.0-70.1)-fold greater in patients with postoperative shift ≥5 mm (P < 0.001)., Conclusion: The ideal surface area for "large" square bone flaps should result in an MLS of <5 mm. Enlargement of the craniectomy edges should be considered for patients in whom MLS ≥5 mm persists according to early postoperative computed tomography scans., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background Aims: Subcutaneous fat represents a valuable reservoir of adipose-derived stem cells (ASCs) in the stromal vascular fraction (SVF), widely exploited in regenerative medicine applications, being easily harvested through lipoaspiration. The lack of standardized procedures for autologous fat grafting guided research efforts aimed at identifying possible differences related to the harvesting site, which may affect cell isolation yield, cell growth properties and clinical outcomes. Subcutaneous fat features a complex architecture: the superficial fascia separates superficial adipose tissue (SAT) from deep layer tissue (DAT). We aimed to unravel the differences between SAT and DAT, considering morphological structure, SVF composition, and ASC properties., Methods: SAT and DAT were collected from female donors and comparatively analyzed to evaluate cellular yield and viability, morphology, immunophenotype and molecular profile. ASCs were isolated in primary culture and used for in vitro differentiation assays. SAT and DAT from cadaver donors were also analyzed through histology and immunohistochemistry to assess morphology and cell localization within the hypoderm., Results: Liposuctioned SAT contained a higher stromal tissue compound, along with a higher proportion of CD105-positive cells, compared with DAT from the same harvesting site. Also, cells isolated from SAT displayed increased multipotency and stemness features. All differences were mainly evidenced in specimens harvested from the abdominal region. According to our results, SAT features overall increased stem properties., Conclusions: Given that subcutaneous adipose tissue is currently exploited as the gold standard source for high-yield isolation of adult stem cells, these results may provide precious hints toward the definition of standardized protocols for microharvesting., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Context: Little is known about activated immune-inflammatory pathways and interleukin-6 (IL-6) in the development of fatigue and/or depression in patients with end-stage renal disease on chronic hemodialysis (HD)., Objectives: To evaluate the possible correlation between fatigue and serum levels of IL-6 in patients on chronic HD., Methods: One hundred HD patients were assessed for the presence of fatigue using the SF-36 Vitality subscale and were administered the Beck Depression Inventory (BDI), the Hamilton Anxiety Rating Scale (HARS), the Mini-Mental State Examination (MMSE), the activities of daily living (ADL), and the instrumental activities of daily living (IADL). We also calculated the time of recovery after hemodialysis (TIRD) and the number/severity of comorbidities using the Charlson Comorbidity Index (CCI). Laboratory parameters were measured as well as serum IL-6., Results: Forty-three patients constituted the fatigued group and 57 the nonfatigued group. Age, CCI, BDI, HARS, and TIRD were significantly higher in fatigued patients than in the nonfatigued patients. Conversely, the scores of ADL, IADL, and MMSE were significantly lower in fatigued than in nonfatigued patients. Serum IL-6 levels (pg/mL) were higher in the fatigued group (5.1 ± 3.4) than in the nonfatigued group (1.6 ± 1.5; P < 0.001); serum albumin and creatinine levels were significantly lower. Twenty-six patients (26%) had no symptoms of depression (BDI score <10), and 74 patients (74%) had symptoms of depression (BDI score >9). Patients with a BDI score >9 were older; had a higher CCI; a lower MMSE; a higher TIRD; lower serum albumin, creatinine, and urea levels; and higher serum IL-6 levels. The correlation analyses showed that the score of the SF-36 Vitality subscale was associated with age, dialytic age, TIRD, ADL, IADL, CCI, BDI, HARS, MMSE, serum urea, creatinine, albumin, and IL-6 levels. On multivariate general linear model analyses, with fatigue as the dependent variable and gender as a second factor, BDI and serum IL-6 levels were independently associated with the score of the SF-36 Vitality subscale. A canonical correlation analysis was performed including in the model fatigue, BDI, and biomarkers; the correlation was 0.679 (R(2) = 0.462). Fatigue, BDI, and IL-6 among biomarkers showed the strongest association with the underlying construct (standardized canonical coefficients = -0.989, 0.015, and 0.852, respectively), thus explaining a correlation of IL-6 with both depression and fatigue., Conclusion: Fatigue was significantly associated with symptoms of depression and serum IL-6 levels in patients receiving chronic HD., (Copyright © 2015 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Sorafenib has become the standard first-line treatment for patients with advanced HCC and acts by inducing alterations in tumor vascularity. We wanted to evaluate the feasibility of dynamic CEUS (D-CEUS) as a predictor of early tumor response to sorafenib and to correlate functional parameters with clinical efficacy end points., Methods: Twenty-eight HCC patients treated with sorafenib 400mg bid were prospectively enrolled. CEUS was performed at baseline (T0) and after 15 (T1) and 30 (T2) days of treatment. Tumor vasculature was assessed in a specific harmonic mode associated with a perfusion and quantification software (Q-Lab, Philips). Variations between T1/T2 and T0 were calculated for five D-CEUS functional parameters (peak intensity, PI; time to PI, TP; area under the curve, AUC; slope of wash in, Pw; mean transit time, MTT) and were compared for responders and non-responders. The correlation between D-CEUS parameters, overall survival (OS), and progression-free survival (PFS) was also assessed. A p value <0.05 was considered statistically significant., Results: The percentage variation at T1 significantly correlated with response in three D-CEUS parameters (AUC, PI and Pw; p=0.002, <0.001, and 0.003, respectively). A decrease of AUC (p=0.045) and an increased/unchanged value of TP (p=0.029) and MTT (p=0.010) were associated with longer survival. Three D-CEUS parameters (AUC, TP, Pw) were significantly associated with PFS., Conclusions: D-CEUS provides a reliable and early measure of efficacy for anti-angiogenic therapies and could be an excellent tool for selecting patients who will benefit from treatment., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Shear stress triggers conformational stretching of von Willebrand factor (VWF), which is responsible for its self-association and binding to the platelet receptor glycoprotein (GP)Ibα. This phenomenon supports primary hemostasis under flow. Type 2B VWF natural mutants are considered to have increased affinity for platelet GPIbα., Objectives: To assess the mechanism responsible for the enhanced interaction of the p.R1306W VWF mutant with the platelet receptor., Methods: The interaction of GPIbα with wild-type (WT) and p.R1306W VWF multimers and A1-A2-A3 constructs was investigated with surface plasmon resonance spectroscopy. Analysis of the static VWF conformation in solution was performed with dynamic light scattering spectroscopy. The shear stress-induced self-association of VWF multimers was investigated with atomic force microscopy (AFM) over a 0-60 dyn cm(-2) range., Results: WT VWF did not interact with GPIbα under static conditions, whereas the mutant at ~ 2 μg mL(-1) already bound to the receptor. By contrast, the WT and p.R1306W-A1-A2-A3 constructs showed comparable affinities for GPIbα (Kd  ~ 20 nm). The hydrodynamic diameter of resting R1306W VWF multimers was significantly greater than that of the wild type (210 ± 60 nm vs. 87 ± 22 nm). At shear forces of < 14 dyn cm(-2) , the p.R1306W multimers rapidly changed conformation, entering a regime of self-aggregation, which, in contrast, was induced for WT VWF by shear forces of > 30 dyn cm(-2) . Mechanical stretching AFM experiments showed that p.R1306W multimers needed less energy per length unit (~ 10 pN) to be stretched than the WT protein., Conclusions: The increased affinity of p.R1306W VWF for GPIbα arises mostly from higher sensitivity to shear stress, which facilitates exposure of GPIbα binding sites., (© 2013 International Society on Thrombosis and Haemostasis.)

Study Objective: To determine the effect of positive end-expiratory pressure (PEEP) on the respiratory system and on cardiac function., Design: Prospective randomized study., Setting: Operating room., Patients: 60 ASA physical status 1 women scheduled for pelvic laparoscopic surgery., Interventions: Patients were ventilated normally during surgery; PEEP was modified depending on patient group allocation. Group A was the control group and did not receive PEEP. Group B received PEEP 5 cmH2O and Group C received PEEP 10 cmH2O., Measurements: Respiratory parameters measured were partial pressure of arterial oxygen (PaO2), partial pressure of carbon dioxide (PaCO2), and end-tidal carbon dioxide tension (ETCO2). Cardiac parameters measured were left ventricular end-diastolic volume index (LVEDVI), ie, ratio of LVEDV/body surface area (BSA; [LVEDVI = end-diastolic volume [EDV]/BSA); left ventricular (LV) systolic function, tricuspid annular plane systolic excursion (TAPSE), right ventricular (RV) fractional area change (FAC), RV dimensions in the apical 4-chamber view, tracing basal and mid-cavity minor dimensions and longitudinal dimension, cardiac index, systolic pulmonary artery pressure (PASP), and systolic RV pressure (RVSP). Respiratory and cardiac measurements were recorded at T0 (baseline); T1 (after anesthesia induction, before pneumoperitoneum induction); at 10 (T2), 20 (T3), and 30 (T4) minutes after CO2 insufflation; and at the end of surgery (T5)., Main Results: Ventilation with PEEP at 10 cm H2O led to significant improvement in both respiratory and cardiac parameters. A reduction in pulmonary vascular resistance and enhanced washout of expiratory CO2 occurred. Ten and, to a lesser extent, 5 cm H2O of PEEP decreased LV stroke work., Conclusions: Ventilation with PEEP (up to 10 cm H2O) recruits the hypoventilated areas of the lungs and reduces cardiac afterload., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: Previous studies have shown a high cardiovascular risk in patients with autoimmune diseases, such as type 1 diabetes mellitus (T1DM). Conversely, few data are available about patients with celiac disease (CD). The aim of our study was to assess carotid intima-media thickness (c-IMT), in patients with T1DM, CD or both (T1DM+CD) as compared with age- and sex-matched healthy individuals (H)., Methods: We enrolled 120 patients, 30 with T1DM, 30 with CD, 30 with T1DM+CD and 30 H. Clinical, metabolic and anthropometric data were collected. All T1DM patients were on insulin while all CD patients were on a gluten-free diet. c-IMT was evaluated by high frequency linear digital ultrasound., Results: c-IMT was significantly greater in patients with T1DM+CD than in patients with T1DM or CD (P<0.001 for both), while no difference was found between T1DM and CD. Moreover, c-IMT was greater in CD than in H (P<0.001). Glycemic control and disease duration were similar between T1DM+CD and T1DM. Lipid and anthropometric parameters were similar among groups. Furthermore, in a pooled multivariate analysis, only age and disease type were significantly correlated with c-IMT (P<0.001 for both)., Conclusion: Our study demonstrates that celiac patients have greater c-IMT as compared with healthy individuals. Thus, non-invasive monitoring of c-IMT in CD might be useful in preventing cardiovascular disease. Moreover, patients with T1DM+CD show more severe subclinical atherosclerosis as compared with those presenting T1DM or CD only, suggesting that the association of these autoimmune diseases might accelerate the atherosclerotic process., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: The cardiac left ventricle responds to pressure overloads with mechanisms culminating in irreversible structural/functional cardiac alterations (left ventricular hypertrophy and/or diastolic dysfunction), inducing myocardial cells to secrete natriuretic peptides (NT-proBNP) antagonists of the renin-angiotensin-aldosterone system. The aim of this study was to evaluate the diagnostic accuracy of serum NT-proBNP levels in order to detect structural/functional cardiac diseases assessed by echocardiography., Methods: A total of 126 consecutive newly diagnosed, never before treated, hypertensive patients (30-67 years) were enrolled, and clinical, echocardiography parameters and biochemical data were collected. Our reference was the presence of structural/functional cardiac disease (CSFD) and our index text was the serum NT-proBNP levels., Results: NT-proBNP levels in CSFD patients were ~2 times higher than in non-CSFD subjects (median 61 vs 29 ng/L, n=50 and 76, respectively); in addition, 60% of CSFD subjects (only 14% of which with pathological levels, >125 ng/L), and 30% without CSFD showed NT-proBNP concentrations higher than 50 ng/L. However, ROC curves demonstrated a low specificity (38%) (calculated at 90% sensitivity at a cut-off of 22.5 ng/L)., Discussion: NT-proBNP levels, as a screening tool for cardiac structural/functional disease, appear to be limited, because of the low specificity. However, the strong association between its concentration and the establishment of irreversible cardiac hypertrophy prompts successive studies aimed to ascertain the use of its serum levels as an early alert indicator of disease severity., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: We aimed to evaluate the relationship between serum leptin and the leptin/body mass index (BMI) ratio with prevalent cardiovascular disease (CVD), and their influence on all-cause and CVD-related mortality in patients on hemodialysis (HD)., Methods: 118 stable HD patients (50 women, median [interquartile range] age, 65.1 [54.7-72.2] years) were studied. All patients had baseline measurement of serum leptin concentrations. Relationships between leptin and all-cause and CVD mortality were studied by means of survival analysis and Cox regression analysis., Results: The leptin/BMI ratio was similar in patients with and without CVD at baseline (0.65 [0.29-2.23] vs. 0.68 [0.29-1.49] ng·m2/ml·kg, respectively, NS). Multiple logistic regression analysis showed that there was not an independent association between leptin/BMI ratio and prevalent CVD. During the follow-up time, 52 (44.1%) patients died. CVD was the cause of death in 27 out of 52 (51.9%) deceased patients. Survival analysis and Cox proportional multivariate regression analysis showed that there were no significant relationships between leptin levels or the leptin/BMI ratio and all-cause and CVD-related mortality., Conclusion: These results do not support that, in stable HD patients, serum leptin concentrations and the leptin/BMI ratio are related with prevalent CVD. Leptin/BMI ratio seems not to be a risk factor for mortality in these patients.

Background/aims: Prognostic scores currently used in cirrhotic patients do not include thrombotic risk factors (TRFs). Predicting factors of portal vein thrombosis (PVT) development are still unknown. We wanted to describe TRFs as a function of liver disease severity using the MELD score and assess the role of local and systemic TRFs as predictors of PVT development in cirrhotic patients., Methods: One hundred consecutive patients with liver cirrhosis were included in the study. TRFs, D-dimers, MELD score, portal vein patency and flow velocity were evaluated in all subjects at baseline and every 6 months thereafter. Variables able to predict PVT development within 1 year were identified by means of multiple logistic regression., Results: The plasma levels of protein C and antithrombin were lower and the concentration of D-dimers was higher in patients with advanced disease. Plasma levels of antithrombin, protein C and protein S resulted significantly lower in PVT group at univariate analysis, but reduced portal vein flow velocity was the only variable independently associated with PVT development., Conclusions: Lower concentrations of natural coagulation inhibitors are frequently detected in patients with liver cirrhosis. A reduced portal flow velocity seems to be the most important predictive variable for PVT development in patients with cirrhosis.

Mass spectrometry-based approaches are the reference techniques for the determination of nitrite and nitrate in plasma and serum. However, due to their simplicity and rapidity, assays based on the Griess reaction or HPLC are generally used in clinical studies, but they generate diverging values for nitrite/nitrate concentration. In this study, particular attention is paid to the optimization of the deproteinization procedure for plasma and serum samples prior to nitrite/nitrate analysis by an enzymatic batch Griess assay, HPLC and GC-MS. A method is reported to verify completeness of deproteinization and to correct for nonspecific contribution to the absorbance of the diazo dye at 540 nm. With the application of such optimized procedures, we were able to significantly improve the correlation between Griess and HPLC method or the GC-MS technique for nitrite+nitrate concentrations in human serum and plasma. Despite remaining potentially interfering pre-analytical and analytical factors, the procedures reported in the present study may be helpful in a critical evaluation of limits and possibilities of the enzymatic batch Griess assay as a large-scale method for nitrite/nitrate determination in human serum in clinical studies.

Histatin-5 is a peptide secreted in the human saliva, which possesses powerful antifungal activity. Previous studies have shown that this peptide exerts its candidacidal activity, through the inhibition of both mitochondrial respiration and the formation of reactive oxygen species. The purpose of the present study was to investigate the biological consequences of histatin-5 action on mammalian mitochondria to verify if the toxic mechanism exerted on mitochondria from Candida albicans is an exclusive for fungal cells. Moreover, hypothesising that the damage exerted on mitochondria may induce programmed cellular death pathways, we evaluated two main markers of apoptosis: the mitochondrial membrane potential (DeltaPsi) and the release of cytochrome c. The results obtained show that exposure of isolated mammalian mitochondria to histatin-5 determines: (i) a large inhibition of the respiratory chain at the level of complex I, (ii) a slight decrease in the mitochondrial membrane potential, and (iii) no release of cytochrome c.

A simple and reliable method for the preparation of biological samples for the evaluation of biochemical parameters representative of the redox and energy states, such as glutathione (GSH), oxidized glutathione (GSSG), oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), coenzyme A (CoASH), oxidized CoASH, ascorbate, malondialdehyde, oxypurines, nucleosides, and energy metabolites, is presented. Fast deproteinization under nonoxidizing conditions is obtained by tissue homogenization in ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4 (3:1; v:v), pH 7.40. After sample centrifugation to pellet precipitated proteins, organic solvent removal is performed on clear supernatants by three washings with large volumes of high-performance liquid chromatography (HPLC)-grade chloroform. The remaining aqueous phase, free of solvent and any lipid-soluble substances that may interfere with the further metabolite analysis, is used for the simultaneous ion-pairing HPLC determination of 39 compounds by means of a Kromasil C-18, 250 x 4.6-mm, 5-microm-particle-size column with tetrabutylammonium hydroxide as the pairing reagent. Results obtained by using the present method to prepare different rat tissue extracts demonstrate that it is possible to perform a single tissue preparation only for monitoring, in the same sample, compounds representative of the redox state (through the direct determination of GSH, GSSG, NAD+, NADH, NADP+, NADPH, CoASH, and oxidized CoASH) and of the cell energy state (by the analysis of oxypurines, nucleosides, and energy metabolites). Applicability of this sample processing procedure to quantify variations of the aforementioned compounds under pathological conditions was effected in rats subjected to moderate closed-head trauma.

The beta-dystroglycan/Grb2 interaction was investigated and a proline-rich region within beta-dystroglycan that binds Grb2-src homology 3 domains identified. We used surface plasmon resonance (SPR), fluorescence analysis, and solid-phase binding assay to measure the affinity constants between Grb2 and the beta-dystroglycan cytoplasmic tail. Analysis of the data obtained from SPR reveals a high-affinity interaction (K(D) approximately 240 nM) between Grb2 and the last 20 amino acids of the beta-dystroglycan carboxyl-terminus, which also contains a dystrophin-binding site. A similar K(D) value (K(D) approximately 280 nM) was obtained by solid-phase binding assay and in solution by fluorescence. Both Grb2-SH3 domains bind beta-dystroglycan but the N-terminal SH3 domain binds with an affinity approximately fourfold higher than that of the C-terminal SH3 domain. The Grb2-beta-dystroglycan interaction was inhibited by dystrophin in a range of concentration of 160-400 nM. These data suggest a highly regulated and dynamic dystrophin/dystroglycan complex formation and that this complex is involved in cell signaling., (Copyright 2000 Academic Press.)

A protein fragment corresponding to the mouse beta-dystroglycan N-terminal extracellular region from position 654 to 750, beta-DG(654-750) was recombinantly expressed in BL21(DE3) Escherichia coli cells. Secondary structure prediction of the protein fragment reveals about 70% of random coil, as confirmed by circular dichroism analysis. Moreover, fluorescence analysis shows that the tryptophan residue in position 659 lays in a solvent-exposed fashion. These data suggest that the beta-DG(654-750) is likely to have a quite flexible structure and to be only partially folded. Interestingly, the protein still retains its biological function since using solid-phase assays we have detected binding of biotinylated beta-DG(654-750) both to native alpha-dystroglycan and to a recombinant fragment which spans the C-terminal region of alpha-dystroglycan., (Copyright 1999 Academic Press.)