The Effects of Multidisciplinary Intensive Rehabilitation on Cognitive and Executive Functions in Parkinson's Disease: A Clinical Database Analysis.

Background/Objectives: This study is based on data collected from a medical health record review to assess whether multidisciplinary intensive rehabilitation treatment in Parkinson's disease (PD) patients can improve global cognitive functioning and executive functions. Methods: The data related to PD patients were extrapolated from a clinical database called "NeuroRehab". A total of 104 PD patients (51 males; 53 females) performed 6 weeks of multidisciplinary intensive rehabilitation treatment in clinical practice from January 2019 to May 2023. This training program was characterized by three daily sessions of 60 min of activities (muscle relaxation and stretching exercises, moderate physical aerobic exercise, and occupational therapy). The patients were classified and stratified according to disease severity (according to the Hoehn and Yahr scale), postural instability and gait difficulty (PIGD) or tremor-dominant (TD) subtypes, disease duration (DD), and the presence of dyskinesias. The effect of multidisciplinary intensive rehabilitation treatment on cognitive and executive functions was evaluated through the administration of cognitive tests, such as the Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), and Frontal Assessment Battery (FAB). All the parameters were evaluated at the baseline (T0) and at the end of the rehabilitation program (T1). Results: The multidisciplinary intensive rehabilitation treatment significantly improved cognitive performance. The MMSE, MoCA, and FAB test scores after the rehabilitation program (T1) were significantly higher compared to the scores obtained at the baseline (T0). Moreover, further analyses on subgroups of the patients who scored below the cut-off in the MMSE showed that at least 50% of patients overcame the cut-off score. Interestingly, the same analyses performed for the MoCA and FAB revealed a higher rate of improvement in cognitive functions, with normal scores in both tests after 6 weeks of multidisciplinary intensive rehabilitation treatment. Conclusions: This study revealed the potential effects of a 6-week multidisciplinary rehabilitation program in improving cognitive status in a PD inpatient cohort.

Dose-response effects of physical exercise standardized volume on peripheral biomarkers, clinical response, and brain connectivity in Parkinson's disease: a prospective, observational, cohort study.

Background: Exercise has been proposed as the "Universal Prescription for Parkinson's Disease"; however, the specificity of exercise dose in terms of frequency, intensity, duration, and type to be prescribed remains to be elucidated. The 2018 US updated guidelines and WHO Guidelines on Physical Activity and Sedentary Behavior recommend older adults (> 65+ years) to achieve weekly minimal activity levels, indicating the intensity of aerobic exercise as the metabolic equivalent of task and duration as minutes/week (150-300 min/week at a moderate intensity of 3-5.9 MET- or 75-150 min/week of a vigorous intensity of ≥6 MET). Translating these recommendations to PD patients, the study aimed to assess the dose-response effects of standardized volume of structured exercise, measured as METs-minutes/week (weekly energy expenditure) of two different rehabilitation settings to quantify the change in neurotrophic factors. The exercise-induced benefits between the two rehabilitation settings will be evaluated based on motor and non-motor symptoms, kinematic parameters of gait, cognitive function, quality of life, and cortical activity and brain connectivity. Methods: METEX-PD is a pilot, prospective, observational, cohort study. The study will enroll consecutively thirty (N = 30) participants with mild-to-moderate Parkinson's disease diagnosis to be assigned to a non-intensive or intensive rehabilitation group. The non-intensive rehabilitation group will achieve a range of 180-270 METs-min/week (90 min/week of low-intensity aerobic exercise, 2-3 METs), while the intensive rehabilitation group will exercise at 1350-1980 METs-min/week (225 min/week of high-intensity aerobic exercise, 6-8.8 METs). The METEX-PD trial will last 12 weeks, including 4 weeks of aerobic training program and two follow-ups. Assessments will be performed at baseline (T0), at the end of the exercise program (T1-end of the program), and 4- and 8 weeks after the end of the training program (FU-1 and FU-2). The primary outcome is the change from baseline in peripheral blood BDNF levels. Secondary outcomes are differences in peripheral biomarkers, functional-motor assessments, clinical-functional evaluations, and brain imaging. Conclusion: METEX-PD trial will enable us to estimate the change in BDNF levels and other peripheral biomarkers under precise exercise-induced energy expenditure. The primary results of the METEX-PD study will allow the development of a larger multicenter randomized controlled trial to investigate the molecular pathways inducing the change in selected neurotrophic factors, such as BDNF, IGF-1, or irisin, and the downstream mechanisms of neuroplasticity in PD patients.

Physical activity and neurotrophic factors as potential drivers of neuroplasticity in Parkinson's Disease: A systematic review and meta-analysis.

Parkinson's disease (PD) is a neurodegenerative disorder, characterized by motor and non-motor symptoms, that still lacks of a disease-modifying treatment. Consistent evidence proved the benefits of physical therapy on motor and non-motor symptoms in PD patients, leading the scientific community to propose physical activity as disease-modifying therapy for PD and suggesting the involvement of neurotrophic factors (NFs) as key mediators of neuroplasticity. However, the lack of standardized exercise training and methodological flaws of clinical trials have limited the evidence demonstrating the exercise-induced changes in serum and plasma neurotrophic factors concentration. A systematic search, covering 20 years of research in this field and including randomized and non-randomized controlled trials (RCTs and non-RCTs), which reported changes in serum and plasma NFs after a specific intervention, were reviewed. Pooled effect sizes (p-ESs) and 95% confidence intervals (95%CIs) were calculated using a random effects model with R software. A total of 18 articles, of which exercise programs of interventions were codified in terms of type, intensity and duration adopting a standardisation methodology, were included in the systematic review. Six papers, describing the effect of different training programs on BDNF and IGF-1 levels, were included and independently analysed in two meta-analyses. Quantitative analysis for BDNF indicated a statistically significant improvement in serum concentration of PD patients (MD: 5.99 ng/mL; 95%IC: 0.15 -11.83; I2 = 77%) performing physical activity compared with control conditions in RCTs. Preliminary evidence supported the hypothesis that a moderate intensity aerobic exercise (MIAE) would be necessary to induce the changes in NFs. However, sensitivity analysis of meta-analysis and the few studies included in subgroup analysis did not support these results. Alongside, meta-analysis followed by sensitivity analysis revealed a potential change in serum IGF-1 (MD: 33.47 ng/mL; 95%IC: 8.09-58.85) in PD patients performing physical activity with respect controls in RCT studies. Considering the limited evidence to support or refute the increase in NFs levels in PD patients performing physical activity, there is a need to develop a rigorous controlled randomized trial, with standardization for loading intensity of physical activity, greater sample size, and a correct stratification of PD patients to establish a well-defined correlation between physical activity and NFs levels.

Therapeutic education for empowerment and engagement in patients with Parkinson's disease: A non-pharmacological, interventional, multicentric, randomized controlled trial.

Background: In 1997 the European Parkinson's Disease Associations launched the Charter for People with Parkinson's disease that stated the right of patients to be informed and trained on the disease, its course, and treatments available. To date, few data analyzed the effectiveness of education program on motor and non-motor symptoms of PD. Objective: The aim of this study was to evaluate the efficacy of an education program as it was a pharmacological treatment, thus choosing as the primary endpoint the change in daily OFF hours, the most widely used outcome in pharmaceutical clinical trials on PD patients with motor fluctuations. Secondary outcomes were change in motor and non-motor symptoms, quality of life and social functioning. The long-term efficacy of the education therapy was also evaluated by analyzing data collected at 12- and 24-weeks follow-up outpatient visits. Methods: One hundred and twenty advanced patients and their caregivers were assigned to the intervention or control group in a single-blind, multicentric, prospective, randomized study evaluating an education program structured in individual and group sessions over a 6-weeks period.At the end of study, the intervention group showed a significant reduction in daily OFF hours compared to control patients (-1.07 ± 0.78 vs. 0.09 ± 0.35, p < 0.0001) and a significant improvement was also reported in most secondary outcomes. Patients retained significant medication adherence and daily OFF hours reduction at 12- and 24-weeks follow-up. Conclusion: The results obtained demonstrated that education programs may translate in a notable improvement in motor fluctuations and non-motor symptoms in advanced PD patients.Clinical Trial Registration:Clinicaltrials.gov, identifier NCT04378127.

The Sesquiterpene Lactone Cynaropicrin Manifests Strong Cytotoxicity in Glioblastoma Cells U-87 MG by Induction of Oxidative Stress.

Cynaropicrin has shown a wide range of pharmacological properties, such as antitumor action. Here, we showed the inhibitory effect of Cyn on human glioblastoma cell U-87 MG growth. According to the IC50 values, Cyn 4, 8 and 10 µM displayed a significant cytotoxicity, as confirmed by the cell count and MTT assay. Furthermore, Cyn completely abolished the ability of U-87 MG to form colonies and induced drastic morphological changes. Interestingly, pretreatment with ROS scavenger N-acetylcysteine 3 mM reversed the cytotoxicity induced by Cyn 25 µM and preserved the cells by morphological changes. Therefore, oxidative stress induction was evaluated at low 8- and high 25-µM concentrations in U-87 MG, as demonstrated by the quantitative and qualitative analysis of ROS. A prolonged increase in ROS generation under Cyn 25 µM exposure was followed by the loss of the mitochondrial membrane potential in treated U-87 MG cells. An acute treatment with Cyn 25 µM induced Cyt c release, as revealed by immunofluorescence staining and the activation of cell death pathways, apoptosis and autophagy. On the other hand, chronic treatment with Cyn 8 µM induced senescence, as revealed by the increase in SA-ß-Gal activity. Moreover, at this concentration, Cyn led to ERK dephosphorylation accompanied by a relevant reduction of the NF-κB p65 subunit. Finally, the combined effect of TMZ and Cyn resulted in synergistic cytotoxicity, as evaluated by the Bliss additivity model. The strong cytotoxicity of Cyn was also confirmed on IDH1 mutant U-87 MG cells and patient-derived IDH wild-type glioblastoma cell lines NULU and ZAR. In conclusion, given the high toxicity at minimal concentrations, the high inhibition of tumor cell growth and synergy with the standard drug for glioblastoma TMZ, Cyn could be proposed as a potential adjuvant for the treatment of glioblastoma.

Ageritin-The Ribotoxin-like Protein from Poplar Mushroom (Cyclocybe aegerita) Sensitizes Primary Glioblastoma Cells to Conventional Temozolomide Chemotherapy.

Here, we propose Ageritin, the prototype of the ribotoxin-like protein family, as an adjuvant treatment to control the growth of NULU and ZAR, two primary human glioblastoma cell lines, which exhibit a pharmacoresistance phenotype. Ageritin is able to inhibit NULU and ZAR growth with an IC50 of 0.53 ± 0.29 µM and 0.42 ± 0.49 µM, respectively. In this study, Ageritin treatment highlighted a macroscopic genotoxic response through the formation of micronuclei, which represents the morphological manifestation of genomic chaos induced by this toxin. DNA damage was not associated with either the deregulation of DNA repair enzymes (i.e., ATM and DNA-PK), as demonstrated by quantitative PCR, or reactive oxygen species. Indeed, the pretreatment of the most responsive cell line ZAR with the ROS scavenger N-acetylcysteine (NAC) did not follow the reverse cytotoxic effect of Ageritin, suggesting that this protein is not involved in cellular oxidative stress. Vice versa, Ageritin pretreatment strongly enhanced the sensitivity to temozolomide (TMZ) and inhibited MGMT protein expression, restoring the sensitivity to temozolomide. Overall, Ageritin could be considered as a possible innovative glioblastoma treatment, directly damaging DNA and downregulating the MGMT DNA repair protein. Finally, we verified the proteolysis susceptibility of Ageritin using an in vitro digestion system, and considered the future perspective use of this toxin as a bioconjugate in biomedicine.

Early Warnings by Liver Organoids on Short- and Long-Chain PFAS Toxicity.

Short-chain per-fluoroalkyl substances (PFAS) have replaced long-chains in many applications, however the toxicity and its mode of action and interactions due to the large number of these compounds and their mixtures is still poorly understood. The paper aims to compare the effects on mouse liver organoids (target organ for bioaccumulation) of two long-chain PFAS (perfluorooctane sulfonate -PFOS-, perfluorooctanoic acid -PFOA) and two short-chain PFAS commonly utilized in the industry (heptafluorobutyric acid -HFBA-, Pentafluoropropionic anhydride-PFPA) to identify the mode of action of these classes of contaminants. Cytomorphological aberrations and ALT/GDH enzyme disruption were identified but no acute toxicity endpoint neither apoptosis was detected by the two tested short-chain PFAS. After cytomorphological analysis, it is evident that short-chain PFAS affected organoid morphology inducing a reduction of cytostructural complexity and aberrant cytological features. Conversely, EC50 values of 670 ± 30 µM and 895 ± 7 µM were measured for PFOS and PFOA, respectively, together with strong ALT/GDH enzyme disruption, caspase 3 and 7 apoptosis activation and deep loss of architectural complexity of organoids in the range of 500-1000 µM. Eventually, biochemical markers and histology analysis confirmed the sensitivity of organoid tests that could be used as a fast and reproducible platform to test many PFAS and mixtures saving time and at low cost in comparison with in vivo tests. Organoids testing could be introduced as an innovative platform to assess the toxicity to fast recognize potentially dangerous pollutants.

Inhibiting effect of p-Coumaric acid on U87MG human glioblastoma cell growth.

p-Coumaric acid (pCA) is a hydroxycinnamic acid derivative commonly found in many natural products that has been extensively studied for its anticancer activity in multiple cell lines. In this report we investigated the effects of this phytochemical as adjuvant therapy to treat glioblastoma, an infaust brain tumour characterized by the acquired or innate resistance to the conventional chemotherapy temozolomide (TMZ). U87Mg glioblastoma cell growth and viability was assessed by growth rate curves and MTT assay incubating cells with 0.5 and 1 mM pCA for 24 h, 48 h and 72 h. Cell cycle analysis, performed by flow cytometry, showed that pCA led the accumulation of GBM cells in G2/M phase. Western blot analysis shows that pCA induced CDK4 cyclin-dependent kinase reduction and p53 increase, followed by induction of the CDK inhibitor p21. Furthermore, pCA treatment mediated the activation of apoptosis and the inhibition of migration of U87Mg cells. Finally, the treatment of glioblastoma cells in vitro with pCA concomitantly with the TMZ revealed a synergistic effect between the natural substance and the chemotherapy. In conclusion, our results demonstrated that pCA acts influencing the cell viability and cell cycle of U87Mg cells by promoting cell cycle arrest in G2/M phase and apoptosis.

Cytotoxicity Effect of Quinoin, Type 1 Ribosome-Inactivating Protein from Quinoa Seeds, on Glioblastoma Cells.

Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblastoma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients' gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), currently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells' growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs.

Gymnema sylvestre Extract Restores the Autophagic Pathway in Human Glioblastoma Cells U87Mg.

Glioblastoma is a brain tumour, characterised by recurrent or innate resistance to conventional chemoradiotherapy. Novel natural molecules and phyto-extracts have been proposed as adjuvants to sensitise the response to Temozolomide (TMZ). In this study, we investigated the effect of GS extract on human glioblastoma cells U87Mg. According to the IC50-values, GS extract displayed a significant cytotoxicity. This was confirmed by cell growth inhibition and alteration in metabolic activity evaluated by cell count and MTT assay. GS induced reduction in Pro-caspase 9, 3, but not PARP cleavage nor DNA fragmentation. Thus, in GS-induced cytotoxicity, cell death is not associated with apoptosis. In this context, short-term treatment of U87Mg cells with GS extract (1 mg/mL) reduced the phosphorylation levels of mTOR and of its downstream target P70 S6 kinase, highlighting the role of GS extract into autophagy induction. The activation of autophagic flux by GS extract was confirmed by Western blot analysis, which revealed the reduction in p62 and the concomitant increase in LC3B II/I ratio. Immunofluorescence evidenced the accumulation of LC3B puncta in U87Mg cells pretreated with autophagy inhibitor Bafilomycin A1. Furthermore, as main key regulators of type II programmed cell death, p53, p21 and CDK4 were also investigated and were inhibited by GS treatment. In conclusion, GS extract could be considered as an autophagy inducer in glioblastoma cells U87Mg.

Implication of Lactucopicrin in Autophagy, Cell Cycle Arrest and Oxidative Stress to Inhibit U87Mg Glioblastoma Cell Growth.

In this study, we propose lactucopicrin (LCTP), a natural sesquiterpene lactone from Lactucavirosa, as a molecule able to control the growth of glioblastoma continuous cell line U87Mg. The IC50 of U87Mg against LCTP revealed a strong cytotoxic effect. Daily administration of LCTP showed a dose and time-dependent reduction of GBM cell growth and viability, also confirmed by inhibition of clonogenic potential and mobility of U87Mg cells. LCTP activated autophagy in U87Mg cells and decreased the phosphorylation of proliferative signals pAKT and pERK. LCTP also induced the cell cycle arrest in G2/M phase, confirmed by decrease of CDK2 protein and increase of p53 and p21. LCTP stimulated apoptosis as evidenced by reduction of procaspase 6 and the increase of the cleaved/full-length PARP ratio. The pre-treatment of U87Mg cells with ROS scavenger N-acetylcysteine (NAC), which reversed its cytotoxic effect, showed the involvement of LCTP in oxidative stress. Finally, LCTP strongly enhanced the sensitivity of U87Mg cells to canonical therapy Temozolomide (TMZ) and synergized with this drug. Altogether, the growth inhibition of U87Mg GBM cells induced by LCTP is the result of several synergic mechanisms, which makes LCTP a promising adjuvant therapy for this complex pathology.

An Effective Multi-Stage Liposomal DNA Origami Nanosystem for In Vivo Cancer Therapy.

DNA origami systems could be important candidates for clinical applications. Unfortunately, their intrinsic properties such as the activation of non-specific immune system responses leading to inflammation, instability in physiological solutions, and a short in vivo lifetime are the major challenges for real world applications. A compact short tube DNA origami (STDO) of 30 nm in length and 10 nm in width was designed to fit inside the core of a stealth liposome (LSTDO) of about 150 nm to remote load doxorubicin. Biocompatibility was tested in three-dimensional (3D) organoid cultures and in vivo. Efficacy was evaluated in different cell lines and in a xenograft breast cancer mouse model. As described in a previous work, LSTDO is highly stable and biocompatible, escaping the recognition of the immune system. Here we show that LSTDO have an increased toleration in mouse liver organoids used as an ex vivo model that recapitulate the tissue of origin. This innovative drug delivery system (DDS) improves the antitumoral efficacy and biodistribution of doxorubicin in tumor-bearing mice and decreases bone marrow toxicity. Our application is an attractive system for the remote loading of other drugs able to interact with DNA for the preparation of liposomal formulations.

Stereoselectivity of Aldose Reductase in the Reduction of Glutathionyl-Hydroxynonanal Adduct.

The formation of the adduct between the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and glutathione, which leads to the generation of 3-glutathionyl-4-hydroxynonane (GSHNE), is one of the main routes of HNE detoxification. The aldo-keto reductase AKR1B1 is involved in the reduction of the aldehydic group of both HNE and GSHNE. In the present study, the effect of chirality on the recognition by aldose reductase of HNE and GSHNE was evaluated. AKR1B1 discriminates very modestly between the two possible enantiomers of HNE as substrates. Conversely, a combined kinetic analysis of the glutathionyl adducts obtained starting from either 4R- or 4S-HNE and mass spectrometry analysis of GSHNE products obtained from racemic HNE revealed that AKR1B1 possesses a marked preference toward the 3S,4R-GSHNE diastereoisomer. Density functional theory and molecular modeling studies revealed that this diastereoisomer, besides having a higher tendency to be in an open aldehydic form (the one recognized by AKR1B1) in solution than other GSHNE diastereoisomers, is further stabilized in its open form by a specific interaction with the enzyme active site. The relevance of this stereospecificity to the final metabolic fate of GSHNE is discussed.

First-of-its-kind STARD3 Inhibitor: In Silico Identification and Biological Evaluation as Anticancer Agent.

STARD3 is a cellular protein that represents an attractive target for cancer therapy, being overexpressed in breast cancer and implied in the development of colorectal, gastric, and prostate cancers. Unfortunately, no STARD3 inhibitor has been identified yet. In this work, an in silico strategy was applied to predict a reliable binding mode of cholesterol into STARD3 and to develop a pharmacophore-based virtual screening protocol that allowed the identification of the first STARD3 inhibitor ever reported. The identified compound VS1 binds STARD3 with micromolar affinity (IC50 = 35 µM) and shows antiproliferative activity in breast (MCF7 and MDA- MB-231) and colon (HCT-116) cancer cell lines in the same concentration range (IC50 = 49.7-105.5 µM). Although VS1 has a moderate potency, we demonstrated that it specifically targets STARD3 in the cells and induces its degradation. Overall, the results confirm the reliability of the computational strategies herein applied and the identification of the first hit compound for the development of novel potent STARD3 inhibitors.

Kinetic features of carbonyl reductase 1 acting on glutathionylated aldehydes.

The attempt to evaluate the human carbonyl reductase 1 (CBR1) activity on 3-glutathionylated-4-hydroxyalkanals through the classical spectrophotometric assay, in which NADPH oxidation is monitored at 340 nm, failed. This was due to the ability of the enzyme to catalyze the reduction of the free aldehyde form and at the same time the oxidation of the hemiacetal structure of this class of substrates, thus leading to the occurrence of a disproportion reaction sustained by a redox recycle of the pyridine cofactor. Making use of glutathionylated alkanals devoid of the 4 hydroxyl group, and thus unable to structurally arrange into a cyclic hemiacetal form, the susceptibility to inhibition of CBR1 to polyphenols was tested. Flavones, that were much more effective than isoflavones, resulted able to modulate the reductase activity of the enzyme on this new peculiar class of substrates.

Human carbonyl reductase 1 as efficient catalyst for the reduction of glutathionylated aldehydes derived from lipid peroxidation.

Human recombinant carbonyl reductase 1 (E.C. 1.1.1.184, hCBR1) is shown to efficiently act as aldehyde reductase on glutathionylated alkanals, namely 3-glutathionyl-4-hydroxynonanal (GSHNE), 3-glutathionyl-nonanal, 3-glutathionyl-hexanal and 3-glutathionyl-propanal. The presence of the glutathionyl moiety appears as a necessary requirement for the susceptibility of these compounds to the NADPH-dependent reduction by hCBR1. In fact the corresponding alkanals and alkenals, and the cysteinyl and γ-glutamyl-cysteinyl alkanals adducts were either ineffective or very poorly active as CBR1 substrates. Mass spectrometry analysis reveals the ability of hCBR1 to reduce GSHNE to the corresponding GS-dihydroxynonane (GSDHN) and at the same time to catalyze the oxidation of the hemiacetal form of GSHNE, generating the 3-glutathionylnonanoic-δ-lactone. These data are indicative of the ability of the enzyme to catalyze a disproportion reaction of the substrate through the redox recycle of the pyridine cofactor. A rationale for the observed preferential activity of hCBR1 on different GSHNE diastereoisomers is given by molecular modelling. These results evidence the potential of hCBR1 acting on GSHNE to accomplish a dual role, both in terms of HNE detoxification and, through the production of GSDHN, in terms of involvement into the signalling cascade of the cellular inflammatory response.

Zolfino landrace (Phaseolus vulgaris L.) from Pratomagno: general and specific features of a functional food.

BACKGROUND: The Zolfino bean is a variety of Phaseolus vulgaris, which is cultivated in a limited area of Tuscany, Italy, and is widely appreciated for its flavor and culinary uses. OBJECTIVES: A yellow Zolfino landrace cultivated in the Leccio-Reggello area was characterized and compared with three other varieties of Phaseolus vulgaris (i.e. the Borlotto, Cannellino, and Corona beans) in terms of its general features and potential as an antioxidant/anti-inflammatory agent. DESIGN: The length, width, thickness, equatorial section surface, weight, volume, and seed coat section were measured in all the beans. The seed surface area was also estimated by an original empirical method. The ability of the different beans to interfere with the enzymes of the polyol pathway (that is, aldose reductase (AR) and sorbitol dehydrogenase) was tested using the supernatant after soaking the beans at room temperature and after thermal treatment, which simulated the bean-cooking process in a controlled fashion. RESULTS: Concerning the general features, Zolfino was comparable with other beans, except Corona, in terms of surface-volume ratio, which possesses the lowest tegument thickness. Moreover, Zolfino appears the most effective in inhibiting AR activity. The inhibitory ability is unaffected by thermal treatment and appears to be associated with compound(s) present in the coat of the bean. CONCLUSIONS: The ability of Zolfino to inhibit AR, thus reducing the flux of glucose through the polyol pathway, highlights the features of Zolfino as a functional food, potentially useful in treating the dysfunctions linked to the hyperactivity of AR, such as diabetic complications or inflammatory responses.

Modulation of aldose reductase activity by aldose hemiacetals.

BACKGROUND: Glucose is considered as one of the main sources of cell damage related to aldose reductase (AR) action in hyperglycemic conditions and a worldwide effort is posed in searching for specific inhibitors of the enzyme. This AR substrate has often been reported as generating non-hyperbolic kinetics, mimicking a negative cooperative behavior. This feature was explained by the simultaneous action of two enzyme forms acting on the same substrate. METHODS: The reduction of different aldoses and other classical AR substrates was studied using pure preparations of bovine lens and human recombinant AR. RESULTS: The apparent cooperative behavior of AR acting on glucose and other hexoses and pentoses, but not on tethroses, glyceraldehyde, 4-hydroxynonenal and 4-nitrobenzaldehyde, is generated by a partial nonclassical competitive inhibition exerted by the aldose hemiacetal on the reduction of the free aldehyde. A kinetic model is proposed and kinetic parameters are determined for the reduction of l-idose. CONCLUSIONS: Due to the unavoidable presence of the hemiacetal, glucose reduction by AR occurs under different conditions with respect to other relevant AR-substrates, such as alkanals and alkenals, coming from membrane lipid peroxidation. This may have implications in searching for AR inhibitors. The emerging kinetic parameters for the aldoses free aldehyde indicate the remarkable ability of the enzyme to interact and reduce highly hydrophilic and bulky substrates. GENERAL SIGNIFICANCE: The discovery of aldose reductase modulation by hemiacetals offers a new perspective in searching for aldose reductase inhibitors to be developed as drugs counteracting the onset of diabetic complications.

NADP(+)-dependent dehydrogenase activity of carbonyl reductase on glutathionylhydroxynonanal as a new pathway for hydroxynonenal detoxification.

An NADP(+)-dependent dehydrogenase activity on 3-glutathionyl-4-hydroxynonanal (GSHNE) was purified to electrophoretic homogeneity from a line of human astrocytoma cells (ADF). Proteomic analysis identified this enzymatic activity as associated with carbonyl reductase 1 (EC 1.1.1.184). The enzyme is highly efficient at catalyzing the oxidation of GSHNE (KM 33 µM, kcat 405 min(-1)), as it is practically inactive toward trans-4-hydroxy-2-nonenal (HNE) and other HNE-adducted thiol-containing amino acid derivatives. Combined mass spectrometry and nuclear magnetic resonance spectroscopy analysis of the reaction products revealed that carbonyl reductase oxidizes the hydroxyl group of GSHNE in its hemiacetal form, with the formation of the corresponding 3-glutathionylnonanoic-δ-lactone. The relevance of this new reaction catalyzed by carbonyl reductase 1 is discussed in terms of HNE detoxification and the recovery of reducing power.

L-Idose: an attractive substrate alternative to D-glucose for measuring aldose reductase activity.

Although glucose is one of the most important physio-pathological substrates of aldose reductase, it is not an easy molecule for in vitro investigation into the enzyme. In many cases alternative aldoses have been used for kinetic characterization and inhibition studies. However these molecules do not completely match the structural features of glucose, thus possibly leading to results that are not fully applicable to glucose. We show how aldose reductase is able to act efficiently on L-idose, the C-5 epimer of D-glucose. This is verified using both the bovine lens and the human recombinant enzymes. While the kcat values obtained are essentially identical to those measured for D-glucose, a significant decrease in KM was observed. This can be due to the significantly higher level of the free aldehyde form present in L-idose compared to D-glucose. We believe that L-idose is the best alternative to D-glucose in studies on aldose reductase.