Ameliorative Effect of Quercetin on Neurochemical and Behavioral Deficits in Rotenone Rat Model of Parkinson's Disease: Modulating Autophagy (Quercetin on Experimental Parkinson's Disease)

Autophagy is necessary for neuronal homeostasis and its dysfunction has been implicated in Parkinson's disease (PD) as it can exacerbate endoplasmic reticulum (ER) stress and ER stress‐induced apoptosis. Quercetin is a flavonoid known for its neuroprotective and antioxidant effects. The present study investigated the protective, autophagy‐modulating effects of quercetin in the rotenone rat model of PD. Rotenone was intraperitoneally injected at dose of 2 ml/kg/day for 4 weeks. Simultaneous intraperitoneal injection of quercetin was given at a dose of 50 mg/kg/day also for 4 weeks. Neurobehavioral changes were studied. Oxidative/antioxidant status, C/EBP homologous protein (CHOP), Beclin‐1, and dopamine levels were assessed. DNA fragmentation and histopathological changes were evaluated. This research work revealed that quercetin significantly attenuated rotenone‐induced behavioral impairment, augmented autophagy, ameliorated ER stress‐ induced apoptosis with attenuated oxidative stress. From the current study, quercetin can act as an autophagy enhancer in PD rat model and modulates the microenvironment that leads to neuronal death.     

Environmentally benign first derivative synchronous spectrofluorimetry for the analysis of two binary mixtures containing duloxetine with avanafil or tadalafil in spiked plasma samples

Antidepressants can cause sexual dysfunction side effects, necessitating the co-administration of phosphodiesterase type 5 inhibitors. The simultaneous determination of these drugs in biological fluids is critical for therapeutic drug monitoring. For the first time, two binary mixtures containing duloxetine with either avanafil or tadalafil were estimated utilizing simple green spectrofluorimetric methods without the need for a previous separation step. The study was based on first derivative synchronous spectrofluorimetry in ethanol using a change in wavelength difference (∆λ) of 20 and 25 nm for the first and second combinations, respectively. Duloxetine and avanafil were estimated at 297.7 and 331 nm in their binary mixture, while duloxetine and tadalafil were determined at 290.3 and 297.7 nm, respectively. The linearity was achieved over the ranges of 0.1–1.5 μg mL⁻¹ for both duloxetine and avanafil and 0.01–0.40 μg mL⁻¹ for tadalafil, with limits of detection of 0.013, 0.022, and 0.004 μg mL⁻¹ for duloxetine, avanafil, and tadalafil, respectively. Successful application of the developed approaches was accomplished for the estimation of the two mixtures in dosage forms as well as human plasma with excellent percentage recoveries (96–103.75% in plasma), which supports their suitability for use in quality control laboratories and pharmacokinetic studies. Moreover, the adopted approaches' greenness was evidenced by applying three tools.     

Peptic ulcers and erosions are common in Israeli children undergoing upper endoscopy

Background: Peptic ulcers and erosions (PU&E) are thought to be uncommon in children. Patients with early exposure to Helicobacter pylori may be at a higher risk for early onset PU&E. Children in Israel have a high prevalence and early acquisition of Helicobacter pylori (H. pylori) and have easy access to pediatric gastroenterologists and endoscopy. Our aim was to describe the prevalence and characteristics of PU&E in this population referred by Pediatric Gastroenterologists for an upper endoscopy. Methods: We conducted a retrospective study over the years January 2003–May 2006. Over these years we had information on 751 diagnostic upper endoscopies. PU&E was regarded as erosive gastritis/duodenitis or ulcer in either the stomach or duodenum. H. pylori status was assessed using rapid urease test and gastric biopsies. Results: PU&E was detected in 169 (22.5%) patients (ulcers 51 (6.8%), erosions 118 (15.7%)). One hundred twenty‐four had gastric PU&E and 58 had duodenal PU&E. H. pylori was positive in 112 (66.3%). H. pylori‐associated PU&E becomes common after age 10 years, with gastric PU&E presenting much earlier than duodenal disease. Most of the H. pylori‐negative PU&E were idiopathic and improved symptomatically on PPI treatment. Interestingly, 43% of patients with PU&E in our cohort were either immigrants from the former Soviet Union or of Israeli Arab origin. Conclusions: PU&E appears to be common in this selected population with a relatively high incidence of gastric PU&E. H. pylori associated PU&E becomes common after age 10 years with gastric PU&E presenting much earlier than duodenal disease. Non H. pylori PU&E in children comprises approximately a third of all PU&E, are mostly idiopathic and appear earlier than H. pylori associated PU&E.     

The ubiquitin ligase itch is auto-ubiquitylated in vivo and in vitro but is protected from degradation by interacting with the deubiquitylating enzyme FAM/USP9X

Itch is a ubiquitin ligase that has been implicated in the regulation of a number of cellular processes. We previously have identified Itch as a binding partner for the endocytic protein Endophilin and found it to be localized to endosomes. Using affinity purification coupled to mass spectrometry, we have now identified the ubiquitin-protease FAM/USP9X as a binding partner of Itch. The association between Itch and FAM/USP9X was confirmed in vitro by glutathione S-transferase pulldown and in vivo through coimmunoprecipation. Itch and FAM partially colocalize in COS-7 cells at the trans-Golgi network and in peripheral vesicles. We mapped the FAM-binding domain on Itch to the WW domains, a region known to be involved in substrate recognition. However, transient overexpression of FAM/USP9X resulted in the deubiquitylation of Itch. Moreover, we show that Itch auto-ubiquitylation leads to its degradation in the proteasome. By examining the amounts of Itch and FAM in various cell lines and rat tissues, a positive correlation was found in the expression of both proteins. This observation suggests that the levels of FAM expression could have an influence on Itch in cells. Experimental decrease in FAM levels by RNA interference leads to a significant reduction in intracellular levels of endogenous Itch, which can be prevented by treatment with the proteasome inhibitor lactacystin. Accordingly, overexpression of FAM/USP9X resulted in a marked increase in endogenous Itch levels. These results demonstrate an intriguing interplay between a ubiquitin ligase and a ubiquitin protease, based on direct interaction between the two proteins.     

Independent of Calorie Intake,Short-term Alternate-day Fasting Alleviates NASH,With Modulation of Markers of Lipogenesis,Autophagy, Apoptosis,and Inflammation in Rats

Non-alcoholic steatohepatitis (NASH) is a worldwide health problem. Alternate-day fasting (ADF), although thought to be aggressive, has proven safety and efficacy. We aimed to evaluate the effect of short-term ADF against already established high-fat-fructose (HFF)-induced NASH, independent of the amount of calorie intake, and to study the effect of ADF on lipogenesis, apoptosis, and hepatic inflammation. Male Sprague Dawley rats were divided into two groups: (1) negative control and (2) NASH group fed on HFF for 9 weeks, and then randomized into two subgroups of either HFF alone or with ADF protocol for 3 weeks. The ADF could improve HFF-related elevation in serum lactate dehydrogenase and could decrease the mRNA expression of lipogenesis genes; acetyl CoA carboxylase, peroxisome proliferator-activated receptor γ, and peroxisome proliferator-activated receptor α; apoptotic genes caspase-3, p53, and inflammatory cyclo-oxygenase 2; and immunohistochemical staining for their proteins in liver with upregulation of LC3 and downregulation of P62 immunoexpression. Moreover, ADF ameliorated HFF-induced steatosis, inflammation, ballooning, and fibrosis through hematoxylin and eosin, Oil Red O, and Sirius Red staining, confirmed by morphometric analysis, without significant weight loss. Significant correlation of morphometric parameters with levels of gene expression was found. These findings suggest ADF to be a safe effective therapeutic agent in the management of NASH     

A general strategy to characterize calmodulin-calcium complexes involved in CaM-target recognition: DAPK and EGFR calmodulin binding domains interact with different calmodulin-calcium complexes

Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four “EF hand” type Ca²⁺ binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca²⁺ ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM-Ca_n (n = 0-4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Muscle-Strengthening and Conditioning Activities and Risk of Type 2 Diabetes: A Prospective Study in Two Cohorts of US Women

Background

It is well established that aerobic physical activity can lower the risk of type 2 diabetes (T2D), but whether muscle-strengthening activities are beneficial for the prevention of T2D is unclear. This study examined the association of muscle-strengthening activities with the risk of T2D in women.

Methods and Findings

We prospectively followed up 99,316 middle-aged and older women for 8 years from the Nurses'' Health Study ([NHS] aged 53–81 years, 2000–2008) and Nurses'' Health Study II ([NHSII] aged 36–55 years, 2001–2009), who were free of diabetes, cancer, and cardiovascular diseases at baseline. Participants reported weekly time spent on resistance exercise, lower intensity muscular conditioning exercises (yoga, stretching, toning), and aerobic moderate and vigorous physical activity (MVPA) at baseline and in 2004/2005. Cox regression with adjustment for major determinants for T2D was carried out to examine the influence of these types of activities on T2D risk. During 705,869 person years of follow-up, 3,491 incident T2D cases were documented. In multivariable adjusted models including aerobic MVPA, the pooled relative risk (RR) for T2D for women performing 1–29, 30–59, 60–150, and >150 min/week of total muscle-strengthening and conditioning activities was 0.83, 0.93, 0.75, and 0.60 compared to women reporting no muscle-strengthening and conditioning activities (p<0.001 for trend). Furthermore, resistance exercise and lower intensity muscular conditioning exercises were each independently associated with lower risk of T2D in pooled analyses. Women who engaged in at least 150 min/week of aerobic MVPA and at least 60 min/week of muscle-strengthening activities had substantial risk reduction compared with inactive women (pooled RR = 0.33 [95% CI 0.29–0.38]). Limitations to the study include that muscle-strengthening and conditioning activity and other types of physical activity were assessed by a self-administered questionnaire and that the study population consisted of registered nurses with mostly European ancestry.

Conclusions

Our study suggests that engagement in muscle-strengthening and conditioning activities (resistance exercise, yoga, stretching, toning) is associated with a lower risk of T2D. Engagement in both aerobic MVPA and muscle-strengthening type activity is associated with a substantial reduction in the risk of T2D in middle-aged and older women. Please see later in the article for the Editors'' Summary