Loss Of Total Protein Research Articles

Standardized aqueous stem bark extract of Gmelina arborea roxb. possesses nephroprotection against adriamycin-induced nephrotoxicity in Wistar rats

Nephrotoxicity is a major limitation of adriamycin (ADR) chemotherapy. We hypothesized that administration of standardized aqueous bark extract of Gmelina arborea Roxb. (GA) (Family; Verbenaceae), a traditional therapeutic agent, may reduce the nephrotoxicity caused by ADR in Wistar rats. The dose-dependent nephroprotective activity of the standardized GA extract was investigated in ADR-induced (20 mg/kg, ip) nephrotoxicity in male Wistar rats (n = 6/group). The lyophilized powder of the aqueous refluxed (4 h) GA extract was administered at 100, 300 and 500 mg/kg doses orally for three consecutive days. Fosinopril sodium (0.09 mg/kg) was used as the positive control. Assessment of biochemical parameters on serum, urine and histopathology on H and E stained kidney sections were done at the end of the intervention. The treatment with GA and fosinopril decreased the elevation of serum creatinine, blood urea nitrogen, cystatin C, β2-microglobulin and loss of total protein in urine in nephrotoxic rats in a dose-dependent manner (p < 0.05). In contrast, serum concentrations of albumin and total protein were increased significantly (p < 0.05). H and E stained kidney sections showed an attenuation of renal parenchymal injury following the treatment. The aqueous extract of GA demonstrated antioxidant potential in vitro. Present findings conclude that the standardized aqueous extract of GA stem bark exerted a dose-dependent protection against ADR-induced nephrotoxicity in vivo and may be a promising adjunct in ADR chemotherapy.

Protective effects of three selected standardized medicinal plant extracts used in Sri Lankan traditional medicine in adriamycin induced nephrotoxic Wistar rats

Ethnopharmacological relevanceAbelmoschus moschatus Medik. (family: Malvaceae), Asparagus falcatus (family: Asparagaceae) and Barleria prionitis Linn. (family: Acanthaceae) have been used in the treatment of kidney diseases in Sri Lankan traditional medicine. Besides the traditional use, scientific scrutinization of safe therapeutic use of these medicinal plants in the management of kidney diseases has not been reported to date. Aim of the studyThe three selected doses of the aqueous extracts of the selected medicinal plants were studied for their protective effects against adriamycin (ADR) induced nephrotoxicity in Wistar rats. Materials and methodsChemically standardized plant materials were used in the study. The nephroprotective activity of the lyophilized powder of the aqueous refluxed (4hr) leaf extracts of A. moschatus, A. falcatus and the whole plant extract of B. prionitis was investigated in adriamycin (20 mg/kg, ip) induced nephrotoxicity in Wistar rats (n = 6/group). The treatment regimens were initiated 24 h after the induction of nephrotoxicity and continued daily as a single dose for three consecutive days at three selected doses (200, 400 and 600 mg/kg). Fosinopril sodium (0.09 mg/kg) was used as the standard drug. Nephroprotective activity was assessed by estimating the selected biochemical parameters and by the assessment of histopathology on H and E stained sections of the kidney. ResultsThe plant extracts at the three selected doses significantly attenuated the elevations in serum creatinine, blood urea nitrogen and the loss of urine total protein in a dose related manner in ADR induced nephrotoxic rats (p < 0.001). The serum concentration of albumin and total protein increased significantly (p < 0.001). Histopathological findings corroborated the biochemical evidence of nephroprotective activity. The aqueous extracts of the three selected medicinal plants exerted a relatively high antioxidant activity in vitro. ConclusionsEvaluation of the protective effects based on biochemical parameters and histopathology assessment revealed that the aqueous leaf extracts of A. moschatus, A. falcatus and the whole plant extract of B. prionitis possess significant nephroprotective activity against ADR induced acute nephrotoxicity. The secondary metabolites present in the plant extracts may attribute to the total antioxidant activities of the selected medicinal plant extracts thereby exerting protective effects against nephrotoxicity in Wistar rats.

Degradation of GRK2 and AKT is an early and detrimental event in myocardial ischemia/reperfusion.

BackgroundIdentification of signaling pathways altered at early stages after cardiac ischemia/reperfusion (I/R) is crucial to develop timely therapies aimed at reducing I/R injury. The expression of G protein-coupled receptor kinase 2 (GRK2), a key signaling hub, is up-regulated in the long-term in patients and in experimental models of heart failure. However, whether GRK2 levels change at early time points following myocardial I/R and its functional impact during this period remain to be established.MethodsWe have investigated the temporal changes of GRK2 expression and their potential relationships with the cardioprotective AKT pathway in isolated rat hearts and porcine preclinical models of I/R.FindingsContrary to the maladaptive up-regulation of GRK2 reported at later times after myocardial infarction, successive GRK2 phosphorylation at specific sites during ischemia and early reperfusion elicits GRK2 degradation by the proteasome and calpains, respectively, thus keeping GRK2 levels low during early I/R in rat hearts. Concurrently, I/R promotes decay of the prolyl-isomerase Pin1, a positive regulator of AKT stability, and a marked loss of total AKT protein, resulting in an overall decreased activity of this pro-survival pathway. A similar pattern of concomitant down-modulation of GRK2/AKT/Pin1 protein levels in early I/R was observed in pig hearts. Calpain and proteasome inhibition prevents GRK2/Pin1/AKT degradation, restores bulk AKT pathway activity and attenuates myocardial I/R injury in isolated rat hearts.InterpretationPreventing transient degradation of GRK2 and AKT during early I/R might improve the potential of endogenous cardioprotection mechanisms and of conditioning strategies.

Suppression of TRPV1 and P2Y nociceptors by honokiol isolated from Magnolia officinalis in 3rd degree burn mice by inhibiting inflammatory mediators

Burn pain is one of the worst imaginable pain, associated with considerable morbidity and mortality worldwide. The management of pain made significant progress; however, more research is needed for burn pain. In the present study, the antinociceptive effect of honokiol extracted from Magnolia officinalis was assessed for 3 consecutive days. The third-degree burns were induced by the hot water method. The honokiol both by intraperitoneal (i.p) and intra plantar (i.pl) route and in combination with tramadol (i.p) was found to be effective in significantly reducing the mechanical allodynia, hyperalgesia, thermal hyperalgesia and paw edema. Honokiol also succeeded in reducing weight loss and spontaneous pain behavior in mice. Honokiol treatment both i.p and ipl decrease significantly the loss of total protein (3.3 and 3.4 g/dl of total protein) and albumin (2.2 and 2.6 g/dl of total albumin) respectively. It also significantly recovers the normal balance of blood electrolytes and normalizes blood profile. Effect of honokiol on cytokines and mRNA expression levels of TRPV1 and P2Y were also assessed. Honokiol significantly decreases the expression of TNF-α, IL-1β and IL-6 and decreases expression level of TRPV1 and P2Y. Additionally, TRPV1 and P2Y proteins expression levels were also assessed by Western blot in paw skin tissue, sciatic nerve and spinal cord which were remarkably down-regulated by honokiol. Histological analysis of vehicle control and drug-treated paws were also performed through hematoxylin and eosin (H&E) staining which exhibited that honokiol significantly reduced the dermal layers distortion and inflammation associated with the burn. The antioxidant enzymes and nitric oxide (NO) were also determined through ELISA. Honokiol treatment also potentiates the expression of reduced glutathione and glutathione S-transferase, and catalase levels and reduced significantly the nitric oxide (NO) as compared to the burn-induced group. It can be concluded on the base of the results that honokiol has a significant analgesic activity through its action on cytokines and by downregulating TRPV1 and P2Y receptors. It also has a protective role against burn damage by upregulation of antioxidants.

Constant Degradation of the Androgen Receptor by MDM2 Conserves Prostate Cancer Stem Cell Integrity.

Prostate cancer stem cells (CSC) are implicated in tumor initiation, cancer progression, metastasis, and the development of therapeutic-resistant disease. It is well known that the bulk of prostate cancer cells express androgen receptor (AR) and that androgens are required for prostate cancer growth, progression, and emergence of castration-resistant disease. In contrast, the small subpopulation of self-renewing CSCs exhibits an AR-negative (AR-) signature. The mechanisms underlying the absence of AR are unknown. Using CSC-like cell models isolated from clinical biopsy tissues, we identify the E3 ligase MDM2 as a key regulator of prostate CSC integrity. First, unlike what has been reported for the bulk of AR+ tumor cells where MDM2 regulates the temporal expression of AR during transcriptional activity, MDM2 in CSCs promoted the constant ubiquitination and degradation of AR, resulting in sustained loss of total AR protein. Second, MDM2 promoted CSC self-renewal, the expression of stem cell factors, and CSC proliferation. Loss of MDM2 reversed these processes and induced expression of full-length AR (and not AR variants), terminal differentiation into luminal cells, and cell death. Selectively blocking MDM2-mediated activity in combination with androgen/AR-targeted therapy may offer a novel strategy for eliminating AR- CSCs in addition to the bulk of AR+ prostate cancer cells, decreasing metastatic tumor burden and inhibiting the emergence of therapeutic resistance.Significance: These findings provide a novel mechanistic aspect of prostate cancer cell stemness that advances our understanding of the diverse transcriptional activity that bypasses AR in contributing to therapeutic resistance, tumor progression, and metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/6/1124/F1.large.jpg.

The role of LRRK2 inhibition in α-synuclein-induced neurotoxicity and neuroinflammation in vivo.

Event Abstract Back to Event The role of LRRK2 inhibition in α-synuclein-induced neurotoxicity and neuroinflammation in vivo. Diego Cabezudo1*, Anke Van Der Perren1, Géraldine Gelders1, Chris Van Den Haute1, Veerle Baekelandt1 and Evy Lobbestael1 1 KU Leuven, Belgium Parkinson’s disease (PD) is the most common neurodegenerative motor disorder, affecting 10 million people worldwide. A major challenge is the development of disease-modifying therapies, for which a better understanding of PD pathogenesis is indispensable. Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein (α-SYN) are key players in PD and previous studies indicate that targeting LRRK2 can modify α-SYN-induced neurotoxicity and neuroinflammation in preclinical PD models. To further explore the functional interaction between both proteins and evaluate the therapeutic potential of LRRK2, we studied the effects of LRRK2 kinase inhibition and total LRRK2 protein loss in different viral vector-mediated α-SYN-based PD models. We used recombinant adeno-associated viral vectors (rAAV2/7) to overexpress human wild-type or pathogenic A53T α-SYN in the substantia nigra of wild-type and LRRK2 knock out rats. By applying different vector doses, we modelled early and late stage neurodegeneration. In addition, by using an in-diet treatment protocol with MLi-2, we assessed the therapeutic potential of LRRK2 kinase inhibition. Effects on motor behaviour deficits were evaluated by the cylinder test, nigrostriatal neurodegeneration and α-SYN pathology were assessed by immunohistochemistry. Given the role of both LRRK2 and α-SYN in neuroinflammation, we also examined whether loss of LRRK2 (kinase activity) can affect α-SYN-induced neuroinflammatory changes in the different models. Keywords: LRRK2, A-synuclein, Neurodegenaration, Parkinson ' s disease, Neuroinflammantion Conference: 13th National Congress of the Belgian Society for Neuroscience , Brussels, Belgium, 24 May - 24 May, 2019. Presentation Type: Poster presentation Topic: Cellular/Molecular Neuroscience Citation: Cabezudo D, Van Der Perren A, Gelders G, Van Den Haute C, Baekelandt V and Lobbestael E (2019). The role of LRRK2 inhibition in α-synuclein-induced neurotoxicity and neuroinflammation in vivo.. Front. Neurosci. Conference Abstract: 13th National Congress of the Belgian Society for Neuroscience . doi: 10.3389/conf.fnins.2019.96.00020 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Mr. Diego Cabezudo, KU Leuven, Leuven, Belgium, diego.cabezudo@kuleuven.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Diego Cabezudo Anke Van Der Perren Géraldine Gelders Chris Van Den Haute Veerle Baekelandt Evy Lobbestael Google Diego Cabezudo Anke Van Der Perren Géraldine Gelders Chris Van Den Haute Veerle Baekelandt Evy Lobbestael Google Scholar Diego Cabezudo Anke Van Der Perren Géraldine Gelders Chris Van Den Haute Veerle Baekelandt Evy Lobbestael PubMed Diego Cabezudo Anke Van Der Perren Géraldine Gelders Chris Van Den Haute Veerle Baekelandt Evy Lobbestael Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Sex-related differences in response to zinc pyrithione shampoo vs. non-anti-dandruff shampoo.

Sex-related differences in skin properties may be expected to impact dandruff formation and treatment. A meta-analysis approach was undertaken to investigate potential differences between males and females in response to zinc pyrithione (ZnPT) treatment vs. non-anti-dandruff (AD) shampoo. A separate pooled statistical analysis of ceramide and total protein loss endpoints was also undertaken to assess potential sex-related differences in stratum corneum properties that might influence response to ZnPT vs. non-AD shampoo in subjects with dandruff. The meta-analysis approach included data from 17 half-head, double-blind, randomized studies (N=2088) undertaken in Asia to assess the effectiveness of 1% ZnPT shampoo and/or non-AD shampoo in reducing dandruff severity, as assessed by Total Weighted Head Score Adherent Flake (TWHS-AF) methodology. Treatment duration was 4weeks, with TWHS-AF measured at weekly intervals. Data from an additional three studies (N=143) conducted in Asia were included in the pooled analysis of ceramide levels and protein loss from scalp skin of subjects with dandruff. Response to 1% ZnPT vs. non-AD shampoo was greater in males than in females at all time points; after 4weeks, the between-treatment difference in TWHS-AF was -17.5 (95% confidence interval [CI] -19.5, -15.5) in males and -11.1 (-13.2, -8.9) in females. Sex-related differences were observed between males and females in response to both 1% ZnPT and non-AD shampoos. Males had a stronger response than females to treatment with 1% ZnPT shampoo, while dandruff decreased to a greater extent in females than in males when using non-AD shampoo. Statistically significant sex-related differences in ceramide levels and total protein loss were observed (both P<0.01). Ceramide levels were 0.76 times lower (95% CI 0.60, 0.97) in males than in females, while total protein loss was 1.4 times greater (95% CI 1.1, 1.9) in males than in females. Males show a greater response than females to 1% ZnPT shampoo, while females show a greater response than males to non-AD shampoo. These findings may in part be explained by the sex-related differences observed in stratum corneum properties, which may make males more prone to dandruff than females.

A One-Step Extraction and Luminescence Assay for Quantifying Glucose and ATP Levels in Cultured HepG2 Cells.

A fluorescence-based enzymatic microplate intracellular glucose assay was designed and fully validated. The method was tested in a hepatocellular cancer cell line (HepG2). Our novel one-step extraction reagent gave stable cell lysates for glucose, adenosine triphosphate (ATP), and total protein determination from the same sample. Limit of detection for glucose was 0.13 µM (26 pmol/well), which is superior to commercially available glucose assays. Both intra- and interday assay imprecision in HepG2 cultures were less than 12% coefficient of variance (CV). In cell lysates spiked with glucose, recovery at two levels varied between 83.70% and 91.81%, and both linearity and stability were acceptable. HepG2 cells treated with agents affecting glucose uptake/metabolism (phloretin, quercetin, quercetin-3′-sulfate, NaF, 3-bromopyruvate, NaN3, oligomycin A, ochratoxin A, cytochalasin B, and anti-GLUT1 antibody) showed dose-dependent changes in glucose and ATP levels without total protein (cell) loss. Finally, we performed flow cytometric glucose uptake measurement in the treated cells using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose fluorescent glucose analog. Glucose uptake did not always mirror the intracellular glucose levels, which most likely reflects the differences between the two methodologies. However, interpreting data obtained by both methods and taking ATP/protein levels at the same time, one can get information on the mode of action of the compounds.

Outcome of Discharge Within 72 Hours of Robotic Gastrectomy Using Enhanced Recovery After Surgery Programs.

To evaluate the safety and outcome of discharge within 72 hours of a robotic gastrectomy together with enhanced recovery after surgery (ERAS) programs. In total, 108 consecutive patients received elective robotic gastrectomy for gastric cancer from April 2017 to September 2017. All patients attended ERAS programs, which do not routinely use nasogastric tubes but include early feeding, early ambulation, and standard discharge criteria, among other items. The mean age was 58.7 ± 10.4 years old, and the mean body mass index was 22.9 ± 2.8 kg/m2. The mean postoperative length of hospital stay was 5.6 ± 8.0 days, and 13 patients (12.0%) exhibited a complication within 30 days with no mortality. A total of 38 patients (35.2%) were discharged within 72 hours of surgery. Patients were grouped based on being discharged within or after 72 hours. The rate of complications was significantly lower in patients discharged within 72 hours than patients discharged after 72 hours (1/38, 2.6% versus 12/70, 17.1%, P = .028). Although patients discharged within 72 hours showed lower readmission numbers, this difference was not statistically significant (1/38, 2.6% versus 8/70, 11.4%, P = .116). One month after surgery, loss of weight, loss of total protein, loss of albumin, and loss of prealbumin in patients discharged within 72 hours were less than those of patients discharged after 72 hours. Complication and readmission rates are low in patients discharged within 72 hours of robotic gastrectomy when ERAS programs and standard discharge criteria are used.

Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

Lipin 1 regulates glycerolipid homeostasis by acting as a phosphatidic acid phosphohydrolase (PAP) enzyme in the triglyceride-synthesis pathway and by regulating transcription factor activity. Mutations in human lipin 1 are a common cause of recurrent rhabdomyolysis in children. Mice with constitutive whole-body lipin 1 deficiency have been used to examine mechanisms connecting lipin 1 deficiency to myocyte injury. However, that mouse model is confounded by lipodystrophy not phenocopied in people. Herein, 2 muscle-specific mouse models were studied: 1) Lpin1 exon 3 and 4 deletion, resulting in a hypomorphic protein without PAP activity, but which preserved transcriptional coregulatory function; and 2) Lpin1 exon 7 deletion, resulting in total protein loss. In both models, skeletal muscles exhibited a chronic myopathy with ongoing muscle fiber necrosis and regeneration and accumulation of phosphatidic acid and, paradoxically, diacylglycerol. Additionally, lipin 1-deficient mice had abundant, but abnormal, mitochondria likely because of impaired autophagy. Finally, these mice exhibited increased plasma creatine kinase following exhaustive exercise when unfed. These data suggest that mice lacking lipin 1-mediated PAP activity in skeletal muscle may serve as a model for determining the mechanisms by which lipin 1 deficiency leads to myocyte injury and for testing potential therapeutic approaches.-Schweitzer, G. G., Collier, S. L., Chen, Z., McCommis, K. S., Pittman, S. K., Yoshino, J., Matkovich, S. J., Hsu, F.-F., Chrast, R., Eaton, J. M., Harris, T. E., Weihl, C. C., Finck, B. N. Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

Protein Losses and Urea Nitrogen Underestimate Total Nitrogen Losses in Peritoneal Dialysis and Hemodialysis Patients

Muscle wasting is associated with increased mortality and is commonly reported in dialysis patients. Hemodialysis (HD) and peritoneal dialysis (PD) treatments lead to protein losses in effluent dialysate. We wished to determine whether changes in current dialysis practice had increased therapy-associated nitrogen losses. Cross-sectional cohort study. Measurement of total protein, urea and total nitrogen in effluent dialysate from 24-hour collections from PD patients, and during haemodiafiltration (HDF) and haemodialysis (HD) sessions. One hundred eight adult dialysis patients. Peritoneal dialysis, high-flux haemodialysis and haemodiafiltration. Total nitrogen and protein losses. Dialysate protein losses were measured in 68 PD and 40 HD patients. Sessional losses of urea (13.9 [9.2-21.1] vs. 4.8 [2.8-7.8] g); protein (8.6 [7.2-11.1] vs. 6.7 [3.9-11.1] g); and nitrogen (11.5 [8.7-17.7] vs. 4.9 [2.6-9.5] g) were all greater for HD than PD, P<.001. Protein-derived nitrogen was 71.9 (54.4-110.4) g for HD and 30.8 (16.1-59.6) g for PD. Weekly protein losses were lower with HD 25.9 (21.5-33.4) versus 46.6 (27-77.6) g/week, but nitrogen losses were similar. We found no difference between high-flux HD and HDF: urea (13.5 [8.8-20.6] vs. 15.3 [10.5-25.5] g); protein (8.8 [7.3-12.2] vs. 7.6 [5.8-9.0] g); and total nitrogen (11.6 [8.3-17.3] vs. 10.8 [8.9-22.5] g). Urea nitrogen (UN) only accounted for 45.1 (38.3-51.0)% PD and 63.0 (55.3-62.4)% HD of total nitrogen losses. Although sessional losses of protein and UN were greater with HD, weekly losses were similar between modalities. We found no differences between HD and HDF. However, total nitrogen losses were much greater than the combination of protein and UN, suggesting greater nutritional losses with dialysis than previously reported.

Investigation of the Presence and Implications of S‐Palmitoylation on Norepinephrine and Serotonin Transporters

The dopamine, norepinephrine and serotonin transporters (DAT, NET and SERT, respectively) are plasma membrane proteins belonging to the SLC6 family of secondary active transporters and are responsible for clearance of their corresponding substrates dopamine (DA), norepinephrine (NE) and serotonin (5‐HT) from the extraneuronal space during neurotransmission. Each monoamine controls distinct behavioral and physiological functions in the nervous system. DA controls motor functions, mood, reward and cognition. NE regulates arousal, mood, attention, stress‐responsiveness and 5‐HT modulates mood, aggression, motivation, appetite, sleep, cognition and sexual function. Extraneuronal monoamine levels are controlled spatially and temporally by transporter mediated reuptake of released transmitter into presynaptic neurons. Abnormalities in transmitter levels and subsequent neurotransmission are linked to neurological disorders including major depression, schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, Tourette syndrome, and Parkinson disease, through mechanisms that are incompletely understood. The transporters are sites of action for therapeutic drugs such as methylphenidate, bupropion, selective serotonin and serotonin‐norepinephrine reuptake inhibitors, used to treat these disorders, and are also targets for addictive drugs including cocaine, amphetamine (AMPH), and methylenedioxy methamphetamine (MDMA) that elevate transmitter levels. Several lines of evidence indicate that modulation of transporter phosphorylation, ubiquitylation, and palmitoylation regulate their cell surface expression, activity, trafficking and degradation. We previously reported that rat, mouse and human DATs are modified by S‐palmitoylation, a post‐translational modification in which C16 saturated palmitic acid is added via a thioester linkage to cysteine. These results indicated that DAT palmitoylation has the capacity to impact dopaminergic signaling acutely by regulating DA transport kinetics independent of surface losses and chronically by opposing DAT degradation. We have extended our examination of transporter palmitoylation using acyl‐biotinyl exchange to include SERT and NET finding both transporters are palmitoylated proteins, suggesting potential palmitoylation regulatory mechanisms for NET and SERT analogous to DAT. Furthermore, treatment with the palmitoyl acyltransferase inhibitor 2‐bromopalmitate (2BP), in a heterologous cell system, resulted in decreased overall palmitoylation of these proteins. Inhibition of palmitoylation by 2BP also revealed acute and long‐term differences between SERT and NET stability where loss of palmitoylation is accompanied by a loss of total transporter protein. Collectively, these results indicate that S‐palmitoylation may serve as a potential regulator for NET and SERT properties, sharing some functional consequences as demonstrated with DAT.Support or Funding InformationSupported by the National Institute on Drug Abuse Grant DA 031991 JDF, P20 RR017699 (to U.N.D.) from the COBRE program of the National Center for Research Resources, and P20 RR016741 (to U.N.D.) from the INBRE program of the National Center for Research Resources.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Precise Quantitative Assessment of the Clinical Performances of Two High-Flux Polysulfone Hemodialyzers in Hemodialysis: Validation of a Blood-Based Simple Kinetic Model Versus Direct Dialysis Quantification.

Highly permeable dialysis membranes with better design filters have contributed to improved solute removal and dialysis efficacy. However, solute membrane permeability needs to be well controlled to avoid increased loss of albumin that is considered to be detrimental for dialysis patients. A novel high-flux dialyzer type (FX CorDiax; Fresenius Medical Care) incorporating an advanced polysulfone membrane modified with nano-controlled spinning technology to enhance the elimination of a broader spectrum of uremic toxins has been released. The aim of this study was to compare in the clinical setting two dialyzer types having the same surface area, the current (FX dialyzer) and the new dialyzer generation (FX CorDiax), with respect to solute removal capacity over a broad spectrum of markers, including assessment of albumin loss based on a direct dialysis quantification method. We performed a crossover study following an A1-B-A2 design involving 10 patients. Phase A1 was 1 week of thrice-weekly bicarbonate hemodialysis with the FX dialyzer, 4 h per treatment; phase B was performed with a similar treatment regimen but with a new FX CorDiax dialyzer and finally the phase A2 was repeated with FX dialyzer as the former phase. Solute removal markers of interest were assessed from blood samples taken before and after treatment and from total spent dialysate collection (direct dialysis quantification) permitting a mass transfer calculation (mg/session into total spent dialysate/ultrafiltrate). On the blood side, there were no significant differences in the solute percent reduction between FX CorDiax 80 and FX 80. On the dialysate side, no difference was observed regarding eliminated mass of different solutes including β2 -microglobulin (143.1 ± 33.6 vs. 138.3 ± 41.9 mg, P = 0.8), while the solute mass removal of total protein (1.65 ± 0.51 vs. 2.14 ± 0.75 g, P = 0.04), and albumin (0.41 ± 0.21 vs. 1.22 ± 0.51 g, P < 0.001) were significantly less for FX CorDiax 80 compared to the FX 80 dialyzer. The results of this cross-over study indicate that the new FX CorDiax dialyzer has highly effective removal of middle molecules, without any concomitant increase in total protein and albumin loss. The clinical relevance and potential benefit of this finding needs to be determined.

Severe Sarcopenia and Increased Fat Stores in Pediatric Patients With Liver, Kidney, or Intestine Failure.

Malnutrition and wasting predict clinical outcomes in children with severe chronic illness. Objectively calculated malnutrition in children with end-stage organ failure has not been well studied. This analysis compares children with kidney, liver or intestine failure to healthy controls to quantitate the disparity in muscle and fat stores. Children younger than 19 years with end-stage liver, kidney, or intestine failure and with pretransplant computed tomography (CT) imaging were selected from the transplant database. Age- and sex-matched healthy controls were selected from the trauma database. Measures of nutrition status included a scaled scoring of core muscle mass, and visceral and subcutaneous fat stores. Analysis was conducted using the pooled and individually matched subject-control differences. There were 81 subjects included in the final analysis (liver [n = 35], kidney [n = 20], and intestine [n = 26]). Children with end-stage liver disease had a 23% reduction in muscle mass, a 69% increase in visceral fat, and a 29% increase in subcutaneous fat. End-stage renal disease patients had a 19% reduction in muscle mass and a 258% increase in subcutaneous fat. Intestine failure patients had a 24% reduction in muscle mass, a 30% increase in visceral fat, and a 46% increase in subcutaneous fat. These results demonstrate significant sarcopenia and increased fat stores in end-stage organ failure patients, which supports the idea of an active physiologic mechanism to store fat while losing muscle mass. Sarcopenia may be related to total protein loss from a catabolic state, or from decreased synthesis (liver), wasting (kidney), or malabsorption (intestine).

A COMPARATIVE STUDY OF THE ANTI-OXIDATIVE AND ANTI-DIABETIC POTENTIAL OF IN VITRO AND IN VIVO ROOT AND LEAF EXTRACTS OF WITHANIA SOMNIFERA ON STREPTOZOTOCIN INDUCED DIABETIC RATS

&lt;p&gt;&lt;strong&gt;Objective: &lt;/strong&gt;The present investigation explores the possibilities of using the &lt;em&gt;in vitro&lt;/em&gt; and &lt;em&gt;in vivo &lt;/em&gt;root and leaf extracts of &lt;em&gt;Withania somnifera &lt;/em&gt;for anti-diabetic and anti-hyperlipidaemic effects on streptozotocin-induced diabetic rats.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods: &lt;/strong&gt;&lt;em&gt;In vitro &lt;/em&gt;shoot cultures of &lt;em&gt;Withania somnifera&lt;/em&gt; were raised by the axillary proliferation in nodal explants from a garden grown plant using Murashige and Skoog medium then &lt;em&gt;in&lt;/em&gt;&lt;em&gt; vitro&lt;/em&gt; raised roots and shoots were used for the anti-hyperglycemic and anti-hyperlipidaemic experiment. After 72 h of STZ administration, the fasting blood glucose levels were measured and the rats showing FBG level&amp;gt;220 mg/dl were considered to be diabetic and were used for the hyperglycemic study. &lt;em&gt;In vitro&lt;/em&gt; and &lt;em&gt;in vivo&lt;/em&gt; methanolic root and leaf extracts were orally administered daily to diabetic rats for eight weeks. After the treatment period, blood glucose and serum enzymes like aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total cholesterol, triglycerides, HDL-c high density lipoprotein-bound cholesterol, LDL-c low density lipoprotein-bound cholesterol, LDH, serum protein level, total phenolics and anti-oxidative analysis (DPPH and FRAP) were determined.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;The levels of blood glucose, AST, ALT, ALP, LDH, HDL-c significantly increased by the use of &lt;em&gt;in vitro&lt;/em&gt; methanolic root extracts compared to normal control rats. However, remarkable loss of total protein, albumin, albumin: globulin (A: G) ratio was reported in streptozotocin-induced diabetic rats by using &lt;em&gt;in vitro&lt;/em&gt; root extracts. Methanolic &lt;em&gt;in vitro&lt;/em&gt; root extract at the dose levels of 300 mg/kg body weight produced a significant decrease in fasting blood glucose (FBG) level by 102.65 with respect to initial fasting blood glucose level after 30 d of the treatment. &lt;em&gt;In vitro&lt;/em&gt; root extract demonstrated highest DPPH and FRAP free radical scavenging activity, i.e. 86.55±1.77 and 48.87±2.55 than other extracts.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusion: &lt;/strong&gt;It may be concluded that methanolic &lt;em&gt;in vitro&lt;/em&gt; root extract &lt;em&gt;W. somnifera &lt;/em&gt;at the dose (300 mg/kg) has more potent anti-hyperglycaemic activity than the other &lt;em&gt;in vitro&lt;/em&gt; and &lt;em&gt;in vivo &lt;/em&gt;extracts of leaf and root on streptozotocin induced diabetic rats and was also found to be similar in effect to that of the standard drug ‘Glibenclamide’.&lt;/p&gt;

Characterization of proteases and nucleases associated with ethylene-induced programmed cell death in immature cucumber fruit

Previous studies demonstrated that continuous 10μLL−1 ethylene exposure induces acute watersoaking of immature beit-alpha cucumber fruit (Cucumis sativus L. cv. Manar). This phenomenon includes increases in nuclease activities and loss of total protein, supporting the idea that watersoaking is a form of programmed cell death (PCD). The present study was designed to further characterize ethylene-induced, PCD-associated nucleases and proteases in immature cucumber fruit. DNases were found to possess bifunctional (DNA/RNA) activities. In addition, four RNA-specific nucleases (17.5, 22.3, 24.4, and 26.1kDa) were detected by in-gel assays, all of which were ethylene inducible. All nucleases were inhibited by β-mercaptoethanol, indicating a requirement for disulfide bond(s) for activity. Both the bifunctional nucleases and the 17.5kDa RNase exhibited Ca2+ dependency. Bifunctional nuclease activities were detected above pH 7, but strongly inhibited at weakly acidic pH (6). By contrast, RNA-specific nuclease activity was optimal at acidic pH and strongly inhibited above pH 8.Ethylene induced significant protein degradation after only 2d of exposure, with total protein levels declining by about 60% after 6d. The decline in total protein was accompanied by significant increases in protease activity. Increased activity was also observed after 2d, reaching levels 9.3-fold higher than initial values after 6d. Total protease activity was greatest at pH 5 and declined sharply through pH 8. In-gel assays revealed three ethylene-responsive proteases with apparent molecular masses of 56, 106, and 180kDa. Levels of the 106 and 180kDa proteases increased after 2d of ethylene exposure, reaching maxima at 4–5d coincident with symptoms of watersoaking. A 56kDa protease was observed only in ethylene-treated fruit and was first detected after 4d of ethylene exposure. Together with up-regulated nuclease activities and reactive oxygen species (ROS)-generating capacity, the early appearance of proteases in response to ethylene provides additional evidence that watersoaking in immature cucumber fruit reflects the execution phase of a PCD pathway.

Advanced hair damage model from ultra-violet radiation in the presence of copper.

Damage to hair from UV exposure has been well reported in the literature and is known to be a highly complex process involving initiation via absorption of UV light followed by formation and propagation of reactive oxygen species (ROS). The objective of this work was to understand these mechanisms, explain the role of copper in accelerating the formation of ROS and identify strategies to reduce the hair damage caused by these reactive species. The location of copper in hair was measured by Transmission electron microscopy-(TEM) X-ray energy dispersive spectroscopy (XEDS) and levels measured by ICP-OES. Protein changes were measured as total protein loss via the Lowry assay, and MALDI ToF was used to identify the biomarker protein fragments. TBARS assay was used to measure lipid peroxide formation. Sensory methods and dry combing friction were used to measure hair damage due to copper and UV exposure and to demonstrate the efficacy of N,N' ethylenediamine disuccinic acid (EDDS) and histidine chelants to reduce this damage. In this work, a biomarker protein fragment formed during UV exposure is identified using mass spectrometry. This fragment originates from the calcium-binding protein S100A3. Also shown is the accelerated formation of this peptide fragment in hair containing low levels of copper absorbed from hair during washing with tap water containing copper ions. Transmission electron microscopy (TEM) X-ray energy dispersive spectroscopy (XEDS) studies indicate copper is located in the sulphur-poor endo-cuticle region, a region where the S100A3 protein is concentrated. A mechanism for formation of this peptide fragment is proposed in addition to the possible role of lipids in UV oxidation. A shampoo and conditioner containing chelants (EDDS in shampoo and histidine in conditioner) is shown to reduce copper uptake from tap water and reduce protein loss and formation of S100A3 protein fragment. In addition, the long-term consequences of UV oxidation and additional damage induced by copper are illustrated in a four-month wear study where hair was treated with a consumer relevant protocol of hair colouring treatments, UV exposure and regular shampoo and conditioning. The role of copper in accelerating UV damage to hair has been demonstrated as well as the ability of chelants such as EDDS and histidine in shampoo and conditioner products to reduce this damage.

Antilithiatic effects of crocin on ethylene glycol-induced lithiasis in rats.

In this study, the antilithiatic potential of crocin, a pharmacologically active constituent of Crocus sativus L. (saffron), was evaluated against ethylene glycol (EG)-induced nephrolithiasis in rats. Negative control rats were provided with EG (1 %) in drinking water for 30 days. crocin (10, 20 and 40 mg/kg, i.p.) was administered simultaneously once daily for 30 days (prophylactic regimen) or 14 days after stone induction (therapeutic study). For biochemical analysis, 24-h urine was collected from all experimental animals at the beginning (day 0) and end of the experiment (day 30). The urine output was evaluated during the first 24 h (day 1). Ethylene glycol feeding resulted in decreased hyperoxaluria (P < 0.01) and total protein loss (P < 0.001), along with decreased excretion of citrate and magnesium (P < 0.01) compared with the intact animals. Treatment with prophylactic regimen of crocin (20 and 40 mg/kg) significantly reduced the elevated oxalate, and increased the citrate and magnesium levels of urine. The attenuation of protein loss was only seen with a high dose of crocin in a prophylactic study. Urine volume was not significantly altered after EG or crocin administration. The increased number of calcium deposits in the kidney tissue of lithiatic rats was decreased after prophylactic treatment with 20 and 40 mg/kg of crocin. The urinary ionic parameters and crystal count were not significantly altered after the therapeutic study. A marked increase in malondialdehyde (MDA, a lipid peroxidation product) level was observed in the EG-given group. Treatment with crocin (20 and 40 mg/kg) reduced the elevated levels of MDA. Results indicate that crocin can be effective in preventing urine calculi formation and recurrence of the disease. The mechanism underlying this effect is mediated possibly through balancing promoter and inhibitor factors and an antioxidant effect.

Direct uptake of copper from plasma ceruloplasmin by cells that do and do not express CTR1 (996.2)

The main Cu‐binding protein of blood plasma, ceruloplasmin (Cp), has mainly been studied for its role in mediating cellular iron efflux but also has other functions, and as shown by i.v. infusion of 67Cu‐Cp, its Cu enters most major organs. To determine whether it delivers Cu directly to cells and the mechanisms involved, we incubated purified radio‐copper‐labeled and unlabeled Cp with cells and measured rates of Cu uptake and accumulation. Uptake was demonstrated for human mammary epithelial cell monolayers (PMC42 cells) with tight junctions, and with mouse embryonic fibroblasts expressing/not expressing Ctr1. Cp‐Cu was internalized. It could not be washed from the cell surface even with acidic buffers, and was found in the cytosol. Inhibitors of endocytosis did not impair uptake. Cu deficient cells exposed to Cp increased their cellular Cu contents; and holo‐Cp was converted to apo‐Cp during cell treatment, with no loss of total Cp protein. Uptake was almost the same in cells not expressing Ctr1. Excess ionic Cu(II) and Cu(I) virtually eliminated Cp‐Cu uptake. The presence of Cu(II) and Fe(III) reductase activities on the cell surface was verified. We conclude that Cp‐Cu delivers Cu to cells by a process involving cell surface reduction of Cp‐Cu(II), release of apo‐Cp, and uptake of the resulting Cu(I) by Ctr1 and an additional as yet unidentified Cu(I) transporter.Grant Funding Source: Supported by PHS Grant R15 GM100464

YES, a Src Family Kinase, Is a Proximal Glucose-specific Activator of Cell Division Cycle Control Protein 42 (Cdc42) in Pancreatic Islet β Cells

Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.