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The gut microbiome plays an essential role in host immune responses, including allergic reactions. However, commensal gut microbiota is extremely sensitive to antibiotics and excessive usage can cause microbial dysbiosis. Herein, we investigated how changes in the gut microbiome induced by ampicillin affected the production of IgG1 and IgG2a antibodies in mice subsequently exposed to Anisakis pegreffii antigens. Ampicillin treatment caused a notable change in the gut microbiome as shown by changes in both alpha and beta diversity indexes. In a 1-dimensional immunoblot using Anisakis-specific anti-mouse IgG1, a 56-kDa band corresponding to an unnamed Anisakis protein was detected using mass spectrometry analysis only in ampicillin-treated mice. In the Anisakis-specific anti-mouse IgG2a-probed immunoblot, a 70-kDa band corresponding to heat shock protein 70 (HSP70) was only detected in ampicillin-treated and Anisakis-immunized mice. A 2-dimensional immunoblot against Anisakis extract with immunized mouse sera demonstrated altered spot patterns in both groups. Our results showed that ampicillin treatment altered the gut microbiome composition in mice, changing the immunization response to antigens from A. pegreffii. This research could serve as a basis for developing vaccines or allergy immunotherapies against parasitic infections. [ABSTRACT FROM AUTHOR]

Copyright of Revista de la Sociedad Química del Perú is the property of Sociedad Quimica del Peru and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Parasite (1252607X) is the property of EDP Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The knowledge of the mechanisms affecting the process of berry fruit ripening is important, not only to correctly determine the appropriate date of harvest, at which the fruit is most palatable and characterized by adequate shelf-life stability, but also, to develop new strategies of regulating the ripening process before harvesting. It is recognized that berry fruit quality and its post-harvest shelf-life depend on the cellular redox homeostasis. We, therefore, decided to conduct a comprehensive analysis of the level of oxidative stress status in the raspberry fruit proper at different stages of fruit ripening, from green to overripe fruit. We assessed both the level of typical oxidative stress markers, i.e. ROS production, expression of antioxidant enzymes, degree of cell damage, as well as the expression of selected proteins involved in the energy, glutathione, and polyphenols metabolism. We found two stage-related peaks of ROS production. The first - in green fruit, which corresponded to the maximum expression of antioxidant enzymes (MnSOD, CAT), PARP-1, as well as proteins related to glutathione biosynthesis (GS, γ-GC), autophagy (ATG-8) and ubiquitination (UBQ-11). The second one, in the overripe fruit, was responsible for the intensification of oxidative modifications of cell components, i.e. lipids, proteins, and DNA, as well as the loss of low molecular-weight antioxidants. The fruit ripening process, in turn, was manifested by a strong increase in the expression of proteins involved in the biosynthesis of polyphenols (PAL, CHS), cellular respiration (ACO-1, Cyt-C-ox, ATPase), ethylene biosynthesis and signaling (SAMs, EIN-2) and increasing amounts of ABA. [Display omitted] • Raspberry ripening is regulated by ROS, ABA and ethylene. • Autophagy and ubiquitination occur most intensively in green fruit. • Fruit ripening is associated with an increase in PAL and CHS expression. • The greatest degree of damage of cell components is in overripe fruit. [ABSTRACT FROM AUTHOR]

Copyright of Revue Francaise d'Allergologie is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

AICAR, an adenosine analog, has been shown to exhibit vascular protective effects through activation of AMP-activated protein kinase (AMPK). However, it remains unclear as to whether adenosine kinase-mediated ZMP formation or adenosine receptor activation contributes to AICAR-mediated AMPK activation and/or vasorelaxant response in vascular smooth muscle. In the present study using endothelium-denuded rat aortic ring preparations, isometric tension measurements revealed that exposure to 1 mM AICAR for 30 min resulted in inhibition of phenylephrine (1 μM)-induced smooth muscle contractility by ∼35%. Importantly, this vasorelaxant response by AICAR was prevented after pretreatment of aortic rings with an AMPK inhibitor (compound C, 40 µM) and adenosine kinase inhibitor (5-iodotubercidin, 1 µM), but not with an adenosine receptor blocker (8-sulfophenyltheophylline, 100 µM). Immunoblot analysis of respective aortic tissues showed that AMPK activation seen during vasorelaxant response by AICAR was abolished by compound C and 5-iodotubercidin, but not by 8-sulfophenyltheophylline, suggesting ZMP involvement in AMPK activation. Furthermore, LC–MS/MS MRM analysis revealed that exposure of aortic smooth muscle cells to 1 mM AICAR for 30 min enhanced ZMP level to 2014.9 ± 179.4 picomoles/mg protein (vs. control value of 8.5 ± 0.6; p<0.01), which was accompanied by a significant decrease in ATP/ADP ratio (1.08 ± 0.02 vs. 2.08 ± 0.06; p<0.01). Together, the present findings demonstrate that AICAR-mediated ZMP elevation and the resultant AMPK activation in vascular smooth muscle contribute to vasorelaxation. [ABSTRACT FROM AUTHOR]

Exercise training (ET) is well established to induce vascular adaptations on the metabolically active muscles. These adaptations include increased function of vascular potassium channels and enhanced endothelium-dependent relaxations. However, the available data on the effect of ET on vasculatures that normally constrict during exercise, such as mesenteric arteries (MA), are scarce and not conclusive. Therefore, this study hypothesized that 10 weeks of moderate-intensity ET would result in adaptations towards more vasoconstriction or/and less vasodilatation of MA. Young Fischer 344 rats were randomly assigned to a sedentary group (SED; n=24) or exercise training group (EXE; n=28). The EXE rats underwent a progressive treadmill ET program for 10 weeks. Isometric tensions of small (SED; 252.9±29.5 μm, EXE; 248.6±34.4 μm) and large (SED; 397.7±85.3 μm, EXE; 414.0±86.95 μm) MA were recorded in response to cumulative phenylephrine concentrations (PE; 0-30 μM) in the presence and absence of the BKCa channel blocker, Iberiotoxin (100 nM). In another set of experiments, tensions in response to cumulative concentration-response curves of acetylcholine (ACh) or sodium nitroprusside (SNP) were obtained, and pEC50s were compared. Immunoblotting was performed to measure protein expression levels of the BKCa channel subunits and eNOS. ET did not alter the basal tension of small and large MA but significantly increased their responses to PE, and reduced the effect of BKCa channels in opposing the contractile responses to PE without changes in the protein expression level of BKCa subunits. ET also elicited a sizedependent functional adaptations that involved reduced endothelium-independent and endothelium-dependent relaxations. In large MA the sensitivity to SNP was decreased more than in small MA suggesting impaired nitric oxide (NO)-dependent mechanisms within the vascular smooth muscle cells of ET group. Whereas the shift in pEC50 of ACh-induced relaxation of small MA would suggest more effect on the production of NO within the endothelium, which is not changed in large MA of ET group. However, the eNOS protein expression level was not significantly changed between the ET and SED groups. In conclusion, our results indicate an increase in contraction and reduced relaxation of MA after 10 weeks of ET, an adaptation that may help shunt blood flow to metabolically active tissues during acute exercise. [ABSTRACT FROM AUTHOR]

[Display omitted] • Protein dot-blot method is suitable to compare expression levels of MMP-8 and MMP-9 in gums. • The relative levels of MMP-8 and MMP-9 in mild and moderate gingival overgrowth were measured. • Biological variability among individuals did not affect assay development. • The protein dot blot was fast, highly sensitive, repeatable and economic. Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in extracellular matrix remodeling of all body tissues, including oral tissues such as gingival tissue. Expression levels of MMPs are widely studied as important biomarkers for explaining the biochemical mechanisms and evolution of many oral diseases. Demonstrate the sensitivity, reproducibility, repeatability, and robustness of the dot blot assay for the relative quantification of MMP-8 and MMP-9 expression levels in patients with GO associated with orthodontic treatment. A validated dot blot assay was used to compare the relative expression levels of MMP-8 and MMP-9 in gingival samples. Methodological variability, reproducibility, sensitivity and robustness were determined with the use of control samples from healthy donors (G1). Next, expression levels were measured in gingival tissue from patients with mild and moderate gingival overgrowth associated with orthodontic treatment (G3 and G4) and patients without gingival overgrowth but with a history of using orthodontic appliances (G2). Dot blot assay demonstrated that MMP-8 and MMP-9 expression levels were higher in patients with gingival overgrowth and distinguished those with moderate clinical grade (G4) from those with mild overgrowth (G3). In addition, patients with a history of orthodontic treatment showed similar expression levels to the control group two years after removing orthodontic appliances. With the assay used, we were able to detect differences in MMP-8 and MMP-9 expression in patients with different levels of severity of gingival overgrowth. Dot blot could be used to measure MMPs during the onset and progression of gingival overgrowth. [ABSTRACT FROM AUTHOR]

The major aim of this study was to demonstrate the influence of UV-C radiation exposure of the edible sprouts on their redox status and the mechanism of production of selected phytochemicals in them. The study has shown that UV-C irradiation of broccoli sprouts and red cabbage for 5 min, and alfalfa for 15 min activates mechanisms involved in the antioxidant protection of the cell, resulting in an increase in the level of low-molecular antioxidants, i.e. polyphenols and glutathione. It was proven that sprouts irradiated with UV-C were characterised by higher MAPK expression, as well as higher level of ABA (ROS-dependent) which contributed to the induction of transcription factors (MYB) and consequently, to increased expression of proteins involved in antioxidants biosynthesis (PAL, CHS, γ-GCS, GS) and antioxidant enzymes (MnSOD, CAT). Moreover, UV-C increased mitochondrial activity, which was observed by higher concentration of ATP, as well as increased expression of SnRK1 protein. [Display omitted] • UV-C treatment as a promising way to improve antioxidant activity of sprouts. • UV-C increases PAL, CHS, γ-GCs and GS protein expression. • UV-C may act through ABA and the MAP kinase cascade. [ABSTRACT FROM AUTHOR]

Background: The detection of anti-mitochondrial antibodies (AMA) is considered a hallmark in diagnosing primary biliary cholangitis (PBC). The most important AMA-subtype is AMA-M2 directed against the E2-subunit of pyruvate dehydrogenase. It is common clinical interpretation that lack of M2 due to immunoblotting (IB) indicates absence of specific auto-reactivity. We aimed to define whether M2-IB confirmation is linked to clinical outcomes.Methods: Our cohort comprised 302 patients who tested positive for AMA with indirect immunofluorescence between 2006 and 2015. One hundred and eighty-four subjects (60.9%; male n = 29 [15.8%]) were tested M2-positive by confirmatory IB, whereas 118 subjects were IB-M2-negative (39.1%; male n = 25 [21.2%]). The natural history of 236 patients (78.1%) was evaluated by clinical follow-up (FU) assessing causes of death, leading health condition and response to PBC standard therapy if applicable.Results: Mean time to FU was 6.8 years. Twenty-eight M2-positive patients (15.2% of 184) and 28 M2-negative patients (23.7% of 118) had died (p = 0.0958). Thirty-four M2-positives (18.5%) and 32 M2-negatives (27.1%) were not available for FU. According to the clinical course by the time of FU, subjects were allocated to one of four groups: a) 34 patients had known PBC with n = 16 having an adequate and 18 an inadequate treatment response, b) 1 de novo PBC was detected, c) 13 were AMA-positive without biochemical evidence of PBC and d) 9 subjects were tested AMA-negative at FU. These numbers were comparable to M2-positive subjects with similar long-term clinical outcome.Conclusion: Our data suggest that the clinical value of confirmatory M2 immunoblotting in the diagnostic routine of PBC is overestimated as the clinical course appears not to be related to the test result. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a neurological condition with selective progressive degeneration of dopaminergic neurons. Routine therapies are symptomatic and palliative. Although, hesperidin (Hsd) is known for its neuroprotective effects, its exact cellular mechanism is still a mystery. Considering the important role of calcium (Ca2+) in cellular mechanisms of neurodegenerative diseases, the present study aimed to investigate the possible effects of Hsd on Ca2+ channels in cellular model of PD and the possible association between the selective vulnerability of neurons in cellular models of PD and expression of the physiological phenotype that changes Ca2+ homeostasis. SH-SY5Y cell line was used in this study; cell damage was induced by 150 µM 6-OHDA and the cells' viability was examined using MTT assay. Intracellular calcium, reactive oxygen species (ROS) and mitochondrial membrane potential were determined by the fluorescence spectrophotometry method. The expressions of calcium channel receptors were determined by gel electrophoresis and immunoblotting. Loss of cell viability and mitochondrial membrane potential were confirmed in 6-OHDA treated cells. In addition, intracellular ROS and calcium levels, calcium channel receptors significantly increased in 6-OHDA-treated cells. Incubation of SH-SY5Y cells with hesperidin showed a protective effect, reduced the biochemical markers of cell damage/death, and balanced calcium hemostasis. Based on our findings, it seems that hesperidin could suppress the progression of the cellular model of PD via acting on intracellular calcium homeostasis. Further studies are needed to confirm the potential benefits of preventive and therapeutic effects of stabilizing cellular calcium homeostasis in neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Autophagy plays an essential role in body homeostasis achievement. One of the main proteins involved in this process is the LC3I, which, after lipidation, leads to the formation of LC3II that participates in the formation and maturation of autophagosome. This descriptive study verified the responses of LC3II to LC3I proteins, as well as the time-course of this ratio in mice livers after different types of acute physical exercise protocols. Eight-week-old male C57BL/6 mice were maintained three per cage with controlled temperature (22±2 °C) on a 12:12-h light-dark normal cycle with food (Purina chow) and water ad libitum. Mice were randomly divided into four groups: control (CT, sedentary mice), resistance (RE, submitted to a single bout of resistance exercise), endurance (EE, submitted to a single bout of endurance exercise), and concurrent (CE, submitted to a single bout of endurance combined with resistance exercise). The mice livers were extracted and used for the immunoblotting technique. The hepatic LC3B II/I ratio for the RE and EE groups were not altered during the different time-points. For the CE group, there was a decrease in this ratio 12h after exercise compared to time 0 and 18h. Also, the hepatic LC3B II/I ratios were not different among the acute physical exercise protocols along the time-course. The hepatic LC3B II/I ratio was not influenced by the endurance and resistance protocols but decreased in response to the concurrent protocol at 12h after the stimulus. [ABSTRACT FROM AUTHOR]

Abstract Gamma irradiation has been reported to modulate the biochemical and molecular parameters associated with the tolerance of plant species under biotic/ abiotic stress. Wheat is highly sensitive to heat stress (HS), as evident from the decrease in the quantity and quality of the total grains. Here, we studied the effect of pre-treatment of wheat dry seeds with different doses of gamma irradiation (0.20, 0.25 and 0.30 kGy) on tolerance level and quality of developing wheat endospermic tissue under HS (38 °C, 1 h; continuously for three days). Expression analysis of genes associated with defence and starch metabolism in developing grains showed maximum transcripts of HSP17 (in response to 0.25 kGy + HS) and AGPase (under 0.30 kGy), as compared to control. Gamma irradiation was observed to balance the accumulation of H 2 O 2 by enhancing the activities of SOD and GPx in both the cvs. under HS. Gamma irradiation was observed to stabilize the synthesis of starch and amylose by regulating the activities of AGPase, SSS and α-amylase under HS. The appearance of isoforms of gliadins (α, β, γ, ω) were observed more in gamma irradiated seeds (0.20 kGy), as compared to control. Gamma irradiation (0.25 kGy in HD3118 & 0.20 kGy in HD3086) was observed to have positive effect on the width, length and test seed weight of the grains under HS. The information generated in present investigation provides easy, cheap and user-friendly technology to mitigate the effect of terminal HS on the grain-development process of wheat along with development of robust seeds with high nutrient density. Highlights • γ-irradiation enhances the expression of HSP17 in developing wheat grains under HS. • γ-irradiation regulate the ROS in grains by triggering antioxidant enzymes under HS. • γ-irradiation stabilize the synthesis of starch and amylose in wheat grain under HS. • Isoforms of gliadins (α, β, γ, ω) were observed more in γ- irradiated wheat seeds. • γ-irradiation (0.25 kGy-HD3118 & 0.20 kGy-HD3086) enhances quality of wheat grains. [ABSTRACT FROM AUTHOR]

Abstract Context Excessive adipose glucocorticoid action is associated with insulin resistance, but the mechanisms linking adipose glucocorticoid action to insulin resistance are still debated. We hypothesized that insulin resistance from excess glucocorticoid action may be attributed in part to increased ectopic lipid deposition in liver. Methods We tested this hypothesis in the adipose specific 11β-hydroxysteroid dehydrogenase-1 (HSD11B1) transgenic mouse, an established model of adipose glucocorticoid excess. Tissue specific insulin action was assessed by hyperinsulinemic-euglycemic clamps, hepatic lipid content was measured, hepatic insulin signaling was assessed by immunoblotting. The role of hepatic lipid content was further probed by administration of the functionally liver-targeted mitochondrial uncoupler, Controlled Release Mitochondrial Protonophore (CRMP). Findings High fat diet fed HSD11B1 transgenic mice developed more severe hepatic insulin resistance than littermate controls (endogenous suppression of hepatic glucose production was reduced by 3.8-fold, P < 0.05); this was reflected by decreased insulin-stimulated hepatic insulin receptor kinase tyrosine phosphorylation and AKT serine phosphorylation. Hepatic insulin resistance was associated with a 53% increase (P < 0.05) in hepatic triglyceride content, a 73% increase in diacylglycerol content (P < 0.01), and a 66% increase in PKCε translocation (P < 0.05). Hepatic insulin resistance was prevented with administration of CRMP by reversal of hepatic steatosis and prevention of hepatic diacylglycerol accumulation and PKCε activation. Conclusions These findings are consistent with excess adipose glucocorticoid activity being a predisposing factor for the development of lipid (diacylglycerol-PKCε)-induced hepatic insulin resistance. Highlights • Adipose glucocorticoid excess drives hepatic insulin resistance (IR) before obesity. • This IR is associated with hepatic steatosis; reversed by ectopic lipid depletion. • Hepatic IR is consistent with diacylglycerol-PKCε mediated IR. • More energy intake and reduced chylomicron clearance may explain hepatosteatosis. [ABSTRACT FROM AUTHOR]

Food allergies are evolving as a major public health problem in modernized regions of the globe that got importance in recent years due to life threatening outcomes. In this regard, the problem of hidden food allergens is a very serious one for persons with allergy. Beside this the advancement in food processing or food production made it difficult for the producer and the consumers to resolve whether a food comprises of aggressive allergens. This is why, food industries are facing problems in developing reliable, rational methods that have the ability to identify and quantify hidden allergens in food. A number of techniques have been developed to address the problem of hidden food allergens, but all the techniques have some shortcomings. This review is therefore focused on the food allergens and various methods used for the detection of these allergens before and after thermal and chemical processing of food. [ABSTRACT FROM AUTHOR]

Highlights • A distinctive miRNA signature between visceral and subcutaneous adipose tissues. • The novel role and function of miR-122 in adipose tissue. • The ratio of miR-122 between visceral and subcutaneous adipose tissues correlates with bariatric surgery outcome. Abstract The physiological roles and clinical impacts of the differences between visceral fat (VF) and subcutaneous fat (SF) are unclear. The present study aimed to compare the miRNA signatures between visceral fat (VF) and subcutaneous fat (SF) and study their influences on outcomes of bariatric surgery. To study the microRNA signatures of the VF and SF in obesity, we performed paired microRNA arrays of the adipose tissues from 20 bariatric surgery patients. The microRNA analysis identified miR-122 as the most significant signature between VF and SF. The tissue distribution, functions, and influences on adipogensis of miR-122 were analysed by Northern blotting, microRNA mimics and inhibitors, and whole-genome microarray analysis. The outcomes of body weight changes after bariatric surgery were analysed and correlated with the miR-122 abundances. Northern blotting confirmed that miR-122 was highly expressed in VF and SF. Bioinformatics analysis of the microarray revealed that proliferator activator receptor-γ (PPAR-γ) signalling was critically affected by miR-122. The modulation of PPAR-γ by miR-122 was confirmed in murine adipocytes and human adipose tissues. Furthermore, the differentiation of preadipocytes was significantly influenced by miR-122. In obese patients receiving bariatric surgery, the ratio of VF and SF miR-122 abundance correlated with 6-month and 1-year % excess body weight loss. Our findings indicate that miR-122 is highly expressed in adipose tissue. The abundance of miR-122 affects PPAR-γ signalling and adipocytes differentiation in vitro and human adipose tissues. Higher miR-122 in VF may be associated with greater body weight loss after bariatric surgery. [ABSTRACT FROM AUTHOR]

Background: Brown adipose tissue (BAT) contains both α- and β-adrenergic receptors. In the literature, the activity of α-adrenoreceptors is less documented, and their functions still remain puzzling. The present investigation has been undertaken to understand α-adrenoreceptors’ activity and their relation between uncoupling protein 1 (UCP1) mRNA expression and cyclic AMP (cAMP) generation in BAT. Methods: BAT precursor cells from young mice were grown in culture. Cells were exposed to norepinephrine (NE) and other agents. RNA was isolated after harvesting the cells, and northern blot was performed. Filters were exposed to film after hybridization with nick-translated complementary DNA probes, and results were evaluated by scanning. Amersham assay kit was used for cAMP measurement. Results: Treatment of prazosin and yohimbine separately with 1 μM of NE shows stimulation of UCP1 mRNA expression 106% and 154%, respectively, whereas with that of both drugs shows only 76%. cAMP generation occurs 282% with prazosin, 100% with yohimbine, and 382% with both drugs with 1 μM of NE, whereas it is 310%, 40%, and 358%, respectively, with 10 μM of NE. Conclusions: Stimulation of thermogenesis after treatment of prazosin and NE may be due to the inhibition of phosphodiesterase enzyme and with yohimbine and NE indicates the possibility of inhibition of the inhibitory effect of α2- and stimulation of α1-receptors. Increase of cAMP concentration with yohimbine and both drugs with NE are not correlated to UCP1 mRNA expression. This indicates that the relationship between cAMP elevation and stimulation of thermogenesis is not simple. This study clearly shows the interaction between β- and α-adrenoreceptor activities. [ABSTRACT FROM AUTHOR]

Cutaneous exposure to food allergens can predispose individuals to food allergies. Soybean, a major allergenic food, is an ingredient in various cosmetic products. However, the types of soybean proteins that are percutaneously sensitizing in humans or animal models remain unknown. In this study, BALB/c mice were dorsally shaved and epicutaneously exposed to a crude soybean extract including sodium dodecyl sulfate or distilled water alone. Specific IgEs secreted in response to 7S globulin (Gly m 5), 11S globulin (Gly m 6), Gly m 3, and Gly m 4 were measured using enzyme-linked immunosorbent assays or immunoblots. Exposure to soybean extract elicited the secretion of soybean-specific IgEs. Of the soybean proteins, 7S and 11S globulins acted as percutaneous sensitizers in 6/9 mice (67%). Additionally, IgE bound specifically and preferentially to the 7S globulin β subunit. In conclusion, this is the first report to identify percutaneously sensitizing soybean allergens in a mouse model. [ABSTRACT FROM AUTHOR]

The levels of food allergens in worm-wounded or non-wounded green soybeans (edamame) and mature soybeans were investigated by immunoblotting and enzyme-linked immunosorbent assay (ELISA), using allergen-specific antibodies. Non-wounded and worm-wounded soybeans showed similar total protein profiles after Coomassie brilliant blue staining, but some protein bands were observed to have been changed by worm wounding. Immunoblotting with specific antibodies for major soybean allergens (Gly m 5, Gly m 6, Gly m Bd 30 K, and Kunitz soybean trypsin inhibitor) revealed that protein band profiles and intensities were not significantly changed by worm wounding. In contrast, levels of the pollen-related soybean allergens Gly m 4 and Gly m 3 were strongly increased by worm wounding in both green and mature soybeans, as detected by immunoblotting and ELISA. These results suggested that the pollen-related food allergen risk (i.e., oral allergy syndrome; OAS) from soybeans might be enhanced by worm wounding of soybeans. [ABSTRACT FROM AUTHOR]

Growth hormone (GH) has been shown to act directly on multiple tissues throughout the body. Historically, it was believed that GH acted directly in the liver and only indirectly in other tissues via insulin‐like growth hormone 1 (IGF‐1). Despite extensive work to describe GH action in individual tissues, a comparative analysis of acute GH signaling in key metabolic tissues has not been performed. Herein, we address this knowledge gap. Acute tissue response to human recombinant GH was assessed in mice by measuring signaling via phospho‐STAT5 immunoblotting. STAT5 activation is an easily and reliably detected early marker of GH receptor engagement. We found differential tissue sensitivities; liver and kidney were equally GH‐sensitive and more sensitive than white adipose tissue, heart, and muscle (gastrocnemius). Gastrocnemius had the greatest maximal response compared to heart, liver, white adipose tissue, and whole kidney. Differences in maximum responsiveness were positively correlated with tissue STAT5 abundance, while differences in sensitivity were not explained by differences in GH receptor levels. Thus, GH sensitivity and responsiveness of distinct metabolic tissues differ and may impact physiology and disease. [ABSTRACT FROM AUTHOR]

We report the first case of an autoimmune hypertriglyceridemia successfully treated with rituximab, an anti-CD20-targeted monoclonal antibody. A 45-year-old man, with prior autoimmune conditions, developed severe, acquired hypertriglyceridemia resistant to traditional triglyceride lowering therapies. After the elimination of secondary or genetic causes, we detected the presence of anti-LPL-IgG by immunoblot. After 3 infusions of rituximab, we observed a marked improvement of his hypertriglyceridemia, concomitant with a reduction in plasma anti-LPL antibody titer and B-lymphocytes counts. The patient has been receiving rituximab maintenance therapy for 5 years without any serious adverse events and with excellent control of his previous, marked hypertriglyceridemia. [ABSTRACT FROM AUTHOR]

Background and Aims. Detection of oligoclonal IgG (o-IgG) in the cerebrospinal fluid (CSF) not found in serum is the principal laboratory test to support a diagnosis of multiple sclerosis. The aim of this study was to compare chemiluminescent and chromogenic detection of oligoclonal immunoglobulins in the cerebrospinal fluid and serum after their separation by means of isoelectric focusing followed by immunoblotting. Methods. A set of experiments was designed to detect oligoclonal immunoglobulins by means of alkaline phosphatase BCIP/NBT substrate and chemiluminescent peroxidase substrate. Results. Based on visual evaluation of signals, chemiluminescent detection requires about a 4 times lower amount of applied protein than very sensitive BCIP/NBT chromogenic detection. Very good correlation between methods has been shown for oligoclonal IgG. Antigen-specific oligoclonal IgG could be demonstrated by both methods although the pattern was clearer using chemiluminescence. In one patient, oligoclonal IgD bands barely visible by BCIP/NBT were convincingly demonstrated by chemiluminescence. Conclusion. Chemiluminescent detection is a feasible option for oligoclonal immunoglobulin detection and could be used in cases when the sensitivity needs to be improved. Further studies and method optimisation are warranted. [ABSTRACT FROM AUTHOR]

We report a case of severe type I hyperlipoproteinemia caused by autoimmunity against lipoprotein lipase (LPL) in the context of presymptomatic Sjögren's syndrome. A 7-year-old mixed race (Caucasian/African American) girl was admitted to the intensive care unit at Vanderbilt Children's Hospital with acute pancreatitis and shock. She was previously healthy aside from asthma and history of Hashimoto's thyroiditis. Admission triglycerides (TGs) were 2191 mg/dL but returned to normal during the hospital stay and in the absence of food intake. At discharge, she was placed on a low-fat, low-sugar diet. She did not respond to fibrates, prescription fish oil, metformin, or orlistat, and during the following 2 years, she was hospitalized several times with recurrent pancreatitis. Except for a heterozygous mutation in the promoter region of LPL, predicted to have no clinical significance, she had no further mutations in genes known to affect TG metabolism and to cause inherited type I hyperlipoproteinemia, such as APOA5, APOC2 , GPIHBP1 , or LMF1 . When her TG levels normalized after incidental use of prednisone, an autoimmune mechanism was suspected. Immunoblot analyses showed the presence of autoantibodies to LPL in the patient's plasma. Autoantibodies to LPL decreased by 37% while patient was on prednisone, and by 68% as she subsequently transitioned to hydroxychloroquine monotherapy. While on hydroxychloroquine, she underwent a supervised high-fat meal challenge and showed normal ability to metabolize TG. For the past 3 years and 6 months, she has had TG consistently <250 mg/dL, and no symptoms of, or readmissions for, pancreatitis. [ABSTRACT FROM AUTHOR]

Copyright of Zentralblatt fuer Arbeitsmedizin, Arbeitsschutz und Ergonomie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In this study, the effects of the heavy metal cadmium on the stress protein HSP70 are investigated in freshwater mollusks Biomphalaria glabrata . Adult snails were exposed for 96 h to CdCl 2 at concentrations ranging from 0.09 to 0.7 mg L −1 (LC 50/96 h =0.34 (0.30–0.37). Time and concentration-dependent increases in the expression of HSP70 were observed at sub-lethal levels in the immunoblotting assay. Further, an increased survival to a lethal heat shock was observed in animals pre-exposed to a nonlethal concentration of cadmium, evidencing the induction of acquired tolerance. The present study demonstrated the inducibility of B. glabrata HSP70 by cadmium, a relevant environmental contaminant, at non-lethal levels, providing evidences that the assessment of HSP70 in B. glabrata can be regarded as a suitable biomarker for ecotoxicological studies. [ABSTRACT FROM AUTHOR]

Introduction and objective. Gluten proteins (gliadins and glutenins) are polymorphic wheat storage proteins of allergenic properties. Significant differences in chemical composition between both protein groups allow to expect highly specific immunological response of individual subunits and fractions in reactions with IgE sera of people allergic to wheat. The aim of these studies was to identify and characterize the most allergenic gluten proteins (GP) and nongluten proteins (NGP) occurred in two closely related wheat hybrid genotypes. Materials and method. 3xC and 3xN wheat hybrids, which differ strongly in regard of gliadin composition, were analyzed. Seven people manifesting different symptoms of wheat allergy donated sera for the experiment. The technique of immunoblotting after SDS-PAGE was used for identification of allergenic subunits and fractions among GP and NGP. Immunologically active protein bands were visualized by chemiluminescence. Results. Great variation of immunodetection spectra was observed. Results of immunoblotting showed LMW glutenins to be of highest, gliadins of medium, while NGP of lowest allergenicity for selected patients. The 43-kDa and 47-kDa LMW glutenin subunits, 40-kDa and 43-kDa γ-gliadin fractions and 49-kDa NGP can be considered as the most immunoreactive among all protein bands separated by SDS-PAGE. Conclusion. The observed differentiation of immunodetection spectra allows to model highly specific IgE-binding profiles of allergenic wheat proteins attributed to individual patients with symptoms of gluten intolerance. Highly immunoreactive subunits and fractions among GP and NGP were identified. The observed immunoreactivity of 49 kDa NGP is worth to emphasize, as it has never been reported as wheat allergenic protein before. [ABSTRACT FROM AUTHOR]

In the present study, we used a proteomics approach based on a two-dimensional electrophoresis (2-DE) reference map to investigate protein expression in the ovarian tissues of pubertal Swiss-Webster mice subjected to carbon ion radiation (CIR). Among the identified proteins, ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is associated with the cell cycle [1] and that it influences proliferation in ovarian tissues. We analyzed the expression of UCH-L1 and the proliferation marker proliferation cell nuclear antigen (PCNA) following CIR using immunoblotting and immunofluorescence. The proteomics and biochemical results provide insight into the underlying mechanisms of CIR toxicity in ovarian tissues. [ABSTRACT FROM AUTHOR]

The present study was undertaken to separate the FAA of seminal plasma and frozen-thawed sperm extracts from 30 buffalo bulls by immunoblotting and determine their relationship with post-thaw sperm function tests visà-vis bull fertility. Eight immunoreactive bands in seminal plasma (60, 55, 45, 33, 31, 18, 16 and 14 kDa) and four in frozen-thawed sperm extracts (65, 55, 48 and 18 kDa) were detected in Western blots. The frozen-thawed semen was evaluated for first service conception rate (FSCR), per cent acrosome reaction, HOST, viability, DNA integrity and total motility and linked to FAA. In seminal plasma, the bulls positive for 60, 31 and 14 kDa FAA had significantly higher FSCR (37.0±3.2 vs 0.0±0.0%, 46.7±3.2 vs 22.5±3.3% and 48.6±3.8 vs 26.0±3.0%), respectively, as compared to their negative counterparts. The FSCR was also higher in detectable FAA-33 than in undetectable FAA-33. Almost all seminal parameters were found to be significantly higher in bulls positive for FAA of 60, 33, 31 and 14 kDa than in their negative contemporary mates. In frozen-thawed sperm extracts, the bulls positive for FAA-65, 48 and 18 had significantly higher FSCR, per cent acrosome reaction and total motility in comparison to their negative herd mates. In conclusion, we have identified buffalo bull seminal FAA that bind to spermatozoa; influence semen quality and subsequent fertility of buffalo bulls. [ABSTRACT FROM AUTHOR]

The incidence of breast cancer in India is on the rise and is rapidly becoming the primary cancer in Indian women. The aldoketo reductase (AKR) family has more than 190 proteins including aldose reductase (AKR1B1) and aldose reductase like protein (AKR1B10). Apart from liver cancer, the status of AKR1B1 and AKR1B10 with respect to their expression and activity has not been reported in other human cancers. We studied the specific activity and expression of AKR1B1 and AKR1B10 in breast non tumor and tumor tissues and in the blood. Fresh post-surgical breast cancer and non-cancer tissues and blood were collected from the subjects who were admitted for surgical therapy. Malignant, benign and pre-surgical chemotherapy samples were evaluated by histopathology scoring. Expression of AKR1B1 and AKR1B10 was carried out by immunoblotting and immunohistochemistry (IHC) while specific activity was determined spectrophotometrically. The specific activity of AKR1B1 was significantly higher in red blood cells (RBC) in all three grades of primary surgical and post-chemotherapy samples. Specific activity of both AKR1B1 and AKR1B10 increased in tumor samples compared to their corresponding non tumor samples (primary surgical and post-chemotherapy). Immunoblotting and IHC data also indicated overexpression of AKR1B1 in all grades of tumors compared to their corresponding non tumor samples. There was no change in the specific activity of AKR1B1 in benign samples compared to all grades of tumor and non-tumors. [ABSTRACT FROM AUTHOR]

Objective: Heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), serine/arginine-rich splicing factor 1 (SRSF1), and SRSF3 are splicing regulators associated with oncogenesis. However, the alterations of SF proteins and their diagnostic values in cervical cancer are unclear. To apply SFs clinically, effective marker selection and characterization of the target organ properties are essential. Materials and Methods: We concurrently analyzed HNRNPA1, SRSF1, SRSF3, and the conventional tumor markers squamous cell carcinoma antigen (SCCA) and carcinoembryonic antigen (CEA) in cervical tissue samples (n = 127) using semiquantitative immunoblotting. In addition, we compared them with p16 (cyclin-dependent kinase inhibitor 2A [CDKN2A]), which has shown high diagnostic efficacy in immunohistochemical staining studies and has been proposed as a candidate protein for point-of-care screening biochemical tests of cervical neoplasia. Results: HNRNPA1, higher molecular weight forms of SRSF1 (SRSF1-HMws), SRSF3, CEA, and p16 levels were higher (P < 0.05) in cervical carcinoma tissue samples than in nontumoral cervical tissue samples. However, the levels of SRSF1-Total (sum of SRSF1-HMws and a lower molecular weight form of SRSF1) and SCCA, a commonly used cervical tumor marker, were not different between carcinoma and nontumoral tissue samples. In paired sample comparisons, HNRNPA1 (94%) showed the highest incidence of up-regulation (carcinoma/nontumor, >1.5) in cervical carcinoma, followed by p16 (84%), SRSF1-HMws (69%), SRSF3 (66%), CEA (66 %), SCCA (32%), and SRSF1-Total (31%). HNRNPA1 (92%) and p16 (91%) presented the two highest diagnostic accuracies for cervical carcinoma, which were superior to those of SRSF3 (75%), SRSF1-HMws (72%), CEA (72%), SCCA (59%), and SRSF1-Total (55%). Conclusions: Our results identified that HNRNPA1 is the best diagnostic marker among the SFs and conventional markers given its excellent diagnostic efficacy for cervical carcinoma, and it has a p16-comparable diagnostic value. We suggest that HNRNPA1 is an additional effective target protein for developing cervical cancer detection tools. [ABSTRACT FROM AUTHOR]

Traditional approaches to vaccine development have failed to identify better vaccines to replace or supplement BCG for the control of tuberculosis (TB). Subunit vaccines offer a safer and more reproducible alternative for the prevention of diseases. In this study, the immunogenicity of bacterially derived polyester beads displaying three different Rv antigens of Mycobacterium tuberculosis was evaluated. Polyester beads displaying the antigens Rv1626, Rv2032, Rv1789, respectively, were produced in an endotoxin-free Escherichia coli strain. Beads were formulated with the adjuvant DDA and subcutaneously administered to C57BL/6 mice. Cytokine responses were evaluated by CBA and antibody responses by ELISA. Specificity of the IgG response was assessed by immunoblotting cell lysates of the vaccine production strains using sera from the vaccinated mice. Mice vaccinated with beads displaying Rv1626 had significantly greater IgG1 responses compared to mice vaccinated with Rv1789 beads and greater IgG2 responses than the group vaccinated with Rv2032 beads (p < 0.05). Immunoblotting of antisera from these mice indicated the antibody responses were Rv1626 antigen-specific and there was no detectable immune response to the polyester component of the vaccine. Overall, this study suggested that selected TB antigens derived from reverse vaccinology approaches can be displayed on polyester beads to produce antigen-specific immune responses potentially relevant to the prevention of TB. [ABSTRACT FROM AUTHOR]

Novel nanomaterials for the intracellular transport of therapeutic cargos have been actively sought to effectively breach cell-membrane barriers. In this study we developed novel self-micellizing anticancer lipid (SMAL)-based pro-apoptotic nanoparticles (NPs) that enhance the accumulation and chemotherapeutic efficacy of oxaliplatin (OL) in colorectal cancer cells (CRCs). We demonstrated that NPs with special affinity to caveolae could be designed and based on this specificity, NPs effectively differentiated between endothelial cells (tumor cells) and epithelial cells, without the need for a cell-specific targeting moiety. We demonstrated a remarkable uptake of OL-loaded SMAL NPs (SMAL-OL) in HCT116 and HT-29 cells via the caveolae-mediated endocytosis (CvME) pathway. The higher accumulation of SMAL-OL in the intracellular environment resulted in a significantly elevated anticancer effect compared to that of free OL. Cell cycle analysis proved G2/M phase arrest, along with substantial presence of cells in the sub-G1 phase. An immunoblot analysis indicated an upregulation of pro-apoptotic markers (Bax; caspase-3; caspase-9; and PARP1) and downregulation of Bcl-xl and the PI3K/AKT/mTOR complex, indicating a possible intrinsic apoptotic signaling pathway. Overall, the ability of SMAL NPs to confer preferential specificity towards the cell surface domain could offer an exciting means of targeted delivery without the need for receptor-ligand-type strategies. Statement of Significance In this work, we developed a novel self-micellizing anticancer lipid (SMAL)-based pro-apoptotic nanoparticles (NPs) that enhance the accumulation and chemotherapeutic efficacy of oxaliplatin (OL) in colorectal cancer cells. We demonstrated that NPs with special affinity to caveolae could be realized and based on this specificity, NPs effectively differentiated between endothelial cells (tumor cells) and epithelial cells, without the need for a cell-specific targeting moiety. In addition, oxaliplatin-loaded SMAL were efficiently endocytosed by the cancer cells and represent a significant breakthrough as an effective drug delivery system with promising potential in cancer therapy. We believe this work holds promising potential for the development of next generation of multifunctional nanocarriers for an exciting means of targeted delivery without the need for receptor-ligand-type strategies. [ABSTRACT FROM AUTHOR]

Background: The immunoprecipitation (IP) assay is a valuable molecular biology tool applied across a breadth of fields. The standard assay couples IP to immunoblotting (IP/IB), a procedure severely limited as it is not easily scaled for high-throughput analysis. Results: Here we describe and characterize a new methodology for fast and reliable evaluation of an immunoprecipitation reaction. FLIP (FLuorescence IP) relies on the expression of the target protein as a chromophore-tagged protein and couples IP with the measurement of fluorescent signal coating agarose beads. We show here that FLIP displays similar sensitivity to the standard IP/IB procedure but is amenable to high-throughput analysis. We applied FLIP to the screening of mouse monoclonal antibodies of unknown behavior in IP procedures. The parallel analysis of the considered antibodies using FLIP and IP/western shows good correlation between the two procedures. We also show application of FLIP using unpurified antibodies (hybridoma supernatant) and we developed a publicly available tool for the easy analysis and quantification of FLIP signals. Conclusions: Altogether, our characterizations of this new methodology show that FLIP is an appealing and reliable tool for any application of high-throughput IP. [ABSTRACT FROM AUTHOR]

Purpose Lower urinary tract symptoms are a common finding in patients with chronic prostatitis/chronic pelvic pain syndrome. We previously reported that the mast cell-tryptase-PAR2 (protease activated receptor 2) axis has a critical role in the development of chronic pain in experimental autoimmune prostatitis, a mouse model of chronic prostatitis/chronic pelvic pain syndrome. Therefore, we examined whether PAR2 activation mediates lower urinary tract dysfunction. Materials and Methods Functional cystometry was done in male B6 mice along with immunoblotting and immunohistochemistry for the expression of COL1A1 (collagen type I α I) and α-SMA (α-smooth muscle actin). Flow cytometry analysis was performed on single cell suspensions of the prostate, bladder, lymph nodes and spleen. Results Experimental autoimmune prostatitis resulted in increased urinary voiding frequency and decreased bladder capacity 30 days after initiation. Concurrently, there was increased expression of COL1A1 and α-SMA in the prostates and bladders. In contrast, induction of experimental autoimmune prostatitis in PAR2 knockout mice did not result in altered urodynamics or increased markers of fibrosis in the prostate or the bladder. Single cell suspensions of the prostate, bladder, lymph nodes and spleen demonstrated that in the absence of PAR2 cellular inflammatory mechanisms were still initiated in experimental autoimmune prostatitis but PAR2 expression may be required to maintain chronic inflammation. Finally, antibody mediated PAR2 neutralization normalized urinary voiding frequency and bladder capacity, and attenuated chronic pelvic pain. Conclusions PAR2 activation in the prostate may contribute to the development of lower urinary tract dysfunction through proinflammatory as well as profibrotic pathways. [ABSTRACT FROM AUTHOR]

The effects of microcurrent application on the elastic cartilage defects in the outer ear of young animals were analyzed. Sixty male Wistar rats were divided into a control (CG) and a treated group (TG). An excisional lesion was created in the right outer ear of each animal. Daily treatment was started after 24 h and consisted of the application of a low-intensity (20 μA) continuous electrical current to the site of injury for 5 min. The animals were euthanized after 7, 14 and 28 days of injury and the samples were submitted to analyses. In CG, areas of newly formed cartilage and intense basophilia were seen at 28 days, while in TG the same observations were made already at 14 days. The percentage of birefringent collagen fibers was higher in CG at 28 days. The number of connective tissue cells and granulocytes was significantly higher in TG. Ultrastructural analysis revealed the presence of chondrocytes in TG at 14 days, while these cells were observed in CG only at 28 days. Cuprolinic blue staining and the amount of glycosaminoglycans were significantly higher in TG at 14 days and 28 days. The amount of hydroxyproline was significantly higher in TG at all time points studied. The active isoform of MMP-2 was higher activity in TG at 14 days. Immunoblotting for type II collagen and decorin was positive in both groups and at all time points. The treatment stimulated the proliferation and differentiation of connective tissue cells, the deposition of glycosaminoglycans and collagen, and the structural reorganization of these elements during elastic cartilage repair. [ABSTRACT FROM AUTHOR]

Sinusoidal endothelial cells (SEC) and hepatic stellate cells (HSC) are closely associated specialized vascular cells residing in the hepatic sinusoid. These cells have been shown to play important roles in many different pathophysiologic processes, in particular in liver fibrosis/cirrhosis and portal hypertension. Caveolin-1 functions as a scaffolding protein, and has a variety of functions including in many disease states, such as liver cirrhosis. Although previous studies have shown that in the injured rat liver, caveolin-1 is upregulated, the precise cells in which remains unclear. Therefore, the purpose of this study was to clarify the cell type (or types) in which caveolin-1 is expressed in normal and injured rat liver. We have utilized both detailed immunohistochemical labeling with cell specific markers as well as cell isolation techniques (isolating sinusoidal endothelial cells, HSCs, and hepatocytes) in normal and injured (bile duct ligation) rat liver. We show here that in the normal liver caveolin-1 is expressed predominantly in HSCs and SECs but after liver injury there is upregulation of caveolin-1 in HSCs, but not in SECs. These data have functional implications for the cells in which caveolin-1 is regulated. [ABSTRACT FROM AUTHOR]

Background-After myocardial infarction, the left ventricle undergoes a wound healing response that includes the robust infiltration of neutrophils and macrophages to facilitate removal of dead myocytes as well as turnover of the extracellular matrix. Matrix metalloproteinase (MMP)-9 is a key enzyme that regulates post-myocardial infarction left ventricular remodeling. Methods and Results-Infarct regions from wild-type and MMP-9 null mice (n=8 per group) analyzed by glycoproteomics showed that of 541 N-glycosylated proteins quantified, 45 proteins were at least 2-fold upregulated or downregulated with MMP-9 deletion (all P<0.05). Cartilage intermediate layer protein and platelet glycoprotein 4 (CD36) were identified as having the highest fold increase in MMP-9 null mice. By immunoblotting, CD36 but not cartilage intermediate layer protein decreased steadily during the time course post-myocardial infarction, which identified CD36 as a candidate MMP-9 substrate. MMP-9 was confirmed in vitro and in vivo to proteolytically degrade CD36. In vitro stimulation of day 7 post-myocardial infarction macrophages with MMP-9 or a CD36-blocking peptide reduced phagocytic capacity. Dual immunofluorescence revealed concomitant accumulation of apoptotic neutrophils in the MMP-9 null group compared with wild-type group. In vitro stimulation of isolated neutrophils with MMP-9 decreased neutrophil apoptosis, indicated by reduced caspase-9 expression. Conclusions-Our data reveal a new cell-signaling role for MMP-9 through CD36 degradation to regulate macrophage phagocytosis and neutrophil apoptosis. [ABSTRACT FROM AUTHOR]

Background: The energy status of the cell is regulated by the energy sensing network constituted by AMP-activated protein kinase (AMPK), the NAD+-dependent type III deacetylase silence information regulator T1 (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). This study investigates the potential effect of 5-aminoimidazole-4-carboximide-1-b-D-ribofuranoside (AICAR), an AMPK activator on insulin signaling and energy sensing network in insulin resistant rats. Methods: Adult male albino Wistar rats with body weight of 150-180 g were fed high-fructose diet (HFD) for 60 days to induce insulin resistance. Rats fed HFD were divided into two and were treated or untreated with AICAR (0.7 mg/kg bw, i.p.) for the last 2 weeks. Results: Insulin resistant rats displayed increased glucose and insulin levels and reduced tyrosine phosphorylation of insulin resistance receptor and insulin receptor substrate 1. The downstream signaling and glucose transport were also affected. Phosphorylation of AMPK, SIRT1 protein abundance and mRNA expression of PGC-1α were reduced. Treatment with AICAR reduced hyperglycemia and hyperinsulinemia and improved the activation of the key molecules of insulin signaling. Improved action of energy sensing network was noted after AICAR treatment. AICAR showed higher binding affinity with Akt (-8.2 kcal/mol) than with AMPK or insulin receptor (-8.0 kcal/mol) in the in silico study. Conclusions: The findings suggest that AICAR, the AMPK activator, influences insulin signaling proteins and molecules involved in energy modulation during insulin resistance. [ABSTRACT FROM AUTHOR]

Background: The hyperpolarization-activated current, If, plays an important role in sinoatrial node (SAN) pacemaking. Surprisingly, the distribution of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in human SAN has only been investigated at the mRNA level. Our aim was to define the expression pattern of HCN proteins in human SAN and different atrial regions. Methods and Results: Entire SAN complexes were isolated from failing (n=5) and nonfailing (n=9) human hearts cardioplegically arrested in the operating room. Three-dimensional intramural SAN structure was identified as the fibrotic compact region around the SAN artery with Connexin 43-negative pacemaker cardiomyocytes visualized in Masson's trichrome and immunostained cryosections. SAN protein was precisely isolated from the adjacent frozen SAN tissue blocks using a 16G biopsy needle. The purity of the SAN protein was confirmed by Connexin 43 immunoblot. All 3 HCN isoform proteins were detected in SAN. HCN1 was predominantly distributed in the human SAN with a 125.1±40.2 (n=12) expression ratio of SAN to right atrium. HCN2 and HCN4 expression levels were higher in SAN than in atria, with SAN to right atrium ratios of 6.1±0.9 and 4.6±0.6 (n=12), respectively. Conclusions: This is the first study to conduct precise 3D molecular mapping of the human SAN by isolating pure pacemaker SAN tissue. All 3 cardiac HCN isoforms had higher expression in the SAN than in the atria. HCN1 was almost exclusively expressed in SAN, emphasizing its utility as a new specific molecular marker of the human SAN and as a potential target of specific treatments intended to modify sinus rhythm. [ABSTRACT FROM AUTHOR]

Background: The Na(+)/Ca(2+) exchanger (NCX) has been implied to cause arrhythmias. To date, information on the role of NCX in arrhythmogenesis derived from models with increased NCX expression, hypertrophy, and heart failure. Furthermore, the exact mechanism by which NCX exerts its potentially proarrhythmic effect, ie, by promoting early afterdepolarization (EAD) or delayed afterdepolarization (DAD) or both, is unknown.Methods and Results: We investigated isolated cardiomyocytes from a murine model with heterozygous knockout of NCX (hetKO) using the patch clamp and Ca(2+) imaging techniques. Action potential duration was shorter in hetKO with IKtot not being increased. The rate of spontaneous Ca(2+) release events and the rate of DADs were unaltered; however, DADs had lower amplitude in hetKO. A DAD triggered a spontaneous action potential significantly less often in hetKO when compared with wild-type. The occurrence of EADs was also drastically reduced in hetKO. ICa activity was reduced in hetKO, an effect that was abolished in the presence of the Ca(2+) buffer BAPTA.Conclusions: Genetic suppression of NCX reduces both EADs and DADs. The following molecular mechanisms apply: (1) Although the absolute number of DADs is unaffected, an impaired translation of DADs into spontaneous action potentials results from a reduced DAD amplitude. (2) EADs are reduced in absolute number of occurrence, which is presumably a consequence of shortened action potential duration because of reduced NCX activity but also reduced ICa the latter possibly being caused by a direct modulation of Ca(2+)-dependent ICa inhibition by reduced NCX activity. This is the first study to demonstrate that genetic inhibition of NCX protects against afterdepolarizations and to investigate the underlying mechanisms. [ABSTRACT FROM AUTHOR]

The article discusses a study which examines if the desmosomal cadherin desmoglein 2 (Dsg2) could cause cardiomyopathy. Topics include the characteristics of arrhythmogenic cardiomyopathy (AC) in such as arrhythmia and intercalated disc dissociation, the use of mice in the study and the electron microscopy method, and analyzing protein extracts and heart tissue with immunoblotting.

The article presents a study which evaluated the utility of analyzing the pattern of bovine leukemia virus (BVL) integration as a marker for enzootic bovine leukosis (EBL) onset in cattle without enlarged superficial lymph nodes. Topics include the clinical signs of EBL, the use of Southern blotting and hybridization in detecting leukosis onset in atypical EBL cases, and how this could serve as a marker for malignant transformation of lymphocytes in cattle with persistent lymphocytosis (PL).

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis. [ABSTRACT FROM AUTHOR]

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochon-drial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis. [ABSTRACT FROM AUTHOR]

Background Distal skin flap necrosis represents a severe complication in surgery. This study investigated angiogenic responses in healthy and impaired pedicled skin flap tissue in normal and diabetic mice. Methods Histologic, qRT-PCR, ELISA and immunoblot techniques determined expression and localization of angiogenesis-related growth factors, receptors and cell types upon skin flap re-integration. Results Skin flap tissue re-integration was severely disturbed in diabetic mice. Impaired skin flap tissue lost early VEGF expression from wound margin keratinocytes and markedly reduced expression of endothelium-specific receptors Tie-2 and FLT-1. Numbers of blood vessels were reduced in impaired flaps. In addition, HIF-1α protein was absent from disturbed skin flap tissue. Reduced VEGF expression and the loss of epithelium in disturbed skin flaps were paralleled by the appearance of VEGF expressing inflammatory infiltrate. Conclusion In summary, our data show a dysregulated spatial and temporal pattern of angiogenic processes during skin flap re-integration in diabetic mice. Our data suggest that reduced expression of angiogenic receptors in skin flap tissue might contribute to a loss of VEGF function in impaired tissue. [ABSTRACT FROM AUTHOR]

Peroxide enzymes are widely used as a reagent in many clinical diagnosis of analysis methods such as ELISA, immunoblotting, and immunochemistry. Water spinach ( Ipomea aquatica Forssk) is a species of plant available abundantly in Indonesia, hence it is a potential source of peroxidase. In this study, the crude extract of water spinach leaves was known to contain peroxidase enzyme which was active toward hydrogen peroxide. A fraction of extract yielded from differential fractionation using ammonium sulfate at concentration 40-65% (F4) was known to be the most active. Characterization of specific activity of peroxidase of F4 for optimum pH and temperature was at pH 5 and 60 °C. The Michaelis-Menten constant (Km) was 0,27 mg/mL and enzyme's catalytic rate (Vmax) was 2,16 mg/mL/min. [ABSTRACT FROM AUTHOR]

Objective Due to their previously identified naturally and chronically increased levels of skeletal muscle pAMPK we hypothesized and now investigated whether the MRL/MpJ (MRL) mice would be resistant to high fat diet (HFD)-induced metabolic changes. Materials/methods Three-week old male MRL and control C57Bl/6 (B6) mice were randomly assigned to 12 weeks of high fat diets (HFD) or control diets (CD). Weekly animal masses and fasting blood glucose measurements were acquired. During the last week of diet intervention, fasted animals were subjected to glucose and insulin tolerance tests. At harvest, tissues were dissected for immunoblots and serum was collected for elisa assays. Results The MRL mouse strain is known for its ability to regenerate ear punch wounds, cardiac cryoinjury, and skeletal muscle disease. Despite gaining weight and increasing their fat deposits the MRL mice were resistant to all other indicators of HFD-induced metabolic alterations assayed. Only the HFD-B6 mice displayed fasting hyperglycemia, hyperinsulinemia and hypersensitivity to glucose challenge. HFD-MRL mice were indistinguishable from their CD-MRL counterparts in these metrics. Skeletal muscles from the HFD-MRL contained heightened levels of pAMPK, even above their CD counterparts. Conclusions The MRL mouse strain is the first naturally occurring mouse strain that we are aware of that is resistant to HFD-induced metabolic changes. Furthermore, the increased pAMPK suggests a proximal mechanism for these beneficial metabolic differences. We further hypothesize that these metabolic differences and plasticity provide the basis for the MRL mouse strain's super healing characteristics. This project's ultimate aim is to identify novel therapeutic targets, which specifically increase pAMPK. [ABSTRACT FROM AUTHOR]

The determination of the accurate immune status of pregnant women is crucial in order to prevent congenital toxoplasmosis. Equivocal results with conventional serological techniques are not uncommon when IgG titers are close to the cut-off value of the test, so that a confirmatory technique is needed. For this purpose, we developed a homemade immunoblot (IB) using soluble extract of Toxoplasma gondii tachyzoites and assessed it by testing 154 positive, 100 negative, and 123 equivocal sera obtained from pregnant women. In order to select the more valuable bands in terms of sensitivity and specificity, we used the Youden Index (YI). The highest YIs were those given by the 32, 36, 98, 21, and 33 bands. The simultaneous presence on the same blot of at least 3 bands showed a much higher YI (0.964) and was adapted as the positivity criterion. The analysis of results showed that our homemade IB correlated well with the commercial LDBIO Toxo II IgG® kit recently recommended as a confirmatory test (96.7% of concordance). [ABSTRACT FROM AUTHOR]

The localization of specific claw beta-proteins among the 40 total corneous beta-proteins present in the lizard Anolis carolinensis is not known. The hardness of claws likely depends on glycine–cysteine-rich beta-proteins content, as suggested by previous immunoblot studies. Previous studies have indicated that glycine–cysteine-rich corneous beta-proteins in addition to cysteine-rich alpha-keratins are present in the claw. In order to detect at the ultrastructural level the presence of claw-specific corneous proteins immunofluorescence and electron microscopy immunogold have been utilized. More intense immunoreactivity is obtained for the HgGC3 beta-protein while less intense immunolabeling is seen for HgGC10 and HgG5 beta-proteins and no labeling for the cysteine-rich beta-protein HgC1. The HgGC3 beta-protein appears the prevalent type present in the claw and its numerous cysteines likely form intermolecular disulphide bonds while glycine contributes hydrophobic properties to the corneous material. Other antibodies tagging the core-box and pre-core box regions of beta-proteins label with less intensity the corneous layer. The presence of cysteine-rich alpha-keratins with high homology to some human hair keratins in the dorsal part of the claw suggests that HgGC3-like beta-proteins form numerous disulphide bonds with the larger alpha-keratins giving rise to the hard corneous material of the claw. [ABSTRACT FROM AUTHOR]

Introduction Recent studies of inflammasome activation have focused on the pathogenesis of diverse inflammatory and autoimmune diseases. Inflammasome activation results in caspase-1 activation, which is required for processing of prointerleukin (IL)-1 beta to its secreted form as well as a proinflammatory cell death (ie, pyroptosis). The purpose of this study was to analyze whether Enterococcus faecalis associated with endodontic infection induces inflammasome activation. Methods THP-1 macrophages were treated with E . faecalis in the presence or absence of caspase-1 inhibitors. Caspase-1 activation, pro–IL-1 beta expression, and IL-1 beta secretion were detected by immunoblotting, real-time reverse-transcription polymerase chain reaction, and enzyme-linked immunosorbent assay, respectively. Cell death was measured by lactate dehydrogenase release and propidium iodide staining. Adenosine triphosphate (ATP) release was measured by an ATP bioluminescence assay kit. Results E. faecalis induced caspase-1 activation and pro–IL-1 beta expression, which resulted in IL-1 beta secretion in macrophages. E. faecalis significantly induced ATP release, which is a mechanism of Nod-like receptor family protein 3 (NLRP3) inflammasome activation, whereas oxATP treatment inhibited E. faecalis –induced caspase-1 activation. E. faecalis significantly increased lactate dehydrogenase release and propidium iodide uptake, which are characteristics of pyroptosis. Conclusions Our results show that E. faecalis may contribute to the progression of pulpal inflammation by stimulating excessive secretion of IL-1 beta and cell death. [ABSTRACT FROM AUTHOR]