Choice of biological source material supersedes oxidative stress in its influence on DJ-1 in vivo interactions with Hsp90

DJ-1 is a small but relatively abundant protein of unknown function that may undergo stress-dependent cellular translocation and has been implicated in both neurodegenerative diseases and cancer. As such, DJ-1 may be an excellent study object to elucidate the relative influence of the cellular context on its interactome and for exploring whether acute exposure to oxidative stressors alters its molecular environment. Using quantitative mass spectrometry, we conducted comparative DJ-1 interactome analyses from in vivo cross-linked brains or livers and from hydrogen peroxide-treated or na?ve embryonic stem cells. The analysis identified a subset of glycolytic enzymes, heat shock proteins 70 and 90, and peroxiredoxins as interactors of DJ-1. Consistent with a role of DJ-1 in Hsp90 chaperone biology, we document destabilization of Hsp90 clients in DJ-1 knockout cells. We further demonstrate the existence of a C106 sulfinic acid modification within DJ-1 and thereby establish that this previously inferred modification also exists in vivo. Our data suggest that caution has to be exerted in interpreting interactome data obtained from a single biological source material and identify a role of DJ-1 as an oxidative stress sensor and partner of a molecular machinery notorious for its involvement in cell fate decisions.     

Protein pathway activation associated with sustained virologic response in patients with chronic hepatitis C treated with pegylated interferon (PEG-IFN) and ribavirin (RBV)

Only half of chronic hepatitis C (CH-C) patients treated with pegylated interferon and ribavirin (PEG-IFN+RBV) achieve sustained virologic response) SVR. In addition to known factors, we postulated that activation of key protein signaling networks in the peripheral blood mononuclear cells (PBMCs) may contribute to SVR due to inherent patient-specific basal immune cell signaling architecture. In this study, we included 92 patients with CH-C. PBMCs were collected while patients were not receiving treatment and used for phosphoprotein-based network profiling. Patients received a full course of PEG-IFN+RBV with overall SVR of 55%. From PBMC, protein lysates were extracted and then used for Reverse Phase Protein Microarray (RPMA) analysis, which quantitatively measured the levels of cytokines and activation levels of 25 key protein signaling molecules involved in immune cell regulation and interferon alpha signaling. Regression models for predicting SVR were generated by stepwise bidirectional selection. Both clinical-laboratory and RPMA parameters were used as predictor variables. Model accuracies were estimated using 10-fold cross-validation. Our results show that by comparing patients who achieved SVR to those who did not, phosphorylation levels of 6 proteins [AKT(T308), JAK1(Y1022/1023), p70 S6 Kinase (S371), PKC zeta/lambda(T410/403), TYK2(Y1054/1055), ZAP-70(Y319)/Syk(Y352)] and overall levels of 6 unmodified proteins [IL2, IL10, IL4, IL5, TNF-alpha, CD5L] were significantly different (P < 0.05). For SVR, the model based on a combination of clinical and proteome parameters was developed, with an AUC = 0.914, sensitivity of 92.16%, and specificity of 85.0%. This model included the following parameters: viral genotype, previous treatment status, BMI, phosphorylated states of STAT2, AKT, LCK, and TYK2 kinases as well as steady state levels of IL4, IL5, and TNF-alpha. In conclusion, SVR could be predicted by a combination of clinical, cytokine, and protein signaling activation profiles. Signaling events elucidated in the study may shed some light into molecular mechanisms of response to anti-HCV treatment.     

Insulin activates epithelial sodium channel (ENaC) via phosphoinositide 3-kinase in mammalian taste receptor cells

Diabetes is a profound disease that results in a severe lack of regulation of systemic salt and water balance. From our earlier work on the endocrine regulation of salt taste at the level of the epithelial sodium channel (ENaC), we have begun to investigate the ability of insulin to alter ENaC function with patch-clamp recording on isolated mouse taste receptor cells (TRCs). In fungiform and vallate TRCs that exhibit functional ENaC currents (e.g., amiloride-sensitive Na(+) influx), insulin (5-20 nM) caused a significant increase in Na(+) influx at -80 mV (EC(50) = 7.53 nM). The insulin-enhanced currents were inhibited by amiloride (30 μM). Similarly, in ratiometric Na(+) imaging using SBFI, insulin treatment (20 nM) enhanced Na(+) movement in TRCs, consistent with its action in electrophysiological assays. The ability of insulin to regulate ENaC function is dependent on the enzyme phosphoinositide 3-kinase since treatment with the inhibitor LY294002 (10 μM) abolished insulin-induced changes in ENaC. To test the role of insulin in the regulation of salt taste, we have characterized behavioral responses to NaCl using a mouse model of acute hyperinsulinemia. Insulin-treated mice show significant avoidance of NaCl at lower concentrations than the control group. Interestingly, these differences between groups were abolished when amiloride (100 μM) was added into NaCl solutions, suggesting that insulin was regulating ENaC. Our results are consistent with a role for insulin in maintaining functional expression of ENaC in mouse TRCs.     

Relationship between thyrotropin receptor hinge region proteolytic posttranslational modification and receptor physiological function

The glycoprotein hormone receptor hinge region is the least conserved component and the most variable in size; the TSH receptor (TSHR) being the longest (152 amino acids; residues 261-412). The TSHR is also unique among the glycoprotein hormone receptor in undergoing in vivo intramolecular cleavage into disulfide-linked A- and B-subunits with removal of an intervening 'C-peptide' region. Experimentally, hinge region amino acids 317-366 (50 residues) can be deleted without alteration in receptor function. However, in vivo, more than 50 amino acids are deleted during TSHR intramolecular cleavage; furthermore, the boundaries of this deleted region are ragged and poorly defined. Studies to determine the extent to which hinge region deletions can be tolerated without affecting receptor function ('minimal hinge') are lacking. Using as a template the functionally normal TSHR with residues 317-366 deleted, progressive downstream extension of deletions revealed residue 371 to be the limit compatible with normal TSH binding and coupling with cAMP signal transduction. Based on the foregoing downstream limit, upstream deletion from residue 307 (307-371 deletion) was also tolerated without functional alteration, as was deletion of residues 303-366. Addressing a related issue regarding the functional role of the TSHR hinge region, we observed that downstream hinge residues 377-384 contribute to coupling ligand binding with cAMP signal transduction. In summary, we report the first evaluation of TSHR function in relation to proteolytic posttranslational hinge region modifications. Deletion of TSHR hinge amino acids 303-366 (64 residues) or 307-371 (65 residues) are the maximum hinge region deletions compatible with normal TSHR function.     

The Study of Chromium and Zinc Contaminated Soil Influence on Iron Content and Protein Profile of Ornamental Cabbage Plant

Biology Bulletin - To evaluate the accumulation potential of zinc (Zn) and chromium (Cr) by Brassica oleracea L. plant, 15-day-old seedlings were exposed to different concentrations of Zn and Cr...     

Oblique circle method for measuring the curvature and twist of mitotic spindle microtubule bundles

The highly ordered spatial organization of microtubule bundles in the mitotic spindle is crucial for its proper functioning. The recent discovery of twisted shapes of microtubule bundles and spindle chirality suggests that the bundles extend along curved paths in three dimensions, rather than being confined to a plane. This, in turn, implies that rotational forces, i.e., torques, exist in the spindle in addition to the widely studied linear forces. However, studies of spindle architecture and forces are impeded by a lack of a robust method for the geometric quantification of microtubule bundles in the spindle. In this work, we describe a simple method for measuring and evaluating the shapes of microtubule bundles by characterizing them in terms of their curvature and twist. By using confocal microscopy, we obtain three-dimensional images of spindles, which allows us to trace the entire microtubule bundle. For each traced bundle, we first fit a plane and then fit a circle lying in that plane. With this robust method, we extract the curvature and twist, which represent the geometric information characteristic for each bundle. As the bundle shapes reflect the forces within them, this method is valuable for the understanding of forces that act on chromosomes during mitosis.     

Mutations of the withered (whd) gene in Drosophila melanogaster confer hypersensitivity to oxidative stress and are lesions of the carnitine palmitoyltransferase I (CPT I) gene.

Since some oxygen defense mutants of Drosophila melanogaster exhibit a crinkled wing phenotype, a screen was performed on strains bearing mutant alleles conferring a visible wing phenotype to determine whether any were hypersensitive to oxidative stress. One mutant, withered (whd), was found to be sensitive to both dietary paraquat and hyperoxia. New alleles of whd were induced on a defined genetic background and strains carrying these alleles were also found to be sensitive to oxidative stress. To identify the product of the whd gene we used a sequence-based positional candidate approach and by this method we determined that whd encodes carnitine palmitoyltransferase I (CPT I), an enzyme of the outer mitochondrial membrane that is required for the import of long-chain fatty acids into the mitochondria for beta-oxidation. Although this function is not vital under laboratory conditions, whd adults were found to be highly sensitive to starvation and to heavy metal toxicity relative to controls. This work uncovers a novel relationship between fatty acid metabolism and reactive oxygen metabolism. Further, these results in conjunction with past research on whd and on mammalian CPT I support the hypothesis that CPT I serves a vital function in the response to thymine supplementation.     

Carbon Quantum Dots as Multi-Purpose Nanomaterial in Stem Cell Therapy

During stem cell therapy, some issues, such as obscure fate of stem cells or their low survival rate in the body, should be addressed to boost their therapeutic efficiency. Nanotechnology offers a suitable solution to combat such limitations. Carbon quantum dots (CQDs) are carbon-based nanomaterials and may be used as multi-purpose compounds in stem cell therapy. CQDs are excellent choices for stem cell labeling thanks to their special features such as optical properties and good biocompatibility. Besides, they can modulate the biological function of stem cells, such as their proliferation, homing ability, and differentiation properties. Considering the charismatic feature of CQDs and their broad unique effect on stem cells, the current review aims to summarize the most advancements in this field. Hence, we first focused on CQDs synthesis and their applications. In the next section, the stem cell categories will be discussed, and the final part is dedicated to the recent research evaluating the impact of CQDs on stem cell therapy.     

Incidence of earlobe ptosis and pseudoptosis in patients seeking facial rejuvenation surgery and effects of aging

The authors have previously described a classification system for earlobe ptosis and have established a criterion for earlobe pseudoptosis. Earlobe heights were characterized based on anatomic landmarks, including the intertragal notch, the otobasion inferius (the most caudal anterior attachment of the earlobe to the cheek skin), and the subaurale (the most caudal extension of the earlobe free margin). The classification system was derived from earlobe height preferences as determined by a survey of North American Caucasians, and it identified the ideal free caudal lobule height range to measure 1 to 5 mm from otobasion inferius to subaurale (grade I ptosis). Also, earlobe pseudoptosis was defined by the attached cephalic lobule height measuring an intertragal notch to otobasion inferius distance greater than 15 mm. In this study, the preoperative earlobe height measurements of 44 patients seeking facial rejuvenation were evaluated. The average attached cephalic segment (intertragal notch to otobasion inferius distance) of patient earlobes measured 11.10 +/- 0.46 mm, and the average free caudal segment (otobasion inferius to subaurale distance) of patient earlobes measured 7.15 +/- 0.49 mm. Assessment of patient groups based on single-decade age differences demonstrated an increase in the free caudal segment (otobasion inferius to subaurale distance) with increasing age (p = 0.003). Assessment of patient groups based on single-decade age differences demonstrated no increase in the attached cephalic segment (intertragal notch to otobasion inferius distances) with increasing age (p = 0.281). When evaluating for the ideal otobasion inferius to subaurale distance, only 22.2 percent of earlobes demonstrated an ideal free caudal earlobe height (grade I ptosis). Moreover, pseudoptosis was detected in 12.3 percent of earlobes. Finally, a majority of earlobes demonstrated intrapatient variability, with only 16.2 percent of patients demonstrating identical attached cephalic segment (intertragal notch to otobasion inferius distances) and 37.8 percent demonstrating identical free caudal segment (otobasion inferius to subaurale distances) when compared with their contralateral ear. Plastic surgeons should be aware that a significant number of patients (77.8 percent of earlobes) may not possess an ideal free caudal segment and that 12.3 percent of earlobes may present with pseudoptosis. Therefore, earlobe height assessment should be an essential aspect of evaluation in patients desiring facial rejuvenation surgery. Evaluation of both ears should be performed independently due to intrapatient earlobe height variations. Finally, patients should be counseled with regard to the ideal earlobe parameters and aging patterns (stable attached cephalic segment versus increasing free caudal segment). With the natural progression of both facial rhytides and caudal segment earlobe ptosis (increasing free lobule segment) with increasing age, independent and accurate assessment of earlobe height is indicated so that the aging ear may be addressed concurrently with the aging face.