Assessment of changes in growth traits,oxidative stress parameters,and enzymatic and non-enzymatic antioxidant defense mechanisms in Lepidium draba plant under osmotic stress induced by polyethylene glycol

Protoplasma - Lepidium draba is a weed with the medicinal properties which few researches have been done on it. In this study, some traits, related to the osmotic stress, in 14-day-old L. draba...     

Calpain activity is essential in skin wound healing and contributes to scar formation

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.     

Phosphorylation and dephosphorylation of calsequestrin on CK2-sensitive sites in heart

Calsequestrin (CSQ) concentrates in junctional sarcoplasmic reticulum (SR) where it functions in regulation of Ca2+ release. When purified from heart tissue, cardiac CSQ contains phosphate on a cluster of C-terminal serine residues, but little is known about the cellular site of kinase action, and the identity of the kinase remains uncertain. To determine basic features of the phosphorylation, we examined the reaction in canine heart preparations. CSQ phosphorylation was observed in [32P]metabolically-labeled heart cells after adenoviral overexpression, and its constitutive phosphorylation was limited to a CK2-sensitive C-terminal serine cluster. The CSQ kinase was oriented intralumenally, as was CSQ, inside membrane vesicles, such that exposure to each required detergent permeabilization. Yet even after detergent permeabilization, CSQ was phosphorylated much less efficiently by protein kinase CK2 in cardiac microsomes than was purified CSQ. Reduced phosphorylation was strongly dependent upon protein concentration, and phosphorylation time courses revealed a phosphatase activity that occurred constitutively as phosphorylated substrate accumulates. Evidence of selective dephosphorylation of CSQ glycoforms in heart homogenates was also seen by mass spectrometry analysis. Molecules with greater mannose content, a feature of early secretory pathway compartments, were more highly phosphorylated, while greater dephosphorylation was apparent in more distal compartments. Taken together, the analyses of CSQ phosphorylation in heart suggest that a constitutive process of phosphate turnover occurs for cardiac CSQ perhaps associated with its intracellular transport.     

Tolerance of low pH in Schizosaccharomyces pombe requires a functioning pub1 ubiquitin ligase

A strain of Schizosaccharomyces pombe carrying a disrupted Na⁺/H⁺ antiporter gene (sod2::sup3-5), in addition to the common auxotrophic mutations, ade6-216, ura4-D18 and leu1-32, is highly sensitive to media adjusted to pH 6.9. Reversion analysis of this strain yielded a group of revertants capable of growth at pH 6.9. Two of the revertants elongated and failed to form colonies at pH 3.5. Genetic characterization of one of the pH-sensitive elongated strains, J227, showed the presence of two independently segregating mutations. One, pub1 (?protein ubiquitin ligase 1), has recently been reported as an E3 protein ubiquitin ligase involved in cdc25 turnover. The second has been named elp3-1 (elongated at low pH). Genetic dissection of the original strain revealed that poor growth at high pH was due to the presence of the auxotrophic markers, suggesting a possible inhibitory effect of high pH on the function of permeases responsible for uptake of the necessary nutrients. Suppression of the high pH sensitivity required the presence of both the pub1-1 and elp3-1 mutations. While the pub1-1 mutation reduced the capacity of cells to tolerate relatively moderate concentrations of LiCl (3?mM) in liquid culture, it was capable of partially suppressing the extreme Li⁺ sensitivity caused by the sod2 disruption. Under these conditions, the growth of pub1-1 sod2::ura4 double mutant cells was improved over that of either pub1-1 or sod2::ura4 cells. The elp3-1 mutation had no effect on the Li⁺ tolerance in either wild-type or sod2::ura4 backgrounds. pub1-1 cells are elongated and incapable of colony formation at pH 3.5. In contrast, elp3-1 cells are elongated at pH 3.5 and pH?5.5 (the normal pH of minimal medium) but can form colonies under both conditions. J227 cells are significantly longer than either single mutant at pH 3.5 and do not form colonies but are visually similar to elp3-1 cells at pH 5.5. Complementation cloning in the J227 background yielded a genomic clone of pub1, allowing us to define the intron-exon structure of the gene. Sequences with high homology to the predicted amino acid sequence of pub1 have been identified in Saccharomyces cerevisiae (RSP5/NPI1), human (hRPF1), mouse (mNedd4), and rat (rNedd4). Based on the nature of our mutant selection, the pH-sensitive phenotype of the strains selected, and the known involvement of RSP5/NPI1 in membrane permease turnover in S. cerevisiae, we hypothesize a role for pub1, either directly or indirectly, in regulating membrane transport processes. This is further supported by the broad range of effects that the pub1-1 mutation exerts on overall performance of cells at high and low external pH, and in the presence of toxic levels of Li⁺.     

Assessment of changes in physiological and biochemical traits in four pistachio rootstocks under drought,salinity and drought + salinity stresses

In this study, 7-month-old UCB-1, Badami, Ghazvini and Kale-Ghouchi pistachio rootstocks were exposed to control, drought, salinity and drought + salinity environments for 60 d. Total chlorophyll and total carotenoid contents decreased in all cultivars under drought, salinity and drought + salinity stresses. Under drought and salinity stresses, alone or in combination, Na⁺ and Cl⁻ ions increased in all four pistachio rootstocks, while K⁺ ion decreased only in Ghazvini and Kaleh-Ghouchi cultivars. The enzyme activities of ascorbate peroxidase, polyphenol oxidase, catalase and guaiacol peroxidase increased in all cultivars when subjected to all three stresses with the exception of the ascorbate peroxidase activity in Kale-Ghouchi cultivar during drought stress. Oxidative stress parameters including electrolyte leakage, malondialdehyde, other aldehydes and hydrogen peroxide increased under all three stress conditions in all genotypes. The content of proline, total free amino acids and total soluble carbohydrates were enhanced under drought, salinity and drought + salinity stresses, whereas the protein content decreased in all pistachio rootstocks. In all evaluated traits, except for the K⁺ ion content and APX activity, the highest impacts was seen for drought + salinity > salinity > drought stresses, respectively. For the first time, we have proven that K⁺ ion content has a positive correlation with the ascorbate peroxidase, polyphenol oxidase, catalase and guaiacol peroxidase enzymes activities under drought + salinity stress. Finally, based on the bi-plot and cluster analyses, we have selected the UCB-1 > Badami > Ghazvini > Kale-Ghouchi cultivars as the most tolerant pistachio rootstocks under drought + salinity stress, respectively.     

Quantitative imaging of alveolar recruitment with hyperpolarized gas MRI during mechanical ventilation

The aim of this study was to assess the utility of (3)He MRI to noninvasively probe the effects of positive end-expiratory pressure (PEEP) maneuvers on alveolar recruitment and atelectasis buildup in mechanically ventilated animals. Sprague-Dawley rats (n = 13) were anesthetized, intubated, and ventilated in the supine position ((4)He-to-O(2) ratio: 4:1; tidal volume: 10 ml/kg, 60 breaths/min, and inspiration-to-expiration ratio: 1:2). Recruitment maneuvers consisted of either a stepwise increase of PEEP to 9 cmH(2)O and back to zero end-expiratory pressure or alternating between these two PEEP levels. Diffusion MRI was performed to image (3)He apparent diffusion coefficient (ADC) maps in the middle coronal slices of lungs (n = 10). ADC was measured immediately before and after two recruitment maneuvers, which were separated from each other with a wait period (8-44 min). We detected a statistically significant decrease in mean ADC after each recruitment maneuver. The relative ADC change was -21.2 ± 4.1 % after the first maneuver and -9.7 ± 5.8 % after the second maneuver. A significant relative increase in mean ADC was observed over the wait period between the two recruitment maneuvers. The extent of this ADC buildup was time dependent, as it was significantly related to the duration of the wait period. The two postrecruitment ADC measurements were similar, suggesting that the lungs returned to the same state after the recruitment maneuvers were applied. No significant intrasubject differences in ADC were observed between the corresponding PEEP levels in two rats that underwent three repeat maneuvers. Airway pressure tracings were recorded in separate rats undergoing one PEEP maneuver (n = 3) and showed a significant relative difference in peak inspiratory pressure between pre- and poststates. These observations support the hypothesis of redistribution of alveolar gas due to recruitment of collapsed alveoli in presence of atelectasis, which was also supported by the decrease in peak inspiratory pressure after recruitment maneuvers.     

Regional correlation of emphysematous changes in lung function and structure: a comparison between pulmonary function testing and hyperpolarized MRI metrics

Regional and global relationships of lung function and structure were studied using hyperpolarized 3He MRI in a rat elastase-induced model of emphysema (n = 4) and healthy controls (n = 5). Fractional ventilation (r) and apparent diffusion coefficient (ADC) of 3He were measured at a submillimeter planar resolution in ventral, middle, and dorsal slices 6 mo after model induction. Pulmonary function testing (PFT) was performed before MRI to yield forced expiratory volume in 50 ms (FEV??), airway resistance (R(I)), and dynamic compliance (C(dyn)). Cutoff threshold values of ventilation and diffusion, r* and ADC*, were computed corresponding to 80% population of pixels falling above or below each threshold value, respectively. For correlation analysis, r* was compared with FEV??/functional residual capacity (FRC), R(I) and C(dyn), whereas ADC* was compared with FEV??/FRC, total lung capacity (TLC), and C(dyn). Regional correlation of r and ADC was evaluated by dividing each of the three lung slices into four quadrants. C(dyn) was significantly larger in elastase rats (0.92 ± 0.16 vs. 0.61 ± 0.12 ml/cmH?O). The difference of R(I) and FEV?? was insignificant between the two groups. The r* of healthy rats was significantly larger than the elastase group (0.42 ± 0.03 vs. 0.28 ± 0.06), whereas ADC* was significantly smaller in healthy animals (0.27 ± 0.04 vs. 0.36 ± 0.01 cm2/s). No systematic difference in these quantities was observed between the three lung slices. A significant 33% increase in ADC* and a significant 31% decline in r* for elastase rats was observed compared with a significant 51% increase in C(dyn) and a nonsignificant 26% decline in FEV??/FRC. Correlation of imaging and PFT metrics revealed that r and ADC divide the rats into two separate clusters in the sample space.     

Regional function-structure relationships in lungs of an elastase murine model of emphysema

Changes in lung function and structure were studied using hyperpolarized (3)He MRI in an elastase-induced murine model of emphysema. The combined analysis of the apparent diffusion coefficient (ADC) and fractional ventilation (R) were used to distinguish emphysematous changes and also to develop a model for classifying sections of the lung into diseased and normal. Twelve healthy male BALB/c mice (26 ± 2 g) were randomized into healthy and elastase-induced mice and studied ～8-11 wk after model induction. ADC and R were measured at a submillimeter planar resolution. Chord length (L(x)) data were analyzed from histology samples from the corresponding imaged slices. Logistic regression was applied to estimate the probability that an imaged pixel came from a diseased animal, and bootstrap methods (1,000 samples) were used to compare the regression results for the morphological and imaging results. Multivariate ANOVA (MANOVA) was used to analyze transformed ADC (ADC(BC)), and R (R(BC)) data and also to control for the experiment-wide error rate. MANOVA and ANOVA showed that elastase induced a statistically measureable change in the average transformed L(x) and ADC(BC) but not in the average R(BC). Marginal mean analysis demonstrated that ADC(BC) was on average 0.19 [95% confidence interval (CI): 0.16, 0.22] higher in the emphysema group, whereas R(BC) was on average 0.05 (95% CI: 0.04, 0.06) lower. Logistic regression supported the hypothesis that ADC(BC) and R(BC), together, were better at differentiating normal from diseased tissue than either measurement alone. The odds ratios for ADC(BC) and R(BC) were 7.73 (95% CI: 5.23, 11.42) and 9.14 × 10(-5) (95% CI: 3.33 × 10(-5), 25.06 × 10(-5)), respectively. Using a 50% probability cutoff, this model classified 70.6% of pixels correctly. The sensitivity and specificity of this model at the 50% cutoff were 74.9% and 65.2%, respectively. The area under the receiver operating characteristic curve was 0.76 (95% CI: 0.74, 0.78). The regression model presented can be used to map MRI data to disease probability maps. These probability maps present a future possibility of using both measurements in a more clinically feasible method of diagnosing this disease.