Sanja Manitašević Jovanović

Lymphocyte glucocorticoid receptor expression level and hormone-binding properties differ between war trauma-exposed men with and without PTSD.

OBJECTIVE Posttraumatic stress disorder (PTSD) has been shown to be associated with altered glucocorticoid receptor (GR) activity. We studied the expression and functional properties of the receptor in peripheral blood mononuclear cells (PBMCs) from non-traumatized healthy individuals (healthy controls; n=85), and war trauma-exposed individuals with current PTSD (n=113), with life-time PTSD (n=61) and without PTSD (trauma controls; n=88). The aim of the study was to distinguish the receptor alterations related to PTSD from those related to trauma itself or to resilience to PTSD. METHODS Functional status of the receptor was assessed by radioligand binding and lysozyme synthesis inhibition assays. The level of GR gene expression was measured by quantitative PCR and immunoblotting. RESULTS Current PTSD patients had the lowest, while trauma controls had the highest number of glucocorticoid binding sites (Bmax) in PBMCs. Hormone-binding potential (Bmax/KD ratio) of the receptor was diminished in the current PTSD group in comparison to all other study groups. Correlation between Bmax and KD that normally exists in healthy individuals was decreased in the current PTSD group. Contrasting Bmax data, GR protein level was lower in trauma controls than in participants with current or life-time PTSD. CONCLUSIONS Current PTSD is characterized by reduced lymphocyte GR hormone-binding potential and by disturbed compensation between Bmax and hormone-binding affinity. Resilience to PTSD is associated with enlarged fraction of the receptor molecules capable of hormone binding, within the total receptor molecule population in PBMCs.

Mineralocorticoid receptor and heat shock protein expression levels in peripheral lymphocytes from war trauma-exposed men with and without PTSD

Alterations in the number and functional status of mineralocorticoid (MR) and glucocorticoid receptors (GR) may contribute to vulnerability to posttraumatic stress disorder (PTSD). Corticosteroid receptors are chaperoned by heat shock proteins Hsp90 and Hsp70. We examined relations between corticosteroid receptor and heat shock protein expression levels, and related them with war trauma exposure, PTSD and resilience to PTSD. Relative levels of MR, Hsp90 and Hsp70 were determined by immunoblotting in lymphocytes from war trauma-exposed men with current PTSD (current PTSD group, n=113), with life-time PTSD (life-time PTSD group, n=61) and without PTSD (trauma control group, n=88), and from non-traumatized healthy controls (healthy control group, n=85). Between-group differences in MR, Hsp90 and Hsp70 levels and in MR/GR ratio were not observed. The level of MR was correlated with both Hsp90 and Hsp70 levels in trauma control and healthy control groups. On the other hand, GR level was correlated only with Hsp90 level, and this correlation was evident in current PTSD and trauma control groups. In conclusion, PTSD and exposure to trauma are not related to changes in lymphocyte MR, Hsp90 or Hsp70 levels, but may be associated with disturbances in corticosteroid receptors interaction with heat shock proteins.

Validation of endogenous controls for gene expression studies in peripheral lymphocytes from war veterans with and without PTSD

BackgroundSelection of appropriate endogenous control is a critical step in gene expression analysis. The aim of this study was to evaluate expression stability of four frequently used endogenous controls: β-actin, glyceraldehyde-3-phosphate dehydrogenase, β2-microglobulin and RNA polymerase II polypeptide A in peripheral blood mononuclear cells from war veterans with and without posttraumatic stress disorder (PTSD). The study was designed as to identify suitable reference gene(s) for normalization of gene expression in peripheral blood mononuclear cells in response to war trauma and/or PTSD.ResultsThe variability in expression of the four endogenous controls was assessed by TaqMan Real-time RT-PCR in peripheral blood mononuclear cells from: war veterans with current PTSD, those with lifetime PTSD, trauma controls and healthy subjects. Expression stability was analyzed by GeNorm and NormFinder software packages, and by direct comparison of Ct values. Both, GeNorm and NormFinder identified β-actin and glyceraldehyde-3-phosphate dehydrogenase as a pair of genes with the lowest stability value.ConclusionsThe combination of β-actin and glyceraldehyde-3-phosphate dehydrogenase appeared to be the most suitable reference for studying alterations in gene expression in peripheral blood mononuclear cells related to vulnerability and resilience to PTSD, as well as to trauma-provoked developing of this disorder and recovery from it. Using glyceraldehyde-3-phosphate dehydrogenase, β-actin and β2-microglobulin as individual endogenous controls would provide satisfactory data, while RNA polymerase II polypeptide A could not be recommended.

How do plants cope with oxidative stress in nature? A study on the dwarf bearded iris (Iris pumila)

Oxidative stress results from incongruity between the generation of toxic reactive oxygen species (ROS) and the availability of their scavengers—antioxidants. Although the short-term effects of this phenomenon are attracting much scientific attention, oxidative stress may influence an organism’s metabolism over the long (evolutionary) time scale as well. To disentangle the impact of strong light intensity from co-occurring abiotic stresses in creating adaptive responses in antioxidants and heat shock proteins (Hsps), an environment manipulation experiment was performed using a xerophyte clonal monocot, Iris pumila, native to semi-arid grasslands at the Deliblato Sands. This species is very tolerant to the combined effect of extreme abiotic stressors such as high light intensity, elevated soil surface temperatures, and scarcity of water, which commonly takes place in its natural habitats during the summer. By shading half of each selected clone, leaving the other half sun-exposed, we contrasted short-term effects of reduced daylight intensity with long-term effects of photo-oxidative stress. In both light treatments, the enzymatic activities of SOD and APX antioxidants were similar in magnitude, whereas those of CAT and POD significantly decreased in exposed compared to shaded leaves. Moreover, exposed leaves expressed a unique CAT isoform that differed biochemically from two CAT isoforms observed in shaded leaves. The content of non-enzymatic antioxidants, carotenoids (Car), remained constant with the reduction of light intensity, but their ratio to total chlorophylls (Chl) significantly decreased compared to that expressed in full sunlight. The abundance of Hsps was considerably greater in exposed than in shaded leaves, especially regarding the inducible isoforms, Hsp70 and Hsp90a, as were their proportions in relation to the constitutively expressed Hsp90b isoform. The presented results, thus, indicate that adaptive metabolic responses of I. pumila leaves to photo-oxidative stress entailed the high activity of two key enzymatic antioxidants, SOD and APX and the expression of a light-resistant CAT—to counteract the stress-mediated ROS accumulation, the increased Car to Chl ratio—to adjust the photosynthetic apparatus to the high light conditions, as well as the accelerated biosynthesis of heat shock proteins Hsp70 and Hsp90—to preserve the cellular proteostasis.

Differential expression of heat-shock proteins Hsp70 and Hsp90 in vegetative and reproductive tissues of Iris pumila

Tissue-specific variation in Hsp70 and Hsp90 expression was studied in vegetative (leaf) and reproductive organs (floral tube, ovary and stamen) of Iris pumila plants originating from a sun-exposed and a shaded natural population, which experienced similar growth conditions in an experimental garden. Western blot analysis revealed the presence of both the Hsps in all examined tissues, but at different amounts. In addition to Hsp90a and Hsp90b that were previously detected in vegetative tissues, three new immunospecific bands, designated herein as Hsp90c, Hsp90d and Hsp90e, were recognized with the same anti-Hsp90 antibody in the reproductive tissues. Apart from showing tissue-specific differences in the relative amount of Hsp70 and Hsp90, our study provides evidence that the degree of Hsps expression within the same tissue also depended on the habitat type that the I. pumila plants were derived from.

Phenotypic plasticity in response to environmental heterogeneity contributes to fluctuating asymmetry in plants: first empirical evidence

Fluctuating asymmetry (FA) is widely used to quantify developmental instability (DI) in ecological and evolutionary studies. It has long been recognized that FA may not exclusively originate from DI for sessile organisms such as plants, because phenotypic plasticity in response to heterogeneities in the environment might also produce FA. This study provides the first empirical evidence for this hypothesis. We reasoned that solar irradiance, which is greater on the southern side than on the northern side of plants growing in the temperate zone of the Northern Hemisphere, would cause systematic morphological differences and asymmetry associated with the orientation of plant parts. We used geometric morphometrics to characterize the size and shape of flower parts in Iris pumila grown in a common garden. The size of floral organs was not significantly affected by orientation. Shape and particularly its asymmetric component differed significantly according to orientation for three different floral parts. Orientation accounted for 10.4% of the total shape asymmetry within flowers in the falls, for 11.4% in the standards and for 2.2% in the style branches. This indicates that phenotypic plasticity in response to a directed environmental factor, most likely solar irradiance, contributes to FA of flowers under natural conditions. That FA partly results from phenotypic plasticity and not just from DI needs to be considered by studies of FA in plants and other sessile organisms.

Does Environmentally Contingent Variation in the Level of Molecular Chaperones Mirror a Biochemical Adaptation to Abiotic Stress

As Hochachka and Somero emphasized in their seminal book “Biochemical Adaptation: Mechanism and Process in Physiological Evolution“, the key question to be posed in the study of biochemical adaptation is: “How have living systems, which are based on a common set of biochemical structures and processes and subject to a common set of physical-chemical lows, been able to adapt to the enormously wide spectrum of environmental conditions found in the biosphere?“ (Hochachka & Somero, 2002). Given that the biosphere encompasses habitats with tremendously diverse combinations of physical, chemical, and biotic environmental factors, it seems reasonably to believe that the diversity of life forms that are observable in these habitats is the outcome of adaptations which have evolved to permit organisms the exploitation of nearly all land and water areas around the globe (Hochachka & Somero, 2002).