Sabera Ruzdijic

Effects of aging, dietary restriction and glucocorticoid treatment on housekeeping gene expression in rat cortex and hippocampus- : Evaluation by real time RT-PCR

Accurate normalization is the prerequisite for obtaining reliable results in the quantification of gene expression. Using TaqMan Real Time RT-PCR, we carried out an extensive evaluation of five most commonly used endogenous controls, gapdh, β-actin, 18S rRNA, hprt and cypB, for their presumed stability of expression, in rat cortex and hippocampus, during aging, under dietary restriction and dexamethasone treatment. Valid reference genes (HKGs) were identified using GeNorm and Norm-Finder software packages and by direct comparison of Ct values. Analysis revealed gapdh and β-actin as the most stable HKGs for all treatments analyzed, combined or separately, in the cortex, while in the hippocampus gapdh/hprt and β-actin/hprt are the combination of choice for the single or combined effects of dietary restriction/dexamethasone, respectively. All treatments significantly influenced expression of 18S rRNA and cypB in both structures. In addition, we used gapdh and normalization factor, calculated by GeNorm, to compare the expression of α-syn in the cortex. Our results demonstrate the importance of the right choice of HKG and suggest the appropriate endogenous control to be used for TaqMan RT-PCR analysis of mRNA expression in rat cortex and hippocampus for selected experimental paradigms.

Identification of genes associated with non-small-cell lung cancer promotion and progression

Lung cancer is the most common cause of neoplasia-related death worldwide. One of the crucial early events in carcinogenesis is the induction of genomic instability and mutator phenotype. We investigated genomic instability in 30 patients with non-small-cell lung cancer (NSCLC) by comparing DNA fingerprints of paired tumor and normal tissues using arbitrarily primed polymerase chain reaction (AP-PCR). Selected 21 DNA bands with altered mobility were isolated from polyacrylamide gels, cloned and sequenced. Obtained sequences were submitted to homology search in GenBank database which revealed the following genes: TSPAN14, CDH12, RDH10, CYP4Z1, KIR, E2F4, PHACTR3, PHF20, PRAME family member and SLC2A13. Following the identification of these genes we examined their relation to the clinicopathological parameters and survival of the patients. Our study revealed that genetic alterations of TSPAN14, SLC2A13 and PHF20 appeared prevalently in tumors of grade 1, stage I suggesting that structural changes of these genes could play a role in NSCLC promotion. Contrary to this CYP4Z1, KIR and RDH10 were prevalently mutated in tumors of grade 3, stage III suggesting that they could play a role in NSCLC progression. E2F4, PHACTR3, PRAME family member and CDH12 most probably play important role in NSCLC geneses. In conclusion, our study revealed altered genes previously not described in regard to this type of cancer.

Propofol-induced changes in neurotrophic signaling in the developing nervous system in vivo.

Several studies have revealed a role for neurotrophins in anesthesia-induced neurotoxicity in the developing brain. In this study we monitored the spatial and temporal expression of neurotrophic signaling molecules in the brain of 14-day-old (PND14) Wistar rats after the application of a single propofol dose (25 mg/kg i.p). The structures of interest were the cortex and thalamus as the primary areas of anesthetic actions. Changes of the protein levels of the brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), their activated receptors tropomyosin-related kinase (TrkA and TrkB) and downstream kinases Akt and the extracellular signal regulated kinase (ERK) were assessed by Western immunoblot analysis at different time points during the first 24 h after the treatment, as well as the expression of cleaved caspase-3 fragment. Fluoro-Jade B staining was used to follow the appearance of degenerating neurons. The obtained results show that the treatment caused marked alterations in levels of the examined neurotrophins, their receptors and downstream effector kinases. However, these changes were not associated with increased neurodegeneration in either the cortex or the thalamus. These results indicate that in the brain of PND14 rats, the interaction between Akt/ERK signaling might be one of important part of endogenous defense mechanisms, which the developing brain utilizes to protect itself from potential anesthesia-induced damage. Elucidation of the underlying molecular mechanisms will improve our understanding of the age-dependent component of anesthesia-induced neurotoxicity.

Synergistic effects of the purine analog sulfinosine and curcumin on the multidrug resistant human non-small cell lung carcinoma cell line (NCI-H460/R)

Multidrug resistance (MDR) is the main obstacle to a successful chemotherapy of lung cancer. We tested the potential of sulfinosine and curcumin, alone and in combination, for modulating MDR in the human resistant, non-small cell lung carcinoma cell line (NCI-H460/R). First, we determined the mutational status of the p53 gene in NCI-H460/R cells by PCR-SSCP and DNA sequencing and identified mutations which could at least partially contribute to the development of the MDR phenotype. The effects of sulfinosine and curcumin were studied, both separately and in combination, at the level of cytotoxicity, cell cycle distribution and gene expression. Sulfinosine displayed dose-dependent growth inhibition in both resistant and control sensitive cell lines, whereas curcumin considerably inhibited their growth only at relatively high doses. When sulfinosine was combined with a low dose of curcumin the drugs exerted a synergistic cytotoxic effect in NCI-H460/R cells. The expression of MDR-related genes mdr1, gst-π and topo ΙΙα, was altered by sulfinosine and curcumin. The most pronounced effect was observed when the agents were applied together. Sulfinosine and curcumin caused perturbations in cell cycle distribution in the NCI-H460/R cell line. The combination of the two drugs induced a more pronounced cell cycle arrest in S and G2/M in NCI-H460/R cells. Our results show that sulfinosine and curcumin overcome MDR in non-small cell lung carcinoma cell line (NSCLC), especially in combination despite the presence of a mutated p53 gene.

Isolation and Biological Evaluation of Jatrophane Diterpenoids from Euphorbia dendroides

From the Montenegrin spurge Euphorbia dendroides, seven new diterpenoids [jatrophanes (1-6) and a tigliane (7)] were isolated and their structures elucidated by spectroscopic techniques. The biological activity of the new compounds was studied against four human cancer cell lines. The most effective jatrophane-type compound (2) and its structurally closely related derivative (1) were evaluated for their interactions with paclitaxel and doxorubicin using a multi-drug-resistant cancer cell line. Both compounds exerted a strong reversal potential resulting from inhibition of P-glycoprotein transport.

α-Synuclein is expressed in different tissues during human fetal development

Abstractα-Synuclein is a small presynaptic protein associated with both normal synaptic plasticity and neurodegenerative processes. Its normal cellular function, however, remains unknown. Even though it is highly enriched in the brain, its presence was reported in other human adult tissues. In the present study, we examined tissue expression of α-synuclein in human and rat prenatal development. Using Western blot analysis, various peripheral tissues from 15 to 23 gestational weeks, human and E19 rat fetuses, along with human and rat adult tissues, were assayed. α-Synuclein expression was observed in all fetal human organs examined. In adult human tissues the high expression of α-synuclein was maintained in the brain, whereas in other organs the expression was greatly reduced. In contrast, both in fetal and adult rat tissues, α-synuclein was only detected in the brain. In addition to a 19-kDa α-synuclein band, 36- and 52-kDa immunoreactive bands were observed in all fetal and adult human organs, with the exception of the brain, but their identity remains to be determined. These findings suggest that apart from its function in development of the nervous system, α-synuclein has an important function in peripheral tissues as well during normal human prenatal development.

Characterization of 9L Glioma Model of the Wistar Rat

The aim of our study was to develop and characterize solid brain tumors in Wistar rats, which could be used in investigations concerning the molecular mechanisms that lay beneath the genesis of the gliomas as well as in the testing of curative potentials of various therapeutics.The tumors were induced by intracerebral inoculation of 9L glioma cells and characterized by morphometrical, histological and immunohistochemical analysis after 7, 14 and 21 postimplantation days. Immunohistochemical characterization included detection of the nuclear antigene Ki-67 as the proliferative cell marker, GFAP as a tracer of reactive gliosis surrounding the tumor mass, and CD4/CD8 and ED1 antigens, as markers of the immunological response.Our results showed that after 7 days all experimental animals developed solid, well-circumcised tumors, which were clearly separated from the surrounding brain tissue. Tumors showed progressive growth from the 7th to the 21st day despite the observed immunological response starting after 14 days. Histologically tumors were hypercellular with neovascularization and necrosis.These results indicate that reproducible morphometric evaluation can be performed on 9L tumors growing in immunocompetent Wistar rats, enabling its use as an animal tumor model for the evaluation of various therapeutic approaches.

GAP-43 mRNA expression in early development of human nervous system

The temporal and spatial distribution of GAP-43 mRNA in early human development, from 6 to 23 gestational weeks (g.w.), was examined by in situ hybridization histochemistry. GAP-43 mRNA was expressed as early as 6 g.w. in all regions of developing nervous system, the spinal cord, brainstem, cerebellum, diencephalic and telencephalic regions. Although the pronounced level of expression persisted during the entire examined period, the intensity of expression varied along the spatial axis over time. Analysis at the cellular level revealed that early on in development (6 g.w.) GAP-43 mRNA was expressed in the entire neuroblast population. With the onset of differentiation, at 13-23 g.w., GAP-43 mRNA expression had switched to the neurons that are in the process outgrowth. The highest level of GAP-43 mRNA expression was localized in the regions consisting of differentiating neurons, such as the cortical plate and intermediate zone of the telencephalic wall, and several delineated subcortical and thalamic nuclei. The spatial and temporal pattern of GAP-43 mRNA expression obtained suggests a possible dual role of GAP-43 in the development of the human nervous system: in the embryonic brain it could be involved in fundamental processes underlying cell proliferation; in the fetal brain its expression is specifically correlated with differentiation and the outgrowth of axons.

Regional and Temporal Profiles of Calpain and Caspase-3 Activities in Postnatal Rat Brain following Repeated Propofol Administration

Exposure of newborn rats to a variety of anesthetics has been shown to induce apoptotic neurodegeneration in the developing brain. We investigated the effect of the general anesthetic propofol on the brain of 7-day-old (P7) Wistar rats during the peak of synaptic growth. Caspase and calpain protease families most likely participate in neuronal cell death. Our objective was to examine regional and temporal patterns of caspase-3 and calpain activity following repeated propofol administration (20 mg/kg). P7 rats were exposed for 2, 4 or 6 h to propofol and killed 0, 4, 16 and 24 h after exposure. Relative caspase-3 and calpain activities were estimated by Western blot analysis of the proteolytic cleavage products of α-II-spectrin, protein kinase C and poly(ADP-ribose) polymerase 1. Caspase-3 activity and expression displayed a biphasic pattern of activation. Calpain activity changed in a region- and time-specific manner that was distinct from that observed for caspase-3. The time profile of calpain activity exhibited substrate specificity. Fluoro-Jade B staining revealed an immediate neurodegenerative response that was in direct relationship to the duration of anesthesia in the cortex and inversely related to the duration of anesthesia in the thalamus. At later post-treatment intervals, dead neurons were detected only in the thalamus 24 h following the 6-hour propofol exposure. Strong caspase-3 expression that was detected at 24 h was not followed by cell death after 2- and 4-hour exposures to propofol. These results revealed complex patterns of caspase-3 and calpain activities following prolonged propofol anesthesia and suggest that both are a manifestation of propofol neurotoxicity at a critical developmental stage.

Caloric Restriction Suppresses Microglial Activation and Prevents Neuroapoptosis Following Cortical Injury in Rats

Traumatic brain injury (TBI) is a widespread cause of death and a major source of adult disability. Subsequent pathological events occurring in the brain after TBI, referred to as secondary injury, continue to damage surrounding tissue resulting in substantial neuronal loss. One of the hallmarks of the secondary injury process is microglial activation resulting in increased cytokine production. Notwithstanding that recent studies demonstrated that caloric restriction (CR) lasting several months prior to an acute TBI exhibits neuroprotective properties, understanding how exactly CR influences secondary injury is still unclear. The goal of the present study was to examine whether CR (50% of daily food intake for 3 months) alleviates the effects of secondary injury on neuronal loss following cortical stab injury (CSI). To this end, we examined the effects of CR on the microglial activation, tumor necrosis factor-α (TNF-α) and caspase-3 expression in the ipsilateral (injured) cortex of the adult rats during the recovery period (from 2 to 28 days) after injury. Our results demonstrate that CR prior to CSI suppresses microglial activation, induction of TNF-α and caspase-3, as well as neurodegeneration following injury. These results indicate that CR strongly attenuates the effects of secondary injury, thus suggesting that CR may increase the successful outcome following TBI.