Volume 20, Issue 2

Human phospholipid scramblase 1 (hPLSCR1) is a Ca 2+ -dependent protein known to scramble phospholipids in the plasma membrane resulting in loss of membrane asymmetry. It has been reported that hPLSCR1 exhibits Ca 2+ - independent activity at low pH. However, the conformational changes induced at low pH leading to functional activation are not known. Our results showed that recombinant hPLSCR1 was functionally activated at low pH, which is similar to the behavior of natively extracted hPLSCR1. Tryptophan fluorescence measurements showed a decrease in Ca 2+ -binding affinity at low pH, although not at pH 5.5. Far and near UV-CD revealed that low pH induced structural changes, with a significant increase in the β-sheet content of the protein. At the physiological level, decreased hPLSCR1 expression was observed after a period of exposure to low pH. The effect occurred at the promoter level. The expression levels of hPLSCR1 directly correlated with the sensitivity of HEK293 to apoptosis. Based on these results, we conclude that the mechanisms of Ca 2+ - and pH-induced functional activation of hPLSCR1 are different and that hPLSCR1 expression regulated by low pH could provide insights into the role of hPLSCR1 in cancer progression.

Chondroitin sulfate (CS) is a ubiquitous component of the cell surface and extracellular matrix of animal tissues. CS chains are covalently bound to a core protein to form a proteoglycan, which is involved in various biological events including cell proliferation, migration, and invasion. Their functions are executed by regulating the activity of bioactive proteins, such as growth factors, morphogens, and cytokines. This review article focuses on the catabolism of CS. This catabolism predominantly occurs in lysosomes to control the activity of CS-proteoglycans. CS chains are fragmented by endo-type glycosidase(s), and the resulting oligosaccharides are then cleaved into monosaccharide moieties from the nonreducing end by exoglycosidases and sulfatases. However, the endo-type glycosidase responsible for the systemic catabolism of CS has not yet been identified. Based on recent advances in studies on hyaluronidases, which were previously considered to be hyaluronan-degrading enzymes, it appears that they recognize CS as their original substrate rather than hyaluronan and acquired hyaluronan-hydrolyzing activity at a relatively late stage of evolution.

Prostaglandin E 2 (PGE 2 ) is generated in various cells, including endothelial cells, and is responsible for various functions, such as vascular relaxation and angiogenesis. Effects of PGE 2 are mediated via receptors EP1-EP4, among which EP2 and EP4 are coupled to Gs protein which activates adenylate cyclase (AC) and cAMP synthesis. The aim of this work was to study the ability of human microvascular endothelial cells (HMEC-1) to synthesize cAMP in the presence of PGE 2 , and to determine the effect of hypoxia on the PGE 2 - stimulated cAMP level. It was decided to evaluate the effect of PGE 2 on the secretion of VEGF, an inducer of angiogenesis. In summary, our findings show that PGE 2 induces cAMP production, but hypoxia may impair PGE 2 -stimulated activity of the AC-cAMP signaling pathway. These results suggest that the cardioprotective effect of PGE 2 /EP4/cAMP may be attenuated during ischemia. Furthermore, this study indicates that the pro-angiogenic effect of PGE 2 is not associated with VEGF secretion in HMEC-1 cells.

Telomerase is involved in the elongation of telomeres. It remains active in very few types of cell in mature organisms. One such cell type is the lymphocytes. In this study, we investigated the activity and expression of telomerase in lymphocytes from renal failure patients and compared it to that for normal controls. Inflammation status was determined at the same time. The enzyme activity was measured using PCR-ELISA with peripheral blood mononuclear cells (PBMCs) from three groups: 53 healthy individuals, 50 patients with chronic kidney disease (CKD) and 50 dialysis patients. In the same cell populations, the expression of the reverse transcriptase of the human telomerase gene (hTERT) was measured via real-time PCR. The inflammationstatus of these individuals was determined by calculating the interleukin 6 (IL-6), IL-10, C-reactive protein (CRP) and tumor necrosis factor alpha (TNF-a) serum concentrations via ELISA. The lowest levels of telomerase activity were detected in CKD, and this group had the highest IL-6 and CRP values and the lowest hTERT expression. The dialysis group showed significant differences in comparison to the normal subjects and to the CKD patients. Further studies are warranted in order to explore the way inflammation influences telomerase activity and hTERT expression.

We previously characterized the β-actin gene promoter of Indian domesticated rohu carp (Labeo rohita) and made a reporter construct via fusion to green fluorescence protein (GFP) cDNA. In this study, the same construct was used to breed transgenic rohu fish. About 20% of the transgenic offspring showed ubiquitous expression of the reporter GFP gene. In a few of the transgenic fish, we documented massive epithelial and/or muscular expression with visible green color under normal light. The expression of GFP mRNA was higher in the muscle tissue of transgenic fish than in that of non-transgenic fish. A highly efficient nucleofection protocol was optimized to transfect proliferating spermatogonial cells of rohu using this reporter construct. The β-actin promoter also drove expressions in HEK293 (derived from human embryonic kidney cells), K562 (human leukemic cells) and SF21 (insect ovarian cells) lines. These findings imply conserved regulatory mechanisms of β-actin gene expression across eukaryotes. Furthermore, the isolated β-actin promoter with consensus regulatory elements has the potential to be used in generating transgenic carp with genes of interest and in basic biology research.

Biological systems are characterized by directional and precisely controlled flow of matter and information along with the maintenance of their structural patterns. This is possible thanks to sequential transformations of information, energy and structure carried out by molecular machines. The new perception of biological systems, including their mechanical aspects, requires the implementation of tools and approaches previously developed for engineering sciences. In this review paper, a biological system is presented in a new perspective as an ensemble of coordinated molecular devices functioning in the limited space confined by the biological membrane. The working of a molecular machine is presented using the example of F 0 F 1 ATPase, and the general conditions necessary for the coordination of a large number of functional units are described.

Pituitary adenylate cyclase activating polypeptide (PACAP) is widely expressed in the central and peripheral nervous system. PACAP can initiate multiple signaling pathways through binding with three class B G-protein coupled receptors, PAC1, VPAC1 and VPAC2. Previous studies have revealed numerous biological activities of PACAP in the nervous system. PACAP acts as a neurotransmitter, neuromodulator and neurotrophic factor. Recently, its neuroprotective potential has been demonstrated in numerous in vitro and in vivo studies. Furthermore, evidence suggests that PACAP might move across the blood-brain barrier in amounts sufficient to affect the brain functions. Therefore, PACAP has been examined as a potential therapeutic method for neurodegenerative diseases. The present review summarizes the recent findings with special focus on the models of Alzheimer’s disease (AD) and Parkinson’s disease (PD). Based on these observations, the administered PACAP inhibits pathological processes in models of AD and PD, and alleviates clinical symptoms. It thus offers a novel therapeutic approach for the treatment of AD and PD.

Pichia guilliermondii strain SO isolated from spoiled orange was developed for use as an alternative expression host by using Pichia pastoris as the model of the experiment. This is the first study to report on the capability of P. guilliermondii SO as a host to express thermostable T1 lipase from Geobacillus zalihae. Alcohol oxidase and formaldehyde dehydrogenase promoters were present in the yeast genome. Interestingly, the recombinant yeast [SO/pPICZαB/T1-2 (SO2)] took only 30 h to reach optimal production with minimal methanol induction [1.5% (v/v)] in YPTM medium, as compared to P. pastoris, which took longer to reach its optimal condition. The purification yield of the His-tagged fusion lipase was 68.58%, with specific activity of 194.58 U/mg. The optimum temperature was 65°C at pH 9 in glycine-NaOH buffer, and it was stable up to 70°C in a wide pH range from pH 5 to 12. In conclusion, a newly isolated yeast from spoiled orange has been proven suitable for use as an expression host.

Lamin A is an intermediate filament protein which is cleaved by the enzyme, FACE 1 at VTRSY↓L. The cleavage is the final step in the production of the mature lamin A protein. The mature lamin A protein localizes in the inner membrane of the nucleus. The mutation in the lamin A gene causes many diseases, including accelerated aging. It is known that the protein is not expressed in neuronal cells of the brain. Many splicing variants of the lamin A gene have been reported. In this study, the amino acid sequence VTRSY (a penta-peptide repeat) was found in three different sites of the C-terminal end of the lamin A protein, the protein expressed in cells of ear cartilage tissues is shorter than the protein expressed in cells of the skin tissues. Using two lamin A antibodies, it was found that the amino acid sequence between penta-peptide 2 and 3 is missing in lamin A protein that was expressed in the cells of mouse ear cartilage tissue, besides the RT-PCR data confirmed that the corresponding coding sequence between the penta repeat 2 and 3 is intact. Cleavage may occur at the penta-peptide (VTRSY) at site 3 in the lamin A tail of mouse ear cartilage.

Previous studies evidenced a significant reduction in serum cholesterol levels during an episode of acute inflammation. The aim of the present study was to verify the hypothesis of a regulatory role of cytokines through an in vitro model that simulates a situation of vascular inflammation and high levels of LDL or lipoperoxides. Human microvascular endothelial cells-1 were used in all experiments. The cells were exposed for 24 h to increasing doses of LDL, oxidized lipoprotein, and 8-isoprostane (in the absence or presence of SQ29.548, a TXA2 receptor antagonist). Moreover, LDL receptor and oxidized lipoprotein receptor expression analyzed after endothelial cells’ incubation with increasing doses of interleukin-6. The ELISA test and quantitative real-time PCR were performed. Endothelial cells showed a significant increase in interleukin-6 medium levels associated with LDL, oxidized LDL and with the degree of oxidation (absence or presence of SQ29.548), while 8-isoprostane did not. Treatment of human microvascular endothelial cells-1 for 24 h with increasing doses of interleukin-6 significantly enhanced LDL receptor and oxidized lipoprotein receptor-1 mRNA expression. Our data suggest the presence of a compensatory mechanism. The induction of a significant increase of IL-6 does not seem to be caused by the presence of the biological activity of 8-isoprostane.

Long non-coding RNAs (lncRNAs) have attracted considerable attention recently due to their involvement in numerous key cellular processes and in the development of various disorders. New high-throughput methods enable their study on a genome-wide scale. Numerous lncRNAs have been identified and characterized as important members of the biological regulatory network, with significant roles in regulating gene expression at the epigenetic, transcriptional and post-transcriptional levels. This paper summarizes the diverse mechanisms of action of these lncRNAs and looks at the study strategies in this field. A major challenge in future study is to establish more effective bioinformatics and experimental methods to explore the functions, detailed mechanisms of action and structures deciding the functional diversity of lncRNAs, since the vast majority remain unresolved.