Search results for: mitochondrial E3 ubiquitin ligase 1

Authors: Dona Pamoda W. Jayatunga, Veer Bala Gupta, Eugene Hone, Ralph N. Martins

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Being a neurodegenerative disorder, Alzheimer’s disease (AD) affects a majority of the elderly demented worldwide. The key risk factors for AD are age, metabolic syndrome, allele status of APOE gene, head injuries and lifestyle. The progressive nature of AD is characterized by symptoms of multiple cognitive deficits exacerbated over time, leading to death within a decade from clinical diagnosis. However, it is revealed that AD originates via a prodromal phase that spans from one to few decades before symptoms first manifest. The key pathological hallmarks of AD brains are deposition of amyloid beta (Aβ) plaques and neurofibrillary tangles (NFT). However, the yet unknown etiology of the disease fails to distinguish mitochondrial dysfunction between a cause or an outcome. The absence of early diagnosis tools and definite therapies for AD have permitted recruits of nutraceutical-based approaches aimed at reducing the risk of AD by modulating lifestyle or be used as preventive tools during AD prodromal state before widespread neurodegeneration begins. The objective of the present study was to investigate beneficial effects of luteolin, a plant-based flavone compound, against AD. The neuroprotective effects of luteolin on amyloid beta (Aβ) (1-42)-induced neurotoxicity was measured using cultured human neuroblastoma BE(2)-M17 cells. After exposure to 20μM Aβ (1-42) for 48 h, the neuroblastoma cells exhibited marked apoptotic death. Co-treatment of 20μM Aβ (1-42) with luteolin (0.5-5μM) significantly protected the cells against Aβ (1-42)-induced toxicity, as assessed by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4sulfophenyl)-2H-tetrazolium, inner salt; MTS] reduction assay and the lactate dehydrogenase (LDH) cell death assay. The results suggest that luteolin prevents Aβ (1-42)-induced apoptotic neuronal death. However, further studies are underway to determine its protective mechanisms in AD including the activity against tau hyperphosphorylation and mitochondrial dysfunction.

Keywords: Aβ (1-42)-induced toxicity, Alzheimer’s disease, luteolin, neuroblastoma cells

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Authors: Kaomud Tyagi, Rajasree Chakraborty, Shantanu Kundu, Devkant Singha, Kailash Chandra, Vikas Kumar

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The melon thrips, Thrips palmi is a serious pest of a wide range of agriculture crops and also act as vectors for plant viruses (genus Tospovirus, family Bunyaviridae). More molecular data on this species is required to understand the cryptic speciation and evolutionary affiliations. Mitochondrial genomes have been widely used in phylogenetic and evolutionary studies in insect. So far, mitogenomes of five thrips species (Anaphothrips obscurus, Frankliniella intonsa, Frankliniella occidentalis, Scirtothrips dorsalis and Thrips imaginis) is available in the GenBank database. In this study, we sequenced the first complete mitogenome T. palmi and compared it with available thrips mitogenomes. We assembled the mitogenome from the whole genome sequencing data generated using Illumina Hiseq2500. Annotation was performed using MITOS web-server to estimate the location of protein coding genes (PCGs), transfer RNA (tRNAs), ribosomal RNAs (rRNAs) and their secondary structures. The boundaries of PCGs and rRNAs was confirmed manually in NCBI. Phylogenetic analyses were performed using the 13 PCGs data using maximum likelihood (ML) in PAUP, and Bayesian inference (BI) in MrBayes 3.2. The complete mitogenome of T. palmi was 15,333 base pairs (bp), which was greater than the genomes of A. obscurus (14,890bp), F. intonsa (15,215 bp), F. occidentalis (14,889 bp) and S. dorsalis South Asia strain (SA1) (14,283 bp), but smaller than the genomes of T. imaginis (15,407 bp) and S. dorsalis East Asia strain (EA1) (15,343bp). Like in other thrips species, the mitochondrial genome of T. palmi was represented by 37 genes, including 13 PCGs, large and small ribosomal RNA (rrnL and rrnS) genes, 22 transfer RNA (tRNAs) genes (with one extra gene for trn-Serine) and two A+T-rich control regions (CR1 and CR2). Thirty one genes were observed on heavy (H) strand and six genes on the light (L) strand. The six tRNA genes (trnG,trnK, trnY, trnW, trnF, and trnH) were found to be conserved in all thrips species mitogenomes in their locations relative to a protein-coding or rRNA gene upstream or downstream. The gene arrangements of T. palmi is very close to T. imaginis except the rearrangements in tRNAs genes: trnR (arginine), and trnE (glutamic acid) were found to be located between cox3 and CR2 in T. imaginis which were translocated between atp6 and CR1 in T. palmi; trnL1 (Leucine) and trnS1(Serine) were located between atp6 and CR1 in T. imaginis which were translocated between cox3 and CR2 in T. palmi. The location of CR1 upstream of nad5 gene was suggested to be ancestral condition of the thrips species in subfamily Thripinae, was also observed in T. palmi. Both the Maximum likelihood (ML) and Bayesian Inference (BI) phylogenetic trees generated resulted in similar topologies. The T. palmi was clustered with T. imaginis. We concluded that more molecular data on the diverse thrips species from different hierarchical level is needed, to understand the phylogenetic and evolutionary relationships among them.

Keywords: thrips, comparative mitogenomics, gene rearrangements, phylogenetic analysis

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Authors: Julia Kapr, Andrea Rossi, Haribaskar Ramachandran, Marius Pollet, Ilka Egger, Selina Dangeleit, Katharina Koch, Jean Krutmann, Ellen Fritsche

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It is widely acknowledged that animal models do not always represent human disease. Especially human brain development is difficult to model in animals due to a variety of structural and functional species-specificities. This causes significant discrepancies between predicted and apparent drug efficacies in clinical trials and their subsequent failure. Emerging alternatives based on 3D in vitro approaches, such as human brain spheres or organoids, may in the future reduce and ultimately replace animal models. Here, we present a human induced pluripotent stem cell (hiPSC)-based 3D neural in a vitro disease model for the Cockayne Syndrome B (CSB). CSB is a rare hereditary disease and is accompanied by severe neurologic defects, such as microcephaly, ataxia and intellectual disability, with currently no treatment options. Therefore, the aim of this study is to investigate the molecular and cellular defects found in neural hiPSC-derived CSB models. Understanding the underlying pathology of CSB enables the development of treatment options. The two CSB models used in this study comprise a patient-derived hiPSC line and its isogenic control as well as a CSB-deficient cell line based on a healthy hiPSC line (IMR90-4) background thereby excluding genetic background-related effects. Neurally induced and differentiated brain sphere cultures were characterized via RNA Sequencing, western blot (WB), immunocytochemistry (ICC) and multielectrode arrays (MEAs). CSB-deficiency leads to an altered gene expression of markers for autophagy, focal adhesion and neural network formation. Cell migration was significantly reduced and electrical activity was significantly increased in the disease cell lines. These data hint that the cellular pathologies is possibly underlying CSB. By induction of autophagy, the migration phenotype could be partially rescued, suggesting a crucial role of disturbed autophagy in defective neural migration of the disease lines. Altered autophagy may also lead to inefficient mitophagy. Accordingly, disease cell lines were shown to have a lower mitochondrial base activity and a higher susceptibility to mitochondrial stress induced by rotenone. Since mitochondria play an important role in neurotransmitter cycling, we suggest that defective mitochondria may lead to altered electrical activity in the disease cell lines. Failure to clear the defective mitochondria by mitophagy and thus missing initiation cues for new mitochondrial production could potentiate this problem. With our data, we aim at establishing a disease adverse outcome pathway (AOP), thereby adding to the in-depth understanding of this multi-faced disorder and subsequently contributing to alternative drug development.

Keywords: autophagy, disease modeling, in vitro, pluripotent stem cells

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Authors: Bhavini Patel, Aslihan Ugun-Klusek, Ellen Billet

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The two main contributing factors to familial and idiopathic form of Parkinson’s disease (PD) are oxidative stress and altered proteolysis. Monoamine oxidase-A (MAO-A) plays a significant role in redox homeostasis by producing reactive oxygen species (ROS) via deamination of for example, dopamine. The ROS generated induces chemical modification of proteins resulting in altered biological function. The ubiquitin-proteasome system, which consists of three different types or proteolytic activity, namely “chymotrypsin-like” activity (CLA), “trypsin-like” activity (TLA) and “post acidic-like” activity (PLA), is responsible for the degradation of ubiquitinated proteins. Defects in UPS are known to be strongly correlated to PD. Herein, the effect of ROS generated by MAO-A on proteasome activity and the effects of proteasome inhibition on MAO-A protein levels in WT, mock and MAO-A overexpressed (MAO-A+) SHSY5Y neuroblastoma cell lines were investigated. The data in this study report increased proteolytic activity when MAO-A protein levels are significantly increased, in particular CLA and PLA. Additionally, 20S proteasome inhibition induced a decrease in MAO-A levels in WT and mock cells in comparison to MAO-A+ cells in which 20S proteasome inhibition induced increased MAO-A levels to be further increased at 48 hours of inhibition. This study supports the fact that MAO-A could be a potential pharmaceutical target for neuronal protection as data suggests that endogenous MAO-A levels may be essential for modulating cell death and survival.

Keywords: monoamine oxidase, neurodegeneration, Parkinson's disease, proteasome

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Authors: Gagandeep Singh Digra, Pawan Kumar, Mandeep Kaur Sidhu

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INTRODUCTION: Chronic Progressive External Ophthalmoplegia (CPEO), also known as Progressive External Ophthalmoplegia (PEO), is a type of eye disorder characterized by a loss of the muscle functions involved in eye and eyelid movement. CPEO can be caused by mutations in mitochondrial DNA. It typically manifests in young adults with bilateral and progressive ptosis as the most common presentation but can also present with difficulty swallowing (dysphagia) and general weakness of the skeletal muscles (myopathy), particularly in the neck, arms, or legs. CASE PRESENTATION: This is a case discussion of 3 cousins who presented to our clinic. A 23-year-old male with past surgical history (PSH) of ptosis repair 2 years ago presented with a chief complaint of nasal intonation for 1.5 years associated with difficulty swallowing. The patient also complained of nasal regurgitation of liquids. He denied any headaches, fever, seizures, weakness of arms or legs, urinary complaints or changes in bowel habits. Physical Examination was positive for facial muscle weakness, including an inability to lift eyebrows (Frontalis), inability to close eyes tightly (Orbicularis Oculi), corneal reflex absent bilaterally, difficulty clenching jaw (Masseter muscle), difficulty smiling (Zygomaticus major), inability to elevate upper lip (Zygomaticus minor). Another cousin of the first patient, a 25-year-old male with no past medical history, presented with complaints of nasal intonation for 2 years associated with difficulty swallowing. He denied a history of nasal regurgitation, headaches, fever, seizures, weakness, urinary complaints or changes in bowel habits. Physical Examination showed facial muscle weakness of the Frontalis muscle, Orbicularis Oculi muscle, Masseter Muscle, Zygomaticus Major, Zygomaticus Minor and absent corneal reflexes. A 28-year-old male, a cousin of the first two patients, presented with chief complaints of ptosis and nasal intonation for the last 8 years. He also complained of difficulty swallowing and nasal regurgitation of liquids. His physical examination showed facial muscle weakness, including frontalis muscle (inability to lift eyebrows), Orbicularis Oculi (inability to close eyes tightly), absent corneal reflexes bilaterally, Zygomaticus Major (difficulty smiling), and Zygomaticus Minor (inability to elevate upper lip). MRI brain and visual field of all the patients were normal. Differential diagnoses, including Grave’s disease, Myasthenia Gravis and Glioma, were ruled out. Due to financial reasons, muscle biopsy could not be pursued. Pedigree analysis revealed only males were affected, likely due to maternal inheritance, so the clinical diagnosis of CPEO was made. The patients underwent symptomatic management, including ptosis surgical correction for the third patient. CONCLUSION: Chronic Progressive External Ophthalmoplegia (CPEO), a rare case entity, occurs in young adults as a manifestation of mitochondrial myopathy. There are three modes of transmission- maternal transmission associated with mitochondrial point mutations, autosomal recessive, and autosomal dominant. CPEO can sometimes be difficult to diagnose, especially in asymmetric presentation. Therefore, it is crucial to keep it in differential diagnosis to avoid delay in diagnosis.

Keywords: neurology, chronic, progressive, ophthalmoplegia

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Authors: Kakali Bhadra

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Harmalol administration caused remarkable reduction in proliferation of HepG₂ cells with GI₅₀ of 14.2 mM, without showing much cytotoxicity in embryonic liver cell line, WRL-68. Data from circular dichroism and differential scanning calorimetric analysis of harmalol-CT DNA complex shows conformational changes with prominent CD perturbation and stabilization of CT DNA by 8 ^oC. Binding constant and stoichiometry was also calculated using the above biophysical techniques. Further, dose dependent apoptotic induction ability of harmalol was studied in HepG₂ cells using different biochemical assays. Generation of ROS, DNA damage, changes in cellular external and ultramorphology, alteration of membrane, formation of comet tail, decreased mitochondrial membrane potential and a significant increase in Sub G_o/G₁ population made the cancer cell, HepG₂, prone to apoptosis. Up regulation of p53 and caspase 3 further indicated the apoptotic role of harmalol.

Keywords: apoptosis, beta carboline alkaloid, comet assay, cytotoxicity, ROS

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Authors: A. S. Vidyashree, C. M. Kalleshwaraswamy, R. Asokan, H. M. Mahadevaswamy

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Termites are very vital ecological thespians in tropical ecosystem, having been designated as “ecosystem engineers”, due to their significant role in providing soil ecosystem services. Despite their importance, our understanding of a number of their basic biological processes in termites is extremely limited. Developing a better understanding of termite biology is closely dependent upon consistent species identification. At present, identification of termites is relied on soldier castes. But for many species, soldier caste is not reported, that creates confusion in identification. The use of molecular markers may be helpful in estimating phylogenetic relatedness between the termite species and estimating genetic differentiation among local populations within each species. To understand this, termites samples were collected from various places of Western Ghats covering four states namely Karnataka, Kerala, Tamil Nadu, Maharashtra during 2013-15. Termite samples were identified based on their morphological characteristics, molecular characteristics, or both. Survey on the termite fauna in Karnataka, Kerala, Maharashtra and Tamil Nadu indicated the presence of a 16 species belongs to 4 subfamilies under two families viz., Rhinotermitidae and Termitidae. Termititidae was the dominant family which was belonging to 4 genera and four subfamilies viz., Macrotermitinae, Amitermitinae, Nasutitermitinae and Termitinae. Amitermitinae had three species namely, Microcerotermes fletcheri, M. pakistanicus and Speculitermes sinhalensis. Macrotermitinae had the highest number of species belonging two genera, namely Microtermes and Odontotermes. Microtermes genus was with only one species i.e., Microtermes obesi. The genus Odontotermes was represented by the highest number of species (07), namely, O. obesus was the dominant (41 per cent) and the most widely distributed species in Karnataka, Karala, Maharashtra and Tamil nadu followed by O. feae (19 per cent), O.assmuthi (11 per cent) and others like O. bellahunisensis O. horni O. redemanni, O. yadevi. Nasutitermitinae was represented by two genera namely Nasutitermes anamalaiensis and Trinervitermes biformis. Termitinae subfamily was represented by Labiocapritermes distortus. Rhinotermitidae was represented by single subfamily Heterotermetinae. In Heterotermetinae, two species namely Heterotermes balwanthi and H. malabaricus were recorded. Genetic relationship among termites collected from various locations of Western Ghats of India was characterized based on mitochondrial DNA sequences (12S, 16S, and COII). Sequence analysis and divergence among the species was assessed. These results suggest that the use of both molecular and morphological approaches is crucial in ensuring accurate species identification. Efforts were made to understand their evolution and to address the ambiguities in morphological taxonomy. The implication of the study in revising the taxonomy of Indian termites, their characterization and molecular comparisons between the sequences are discussed.

Keywords: isoptera, mitochondrial DNA sequences, rhinotermitidae, termitidae, Western ghats

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Authors: Svitlana Antonenko, Gennady Telegeev

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Introductive statement: The development of chronic myeloid leukemia (CML) is caused by Bcr-Abl oncoprotein. Modern treatments with tyrosine kinase inhibitors are greatly complicated by the mutational variability of the Bcr-Abl oncoprotein, which causes drug resistance. Therefore, there is an urgent need to develop new approaches to the treatment of the disease, which will allow modeling the level of Bcr-Abl oncoprotein in the cell. Promising in this direction is the identification of proteases that can selectively promote cellular proteolysis of oncoproteins. The aim of the study was to study the effect of the interaction of Bcr-Abl with deubiquitinase USP1 on the level of oncoprotein in CML cells. Methodology: K562 cells were selected for the experiment. Сells were incubated with ML323 inhibitor for 24 hours. Precipitation of endogenous proteins from K562 cell lysate was performed using anti-Bcr-Abl antibodies. Cell lysates and precipitation results were studied by Western blot. Subcellular localization of proteins was studied by immunofluorescence analysis followed by confocal microscopy. The results were analyzed quantitatively and statistically. Major findings: The Bcr-Abl/USP1 protein complex was detected in CML cells, and it was found that inhibition of USP1 deubiquitinating activity by the compound ML323 leads to disruption of this protein complex and a decrease in the level of Bcr-Abl oncoprotein in cells. The interaction of Bcr-Abl with USP1 may result in deubiquitination of the oncoprotein, which disrupts its proteasomal degradation and leads to the accumulation of CML in cells. Conclusion: We believe that the interaction of oncoprotein with USP1 may be one of the prerequisites that contribute to malignant cell transformation due to the deubiquitination of oncoprotein, which leads to its accumulation and disease progression. A correlation was found between the deubiquitinating activity of USP1 and the level of oncoprotein in CML cells. Thus, we identify deubiquitinase USP1 as a promising therapeutic target for the development of a new strategy for the treatment of CML by modulating the level of Bcr-Abl in the cell.

Keywords: chronic myeloid leukemia, Bcr-Abl, USP1, deubiquitination Bcr-Abl, K562 cell

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Authors: Othman E. Othman, Agnés Germot, Daniel Petit, Abderrahman Maftah

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Threats to the biodiversity are increasing due to the loss of genetic diversity within the species utilized in agriculture. Due to the progressive substitution of the less productive, locally adapted and native breeds by highly productive breeds, the number of threatened breeds is increased. In these conditions, it is more strategically important than ever to preserve as much the farm animal diversity as possible, to ensure a prompt and proper response to the needs of future generations. Mitochondrial (mtDNA) sequencing has been used to explain the origins of many modern domestic livestock species. Studies based on sequencing of sheep mitochondrial DNA showed that there are five maternal lineages in the world for domestic sheep breeds; A, B, C, D and E. Because of the eastern location of Egypt in the Mediterranean basin and the presence of fat-tailed sheep breeds- character quite common in Turkey and Syria- where genotypes that seem quite primitive, the phylogenetic studies of Egyptian sheep breeds become particularly attractive. We aimed in this work to clarify the genetic affinities, biodiversity and phylogeny of five Egyptian sheep breeds using cytochrome B sequencing. Blood samples were collected from 63 animals belonging to the five tested breeds; Barki, Rahmani, Ossimi, Saidi and Sohagi. The total DNA was extracted and the specific primer allowed the conventional PCR amplification of the cytochrome B region of mtDNA (approximately 1272 bp). PCR amplified products were purified and sequenced. The alignment of Sixty-three samples was done using BioEdit software. DnaSP 5.00 software was used to identify the sequence variation and polymorphic sites in the aligned sequences. The result showed that the presence of 34 polymorphic sites leading to the formation of 18 haplotypes. The haplotype diversity in five tested breeds ranged from 0.676 in Rahmani breed to 0.894 in Sohagi breed. The genetic distances (D) and the average number of pairwise differences (Dxy) between breeds were estimated. The lowest distance was observed between Rahmani and Saidi (D: 1.674 and Dxy: 0.00150) while the highest distance was observed between Ossimi and Sohagi (D: 5.233 and Dxy: 0.00475). Neighbour-joining (Phylogeny) tree was constructed using Mega 5.0 software. The sequences of the 63 analyzed samples were aligned with references sequences of different haplogroups. The phylogeny result showed the presence of three haplogroups (HapA, HapB and HapC) in the 63 examined samples. The other two haplogroups described in literature (HapD and HapE) were not found. The result showed that 50 out of 63 tested animals cluster with haplogroup B (79.37%) whereas 7 tested animals cluster with haplogroup A (11.11%) and 6 animals cluster with haplogroup C (9.52%). In conclusion, the phylogenetic reconstructions showed that the majority of Egyptian sheep breeds belonging to haplogroup B which is the dominant haplogroup in Eastern Mediterranean countries like Syria and Turkey. Some individuals are belonging to haplogroups A and C, suggesting that the crosses were done with other breeds for characteristic selection for growth and wool quality.

Keywords: cytochrome B, diversity, phylogheny, Egyptian sheep breeds

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Authors: Omar S. Sharaf

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The mitochondrial 12s rRNA (mt-12s rDNA) gene for pig-specific was developed to detect material from pork species in different products collected from Jordanian market. The amplification PCR products of 359 bp and 531 bp were successfully amplified from the cyt b gene of pig the amplification product using mt-12S rDNA gene were successfully produced a single band with a molecular size of 456 bp. In the present work, the PCR amplification of mtDNA of cytochrome b has been shown as a suitable tool for rapid detection of pig DNA. 100 samples from different dairy, gelatin and chocolate based products and 50 samples from baby food formula were collected and tested to a presence of any pig derivatives. It was found that 10% of chocolate based products, 12% of gelatin and 56% from dairy products and 5.2% from baby food formula showed single band from mt-12S rDNA gene.

Keywords: halal food, baby infant formula, chocolate based products, PCR, Jordan

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Authors: M. Rajasekar, K. Suresh

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Diosmin is a flavonoid, most abundantly found in many citrus fruits. As a flavonoid, it possesses a multitude of biological activities including anti-hyperglycemic, anti-lipid peroxidative, anti-inflammatory, antioxidant, and anti-mutagenic properties. At this point, we established the anti-proliferative and apoptosis-inducing activities of diosmin in Hep-2 and KB cells. Diosmin has cytotoxic effects through inhibiting cellular proliferation of Hep-2 and KB cells, which leads to the induction of apoptosis, as apparent by an increase in the fraction of cells in the sub-G1phase of the cell cycle. Results exposed that inhibition of cell proliferation is associated with regulation of the Interleukins/STAT pathway. In addition, Diosmin treatment with Hep-2 and KB cells actively stimulated reactive oxygen species (ROS) and mitochondrial membrane depolarization. And also an imbalance in the Bax/Bcl-2 ratio triggered the caspase cascade and shifting the balance in favor of apoptosis. These observations conclude that Diosmin induce apoptosis via Interleukins /STAT-mediated pathway.

Keywords: diosmin, apoptosis, antioxidant, STAT pathway

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Authors: B. M. Md-Zain, N. A. Rahman, M. A. B. Abdul-Latiff, W. M. R. Idris

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We conducted molecular research to portray phylogenetic relationships of Malaysian primates particularly in the genus of Macaca. We have sequenced cytochrome C oxidase subunit I (COI) of mitochondrial DNA of several individuals from M. fascicularis and M. arctoides. PCR amplifications were performed and COI DNA sequences were aligned using ClustalW. Phylogenetic trees were constructed using distance analyses by employing neighbor-joining algorithm (NJ). We managed to sequence 700 bp of COI DNA sequences. The tree topology showed that M. fascicularis did not clump based on phyleogeography division in Peninsular Malaysia. Individuals from Negeri Sembilan merged together with samples from Perak and Penang into one clade. In addition, phylogenetic analyses indicated that M. arctoides was classified into sinica group instead of fascicularis group supported by genetic distance data. COI gene is an effective locus to clarify phylogenetic position of M. arctoides but not in discriminating M. fascicularis population in Peninsular Malaysia.

Keywords: cercopithecine, long-tailed macaque, Macaca fascicularis, Macaca arctoides

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Authors: Carmen E. Zapata, Juan Bautista, Olga Montoya, Claudia Moreno, Marisol Suarez, Alejandra Betancur, Duvan Nanclares, Natalia A. Cano

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This study aims to evaluate the diversity of microorganisms in filters PM2.5 and PM10; and determine the genotoxic and cytotoxic activity of the complex mixture present in PM2.5 filters used in the Aburrá Valley Air Quality Monitoring Network (Colombia). The research results indicate that particulate matter PM2.5 of different monitoring stations are bacteria; however, this study of detection of bacteria and their phylogenetic relationship is not complete evidence to connect the microorganisms with pathogenic or degrading activities of compounds present in the air. Additionally, it was demonstrated the damage induced by the particulate material in the cell membrane, lysosomal and endosomal membrane and in the mitochondrial metabolism; this damage was independent of the PM2.5 concentrations in almost all the cases.

Keywords: cytotoxic, genotoxic, microbiological analysis, PM10, PM2.5

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Authors: Arindam Pramanik, Parimal Karmakar

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We have explored the synergistic anti-cancer activity of copper ion and acetylacetone complex containing 1,3 diketone group (like curcumin) in metallorganic compound “Copper acetylacetonate” (CuAA). The cytotoxicity mechanism of CuAA complex was evaluated on various cancer cell lines in vitro. Among these, reactive oxygen species (ROS), glutathione level (GSH) in the cell was found to increase. Further mitochondrial membrane damage was observed. The fate of cell death was found to be induced by apoptosis. For application purpose, we have developed a novel biodegradable, non-toxic polymer-based nanoparticle which has hydrophobically modified core for loading of the CuAA. Folic acid is conjugated on the surface of the polymer (chitosan) nanoparticle for targeting to cancer cells for minimizing toxicity to normal cells in-vivo. Thus, this novel drug CuAA has an efficient anticancer activity which has been targeted specifically to cancer cells through polymer nanoparticle.

Keywords: anticancer, apoptosis, copper nanoparticle, targeted drug delivery

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Authors: Marcel Verwey, Anna M. Joubert, Elsie M. Nolte, Wolfgang Dohle, Barry V. L. Potter, Anne E. Theron

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A tetrahydroisoquinolinone (THIQ) core can be used to mimic the A,B-ring of colchicine site-binding microtubule disruptors such as 2-methoxyestradiol in the design of anti-cancer agents. Steroidomimeric microtubule disruptors were synthesized by introducing C'2 and C'3 of the steroidal A-ring to C'6 and C'7 of the THIQ core and by introducing a decorated hydrogen bond acceptor motif projecting from the steroidal D-ring to N'2. For this in vitro study, four non-steroidal THIQ-based analogues were investigated and comparative studies were done between the non-sulphamoylated compound STX 3450 and the sulphamoylated compounds STX 2895, STX 3329 and STX 3451. The objective of this study was to investigate the modes of cell death induced by these four THIQ-based analogues in A549 lung carcinoma epithelial cells and metastatic breast adenocarcinoma MDA-MB-231 cells. Cytotoxicity studies to determine the half maximal growth inhibitory concentrations were done using spectrophotometric quantification via crystal violet staining and lactate dehydrogenase (LDH) assays. Microtubule integrity and morphologic changes of exposed cells were investigated using polarization-optical transmitted light differential interference contrast microscopy, transmission electron microscopy and confocal microscopy. Flow cytometric quantification was used to determine apoptosis induction and the effect that THIQ-based analogues have on cell cycle progression. Signal transduction pathways were elucidated by quantification of the mitochondrial membrane integrity, cytochrome c release and caspase 3, -6 and -8 activation. Induction of autophagic cell death by the THIQ-based analogues was investigated by morphological assessment of fluorescent monodansylcadaverine (MDC) staining of acidic vacuoles and by quantifying aggresome formation via flow cytometry. Results revealed that these non-steroidal microtubule disrupting analogues inhibited 50% of cell growth at nanomolar concentrations. Immunofluorescence microscopy indicated microtubule depolarization and the resultant mitotic arrest was further confirmed through cell cycle analysis. Apoptosis induction via the intrinsic pathway was observed due to depolarization of the mitochondrial membrane, induction of cytochrome c release as well as, caspase 3 activation. Potential involvement of programmed cell death type II was observed due to the presence of acidic vacuoles and aggresome formation. Necrotic cell death did not contribute significantly, indicated by stable LDH levels. This in vitro study revealed the induction of the intrinsic apoptotic pathway as well as possible involvement of autophagy after exposure to these THIQ-based analogues in both MDA-MB-231- and A549 cells. Further investigation of this series of anticancer drugs still needs to be conducted to elucidate the temporal, mechanistic and functional crosstalk mechanisms between the two observed programmed cell deaths pathways.

Keywords: apoptosis, autophagy, cancer, microtubule disruptor

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Authors: Imad H. Hameed, Mohammad A. Jebor, Ammera J. Omer

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The aim of this research is to study the mitochonderial coding region by using the Sanger sequencing technique and establish the degree of variation characteristic of a fragment. FTA® Technology (FTA™ paper DNA extraction) utilized to extract DNA. Portion of coding region encompassing positions 11719 –12384 amplified in accordance with the Anderson reference sequence. PCR products purified by EZ-10 spin column then sequenced and Detected by using the ABI 3730xL DNA Analyzer. Five new polymorphic positions 11741, 11756, 11878, 11887 and 12133 are described may be suitable sources for identification purpose in future. The calculated value D= 0.95 and RMP=0.048 of the genetic diversity should be understood as high in the context of coding function of the analysed DNA fragment. The relatively high gene diversity and a relatively low random match probability were observed in Iraq population. The obtained data can be used to identify the variable nucleotide positions characterized by frequent occurrence which is most promising for various identifications.

Keywords: coding region, Iraq, mitochondrial DNA, polymorphic positions, sanger technique

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Authors: Ayesha Imtiaz, Darlina Md. Naim

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Family Nemipteridae is among the most abundantly distributed family in Malaysian fish markets due to its high contribution to landing sites of Malaysia. Using an advanced molecular approach that used two mitochondrial (Cytochrome oxidase c I and Cytochrome oxidase b) and one nuclear gene (Recombination activating gene, RAGI) to expose cryptic diversity and phylogenetic relationships among commercially important species of family Nemipteridae. Our research covered all genera (including 31 species out total 45 species) of family Nemipteridae, distributed in Malaysia. We also found certain type of geographical barriers in the South China sea that reduces dispersal and stops a few species to intermix. Northside of the South China Sea (near Vietnam) does not allow genetic diversity to mix with the Southern side of the South China sea (Sarawak) and reduces dispersal. Straits of Malacca reduce the intermixing genetic diversity of South China Sea and the Indian Ocean.

Keywords: Nemipteridae, RAG I, south east Asia, Malaysia

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Authors: K. Suresh, R. Arunkumar

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The objective of this study is to investigate the preparation of gramine nano suspension and protective effect of Gramine on the apoptosis of laryngeal cancer cells cell line (HEp-2 and KB). The growth inhibition rate of Hep-2 and KB cells in vitro were measured by MTT assay and apoptosis by, levels of reactive oxygen species, mitochondrial membrane potential, morphological changes and flowcytometry. Based on the results, we determined the effective doses of gramine as 127.23µm/ml for 24 hr and 119.81 µm/ml for 48hr in hep-2 cell line and 147.58 µm ml for 24 hr and 123.74µm µm/ml for 48hr in KB cell line. cytotoxicity effects of gramine were confirmed by treatment of HEp-2 cell and KB cell with IC50 concentration of gramine resulted in sequences of events marked by the enhance the apoptosis accompanied by loss of cell viability, modulation of reactive oxygen species and cell cycle arrest through the induction of G0/G1 phase arrest on HEp-2 cells. Our study suggests that the nanosuspension of gramine possesses the more cytotoxic effect of cancer cells and a novel candidate for cancer chemoprevention.

Keywords: apoptosis, HEp-2 cell line, KB cell line mitochondria, gramine, nanosuspension

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Authors: Welder A. Baldassini, Jon J. Ramsey, Marcos R. Chiaratti, Amália S. Chaves, Renata H. Branco, Sarah F. M. Bonilha, Dante P. D. Lanna

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With increased production costs there is a need for animals that are more efficient in terms of meat production. In this context, the role of mitochondrial DNA (mtDNA) on physiological processes in liver, muscle and adipose tissues may account for inter-animal variation in energy expenditures and heat production. The purpose this study was to investigate if the amounts of mtDNA in liver, muscle and adipose tissue (subcutaneous and visceral depots) of Nellore bulls are associated with residual feed intake (RFI) and estimated heat production (EHP). Eighteen animals were individually fed in a feedlot for 90 days. RFI values were obtained by regression of dry matter intake (DMI) in relation to average daily gain (ADG) and mid-test metabolic body weight (BW). The animals were classified into low (more efficient) and high (less efficient) RFI groups. The bulls were then randomly distributed in individual pens where they were given excess feed twice daily to result in 5 to 10% orts for 90 d with diet containing 15% crude protein and 2.7 Mcal ME/kg DM. The heart rate (HR) of bulls was monitored for 4 consecutive days and used for calculation of EHP. Electrodes were fitted to bulls with stretch belts (POLAR RS400; Kempele, Finland). To calculate oxygen pulse (O2P), oxygen consumption was obtained using a facemask connected to the gas analyzer (EXHALYZER, ECOMedics, Zurich, Switzerland) and HR were simultaneously measured for 15 minutes period. Daily oxygen (O2) consumption was calculated by multiplying the volume of O2 per beat by total daily beats. EHP was calculated multiplying O2P by the average HR obtained during the 4 days, assuming 4.89 kcal/L of O2 to measure daily EHP that was expressed in kilocalories/day/kilogram metabolic BW (kcal/day/kg BW0.75). Blood samples were collected between days 45 and 90th after the beginning of the trial period in order to measure the concentration of hemoglobin and hematocrit. The bulls were slaughtered in an experimental slaughter house in accordance with current guidelines. Immediately after slaughter, a section of liver, a portion of longissimus thoracis (LT) muscle, plus a portion of subcutaneous fat (surrounding LT muscle) and portions of visceral fat (kidney, pelvis and inguinal fat) were collected. Samples of liver, muscle and adipose tissues were used to quantify mtDNA copy number per cell. The number of mtDNA copies was determined by normalization of mtDNA amount against a single copy nuclear gene (B2M). Mean of EHP, hemoglobin and hematocrit of high and low RFI bulls were compared using two-sample t-tests. Additionally, the one-way ANOVA was used to compare mtDNA quantification considering the mains effects of RFI groups. We found lower EHP (83.047 vs. 97.590 kcal/day/kgBW0.75; P < 0.10), hemoglobin concentration (13.533 vs. 15.108 g/dL; P < 0.10) and hematocrit percentage (39.3 vs. 43.6 %; P < 0.05) in low compared to high RFI bulls, respectively, which may be useful traits to identify efficient animals. However, no differences were observed between the mtDNA content in liver, muscle and adipose tissue of Nellore bulls with high and low RFI.

Keywords: bioenergetics, Bos indicus, feed efficiency, mitochondria

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Authors: Jale Çatak, Bayram Yılmaz, Mustafa Ozilgen

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The second law muscle work efficiency is obtained by multiplying the metabolic and mechanical work efficiencies. Thermodynamic analyses are carried out with 19 sets of arms and legs exercise data which were obtained from the healthy young people. These data are used to simulate the changes occurring during aging. The muscle work efficiency decreases with aging as a result of the reduction of the metabolic energy generation in the mitochondria. The reduction of the mitochondrial energy efficiency makes it difficult to carry out the maintenance of the muscle tissue, which in turn causes a decline of the muscle work efficiency. When the muscle attempts to produce more work, entropy generation and exergy destruction increase. Increasing exergy destruction may be regarded as the result of the deterioration of the muscles. When the exergetic efficiency is 0.42, exergy destruction becomes 1.49 folds of the work performance. This proportionality becomes 2.50 and 5.21 folds when the exergetic efficiency decreases to 0.30 and 0.17 respectively.

Keywords: aging mitochondria, entropy generation, exergy destruction, muscle work performance, second law efficiency

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Authors: Maryam Zahedi Fard, Nazia Abdul Majid, Hapipah Mohd Ali, Mahmood Ameen Abdulla

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New quinazoline schiff base 3-(5-bromo-2-hydroxy-3-methoxybenzylideneamino)-2-(5-bromo-2-hydroxy-3-methoxyphenyl)-2,3-dihydroquinazolin-4(1H)-one was investigated for anticancer activity against MCF-7 human breast cancer cell line with involved mechanism of apoptosis. The compound demonstrated a remarkable antiproliferative effect, with an IC50 value of 3.41 ± 0.34, after 72 hours of treatment. Morphological apoptotic features in treated MCF-7 cells were observed by AO/PI staining. Furthermore, treated MCF-7 cells subjected to apoptosis death, as exhibited by perturbation of mitochondrial membrane potential and cytochrome c release as well as increase in ROS generation. We also found activation of caspases 3/7 and -9. Moreover, acute toxicity test demonstrated the nontoxic nature of the compound in mice. Our results showed the selected compound significantly induce apoptosis in MCF-7 cells via intrinsic pathway, which might be considered as a potent candidate for further in vivo and clinical breast cancer studies.

Keywords: antiproliferative effect, MCF-7 human breast cancer cell line, apoptosis, caspases

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Authors: Boris Melnikov, Ye Zhang, Dmitrii Chaikovskii

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We continue to consider one of the cybernetic methods in computational biology related to the study of DNA chains. Namely, we are considering the problem of reconstructing the not fully filled distance matrix of DNA chains. When applied in a programming context, it is revealed that with a modern computer of average capabilities, creating even a small-sized distance matrix for mitochondrial DNA sequences is quite time-consuming with standard algorithms. As the size of the matrix grows larger, the computational effort required increases significantly, potentially spanning several weeks to months of non-stop computer processing. Hence, calculating the distance matrix on conventional computers is hardly feasible, and supercomputers are usually not available. Therefore, we started publishing our variants of the algorithms for calculating the distance between two DNA chains; then, we published algorithms for restoring partially filled matrices, i.e., the inverse problem of matrix processing. In this paper, we propose an algorithm for restoring the distance matrix for DNA chains, and the primary focus is on enhancing the algorithms that shape the greedy function within the branches and boundaries method framework.

Keywords: DNA chains, distance matrix, optimization problem, restoring algorithm, greedy algorithm, heuristics

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Authors: Moon Jung Kim, Guil Rhim

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The clinical sensitivity of the “VEUPLEXTM TBI assay”, a clinical trial medical device, in mild traumatic brain injury was 28.6% (95% CI, 19.7%-37.5%), and the clinical specificity was 94.0% (95% CI, 89.3%). -98.7%). In addition, when the results analyzed by marker were put together, the sensitivity was higher when interpreting the two tests together than the two tests, UCHL1 and GFAP alone. Additionally, when sensitivity and specificity were analyzed based on CT results for the mild traumatic brain injury patient group, the clinical sensitivity for 2 CT-positive cases was 50.0% (95% CI: 1.3%-98.7%), and 19 CT-negative cases. The clinical specificity for cases was 68.4% (95% CI: 43.5% - 87.4%). Since the low clinical sensitivity for the two CT-positive cases was not statistically significant due to the small number of samples analyzed, it was judged necessary to secure and analyze more samples in the future. Regarding the clinical specificity analysis results for 19 CT-negative cases, there were a large number of patients who were actually clinically diagnosed with mild traumatic brain injury but actually received a CT-negative result, and about 31.6% of them showed abnormal results on VEUPLEXTM TBI assay. Although traumatic brain injury could not be detected in 31.6% of the CT scans, the possibility of actually suffering a mild brain injury could not be ruled out, so it was judged that this could be confirmed through follow-up observation of the patient. In addition, among patients with mild traumatic brain injury, CT examinations were not performed in many cases because the symptoms were very mild, but among these patients, about 25% or more showed abnormal results in the VEUPLEXTM TBI assay. In fact, no damage is observed with the naked eye immediately after traumatic brain injury, and traumatic brain injury is not observed even on CT. But in some cases, brain hemorrhage may occur (delayed cerebral hemorrhage) after a certain period of time, so the patients who did show abnormal results on VEUPLEXTM TBI assay should be followed up for the delayed cerebral hemorrhage. In conclusion, it was judged that it was difficult to judge mild traumatic brain injury with the VEUPLEXTM TBI assay only through clinical findings without CT results, that is, based on the GCS value. Even in the case of CT, it does not detect all mild traumatic brain injury, so it is difficult to necessarily judge that there is no traumatic brain injury, even if there is no evidence of traumatic brain injury in CT. And in the long term, more patients should be included to evaluate the usefulness of the VEUPLEXTM TBI assay in the detection of microscopic traumatic brain injuries without using CT.

Keywords: brain injury, traumatic brain injury, GFAP, UCHL1

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Authors: M. Omer Faruk, A. M. A. M. Zonaed Siddiki, M. Fazal Karim, Md. Masuduzzaman, S. Chowdhury, Md. Shafiqul Islam, M. Alamgir Hossain

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The dog tapeworms Echinococcus granulosus develop hydatid cysts in various organs in human and domestic animals worldwide including Bangladesh. The aim of this study was to identify and characterize the genotype of E. granulosus isolated from cattle using 12S rRNA and Cytochrome oxidase 1 (COX 1) genes. A total of 43 hydatid cyst samples were collected from 390 examined cattle samples derived from slaughterhouses. Among them, three cysts were fertile. Genomic DNA was extracted from germinal membrane and/or protoscoleces followed by PCR amplification of mitochondrial 12S rRNA and Cytochrome oxidase 1 gene fragments. The sequence data revealed existence of G1 (64.28%) and possible G3 (21.43%) genotypes for the first time in Bangladesh. The study indicates that common sheep strain G1 is the dominant subtype of E. granulosus in Chittagong region of Bangladesh. This will increase our understanding of the epidemiology of hydatidosis in the southern part of the country and will be useful to plan suitable control measures in the long run.

Keywords: Echinococcus granulosus, Cox1, 12S rRNA, molecular characterization, Bangladesh

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Authors: C. I. Boshoff, S. Steenkamp-Jonker

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Cystic echinococcosis (CE), caused by Echinococcus granulosus is an important parasitic infection in livestock worldwide, with severe zoonotic potential. It is important to understand the variability of Echinococcus granulosus, as genotype variations may influence lifecycle patterns, development rate, and transmission. Cystic Echinococcus samples were collected from domestic animals in Eastern Cape Province, South Africa. A molecular study was performed on 14 hydatid cysts obtained from caprine, ovine and bovine livers in order to determine the Echinococcus granulosus strain present in these hosts. The sequencing of the mitochondrial cytochrome C oxidase subunit I (coxI) gene of the hydatid cysts produced sequences of 400 bp for each sample analysed. These sequences were aligned with those present in GenBank and a phylogenetic tree was constructed. Based on coxI genotype the isolates could be grouped into E. granulosus sensu stricto. The findings of the study represent a pilot molecular study on Echinococcus from domestic animals undertaken in South Africa.

Keywords: Echinococcus granulosus, genotypes, livestock, South Africa

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Authors: Rajagopal Kavitha, Van Lun Low, Mohd Sofian-Azirun, Chee Dhang Chen, Mohd Yusof Farida Zuraina, Mohd Salleh Ahmad Firdaus, Navaratnam Shanti, Abdul Haiyee Zaibunnisa

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This study investigated the hidden genetic lineages in the oriental latrine fly Chrysomya megacephala (Fabricius) across four states (i.e., Johore, Pahang, Perak and Selangor) and a federal territory (i.e., Kuala Lumpur) in Malaysia using Cytochrome b (Cyt b) genetic marker. The Cyt b phylogenetic tree and haplotype network revealed three distinct genetic lineages of Ch. megacephala. Lineage A, the basal clade was restricted to flies that originated from Kuala Lumpur and Selangor, while Lineages B and C, comprised of flies from all studied populations. An overlap of the three genetically divergent groups of Ch. megacephala was observed. However, the flies from both Kuala Lumpur and Selangor populations consisted of three different lineages, indicating that they are genetically diverse compared to those from Pahang, Perak and Johore.

Keywords: forensic entomology, calliphoridae, mitochondrial DNA, cryptic lineage

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Authors: Bhargavi Santebennur Dwarakanath, Praveen Vadakke Kamath, Savitha Janakiraman

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Cervical cancer is one of the leading causes of mortality in women and is the second most common malignancy worldwide. Lovastatin, a non polar, anticholesterol drug which also exerts antitumour activity in vitro. In the present study, lovastatin from Aspergillus terreus (KM017963) was purified by adsoprtion chromatography and evaluated for its anticancer and anti-oxidant properties in human cervical cancer cell lines (HeLa). The growth inhibitory and proapoptotic effects of purified lovastatin on HeLa cell lines were investigated by determining its influence on cytotoxicity, Mitochondrial Membrane Potential (MMP), DNA fragmentation and antioxidant property (Hydroxy radical scavenging effect and the levels of total reduced glutathione). Flow cytometry analysis by propidium iodide staining confirmed the induction of apoptotic cell death and revealed cell cycle arrest at G0/G1 phase. Results of the study give leads for anticancer effects of lovastatin and its potential efficacy in the chemotherapy of cervical cancer.

Keywords: apoptosis, Aspergillus terreus, cervical cancer, lovastatin

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Authors: Hasmik V. Zanginyan, Gayane S. Ghazaryan, Laura M. Hovsepyan

Abstract:

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system that is induced in laboratory animals by developing an immune response against myelin epitopes. The typical clinical course is ascending palsy, which correlates with inflammation and tissue damage in the thoracolumbar spinal cord, although the optic nerves and brain (especially the subpial white matter and brainstem) are also often affected. With multiple sclerosis, there is a violation of lipid metabolism in myelin. When membrane lipids (glycosphingolipids, phospholipids) are disturbed, metabolites not only play a structural role in membranes but are also sources of secondary mediators that transmit multiple cellular signals. The purpose of this study was to investigate the effect of ganglioside as a therapeutic agent in experimental multiple sclerosis. The biological activity of a ganglioside-containing medicinal preparation (Cronassial) was evaluated in an experimental model of multiple sclerosis in laboratory animals. An experimental model of multiple sclerosis in rats was obtained by immunization with myelin basic protein (MBP), as well as homogenization of the spinal cord or brain. EAE was induced by administering a mixture of an encephalitogenic mixture (EGM) with Complete Freund’s Adjuvant. Mitochondrial fraction was isolated in a medium containing 0,25 M saccharose and 0, 01 M tris buffer, pH - 7,4, by a method of differential centrifugation on a K-24 centrifuge. Glutathione peroxidase activity was assessed by reduction reactions of hydrogen peroxide (H₂O₂) and lipid hydroperoxides (ROOH) in the presence of GSH. LPO activity was assessed by the amount of malondialdehyde (MDA) in the total homogenate and mitochondrial fraction of the spinal cord and brain of control and experimental autoimmune encephalomyelitis rats. MDA was assessed by a reaction with Thiobarbituric acid. For statistical data analysis on PNP, SPSS (Statistical Package for Social Science) package was used. The nature of the distribution of the obtained data was determined by the Kolmogorov-Smirnov criterion. The comparative analysis was performed using a nonparametric Mann-Whitney test. The differences were statistically significant when р ≤ 0,05 or р ≤ 0,01. Correlational analysis was conducted using a nonparametric Spearman test. In the work, refrigeratory centrifuge, spectrophotometer LKB Biochrom ULTROSPECII (Sweden), pH-meter PL-600 mrc (Israel), guanosine, and ATP (Sigma). The study of the process of lipid peroxidation in the total homogenate of the brain and spinal cord in experimental animals revealed an increase in the content of malonic dialdehyde. When applied, Cronassial observed normalization of lipid peroxidation processes. Reactive oxygen species, causing lipid peroxidation processes, can be toxic both for neurons and for oligodendrocytes that form myelin, causing a violation of their lipid composition. The high content of lipids in the brain and the uniqueness of their structure determines the nature of the development of LPO processes. The lipid layer of cellular and intracellular membranes performs two main functions -barrier and matrix (structural). Damage to the barrier leads to dysregulation of intracellular processes and severe disorders of cellular functions.

Keywords: experimental autoimmune encephalomyelitis, multiple sclerosis, neuroinflammation, therapy

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Authors: Sehrish Iftikhar, Ahmad Ali Shahid, Kiran Nawaz, Waheed Anwar

Abstract:

Discovery and development of new compounds require intense studies in chemistry, biochemistry. Numerous experiments under laboratory-, greenhouse- and field conditions can be performed to select suitable candidates and to understand their full potential. Novel fungicides are fundamental to combat plant diseases. Fusarium solani is important plant pathogen. New broad spectrum foliar fungicides against complex II were designed in this study. Complex II, namely succinate dehydrogenase (SDH), or succinate quinone oxidoreductase (SQR) is a multi-subunit enzyme at the crossroads of TCA and ETC at the inner mitochondrial membrane. The need for new and innovative fungicides is driven by resistance management, regulatory hurdles and increasing customer expectations amongst others. Fungicidal activity was assessed for the effect on mycelial growth and spore germination of the fungi using fungicide amended media assay. In mycelial growth assay compounds C10 and C6 were highly active against all the isolates. The compounds C1 and C10 were found most potent in spore germination test. It fully proved that the SDHIs designed in this paper displayed as good inhibitory effects against Fusarium solani.

Keywords: Wilt, Fusarium, SDH, antifungal

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Authors: Ozlem Oral, Emre Taskin, Aysel Yuce, Serap Dokmeci, Devrim Gozuacik

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Autophagy is an evolutionary conserved lysosome-dependent catabolic pathway, responsible for the degradation of long-lived proteins, abnormal aggregates and damaged organelles which cannot be degraded by the ubiquitin-proteasome system. Lysosomes degrade the substrates through the activity of lysosomal hydrolases and lysosomal membrane-bound proteins. Mutations in the coding region of these proteins cause malfunctional lysosomes, which contributes to the pathogenesis of lysosomal storage diseases. Gaucher disease is a lysosomal storage disease resulting from the mutation of a lysosomal membrane-associated glycoprotein called glucocerebrosidase and its cofactor saposin C. The disease leads to intracellular accumulation of glucosylceramide and other glycolipids. Because of the essential role of lysosomes in autophagic degradation, Gaucher disease may directly be linked to this pathway. In this study, we investigated the expression of autophagy and/or lysosome-related genes and proteins in fibroblast cells isolated from patients with different mutations. We carried out confocal microscopy analysis and examined autophagic flux by utilizing the differential pH sensitivities of RFP and GFP in mRFP-GFP-LC3 probe. We also evaluated lysosomal pH by active lysosome staining and lysosomal enzyme activity. Beside lysosomes, we also performed proteasomal activity and cell death analysis in patient samples. Our data showed significant attenuation in the expression of key autophagy-related genes and accumulation of their proteins in mutant cells. We found decreased the ability of autophagosomes to fuse with lysosomes, associated with elevated lysosomal pH and reduced lysosomal enzyme activity. Proteasomal degradation and cell death analysis showed reduced proteolytic activity of the proteasome, which consequently leads to increased susceptibility to cell death. Our data indicate that the major degradation pathways are affected by multifunctional lysosomes in mutant patient cells and may underlie in the mechanism of clinical severity of Gaucher patients. (This project is supported by TUBITAK-3501-National Young Researchers Career Development Program, Project No: 112T130).

Keywords: autophagy, Gaucher's disease, glucocerebrosidase, mutant fibroblasts

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