Eukaryotic Multi-subunit DNA Dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism | Chapter 01 | Emerging Trends and Research in Biological Science Vol. 1

Aim: To analyze the most complex multi-subunit (MSU) DNA dependent RNA polymerases (RNAPs) of eukaryotic organisms and find out conserved motifs, metal-binding sites and catalytic regions and propose a plausible mechanism of action for these complex eukaryotic MSU RNAPs, using yeast (Saccharomyces cerevisiae) RNAP II, as a model enzyme.

Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis and X-ray crystallographic data were analyzed.

Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India, between 2007- 2013.

Methodology: Bioinformatics, Biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data of the enzyme were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNAPs from various eukaryotic sources. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical and SDM experiments and X-ray crystallographic analysis of these enzymes were used to confirm the possible amino acids involved in the active sites and catalysis.

Results: Multiple sequence alignment (MSA) of RNAPs from different eukaryotic organisms showed a large number of highly conserved motifs among them. Possible catalytic regions in the catalytic subunits of the yeast Rpb2 (= β in eubacteria) and Rpb1 (= β’ in eubacteria) consist of an absolutely conserved amino acid R, in contrast to a K that was reported for DNA polymerases and single subunit (SSU) RNAPs. However, the invariant ‘gatekeeper/DNA template binding’ YG pair that was reported in all SSU RNAPs, prokaryotic MSU RNAPs and DNA polymerases is also highly conserved in eukaryotic Rpb2 initiation subunits, but unusually a KG pair is found in higher eukaryotes including the human RNAPs. Like the eubacterial initiation subunits of MSU RNAPs, the eukaryotic initiation subunits, viz. Rpb2, exhibit very similar active site and catalytic regions but slightly different distance conservations between the template binding YG/KG pair and the catalytic R. In the eukaryotic initiation subunits, the proposed catalytic R is placed at the -9th position from the YG/KG pair and an invariant R is placed at -5 which are implicated to play a role in nucleoside triphosphate (NTP) selection as reported for SSU RNAPs (viral family) and DNA polymerases. Similarly, the eukaryotic elongation subunits (Rpb1) are also found to be very much homologous to the elongation subunits (β’) of prokaryotes. Interestingly, the catalytic regions are highly conserved, and the metal-binding sites are absolutely conserved as in prokaryotic MSU RNAPs. In eukaryotes, the template binding YG pair is replaced with an FG pair. Another interesting observation is, similar to the prokaryotic β’ subunits, in the eukaryotic Rpb1 elongation subunits also, the proposed catalytic R is placed double the distance, i.e., -18 amino acids downstream from the FG pair unlike in the SSU RNAPs and DNA polymerases where the distance is only -8 amino acids downstream from the YG pair. Thus, the completely conserved FG pair, catalytic R with an invariant R, at -6th position are proposed to play a crucial role in template binding, NTP selection and polymerization reactions in the elongation subunits of eukaryotic MSU RNAPs. Moreover, the Zn binding motif with the three completely conserved Cs is also highly conserved in the eukaryotic elongation subunits also. A plausible proof-reading mechanism during the elongation process is also proposed based on the MSA and experimental data. Another important difference is that the catalytic region is placed very close to the N-terminal region in eukaryotes.

Conclusions: Unlike reported for the DNA polymerases and SSU RNA polymerases, the of eukaryotic MSU RNAPs use an R as the catalytic amino acid and exhibit a different distance conservation in the initiation and elongation subunits. An invariant Zn2+ binding motif, found in the Rpb1 elongation subunits is proposed to participate in the proof-reading function. Differences in the active sites of bacterial and human RNA polymerases may pave the way for the design of new and effective drugs for many bacterial infections, including the multidrug-resistant strains which are a global crisis at present.

Author(s) Details

Dr. Peramachi Palanivelu
Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625021, India.

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Studies on Alzheimer Disease – Metformin Connection – A Brief Review | Chapter 02 | Emerging Trends and Research in Biological Science Vol. 1

Metformin is the widely prescribed first line oral antidiabetic drug used in diabetes mellatus, type 2. The global sales turnover of metformin runs into millions of dollars. The Increased risk of metformin (Met) users for developing Alzheimer disease (AD) is reported first in a study conducted in 2011.  Since then, the subject has attracted the attention   of the researchers as well as the pharmaceutical industry, resulting in a number of studies, both clinical as well as experiments on animals. Confusing results poured in, ranging from confirmation of the risk of AD to protection against developing AD, making the scenario, all the more intriguing. Added to the confusion, is the diversity of various studies as well as the parameters interpreting their results. Of the many clinical trials, some are retrospective cohort studies (Tseng Chin-Hsiao [1]), case control studies (Imfeld P, et al. [2]) Randomised studies (Hsu CC, et al. [3]), double blind, cross over pilot studies. (Aaron Koenig et al. [4]) and some longitudinal studies (Ng TP, et al. [5]), besides studies doing meta analysis. Of these studies most of the trials estimate the risk of development of dementia with metformin alone (Tseng Chin-Hsiao [1]) or in comparison with other OHAs (Hsu et al. [3], Cheng et al. [6]). The other studies studied the effect of metformin on the cognition (Moor EM, et al. [7]). These trials have different out come measures, (like Hazard ratio, (HR) Odds (OR) ratio, relative risk (RR) etc.) which don’t mean one and the same. So the multiplicity of the types of studies and different out- comes with different conclusions will be surely baffling to an average reader who tries to take cognisance of the involved issues. The article attempts to take stock of the overall developments in this regard. The author adopted a reader friendly approach which is discussed in the article, at the outset. Finally, it is reiterated that future prospective studies only can resolve the conflict of opinion on the nexus between metformin and Alzheimer’s disease.

Author(s) Details

Dr. A. S. V. Prasad
Department of Internal Medicine, G.I.T.A.M Dental College, Rushikonda, Visakhapatnam, Andhra Pradesh, India.

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Ethanol Affects Liver Oxidative Stress and Methylation Status in HCV-infection: study on NS5A-Transgenic Mice | Chapter 03 | Emerging Trends and Research in Biological Science Vol. 1

Background: Alcohol consumption accelerates the progression and worsens the outcomes of hepatitis C viral (HCV) infection in heavy and moderate drinkers. Non-structural NS5A protein is a known inducer of oxidative stress and carcinogenesis. Although alcohol consumption exacerbates the course of HCV-infection, the combined effects of NS5A protein and alcohol have not been studied and experimental animal HCV models as well as ways of ethanol administration to animals are not optimized.   Our aim was to investigate the effects of two modes of oral ethanol feeding on induction of oxidative stress, methylation status and changes in proteasome activity in livers of NS5A-transgenic (Tg) mice.

Methods: Ethanol was administered either in drinking water (chow- fed mice given 20% ethanol in water; designated chow-EtOH) or in Lieber DeCarli liquid diet (LCD-EtOH). Appropriate controls were used.  The mechanisms of alcohol and NS5A-induced changes in oxidative stress, liver methylation status and 20S proteasome activity were determined after 5 weeks of the feeding regimen.

Results: Ethanol administration using both feeding regimens induced oxidative stress and suppressed cytosolic proteasome activity. However, only LCD-EtOH diet induced fatty changes in the liver, which correlated with higher levels of oxidative stress, impaired methylation potential and reduced cytosolic and nuclear proteasome activity. Importantly, LCD diet administration by itself promoted lipid peroxidation in NS5A-expressing mice.

Conclusion: We conclude that both modes of oral ethanol feeding (chow and LCD-based) induce oxidative stress in NS5A-Tg mice that suppresses proteasome activity. Nonetheless, impaired methylation potential, higher level of oxidative stress and suppression of nuclear proteasome was observed only in LCD-EtOH mice.  However, the effects of LCD-control liquid diet in inducing lipid peroxidation in NS5A-Tg mice, in certain cases, tended to mask the effects of ethanol, indicating that fatty diet serves as a second hit for NS5A-protein-induced stress response of liver cells.

Author(s) Details

Natalia A. Osna [M.D., Ph.D.]
Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System Omaha, NE, USA and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE  68105, USA.

Murali Ganesan [Ph.D.]
Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System Omaha, NE, USA and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE  68105, USA.

Larisa Y. Poluektova [M.D., Ph.D.]
Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE  68105, USA.

Kusum K. Kharbanda [Ph.D.]
Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System Omaha, NE, USA and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE  68105, USA and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE  68105, USA.

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Rare Occurrence of the Young Basking Shark Cetorhinus maximus (Gunnerus, 1765) in the Northeastern Mediterranean | Chapter 04 | Emerging Trends and Research in Biological Science Vol. 1

One female specimen of Basking shark Cetorhinus maximus (TL – total length = 245 cm; W – weight = 75 kg) was incidentally captured by a bottom trawler at a depth of about 25 m on 20 March 2014 from the off Yesilovacık coast (Mersin Bay, Turkey). Morphometric measurements presented in this paper. The present article reports the young occurrence of C. maximus in the northeastern Mediterranean, Turkey. The basking shark was not very abundant throughout the eastern Mediterranean Sea. This species is infrequent in the northern part of the Mediterranean, Turkey. The occurrence of this species not only extends the geographical distribution of its known range but also represents the first presentation of a young female specimen from the northeastern Mediterranean (Mersin Bay, Turkey). Due to their slow maturity, extended longevity, and low reproduction rate, the basking shark is considered to be extremely vulnerable to overfishing. Their population numbers declining significantly in the past decades. Thus, the basking shark species is currently listed in Appendix I and II of CMS, Annex I of the CMS Sharks MOU, and Annex I of UNCLOS.

Author(s) Details

Deniz Ergüden
Department of Marine Sciences, Faculty of Marine Sciences and Technology, Iskenderun Technical University, TR 31220 Iskenderun, Hatay, Turkey.

Deniz Ayas
Faculty of Fisheries, Mersin University, Yenişehir Campus, TR 33160, Mersin, Turkey.

Sibel Alagöz Ergüden
Imamoglu Vocational School, Cukurova University, TR 01700, Imamoglu, Adana, Turkey.

Hasan Deniz Akbora
Faculty of Fisheries, Mersin University, Yenişehir Campus, TR 33160, Mersin, Turkey and Department of Biological Sciences, Faculty of Arts and Sciences, Eastern Mediterranean University, Famagusta 99628, North Cyprus via Mersin 10, Turkey.

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Digestibility of Leaf Proteins of Gnetum spp Vegetables in Rats and Effects of Some Antinutrients | Chapter 05 | Emerging Trends and Research in Biological Science Vol. 1

Aims: Study leaf protein digestibility and effect of some antinutritional factors of Gnetum africanum and Gnetum buchholzianum on young male rats Wistar albinos.

Study Design: Nutritional enhancement of use of Gnetum spp leafy vegetables.

Place and Duration of Study: Limbe botanical Garden of Cameroon, between November 2013 and February 2014.

Methodology: Proteins and antinutritional factors were determined using standard analytical methods. Standard diet AIN-93 was used as reference for preparation of experimental diets used for in vivo digestibility of these leafy vegetables. Rats were fed with diets containing respectively 5% or 10% leaf proteins of G. africanum (GA5P or GA10P) or G. buchholzianum (GB5P or GB10P).

Results: Results showed that mean proteins contents was 16.70 mg/100g. Average contents of crude fibers, Neutral Detergent Fibers, Acid Detergent Fibers, crude phenolic compounds, tannins and phytates were respectively 36.17, 41.97, 37.80 g/100 g and 478.80, 244.94, 215.64 mg/100. Rats’ growth was more promoted by diets containing 5% proteins. Protein Efficiency and Net Protein Efficiency Ratio were low. Digestibility Coefficient, Biological Value and Net Protein Utilization were high for 5% proteins diets. Principal Component Analyses revealed that fibers and antinutrients reduced growth and nitrogen retention from diet containing 10% proteins.

Conclusion: Gnetum spp. leafy vegetables have high contents of fiber, phenolic compounds and phytates which contribute to reduction of digestibility of theirs proteins.

Author(s) Details

Ndomou Mathieu
Department of Biochemistry, Faculty of Science, University of Douala, P.O.Box 24157, Douala, Cameroon.

Mezajoug Kenfack Laurette Blandine
University Institute of Technology, Laboratory of Bioprocess, Food Biochemistry and Nutrition Unit, University of Ngaoundere, Cameroon.

Gouado Inocent
Department of Biochemistry, Faculty of Science, University of Douala, P.O.Box 24157, Douala, Cameroon.

Tchiégang Clergé
University Institute of Technology, Laboratory of Bioprocess, Food Biochemistry and Nutrition Unit, University of Ngaoundere, Cameroon.

Professor Ngogang Yonkeu Jeanne
Faculty of Medicine, Université des Montagnes, Bagangté, Cameroon.

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Phytochemical Screening and Antimicrobial Activity of Plant Extracts for Textile Applications | Chapter 06 | Emerging Trends and Research in Biological Science Vol. 1

Aims: To carry out the qualitative and quantitative phytochemical screening and assess the antimicrobial activity of banyan (Ficus benghalensis), castor (Ricinus communis) and clerodendron (Clerodendron inerme) leaf extracts.

Place and Duration of Study: Department of Textile and Apparel Designing, College of Community Science, University of Agricultural Sciences, Dharwad, Karnataka, between July 2014 and June 2017.

Methodology: Extraction of phytochemicals was carried out by different solvents viz., 70% ethanol, 70% methanol and distilled water. The phytochemical screening was carried out for the presence of various bio-active constituents according to standard procedures. Total phenolic content (TPC) was determined by Folin-Ciocalteu reagent assay method. Antimicrobial activity of crude extracts of plants was assessed by agar well diffusion method.

Results: The qualitative screening revealed the presence of constituents such as flavonoids, alkaloids, tannins, phenols and saponins in all the leaf extracts. However, terpenoids were absent in banyan leaf extracts. Irrespective of solvents, castor extracts yielded higher total phenols followed by banyan and clerodendron extracts. Further, the antibacterial activity of the crude ethanol extract of castor against bacterial species viz., Staphylococcus aureus, Escherichia coli and fungal strain, Aspergillus niger showed highest antibacterial and antifungal activity compared to banyan and clerodendron extracts.

Conclusion: It can be concluded that the results has provided the basis for use of banyan, castor and clerodendron extracts as potential agents for applying antimicrobial finish to textiles. Hence, there is a need to explore the applicability of these plant resources which are rich in phytochemicals/phenolics and may have beneficial effects on health.

Author(s) Details

Dr. Shameembanu A. Byadgi
Department of Textile and Apparel Designing, College of Community Science, University of Agricultural Sciences, Dharwad, India.

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Molecular Basis of Pathogenesis of Ectopic Fat Deposition in DM2 – An Overview | Chapter 07 | Emerging Trends and Research in Biological Science Vol. 1

The presence of fat, beyond physiological limits, in organs, other than the adipose tissue, like the liver, the skeletal muscle, the heart and the pancreas etc. is called ectopic fat. It causes specific organ dysfunction in the tissues concerned. The importance of the ectopic fat is that it is connected to peripheral tissue insulin resistance, obesity, metabolic syndrome etc. Though the molecular mechanisms underlying the specific organ dysfunctions are understood, still grey areas exists as to the source of the ectopic fat and how it finds it’s way to the specific sites of the target organs (intra- myocellular in skeletal muscle, hepatocyte cytoplasm of liver, epicardial surface and coronary arteries of heart etc.). The molecular mechanisms involving the actual ectopic deposition fat, are not clear. This article focuses on some of the grey areas in the pathogenesis of the ectopic fat deposition, besides reviewing briefly the facts already known in the literature about ectopic fat deposition.

Author(s) Details

Dr. A. S. V. Prasad
Department of Internal Medicine, G.I.T.A.M Dental College, Rishikonda, Visakhapatnam, Andhra Pradesh, India.

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In vivo Propagation of Simmondsia chinensis (Jojoba) | Chapter 08 | Emerging Trends and Research in Biological Science Vol. 1

Simmondsia chinensis (jojoba) is a semi-hard wood perennial shrub grown on arid and semi-arid regions of the world. The stem cutting method is simple and efficient method for semi-hard wood plants to propagate vegetatively. The aim of this study was to investigate the best method of in vivo propagation through stem cutting of Simmondsia chinensis. The healthy branches of mature male and female plants were cut into 10 to 15 cm length having 4 to 5 nodes stem. The basal part of cuttings was dipped in the 500, 1000, 2000 and 3000 ppm of IBA (Indole-3-butyric acid) and NAA (2-Naphthalene acetic acid) respectively for one hour. After which the stem cuttings were planted in the polypots trays filled with rooting media contained perlite and vermiculite (1:1). Among all concentrations of both auxins (IBA and NAA), the highest rooting 68.9% in male stem cutting and 66.5% in female stem cutting were observed at 2000 ppm concentration of IBA. At this concentration, the maximum number 12.5 and 13.2 of roots in male and female cuttings were obtained respectively. Vegetatively, in vivo propagation method through stem cuttings of male and female plants of Simmondsia chinensis proves to be ideal solution for production of large scale plant materials at commercial level.

Author(s) Details

Raman Bala
Department of Environmental Science, Maharshi Dayanand University, Rohtak, 124001 (Haryana), India.

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Biochemistry and Molecular Biology of Mechanisms of Action of Fibrates – An Overview | Chapter 09 | Emerging Trends and Research in Biological Science Vol. 1

Fibrates are a class of medication that mainly lowers the blood triglyceride levels. They reduce the LDL and increase the levels of HDL C, in the blood. Clofibrate, the first member to be discovered                 in 1962, and introduced in USA in 1967, is withdrawn in 2002, due to unexplained hepatomegaly, hepato-toxicity and possible risk of hepatic cancer. Other fibrates are introduced in the late 1970s and early 1980s, such as gemfibrozil in the United States and bezafibrate and ciprofibrate in Europe. Their lipid lowering effects are found to decrease CVS risk, progression of atherosclerosis and metabolic syndrome, macrovascular and microvascular diabetic complications like stroke, myocardial infarction, peripheral vascular disease and diabetic retinopathy. Various clinical trials like VA-HIT trial (Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial), FIELD trail. (The Fenofibrate Intervention and Event Lowering in Diabetes) Helsinki Heart Study, ACCORD -Lipid trial (The lipid component of the Action to Control Cardiovascular Risk in Diabetes trial) and  BIP (Bezafibrate Infarction Prevention Study)  trial and angiography trials, like LOCAT(Lopid Coronary Angiography Trial) and BECLAIT(Bezafibrate Coronary Atherosclerosis Intervention Trial) demonstrated the  beneficial effects of gemfibrozil and fenofibrate. Their mechanism of action remained obscure for three decades, ie till 1990s, when their mode of action was found. The Mechanism of action of fibrates include limitation of substrate availability for triglyceride synthesis in the liver, promotion of the action of lipoprotein lipase, (LPL) modulation of low density lipoprotein receptor/ligand interaction and stimulation of reverse cholesterol transport The biochemical and molecular mechanisms involving  the various  enzymes like LCAT (Lecithin-cholesterol acyl transferase)andCYP7A1 etc. (cholesterol 7-alpha-monooxygenase or cytochrome P450 7A1 (CYP7A1)), transporters like ABC, CETP (ATP-binding cassette transporter, Cholesterol ester binding protein) and NTCP, OATP (Na+-dependent taurocholate transporter / organic anion transporters). These are the and nuclear factors like LXR, PPAR alfa etc. (liver orphan receptor α, and peroxisome proliferative nuclear factor), in relation to the mechanisms of action of fibrates are discussed. Areas of current interests in literature are briefed.

Author(s) Details

Dr. A. S. V. Prasad
Department of Internal Medicine, G.I.T.A.M Dental College, Rishikonda, Visakhapatnam, Andhra Pradesh, India.

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