Identification of novel components and links in ubiquitin dependent protein degradation pathways of Arabidopsis thaliana

The canonical ubiquitin 26S proteosome dependent protein degradation pathway and its sub-branch N-end rule pathway are important ubiquitin dependent processes in eukaryotes. The majority of substrates are predominantly targeted for degradation by the proteosome. Expression of a ubiquitin variant with Arg instead of Lys at position 48 (ubK48R) in the Arabidopsis RV86-5 line leads to cell death. In order to understand the downstream effects of this pathway, the ubK48R expressing line RV86-5 and the suppressor line of ubiquitin variant induced cell death, sud2, were used as tools. Fine mapping with 1239 recombinants narrowed down the sud2 mutant locus to the south arm of chromosome III, between loci At3g44400 and At3g44900. Problems caused by low recombination and repeated sequences were overcome by sub-genomic PCR-based amplification of a 350 kb region and subsequent Solexa sequencing of this region of interest. The data analysis tailored for nucleotide based comparison to reference sequence identified 15 candidates, 5 of which could be verified by conventional sequencing. In an alternative approach, microarray-based transcriptional expression differences between RV86-5 and sud2 identified 10 additional candidate suppressor genes, the majority of which are of unknown function. Among mutations in 9 of the tested candidates, 8 were able to prevent the lethal phenotype of RV86-5, indicating their involvement in the cell death process. The main interest of the ubiquitin research field is to identify E3-ligases and their interacting substrates. The second part of this work involved the search for novel E3 ligases that modify a known test protein with an aliphatic hydrophobic amino-terminal residue, Leu, which is targeted by none of the known plant N-end rule E3 ligases, PRT6 and PRT1. EMS mutagenesis on a plant line expressing a test protein with L-GUS followed by live tissue GUS staining, to screen for transgene stabilization, identified the 2 complementation groups PRT8 and PRT9, representing candidates for putative E3-ligases involved in destabilization of test proteins with amino-terminal Leu. The prt8 mutant shows delayed development. With the creation of a mapping population, the basis for the identification of locus was laid in this work. Arabidopsis mutants in the functionally unknown UBR domain proteins BIG and PRT7, which share homology with the mammalian N-end rule pathway components UBR4 and UBR7, were analyzed. A mutant in PRT7, isolated by T-DNA library screening, showed premature leaf senescence. In contrast, the big mutant showed delayed senescence and in addition no enzymatic affinity to test substrates with a basic N-terminus. Mutants were 9 isolated in two putative Arabidopsis deamidases, NTAN and NTAQ that are distantly related to mammalian deamidases. These were crossed into reporter lines expressing N-GUS and Q-GUS test proteins to deduce whether these enzymes provide substrates to Arg-t-RNA protein transferase as in mammals. These created mutants have laid the basis to analyse unknown functions of N-end rule pathway components in Arabidopsis. The importance of NO in signaling in plants has been long studied, but its molecular mechanism is still not well understood. In this work, it was found that in the Arabidopsis N-end rule pathway, NO targets test substrates with N-terminal Cys for degradation in a proteosome dependent manner and that this process is dependent on O2. With these results, strong evidence was obtained that the N-end rule pathway has a role in NO signaling and sensing. This finding has brought new insights into the plant N-end rule pathway. Taken together, the research work of this Thesis has developed new methods to overcome the low recombination problem during the mapping process, identified candidates that could potentially link the cell death processes to the ubiquitin dependent degradation pathway and identified putative E3-ligases of the N-end rule pathway by a novel way of EMS mutant screening supported by live tissue GUS assay. This research work found a connection between NO and the N-end rule pathway in A. thaliana. A complete set of mutants in all known plant N-end rule pathway components has been created, opening a window of possibility to further find natural substrates of this pathway

Iron Deficiency Anemia in Growing Years and its Effect on Cognition: A Review

Iron deficiency anemia is a widespread nutritional problem. Children are particularly vulnerable due to poor maternal iron status and inadequate diet. Since iron plays a vital role in neurological development, its deficiency in early life can lead to altered cognition and motor development. Severe iron deficiency during infancy can affect the major processes such as myelination, development of neurotransmitter pathways, neural metabolism and neural plasticity. The present review intends to provide information on the various effects of iron deficiency during different stages of life cycle. Clinical trials conducted on both humans and animals have established that infants who suffer iron deficiency during the early years of life are known to be under risk for experiencing the long lasting effect of early iron deficiency in future years also. Follow-up studies conducted on infants showed that the anemic children continued to exhibit lower cognitive development compared to their non-anemic counterparts and also had difficulty in the development of motor control tasks. Hence, there is a need to identify and correct iron deficiency anemia during the early years of life to prevent possible future complications

Rearrangements of nitrogen fixation (nif) genes in the heterocystous cyanobacteria

In the vegetative cells of heterocystous cyanobacteria, such as Anabaena, two Operons harbouring the nitrogen fixaton (nif) genes contain two separate intervening DNA elements resulting in the dispersion of genes and impaired gene expression. A 11 kb element disrupts thenifD gene in thenifH, D-K operon. It contains a 11 bp sequence (GGATTACTCCG) directly repeated at its ends and harbours a gene,xisA, which encodes a site-specific recombinase. A large 55 kb element interrupts thefdxN gene in thenifB fdxN-nifS-nifU operon. It contains two 5 bp direct repeats (TATTC) at its ends and accommodates at least one gene,xisF, which encodes another site-specific recombinase. During heterocyst differentiation both the discontinuities are precisely excised by two distinct site-specific recombination events. One of them is brought about by the XisA protein between the 11 bp direct repeats. The second one is caused by the XisF protein and occurs between the 5 bp direct repeats. As a consequence the 11kb and 55 kb elements are removed from the chromosome as circles and functional nif Operons are created. Nitrogenase proteins are then expressed from the rearranged genes in heterocysts and aerobic nitrogen fixation ensues. How these elements intruded thenif genes and how and why are they maintained in heterocystous cyanobacteria are exciting puzzles engaging considerable research effort currently. The unique developmental regulation of these gene rearrangements in heterocystous cyanobacteria is discussed

Impact of Elevated Temperature and Carbon dioxide on Seed Physiology and Yield

Food security is of utmost priority to humankind. This is the implication of various interconnected factors that lead to climate change. Elevated temperature and carbon dioxide levels are just 2 of these. The nutrient is an inseparable aspect of food. The change in climate is posing threat not only to the amount of available food but also to the nutrients laden in the food items. Seeds are the miniature form of plants and are a reflection of their future health and nutritional status. The changes in environmental factors predominantly challenge the growth and development of a seed. This review is an attempt to understand the impact of elevated CO2 and temperature on seed germination, the nutritional status of the seed and the yield in form of total seed production. It gives a direction for analysis and future studies that may use the latest available tools like gene editing to tackle and counteract the retarding effect of climate change on these parameters of seed, thereby offering a climate resilient agriculture

La protein binding at the GCAC site near the initiator AUG facilitates the ribosomal assembly on the hepatitis C virus RNA to influence internal ribosome entry site-mediated translation

Human La autoantigen has been shown to influence internal initiation of translation of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that, among the three RRMs of La protein, the RRM2 interacts with HCV internal ribosome entry site (IRES) around the GCAC motif near the initiator AUG present in the stem region of stem-loop IV (SL IV) (Pudi, R., Abhiman, S., Srinivasan, N., and Das S. (2003) J. Biol. Chem. 278, 12231-12240). Here, we have demonstrated that the mutations in the GCAC motif, which altered the binding to RRM2, had drastic effect on HCV IRES-mediated translation, both in vitro and in vivo. The results indicated that the primary sequence of the stem region of SL IV plays an important role in mediating internal initiation. Furthermore, we have shown that the mutations also altered the ability to bind to ribosomal protein S5 (p25), through which 40 S ribosomal subunit is known to contact the HCV IRES RNA. Interestingly, binding of La protein to SL IV region induced significant changes in the circular dichroism spectra of the HCV RNA indicating conformational alterations that might assist correct positioning of the initiation complex. Finally, the ribosome assembly analysis using sucrose gradient centrifugation implied that the mutations within SL IV of HCV IRES impair the formation of functional ribosomal complexes. These observations strongly support the hypothesis that La protein binding near the initiator AUG facilitates the interactions with ribosomal protein S5 and 48 S ribosomal assembly and influences the formation of functional initiation complex on the HCV IRES RNA to mediate efficient internal initiation of translation

Shami (Prosopis cineraria (L) Druce) - A Medicinal Benison

Shami (Prosopis cineraria (L) Druce) belongs to family Fabaceae known for its spiritual uses in India mentioned in almost all the Nighantu’s of Ayurveda. It is endemic to Hot, Dry and Arid regions of India. Even though almost all the parts of Shami are having pharmacological actions specially the Bark and Fruit but these remain unexplored. It mainly contains tannins (gallic acid), alkaloids (spicigerine, prosophylline), Flavone derivatives (prosogerin A, B, C, D and E) and quercetin are widely used as anti-oxidant, anti-microbial, anti-bacterial, anti-convulsant, nootropic and antidepressant activity. Thus, the current article reviews on Ayurveda literature, botanical description, varieties and powder microscopy of Shami

A Study of Somatic Status and Complications Among Female Diabetic Patients from Mysore Urban Area

In recent years, India has undergone rapid urbanization and socioeconomic development. Changes in time trends have resulted in erratic lifestyle, characterized by physical inactivity, unhealthy eating habits and resultant increase in obesity and diabetes. Diabetes is a major cause of mortality and morbidity in India and its prevalence is increasing at an alarming rate. Chronic complications of diabetes, especially coronary artery diseases and chronic renal diseases results in frequent hospitalization. The main aim of the investigation was to study the somatic status and diabetic complications among the female hospitalized and non-hospitalized patients. A total of 80 female volunteers (40 hospitalized and 40 nonhospitalized) of a private hospital in Mysore, with known history of type 2 diabetes mellitus for more than 2 years, were recruited for the study. The tools were developed to collect information on personal history, demography, socioeconomic status, dietary habits and anthropometric measurements. Suitable statistical analysis was applied to the data. The resultsprojected that majority of the patients were hospitalized on an average of at least three times a year. More than 90% of the subjects exhibited blood sugar >300 mg on admission. The reason for high morbidity status included poor dietary habits and erratic lifestyle practices among the female hospitalized patients as compared to non-hospitalized patients. Adapting a healthy lifestyle and maintenance of normal blood sugar level can reduce the incidence of complications and hospitalization among the subjects

The use of biopolymer functionalized single walled carbon nanotubes for effective targeted photodynamic therapy of colon cancer stem cells

D.Tech. (Biomedical Technology)Abstract

Investigation of Volatile Products from Wood Pyrolysis

In this research we are following the thermo-chemical degradation of wood in the absence of oxygen. The objectives are to evaluate the influence of heating rates on pyrolysis products obtained from wood pyrolysis and to evaluate the influence of acid pre-treatment on pyrolysis products. Depending on the wood heating rates, pyrolysis can be categorized as Flash pyrolysis, Fast pyrolysis, and Slow pyrolysis. We have evaluated the volatile products obtained at different heating rates and the volatile products obtained from sulfuric acid pre-treatment by using gas chromatography- mass spectrometry (GC-MS). We have also performed thermo-gravimetric analysis (TGA) of raw wood samples and sulfuric acid pre-treated wood samples of Yellow Pine to determine the changes in weight in relation to change in temperature. Our results indicated that by using the Flash, Fast, and Slow heating rates, the overall volatile products obtained from wood pyrolysis (i.e. the overall list of all the compounds obtained from different temperature ranges in wood pyrolysis by using different heating rates) were the same, but the volatile products obtained at different temperature ranges like Room temperature-300°C, 300°C - 400°C, and 400°C -500°C in Flash, Fast, and Slow pyrolysis were different. Most of the volatile products obtained from the pyrolysis of untreated wood were phenols. Our results also indicated that the pretreatment of wood with sulfuric acid alters the charcoal properties and releases gaseous products including furan derivatives that are useful as fuels or fuel additives. The sulfuric acid (10%) pretreatment of wood followed by slow pyrolysis produced maximum yield of charcoal, indicated by the lowest mass % decrease of 58.234. The production of furan derivatives increased by using sulfuric acid pre-treatment, which is a good improvement for the production of Furanics, the furan based biofuels. The furan based biofuels are of increasing research interest because of their significant advantages over the first generation biofuels. The thermogravimetric analysis (TGA) results indicated that the acid pre-treatment altered the decomposition rate of pyrolysis and lowered the onset of temperature for decomposition. The use of thermal degradation of plants for creating chemicals and fuels is seeing renewed interest across the globe as it is considered carbon-neutral and it uses a renewable feedstock. The information obtained from this research work will also be valued by industries, such as charcoal and activated carbon producers, which currently perform biomass pyrolysis, by allowing them to form approaches that optimize their energy use and minimize waste