In this study, we have characterized some biochemical and electrophoretic features of recombinant 20S Proteasome from a thermoacidophilic archaeon Thermoplasma volcanium. As revealed by SDS-PAGE the 20S Proteasome was composed of two subunits, &amp / #945 / - and &amp / #946 / - subunits with estimated molecular masses of 24 kDa and 23 kDa, respectively. The highest chymotryptic activity was observed over an alkaline pH range (pH 8.0 &ndash / pH 9.0) and the optimum temperature for the activity was determined as 85oC. The heat stability of proteasome was quite high after treatment at 98oC for 30 minutes, 64 % of the activity has still been retained. The highest activity associated with the Thermoplasma volcanium proteasome was found to be peptidylglutamyl peptidase activity. Within the scope of this project, also, we have cloned a 26S Proteasome related Regulatory Subunit gene of Thermoplasma volcanium. For cloning we have followed a PCR based approach. Amplification of 26S Proteasome Regulatory Subunit gene from chromosomal DNA of Tp. volcanium yielded a product of 1419 bp containing an open reading frame of 1128 bp comprising the structural gene. The PCR amplicon was cloned using pDrive vector in E.coli TG-1 cells. Out of ten putative recombinants, three plasmids, E.coli pD1-6, E.coli pD2-3, E.coli pD3-1, were proved to be true recombinants and selected for further characterization by restriction mapping and expression studies. ATPase activities of cell free extracts from both recombinant and non-recombinant E.coli strains were measured and found that ATPase activities in cell free extracts of recombinant strains were 10 times higher than non-recombinants. This result indicates sucessful expression of the cloned regulatory subunit gene with ATPase activity in E.coli.

No description available.

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable aß? heterotrimer comprising a catalytic a subunit and two non-catalytic subunits, ß and ?. The ß subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here I show that the conserved C-terminal 85-residue sequence of the ß subunit, ß1(186-270), is sufficient to form an active AMP-dependent heterotrimer a1ß1(186-270)?1, whereas the 25-residue ß1 C-terminal (246-270) sequence is sufficient to bind ?1, ?2, or ?3 but not the a subunit. Within this sequence (246-270), two residues were essential for ß? association based on Ala scanning mutagenesis. / Substitution of ß1 Tyr-267 for Ala precludes ß? but not aß association suggesting independent binding requirements. Substitution of Tyr-267 for Phe or His but not Ala or Ser can rescue ß? binding. Substitution of Thr-263 for Ala also resulted in decreased ß? but not aß association. Truncation of the a subunit reveals that ß1 binding requires the a1(313-473) sequence while the remainder of the a C-terminus is required for ? binding. The conserved C-terminal 85-residue sequence of the ß subunit (90% between ß1 and ß2) is the primary a? binding sequence responsible for the formation of the AMPK aß? heterotrimer. The ? subunits contain four repeat CBS sequences with variable N-terminal extensions and the ?1 isoform is N-terminally acetylated. The ?2 subunit can be multiply phosphorylated by protein kinase C (PKC) in vitro, with Ser-32 identified as a minor site. A detailed understanding of the structure and regulation of AMPK will enable rational drug design for treatment of such linked diseases as obesity, insulin resistance and type 2 diabetes.

Diss. (sammanfattning) Göteborg : Univ., 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / "May 2007" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. "August 2008" Includes bibliographical references.

Dissertation (Ph.D.)--University of Toledo, 2009. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Table of contents (p. iii) incorrect starting page number for third bibliography (says p. 123, actually is p. 121) and incorrect starting page number for abstract (says p. 156, actually is p. 154). Bibliography: p. 76-83, 108-111, 121-153.

Thesis (Ph.D.)--Medical University of Ohio, 2006. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Han-Fei Ding. Includes abstract. Document formatted into pages: iv, 163 p. Title from title page of PDF document. Title at ETD Web site: NF-KappaB2 is an autoimmunity regulator and its mutation leads to lymphomagenesis in mice. Bibliography: pages 67-77,106-112,134-161.

Microsporidia are a unique phylum of ubiquitous fungal pathogens that are able to infect a wide variety of hosts, including economically and ecologically important organisms. Recently, global declines of the Western honeybee (Apis mellifera) have been associated with infections of the microsporidian pathogen Nosema ceranae. This species was originally described in the Asiatic honeybee (A. cerana), and its identification in global A. mellifera hives could result from a recent host transfer. Recent genome studies have found that global populations of this parasite from A. mellifera hives are polyploid and that humans may have fueled their global expansion. In this thesis, I investigate the genetic diversity of N. ceranae populations from within their native range (Thailand) and among different hosts (A. mellifera, A. cerana), putting them in context with other previously sequenced global populations. Using both PCR and genome-based methods, my findings reveal that Thai populations of N. ceranae exhibit interesting genetic differences from other global pathogen populations but also have some similarities. Thai N. ceranae populations share many single nucleotide polymorphisms (SNPs) with other global populations and appear to be clonal. However, in stark contrast with previous studies, these populations carry many SNPs not found in other global populations of this parasite, indicating that these populations have evolved in their current geographic location for some time. This genome analysis also indicates the potential presence of diploidy within Thai populations of N. ceranae and possible host-specific loss of heterozygosity. Overall, my findings begin to reveal interesting patterns of genetic diversity in N. ceranae populations that bring us one step closer to understanding the biology and genetics of this important honeybee pathogen.