Azotobacter genomes: the genome of Azotobacter chroococcum NCIMB 8003 (ATCC 4412)

The genome of the soil-dwelling heterotrophic N2-fixing Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 (ATCC 4412) (Ac-8003) has been determined. It consists of 7 circular replicons totalling 5,192,291 bp comprising a circular chromosome of 4,591,803 bp and six plasmids pAcX50a, b, c, d, e, f of 10,435 bp, 13,852, 62,783, 69,713, 132,724, and 311,724 bp respectively. The chromosome has a G+C content of 66.27% and the six plasmids have G+C contents of 58.1, 55.3, 56.7, 59.2, 61.9, and 62.6% respectively. The methylome has also been determined and 5 methylation motifs have been identified. The genome also contains a very high number of transposase/inactivated transposase genes from at least 12 of the 17 recognised insertion sequence families. The Ac-8003 genome has been compared with that of Azotobacter vinelandii ATCC BAA-1303 (Av-DJ), a derivative of strain O, the only other member of the Azotobacteraceae determined so far which has a single chromosome of 5,365,318 bp and no plasmids. The chromosomes show significant stretches of synteny throughout but also reveal a history of many deletion/insertion events. The Ac-8003 genome encodes 4628 predicted protein-encoding genes of which 568 (12.2%) are plasmid borne. 3048 (65%) of these show > 85% identity to the 5050 protein-encoding genes identified in Av-DJ, and of these 99 are plasmid-borne. The core biosynthetic and metabolic pathways and macromolecular architectures and machineries of these organisms appear largely conserved including genes for CO-dehydrogenase, formate dehydrogenase and a soluble NiFe-hydrogenase. The genetic bases for many of the detailed phenotypic differences reported for these organisms have also been identified. Also many other potential phenotypic differences have been uncovered. Properties endowed by the plasmids are described including the presence of an entire aerobic corrin synthesis pathway in pAcX50f and the presence of genes for retro-conjugation in pAcX50c. All these findings are related to the potentially different environmental niches from which these organisms were isolated and to emerging theories about how microbes contribute to their communities

Agrin: Soluble and Substrate -Attached Studies of a Synaptogenic Protein

212 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2004.During the development of the nervous system, environmental information affects neuronal phenotype, pathfinding, and synaptic connections. At the neuromuscular junction (NMJ), the extracellular matrix (ECM) protein agrin is secreted into the synaptic cleft by the presynaptic motor neuron. Agrin interacts with an unknown receptor to induce local aggregation of acetylcholine receptors (AChRs) and other postsynaptic molecules. It is known that agrin's AChR clustering activity resides wholly in its carboxyl-terminal half, which is composed of laminin-like globular domains (G1, G2 and G3) and EGF-like domains. Furthermore, this activity is modulated by tissue-specific alternative mRNA splicing in two of these G-domains. This study employs a two-fold approach to analyze agrin's structure-function relationship: (1) isolate and recombine agrin's structural domains to analyze their contribution to agrin's AChR clustering activity in the traditional soluble context and (2) examine agrin isoforms in a novel, substrate-attached assay that more closely mimics the ECM-attached nature of agrin in vivo. Briefly, the first portion of this study has revealed the following: (1) while only the G3(8) domain exhibits detectable activity by itself, all G-domains studied (G1, G2(0), G2(4), G3(0) and G3(8)) enhance G3(8) activity when physically linked to G3(8). This effect is most pronounced when G2(4) is linked to G3(8) and is independent of the order of the G-domains. In contrast, simple mixing of equimolar soluble G2(4) and G3(8) domains does not augment G3(8) activity. (2) The deletion of EGF-like repeats enhances activity. (3) Increasing the physical separation between linked G1 and G3(8) domains significantly increases activity; similar alterations to linked G2 and G3(8) domains are without effect. (4) AChR clusters induced by concatenated G3(8) domains are significantly smaller than those induced by all other agrin forms studied. The second portion of this study has demonstrated that: (1) microprinted agrin induces AChR colocalization in cultured muscle cells. (2) Both agrin(4,8) and agrin(4,0) are active when printed, while control proteins are not. (3) Clustering induced by agrin(4,8) extends beyond the printed pattern, while that induced by agrin (4,0) does not. Additionally, we have demonstrated that microcontact printing is a viable method for studying cellular biological phenomena such as synaptogenesis.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

HPASubC: A suite of tools for user subclassification of human protein atlas tissue images

Background: The human protein atlas (HPA) is a powerful proteomic tool for visualizing the distribution of protein expression across most human tissues and many common malignancies. The HPA includes immunohistochemically-stained images from tissue microarrays (TMAs) that cover 48 tissue types and 20 common malignancies. The TMA data are used to provide expression information at the tissue, cellular, and occasionally, subcellular level. The HPA also provides subcellular data from confocal immunofluorescence data on three cell lines. Despite the availability of localization data, many unique patterns of cellular and subcellular expression are not documented. Materials and Methods: To get at this more granular data, we have developed a suite of Python scripts, HPASubC, to aid in subcellular, and cell-type specific classification of HPA images. This method allows the user to download and optimize specific HPA TMA images for review. Then, using a playstation-style video game controller, a trained observer can rapidly step through 10′s of 1000′s of images to identify patterns of interest. Results: We have successfully used this method to identify 703 endothelial cell (EC) and/or smooth muscle cell (SMCs) specific proteins discovered within 49,200 heart TMA images. This list will assist us in subdividing cardiac gene or protein array data into expression by one of the predominant cell types of the myocardium: Myocytes, SMCs or ECs. Conclusions: The opportunity to further characterize unique staining patterns across a range of human tissues and malignancies will accelerate our understanding of disease processes and point to novel markers for tissue evaluation in surgical pathology

Morphologic Characterization of Syndromic Gastric Polyps

The morphology of gastric hamartomatous polyps from patients with juvenile polyposis syndrome (JuvPS) and Peutz-Jeghers' Syndrome (PJS) is poorly characterized. We investigated the histologic features of gastric polyps in patients with established JuvPS or PJS to develop improved histologic criteria to distinguish these from gastric hyperplastic (HP) polyps. The patients with clinically confirmed hamartomatous polyposis syndromes were identified, including 26 patients with JuvPS (both familial and sporadic) and 17 patients with PJS. All gastric polyps (n = 30) from these patients were intermixed with gastric HP polyps from nonsyndromic patients (n = 26) and subsequently blindly reviewed by a panel of gastrointestinal pathologists. A consensus diagnosis was rendered. The panel then reviewed the slides in the context of clinical data and identified histologic features for distinguishing JuvPS, PJS, and HP gastric polyps based on epithelial changes, pit architecture, lamina propria features, and smooth muscle qualities. A sleeping period of 6 months lapsed before the same cases were renumbered and blindly rereviewed independently. Diagnoses were then rendered while adhering to the suggested criteria. Cases that the reviewers recalled were discarded from the study (n = 8). On initial review, accuracy in diagnosis of gastric polyps in JuvPS was 50% and was 18% in PJS compared with 92% for HP gastric polyps. Adherence to the recommended histologic criteria resulted in diagnostic accuracy of 41% for JuvPS and 54% for PJS, compared with 73% for HP gastric polyps. Accuracy in diagnosis in antral mucosa was 66%, oxyntic mucosa 71%, and transitional-type mucosa (mixed antral and oxyntic) 32%. The diagnostic accuracy based on polyp size was 59% for polyps which were less than equal to 3 mm, 56% for those 4 to 9 mm, and 81% for polyps which were more than equal to 10 mm. The identification of gastric polyps from JuvPS and PJS patients without the context of clinical history of these syndromes remains poor, even with adherence to a set of morphologic criteria. Abiding by such criteria improved recognition of PJS polyps by more than double (P < 0.19), but yielded an accuracy of only 54%. The accuracy did not improve when results were stratified for polyp location but did with biopsy size which were more than equal to 10 mm. Whereas these syndromic polyps are readily diagnosed in the small bowel and colon, histologic features to distinguish gastric JuvPS and PJS from gastric HP polyps are unreliable

Gastric Lesions in Patients With Autoimmune Metaplastic Atrophic Gastritis (AMAG) in a Tertiary Care Setting

Autoimmune metaplastic atrophic gastritis (AMAG) is an early manifestation of pernicious anemia that precedes the hematologic changes by years to decades. It is associated with metaplastic changes and neoplasms, including pyloric gland adenomas (PGAs). We investigated the frequency of PGAs and other lesions in all nonconsultation gastric biopsies and resections (1988 to 2008) diagnosed as AMAG. We further selected cases confirmed as AMAG by immunohistochemical identification of the gastric body (negative gastrin) and linear and nodular enterochromaffin-like cell hyperplasia (chromogranin). From this subset, all polyps and neoplasms were reviewed. We identified a total of 41,245 patients with gastric biopsies or resections from 46.7% males and 53.3% females comprising patients self-identified as 67.0% white, 23.6% African-American, 1.4% Asian, 0.8% non-White Hispanic, and 7.2% other or unknown. AMAG was diagnosed in 461 patients (1.1%), and had the following percentages based on race: 1.1% White, 1.3% African-American, 1.4% Asian, and 2.7% non-White Hispanic. The female: male ratio was 2: 1 with an overall median age at presentation of 67.0 years. Of the 461 patients with AMAG, 143 had endoscopically identifiable lesions. These lesions (n = 240) consisted of 179 polyps (138 hyperplastic polyps, 20 oxyntic mucosa pseudopolyps, 18 intestinal-type gastric adenomas, and 3 PGAs), 46 well-differentiated neuroendocrine neoplasms (carcinoid), 1 gastrointestinal stromal tumor, 3 lymphomas, and 11 adenocarcinomas. In summary, AMAG occurred with similar frequency across all racial groups. Although PGAs are associated with AMAG, they remain rare in the setting of AMAG