Post-translational signal peptide cleavage controls differential epitope recognition in the QP-rich domain of recombinant Theileria parva PIM

The presence of the schizont stage of the obligate intracellular parasites Theileria parva or T. annulata in the cytoplasm of an infected leukocyte results in host cell transformation via a mechanism that has not yet been elucidated. Proteins, secreted by the schizont, or expressed on its surface, are of interest as they can interact with host cell molecules that regulate host cell proliferation and/or survival. The major schizont surface protein is the polymorphic immunodominant molecule, PIM, which contains a large glutamine- and proline-rich domain (QP-rd) that protrudes into the host cell cytoplasm. Analyzing QP-rd generated by in vitro transcription/translation, we found that the signal peptide was efficiently cleaved post-translationally upon addition of T cell lysate or canine pancreatic microsomes, whereas signal peptide cleavage of a control protein only occurred cotranslationally and in the presence of microsomal membranes. The QP-rd of PIM migrated anomalously in SDS-PAGE and removal of the 19 amino acids corresponding to the predicted signal peptide caused a decrease in apparent molecular mass of 24kDa. The molecule was analyzed using monoclonal antibodies that recognize a set of previously defined PIM epitopes. Depending on the presence or the absence of the signal peptide, two conformational states could be demonstrated that are differentially recognized, with N-terminal epitopes becoming readily accessible upon signal peptide removal, and C-terminal epitopes becoming masked. Similar observations were made when the QP-rd of PIM was expressed in bacteria. Our observations could also be of relevance to other schizont proteins. A recent analysis of the proteomes of T. parva and T. annulata revealed the presence of a large family of potentially secreted proteins, characterized by the presence of large stretches of amino acids that are also particularly rich in QP-residues.

Leiomyomatoid angiomatous neuroendocrine tumor (LANT) of the pituitary: a distinctive biphasic neoplasm with primitive secretory phenotype and smooth muscle-rich stroma

We describe a hitherto undocumented variant of dimorphic pituitary neoplasm composed of an admixture of neurosecretory cells and profuse leiomyomatous stroma around intratumoral vessels. Radiologically perceived as a macroadenoma of 3.8 cm in diameter, this pituitary mass developed in an otherwise healthy 43-year-old female. At the term of a yearlong history of amenorrhea and progressive bitemporal visual loss, subtotal resection was performed via transsphenoidal microsurgery. Discounting mild hyperprolactinemia, there was no evidence of excess hormone production. Histologically, solid sheets, nests and cords of epithelial-looking, yet cytokeratin-negative cells were seen growing in a richly vascularized stroma of spindle cells. While strong immunoreactivity for NCAM, Synaptophysin and Chromogranin-A was detected in the former, the latter showed both morphological and immunophenotypic hallmarks of smooth muscle, being positive for vimentin, muscle actin and smooth muscle actin. Architectural patterns varied from monomorphous stroma-dominant zones through biphasic neuroendocrine-leiomyomatous areas, to pseudopapillary fronds along vascular cores. Only endothelia were labeled with CD34. Staining for S100 protein and GFAP, characteristics of sustentacular cells, as well as bcl-2 and c-kit was absent. Except for alpha-subunit, anterior pituitary hormones tested negative in tumor cells, as did a panel of peripheral endocrine markers, including serotonin, somatostatin, calcitonin, parathormone and vasoactive intestinal polypeptide. Mitotic activity was absent and the MIB-1 labeling index low (1-2%). While assignment of this lesion to any established neoplastic entity is not forthcoming, we propose it is being considered as a low-grade neuroendocrine tumor possibly related to null cell adenoma.

Adaptations of intestinal macrophages to an antigen-rich environment

Intestinal macrophages, preferentially located in the subepithelial lamina propria, represent in humans the largest pool of tissue macrophages. To comply with their main task, i.e. the efficient removal of microbes and particulate matter that might have gained access to the mucosa from the intestinal lumen while maintaining local tissue homeostasis, several phenotypic and functional adaptations evolved. Most notably, microbe-associated molecular pattern (MAMP) receptors, including the lipopolysaccharide receptors CD14 and TLR4, but also the Fc receptors for IgA and IgG are absent on most intestinal Mø. Here we review recent findings on the phenotypic and functional adaptations of intestinal Mø and their implications for the pathogenesis of inflammatory bowel diseases.

A family of stage-specific alanine-rich proteins on the surface of epimastigote forms of Trypanosoma brucei

A 'two coat' model of the life cycle of Trypanosoma brucei has prevailed for more than 15 years. Metacyclic forms transmitted by infected tsetse flies and mammalian bloodstream forms are covered by variant surface glycoproteins. All other life cycle stages were believed to have a procyclin coat, until it was shown recently that epimastigote forms in tsetse salivary glands express procyclin mRNAs without translating them. As epimastigote forms cannot be cultured, a procedure was devised to compare the transcriptomes of parasites in different fly tissues. Transcripts encoding a family of glycosylphosphatidyl inositol-anchored proteins, BARPs (previously called bloodstream alanine-rich proteins), were 20-fold more abundant in salivary gland than midgut (procyclic) trypanosomes. Anti-BARP antisera reacted strongly and exclusively with salivary gland parasites and a BARP 3' flanking region directed epimastigote-specific expression of reporter genes in the fly, but inhibited expression in bloodstream and procyclic forms. In contrast to an earlier report, we could not detect BARPs in bloodstream forms. We propose that BARPs form a stage-specific coat for epimastigote forms and suggest renaming them brucei alanine-rich proteins.

Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA

The mRNA stabilizing factor HuR is involved in the posttranscriptional regulation of many genes, including that coding for cyclooxygenase 2 (COX-2). Employing RNA interference technology and actinomycin D experiments, we demonstrate that in human mesangial cells (hMC) the amplification of cytokine-induced COX-2 by angiotensin II (AngII) occurs via a HuR-mediated increase of mRNA stability. Using COX-2 promoter constructs with different portions of the 3' untranslated region of COX-2, we found that the increase in COX-2 mRNA stability is attributable to a distal class III type of AU-rich element (ARE). Likewise, the RNA immunoprecipitation assay showed AngII-induced binding of HuR to this ARE. Using the RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeleton-bound polysomes indicative of an active RNP complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by increased nucleocytoplasmic HuR shuttling and depends on protein kinase Cdelta (PKCdelta), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCdelta-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCdelta represents an important novel mode of HuR activation implied in renal COX-2 regulation.

The Rate of Precipitation of Copper Aluminide in the Silver Rich Silver-Copper-Aluminum Alloys

In order to determine the best annealing temperature at which to age-harden the alloys, hardness tests on speci­men annealed for different lengths of time at different temperatures were made.

Legislative Legacy: Big Changes in Legislative Article of 1972 Constitution -- Jerry Loendorf, Arlyne Reichert & Rich Bechtel “In the Crucible of Change”

The fulcrum upon which were leveraged many of the dramatic progressive changes in Montana that are documented "In the Crucible of Change" series was the lead up to, preparation, writing and adoption of the 1972 Montana Constitution. As Montana citizens exhibited their concern over the dysfunctional state government in MT under its 1889 Constitution, one of the areas that stood out as needing serious change was the Montana Legislature. Meeting for only sixty calendar days every two years, the Legislature regularly tried to carry off the subterfuge of stopping the wall clock at 11:59 PM on the sixtieth day and placing a shroud over it so they could continue to conduct business as if it were still the 60th day. Lawyers hired by the Anaconda Company drafted most bills that legislators wanted to have introduced. Malapportionment, especially in the State Senate where each county had one Senator regardless of their population, created a situation where Petroleum County with 800 residents had one senator while neighboring Yellowstone County with 80,000 people also had one senator -- a 100-1 differential in representation. Reapportionment imposed by rulings of the US Supreme Court in the mid-1960s created great furor in rural Montana to go along with the previous dissatisfaction of the urban centers. Stories of Anaconda Company “thumbs up – thumbs down” control of the votes were prevalent. Committee meeting and votes were done behind closed doors and recorded votes were non-existent except for the nearly meaningless final tally. People were in the dark about the creation of laws that affected their daily lives. It was clear that change in the Legislature had to take the form of change in the Constitution and, because it was not likely that the Legislature would advance Constitutional amendments on the subject, a convention seemed the only remedy. Once that Convention was called and went to work, it became apparent that the Legislative Article provided both opportunity for change and danger that too dramatic a change might sink the whole new document. The activities of the Legislative Committee and the whole Convention when acting upon Legislative issues provides one of the more compelling stories of change. The story of the Legislative Article of the Montana Constitution is discussed in this episode by three major players who were directly involved in the effort: Jerry Loendorf, Arlyne Reichert and Rich Bechtel. Their recollections of the activities surrounding the entire Constitutional Convention and specifically the Legislative Article provide an insider’s perspective of the development of the entire Constitution and the Legislative portion which was of such a high degree of interest to the people of Montana during the important period of progressive change documented “In the Crucible of Change.” Jerry Loendorf, who served as Chair of the Legislative Committee at the 1972 Montana Constitutional Convention, received a BA from Carroll College in 1961 and a JD from the University of Montana Law School in 1964. Upon graduation he served two years as a law clerk for the Montana Supreme Court after which he was for 34 years a partner in the law firm of Harrison, Loendorf & Posten, Duncan. In addition to being a delegate to the Constitutional Convention, Jerry served on the Board of Labor Appeals from 2000 to 2004. He was designated a Montana Special Assistant Attorney General to represent the state in federal court on the challenge to the results of the ratification election of Montana's Constitution in 1972. Jerry served on the Carroll College Board of Directors in the late 1960s and then again as a member of the Board of Trustees of Carroll College from 2001 to 2009. He has served on the Board of Directors of the Rocky Mountain Development Council since 1970 and was on the board of the Helena YMCA from 1981 to 1987. He also served on the board of the Good Samaritan Ministries from 2009 to 2014. On the business side, Jerry was on the Board of Directors of Valley Bank to Helena from 1980 to 2005. He is a member of the American Bar Association, State Bar of Montana, the First Judicial District Bar Association, and the Montana Trial Lawyers Association. Carroll College awarded Jerry the Warren Nelson Award 1994 and the Insignias Award in 2007. At Carroll College, Jerry has funded the following three scholarship endowments: George C and Helen T Loendorf, Gary Turcott, and Fr. William Greytek. Arlyne Reichert, Great Falls Delegate to the Constitutional Convention and former State Legislator, was born in Buffalo, NY in 1926 and attended University of Buffalo in conjunction with Cadet Nurses Training during WWII. She married a Montanan in Great Falls in 1945 and was widowed in 1968. She is mother of five, grandmother of seven, great-grandmother of four. Arlyne was employed by McLaughlin Research Institute in Great Falls for 23 years, serving as Technical Editor of Transplantation Journal in 1967, retiring as Assistant Director in 1989. In addition to being a state legislator (1979 Session) and a delegate to the 1972 Montana Constitutional Convention, she has filled many public roles, including Cascade County Study Commissioner (1974), MT Comprehensive Health Council, US Civil Rights Commission MT Advisory Committee, MT Capitol Restoration Committee, and Great Falls Public Library Trustee. Arlyne has engaged in many non-profit activities including League of Women Voters (State & Local Board Officer – from where her interest in the MT Constitutional change developed), Great Falls Public Radio Association (President & Founder), American Cancer Society (President Great Falls Chapter), Chair of MT Rhodes Scholarship Committee, and Council Member of the National Civic League. She also served a while as a Television Legislative Reporter. Arlyne has been recipient of numerous awards, the National Distinguished Citizens Award from the National Municipal League, two Women of Achievement Awards from Business & Professional Women, the Salute to Women Award by YWCA, Heritage Preservation Award from Cascade County Historical Society and the State of Montana, and the Heroes Award from Humanities Montana. She remains active, serving as Secretary-Treasurer of Preservation Cascade, Inc., and as Board Member of the McLaughlin Research Institute. Her current passion is applied to the preservation/saving of the historic 10th Street Bridge that crosses the Missouri River in Great Falls. Rich Bechtel of Helena was born in Napa, California in 1945 and grew up as an Air Force brat living in such places as Bitberg, Germany, Tripoli, Libya, and Sevilla, Spain. He graduated from Glasgow High School and the University of Montana. Rich was a graduate assistant for noted Montana History professor Professor K. Ross Toole, but dropped out of graduate school to pursue a real life in Montana politics and government. Rich has had a long, varied and colorful career in the public arena. He currently is the Director of the Office of Taxpayer Assistance & Public Outreach for MT’s Department of Revenue. He previously held two positions with the National Wildlife Federation in Washington, DC (Sr. Legislative Representative [1989-91] and Sr. Legislative Representative for Wildlife Policy [2004-2006]). While in Washington DC, he also was Assistant for Senator Lee Metcalf (D-MT), 1974-1976; Federal-State Coordinator for State of Montana, 1976-1989; Director of the Western Governors’ Association Washington Office, 1991-2000; and Director of Federal Affairs for Governor Kitzhaber of Oregon, 2001- 2003. Earlier in Montana Government, between 1971 and 1974, Rich was Research Analyst for MT Blue Ribbon Commission on Postsecondary Education, Legislative Consultant and Bill Drafter for MT Legislative Council, Research Analyst for the MT Constitutional Convention Commission where he provided original research on legislatures, as well as Researcher/Staff for the MT Constitutional Convention Legislative Committee, from where he drafted the various provisions of the Legislative Article and the majority and minority reports on behalf of the Committee members. Rich has represented Montana’s Governor on a trade and cultural mission to Republic of China and participated in US-German Acid Rain Committee sessions in Germany and with European Economic Community environmental officials in Belgium. He is married to Yvonne Seng (Ph.D.) - T’ai Chi apprentice; author and birder.

Effect of platelet-rich plasma on the healing of intrabony defects treated with an anorganic bovine bone mineral: a pilot study

BACKGROUND: Periodontal therapy using the combination of platelet-rich plasma (PRP) and different grafting materials has been suggested as a modality to enhance the outcome of regenerative surgery. In most clinical studies, a barrier membrane was used to cover the defects, and thus, the effects of PRP may have been masked by the effects of the barrier. The data from controlled clinical studies evaluating the effect of regenerative therapy using various grafting materials with or without PRP are still limited. The purpose of this study was to clinically compare the healing of intrabony defects treated with either a combination of an anorganic bovine bone mineral (ABBM) and PRP to those obtained with ABBM alone. METHODS: Thirty patients with advanced chronic periodontal disease and displaying one intrabony defect were randomly treated with PRP + ABBM or ABBM alone. The following clinical parameters were evaluated at baseline and 1 year after treatment: plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD), gingival recession (GR), and clinical attachment level (CAL). The primary outcome variable was CAL. RESULTS: No statistical significant differences in any of the investigated parameters between the two groups were observed at baseline. Healing was uneventful in all patients. In the PRP + ABBM group, mean PD decreased from 8.6 +/- 1.8 mm to 3.4 +/- 1.4 mm (P <0.001) and mean CAL changed from 9.9 +/- 1.7 mm to 5.3 +/- 1.8 mm (P <0.001). In the ABBM group, mean PD decreased from 8.5 +/- 2.0 mm to 3.2 +/- 1.3 mm (P <0.001) and mean CAL changed from 9.6 +/- 1.9 mm to 4.9 +/- 1.5 mm (P <0.001). CAL gains >or=3 mm were measured in 80% (12 of 15 defects) of cases treated with PRP + ABBM and in 87% (13 of 15 defects) of cases treated with ABBM alone. No statistically significant differences in any of the investigated parameters were observed between the two groups at the 1-year reevaluation. CONCLUSIONS: Within the limits of the present study, it can be concluded that 1) at 1 year after regenerative surgery with PRP + ABBM and ABBM alone, significant PD reductions and CAL gains were found, and 2) the use of PRP failed to improve the results obtained with ABBM alone.