UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase from Dictyostelium discoideum recognizes serine-containing peptides and eukaryotic cysteine proteinases

Phosphoglycosylation catalyzed by UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase (Ser:GlcNAc phosphotransferase) adds GlcNAcalpha-1-P to peptidyl-Ser of selected Dictyostelium discoideum proteins. Lysosomal cysteine proteinase (CP), proteinase-1(CP7), is the major phosphoglycosylated protein in bacterially grown amoebae. GlcNAc-1-P is added within a Ser-rich domain containing SSS, SGSG, or SGSQ repeated motifs that are not found in other papain-like CPs. We studied the substrate specificity of the transferase using peptides containing these motifs and 12 other peptides with one or more Ser residues. Phosphoglycosylation is comparable for all three Dictyostelium CP motifs, but it is not restricted to them. Flanking residues in the other peptides strongly influence phosphoglycosylation efficiency. Dictyostelium microsomal membranes also phosphoglycosylate endogenous acceptors, and some of these acceptors occur as an 18 S complex with the transferase. CP-serine motif peptides inhibit endogenous acceptor phosphoglycosylation weakly (30-40%) at 800 microM, whereas catalytically inactive proteinase-1(CP7) and other non-phosphoglycosylated eukaryotic CPs, lacking the serine domain, inhibit transferase activity at 1-4 microM. SDS denaturation destroys the inhibitory potential of all CPs showing that transferase recognizes a conformation-dependent feature that is shared by all. Proteinase-1(CP7) expressed in Escherichia coli lacks GlcNAc-1-P, but it is a substrate for Ser:GlcNAc phosphotransferase, Km = 5.6 microM. Thus, Ser:GlcNAc phosphotransferase recognizes both acceptor peptide sequences and a conformational feature of eukaryotic CPs. This may be physiologically important for establishing or maintaining non-overlapping groups of GlcNAc-1-P- and Man-6-P-modified Dictyostelium proteins that reside in functionally distinct endo-lysosomal vesicles.     

Production of human normal adult and fetal hemoglobins in Escherichia coli

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha- globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma- globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.      

Power from Perspective: Potential future United States energy portfolios

This paper presents United States energy portfolios for the year 2030, developed from seven different Perspectives. The Perspectives are characterized by different weights placed on fourteen defining values (e.g., cost, social acceptance). The portfolios were constructed to achieve three primary goals, energy independence, energy security, and greenhouse gas reductions. The portfolios are also evaluated over a comprehensive set of secondary criteria (e.g., economic growth, technical feasibility). It is found that very different portfolios based on very different defining values can achieve the three primary goals. Commonalities among the portfolios include reliance upon cellulosic ethanol, nuclear power, and energy efficiency to meet year 2030 energy demands. It is concluded that the US energy portfolio must be diverse and to achieve national energy goals will require an explicit statement of goals, a strong role for government, and coordinated action across society.     

Event-related brain potentials during semantic categorization in normal aging and senile dementia of the Alzheimer's type

To assess the effects of normal aging and senile dementia of the Alzheimer's type (SDAT) on semantic analysis of words, we examined the N400 component of the event-related potential (ERP) elicited during the processing of highly constrained (opposites) and less constrained materials (category-category exemplars) in 12 young control subjects, 12 elderly control subjects and 12 patients with SDAT. We employed a priming paradigm in which a context phrase was spoken and a target word (congruent or incongruent) was presented visually. The N400 effect was reduced in amplitude and delayed in the elderly control group relative to that of the younger subjects, and was further attenuated in amplitude, delayed in latency and somewhat flatter in its distribution across the scalp in the SDAT patients. These findings are consistent with less efficient processing and integration of lexical items with semantic context in normal aging, which is further exacerbated by SDAT. Differences in the N400 range associated with the opposite and category conditions were observed only in the young subjects, suggesting less use of controlled attentional resources or perhaps weaker associative links with age.     

Activation of T lymphocytes by syngeneic murine intestinal smooth muscle cells

BACKGROUND & AIMS: Intestinal smooth muscle cells (ISMCs) express major histocompatibility complex II (MCH II) and intercellular adhesion molecule 1 (ICAM-1) after exposure to interferon gamma (IFN-gamma). T lymphocytes invade the intestinal musculature during Crohn's disease or pseudoobstruction. The aim of this study was to determine whether ISMCs activate syngeneic T cells via MHC II and ICAM-1. METHODS: Cultured murine ISMCs were exposed to IFN-gamma for 72 hours and analyzed for Mac-1 (CD11B CD18) antigen, MHC II, and ICAM-1 expression using enzyme-linked immunosorbent assay and fluorescence-activated cell sorter scan. T lymphocytes from mesenteric lymph nodes of ovalbumin-sensitized mice were examined for their ability to proliferate after coculture with IFN-gamma-pretreated and ovalbumin-pretreated ISMCs using [3H]thymidine incorporation. RESULTS: ISMCs expressed smooth muscle alpha-actin before and after IFN-gamma exposure. No macrophages were identified in these cultures. Exposure to IFN-gamma and ovalbumin for 72 hours induced MHC II and ICAM-1 expression; these treated ISMCs induced T-cell proliferation, whereas untreated ISMCs did not. T-cell proliferation was markedly enhanced by adding interleukin 2 and was blocked by antibodies against MHC II and ICAM-1. CONCLUSIONS: ISMCs activate T lymphocytes in an MHC II-linked manner and thus possess the ability to modulate immune function in the gut.     

Expression, purification, and bioassay of human stanniocalcin from baculovirus-infected insect cells and recombinant CHO cells

Pustulosis palmoplantaris (PPP) is a common chronic skin disease, which is very resistant to treatment. It is not known why the lesions are located in the palms and soles. There are few studies of the disease and in particular studies of the histology. Fifty-nine patients with PPP answered a questionnaire concerning their medical history and 39 of them were clinically examined. Biopsy specimens were taken from involved skin in 22 of the 39 patients and studied immunohistologically for tryptase+ mast cells, EG2+ eosinophils, lipocalin+ neutrophils and CD3+ T lymphocytes. The sweat gland and sweat duct were visualized with AE1/AE3 antibody (cytokeratins 1-8, 10, 14/15, 16, 19). In addition to neutrophils in the pustule and lymphocytes in the upper dermis, there were also large numbers of mast cells and eosinophils in the subpustular area. Numerous eosinophils were present in the pustule. The epidermal part of the eccrine duct was not detectable in any of the specimens from patients with PPP but was present in all of the nine control persons (including two smokers). The results indicate that the acrosyringium is involved in the inflammation and also that mast cells and eosinophils participate in a hitherto unknown way. Of the 39 patients clinically examined, two had previously diagnosed thyroid disease and two had gluten hypersensitivity. Seventeen had one or several abnormal serum concentrations of thyroid-stimulating hormone, thyroxin, antibodies against thyroglobulin or thyroperoxidase and 10 had immunoglobulin (Ig) A antibodies to gliadin. The mean +/- SD for serum IgA and for eosinophil cationic protein was increased. From the questionnaire the most notable finding was that 56 of the 59 patients had been or still were smokers, all of whom had started smoking before the first signs of PPP. We hypothesize that the acrosyringium might be the target for the inflammation and that PPP is linked to autoimmune thyroid disease and smoking.