William N. Flemming, A.B. in Speech, Class of 1949

2 scans: first with all captions, 2nd just image - same housescan #, suffix 1of2 and 2of2

Efficiency test on power plant of Jenckes spinning company, Pawtucket, Rhode Island; a graduating thesis presented by Ward N. Gere, Richard B. Cross, Benj. S. Munch, C.M. Loeb, students in the class of 1913 at the Massachusetts institute of technology ...

Mode of access: Internet.

B cell chronic lymphocytic leukaemia cells have reduced capacity to upregulate expression of MHC class I in response to interferon-gamma

Aims: An important consideration in the design of a tumour vaccine is the ability of tumour-specific cytotoxic T lymphocytes (CTL) to recognise unmanipulated tumour cells in vivo. To determine whether B-CLL might use an escape strategy, the current studies compared B-CLL and normal B cell MHC class I expression. Methods: Flow cytometry, TAP allele PCR and MHC class I PCR were used. Results: While baseline expression of MHC class I did not differ, upregulation of MHC class I expression by B-CLL cells in response to IFN-gamma was reduced. No deletions or mutations of TAP 1 or 2 genes were detected. B-CLL cells upregulated TAP protein expression in response to IFN-gamma. Responsiveness of B-CLL MHC class I mRNA to IFN-gamma was not impaired. Conclusions: The data suggest that MHC class I molecules might be less stable at the cell surface in B-CLL than normal B cells, as a result of the described release of beta(2)m and beta(2)m-free class I heavy chains from the membrane. This relative MHC class I expression defect of B-CLL cells may reduce their susceptibility to CTL lysis in response to immunotherapeutic approaches.

Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin

Mannose-binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K-12 strain, and affinity measurements with mannose, common mono- and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl alpha- D-mannoside, bound in the crystal structures, exhibits a significantly better affinity for FimH (K-d = 0.15 muM) than mannose (K-d = 2.3 muM). Exploration of the binding affinities of alpha-D-mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100-fold improvement and 15-fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti-adhesive drugs to prevent anticipated and recurrent UTIs.

Higher-order CpG-DNA stimulation reveals distinct activation requirements for marginal zone and follicular B cells in lupus mice

Mouse follicular B cells express TLR9 and respond vigorously to stimulation with single-stranded CpG-oligodeoxynucleotides (ODN). Surprisingly, follicular B cells do not respond to direct stimulation with other TLR9 ligands, such as bacterial DNA or class A(D) CpG-ODN capable of forming higher-order structures, unless other cell types are present. Here, we show that priming with interferons or with B cell-activating factor, or simultaneous co-engagement of the B cell receptor for antigen (BCR), can overcome this unresponsiveness. The effect of interferons occurs at the transcriptional level and is mediated through an autocrine/paracrine loop, which is dependent on IRF-1, IL-6 and IL-12 p40. We hypothesize that the lack of bystander activation of follicular B cells with more complex CpG ligands may be an important safety mechanism for avoiding autoimmunity. This will prevent resting B cells from responding to foreign or self-derived hypomethylated double-stranded CpG ligands unless these ligands are either delivered through the B cell receptor or under conditions where B cells are simultaneously co-engaged by activated plasmacytoid dendritic cells or TH1 cells. A corollary is that the heightened responsiveness of lupus B cells to TLR9-induced stimulation cannot be ascribed to unprimed follicular B cells, but is rather mediated by hypersensitive marginal zone B cells.

Alpha-selenoconotoxins, a new class of potent alpha(7) neuronal nicotinic receptor antagonists

Disulfide bonds are important structural motifs that play an essential role in maintaining the conformational stability of many bioactive peptides. Of particular importance are the conotoxins, which selectively target a wide range of ion channels that are implicated in numerous disease states. Despite the enormous potential of conotoxins as therapeutics, their multiple disulfide bond frameworks are inherently unstable under reducing conditions. Reduction or scrambling by thiol-containing molecules such as glutathione or serum albumin in intracellular or extracellular environments such as blood plasma can decrease their effectiveness as drugs. To address this issue, we describe a new class of selenoconotoxins where cysteine residues are replaced by selenocysteine to form isosteric and non-reducible diselenide bonds. Three isoforms of alpha-conotoxin ImI were synthesized by t-butoxycarbonyl chemistry with systematic replacement of one([ Sec(2,8)] ImI or [Sec(3,12)] ImI), or both([Sec(2,3,8,12)] ImI) disulfide bonds with a diselenide bond. Each analogue demonstrated remarkable stability to reduction or scrambling under a range of chemical and biological reducing conditions. Three-dimensional structural characterization by NMR and CD spectroscopy indicates conformational preferences that are very similar to those of native ImI, suggesting fully isomorphic structures. Additionally, full bioactivity was retained at the alpha(7) nicotinic acetylcholine receptor, with each seleno-analogue exhibiting a dose-response curve that overlaps with wild-type ImI, thus further supporting an isomorphic structure. These results demonstrate that selenoconotoxins can be used as highly stable scaffolds for the design of new drugs.

A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling:implications for family B G-protein-coupled receptors

Calcitonin receptor like-receptor is a family B G-protein coupled receptor (GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide (CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in alpha-helices. Therefore, all proline residues within the transmembrane helices of the receptor (Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241 Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC(50) for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP(8-37) and 1-piperidinecarboxamide N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3 5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quina zolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC(1) receptor.

Oxidative modification of a specific apolipoprotein B lysine residue confers altered receptor specificity on LDL

During inflammation, many cell types release reactive oxygen species (ROS) via the respiratory burst. These ROS are potent oxidants of LDL and its major protein, apolipoprotein B. Whilst native LDL is taken up by endothelial cells via a feedback controlled receptor-regulated process, oxidative modification of LDL renders it a ligand for many scavenger receptors. Scavenger receptors include CD-36, LOX-1 and the prototypic macrophage SR A I/II, all of which are variably expressed. Uncontrolled uptake of oxidised LDL is implicated in the pathogenesis of atherosclerosis. In addition, oxidised LDL increases CCR2 protein and mRNA expression on monocytes, and thus may contribute to monocyte retention and perpetuation in inflammatory, unstable atherosclerotic lesions. However, little data are available on the effects of specific minor modifications to apolipoprotein B. In order to identify the sequence specificity and nature of oxidative modifications which confer altered properties on LDL, we have investigated the effects of modified peptides (which correspond to the putative LDLR binding domain) on LDL uptake by HUVECs and U937 monocytes.

Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR

G-protein coupled receptors (GPCRs) typically have a functionally important C-terminus which, in the largest subfamily (family A), includes a membrane-parallel eighth helix. Mutations of this region are associated with several diseases. There are few C-terminal studies on the family B GPCRs and no data supporting the existence of a similar eighth helix in this second major subfamily, which has little or no sequence homology to family A GPCRs. Here we show that the C-terminus of a family B GPCR (CLR) has a disparate region from N400 to C436 required for CGRP-mediated internalization, and a proximal region of twelve residues (from G388 to W399), in a similar position to the family A eighth helix, required for receptor localization at the cell surface. A combination of circular and linear dichroism, fluorescence and modified waterLOGSY NMR spectroscopy (SALMON) demonstrated that a peptide mimetic of this domain readily forms a membrane-parallel helix anchored to the liposome by an interfacial tryptophan residue. The study reveals two key functions held within the C-terminus of a family B GPCR and presents support for an eighth helical region with striking topological similarity to the nonhomologous family A receptor. This helix structure appears to be found in most other family B GPCRs.