Cys-92, Cys-95, and the C-terminal 12 residues of the Vibrio harveyi ferric uptake regulator (Fur) are functionally inessential

Ferric uptake regulator (Fur) is a global regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (Fur(vh)) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. Furvh shares 77% overall sequence identity with the Escherichia coli Fur (Fur(Ec)) and could complement a mutant of Fur(Ec). Like Fur(Ec), Fur(Vh), possesses two cysteine residues at positions 92 and 95, yet unlike Fur(Ec), in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of Fur(Vh) proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of Fur(vh) are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of Fur(Vh) are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of Fur(Vh) is possibly different from that of Fur(Ec); and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of Fur(Vh); and (iii) provided insights into the potential function of the local structure involving C137 and K138.

Paleoenvironment evolution of the East Philippine Sea recorded in the new-type ferromanganese crust since the terminal Late Miocene

From systemic research of microstructure, geochemistry, uranium-series and Be-10 isotope dating on a new-type deepwater ferromanganese crust from the East Philippine Sea, the paleoenvironment evolution of the target area since the terminal Late Miocene was recovered. The vertical section changes of microstructure and chemical composition are consistent in the studied crust, which indicate three major accretion periods and corresponding paleoenvironment evolution of the crust. The bottom crust zone was formed in the terminal Late Miocene (5.6 Ma) with loose microstructure, high detritus content and high growth rate. Reductions of mineral element content, accretion rate and positive Ce-anomaly degree at 4.6 Ma indicate temporal warming, which went against the crust accretion and finally formed an accretion gap in the terminal Middle Pliocene (2.8-2.7 Ma). The more active Antarctic bottom seawaters in the Late Pliocene (2.7 Ma) facilitated the fast transfer to the top pure crust zone. Hereafter, with the further apart of volcanic source and the keeping increase of eolian material (1.0 Ma), although surrounding conditions were still favorable, mineral element content still shows an obvious reducing trend. It thereby offers new carrier and data for the unclear paleoceanographic research of the target area since the terminal Late Miocene.

Extremely active catalysts for the hydrogenation of terminal alkenes

Titanocene complexes combined with nanometer-size sodium hydride are extremely active and selective catalysts for the hydrogenation of terminal alkenes under normal pressure. The initial turnover frequencies (TOFinitial) may reach 100-300 s(-1) in the hydrogenation of 1-hexene. The highest catalytic efficiency turnover (TO) reaches 1.5 x 10(5) in 2 h for the hydrogenation of styrene. These catalytic systems exhibit specific selectivity toward alkene substrates. Only terminal alkenes can be hydrogenated. No isomerization of carbon-carbon double bonds occurs during hydrogenation. A suitable substituent on the cyclopentadienyl ring of titanocene and the use of nanometric sodium hydride are key factors in the high efficiency of these catalytic systems. (C) 2002 Elsevier Science.

Highly enantioselective resolution of terminal epoxides using polymeric catalysts

Poly-salen-Co(III) complexes were employed in the hydrolytic kinetic resolution (HKR) of terminal epoxides and ee's up to 98% were obtained. In the HKR of epichlorohydrin, the polymeric catalysts can be recovered and modified for recycling. The recovered polymer catalyst shows good activity and selectivity. (C) 2002 Elsevier Science Ltd. All rights reserved.

The Principle of Double Effect and Terminal Sedation

Williams, Glenys, 'The Principle of Double Effect and Terminal Sedation', Medical Law Review, 9 (2001), pp.41-53 RAE2008

Detection of amino-terminal extracellular domain of somatostatin receptor 2 by specific monoclonal antibodies and quantification of receptor density in medulloblastoma.

Somatostatin receptor 2 (SSTR2) is expressed by most medulloblastomas (MEDs). We isolated monoclonal antibodies (MAbs) to the 12-mer (33)QTEPYYDLTSNA(44), which resides in the extracellular domain of the SSTR2 amino terminus, screened the peptide-bound MAbs by fluorescence microassay on D341 and D283 MED cells, and demonstrated homogeneous cell-surface binding, indicating that all cells expressed cell surface-detectable epitopes. Five radiolabeled MAbs were tested for immunoreactive fraction (IRF), affinity (KA) (Scatchard analysis vs. D341 MED cells), and internalization by MED cells. One IgG(3) MAb exhibited a 50-100% IRF, but low KA. Four IgG(2a) MAbs had 46-94% IRFs and modest KAs versus intact cells (0.21-1.2 x 10(8) M(-1)). Following binding of radiolabeled MAbs to D341 MED at 4 degrees C, no significant internalization was observed, which is consistent with results obtained in the absence of ligand. However, all MAbs exhibited long-term association with the cells; binding at 37 degrees C after 2 h was 65-66%, and after 24 h, 52-64%. In tests with MAbs C10 and H5, the number of cell surface receptors per cell, estimated by Scatchard and quantitative FACS analyses, was 3.9 x 10(4) for the "glial" phenotype DAOY MED cell line and 0.6-8.8 x 10(5) for four neuronal phenotype MED cell lines. Our results indicate a potential immunotherapeutic application for these MAbs.

Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells.

We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX and BAK expression as well as cleaved caspases-3, -7, -8 and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.

HDAC6 and Ubp-M BUZ domains recognize specific C-terminal sequences of proteins.

The BUZ/Znf-UBP domain is a protein module found in the cytoplasmic deacetylase HDAC6, E3 ubiquitin ligase BRAP2/IMP, and a subfamily of ubiquitin-specific proteases. Although several BUZ domains have been shown to bind ubiquitin with high affinity by recognizing its C-terminal sequence (RLRGG-COOH), it is currently unknown whether the interaction is sequence-specific or whether the BUZ domains are capable of binding to proteins other than ubiquitin. In this work, the BUZ domains of HDAC6 and Ubp-M were subjected to screening against a one-bead-one-compound (OBOC) peptide library that exhibited random peptide sequences with free C-termini. Sequence analysis of the selected binding peptides as well as alanine scanning studies revealed that the BUZ domains require a C-terminal Gly-Gly motif for binding. At the more N-terminal positions, the two BUZ domains have distinct sequence specificities, allowing them to bind to different peptides and/or proteins. A database search of the human proteome on the basis of the BUZ domain specificities identified 11 and 24 potential partner proteins for Ubp-M and HDAC6 BUZ domains, respectively. Peptides corresponding to the C-terminal sequences of four of the predicted binding partners (FBXO11, histone H4, PTOV1, and FAT10) were synthesized and tested for binding to the BUZ domains by fluorescence polarization. All four peptides bound to the HDAC6 BUZ domain with low micromolar K(D) values and less tightly to the Ubp-M BUZ domain. Finally, in vitro pull-down assays showed that the Ubp-M BUZ domain was capable of binding to the histone H3-histone H4 tetramer protein complex. Our results suggest that BUZ domains are sequence-specific protein-binding modules, with each BUZ domain potentially binding to a different subset of proteins.

Display of cell surface sites for fibronectin assembly is modulated by cell adherence to (1)F3 and C-terminal modules of fibronectin.

BACKGROUND: Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules (7)F3-(10)F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules (7)F3-(10)F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. METHODOLOGY/PRINCIPAL FINDINGS: To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to (7)F3-(10)F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules (2)F3-(14)F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module (1)F3 or the C-terminal modules to modules (2)F3-(14)F3 resulted in some activity, and addition of both (1)F3 and the C-terminal modules resulted in a construct, (1)F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs (1)F3-C V0, (1)F3-C V64, and (1)F3-C Delta(V(15)F3(10)F1) were all able to support fibronectin assembly, suggesting that (1)F3 through (11)F1 and/or (12)F1 were important for activity. Coatings in which the active parts of (1)F3-C were present in different proteins were much less active than intact (1)F3-C. CONCLUSIONS: These results suggest that (1)F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.

A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.

The Na+/H+ exchanger regulatory factor (NHERF) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of Na+/H+ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the beta2 receptor. Mutagenesis studies of the beta2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the beta2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.

HIV-1 envelope gp41 antibodies can originate from terminal ileum B cells that share cross-reactivity with commensal bacteria.

Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.

Guidelines on artificial nutrition versus hydration in terminal cancer patients

Whether a terminally ill cancer patient should be actively fed or simply hydrated through subcutaneous or intravenous infusion of isotonic fluids is a matter of ongoing controversy among clinicians involved in the care of these patients. Under the auspices of the European Association for Palliative Care, a committee of experts developed guidelines to help clinicians make a reasonable decision on what type of nutritional support should be provided on a case-by-case basis. It was acknowledged that part of the controversy related to the definition of the terminal cancer patient, since this is a heterogeneous group of patients with different needs, expectations, and potential for a medical intervention. A major difficulty is the prediction of life expectancy and the patient's likely response to vigorous nutritional support. In an attempt to reach a decision on the type of treatment support (artificial nutrition vs. hydration) which would best meet the needs and expectations of the patient, we propose a three-step process: Step I: define the eight key elements necessary to reach a decision: Step II: make the decision; and Step III: reevaluate the patient and the proposed treatment at specified intervals. Step I involves assessing the patient concerning the following: 1) oncological/clinical condition; 2) symptoms; 3) expected length of survival; 4) hydration and nutritional status; 5) spontaneous or voluntary nutrient intake; 6) psychological profile; 7) gut function and potential route of administration; and 8) need for special services based on type of nutritional support prescribed. Step II involves the overall assessment of pros and cons, based on information determined in Step I, in order to reach an appropriate decision based on a well-defined end point (i.e. improvement of quality of life; maintaining patient survival; attaining rehydration). Step III involves the periodic reevaluation of the decision made in Step II based on the proposed goal and the attained result.

Solution structure of the N-terminal transactivation domain of ERM modified by SUMO-1.

ERM is a member of the PEA3 group of the Ets transcription factor family that plays important roles in development and tumorigenesis. The PEA3s share an N-terminal transactivation domain (TADn) whose activity is inhibited by small ubiquitin-like modifier (SUMO). However, the consequences of sumoylation and its underlying molecular mechanism remain unclear. The domain structure of ERM TADn alone or modified by SUMO-1 was analyzed using small-angle X-ray scattering (SAXS). Low resolution shapes determined ab initio from the scattering data indicated an elongated shape and an unstructured conformation of TADn in solution. Covalent attachment of SUMO-1 does not perturb the structure of TADn as indicated by the linear arrangement of the SUMO moiety with respect to TADn. Thus, ERM belongs to the growing family of proteins that contain intrinsically unstructured regions. The flexible nature of TADn may be instrumental for ERM recognition and binding to diverse molecular partners.