Tryptophan-Mediated Interactions between Tristetraprolin and the CNOT9 Subunit Are Required for CCR4-NOT Deadenylase Complex Recruitment.

The zinc-finger protein tristetraprolin (TTP) binds to AU-rich elements present in the 3' untranslated regions of transcripts that mainly encode proteins of the inflammatory response. TTP-bound mRNAs are targeted for destruction via recruitment of the eight-subunit deadenylase complex "carbon catabolite repressor protein 4 (CCR4)-negative on TATA-less (NOT)," which catalyzes the removal of mRNA poly-(A) tails, the first obligatory step in mRNA decay. Here we show that a novel interaction between TTP and the CCR4-NOT subunit, CNOT9, is required for recruitment of the deadenylase complex. In addition to CNOT1, CNOT9 is now included in the identified CCR4-NOT subunits shown to interact with TTP. We find that both the N- and C-terminal domains of TTP are involved in an interaction with CNOT9. Through a combination of SPOT peptide array, site-directed mutagenesis, and bio-layer interferometry, we identified several conserved tryptophan residues in TTP that serve as major sites of interaction with two tryptophan-binding pockets of CNOT9, previously found to interact with another modulator GW182. We further demonstrate that these interactions are also required for recruitment of the CCR4-NOT complex and TTP-directed decay of an mRNA containing an AU-rich element in its 3'-untranslated region. Together the results reveal new molecular details for the TTP-CNOT interaction that shape an emerging mechanism whereby TTP targets inflammatory mRNAs for deadenylation and decay.

Quantitative analysis of MRP-8 in gingival crevicular fluid in periodontal health and disease using microbore HPLC.

BACKGROUND: The protein components of GCF can be separated by reverse-phase microbore HPLC on a C18 column with detection on the basis of 214 nm absorbance. A single major symmetrical protein peak eluting with a retention time of 26 min (50% acetonitrile) was evident in gingival crevicular fluid (GCF) from periodontitis patients but not in healthy GCF. This protein was identified as human MRP-8 by N-terminal amino acid sequencing and liquid chromatography quadropole mass spectrometry. AIMS: To quantify the amount of MRP-8 detectable in GCF from individual healthy, gingivitis and periodontitis affected sites and to study the relationship, if any, between the levels of this responsive protein and periodontal health and disease. METHODS: GCF was sampled (30 s) from healthy, gingivitis, and periodontitis sites in peridontitis subjects (n=15) and from controls (n=5) with clinically healthy gingiva and no periodontitis. Purified MRP-8 was sequenced by Edmann degradation and the phenylthiohydantoin (PTH) amino acid yield determined (by comparison of peak area with external PTH amino acid standards). This value was subsequently used to calculate the relative amount of protein in the peak eluting with a retention time of 26.0 min (MRP-8) in individual GCF chromatograms. RESULTS: Higher levels of MRP-8 were detected in inflammatory sites: periodontitis 457.0 (281.0) ng; gingivitis 413.5 (394.5) ng compared with periodontally healthy sites in diseased subjects 14.6 (14.3) ng and in controls 18.6 (18.5) ng, p=0.003. There was at least 20-fold more MRP-8 in the inflammatory compared with the healthy sites studied. CONCLUSIONS: The preliminary data indicate that MRP-8 is present in GCF, with significantly greater amounts present at diseased than healthy sites. A systematic study of the relationship of this protein to periodontal disease could prove useful in further clarifying whether MRP-8 could be a reliable GCF biomarker of gingivitis and periodontitis.

Identification of MRP-8 (calgranulin A) as a major responsive protein in chronic periodontitis.

The purpose of the study was to analyse how the protein composition of the inflammatory exudate associated with chronic periodontitis differed from the exudate in periodontal health. Gingival crevicular fluid (GCF) was collected from sites with chronic periodontal inflammation and from non-diseased sites in healthy control subjects. Microbore HPLC analysis revealed one major difference in GCF protein profiles between healthy controls and periodontitis patients. The protein enhanced in periodontitis patients was identified as migration inhibitory factor-related protein-8 (MRP-8) by a combination of N-terminal amino acid sequencing, mass spectrometry, and SDS-PAGE. Together, these data demonstrate, for the first time, the presence of monomeric MRP-8 in an inflammatory exudate. Whether monomeric MRP-8 is a unique feature of chronic periodontal inflammation is not yet clear, but the chemotactic properties of this peptide support a functional role for MRP-8 in periodontal inflammation.

Calling the shots: immunization finance policies and practices. Executive summary of the report of the Institute of Medicine.

Federal, state, and private-sector investments in vaccine purchases and immunization programs are lagging behind emerging opportunities to reduce the risks of vaccine-preventable disease. Although federal assistance to the states for immunization programs and data collection efforts rapidly expanded in the early part of the 1990s, significant cutbacks have occurred in the last 5 years that have reduced the size of state grant awards by more than 50% from their highest point. During this same period, the vaccine delivery system for children and adults has become more complex and fragmented. This combination of new challenges and reduced resources has led to instability in the public health infrastructure that supports the U. S. immunization system. Many states have reduced the scale of their immunization programs and currently lack adequate strength in areas such as data collection among at-risk populations, strategic planning, program coordination, and assessment of immunization status in communities that are served by multiple health care providers. If unmet immunization needs are not identified and addressed, states will have difficulty in achieving the national goal of 90% coverage by the year 2010 for completion of the childhood immunization series for young children. Furthermore, state and national coverage rates, which reached record levels for vaccines in widespread use (79%, 1998), can be expected to decline and preventable disease outbreaks may occur as a result, particularly among persons who are vulnerable to vaccine-preventable disease because of their underimmunization status. The Institute of Medicine (IOM) Committee on Immunization Finance Policies and Practices has therefore concluded that a renewal and strengthening of the federal and state immunization partnership is necessary. The goal of this renewed partnership is to prevent infectious disease; to monitor, sustain, and improve vaccine coverage rates for child and adult populations within more numerous and increasingly diversified health care settings; and to respond to vaccine-safety concerns. To achieve this renewal, states require a consistent strategy, additional funds, and a multiyear finance plan that can help expedite the delivery of new vaccines; strengthen the immunization assessment, assurance, and policy development functions in each state; and adapt childhood immunization programs to serve the needs of new age groups (especially adults with chronic diseases) in different health care environments. The IOM committee recommends that federal and state governments adopt a national finance strategy that would allocate $1.5 billion in federal and state resources over the first 5 years to strengthen the infrastructure for child and adult immunization-an annual increase of $175 million over current spending levels. These resources would consist of $200 million per year in state infrastructure grants awarded by the Centers for Disease Control and Prevention (the Section 317 program) and an additional $100 million per year in increased state contributions. The committee also recommends that the Congress replace the current discretionary Section 317 grants with a formula approach for state immunization grant awards to improve the targeting and stability of federal immunization grants. The formula should provide a base level of support to all states, as well as additional amounts related to each state's need, capacity, and performance. The committee further recommends that Congress introduce a state match requirement for the receipt of increased federal funds to help strengthen and stabilize the infrastructure that supports long-term public health assessment, assurance, and policy development efforts. (ABSTRACT TRUNCATED)

Trityl mass-tags for encoding in combinatorial oligonucleotide synthesis.

Combinatorial libraries of oligonucleotides on beads were synthesised by a split-and-mix strategy using 5'-DMTr- or 5'-Fmoc- nucleoside phosphoramidites. Trityl moieties with different masses were used to encode for the bases coupled at each step in the synthesis of oligonucleotides selected by hybridisation from the libraries. Tags orthogonal to the nucleotides added were produced by coupling amines of different MW to an activated carboxyl group(s) on the trityl moiety. Tags can be released from the support by laser irradiation and measured directly by TOF without matrix. Alternatively, they may be released by an acidic treatment and then analysed by (MA)LDI-TOF.

Full sequence and characterization of two insect defensins: immune peptides from the mosquito Aedes aegypti.

We report the complete amino acid sequence and biological activity of two immune peptides, from the yellow fever mosquito Aedes aegypti, that are induced in response to infection. Both peptides display biological activity against the Gram positive microbe Micrococcus luteus and substantial sequence homology to insect defensins, small heat-stable, antibiotic peptides previously described from several non-vector insects. These mosquito peptides, designated Ae. aegypti defensins A and B, are isoforms. Defensin B is the most abundant antibacterial peptide in this species whereas defensin A is much less abundant and carries two amino acid substitutions compared to defensin B, making it more basic in character. Apparent convergence between isoforms from Ae. aegypti and the fleshfly Phormia terranovae is discussed. The synergistic activity previously described between Ae. aegypti immune haemolymph and lysozyme is not caused by these peptides because synergy occurred only at concentrations far outside the physiological range seen in Ae. aegypti.

Brugia pahangi: the effects of cecropins on microfilariae in vitro and in Aedes aegypti.

Synthetic cecropins, antibacterial peptides from insect haemolymph, have been tested for their ability to attenuate the motility of microfilariae of the filarial nematode Brugia pahangi in an in vitro assay. Fifty micromolar concentrations of these peptides, equivalent to physiological concentrations in immune-stimulated insects, cause significant attenuation of motility compared with untreated microfilariae. Similar results were obtained with cecropins A and B. This is the lowest concentration for which cecropin has been reported to be active against eukaryote organisms. Antiserum to the cecropin homologue sarcotoxin 1A successfully blocked the observed activity. When the same concentration of cecropin B was coinjected with B. pahangi microfilariae into adult females of the mosquito, Aedes aegypti, a significant reduction in the numbers of developing larvae was observed.

Purification of an insect defensin from the mosquito, Aedes aegypti.

Using a new, sensitive assay of bacterial growth inhibition, inducible antibacterial activity has been identified in the haemolymph of the mosquito, Aedes aegypti following inoculation with bacteria or with microfilariae of the filarial nematode Brugia pahangi, but not after inoculation with sterile culture medium. A lower level of antibacterial activity has also been observed in untreated individual mosquitoes. Following bacterial inoculation, a basic, inducible antibacterial peptide has been detected using native PAGE at pH 4, which corresponds with a 4.5 kDa peptide detected by tricine SDS-PAGE followed by silver staining. A peptide has been purified from immune haemolymph by ultrafiltration, followed by reversed-phase HPLC, yielding a single major peak with antibacterial activity. Partial amino acid sequence analysis of this fraction has revealed substantial homology with insect defensins. The data are consistent with the peptide being another member of this family, and we propose the name Aedes aegypti defensin.

Approaches to vector control: new and trusted. 1. Humoral immune responses in blackfly and mosquito vectors of filariae.

The vectors of filariasis, mosquitoes and blackflies, are capable of mounting a defence response to the infection. This selective review describes the molecules that are involved in these immune systems. Several antibacterial peptides are known to be induced and secreted into the haemolymph by the fat body and the circulating haemocytes. In addition, haemagglutinating lectins with carbohydrate specificities to the surface of the developing filarial larvae appear. Activation of a range of proteases occurs rapidly as does activation of the prophenoloxidase pathway. The possible roles of these and other molecules is discussed, together with mention of a working hypothesis as to how these molecules may be regulated.

Antibacterial peptides in insect vectors of tropical parasitic disease.

The induction and characterization of immune peptides in two groups of medically important insects, the mosquitoes and blackflies, is currently an important research area. Mosquitoes transmit a variety of viral and parasitic diseases including yellow fever, dengue, malaria and lymphatic filariasis. Simuliid black flies are vectors of river blindness. The diseases are together responsible for death and morbidity in millions of people each year. The relationship between inducible peptides and bacterial and parasitic infections in these insects is proving to be a complex one. The identification of an insect defensin (4 kDa) in Aedes aegypti, the yellow fever mosquito, has proved to be the first peptide characterized in a vector of human disease. This inducible molecule appears in the haemolymph in response to bacterial and to a lesser extent filarial infection. The characterization of inducible blackfly peptides has revealed potent inducible anti-Gram-positive as well as anti-Gram-negative activity. In addition, non-self recognition molecules such as phenoloxidase may play a part in differentiating one species of eukaryotic pathogen from another of the same genus. The interactions between the peptides and these other proteins are likely to be important in the establishment of a successful immune response against a parasitic pathogen, particularly as we now know these peptides to have anti-eukaryotic activity (against a range of parasite species). As well as being of fundamental interest in our understanding of host-parasite relationships, the indication that antibacterial peptides are toxic to parasitic organisms has implications for their possible use in the disease vector control strategies of the future. It may also mean that a revision in our understanding of their mode of action, loose as it is, has to take place.

Synthesis of 6,1',6'-tri-O-(mesitylenesulfonyl)sucrose, further examination of "tri-O-tosylsucrose", and the chemistry of 3,6:1',4':3',6'-trianhydrosucrose.

Selective trimolar mesitylenesulfonylation of sucrose resulted in the formation of a highly crystalline trimesitylenesulfonate (1), which was isolated in greater than 50% yield without recourse to chromatography. As anticipated, the sulfonyl groups in 1 were located at the primary positions, as treatment with alkali afforded 3.6:1',4':3',6'-trianhydrosucrose (4) in high yield. Fractionation of "tri-O-tosyl-sucrose" by high-pressure liquid chromatography effected separation of the minor isomer from the known, preponderant 6,1',6'-isomer 3. 13C-N.m.r. spectroscopy indicated that the minor isomer was 2,6,6'-tri-O-p-tolylsulfonylsucrose (2). The trianhydride 4 was found to be dimorphous and was further characterized as the diacetate (5), the dibenzoate (6), the di-p-toluenesulfonate (7), and the dimethyl ether (8). Considerable differences in the reactivities toward acylation and etherification of the two axial hydroxyl groups in 4 permitted the preparation, in good yields, of the 4-acetate (9) and of the 4-methyl ether (12). Several derivatives of methyl 3,6-anhydro-alpha-D-glucopyranoside (13) were prepared for comparison with corresponding derivatives of 4, and the hydroxyl groups in 13 also showed differences in reactivities analogous with those of 4.