Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5 untranslated regions (5 UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins (IMPs) that exhibit multiple attachments to the 5 UTR from the translationally regulated IGF-II leader 3 mRNA but are unable to bind to the 5 UTR from the constitutively translated IGF-II leader 4 mRNA. IMPs contain the unique combination of two RNA recognition motifs and four hnRNP K homology domains and are homologous to the Xenopus Vera and chicken zipcode-binding proteins. IMP localizes to subcytoplasmic domains in a growthdependent and cell-specific manner and causes a dose-dependent translational repression of IGF-II leader 3 -luciferase mRNA. Mouse IMPs are produced in a burst at embryonic day 12.5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5 UTR-binding proteins control IGF-II biosynthesis during late mammalian development.Specific RNA-binding proteins are emerging as regulators of cytoplasmic mRNA events such as translatability, stability, and localization. Several examples of these types of regulatory events have been reported in studies of invertebrate embryogenesis and amphibian oogenesis, in which the 3Ј untranslated region (3Ј UTR) has been identified as a repository of regulatory elements (reviewed in reference 35). It is anticipated that similar mechanisms operate during mammalian development, since important physiological roles for RNA-binding proteins have been discerned from deletions of the DAZ and RBM genes leading to azoospermia (9, 26) and from a point mutation in the FMR1 gene resulting in the fragile X mental retardation syndrome (7). RNA-binding proteins often contain one or more RNA-binding motif such as the RNA recognition motif (RRM) and the K homology (KH) domain (reviewed in reference 28), which may either ensure increased specificity towards a single RNA molecule or provide an ability to bind different molecules simultaneously. Moreover, solution structures of the N-terminal RRM domain of the human U1A protein in complex with its own pre-mRNA and of the first KH domain of FMR1 suggest that flexible loop regions provide discriminating binding surfaces for RNA recognition (1, 20).Insulin-like growth factor II (IGF-II) is a fetal growth factor with auto-and paracrine modes of action. In the mouse, lack of IGF-II results in a small but apparently normal progeny (3), whereas an increased IGF-II dose is more detrimental (13, 30). In humans, increased levels of IGF-II are associated with the Beckwith-Wiedemann syndrome, which is characterized by a disproportionate overgrowth of the fetus and malformations (32). IGF-II expression is controlled by parental imprinting, since only the paternal allele is expressed in most tissues (8). However,...

Cathelicidins are a family of antimicrobial proteins found in the peroxidase-negative granules of neutrophils. The known biologic functions reside in the C-terminus, which must be cleaved from the holoprotein to become active. Bovine and porcine cathelicidins are cleaved by elastase from the azurophil granules to yield the active antimicrobial peptides. The aim of this study was to identify the physiological setting for cleavage of the only human cathelicidin, hCAP-18, to liberate the antibacterial and cytotoxic peptide LL-37 and to identify the protease responsible for this cleavage. Immunoelectron microscopy demonstrated that both hCAP-18 and azurophil granule proteins were present in the phagolysosome. Immunoblotting revealed no detectable cleavage of hCAP-18 in cells after phagocytosis. In contrast, hCAP-18 was cleaved to generate LL-37 in exocytosed material. Of the 3 known serine proteases from azurophil granules, proteinase 3 was solely responsible for cleavage of hCAP-18 after exocytosis. This is the first detailed study describing the generation of a human antimicrobial peptide from a promicrobicidal protein, and it demonstrates that the generation of active antimicrobial peptides from common proproteins occurs differently in related species. (Blood. 2001; 97:3951-3959)

The leading cause of mortality in patients with cystic fibrosis (CF) is respiratory failure due in large part to chronic lung infection with Pseudomonas aeruginosa strains that undergo mucoid conversion, display a biofilm mode of growth in vivo and resist the infiltration of polymorphonuclear leukocytes (PMNs), which release free oxygen radicals such as H202. The mucoid phenotype among the strains infecting CF patients indicates overproduction of a linear polysaccharide called alginate. T o mimic the inflammatory environment o f the CF lung, P. aeruginosa PAO1, a typical non-mucoid strain, was grown in a biofilm. This was treated with low levels of H202, as if released by the PMNs, and the formation of mucoid variants was observed. These mucoid variants had mutations in mucA, which encodes an anti-o factor; this leads t o the deregulation of an alternative c factor (022, AlgT or AlgU) required for expression of the alginate biosynthetic operon. All of the mucoid variants tested showed the same mutation, the mucA22 allele, a common allele seen in CF isolates. The mucoid mucA22 variants, when compared to the smooth parent strain PAO1, (i) produced 2-6-fold higher levels of alginate, (ii) exhibited no detectable differences in growth rate, (iii) showed an unaltered LPS profile, (iv) were -72% reduced in the amount of inducible-P-lactamase and (v) secreted little or no LasA protease and only showed 44% elastase activity. A characteristic -54 kDa protein associated with alginate overproducing strains was identified as AlgE (Alg76) by N-terminal sequence analysis. Thus, the common phenotype of the mucoid variants, which included a genetically engineered mucA22 mutant, suggested that the only mutation incurred as a result of H202 treatment was in mucA. When a P. aeruginosa biofilm was repeatedly exposed to activated PMNs in vitro, mucoid variants were also observed, mimicking in vivo observations. Thus, PMNs and their oxygen by-products may cause P. aeruginosa t o undergo the typical adaptation to the intractable mu-coid form in the CF lung. These findings indicate that gene activation in bacteria by toxic oxygen radicals, similar to that found in plants and mammalian cells, may serve as a defence mechanism for the bacteria. This suggests that mucoid conversion is a response to oxygen radical exposure and that this response is a mechanism of defence by the bacteria. This is the f i r s t report to show that PMNs and their oxygen radicals can cause this phenotypic and genotypic change which is so typical of the intractable form o f P. aeruginosa in the CF lung. These findings may provide a basis for the development of anti-oxidant and anti-inflammatory therapy for the early stages of infection in CF patients.

The metabolism of glucagon-like peptide-1 (GLP-1) has not been studied in detail, but it is known to be rapidly cleared from the circulation. Measurement by RIA is hampered by the fact that most antisera are side-viewing or C-terminally directed, and recognize both intact GLP-1 and biologically inactive. N-terminally truncated fragments. Using high pressure liquid chromatography in combination with RIAs, methodology allowing specific determination of both intact GLP-1 and its metabolites was developed. Human plasma was shown to degrade GLP-1-(7-36)amide, forming an N-terminally truncated peptide with a t1/2 of 20.4 +/- 1.4 min at 37 C (n = 6). This was unaffected by EDTA or aprotinin. Inhibitors of dipeptidyl peptidase-IV or low temperature (4 C) completely prevented formation of the metabolite, which was confirmed to be GLP-1-(9-36)amide by mass spectrometry and sequence analysis. High pressure liquid chromatography revealed the concentration of GLP-1-(9-36)amide to be 53.5 +/- 13.7% of the concentration of endogenous intact GLP-1 in the fasted state, which increased to 130.8 +/- 10.0% (P < 0.01; n = 6) 1 h postprandially. Metabolism at the C-terminus was not observed. This study suggests that dipeptidyl peptidase-IV is the primary mechanism for GLP-1 degradation in human plasma in vitro and may have a role in inactivating the peptide in vivo.

Kaposi's sarcoma-associated herpesvirus encodes a chemokine called vMIP-II. This protein displayed a broader spectrum of receptor activities than any mammalian chemokine as it bound with high affinity to a number of both CC and CXC chemokine receptors. Binding of vMIP-II, however, was not associated with the normal, rapid mobilization of calcium from intracellular stores; instead, it blocked calcium mobilization induced by endogenous chemokines. In freshly isolated human monocytes the virally encoded vMIP-II acted as a potent and efficient antagonist of chemotaxis induced by chemokines. Because vMIP-II could inhibit cell entry of human immunodeficiency virus (HIV) mediated through CCR3 and CCR5 as well as CXCR4, this protein may serve as a lead for development of broad-spectrum anti-HIV agents.

A 19 kDa protein was identified in specific granules of human neutrophils. A full-length cDNA clone was isolated from a human CML cDNA library, based on amino-acid sequences of isolated tryptic fragments. This clone includes the recently identified cDNA for FALL-391CAP-18. Aminoacid sequences of proteolytic fragments derived both from the conserved N-terminal cathelin-like region and the highly variable C-terminal region characteristic of this family of bactericidal, LPS binding proteins, were in complete agreement with the sequence deduced from the cDNA. Thus, the 19 kDa protein is hCAP-18, stored as a 'pro-peptide' in specific granules. Materials and methods Subcellular fractionationHuman neutrophils were isolated from freshly drawn blood and disrupted by nitrogen cavitation following treatment with 5 mM DFP (Aldrich) [6]. The post-nuclear supernatant (10 ml), was loaded on a 3-layer Percoll (Pharmacia) density gradient (gradient volume 27 ml) and fractionated [5]. Azurophil granules were identified by their content of myeloperoxidase [7], specific granules by lactoferrin [7], gelatinase granules by gelatinase [5,8], and plasma membranes by HLA, all assayed by ELISA [5,9,10]. Secretory vesicles were identified by their content of latent alkaline phosphatase [4], assayed as described [11] and by their content of albumin, assayed by ELISA [12].

Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in motile cells IMP-containing granules are dispersed around the nucleus and in cellular protrusions. We isolated the IMP1-containing RNP granules and found that they represent a unique RNP entity distinct from neuronal hStaufen and/or fragile X mental retardation protein granules, processing bodies, and stress granules. Granules were 100 -300 nm in diameter and consisted of IMPs, 40 S ribosomal subunits, shuttling heterologous nuclear RNPs, poly ( Moreover the exon junction complex, which is deposited during splicing, is removed during the so-called pioneering round of translation (for reviews, see Refs. 1 and 2). Finally a particular mRNA becomes embroidered with nuclear RNA-binding proteins, and the specific ensemble may determine cytoplasmic events such as RNA localization, translation, and stability (for a review, see Ref.3). Cytoplasmic mRNPs may become destined for local translation. In support of this possibility, RNAs have been found in large mRNP granules, which are transported along cytoskeletal structures and anchored at their final destination. Messenger RNA localization has mainly been examined in polarized oocytes and neurons, and it has been proposed that local postsynaptic protein synthesis is required for synaptic plasticity (4). Previous studies have identified neuronal Staufen (5) and FMRP granules (6, 7), containing mRNAs, small and large ribosomal subunits, translation initiation factors including eIF4E and eIF2␣, and RNA-binding proteins (Refs. 8 -11; for a review, see Ref. 12). The protein composition of neuronal mRNP granules is to some degree overlapping with stress granules and processing bodies (P-bodies). The hallmark of stress granules is the presence of stalled 48 S initiation complexes and stress-dependent RNA-binding factors such as G3BP (13,14), whereas P-bodies contain components of the 5Ј-3Ј mRNA decay machinery and factors involved in nonsense-mediated decay (15).The zip code-binding proteins IMP1, -2, and -3 (human), ZBP1 (chicken), Vg1-RBP/Vera (Xenopus), and coding region determinant-binding protein (mouse) are members of the From the ‡Department

Apolipoprotein M (apoM) is a novel apolipoprotein with unknown function. In this study, we established a method for isolating apoM-containing lipoproteins and studied their composition and the effect of apoM on HDL function. ApoM-containing lipoproteins were isolated from human plasma with immunoaffinity chromatography and compared with lipoproteins lacking apoM. The apoMcontaining lipoproteins were predominantly of HDL size; z5% of the total HDL population contained apoM. Mass spectrometry showed that the apoM-containing lipoproteins also contained apoJ, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, paraoxonase 1, and apoB. ApoM-containing HDL (HDL apoM1 ) contained significantly more free cholesterol than HDL lacking apoM (HDL apoM2 ) (5.9 6 0.7% vs. 3.2 6 0.5%; P , 0.005) and was heterogeneous in size with both small and large particles. HDL apoM1 inhibited Cu 21 -induced oxidation of LDL and stimulated cholesterol efflux from THP-1 foam cells more efficiently than HDL apoM2 . In conclusion, our results suggest that apoM is associated with a small heterogeneous subpopulation of HDL particles. Nevertheless, apoM designates a subpopulation of HDL that protects LDL against oxidation and stimulates cholesterol efflux more efficiently than HDL lacking apoM.