Volume 388, Issue 3

We suggest an extension of connexin orthology relationships across the major vertebrate lineages. We first show that the conserved domains of mammalian connexins (encoding the N-terminus, four transmembrane domains and two extracellular loops) are subjected to a considerably more strict selection pressure than the full-length sequences or the variable domains (the intracellular loop and C-terminal tail). Therefore, the conserved domains are more useful for the study of family relationships over larger evolutionary distances. The conserved domains of connexins were collected from chicken, Xenopus tropicalis , zebrafish, pufferfish, green spotted pufferfish, Ciona intestinalis and Halocynthia pyriformis (two tunicates). A total of 305 connexin sequences were included in this analysis. Phylogenetic trees were constructed, from which the orthologies and the presumed evolutionary relationships between the sequences were deduced. The tunicate connexins studied had the closest, but still distant, relationships to vertebrate connexin36 , 39.2 , 43.4 , 45 and 47 . The main structure in the connexin family known from mammals pre-dates the divergence of bony fishes, but some additional losses and gains of connexin sequences have occurred in the evolutionary lineages of subsequent vertebrates. Thus, the connexin gene family probably originated in the early evolution of chordates, and underwent major restructuring with regard to gene and subfamily structures (including the number of genes in each subfamily) during early vertebrate evolution.

M.EcoHK31I is a naturally occurring mC5-methyltransferase with a large α polypeptide and a small β polypeptide. Polypeptide α contains conserved motifs I–VIII and X, and polypeptide β contains motif IX. To understand how polypeptide α carries out its function, a molecular model of the large domain of polypeptide α was generated using M.HhaI and M.HaeIII as templates. The large domain is a mixed α/β structure. Residues 15–19 in motif I (Phe-Naa-Gly-Naa) are conserved for cofactor binding. The key catalytic residue Cys-79 in motif IV is also conserved in comparison with other C-5 MTases. Comparing polypeptide α with M.HhaI and M.HaeIII revealed a unique region upstream of motif X. To understand the role of this region, 14 charged residues between R224 and E271 in the putative small domain were mutated. Activity assays indicated that most of these charges can be eliminated or changed conservatively. Among these charged residues, R224, E240, D245 and D251 may take part in proper interaction with DNA in the presence of polypeptide β.

A novel basic protein with antifungal activity was isolated from the seeds of Ginkgo biloba and purified to homogeneity. The protein inhibited the growth of some fungi ( Fusarium oxysporum , Trichoderma reesei , and Candida albicans ) but did not exhibit antibacterial action against Escherichia coli . Furthermore, this protein showed weak inhibitory activity against the aspartic protease pepsin. To design primers for gene amplification, the NH 2 -terminal and partial internal amino acid sequences were determined using peptides obtained from a tryptic digest of the oxidized protein. The full-length cDNA of the antifungal protein was cloned and sequenced by RT-PCR and rapid amplification of cDNA ends (RACE). The cDNA contained a 402-bp open reading frame encoding a 134-aa protein with a potential signal peptide (26 residues), suggesting that this protein is synthesized as a preprotein and secreted outside the cells. The antifungal protein shows approximately 85% identity with embryo-abundant proteins from Picea abies and Picea glauca at the amino acid level; however, there is no homology between this protein and other plant antifungal proteins, such as defensin, and cyclophilin-, miraculin- and thaumatin-like proteins.

Using bovine heart sarcolemma vesicles we studied the effects of protons and phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) on the affinity of the mammalian Na + /Ca 2+ exchanger (NCX1) for intracellular Ca 2+ . By following the effects of extravesicular ligands in inside-out vesicles, their interactions with sites of NCX1 facing the intracellular medium were investigated. Two Na + -gradient-dependent fluxes were studied: Ca 2+ uptake and Ca 2+ release. PtdIns-4,5-P2 binding to NCX1 was investigated in parallel. Without MgATP (no ‘ de novo ’ synthesis of PtdIns-4,5-P2), alkalinization increased the affinity for Ca 2+ and the PtdIns-4,5-P2 bound to NCX1. Vesicles depleted of phosphoinositides were insensitive to alkalinization, but became responsive following addition of exogenous PtdIns-4,5-P2 or PtdIns plus MgATP. Acidification reduced the affinity for Ca 2+ ev ; this was only partially reversed by MgATP, despite the increase in bound PtdIns-4,5-P2 to levels observed with alkalinization. Inhibition of Ca 2+ uptake by increasing extravesicular [Na + ] indicates that it is related to H + i and Na + i synergistic inhibition of the Ca 2+ i regulatory site. Therefore, the affinity of the NCX1 Ca 2+ i regulatory site for Ca 2+ was maximal when both intracellular alkalinization and an increase in PtdIns-4,5-P2 bound to NCX1 (not just of the total membrane PtdIns-4,5-P2) occurred simultaneously. In addition, protons influenced the distribution, or the exposure, of PtdIns-4,5-P2 molecules in the surroundings and/or on the exchanger protein.

In this study we characterize a novel gene on human chromosome 9 and its translation product, PC3-secreted microprotein (PSMP). The gene contains three exons that encode a protein of 139 amino acid residues, including a predicted signal peptide of 36 residues. The molecule is homologous to β-microseminoprotein (MSP), a protein of unknown function, secreted at high concentration by the prostate gland. These two proteins have only 23% sequence identity, but their common origin is revealed by a preserved pattern of Cys residues. In contrast to MSP, which shows poor conservation between species, PSMP is very conserved. High transcript levels were detected in the prostate cancer cell line PC-3. Antiserum raised against PSMP detected a protein with an apparent molecular mass of 18 kDa in culture medium conditioned by PC-3 cells, but in cell lysates the antiserum also recognized a molecular species of 16 kDa, suggesting that PSMP undergoes post-translational modification. Xenografted PC-3 cell tumors in athymic nude mice showed strong staining for both PSMP protein and mRNA. Studies on human prostate cancer specimens showed immunohistochemical staining of both tumor and benign glandular cells. Our results suggest that PSMP is an important protein with significance in prostate cancer.

The O -acetylation of sialic acids in various positions is a frequent modification of these residues in glycoproteins and glycolipids of higher animals and some bacteria. Sialic acid O -acetylation is involved in the regulation of many cell biological and pathophysiological events. Since the properties and the structural and molecular genetic aspects of the eukaryotic sialate O -acetyltransferases are not yet known, we attempted to isolate the enzyme from bovine submandibular glands. O -Acetyltransferase was solubilised from its microsomal location with a zwitterionic detergent and enriched by approximately 50-fold in three steps, including affinity chromatography on coenzyme A. It exhibits a molecular mass of 150–160 kDa. Evidence was obtained for the putative existence of a low-molecular-mass, dialysable enzyme activator. The enzyme showed best activity with CMP- N -acetylneuraminic acid (CMP-Neu5Ac), followed by N -acetylneuraminic acid (Neu5Ac). These compounds, as well as AcCoA, have high affinity for both the microsome-bound and the partially purified O -acetyltransferase. CoA is a strong inhibitor. N -Acetyl-9- O -acetylneuraminic acid was found to be the main reaction product. No evidence was obtained for the involvement of an isomerase that might be responsible for the migration of O -acetyl groups within the sialic acid side chain.

Lipid rafts are liquid ordered platforms that dynamically compartmentalize membranes. Caveolins and flotillins constitute a group of proteins that are enriched in these domains. Caveolin-1 has been shown to be an essential component of caveolae. Flotillins were also discovered as an integral component of caveolae and have since been suggested to interact with caveolins. However, flotillins are also expressed in non-caveolae-containing cells such as lymphocytes and neuronal cells. Hence, a discrepancy exists in the literature regarding the caveolin dependence of flotillin expression and their subcellular localization. To address this controversy, we used mouse embryonic fibroblasts (MEFs) from caveolin-1 knockout (Cav-1 -/- ) and wild-type mice to study flotillin expression and localization. Here we show that both membrane association and lipid raft partitioning of flotillins are not perturbed in Cav-1 -/- MEFs, whereas membrane targeting and raft partitioning of caveolin-2, another caveolin family protein, is severely impaired. Moreover, we demonstrate that flotillin-1, but not flotillin-2, associates with lipid droplets upon oleic acid treatment and that this association is completely independent of caveolin. Taken together, our results show that flotillins are localized in lipid rafts independent of caveolin-1 and that translocation of flotillin-1 to lipid droplets is a caveolin-independent process.

C 2 -ceramide ( N -acetyl-sphingenine) is often used as an analog to study ceramide-mediated cellular processes. According to Lee et al. [J. Biol. Chem. 271 (1996), 209–217], C 2 -ceramide is formed by an acetyl transfer from platelet-activating factor (PAF, 1- O -alkyl-2-acetyl- sn -glycero-3-phosphocholine) to sphingenine. To substantiate these unconfirmed findings, we (i) developed a method to quantify C 2 -ceramide and (ii) analyzed C 2 -ceramide levels in Pex5 -/- mice, a model for Zellweger syndrome, in which the synthesis of ether lipids such as PAF is impaired. The presence of C 2 -ceramide could be established in brain (±10 pmol/g) and liver (±25 pmol/g) from control mice, and was approximately 5000-fold less than the main long-chain ceramide species. In Pex5 -/- mice, C 2 -ceramide levels did not differ significantly compared to control tissues. Given the presence of a ceramide kinase in mammals, phosphorylation of C 2 -ceramide by human ceramide kinase ( Hs CERK) was tested. C 2 -ceramide appears to be a good substrate when albumin is used as carrier. In CHO cells overexpressing Hs CERK, phosphorylation of exogenously added C 2 -ceramide could also be demonstrated. Our data indicate that C 2 -ceramide is present in mammalian tissues and can be converted to C 2 -ceramide-1-phosphate, in addition to other documented metabolic alterations, but does not seem to be linked to ether lipid metabolism.

Interleukin-13 (IL-13) is a T-cell-derived pleiotropic cytokine of particular medical importance because of its critical role in the development of allergic asthma. The effects of IL-13 on its target cells are mediated through a dimeric transmembrane receptor (IL-13R), which shares the IL-4Rα subunit with the IL-4R system, but contains as a specific component the IL-13Rα1 chain. We have generated a set of single-chain Fv fragments with specific binding capacity to the extracellular domain of the human IL-13Rα1 receptor. Bacteriophage clones displaying receptor-binding antibody domains were selected from both naive and synthetic libraries by repetitive panning on recombinant and cell surface-expressed recombinant IL-13Rα1. Their specific reactivity with native human IL-13Rα1 expressed on the surface of transfected cells was demonstrated by flow cytometry. One binder that specifically interfered with cell activation by IL-13 was extensively characterized. This scFv inhibited IL-13-driven gene transcription and cell proliferation in test cell lines, as well as IL-13-induced activation of primary human monocytes in a dose-dependent manner, with an IC 50 below 300 nM. This novel reagent thus constitutes a valuable tool for the further elucidation of IL-13 function in disease and offers potential therapeutic perspectives.

Rheumatoid arthritis is an inflammatory and disabling joint disease affecting 0.5–1.5% of the population. Although various anti-inflammatory (NSAIDs) and disease-modifying (DMARDs) drugs are in clinical use, their precise mechanisms of action are not always defined. In this report, we discuss the effects of widely used DMARDs such as gold derivatives and chloroquine on cathepsins K and S, which have been implicated as critical mediators of inflammation and joint erosion in rheumatoid arthritis. We demonstrate that clinically potent gold derivatives inhibit cathepsins K and S in in vitro and cell-based assays. An X-ray analysis of the gold thiomalate/cathepsin K complex reveals that the inhibitor is bound to the active-site cysteine residue of the protease. Chloroquine, a lysosomotropic agent of lower clinical potency than gold derivatives, inhibits neutral pH-labile cathepsins intracellularly, but does not affect the neutral pH-stable cathepsin S. The potent inhibition of cathepsins implicated in the pathogenesis of rheumatoid arthritis by gold derivatives may explain the therapeutic efficacy of these drugs.

Epithelial cells in the human small intestine express meprin, an astacin-like metalloprotease, which accumulates normally at the brush border membrane and in the gut lumen. Therefore, meprin is targeted towards luminal components. In coeliac disease patients, peptides from ingested cereals trigger mucosal inflammation in the small intestine, disrupting epithelial cell differentiation and function. Using in situ hybridisation on duodenal tissue sections, we observed a marked shift of meprin mRNA expression from epithelial cells, the predominant expression site in normal mucosa, to lamina propria leukocytes in coeliac disease. Meprin thereby gains access to the substrate repertoire present beneath the epithelium.

Dipeptidyl peptidase III (DPP III) is a zinc exopeptidase with an implied role in the mammalian pain-modulatory system owing to its high affinity for enkephalins and localisation in the superficial laminae of the spinal cord dorsal horn. Our study revealed that this human enzyme hydrolyses opioid peptides belonging to three new groups, endomorphins, hemorphins and exorphins. The enzymatic hydrolysis products of endomorphin-1 were separated and quantified by capillary electrophoresis and the kinetic parameters were determined for human DPP III and rat DPP IV. Both peptidases cleave endomorphin-1 at comparable rates, with liberation of the N-terminal Tyr-Pro. This is the first evidence of DPP III acting as an endomorphin-cleaving enzyme.

Rapid BSE tests are widely used diagnostics in veterinary medicine and more than 11 million tests are applied worldwide. The evaluation of new rapid BSE tests and the quality assurance of approved BSE tests pose a challenge owing to the natural scarcity of BSE-infected bovine brainstems and regional variations in prion titer. Transgenic mice expressing bovine prion protein (Tg4092) offer an alternative approach to these problems. To determine whether BSE-infected Tg4092 mouse brains could serve as a general standard for rapid BSE tests, we inoculated Tg4092 mice intracerebrally with BSE prions, harvested brains at defined time points post-infection and analyzed cerebral hemispheres with several approved rapid BSE tests. The results show that de novo formation of the disease-causing prion protein isoform, PrP Sc , can be monitored during the course of infection. We demonstrate that BSE-infected Tg4092 mouse brains provide a renewable and controllable source of reference samples and suggest that such samples can generally be used for the evaluation and quality control of rapid BSE tests.