Staphylococci express a receptor for human transferrin: identification of a 42-kilodalton cell wall transferrin-binding protein

Staphylococcus aureus and the coagulase-negative staphylococci are commonly responsible for peritonitis in renal patients undergoing continuous ambulatory peritoneal dialysis. To simulate growth conditions in vivo, staphylococci isolated from peritoneal infections were cultured in used human peritoneal dialysate (HPD). Immunoblotting experiments using cell wall preparations from these staphylococci revealed the presence of the host iron-binding glycoprotein transferrin bound to S. aureus, S. epidermidis, S. capitis, S. haemolyticus, and S. hominis but not to S. warneri or S. saprophyticus. Similar results were obtained by incubating broth-grown staphylococci with human transferrin, although, in contrast to S. aureus, the coagulase-negative staphylococci bound more transferrin after growth in iron-restricted broth. To determine whether the staphylococci express a saturable specific receptor for human transferrin, the interaction of human 125I-transferrin with the staphylococci was examined. Both S. aureus and S. epidermidis bound the radiolabelled iron-saturated ligand in a time- and concentration-dependent manner. From competition binding assays, the affinity (Kd) and number of receptors were estimated for S. epidermidis (Kd, 0.27 microM; 4,200 receptors per cell) and S. aureus (Kd, 0.28 microM; 4,200 receptors per cell). S. epidermidis but not S. aureus receptor activity was partially iron regulated. Human apotransferrin and iron-saturated transferrin and rabbit and rat transferrins competed equally well for the staphylococcal receptor. Bovine and porcine transferrins and ovotransferrin as well as human and bovine lactoferrins were much less effective at competing with human transferrin. Treatment of whole staphylococci with protease abolished transferrin binding, indicating the involvement of cell surface protein. Western blots (immunoblots) of cell wall preparations probed with human transferrin revealed the presence of a 42-kDa transferrin-binding protein common to both S. aureus and S. epidermidis. On Western strip blots, the binding of human transferrin to this protein was blocked by labelled human transferrin but not by albumin, immunoglobulin G, or bovine transferrin or ovotransferrin. To assess the conservation of the 42-kDa transferrin-binding protein, cell wall proteins of S. epidermidis, S. haemolyticus, S. capitis, S. hominis, S. warneri, and S. saprophyticus were Western blotted and probed with human transferrin. Only S. warneri and S. saprophyticus lacked the 42-kDa wall protein, consistent with their inability to bind transferrin. These data show that the staphylococci express a specific receptor for human transferrin based at least in part on a common 42-kDa cell wall protein.     

Molecular cloning and functional characterization of the Paracoccus denitrificans porin

Bacterial porins facilitate the passive uptake of small solutes across the outer membrane of the cell. The channel properties and the primary structure of the porin from Paracoccus denitrificans were investigated. As judged from single-channel conductance experiments, this porin forms trimeric pores that show no ion selectivity in potassium chloride solution, which indicates that the charges within or near the channel are balanced. Based on peptide fragment sequence, the gene porG, which codes for this general pore protein, was cloned and analyzed. Its primary translation product contains a 20-residue signal sequence, followed by the 295 amino acids of the mature protein with a molecular mass of 31.9 kDa. Sequence alignments with porins from Rhodopseudomonas blastica and Rhodobacter capsulatus and secondary structure predictions suggest a typical rigid barrel structure consisting of 16 antiparallel beta-strands.     

Molecular cloning and biochemical characterization of bovine spleen myristoyl CoA:protein N-myristoyltransferase

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. We have isolated full-length cDNA encoding bovine spleen NMT (sNMT). The single long open reading frame of 1248 bp of sNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da. The protein coding sequence was expressed in Escherichia coli resulting in the production of functionally active 50-kDa NMT. Deletion mutagenesis showed that the C-terminus is essential for activity whereas up to 52 amino acids can be deleted from the N-terminus without affecting the function. One of the N-terminal deletions resulted in threefold higher NMT activity. Genomic Southern analysis indicated the presence of two strong hybridizing bands with three different restriction enzyme digests suggesting the possibility of two copies of the NMT gene in the bovine genome. RNA blot hybridization analysis of total cellular RNA prepared from bovine brain, heart, spleen, lung, liver, kidney, and skeletal muscle probed with bovine sNMT cDNA revealed a single 1.7-kb mRNA. Western blot analysis of various bovine tissues with human NMT peptide antibody indicated a common prominent immunoreactive band with an apparent molecular mass of 48.5-50 kDa in all tissues. Additional immunoreactive bands were observed in brain (84 and 50 kDa), lung (58 kDa), and skeletal muscle (58 kDa). Activity measurements demonstrated that brain contained the highest NMT activity followed by spleen, lung, kidney, heart, skeletal muscle, pancreas, and liver. It appears therefore that mRNA and protein expression do not correlate with NMT activity, suggesting the presence of regulators of the enzyme activity.     

Cloning and characterization of AtRGP1. A reversibly autoglycosylated arabidopsis protein implicated in cell wall biosynthesis

A reversibly glycosylated polypeptide from pea (Pisum sativum) is thought to have a role in the biosynthesis of hemicellulosic polysaccharides. We have investigated this hypothesis by isolating a cDNA clone encoding a homolog of Arabidopsis thaliana, Reversibly Glycosylated Polypeptide-1 (AtRGP1), and preparing antibodies against the protein encoded by this gene. Polyclonal antibodies detect homologs in both dicot and monocot species. The patterns of expression and intracellular localization of the protein were examined. AtRGP1 protein and RNA concentration are highest in roots and suspension-cultured cells. Localization of the protein shows it to be mostly soluble but also peripherally associated with membranes. We confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates. Possible sites for UDP-sugar binding and glycosylation are discussed. Our results are consistent with a role for this reversibly glycosylated polypeptide in cell wall biosynthesis, although its precise role is still unknown.     

Ribotyping: a tool for taxonomy and identification of the Nocardia asteroides complex species

Ribotyping has been evaluated as a taxonomic tool for studying bacteria belonging to the Nocardia asteroides complex. The probe used was obtained by PCR from DNA extracted from the type strain Nocardia asteroides ATCC 19247, a sequence that codes partly for 16S rRNA. Interpretation of hybridization after EcoRI restriction of total DNA of 21 strains of the N. asteroides complex allowed for the identification of only four different ribotypes: two related to N. asteroides sensu stricto, one related to N. farcinica and one related to N. nova. These results confirm the classification established by Tsukamura in 1982 (M. Tsukamura, Microbiol. Immunol. 26:1101-1119, 1982) as well as the heterogeneity of N. asteroides sensu stricto species. Ribotyping may also provide a major identification technique for the species belonging to the N. asteroides complex.     

Molecular and biochemical characterization of the protein template controlling biosynthesis of the lipopeptide lichenysin

To assess the duration of immunosuppression in FK506-dosed pigs, an undiluted whole blood assay was established to measure reactivities of T cells in their physiological milieu. PMA and ionomycin were shown to induce IL-2 production in swine blood. The IC50 of FK506 in inhibiting IL-2 production in whole blood and isolated PBMC stimulated with PMA and ionomycin measured 1.2 and 0.04 nM, respectively. These data underscore the influence of red blood cells and plasma proteins on drug potency. IL-2 levels were determined in blood drawn immediately before and 1, 24, 48, and 72 h after iv dosing. For pigs dosed with 0.05 mg/kg, 50% recovery of IL-2 production was observed at 16 h and 100% at 35 h after dosing. For pigs dosed with 0.15 mg/kg, 50% recovery was observed at 38 h and 100% at 72 h. Blood concentrations of FK506 at 50 and 100% recovery of IL-2 production measured 10.8 and 2.2 nM for pigs dosed with 0.05 mg/kg and 6.1 and 1.1 nM for pigs dosed with 0.15 mg/kg, respectively. These concentrations are severalfold higher than predicted from the IC50 of FK506 for inhibiting IL-2 production in the whole blood assay. These data suggest that the true potency of FK506 in blood after dosing is influenced by additional factors, which could include plasma protein binding, the presence of active or interfering metabolites, serum interfering factors, and sequestration of drug in blood cells. Our results demonstrate the utility of an undiluted whole blood assay for assessing the duration of immunosuppression in drug-dosed animals and emphasize the importance of assessing drug potency in the whole blood environment ex vivo.     

Biochemical and biophysical characterization of human recombinant lecithin: cholesterol acyltransferase

We established a Chinese hamster ovary cell line that constitutively expresses up to 5 mg/L of human recombinant lecithin: cholesterol acyltransferase (rLCAT). We purified the rLCAT to > 96% purity, and characterized it along with plasma LCAT (pLCAT) biochemically and biophysically. The recombinant enzyme is more heavily glycosylated and more heterogeneous in its carbohydrate content than the plasma enzyme, as revealed by differences in molecular weight and pI isoforms, determined by mass spectrometry and isoelectric focusing. Recombinant LCAT is half as active enzymatically as pLCAT. The difference in activity is due to differences in the catalytic rates rather than in the apparent K(m) values, suggesting that the binding of the rLCAT to interfaces is not altered by its different glycosylation pattern. Despite these differences, rLCAT has essentially the same intrinsic tryptophan fluorescence emission spectrum and far-UV CD spectrum as pLCAT, indicating that the tertiary and secondary structures of both enzyme forms are very similar. Both enzyme forms have a propensity to self-associate, and their multimers appear resistant to dissociation by SDS and dilution. The free energies of unfolding (delta G(H2O)) of rLCAT and pLCAT are 3.4 +/- 0.2 and 3.2 +/- 0.2 kcal/mol, respectively, as determined by guanidine hydrochloride denaturation monitored by fluorescence. These relatively low delta G(H2O) values support the notion that LCAT is capable of undergoing major conformational changes upon interaction with interfacial substrates.     

Diameter of the mammalian porin channel in open and "closed" states: direct measurement at the single channel level in planar lipid bilayer

Porin isolated from bovine skeletal muscle was reconstitute in planar lipid bilayers under voltage clamp conditions. A set of non-electrolytes were used as molecular probes for determining the pore diameter. The maximal diameter of the open channel was estimated to be 3.02 +/- 0.26 nm. As observed for other porin channels, a large transmembrane potential drove the channel into a "closed" state. The channel transition to the low conductance (closed) state was followed by a decrease in the maximal diameter of the channel to 2.4 +/- 0.08 nm.     

The ultrastructure of yeast: cell wall structure and formation

Yeasts are unicellular eukaryotes, and are used widely as a model system in basic and applied fields of life science, medicine, and biotechnology. The ultrastructure of yeast cells was first studied in 1957 and the techniques used have advanced greatly in the 40 years since then; an overview of these methods is first presented in this review. The ultrastructure of budding and dimorphic yeast cells observed with a scanning electron microscope (SEM) and a transmission electron microscope (TEM) after thin sectioning and freeze-etching are then described, followed by discussion of the regeneration of the cell wall of Candida albicans protoplasts detected by cryosectioning. C. albicans protoplasts are regenerated to synthesize microfibrils on their surface. They are aggregated into thicker bundles which are intermeshed, forming a wide-meshed network of long fibrils. These microfibrillar structures are chains of beta-1,3-glucan which are broken down after treatment with beta-1,3-glucanase. Morphologically identical microfibrils are synthesized in vitro by a cell-free system in which the active cell membrane fraction as a source of beta-1,3-glucan synthetase and UDP glucose as the sole substrate are used. The diameter of an elemental fibril of beta-glucan is estimated to be 2.8 nm from the pattern of autocorrelation of the image obtained by computer processing. In contrast, in the presence of aculeacin A the formation of normal fibrillar nets or bundles is significantly inhibited, resulting in the occurrence of short fibrils. These electron microscopic data suggest that aculeacin A inhibits not only the synthesis of beta-1,3-glucan but the aggregation of microfibrils of this polysaccharide, allowing formation of the crystalline structure. On the basis of the cumulative data obtained from the electron microscopic studies, we are led to the assumption that de novo synthesized beta-glucan chains might initially form fine particles which are then transformed into thin fibrils with single to multiple strands which appear to be oriented parallel to each other so that they develop into fibrillar structures. This process of assembly of beta-glucan molecules leads to the development of a fibrous network within the regenerating Candida cell wall. Third, the mechanism of cell wall formation is shown by low-voltage (LV) SEM and TEM, using various techniques and computer graphics, of the regeneration system of Schizosaccharomyces pombe protoplasts: after 10 min of regeneration, the protoplasts begin to grow fibrillar substances of a beta-glucan nature, and a fibrillar network covers the surface of all protoplasts. The network is originally formed as fine particles on the protoplast surface and these are subsequently lengthened to microfibrils 2 nm thick. The microfibrils twist around each other and develop into 8 nm thick fibrils forming flat bundles 16 nm thick. Interfibrillar spaces are gradually filled with amorphous particles of an alpha-galactomannan nature and, finally, the complete cell wall is formed after 12 h. Treatment of reverting protoplasts with RuO4 provided clear TEM images of glucan fibrils with high electron density. The relationship between cell wall regeneration and intracellular organelles was examined by using serial thin sections stained with PATAg and computer-aided three-dimensional reconstruction. The secretory vesicles in a protoplast had increased markedly by 1.4, 3.4, and 5.8 times at 1.5, 3.0, and 5 h, respectively. Three-dimensional analysis indicates that Golgi apparatuses are located close together in the nucleus of the protoplast and are dispersed into the cytoplasm during the progress of cell wall formation.     

Catalytic and biophysical properties of a nitrogenase Apo-MoFe protein produced by a nifB-deletion mutant of Azotobacter vinelandii

A Zn-immobilized metal-affinity chromatography technique was used to purify a poly-histidine-tagged, FeMo-cofactorless MoFe protein (apo-MoFe protein) from a nifB-deletion mutant of Azotobacter vinelandii. Apo-MoFe protein prepared in this way was obtained in sufficient concentrations for detailed catalytic, kinetic, and spectroscopic analyses. Metal analysis and electron paramagnetic resonance spectroscopy (EPR) were used to show that the apo-MoFe protein does not contain FeMo-cofactor. The EPR of the as-isolated apo-MoFe protein is featureless except for a minor S = 1/2 signal probably arising from the presence of either a damaged P cluster or a P cluster precursor. The apo-MoFe protein has an alpha2beta2 subunit composition and can be activated to 80% of the theoretical MoFe protein value by the addition of isolated FeMo-cofactor. Oxidation of the as-isolated apo-MoFe protein by indigodisulfonate was used to elicit the parallel mode EPR signal indicative of the two-electron oxidized form of the P cluster (P2+). The midpoint potential of the PN/P2+ redox couple for the apo-MoFe protein was shown to be shifted by -63 mV when compared to the same redox couple for the intact MoFe protein. Although the apo-MoFe protein is not able to catalyze the reduction of substrates under turnover conditions, it does support the hydrolysis of MgATP at 60% of the rate supported by the MoFe protein when incubated in the presence of Fe protein. The ability of the apo-MoFe protein to specifically interact with the Fe protein was also shown by stopped-flow techniques and by formation of an apo-MoFe protein-Fe protein complex. Finally, the two-electron oxidized form of the apo-MoFe protein could be reduced to the one-electron oxidized form (P1+) in a reaction that required Fe protein and MgATP. These results are interpreted to indicate that the apo-MoFe protein produced in a nifB-deficient genetic background [corrected] contains intact P clusters and P cluster polypeptide environments. Small changes in the electronic properties of P clusters contained within the apo-MoFe protein are most likely caused by slight perturbations in their polypeptide environments.     

Immunochemical and biochemical identification of the rice seed protein encoded by cDNA clone A3-12

Previously isolated cDNA clone A3-12 that was expressed in E. coli as the fusion protein with Trp E showed immunoreactivity with the mouse antibody raised against isolated alpha-globulin from rice seed. The N-terminal amino acid sequences determined for the purified alpha-globulin and its tryptic peptides were identical with the deduced amino acid sequence reported, except for two residues at the protein N terminus. An error in the reported sequence was confirmed by re-sequencing the cDNA, the nucleotide sequence for the two N-terminal residues being shown to be CAGCTG and not CACGTG. Thus, the protein encoded by cDNA clone A3-12 was identified to be the major rice seed globulin, alpha-globulin, with an apparent molecular mass of 26kDa.     

Changes in the mechanical properties, biochemical contents and wall structure of the human coronary arteries with age and sex

An experimental study of the mechanical properties, biochemical composition and structure was carried out on the proximal and distal parts of the right and the anterior descending branch of left human coronary arteries. The vessels were removed during an autopsy of 121 males and 84 females being 1 day to 80 years old. The material was divided into six age groups. Branchless segments of vessels 15-20 mm long were cut from proximal and distal parts. The mechanical properties of the coronary arteries were determined by passing fluid at pressures ranging from 0 to 240 mmHg. It was found that the part of the wall of the coronary artery adjacent to the myocardium is thicker in all cases than the other part of the arterial wall. With increasing age the mean thickness of the wall of both coronary arteries increases but the wall-thickening process is non-uniform in nature in both the proximal and distal parts and in the individual layers. The changes in the stretch ratio and tangential modulus in circumferential direction with age and sex were investigated. The greatest changes in the wall thickness and in the mechanical parameters were found for the left coronary artery wall in men over 40 years of age and for the right coronary artery wall in women over 50 years of age. The results of biochemical and densitometric investigations were compared.