rhG-CSF resistant neutropenia in SLE.
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Child↗
Biomedical subjects
M Frosch
Publications and source records attributed to M Frosch.
Autocatalytic polysialylation of polysialyltransferase-1.
Polysialic acid (PSA) is a specific and highly regulated post-translational modification of the neural cell adhesion molecule NCAM. Synthesis of PSA depends on the activity of a single enzyme, the polysialyltransferase-1 (PST-1), recently cloned from three mammalian species. The present study was carried out to investigate the catalytic mechanism of PST-1. Using a newly developed in vitro assay system, we demonstrate autopolysialylation for PST-1. The synthesis of PSA chains, which involved N-glycosylation sites, occurred immediately after contact with the activated sugar donor CMP-Neu5Ac. In contrast to the polysialylation of NCAM, where terminal sialylation in either the alpha2,3 or alpha2,6 position is required, the autopolysialylation could be started in the asialo-PST-1 isolated from CHO cells of the Lec2 complementation group. Pre-formed PSA chains were not transferred to NCAM. Nevertheless, the autocatalytic step is likely to be a prerequisite for enzymatic activity, since agalacto-PST-1 isolated from Lec8 cells was functionally inactive. Our data describe a novel route of autocatalytic maturation of a glycosyltransferase and thereby provide a new basis for studies aimed at elucidating and influencing the catalytic functions of PST-1.
Animals↗
Polysialylation of NCAM by a single enzyme.
The addition of poly-alpha2,8-N-acetylneuraminic acid (polysialic acid; PSA) to the neural cell adhesion molecule NCAM plays a crucial role in neural development [1-3], neural regeneration [4], and plastic processes in the vertebrate brain associated with neurite outgrowth [5], axonal pathfinding [6], and learning and memory [7,-9]. PSA levels are decreased in people affected by schizophrenia [10], and PSA has been identified as a specific marker for some neuroendocrine and lymphoblastoid tumours [11-13]; expression of PSA on the surface of these tumour cells modulates their metastatic potential [11-13]. Studies aimed at understanding PSA biosynthesis and the dynamics of its production have largely been promoted by the cloning of polysialyltransferases (PST-1 in hamster; PST in human and mouse) [14-16]. However, the number of enzymes involved in the biosynthesis of PSA has not been identified. Using incompletely glycosylated NCAM variants and soluble recombinant glycosyltransferases, we reconstituted the site at which PST-1 acts to polysialylate NCAM in vitro. The data presented here clearly demonstrate that polysialylation of NCAM is catalyzed by a single enzyme, PST-1, and that terminal sialylation of the N-glycan core is sufficient to generate the PSA acceptor site. Our results also show that PST-1 can act on core structures with the terminal sialic acid connected to galactose via an alpha2,3 or alpha2,6 linkage.
Animals↗
Modulation of cell surface sialic acid expression in Neisseria meningitidis via a transposable genetic element.
Cell surface-located sialic acids of the capsule and the lipooligosaccharide (LOS) are both pivotal virulence factors in Neisseria meningitidis, promoting survival and dissemination of this pathogen which can cause both sepsis and meningitis. With the aid of a unique set of isogenic meningococcal mutants defective in the expression of cell surface-located sialic acids, we have demonstrated that encapsulation hinders the primary event in the development of the disease, but the spontaneous switching of encapsulated wild-type bacteria to a capsule-negative phenotype promotes meningococcal adherence and invasion into mucosal epithelial cells. Genetic analysis of the capsule-negative, invasive bacteria revealed a unique mechanism for modulation of capsule expression based on the reversible inactivation of an essential sialic acid biosynthesis gene, siaA, by insertion/excision of a naturally occurring insertion sequence element, IS1301. Inactivation of siaA regulates both capsule expression and endogenous LOS sialylation. This is the first example of an insertion sequence element-based genetic switch mechanism in the pathogenic bacterium and is an important step in the understanding of bacterial virulence.
Amino Acid Sequence↗
Development of an amplification and hybridization assay for the specific and sensitive detection of Mycoplasma fermentans DNA.
A polymerase-chain-reaction-based detection system for Mycoplasma fermentans was established. The highly conserved tuf gene, which encodes elongation factor Tu of prokaryotes, served as target sequence for the PCR. With two PCR oligodeoxynucleotides, which were selected from M. fermentans specific sequences of the tuf gene, we amplified a 850 base pair DNA fragment. Via the biotin-moiety of one primer the PCR fragments were immobilized on streptavidin-coated microtitre plates. After alkaline denaturation a digoxigenin-labelled M. fermentans specific DNA probe was hybridized to the single stranded immobilized PCR fragment. Detection was performed by addition of an alkaline phosphatase conjugated anti-digoxigenin antibody. 4-methyl-umbelliferyl-phosphate was used as a fluorogenic substrate. Amplification of 10 fg chromosomal target DNA was detected by this 'DNA enzyme immuno assay (DEIA)' technique, corresponding to seven genome copies. Our study supports the presumption that the tuf gene proves to be a suitable target sequence for the PCR based detection of any bacterial species. Furthermore, hybridization of PCR fragments with radio-labelled DNA probes should no longer be necessary, because a very sensitive non-radioactive test system can easily be established with the 'DEIA' technique.
Base Sequence↗
Avoiding artifacts in the infant rat model for bacterial meningitis: use of Sangur test strips for the rapid quantification of blood contamination in cerebrospinal fluid.
The infant rat model is widely used to study the pathogenesis of meningitis caused by a variety of gram-positive and gram-negative bacteria. However, the interpretation of published results concerning meningitis is difficult in many records because the fact that blood contamination of the cerebrospinal fluid (CSF) cannot be avoided during the traumatic puncture procedure has not been taken into consideration. Since bacterial invasion of the central nervous system develops following bacteremia in this model, blood contamination of the CSF leads to a falsification of the CSF bacterial counts. Here we present an evaluation of a rapid and quantitative test for CSF blood contamination using Sangur test strips. The procedure requires minimal amounts of CSF and allows direct calculation of the CSF bacterial load due to blood contamination and, thus, provides refined criteria for the presence of bacterial meningitis in the infant rat model. It is superior to the detection of erythrocytes using a hemocytometer since it is less time consuming. Furthermore, we demonstrate the value of this method for the experimental infection of rats with Neisseria meningitis.
Animals↗
Sialic acids of both the capsule and the sialylated lipooligosaccharide of Neisseria meningitis serogroup B are prerequisites for virulence of meningococci in the infant rat.
We investigated the contribution of the polysialic acid capsule and of terminal lipooligosaccharide (LOS) sialylation to the pathogenicity of Neisseria meningitidis in vivo using a set of defined isogenic mutants of the N. meningitidis strain B 1940 deficient in either capsule synthesis or LOS sialylation. Furthermore a spontaneous capsule-deficient variant was investigated, which was capable of switching on the capsule synthesis at a frequency of 3 x 10(-3) in vitro. Infection of infant rats with the wild-type strain revealed a high potential to cause bacteremia. This potential was attenuated in the capsule-phase variable mutant (LOS sialylation+). However, using a mutant irreversibly deficient in capsule synthesis, but expressing a sialylated LOS, bacteremia could only be achieved using 10(6) times higher numbers of bacteria when compared to the wild-type. The unencapsulated bacteria were located extracellularly upon examination of blood smears, suggesting that defense mechanisms, i.e. phagocytosis, directed against unencapsulated meningococci were exhausted using very high infecting doses. Interestingly, when infant rats were infected with encapsulated meningococci which were unable to sialylate the LOS, bacteremia could never be achieved, even with an infective dose as high as 10(8) colony forming units (CFU). Despite the presence of capsular polysaccharide this mutant was phagocytosed by peritoneal phagocytes, as was the unencapsulated, LOS-sialylated mutant, suggesting that the inability to cause bacteremia was due to a higher susceptibility to the action of the complement system, which is virtually unsaturable. We conclude that in the infant rat model of meningococcal infection both forms of sialic acid on the bacterial cell surface are indispensable for systemic survival.
Animals↗
The influence of capsulation and lipooligosaccharide structure on neutrophil adhesion molecule expression and endothelial injury by Neisseria meningitidis.
Host inflammatory response to meningococcal infection is believed to be a major determinant of disease severity. Isogenic mutants of Neisseria meningitidis serogroup B1940, which differ in expression of capsular polysaccharide and lipooligosaccharide (LOS), were used to examine host responses in a whole blood model of bacteremia and a model of endothelial injury. The parent organism caused significantly less neutrophil shedding of the adhesion molecule, L-selectin, than the three mutant organisms (P < .01) and was most resistant to the bactericidal activity of whole blood. Despite marked differences in bacterial adhesion to endothelial cells (P < .05), no damage was induced by organisms alone. Endothelial injury was observed when neutrophils were incubated with adherent, capsule-deficient organisms (P < .05). The degree of endothelial damage was related to the number of neutrophils adherent to the endothelium. Thus, bacterial capsulation and LOS structure can influence neutrophil activation and endothelial injury and, as such, may be important in the pathogenesis of meningococcal sepsis.
Antigens, Bacterial↗
Capsule phase variation in Neisseria meningitidis serogroup B by slipped-strand mispairing in the polysialyltransferase gene (siaD): correlation with bacterial invasion and the outbreak of meningococcal disease.
A mechanism of capsular polysaccharide phase variation in Neisseria meningitidis is described. Meningococcal cells of an encapsulated serogroup B strain were used in invasion assays. Only unencapsulated variants were found to enter epithelial cells. Analysis of one group of capsule-deficient variants indicated that the capsular polysaccharide was re-expressed at a frequency of 10(-3). Measurement of enzymatic activities involved in the biosynthesis of the alpha-2,8 polysialic acid capsule showed that polysialyltransferase (PST) activity was absent in these capsule-negative variants. Nucleotide sequence analysis of siaD revealed an insertion or a deletion of one cytidine residue within a run of (dC)7 residues at position 89, resulting in a frameshift and premature termination of translation. We analysed unencapsulated isolates from carriers and encapsulated case isolates collected during an outbreak of meningococcal disease. Further paired blood-culture isolates and unencapsulated nasopharyngeal isolates from patients with meningococcal meningitis were examined. In all unencapsulated strains analysed we found an insertion or deletion within the oligo-(dC) stretch within siaD, resulting in a frameshift and loss of capsule formation. All encapsulated isolates, however, had seven dC residues at this position, indicating a correlation between capsule phase variation and bacterial invasion and the outbreak of meningococcal disease.
Amino Acid Sequence↗
Hepatic tissue culture model for study of host-parasite interactions in alveolar echinococcosis.
An in vitro model for growth and differentiation of the metacestode tissue of the tapeworm Echinococcus multilocularis is described. This model simulates the organotropism of the parasite toward the liver of the intermediate host. In the presence of collagen-embedded primary hepatocytes from rats and humans, which can be kept in culture for 2 to 3 months, the parasitic vesicles grew by exogenous budding and multiplied about 12-fold within 3 weeks. In contrast, without the hepatocytes, the metacestodes rapidly degenerated. Development of protoscolices was seen only in the presence of rat hepatocytes but not in coculture of the metacestodes with hepatocytes of human origin, thus reflecting the in vivo situation during infection of rodents and in alveolar echinococcosis in humans. The experiments indicated that growth of the metacestodes and development of protoscolices depended on soluble low-molecular-weight factors released by the hepatocytes. The in vitro-grown metacestodes did not differ morphologically from the larvae found in infected intermediate hosts, and their infectivity was completely maintained. This report describes the first in vitro model of alveolar echinococcosis and will be the basis for future studies on host-parasite interactions of this important zoonosis.
Animals↗
Genes associated with meningococcal capsule complex are also found in Neisseria gonorrhoeae.
A homolog of the meningococcal cps locus region E has been identified in Neisseria gonorrhoeae immediately upstream of the gonococcal region D locus. Region E has no detectable function in capsule biosynthesis in Neisseria meningitidis or in lipopolysaccharide biosynthesis in either organism. The open reading frame is homologous to proteins of unknown function in Escherichia coli and Haemophilus influenzae. Further analysis of the N. meningitidis cps cluster has identified a second copy of region D encoding three additional open reading frames, including homologs of DNA methyltransferases. The organization of the region D and E genes in N. gonorrhoeae and N. meningitidis in relation to the cps genes provides some insight into the evolutionary origin of encapsulation in N. meningitidis.
Bacterial Capsules↗
Site-specific insertion of IS1301 and distribution in Neisseria meningitidis strains.
The insertion element IS1301 has been shown to mediate capsule phase variation in Neisseria meningitidis found in N. serogroup B by reversible insertional inactivation of the siaA gene. We have determined the target site specificity of this element by cloning and sequencing the insertion sites of 12 identical IS1301 copies found in N. meningitidis B1940. A target consensus core of 5'-AYTAG-3' was identified, with the central TA being duplicated following insertion. Additional features around the target sites, including extended palindromic symmetry, stem-loop formation, and the high incidence of AT tracts, indicate that other factors, such as DNA secondary structure, are involved in target recognition. The left inverted repeat of an IS1016-like element acts as a hot spot for insertion, with one insertion element combination located upstream of their gene. According to further sequence analysis, we were able to place IS1301 in the IS5 subgroup within the IS4 family of elements. A survey of 135 Neisseria strains indicated the presence of IS1301 in 27.9 to 33.3% of N. meningitides serogroup B, C, and W135 strains and in 86.7% of serogroup Y strains. IS1301 did not occur in serogroup A strains, in Neisseria gonorrhoeae, or in apathogenic Neisseria spp.
Amino Acid Sequence↗
Evidence for the extended helical nature of polysaccharide epitopes. The 2.8 A resolution structure and thermodynamics of ligand binding of an antigen binding fragment specific for alpha-(2-->8)-polysialic acid.
The antigen binding fragment from an IgG2a kappa murine monoclonal antibody with specificity for alpha-(2-->8)-linked sialic acid polymers has been prepared and crystallized in the absence of hapten. Crystals were grown by vapor diffusion equilibrium with 16-18% polyethylene glycol 4000 solutions. The structure was solved by molecular replacement methods and refined to a conventional R factor of 0.164 for data to 2.8 A. The binding site is observed to display a shape and distribution of charges that is complementary to that of the predicted conformation of the oligosaccharide epitope. A thermodynamic description of ligand binding has been compiled for oligosaccharides ranging in length from 9 to 41 residues, and the data for the largest ligand has been used in a novel way to estimate the size of the antigen binding site. A model of antigen binding is presented that satisfies this thermodynamic data, as well as a previously reported requirement of conformational specificity of the oligosaccharide. X-ray crystallographic and thermodynamic evidence are consistent with a binding site that accommodates at least eight sialic acid residues.
Antibodies, Monoclonal↗
Molecular characterization of eukaryotic polysialyltransferase-1.
Polysialic acid (PSA) is a dynamically regulated product of post-translational modification of the neural cell adhesion molecule, NCAM. Presence of the large anionic carbohydrate modulates NCAM binding properties and, by increasing the intercellular space, influences interactions between other cell surface molecules. PSA expression underlies cell type- and developmental-specific alterations and correlates with stages of cellular motility. In the adult, PSA becomes restricted to regions of permanent neural plasticity and regenerating neural and muscle tissues. Recent data implicate its important function in spatial learning and memory, and in tumour biology. Here we describe the molecular characterization of polysialyltransferase-1, the key enzyme of eukaryotic PSA synthesis. In reconstitution experiments, the newly cloned enzyme induces PSA synthesis in all NCAM-expressing cell lines. Our data therefore represent convincing evidence that the polycondensation of alpha-2,8-linked sialic acids in mammals is the result of a single enzymatic activity and provide a new basis for studying the functional role of PSA in neuro- and tumour biology.
3T3 Cells↗
Diagnosis of Echinococcus multilocularis infection by reverse-transcription polymerase chain reaction.
Alveolar echinococcosis is a life-threatening parasitosis occurring in countries in the Northern hemisphere. The diagnosis of an Echinococcus multilocularis infection is routinely performed by radiological techniques and by the detection of specific antibodies in the sera of infected patients. However, because serodiagnosis fails in 5%-10% and radiological techniques are difficult to interpret in some cases, we developed a polymerase chain reaction for the detection of echinococcal-specific messenger RNA from fine-needle biopsy specimens. This technique was applied to the diagnosis of alveolar echinococcosis in a 20-year-old seronegative woman. Detection of messenger RNA not only allowed the diagnosis of echinococcal disease but also proved to be a reliable measure for determining the efficacy of an antiparasitic therapy.
Adult↗
Molecular cloning and characterization of an Echinococcus multilocularis and Echinococcus granulosus stress protein homologous to the mammalian 78 kDa glucose regulated protein.
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Amino Acid Sequence↗
Intact survival with transfusion-associated graft-versus-host disease proved by human leukocyte antigen typing of lymphocytes in skin biopsy specimens.
A transient transfusion-associated graft-versus-host disease occurred in a premature infant of 30 weeks of gestation. We demonstrated donor lymphocytes in a skin biopsy specimen with a two-step immunoperoxidase technique using monoclonal antibodies against human leukocyte antigen determinants specific for the donor. The girl survived and is immunocompetent.
Antibodies, Monoclonal↗
Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE.
Homopolymeric alpha-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5' end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.
Amino Acid Sequence↗