Mechanisms of neisserial serum resistance

Pathogenic Neisseria use a variety of mechanisms to survive the bactericidal action of the complement system. Serum resistance is a crucial virulence factor for the development of severe meningococcal disease, meningococcal meningitis and disseminated gonococcal infection. Furthermore, local inflammation at the site of gonococcal infection exposes the bacteria to moderate concentrations of complement factors. We review current concepts of neisserial serum resistance with emphasis on porins and polysaccharides exposed on the neisserial surface and their interaction with components of normal human serum.     

Sequence and functional analysis of the cloned Neisseria meningitidis CMP-NeuNAc synthetase

The CMP-N-acetylneuraminic acid (CMP-NeuNAc) synthetase gene of Neisseria meningitidis group B is located on a 2.3-kb EcoRI fragment within the cps gene cluster. Nucleotide sequence determination of the gene encoding the CMP-NeuNAc synthetase revealed a 515-bp open reading frame that can encode a 18.9-kDA protein. A computer data base scan revealed a 59.4% identity to the CMP-NeuNAc synthetase gene of E. coli K1. Enzymatic activity was confirmed in vitro and in vivo. Transformation of the CMP-NeuNAc defective E. coli K1 strain EV5 with the meningococcal CMP-NeuNAc synthetase could complement the defect in E. coli.     

Granulocyte-macrophage colony-stimulating factor activates macrophages derived from bone marrow cultures to synthesis of MHC class II molecules and to augmented antigen presentation function

The effects of granulocyte-macrophage (GM)-CSF on the synthesis of MHC class II molecules and on the Ag presentation capacity by bone marrow derived macrophages (BMM phi) was investigated. BMM phi obtained by in vitro culture in the presence of macrophage-CSF were negative for synthesis of I-A molecules and induced the Ag-mediated proliferation of insulin-specific T clone cells with lower efficiency than splenic accessory cells. After pulse treatment with GM-CSF for 24 to 48 h, day 12 BMM phi exhibited highly efficient Ag presentation function which was superior to that induced by IFN-gamma. Expression of membrane-bound IL-1 was augmented significantly by GM-CSF, but not by IFN-gamma. However, the T cell clone used to probe for accessory cell function of BMM phi was not dependent on IL-1 for optimal proliferation. Concomitantly, GM-CSF induced the de novo synthesis of I-A molecules, although to a lesser extent than optimal doses of IFN-gamma. Thus GM-CSF appears to elicit properties in addition to Ia molecule synthesis and membrane IL-1 expression in BMM phi being essential for efficient accessory cell function to the T clone cells. The activation of BMM phi by GM-CSF was reversible and could be repeated. These data show that GM-CSF exerts a modulatory influence on preformed BMM phi, reversibly activating cells to Ia biosynthetic potential and pronounced accessory cell capacity, thus rendering the explanation unlikely that differentiation of precursor cells into a constitutively functional state had occurred.     

Ribulose bisphosphate carboxylase capacity and chlorophyll content in developing seedlings of Chenopodium rubrum L. growing under light of different qualities and fluence rates

In order to evaluate the aclimation of Chenopodium seedlings to different quantum fluence rates of R and BL, kinetics of Rubisco capacity, Chl content and chloroplast structure were studied. Under monochromatic light photoreceptors are stimulated selectively and their influence on biosynthetis capacities during chloroplast development can be studied.R irradiations saturate Rubisco capacity even at the lowest quantum fluence rates applied, whereas Chl a+b synthesis depends strongly upon fluence rate of R. Under BL irradiations, both Rubisco capacity and Chl content are fluence rate dependent. R irradiations favour Chl b synthesis relative to Chl a, whereas under BL Chl a content is high relative to Chl b. Under R irradiation Pfr is the main photoreceptor involved in regulation of Rubisco capacity whereas under BL a specific BL absorbing photoreceptor may control the response. From the fluence rate dependency under BL irradiations it is concluded that the blue region of the day light spectrum may be the sensor for monitoring fluence rate and causing the characteristic changes in shade and high/low WL adaptation with respect to Rubisco levels in Chenopodium.     

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.     

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.     

Subunits of forming pollen exine and Ubisch bodies as seen in freeze substitutedLedebouria socialis Roth (Hyacinthaceae)

Summary High-pressure freezing/freeze substitution/TEM was employed to investigate anthers of the monocotyledonous angiospermLedebouria socialis Roth (Hyacinthaceae) during early tetrad stage. The initials of the outer sporopollenous pollen wall stratum (=sexine) and of the homologous tapetal products (=Ubisch bodies) are composed of highly regular subunits: clustered globules with a constant diameter of approximately 28 nm. The clusters develop within diffuse accumulations of electron-dense material. This process, interpreted as sporopollenin polymerization, does not necessarily depend on the presence of membrane-bound enzymes. Immunogold labeling with JIM 5 and JIM 7 antibodies revealed that the primexine as well as the dissolving tapetal cell walls, the sites of sexine and Ubisch body formation, respectively, contain un-esterified and methyl-esterified pectins.Abbreviations E-PTA ethanolic phosphotungstic acid - PA periodic acid - UA/Pb uranyl acetate/lead     

Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE

Homopolymeric α-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.     

Complete nucleotide and deduced protein sequence of CMP-NeuAc: poly-alpha-2,8 sialosyl sialyltransferase of Escherichia coli K1.

Poly-alpha-2,8 N-acetylneuraminic acid (polySia) is an important virulence factor in infections caused by Escherichia coli K1 and Neisseria meningitidis B. In E. coli K1 a membranous CMP-NeuAc: poly-alpha-2,8 sialosyl sialyltransferase (polysialyltransferase) complex catalyses the synthesis of linear polySia chains. The complex also elongates sialyl oligomers that serve as exogenous acceptors. The gene encoding a polysialyltransferase of E. coli has been identified by subcloning and DNA sequence analysis. The subcloned DNA fragment codes for a polypeptide with a molecular mass of 47 kDa catalysing the in vitro synthesis of polySia by elongation of exogenous acceptors.     

A sensitive and specific multiplex PCR approach for sex identification of ursine and tremarctine bears suitable for non‐invasive samples

We report a new approach for molecular sex identification of extant Ursinae and Tremarctinae bears. Two Y‐specific fragments (SMCY and 318.2) and one X‐specific fragment (ZFX) are amplified in a multiplex PCR, yielding a double test for male‐specific amplification and an internal positive control. The primers were designed and tested to be bear‐specific, thereby minimizing the risk of cross‐amplification in other species including humans. The high sensitivity and small amplicon sizes (100, 124, 160 base pairs) facilitate analysis of non‐invasively obtained DNA material. DNA from tissue and blood as well as from 30 non‐invasively collected hair and faeces yielded clear and easily interpretable results. The fragments were detected both by standard gel electrophoresis and automated capillary electrophoresis.