Identification of a locus involved in meningococcal lipopolysaccharide biosynthesis by deletion mutagenesis

A novel method for insertion/deletion mutagenesis in meningococci was devised. This consisted of ligating a digest of total chromosomal DNA to a 1.1 kb restriction fragment containing an erythromycin-resistance marker ( ermC ), and subsequent transformation of the ligation mixture into the homologous meningococcal strain H44/76. Southern blotting of a number of the resulting erythromycin-resistant transformants demonstrated that all carried the ermC gene inserted at different positions in the chromosome. Mutants with a specific phenotype were identified by screening with the anti-lipopolysaccharide (LPS) monoclonal antibody MN4A8B2, which is specific for immunotype L3. In this way, two independent L3-negative mutant strains were isolated. In transformation experiments with chromosomal DNA from these mutants, erythromycin-resistance and lack of MN4A8B2 reactivity were always linked, showing that the insertion/deletion was in a locus involved in LPS biosynthesis. On SDS–PAGE, the mutant LPS displayed an electrophoretic mobility intermediate between that produced by the previously isolated galE and rfaF mutant strains. Chemical analysis of the mutant LPS revealed that the structure was probably lipid A–(KDO)₂–(Hep)₂. Chromosomal DNA flanking the ermC insertion in these two mutant strains was cloned, and used as probe for the isolation of the corresponding region of the wild-type strain. From hybridization and polymerase chain reaction (PCR) analysis, it could be concluded that both mutations map to the same locus. The affected gene probably encodes the glycosyltransferase necessary for adding N -acetylglucosamine to heptose.     

Tandem repeats of the tetramer 5'-CAAT-3'present in lic2A are required for phase variation but not lipopolysaccharide biosynthesis in Haemophilus influenzae

A novel lipopolysaccharide (LPS) biosynthesis gene, lic2B, which is required for the biosynthesis of a phase-variable LPS structure expressed by Haemo philus influenzae RM7004 is described. The product of this gene is homologous to Lic2A and the recently described LPS biosynthetic enzymes, LgtB from Neis seria gonorrhoea and LgtE from Neisseria meningitidis, and LpsA from Pasteurella haemolytica. Of this family of enzymes only Lic2A contains the repetitive tetrapeptide motif (SINQ)ⁿ encoded by multiple tandem repeats of 5′-CAAT-3′. This observation suggested that (SINQ)ⁿ might not be a prerequisite for the catalytic activity of this protein. To address this hypothesis, we deleted the 5′-CAAT-3’repeats from lic2A so that the protein encoded by the modified gene was analogous to Lic2B. This mutation had no apparent effect on the overall apparent molecular weight of LPS as judged by Tricine-SDS-PAGE and did not affect ability to react with monoclonal antibody 4C4. It was therefore concluded that (SINQ)ⁿ is not a prerequisite for the enzymatic function of Lic2A and that the 5′-CAAT-3’repeats in lic2A function solely as a mechan ism for generating phase variation. This observation suggested that wide variation in the number of 5’-CAAT-3’repeats might be tolerated in lic2A, and this was confirmed by surveying the number of 5′-CAAT-3’repeats in a range of different H. influenzae strains. The predicted secondary structure of (SINQ)ⁿ indicates that it forms a highly flexible random coiled structure, which is unlikely to impede formation of the domains that may be required for catalytic activity. This characteristic is also a feature of repetitive tetrapeptides encoded by other tetrameric repeats located within coding sequences present on the chromosome of H. influenzae Rd.     

In vitro activity of minimised hammerhead ribozymes.

A number of minimised hammerhead ribozymes (minizymes) which lack stem II have been kinetically characterised. These minizymes display optimal cleavage activity at temperatures around 37 degrees C. The cleavage reactions of the minizymes are first order in hydroxide ion concentration up to around pH 9.3 above which the cleavage rate constants decline rapidly. The reactions show a biphasic dependence on magnesium-ion concentration; one of the interactions has an apparent dissociation constant of around 20 mM while the other appears to be very weak, showing no sign of saturation at 200 mM MgCl2. The minizymes are significantly less active than comparable, full-size ribozymes when cleaving short substrates. However, at a particular site in a transcribed TAT gene from HIV-1, minizymes are more effective than ribozymes.     

Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4-diacetamido-2,4,6-trideoxyhexose

Neisseria meningitidis pili are filamentous protein structures that are essential adhesins in capsulate bacteria. Pili of adhesion variants of meningococcal strain C311 contain glycosyl residues on pilin (PilE), their major structural subunit. Despite the presence of three potential N -linked glycosylation sites, none appears to be occupied in these pilins. Instead, a novel O -linked trisaccharide substituent, not previously found as a constituent of glycoproteins, is present within a peptide spanning amino acid residues 45 to 73 of the PilE molecule. This structure contains a terminal 1-4-linked digalactose moiety covalently linked to a 2,4-diacetamido-2,4,6-trideoxyhexose sugar which is directly attached to pilin. Pilins derived from galactose epimerase ( galE ) mutants lack the digalactosyl moiety, but retain the diacetamidotrideoxyhexose substitution. Both parental (#3) pilins and those derived from a hyper-adherent variant (#16) contained identical sugar substitutions in this region of pilin, and galE mutants of #3 were similar to the parental phenotype in their adherence to host cells. These studies have confirmed our previous observations that meningococcal pili are glycosylated and provided the first structural evidence for the presence of covalently linked carbohydrate on pili. In addition, they have revealed a completely novel protein/saccharide linkage.     

Opc- and pilus-dependent interactions of meningococci with human endothelial cells: molecular mechanisms and modulation by surface polysaccharides

The interplay between four surface-expressed virulence factors of Neisseria meningitidis (pili, Opc, capsule and lipopolysaccharide (LPS)) in host cell adhesion and invasion was examined using derivatives of a serogroup B strain, MC58, created by mutation (capsule, Opc) and selection of variants. To examine the role of Opc and of additional expression of pili, bacteria lacking the expression of Opa proteins were used. The effects of different LPS structures were examined in variants expressing either sialylated (L3 immunotype) or truncated non-sialylated (L8 immuno-type) LPS. Studies showed that (i) pili were essential for meningococcal interactions with host cells in both capsulate and acapsulate bacteria with the sialylated L3 LPS immunotype, (ii) the Opc-mediated invasion of host cells by piliated and non-piliated bacteria was observed only in acapsulate organisms with L8 LPS immunotype, and (iii) expression of pili in Opc-expressing bacteria resulted in increased invasion. Investigations on the mechanisms of cellular invasion indicated that the Opc-mediated invasion was dependent on the presence of serum in the incubation medium and was mediated by serum proteins with arginine-glycine-aspartic acid (RGD) sequence. Cellular invasion in piliated Opc⁺ phenotype also required bridging molecules containing the RGD recognition sequence and appeared to involve the integrin αvβ3 as a target receptor on endothelial cells. These studies extend the previous observations on variants of a serogroup A strain (C751) and show that Opc mediates cellular invasion in distinct meningococcal strains and provide confirmation of its mechanism of action. This is the first investigation that evaluates, using derivatives of a single strain, the interplay between four meningococcal surface virulence factors in host cell invasion.     

Identification of two molecular-mass forms of phenylalanine hydroxylase that segregate independently in rats. Specific association of each form with certain rat strains

The nature of the different molecular-mass forms of phenylalanine hydroxylase in rat livers was examined by immunoprecipitation of the enzyme from crude liver extracts that had been radiolabelled by reductive methylation. The two forms of the enzyme were resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and detected by fluorography. Segregation of the two forms of the enzyme was demonstrated in Sprague-Dawley rats, as would be expected if the two forms were products of allelic genes. In addition, hooded and albino Wistar rat livers contained only the slower-migrating form and Lewis rat livers contained only the faster-migrating form, and hence we suggest that the forms be referred to as W (for Wistar) and L (for Lewis). Peptide mapping showed that the W and L forms are closely related, and the difference between them appears to reside at one or other end of the polypeptide chain. The kidney contained the same forms as the liver in one-tenth the quantity, providing further evidence that the same phenylalanine hydroxylase gene is expressed in liver and kidney.     

Role of exercise testing early after myocardial infarction in identifying candidates for coronary surgery.

It has been suggested that ST depression in lead V5 or equivalent on early exercise testing after acute myocardial infarction predicts a high risk of death. To evaluate exercise testing and radionuclide ventriculography in this context 103 consecutive patients with myocardial infarction who were able to undertake a limited exercise test before discharge from hospital were exercised and underwent gated blood pool scanning. No serious complications resulted from exercise testing. Twenty nine patients developed ST depression in lead V5, 19 had exertional hypotension, 31 developed a heart rate of greater than or equal to 130 beats/min, and 15 had complex ventricular arrhythmias. Death during the first year after discharge from hospital was associated with exertional hypotension (p less than 0.001) and a heart rate on exercise testing of greater than or equal to 130 beats/min (p less than 0.05); these two variables identified all nine deaths. Inability to complete the exercise protocol for any reason was also predictive of death (p less than 0.01). Ventricular arrhythmias and ST depression in lead V5 induced by exercise were not significantly associated with an increased risk of death. The mean (SD) radionuclide ejection fraction in the patients who died was 29 (16%) compared with 43 (11)% in the patients who survived (p less than 0.001). ST changes on exercise testing after myocardial infarction appear to be less predictive of later complications than haemodynamic signs, which may indicate left ventricular damage rather than ischaemia.     

Purification of glycoproteins IIb and III from human platelet plasma membranes and characterization of a calcium-dependent glycoprotein IIb-III complex

Human platelet membrane glycoproteins IIb and III are two major integral membrane components that have been identified as sites mediating thrombin-induced aggregation. For purposes of our study, glycoproteins IIb and III were solubilized by extracting platelet plasma membranes with a buffer containing 0.1% Triton X-100 and were separated by gel filtration chromatography on Sephacryl S-300, employing Triton X-100-containing column buffers with or without urea or guanidine hydrochloride. The physical properties of the purified glycoproteins were: for glycoprotein IIb, Rs = 61 A, s20.w = 4.7, f/f0 = 1.7, Mr = 125,000 (hydrodynamic values), Mr = 136,000 (sodium dodecyl sulfate gels); for glycoprotein III, Rs = 67 A, s20,w = 3.2 f/f0 = 2.1, Mr = 93,000 (hydrodynamic values), Mr = 95,000 (sodium dodecyl sulfate gels). Although the amino acid compositions of the two glycoproteins were similar, antibodies raised against glycoprotein IIb did not crossreact with glycoprotein III. If divalent cations were not chelated in the Triton extract, glycoproteins IIb and III coeluted during gel filtration chromatography (apparent Stokes radius of 71 A) and co-sedimented on sucrose gradients (apparent s20.w of 8.6), from which Mr = 265,000 was calculated. Glycoproteins IIb and III were coprecipitated by an antibody monospecific for glycoprotein IIb. The two glycoproteins dissociated into monomers when EDTA was added to Triton lysates. Readdition of Ca2+ caused them to reassociate into a complex with physical properties similar to those of the complex in the original Triton lysate. The data show that glycoproteins IIb and III are a heterodimer complex, that complex formation depends upon the presence of Ca2+, and that chelation of Ca2+ causes dissociation into monomeric glycoproteins.     

Polarized fluorescence studies of electrically oriented DNA-dye solutions

Transient changes have been recorded in each of the four polarized components of fluorescence, when dilute solutions of dye-tagged DNA are subjected to short electric pulses. The directions of the absorption and emission transition moments, and hence of the plane of the dye molecules, relative to the DNA geometry have been estimated for eleven dyes. Data obtained for ethidium bromide and five acridine derivatives are consistent with the intercalation model generally accepted for these dyes. In addition, it is shown that neutral red, acridine red and probably auramine O also bind with their molecular planes essentially perpendicular to the long helical axis. The remaining two, hydroxystilbamidine and the bibenzimidazole derivative Hoechst 33258, give rise to effects which indicate that these molecules bind in such a manner that the absorption and emission transitions are closely associated with the grooves of the DNA helix.