Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I-69 Rd−/b+ of Haemophilus influenzae

Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I-69 Rd⁻/b⁺ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de- O -acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid-phase antigens and by haemagglutination with sheep erythrocytes coated with de- O -acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3-deoxy- d - manno -octulopyranosonic acid- (Kdo) 5-phosphate, one for Kdo-4-phosphate, and one required, in addition to a Kdo-phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole-cell lysates or isolated LPS separated by SDS–PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo-4- and Kdo-5-phosphate are synthesized by the bacteria and are not the result of phosphate migration.     

Regulation of O-antigen chain length is required for Shigella flexneri virulence

It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O-antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O-antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)-resistance cassette into the rol gene (controlling the modal O-antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP-rhamnose (the precursor of rhamnose in the O-antigen). The mutations had the expected effect on LPS structure. The rol ::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol ::Km and rfbD ::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria-infected HeLa cells stained with fluorescein isothiocyanate (FITC)-phalloidin demonstrated that both the rol ::Km and rfbD ::Km mutants were defective in F-actin tail formation: the latter mutant showed distorted F-actin tails. Plasma-membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F-actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell-surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O-antigen chains of the rol ::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O-antigen chains, and comparing untreated and Sf6c-treated cells by immunofluorescence with anti-IcsA serum.     

Evidence for two chemosensory pathways in Rhodobacter sphaeroides

In contrast to enteric bacteria, chemotaxis in Rhodobacter sphaeroides requires transport and partial metabolism of chemoattractants. Although a chemotaxis operon has been identified containing homologues of the enteric cheA, cheW, cheR genes and two homologues of the cheY gene, deletion of the entire chemotaxis operon had only minor effects on chemotactic behaviour under the conditions tested. Responses to sugars were enhanced in tethered cells but in all other chemotaxis assays behaviour of the operon deletion mutant was wild type. The mutant also showed wild-type responses to weak organic acids such as acetate and propionate, the dominant chemoattractants for this organism, under all conditions. This is in direct contrast to the enterics in which CheA, CheW and CheY are absolutely essential for taxis to PTS sugars, oxygen and MCP-dependent chemoeffectors. The operon deletion mutant was subjected to Tn5 transposon mutagenesis and new mutants selected using a chemotaxis and phototaxis screen. One mutant, JPA203, was non-chemotactic on swarm plates and showed inverted responses when tethered or subjected to changes in light intensity. Characterization of the Tn5 insertion in JPA203 identified a second chemotaxis operon in R. sphaeroides that contains homologues of cheY, cheA and cheR, the first homologue of cheB and two homologues of cheW. The new genes were labelled orf10, cheY_III, cheA_II, cheW_II, cheW_III, cheR_II, cheB and tlpC. When introduced into a wild-type background, deletion of cheA_II produced a chemotaxis minus phenotype in R. sphaeroides, suggesting that cheA_II forms part of a dominant chemotactic pathway, whereas the earlier identified operon plays only a minor role under laboratory conditions. The data presented here support the existence of two chemosensory pathways in R. sphaeroides, a feature that so far is unique in bacterial chemotaxis.     

Mutational analysis of the RecA protein L1 region identifies this area as a probable part of the co-protease substrate binding site

Previous mutational analysis of the L1 region of the RecA protein suggested that Gly-157 and Glu-158 are 'hot-spots' for the occurrence of constitutive LexA co-protease mutants (coprt^c). In the present study, we clearly establish that position 157 is a hot-spot for the occurrence of such mutants, as 12 of 14 and 10 of 14 substitutions result in this phenotype for UmuD and LexA cleavage respectively. The frequency of such mutations at position 158 is somewhat lower, 8 of 13 and 5 of 13 for UmuD and LexA respectively. Comparison of the UmuD vs. LexA co-protease activity for all single mutants with substitutions at positions 154, 155, 156, 157 and 158 (47 in total) reveals that, although there is good agreement among most mutants regarding their ability to cleave both LexA and UmuD, there are two in particular (Glu-154→Asp and Glu-154→Gln) that show a clear preference for cleavage of UmuD. We also show that three second-site mutations that completely suppress coprt^c activity toward LexA have little or no effect on the coprt^c activity of the primary mutant toward UmuD. In addition, we observe a high frequency of second-site suppressor mutations, suggesting a functional interaction among side-chains in this region. Together, these results support the idea that the L1 region of RecA makes up part of the co-protease substrate-binding site.     

Immune response to a new hepatitis B vaccine in healthcare workers who had not responded to standard vaccine: randomised double blind dose-response study.

OBJECTIVE: To evaluate the immunogenicity and reactogenicity of a new triple S recombinant hepatitis B vaccine in a cohort of healthy people in whom currently licensed hepatitis B vaccines had persistently not induced an immune response. DESIGN: Single centre, randomised, double blind, dose-response study. SETTING: Research vaccine evaluation centre at a teaching hospital. SUBJECTS: 100 healthcare workers aged 18-70 years with a history of failure to seroconvert after at least four doses of a licensed hepatitis B vaccine containing the S component. INTERVENTION: Each subject was randomly allocated two doses of 5, 10, 20, or 40 micrograms of a new hepatitis B vaccine two months apart. MAIN OUTCOME MEASURES: Immunogenicity of the four doses. Seroconversion and seroprotection were defined as an antibody tire > 10 IU/l and > 100 IU/l respectively against an international antibody standard. RESULTS: 69 subjects seroconverted after a single dose of the vaccine. After the booster vaccination one other subject seroconverted, bringing the overall seroconversion rate to 70%. Fifteen subjects given 5 micrograms of vaccine, 19 given 10 micrograms, 16 given 20 micrograms, and 20 given 40 micrograms seroconverted. Seroconversion rates in the four antigen dose groups were 60% (15/25), 76% (19/25), 64% (16/25), and 80% (20/25). After the booster dose there was no significant dose-response effect on the overall seroconversion rate, although the small sample size meant that a clinically important dose-response could not be ruled out. CONCLUSION: A single dose of 20 micrograms of the vaccine was as effective as two doses of either 40 micrograms or 20 micrograms of this vaccine formulation in terms of seroconversion, seroprotection, and geometric mean titres.     

Origin of deoxycorticosterone sulfate (DOC-SO4) in plasma of pregnant women: Pregnenolone-3,21-disulfate is a placental precursor of DOC-SO4

The plasma levels of deoxycorticosterone sulfate (DOC-SO₄) in near-term pregnant women are 100 times those in plasma of men or non-pregnant women. Yet, neither the tissue site of synthesis nor the precursor of DOC-SO₄ that enters maternal plasma is known. Several potential sources have been excluded: plasma DOC-SO₄ is not derived from plasma DOC; and the secretion of C₂₁-steroids (other than aldosterone) from the maternal adrenals during human pregnancy is not increased. Similarly, the transfer of DOC-SO₄ from fetal plasma cannot account for the high level of DOC-SO₄ in the maternal compartment, and a reduced clearance of plasma DOC-SO₄ during pregnancy cannot account for the high levels of DOC-SO₄. Indeed, the rate of clearance of DOC-SO₄ from plasma is 10–100 times that of most other steroid sulfates. To address this question further, we evaluated the possibility that fetal plasma pregnenolone-3,21-disulfate serves as a precursor for DOC-SO₄ formation in the placenta. The preferential hydrolysis of the 3β-sulfate of pregnenolone-3,21-disulfate in placenta would give rise to pregnenolone-21-monosulfate, which, if acted upon by placental 3β-hydroxysteroid dehydrogenase/Δ^{5 → 4} isomerase, could give DOC-SO₄. [³H]Pregnenolone-3,21-disulfate was incubated with minces of human placental tissue for 5, 20, 60 and 120 min. Radiolabelled DOC-SO₄, DOC, and pregnenolone-21-monosulfate were isolated from the incubation media and quantified. After a 5 min incubation, 7.5% of substrate was converted to DOC-SO₄; and after 20, 60 and 120 min 30% of the [³H]pregnenolone-3,21-disulfate was recovered from the media of these incubations as [³H]DOC-SO₄. [³H]DOC was also present in the incubation media and the concentrations of this product increased as a function of incubation time. Therefore, pregnenolone-3,21-disulfate, which is present in very high concentrations in fetal plasma (1000 ng/ml), is metabolized in the placenta to DOC-SO₄. Because of the fetal and maternal vascular arrangements of the hemochorioendothelial placenta of human pregnancy, steroids produced in syncytiotrophoblasts preferentially enter the intervillous space; thus, fetal plasma pregnenolone-3,21-disulfate may serve as a placental precursor of maternal plasma DOC-SO₄.     

Image analysis, neural networks, and the taxonomic impediment to biodiversity studies

The taxonomic impediment to biodiversity studies may be influenced radically by the application of new technology, in particular, desktop image analysers and neural networks. The former offer an opportunity to automate objective feature measurement processes, and the latter provide powerful pattern recognition and data analysis tools which are able to 'learn' patterns in multivariate data. The coupling of these technologies may provide a realistic opportunity for the automation of routine species identifications. The potential benefits and limitations of these technologies, along with the development of automated identification systems are reviewed.     

Identification of a 175 kDa protein as the ligand-binding subunit of the rat liver sinusoidal endothelial cell hyaluronan receptor

The rat liver sinusoidal endothelial cell (LEC) hyaluronan (HA)receptor was previously identified using a photoaffinity HAderivative (J. BioL Chem., 267, 20451–20456, 1992). Twopolypeptides with M_r = 175,000 and 166,000, were consistentlycrosslinked, suggesting that the LEC HA receptor is an oligomer.Whether one or both subunits participate in HA binding, wasnot determined. Here we investigate the HA-subunit interactionsand the potential oligomeric nature of the LEC HA receptor.When Sephacryl-400 gel filtration chromatography was used toenrich the HA receptor, the 175 kDa polypeptide was the majorband seen by SDS-PAGE analysis. Little staining was seen at166 kDa, suggesting that the 175 kDa protein could be separatedfrom the 166 kDa protein and still retain HA-binding activity.A ligand blot assay was used to determine if each individualsubunit could bind HA. LEC proteins were separated by nonreducingSDS-PAGE, and then immobilized onto nitrocellulose. ¹²⁵I-HAbound to a 175 kDa polypeptide but not to the 166 kDa protein.A high molecular weight band of