The (alpha2-->8)-linked polysialic acid capsule and lipooligosaccharide structure both contribute to the ability of serogroup B Neisseria meningitidis to resist the bactericidal activity of normal human serum

The molecular basis for the resistance of serogroup B Neisseria meningitidis to the bactericidal activity of normal human sera (NHS) was examined with a NHS-resistant, invasive serogroup B meningococcal isolate and genetically and structurally defined capsule-, lipooligosaccharide (LOS)-, and sialylation-altered mutants of the wild-type strain. Expression of the (alpha2-->8)-linked polysialic acid serogroup B capsule was essential for meningococcal resistance to NHS. The very NHS-sensitive phenotype of acapsular mutants (99.9 to 100% killed in 10, 25, and 50% NHS) was not rescued by complete LOS sialylation or changes in LOS structure. However, expression of the capsule was necessary but not sufficient for a fully NHS-resistant phenotype. In an encapsulated background, loss of LOS sialylation by interrupting the alpha2,3 sialyltransferase gene, lst, increased sensitivity to 50% NHS. In contrast, replacement of the lacto-N-neotetraose alpha-chain (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) with glucose extensions (GlcN) in a galE mutant resulted in a strain resistant to killing by 50% NHS at all time points. Encapsulated meningococci expressing a Hep2(GlcNAc)-->KDO2-->lipid A LOS without an alpha-chain demonstrated enhanced sensitivity to 50% NHS (98% killed at 30 min) mediated through the antibody-dependent classical complement pathway. Encapsulated LOS mutants expressing truncated Hep2-->KDO2-->lipid A and KDO2-->lipid A structures were also sensitive to 50% NHS (98 to 100% killed at 30 min) but, unlike the wild-type strain and mutants with larger oligosaccharide structures, they were killed by hypogammaglobulinemic sera. These data indicate that encapsulation is essential but that the LOS structure contributes to the ability of serogroup B N. meningitidis to resist the bactericidal activity of NHS.     

Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the alpha1,2 N-acetylglucosamine transferase (RfaK)

A lipooligosaccharide (LOS) mutant of Neisseria meningitidis serogroup B strain NMB (immunotype L3,7,9) was identified in a Tn916 (tetM) mutant bank by loss of reactivity with monoclonal antibody 3F11, which recognizes the terminal Galbeta1-->4GlcNAc epitope in the lacto-N-neotetraose moiety of the wild-type LOS structure. The mutant, designated 559, was found to express a truncated LOS of 3.0 kDa. Southern and PCR analyses demonstrated that there was a single intact Tn916 insertion (class I) in the mutant 559 chromosome. Linkage of the LOS phenotype and the Tn916 insertion was confirmed by transformation of the wild-type parent. Nucleotide sequence analysis of the region surrounding the transposition site revealed a 1,065-bp open reading frame (ORF). A homology search of the GenBank/EMBL database revealed that the amino acid sequence of this ORF had 46.8% similarity and 21.2% identity with the alpha1,2 N-acetylglucosamine transferase (RfaK) from Salmonella typhimurium. Glycosyl composition and linkage analysis of the LOS produced by mutant 559 revealed that the lacto-N-neotetraose group which is attached to heptose I (HepI) and the N-acetylglucosamine and glucose residues that are attached to HepII in the inner core of the parental LOS were absent. These analyses also showed that the HepII residue in both the parent and the mutant LOS molecules was phosphorylated, presumably by a phosphoethanolamine substituent. The insertion of nonpolar and polar antibiotic resistance cartridges into the parental rfaK gene resulted in the expression of LOS with the same mobility as that produced by mutant 559. This result indicated that the inability to add the lacto-N-neotetraose group to the 559 LOS is not due to a polar effect on a gene(s) downstream of rfaK. Our data indicate that we have identified the meningococcal alpha1,2 N-acetylglucosamine transferase responsible for the addition of N-acetylglucosamine to HepII. We propose that the lack of alpha-chain extension from HepI in the LOS of mutant 559 may be due to structural constraints imposed by the incomplete biosynthesis of the LOS inner core.     

Identification of an alpha3-fucosyltransferase and a novel alpha2-fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-->3[Gal(NAc)beta1-->4]GlcNAc sequence

Knowledge of the genealogical relationships of cells during development can allow one to gain insight into when and where developmental decisions are being made. Genealogical relationships can be revealed by a variety of methods, all of which involve marking a progenitor cell and/or a group of cells and then following the progeny. The use of replication-incompetent retroviral vectors for the analysis of lineal relationships in developing vertebrate tissues is described. An overview of the relevant aspects of the retroviral life cycle is given, and the strategies and current methods in use in our laboratory are described.     

Cloning and characterization of the ponA gene encoding penicillin-binding protein 1 from Neisseria gonorrhoeae and Neisseria meningitidis

The ponA gene encoding penicillin-binding protein 1 (PBP 1) from Neisseria gonorrhoeae was cloned by a reverse genetic approach. PBP 1 was purified from solubilized membranes of penicillin-susceptible strain FA19 by covalent ampicillin affinity chromatography and used to obtain an NH2-terminal amino acid sequence. A degenerate oligonucleotide based on this protein sequence and a highly degenerate oligonucleotide based on a conserved amino acid motif found in all class A high-molecular-mass PBPs were used to isolate the PBP 1 gene (ponA). The ponA gene encodes a protein containing all of the conserved sequence motifs found in class A PBPs, and expression of the gene in Escherichia coli resulted in the appearance of a new PBP that comigrated with PBP 1 purified from N. gonorrhoeae. A comparison of the gonococcal ponA gene to its homolog isolated from Neisseria meningitidis revealed a high degree of identity between the two gene products, with the greatest variability found at the carboxy terminus of the two deduced PBP 1 protein sequences.     

Conformational properties of a cycle oligosaccharide: cyclotrikis-(1-->6)-[alphaD-glucopyranosyl-(1-->4)-beta-D-glucopyranosy l]

Within a larger environmental health screening program neurobehavioral measures were taken in 384 6-year-old children (mean age 74 months) in the cities of Leipzig, Gardelegen, and Duisburg. Lead concentrations in venous blood samples (PbB) and urinary mercury excretion in 24-h samples (HgU) were measured as markers of environmental exposure by electrothermal AAS. Dependent variables included two subtests from the WISC [vocabulary (V) and block design (BD)] as well as five tests from the NES2 [pattern comparison, pattern memory, tapping, simple reaction time, and the continuous performance test (CPT; child version)]. In addition, visual functions [visual acuity (TITMUS-test) and contrast sensitivity (FACT)] were tested as covariates. The overall average PbB (geometric mean) was 42.5 microg/l (upper 95% value = 89 microg/l). The overall average mercury excretion (HgU) was 0.16 microg/24 h. Whereas no significant or borderline associations between HgU and any of the target variables was found, significant negative associations were observed between PbB and verbal intelligence (WISC vocabulary but not WISC Block Design) and false-positive responses (false alarms), as well as false-negative responses (miss) in the CPT. Whereas parental education was the most important confounder for WISC performance, visual contrast sensitivity and computer familiarity also proved predictive for performance in several computer-based NES subtests. It is concluded that non-IQ measures, namely measures of sustained attention, are negatively affected in children with 95% of blood-lead levels below 90 microg/l, even after adjustment for intelligence and contrast sensitivity, whereas the causative role of lead in altering IQ functions remains somewhat equivocal, because important covariates could not be controlled for.     

beta Gal(1-->S-4)beta GlcNac--OR: a galactosidase-stable substrate for alpha(1-->3)fucosyltransferase

1'-4 Thio-N-acetyllactosamine was chemically synthesized as a galactosidase-stable substrate for alpha(1-->3)fucosyltransferase. The product of enzymatic fucose addition was confirmed to be the thio-Le X analog.     

Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3-glucan, and chitin

In a previous study (Kollár, R., Petráková, E., Ashwell, G., Robbins, P. W., and Cabib, E. (1995) J. Biol. Chem. 270, 1170-1178), the linkage region between chitin and beta(1-->3)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with beta(1-->3)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, beta(1-->3)-glucan, beta(1-->6)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to beta(1-->6)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five alpha-linked mannosyl residues. The beta(1-->6)-glucan has some beta(1-->3)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a beta(1-->4) or beta(1-->2) linkage. Finally, the reducing end of beta(1-->6)-glucan is connected to the nonreducing terminal glucose of beta(1-->3)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and beta(1-->6)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.     

Synthesis of 6(alpha, beta)-aminocholestanols as ergosterol biosynthesis inhibitors

delta 7-5-Desaturase catalyses one of the last steps in ergosterol biosynthesis in fungi. Moreover delta 5-unsaturation is necessary for the sparking function. Synthesis of three pairs of C-6 epimeric cholestanol derivatives are described as potential growth inhibitors. Preliminary results suggest that 6 beta-aminocholestanol is a potent antifungal agent.     

Characterization of a cooperativity domain mutant Lys3 --> Ala (K3A) T4 gene 32 protein

The N-terminal "B" domain of T4 gene 32 protein contains a Lys3-Arg4-Lys5 sequence that has been postulated to provide a major determinant for cooperative binding. In this report, the equilibrium binding properties of a Lys3 --> Ala substitution mutant of gp32 (K3A gp32) and described and compared to a set of substitution mutants of Arg4 previously described (Villemain, J. L., and Giedroc, D. P. (1993) Biochemistry 32, 11235-11246) and further characterized here. K3A gp32 exhibits binding behavior which mirrors that of R4Q gp32. Despite an 6-8-fold decrease in overall binding affinity (Kapp = Kint x omega) at pH 8.1, 0.20 M NaCl, 20 degrees C, the magnitude of the cooperativity parameter is at most 2-3-fold smaller than that of the wild-type protein. The magnitude of omega is independent of salt concentration and type over the range in [NaCl] from 0.125 to 0. 225 M and [NaF] between 0.20 and 0.32 M (log omega = 2.86 +/- 0.19). For comparison, log omega for wild-type gp32 is 2.91 (+/- 0.21) resolved at 0.275 M NaCl and 3.39 +/- 0.11 in [NaF] between 0.40 and 0.45 M. In contrast to omega, the [NaCl] dependence of Kapp is large and markedly nonlinear for both wild-type and K3A gp32s over a [NaCl] range extending from 0.05 M to 0.40 M NaCl. Modeling of the complete salt dependence of Kapp for wild-type, K3A, and R4T gp32s in NaCl and NaF with a simple ion-exchange model suggests that substitutions within the basic Lys3-Arg4-Lys5 sequence do not strongly modulate the net displacement of cations and anions upon poly(A) complex formation by gp32.     

Sp1 is required for the early response of alpha2(I) collagen to transforming growth factor-beta1

It is currently debated whether AP1 or Sp1 is the factor that mediates transforming growth factor beta1 (TGF-beta) stimulation of the human alpha2(I) collagen (COL1A2) gene by binding to an upstream promoter element (TbRE). The present study was designed to resolve this controversy by correlating expression of COL1A2, AP1, and Sp1 in the same cell line and under different experimental conditions. The results strongly indicate that Sp1 is required for the immediate early response of COL1A2 to TGF-beta and AP1 is not. The Sp1 inhibitor mithramycin blocked stimulation of alpha2(I) collagen mRNA accumulation by TGF-beta, whereas the AP1 inhibitor curcumin had no effect. Furthermore, antibodies against Jun-B and c-Jun failed to identify immunologically related proteins in the TbRE-bound complex, irrespective of whether they were purified from untreated or TGF-beta-treated cells. AP1 did bind to the TbRE probe in vitro, but only in the absence of the upstream Sp1 recognition sequence. Based on this finding and DNA transfection results, we conclude that the AP1 sequence of the TbRE represents a cryptic site used under experimental conditions that either eliminate the more favorable Sp1 binding site or force the balance toward the less probable. Finally, a combination of cell transfections and DNA-binding assays excluded that COL1A2 transactivation involves the retinoblastoma gene product (pRb), an activator of Sp1, the pRb-related protein p107, an inhibitor of Sp1, or the Sp1-related repressor, Sp3.     

The GABAA receptor alpha 6 subunit gene (Gabra6) is tightly linked to the alpha 1-gamma 2 subunit cluster on mouse chromosome 11

Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by hyperplasia and neoplasia in several endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a 900-kb region on chromosome 11q13. The human phosphatidylinositol-specific phospholipase C beta 3 (PLC beta 3) gene, which is located within this region, was considered to be a good candidate for the MEN 1 gene. In this study, the structure and expression of the PLC beta 3 gene in MEN 1 patients were investigated in more detail, to determine its potential role in MEN 1 tumorigenesis. Southern blot analysis, using blood and tumor DNA from affected persons from seven different MEN 1 families, did not reveal structural abnormalities in the PLC beta 3 gene. To detect possible point mutations, or other small structural aberrations, direct sequencing of PLC beta 3 cDNAs from two affected persons from two different MEN 1 families was performed, but no MEN 1-specific abnormalities were revealed. Several common nucleotide sequence polymorphisms were detected in these cDNAs, proving that both alleles of the PLC beta 3 gene were expressed and analyzed. In conclusion, these results exclude the PLC beta 3 gene as a candidate gene for MEN 1.     

Detection of two loci involved in (1-->3)-beta-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene

A 28-item questionnaire was returned by 291 psychiatrists who had completed training between 1962 and 1992. There were positive correlations between the amount of couple and family therapy training (CFTT) they received and the following: the extent to which graduate psychiatrists practice CFT; their involvement as supervisors, teachers, teaching program directors, or researchers; the extent to which they seek continuing education in CFT; their positive attitude toward CFT; and the extent to which they feel that their attitude to and interest in CFT has had a positive effect on the milieu in which they practice and on their personal lives.     

A low renal threshold for glucose in diabetic patients with a mutation in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene

Cartilage-hair hypoplasia (CHH), an autosomal recessive chondrodysplasia, results in severe growth failure, sparse hair and impaired cellular immunity. Lymphocyte subpopulations and proliferative responsiveness in mitogen stimulation were analysed in 35 patients of whom 31% had an increased incidence of infections the year prior to the evaluation. Of the patients, 57% had a decreased CD4+ cell count which led to a decreased total count of T-lymphocytes in 52% and a subnormal CD4 +/CD8 + cell ratio in 32%. The B-lymphocyte count was usually normal. The natural killer cell count was above reference values in 40% of the patients. The lymphocyte stimulation indices as studied with phytohaemagglutinin , Concanavalin A and pokeweed mitogen were subnormal in 69%, 69% and 83% of the patients, respectively. The numbers of lymphocytes, T-lymphocytes and CD4+ cells, and the pokeweed mitogen stimulation index. but not the other measured parameters, correlated significantly with the proness to infections during the preceding year. However, the correlation was reverse with higher counts in the patients who had had recurring infections. The observed significant correlations may reflect immunological stimuli caused by recurring infections. CONCLUSION: The presently used parameters of cellular immunity poorly predict the clinical outcome of an individual cartilage-hair hypoplasia patient. All patients, irrespective of their in vitro immunological competence, have to be carefully followed because of possibility of serious infections and malignancies.     

A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis

Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-D-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-D-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-D-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E. coli and found to catalyze the formation of 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+ as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-D-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E. coli.     

Crystal structure of phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with glucosaminyl(alpha 1-->6)-D-myo-inositol, an essential fragment of GPI anchors

Numerous proteins on the external surface of the plasma membrane are anchored by glycosylated derivatives of phosphatidylinositol (GPI), rather than by hydrophobic amino acids embedded in the phospholipid bilayer. These GPI anchors are cleaved by phosphatidylinositol-specific phospholipases C (PI-PLCs) to release a water-soluble protein with an exposed glycosylinositol moiety and diacylglycerol, which remains in the membrane. We have previously determined the crystal structure of Bacillus cereus PI-PLC, the enzyme which is widely used to release GPI-anchored proteins from membranes, as free enzyme and also in complex with myo-inositol [Heinz, D.W., Ryan, M. Bullock, T.L., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863]. Here we report the refined 2.2 A crystal structure of this enzyme complexed with a segment of the core of all GPI anchors, glucosaminyl(alpha 1-->6)-D-myo-inositol [GlcN-(alpha 1-->6)Ins ]. The myo-inositol moiety of GlcN(alpha 1-->6)Ins is well-defined and occupies essentially the same position in the active site as does free myo-inositol, which provides convincing evidence that the enzyme utilizes the same catalytic mechanism for cleavage of PI and GPI anchors. The myo-inositol moiety makes several specific hydrogen bonding interactions with active site residues. In contrast, the glucosamine moiety lies exposed to solvent at the entrance of the active site with minimal specific protein contacts. The glucosamine moiety is also less well-defined, suggesting enhanced conformational flexibility. On the basis of the positioning of GlcN(alpha 1-->6)Ins in the active site, it is predicted that the remainder of the GPI-glycan makes little or no specific interactions with B. cereus PI-PLC. This explains why B. cereus PI-PLC can cleave GPI anchors having variable glycan structures.     

Usefulness of (1-->3)-beta-D-glucan measurement for diagnosis of deep mycosis

The number of deep mycosis has been increasing because of increases in immunocompromised hosts and in fungal colonization associated with increasing use of broad-spectrum antibacterial antibiotics. Based on these phenomenon, a simple test method for an early diagnosis of deep mycosis is urgently desired. We therefore investigated the usefulness of assaying a fungal cell component, (1-->3)-beta-D-glucan (beta-glucan). The amount of beta-glucan was obtained from the difference between the amounts determined using Toxicolor and Endospecy, and the serum levels of more than 10 pg/ml were considered positive signs for beta-glucan. The following results were obtained: We found that beta-glucan was positive in 75% of the patients who had been definitely diagnosed to have mycosis, and in 58.3% of the patients strongly suspected of mycosis. The numbers of beta-glucan positive patients' in these 2 groups of patients were significantly different from that in those without mycosis (14.7%, P < 0.05). Thus a usefulness of beta-glucan measurement for the diagnosis of mycosis was demonstrated. However, beta-glucan was sometimes negative even in patients with fungemia at an early phase of the disease and turned positive several days later. Even in a patient with definite lung mycosis, who had a latent circumscribed lesion (afebrile and CRP-negative), beta-glucan was also negative. From these findings, one should be aware that the beta-glucan test produces false negatives even in patients with definite mycosis and that the test should be repeated during the course of the disease.