Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen

We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus. Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----6 )]- beta-D-GlcpNAc-(1----3)-beta-D-Gal. The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit. High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58, 112-120). This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide. Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic. Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.     

Signal peptidase can cleave inside a polytopic membrane protein

The signal peptides of most proteins targeted to the endoplasmic reticulum are specifically cleaved by signal peptidase. Although potential cleavage sites occur frequently in polytopic proteins after membrane-spanning segments, processing is restricted to the first hydrophobic domain, suggesting that signal peptidase might not have access to subsequently translocated, internal domains. To test this hypothesis, we replaced the third transmembrane segment of an artificial threefold membrane-spanning protein by a sequence which is normally an amino-terminal signal. Upon in vitro translation and insertion into microsomes, efficient cleavage at this sequence was observed, thus demonstrating the ability of signal peptidase to cleave within polytopic membrane proteins.     

Rapid detection of chromosome 16 inversion in acute nonlymphocytic leukemia, subtype M4: regional localization of the breakpoint in 16p

The pericentric inversion of chromosome 16 characteristic for acute nonlymphocytic leukemia, subtype M4, was detected in five patients by means of nonradioactive in situ hybridization of complete cosmids. First, five cosmids situated along the short arm of chromosome 16 were used to map the breakpoint of the inversion distal to the rare folate-sensitive fragile site FRA16A. Then, the use of two cosmids on either side of the breakpoint, combined with a probe specific for the centromeric region of chromosome 16, readily detected the inversion, even in poor metaphase spreads.     

Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence

To study the insertion of multispanning membrane proteins into the endoplasmic reticulum, we constructed novel proteins on the cDNA level by repeating, up to four times, the internal signal-anchor domain of the asialoglycoprotein receptor H1. Upon in vitro translation in the presence of microsomes, these polypeptides are indeed inserted as polytopic membrane proteins. The first hydrophobic domain functions as a signal and the second as a stop-transfer sequence, while the third initiates a second translocation process, halted again by the fourth. We were able to demonstrate that insertion occurs sequentially, starting with the first apolar segment from the amino terminus. By replacing the original signal-anchor domains by a mutant sequence not recognized by signal recognition particle (SRP), it was shown that only the first hydrophobic domain needs to be a signal sequence and that the second translocation event does not require SRP.     

Immunochemical analysis of the types Ia and Ib group B streptococcal polysaccharides

The types Ia and Ib group B streptococcal type-specific polysaccharides have remarkable immunologic differences despite a great deal of structural similarity. Although these two complex polysaccharides differ only by a single glycosidic linkage, they are antigenically distinct. Furthermore, terminal sialic acid residues appear to be critical to the immunodeterminant on the type Ia polysaccharide, whereas the antigenicity of the type Ib polysaccharide does not show this dependence on sialic acid. In the current investigation we defined better the immunodeterminant of these polysaccharides. With homologous rabbit antiserum, the type Ia native and core polysaccharides demonstrated partial serologic identity, whereas the type Ib native and core polysaccharides demonstrated complete serologic identity. Surprisingly, the type I degalactosylated polysaccharide, degraded structure, was capable of reacting with a population of antibodies present in type Ia antiserum similar to the complete type Ia native polysaccharide, although demonstrating a reduced level of immunodeterminant expression. Unlike the reactions of the type Ia polysaccharides with homologous rabbit antiserum, the Ib native and core polysaccharides were able to react with identical populations of antibodies in type Ib-specific antiserum. A minor population of antibodies was demonstrated in the type Ib antiserum, which was reactive with the degalactosylated polysaccharide. That a population of antibodies reactive toward the degalactosylated polysaccharide is present in both type Ia and type Ib antisera suggests that the Iabc cross-reacting determinant is due to the presence of serum antibodies reactive with this trisaccharide repeating unit, which is shared by both the type Ia and the type Ib native and core polysaccharides.     

Flow cytometric evaluation of cell dispersion from human head and neck tumors

The preparation of single-cell suspensions from 25 human head and neck tumors is described. Dispersal was performed overnight at 4 degrees C under slight agitation of the tissue suspensions using various combinations of enzymes and additives. The cell suspensions were examined for number of cells released, viability, amount of debris, and DNA distribution by means of flow cytometry (FCM). It was shown that both trypsin/dithioerythritol (TD) and collagenase/D Nase (CDse) were of value in dispersing single cells from tumor tissue. In contrast to CDse, incubation with TD appeared to be cytolytic to normal lymphocytes. In a number of cases, DNA-FCM revealed ploidy abnormalities in a TD-suspension, which were not discernible in the concurrent CDse-suspension. Cell culture of primary cell suspensions corroborated the reliability of the DNA-FCM measurements. Pretreatment with CDse improved tumor disaggregation by TD and indicated a different dispersal capacity. Addition of Ca2+ and Mg2+ ions to the dispersal mixtures and preincubation of tumor slices in complete medium for 1 day before initiation of cell dispersion influenced favorably the quality of the cell suspension.     

Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin resistance determinant

The replication region of a 28-kilobase-pair (kbp) cryptic plasmid from Lactococcus lactis subsp. lactis biovar diacetylactis SSD207 was cloned in L. lactis subsp. lactis MG1614 by using the chloramphenicol resistance gene from the streptococcal plasmid pGB301 as a selectable marker. The resulting 8.1-kbp plasmid, designated pVS34, was characterized further with respect to host range, potential cloning sites, and location of replication gene(s). In addition to lactococci, pVS34 transformed Lactobacillus plantarum and, at a very low frequency, Staphylococcus aureus but not Escherichia coli or Bacillus subtilis. The 4.1-kbp ClaI fragment representing lactococcal DNA in pVS34 contained unique restriction sites for HindIII, EcoRI, XhoII, and HpaII, of which the last three could be used for molecular cloning. A region necessary for replication was located within a 2.5-kbp fragment flanked by the EcoRI and ClaI restriction sites. A 3.8-kbp EcoRI fragment derived from a nisin resistance plasmid, pSF01, was cloned into the EcoRI site of pVS34 to obtain a nisin-chloramphenicol double-resistance plasmid, pVS39. From this plasmid, the streptococcal chloramphenicol resistance region was subsequently eliminated. The resulting plasmid, pVS40, contains only lactococcal DNA. Potential uses for this type of a nisin resistance plasmid are discussed.