Characterization of fourteen strains of Neisseria gonorrhoeae: structural analyses and serum reactivities

Resistance to normal human serum (NHS) killing in Neisseria gonorrhoeae has been associated with particular types of Protein I (PI) and lipopolysaccharide (LPS), but many exceptions exist, and the role of these structures in determining serum reactivities remains controversial. In reality, the response of the gonococcus to NHS is probably governed by several parameters involving a number of outer-membrane (OM) components. We surveyed the serum reactivities of 14 strains of N. gonorrhoeae and characterized each of their major OM components. The strains presented a spectrum of sensitivity to pooled NHS. As assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, immunoblotting, and peptide mapping, the strains were also quite heterogeneous in terms of PI, H.8 antigen, and LPS type, and the presence of the 2-1-L8 epitope. Five of the strains had identical PIAs in varying LPS and H.8 backgrounds, and four had identical PIBs in varying LPS and H.8 backgrounds. As assessed by electrophoretic migration and monoclonal antibody binding, Protein III and the 44,000 Dalton protein were identical in these strains. We found no association between PI subclass and serum sensitivity, while H.8 and LPS variation appeared to be related to bactericidal responses. The diversity and close interaction of gonococcal components in the OM are undoubtedly involved in differential abilities to survive NHS killing.     

The genetics of glutamic-pyruvic transaminase in mice: Inheritance,electrophoretic phenotypes,and postnatal changes

Glutamic-pyruvic transaminase (GPT, E.C. 2.6.1.2) from 18 inbred strains of mice was subjected to starch gel electrophoresis. Two electrophoretic phenotypes were observed: a fast-migrating pattern in 16 strains and a slower-migrating pattern in two strains. A comparison of electrophoretic patterns of F₁ and backcross progeny of two strains of mice showed that the inheritance of GPT is autosomal with two codominant alleles. The genetic locus for GPT is designated Gpt-1, and its two alleles are designated Gpt-1 ^a and Gpt-1 ^b to represent the fast-migrating (A) and slow-migrating (B) patterns. The GPT was expressed in 11 tissues with different amounts of enzyme activity. Developmental studies of GPT activity in liver showed that between 5 and 12 days after birth the mean activity was 10 units/g protein. Between 12 and 19 days, a dramatic rise in activity occurred and adult values of 300 units/g protein were reached by 26 days.This research was supported by The National Foundation (CRBS-258) and the National Institutes of Health (GM15253).Preliminary results were reported at the Annual Meeting of the American Society of Human Genetics, October 11–14, 1972, in Philadelphia.R. P. D. is an investigator of the Howard Hughes Medical Institute.     

Studies on the role of the oligomeric state and subunit III of cytochrome oxidase in proton translocation

Anion-exchange fast protein liquid chromatography in the presence of lauryldimethylamine N-oxide (LDAO) was introduced to separate cytochrome oxidase into different complexes that either did or did not contain subunit III. Both kinds of enzyme complex exhibited H+ translocation after reconstitution into phospholipid vesicles, but with a significantly (approx. 50-60%) reduced H+/e- ratio as compared with unchromatographed enzyme. The anion-exchange FPLC fractions of the enzyme (with or without subunit III) sedimented more slowly than the control enzyme upon sucrose gradient centrifugation in the presence of cholate and a high potassium phosphate concentration. When the control enzyme was subjected to the sucrose gradient centrifugation in the presence of LDAO or Triton X-100, instead of cholate, one band containing all subunits was observed, which sedimented slowly like the FPLC fractions. Transfer of this band to cholate medium, and reapplication on the sucrose gradient (with cholate), yielded both a slow- and a fast-migrating band after centrifugation. Enzyme complexes that sedimented slowly or rapidly in the sucrose gradients revealed longer and shorter elution times, respectively, in gel filtration FPLC. This suggests that these complexes corresponds to monomers and dimers of cytochrome oxidase. Solubilization of proteoliposomes and subsequent sucrose gradient centrifugation in cholate yielded one fast-migrating band for the untreated enzyme, but both a fast- and a slow-migrating band for the anion-exchange FPLC-treated enzyme, which was exclusively slow-migrating before reconstitution into liposomes. It is suggested that dimerisation of monomeric cytochrome oxidase may be favoured when the enzyme encounters a membranous milieu, and that the dimeric structure might be necessary for proton translocation.     

BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication

We have recently cloned and characterized a human member (BM28) of the MCM2-3-5 family of putative relication factors (Todorov, I.T., R. Pepperkok, R.N. Philipova, S. Kearsey, W. Ansorge, and D. Werner. 1994. J. Cell Sci. 107:253-265). While this protein is located in the nucleus throughout interphase, we report here a dramatic alteration in its nuclear binding during the cell cycle. BM28 is retained in the nucleus after Triton X-100 extraction in G1 and early S phase cells, but is progressively lost as S phase proceeds, and little BM28 is retained in detergent-extracted G2 nuclei. BM28 that is resistant to extraction in G1 nuclei is removed by DNase I digestion, suggesting that the protein is chromatin associated. In addition, we present evidence for variations in the electrophoretic mobility of BM28 that may reflect posttranslational modifications of BM28 during the cell cycle. During mitosis, BM28 is present as a fast-migrating form, but on entry into G1, the protein is converted into a slow-migrating form. With the onset of S phase, the slow-migrating form is progressively converted into the fast form. BM28 is phosphorylated at all stages of the cell cycle, but during interphase the fast form is hyperphosphorylated compared with the slow form. These apparent changes in modification may reflect or effect changes in the nuclear binding of BM28. The behavior of BM28 is not dissimilar to related proteins in Saccharomyces cerevisiae, such as Mcm2p, which are excluded from the nucleus after DNA replication. We speculate that BM28 may be involved in the control that limits eukaryotic DNA replication to one round per cell cycle.     

Subpopulations of liver coated vesicles resolved by preparative agarose gel electrophoresis

Rat liver clathrin coated vesicles (CVs) were separated into several distinct subpopulations using non-sieving concentrations of agarose, which allowed the separation of species differing primarily in surface charge. Using preparative agarose electrophoresis (Kedersha, N. L., and L. H. Rome, 1986, Anal. Biochem., in press), the CVs were recovered and analyzed for differences in morphology, coat protein composition, and stripped vesicle protein composition. Coat proteins from different populations appeared identical on SDS PAGE, and triskelions stripped from the different populations showed the same mobility on the agarose gel, suggesting that the mobility differences observed in intact CVs were due to differences in the surface charge of underlying vesicles rather than to variations in their clathrin coats. Several non-coat polypeptides appeared to segregate exclusively with different populations as resolved by two-dimensional electrophoresis. Stripped CVs also exhibited considerable heterogeneity when analyzed by Western blotting: the fast-migrating population was enriched in the mannose 6-phosphate receptor, secretory acetylcholine esterase, and an Mr 195,000 glycoprotein. The slow-migrating population of CVs was enriched in the asialoglycoprotein receptor, and it appeared to contain all detectable concanavalin A-binding polypeptides as well as the bulk of detectable WGA-binding proteins. When CVs were prepared from 125I-asialoorosomucoid-perfused rat liver, ligand was found in the slow-migrating CVs, suggesting that these were endocytic in origin. Morphological differences were also observed: the fast-migrating population was enriched in smaller CVs, whereas the slow-migrating population exhibited an enrichment in larger CVs. As liver consists largely of hepatocytes, these subpopulations appear to originate from the same cell type and probably represent CVs of different intracellular origin and destination.     

Cellular location of rat muscle ferritins and their preferential loss during cell isolation

Heart and other muscles of the rat contain two forms of ferritin separable in polyacrylamide gel electrophoresis. The cellular location of the fast- and slow-migrating ferritins was investigated using primary cultures of hindlimb skeletal muscle, and isolated myocardial cell populations. Muscle and non-muscle cells were isolated in good yield from hearts of adult rats pretreated with large doses of iron to increase their ferritin content. In virtually all cases, the isolated muscle cells contained traces only of the fast-migrating species and the non-muscle cells contained small amounts of the slow-migrating ferritin. During cell isolation, 90-100% of both ferritins was lost and could be recovered in the perfusates and solutions employed, while one third of the total tissue protein, and a larger percentage of creatine phosphokinase, was recovered in the isolated cells. Primary cultures of thigh muscle from adult rats which had differentiated into multi-nucleated myotubes, were incubated for 1-3 days with chelated iron. These cells contained substantial amounts of the electrophoretically fast migrating ferritin, with its characteristic larger Stokes' radius (determined by quantitative polyacrylamide gel electrophoresis). None of the slow-migrating ferritin species was detected, although hindlimb muscle from iron-treated rats contained both forms. It is concluded that the fast-migrating ferritin of muscle, which is much larger and more asymmetric than other ferritins, is confined to the muscle cell population, while the other form is predominantly or exclusively in the non-muscle cells. Both ferritins are lost preferentially over other proteins during procedures which injure muscle tissue.     

几种鸡传染性支气管炎病毒活疫苗对中国流行毒株CK/CH/LDL/97I的保护作用评价

选择我国应用的五株鸡传染性支气管炎活疫苗毒株(JAAS、IBN、Jlin、J9和H120)和当地流行毒株(CK/CH/LDL/97 Ⅰ)作为研究对象,对其S1基因进行序列比对分析,结果表明疫苗株与流行毒株的核昔酸序列及推导的氨基酸序列同源性分别不超过76.4%和78.7%.S1基因的核苷酸系统发育树显示,疫苗株与流行毒株分属不同进化群,亲缘关系较远,属于不同的基因型.用这五株活疫苗进行针对强毒株CK/CH/LDL/97Ⅰ株的免疫保护实验,可见临床发病率为30%～100%;攻毒5d后每组随机扑杀10只鸡,采集器官,应用RT-PCR法检测病毒,气管样品病毒检出率为50%～90%,肾脏样品病毒检出率为10%～30%.由此可见:我国目前使用的主要活疫苗对异种IBV分离株的感染不能提供完全的保护作用.     

Macrophage interaction of HDL subclasses separated by free flow isotachophoresis

Preparative isotachophoresis (ITP) was used for the fractionation of fasting and postprandial high density lipoproteins (HDL) according to their net charge in the absence of molecular sieve effects. Three major HDL subpopulations with fast, intermediate, and slow mobility have been recognized. Particle size analysis by gradient gel electrophoresis has shown that in the fast-migrating subpopulation particles dominate with a size of HDL3a and HDL2b. The subpopulation with intermediate mobility contains particles with a size between HDL2a and HDL3b, while in the slow migrating subpopulation particles dominate with a size of HDL2b, HDL3a, and HDL3c. The fast-migrating subpopulation is rich in apoA-I and phosphatidylcholine. The particles of this fraction bind at 4 degrees C to HDL receptors on macrophages with high affinity (KD = 7.71 micrograms/ml; Bmax = 245.6 ng). The subpopulations with intermediate mobility is rich in apoA-II, apoE, C apolipoproteins, cholesteryl esters, and sphingomyelin. Its affinity to HDL receptors (KD = 17.7 micrograms/ml; Bmax = 198.4 ng) is lower than that of the HDL particles in the fast-migrating subfraction. The slow-migrating subpopulation consists of particles rich in apoA-IV and is associated with a high LCAT activity. This fraction expresses the highest nonspecific binding to mouse peritoneal macrophages compared to the other HDL fractions and contains only a small amount of particles that interact with HDL receptors by high affinity binding (KD = 7.3 micrograms/ml; Bmax = 95.9 ng). In 37 degrees C binding experiments the fast-migrating subfraction reveals the highest total cell-associated activity. 72% of which is trypsin-resistant. The other subfractions express a lower total cell-associated activity and 45% of the activity of the intermediate- and 43% of the activity of the slow-migrating fraction is trypsin-sensitive. When the HDL fractions are isolated from postprandial sera of the same donor, the fast-migrating particles bind at 4 degrees C with a higher affinity (KD = 4.6 micrograms/ml) while no significant changes are observed in the intermediate- and slow-migrating subpopulations. The slow- and the fast-migrating HDL subpopulations isolated from fasting serum have a high capacity to promote cholesterol removal from macrophages. We hypothesize that the HDL subpopulations rich in apoA-I promote cholesterol removal predominantly via the interaction with HDL receptors, while apoA-IV-rich HDL particles receive their driving force for cholesterol efflux from the concomitant action of LCAT via a predominantly nonspecific interaction of the particles with the cell surface.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of the rol gene encoding the regulator of lipopolysaccharide O-chain length in Escherichia coli and its influence on the expression of group I capsular K antigens.

The rol (cld) gene encodes a protein involved in the expression of lipopolysaccharides in some members of the family Enterobacteriaceae. Rol interacts with one or more components of Rfc-dependent O-antigen biosynthetic complexes to regulate the chain length of lipopolysaccharide O antigens. The Rfc-Rol-dependent pathway for O-antigen synthesis is found in strains with heteropolysaccharide O antigens, and, consistent with this association, rol-homologous sequences were detected in chromosomal DNAs from 17 different serotypes with heteropolysaccharide O antigens. Homopolymer O antigens are synthesized by a pathway that does not involve either Rfc or Rol. It was therefore unexpected when a survey of Escherichia coli strains possessing mannose homopolymer O8 and O9 antigens showed that some strains contained rol. All 11 rol-positive strains coexpressed a group IB capsular K antigen with the O8 or O9 antigen. In contrast, 12 rol-negative strains all produced group IA K antigens in addition to the homopolymer O antigen. Previous research from this and other laboratories has shown that portions of the group I K antigens are attached to lipopolysaccharide lipid A-core, in a form that we have designated K(LPS). By constructing a hybrid strain with a deep rough rfa defect, it was shown that the K40 (group IB) K(LPS) antigen exists primarily as long chains. However, a significant amount of K40 antigen was surface expressed in a lipid A-core-independent pathway. The typical chain length distribution of the K40 antigen was altered by introduction of multicopy rol, suggesting that the K40 group IB K antigen is equivalent to a Rol-dependent O antigen. The prototype K30 (group IA) K antigen is expressed as short oligosaccharides (primarily single repeat units) in K(LPS), as well as a high-molecular-weight lipid A-core-independent form. Introduction of multicopy rol into the K30 strain generated a novel modal pattern of K(LPS) with longer polysaccharide chains. Collectively, these results suggested that group IA K(LPS) is also synthesized by a Rol-dependent pathway and that the typically short oligosaccharide K(LPS) results from the absence of Rol activity in these strains.     

Hormonal effect on glycosaminoglycans and glycoproteins in uteri of ovariectomized rats

1. Glycosaminoglycans such as chondroitin sulfate A (or C), chondroitin sulfate B (dermatan sulfate), heparitin sulfate (heparan sulfate) and hyaluronic acid were identified as major glycosaminoglycan components in whole uteri as well as in uterine stroma of rats. Two types of sialoglycoproteins with different electrophoretic mobilities (fast- and slow-migrating) were detected in the glycosaminoglycan fraction from the luminal epithelia. 2. Treatment of ovariectomized rats with estradiol-17beta markedly increased the uterine contents of glycosaminoglycans. Chondroitin sulfate A (or C) was found to increase more than chondroitin sulfate B. Furthermore, it was found that the estrogen treatment specifically increases the fast-migrating sialoglycoprotein level in the luminal epithelia and results in the appearance of it in the uterine fluid. 3. Administration of progesterone to ovariectomized rats slightly increased the uterine glycosaminoglycan content without appreciable alteration of the uterine glycosaminoglycan pattern.     

Correlation of myosin isoforms with anatomical divisions in equine musculus biceps brachii.

The biceps brachii of horses is subdivided into a lateral and medial head. Electrophoresis of samples from the lateral head revealed three slow-migrating native myosin isoforms, including one that does not correspond to slow myosin isoforms described for other mammalian muscles. In contrast, the medial head contained a single slow isoform. Both the lateral and medial heads contained three fast-migrating isoforms corresponding with the FM-2, FM-3 and FM-4 isoforms reported for other mammalian fast-twitch muscle fibers. Electrophoresis of myosin heavy chains (MHCs) revealed only two MHC bands, one fast-migrating band that comigrates with rat type I MHC and a second slower-migrating band that comigrates with rat type IIa MHC. Quantitation of the histochemical data is correlated with densitometric analysis of MHCs in the medial and lateral heads of biceps brachii and is consistent with previously hypothesized functional specializations of this muscle.     

An approach to understand the complexation of supramolecular dye Congo red with immunoglobulin L chain lambda

Congo red, a dye of high self-assembling tendency, has been found to form complexes with proteins by adhesion of the ribbon-like supramolecular ligand to polypeptide chains of beta-conformation. Complexation is allowed by local or global protein instability, facilitating penetration of the dye to the locus of its binding. At elevated temperatures, L chain lambda of myeloma origin was found to form two distinct complexes with Congo red, easily differentiated in electrophoresis as slow- and fast-migrating fractions, bearing four- and eight-dye-molecule ligands, respectively, in the V domain of each individual chain. The slow-migrating complex is formed after displacement of the N-terminal polypeptide chain fragment (about 20 residues) from its packing locus, thereby exposing the entrance to the binding cavity. In this work the formation and stability of this complex was studied by molecular dynamics (MD) simulations. The effect of three- and five-molecule ligands introduced to the site binding the dye was also analyzed in an attempt to understand the formation of fast-migrating complexes. The wedging of the ligand containing five dye molecules, hence longer than established experimentally as the maximum for the slow-migrating complex, was found to generate significant structural changes. These changes were assumed to represent the crossing of the threshold on the way to forming a fast-migrating complex more capacious for dyes. They led to almost general destabilization of the V domain, making it susceptible to extra dye complexation. Theoretical studies were designed in close reference to experimental findings concerning the number of dye molecules in the ligand inserted to the site binding the dye, the location of the site in the domain, and the conditions of formation of the complexes. The results of the two kinds of studies appeared coherent.     

Biotyping of Propionibacterium acnes isolated from normal human facial skin

Biochemical and serological characteristics of 128 strains of Propionibacterium acnes isolated from the facial skin of healthy Japanese volunteers were compared with the three standard strains of the American Type Culture Collection, ATCC 6919, 11827, and 11828. Accordingly, the isolated strains of P. acnes were classified into five biotypes (B1 to B5) on the basis of fermentation tests of ribose, erythritol, and sorbitol. Two serotypes were distinguished by the agglutination test. P. acnes belonging to serotype I had galactose as a cell wall sugar, whereas those of serotype II lacked galactose. The strains of serotype I were distributed among all five biotypes (B1 to B5); however, those of serotype II consisted only of one biotype (B2). A term "sero-biotype" was introduced to differentiate and carefully classify the isolates. The predominant sero-biotypes differed with the individual and region of the facial skin. In general, strains of sero-biotype IB1, IB3, IB4, and IIB2 were more frequently isolated than those of sero-biotype IB2 and IB5. Thus, for routine assay work, serotyping of P. acnes as based on erythritol and sorbitol fermentation is both practical and applicable.     

Ly49I NK cell receptor transgene inhibition of rejection of H2b mouse bone marrow transplants

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB. Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/- mice were crossed and back-crossed with 129 mice-H2b, 5E6-, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.     

Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides.

In Escherichia coli K-12, RcsC and RcsB are thought to act as the sensor and effector components, respectively, of a two-component regulatory system which regulates expression of the slime polysaccharide colanic acid (V. Stout and S. Gottesman, J. Bacteriol. 172:659-669, 1990). Here, we report the cloning and DNA sequence of a 4.3-kb region containing rcsC and rcsB from E. coli O9:K30:H12. This strain does not produce colanic acid but does synthesize a K30 (group I) capsular polysaccharide. The rcsB gene from E. coli K30 (rcsBK30) is identical to the rcsB gene from E. coli K-12 (rcsBK-12). rcsCK30 has 16 nucleotide changes, resulting in six amino acid changes in the predicted protein. To examine the function of the rcs regulatory system in expression of the K30 capsular polysaccharide, chromosomal insertion mutations were constructed in E. coli O9:K30:H12 to independently inactivate rcsBK30 and the auxiliary positive regulator rcsAK30. Strains with these mutations maintained wild-type levels of K30 capsular polysaccharide expression and still produced a K30 capsule, indicating that the rcs system is not essential for expression of low levels of the group I capsular polysaccharide in lon+ E. coli K30. However, K30 synthesis is increased by introduction of a multicopy plasmid carrying rcsBK30. K30 polysaccharide expression is also markedly elevated in an rcsBK30-dependent fashion by a mutation in rcsCK30, suggesting that the rcs system is involved in high levels of synthesis. To determine whether the involvement of the rcs system in E. coli K30 expression is typical of group I (K antigen) capsules, multicopy rcsBK30 was introduced into 22 additional strains with structurally different group I capsules. All showed an increase in mucoid phenotype, and the polysaccharides produced in the presence and absence of multicopy rcsBK30 were examined. It is has been suggested that E. coli strains with group I capsules can be subdivided based on K antigen structure. For the first time, we show that strains with group I capsules can also be subdivided by the ability to produce colanic acid. Group IA contains capsular polysaccharides (including K30) with repeating-unit structures lacking amino sugars, and expression of group IA capsular polysaccharides is increased by multicopy rcsBK30. Group IB capsular polysaccharides all contain amino sugars. In group IB strains, multicopy rcsBK30 activates synthesis of colanic acid.     

Immobilization antigens from Tetrahymena thermophila are glycosyl-phosphatidylinositol-linked proteins.

We have studied four strains of Tetrahymena thermophila, each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [3H]ethanolamine or [3H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide electrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [3H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [3H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-linked glycosyl-phosphatidylinositol anchors.     

An enzyme-linked immunosorbent assay for detection of chronic subclinical Pasteurella pneumotropica infection in mice.

Serum samples from seventy-five, 3- to 12-week-old and 16 retired breeder male Swiss mice from a conventional colony with enzootic chronic subclinical Pasteurella pneumotropica infection were tested by enzyme-linked immunosorbent assay (ELISA) and Western blots for IgG antibodies to whole cell (WC) and lipooligosaccharide (LOS) antigens of P. pneumotropica. In 3- to 12-week-old mice, serum antibody levels to LOS exceeded those to the WC preparation. Western blots of sera from mice in this age group substantiated that a major component of the early IgG antibody response was directed against LOS antigens. Higher antibody levels to both antigen preparations in 3-week-old mice compared to mice 4 and 6 weeks old were interpreted as reflecting a decline in antibodies acquired from the dam. Active immunity indicative of infection was first detected at 8 weeks of age. Serum samples from retired breeder mice (28 weeks of age) also had substantial antibody titers to LOS but, in contrast to sera from mice in the younger age groups, retired breeders had significantly greater IgG reactivity to WC preparations than to LOS antigens. The superior specificity of the LOS antigen compared to the WC preparation in the ELISA was demonstrated by testing serum samples from retired breeder mice against WC and LOS antigens from P. ureae, P. multocida, and P. hemolytica. The reactivity of IgG against LOS antigens from these organisms was negligible, whereas substantial titers were evident to WC antigens. This ELISA, using LOS preparations as antigen, is a useful serologic assay for the detection of subclinical P. pneumotropica infection in mice.     

Epithelial cell contact-induced alterations in Salmonella enterica serovar Typhi lipopolysaccharide are critical for bacterial internalization

The invasion of Pseudomonas aeruginosa and Salmonella enterica serovar Typhi into epithelial cells depends on the cystic fibrosis transmembrane conductance regulator (CFTR) protein as an epithelial receptor. In the case of P. aeruginosa , the bacterial ligand for CFTR is the outer core oligosaccharide portion of the lipopolysaccharide (LPS). To determine whether serovar Typhi LPS is also a bacterial ligand mediating internalization, we used both P. aeruginosa and serovar Typhi LPS as a competitive inhibitor of serovar Typhi invasion into the epithelial cell line T84. P. aeruginosa LPS containing a complete core efficiently inhibited serovar Typhi invasion. However, neither killed wild-type Typhi cells nor purified LPS were effective inhibitors. LPS from mutant Typhi strains defective in O side-chain synthesis, but with an apparently normal core, was capable of inhibiting invasion, but LPS obtained from a deeper rough mutant strain with alterations in fast-migrating core oligosaccharide failed to inhibit invasion. Lastly, exposure of wild-type serovar Typhi to T84 cultures before heat killing resulted in a structural alteration in its LPS that allowed the heat-killed cells to inhibit invasion of wild-type serovar Typhi. These data indicate that the serovar Typhi LPS core, like the P. aeruginosa LPS core, is a ligand mediating internalization of bacteria by epithelial cells, and that exposure of this ligand on wild-type Typhi is induced by the bacteria's interaction with host cells.     

Extended type 1 chain glycosphingolipids: dimeric Lea (III4V4Fuc2Lc6) as human tumor-associated antigen

Glycolipid extracts from various human cancer tissues and cell lines showed the presence of a slow-migrating glycolipid component which was strongly reactive with monoclonal antibody (mAb) NCC-ST-421 (raised against human gastric adenocarcinoma) and weakly cross-reactive with anti-Lea mAbs. The slow-migrating glycolipid was isolated from human colonic adenocarcinoma cell line Colo205 grown in nude mice, and was purified by high-performance liquid chromatography followed by preparative thin-layer chromatography. Its structure was elucidated by sequential enzymatic degradation and thin-layer chromatography immunostaining of the degradation products with various mAbs, 1H NMR spectroscopy, positive-ion fast atom bombardment mass spectrometry, and methylation analysis. The major slow-migrating component reacting with mAb ST-421 was identified as dimeric Lea, with the structure as follows. [formula: see text] Antigens containing this structure and various analogous structures (including enzymatically synthesized Lea/Lex hybrid antigen) were tested with ST-421. While the mAb was equally reactive with dimeric Lea and Lea/Lex, only the former was chemically detectable as the slow-migrating glycolipid from the tumor extract. ST-421 showed less reactivity with simple Lea (III4FucLc4) or extended Lea (V4FucLc6, and/or IV3Gal beta 1----3[Fuc alpha 1----4]GlcNAcnLc4), and was not reactive with Lex/Lex (dimeric Lex). It was concluded, therefore, that the major tumor-associated slow-migrating glycolipid reacting with ST-421 has the dimeric Lea structure shown above. Since extension of lacto-series structure has been shown to be limited to type 2 chain in normal cells and tissues, extended elongation of type 1 chain as shown in this structure represents a novel tumor-associated epitope.