Analysis of the chromosomal location of two copies of a Bordetella pertussis insertion sequence

IS481v1 and IS481v2 are two copies of a Bordetella pertussis insertion sequence element. We have shown that IS481v1 is located within 3 kbp of the start of the adenylate cyclase gene whilst IS481v2 is immediately adjacent to the end of the agglutinogen 2 gene and provides the stop codon for that gene. In addition, IS481v1 and IS481v2 were present at these two specific sites in nine strains of B. pertussis, including two Phase IV strains which expressed neither adenylate cyclase nor agglutinogen 2 and three Phase I strains which did not express agglutinogen 2. The loss of expression in these strains is not the result of DNA rearrangements at the sites of IS481v1 or IS481v2.     

Effect of monoclonal antibodies on the adherence of Bordetella pertussis to Vero cells

Abstract An in vitro assay involving the adherence of Bordetella pertussis to Vero cells was used to quantify the inhibitory effects of monoclonal antibodies (McAb) on adherence. McAbs to agglutinogens (fimbriae), filamentous haemagglutinin (FHA), lymphocytosis promoting factor (LPF) and to an X-mode specific outer membrane protein were tested. X-mode cells adhered to the Vero cells to a greater extent than C-mode cells. McAb to agglutinogen 2 inhibited the adherence of B. pertussis bearing homologous agglutinogen. McAbs to LPF, FHA, agglutinogen 3 and X-mode specific outer membrane protein also partially inhibited the adherence of B. pertussis to Vero cells.     

Bordetella pertussis agglutinogens in cultivation dynamics

Bordetella pertussis growth phases during homogenous batch dynamic cultivation in the liquid medium as well as during the static cultivation on the solid medium were established. The maximal activity of agglutination reaction with antisera to B. pertussis agglutinogens 1, 2, and 3 was detected in bacterial culture at the end of exponential phase of growth. The activity of agglutination reaction decreased when cultures in stationary and death phases were used. During transition from exponential to death phase level of antibodies to agglutinogen 2 decreased by4 - 32 times. 2 - 4-fold decrease of antibodies level was observed when antiserum to agglutinogen 3 was used. Activity of agglutination reaction with antiserum to agglutinogen 1 was high and did not depend from phase of growth. When polyvalent antiserum to B. pertussis was used 4-fold decrease of antibody titers was observed in parallel with change of growth phases. Sera from rabbits immunized with B. pertussis cultures from the middle of exponential growth phase, the end of this phase, and begin of the death phase had high (maximal) level of agglutinating antibodies (6400), which was detected on 101 day after immunization with the former culture and on 31 day after immunization with either of the two latter cultures. To the end of experiment (292 day) titers decreased to 800, 3200, and 1600 respectively. These findings confirm an advisability of use of exponential growth culture for immunization of rabbits in order to obtain highly active diagnostic antisera to B. pertussis.     

Fixation of the agglutinogen fromHemophilus pertussis on the bacterial surface

Summary The agglutinogen ofHemophilus pertussis is rather soluble in some suspending media but can be fixed on the bacterial surface with formaldehyde in saline.     

百日咳杆菌有效免疫原的发酵培养

为扩大生产,采用５００立升发酵罐培养无细胞百日咳菌苗,发现随着培养时间的延续,细胞浓度增高,培养液的ｐＨ值上升,ＰＴ、ＦＨＡ活性、血凝效价逐渐增加、Ｏ２溶压下降、ＣＯ２溶压上升。ｐＨ值达８２时,ＰＴ活性最高为３００ＥＵ／ｍｌ,较现用扁瓶培养方法高５倍。ｐＨ值继续上升时,ＰＴ活性开始下降。ＦＨＡ活性及血凝效价具有相似的变化。通过测定培养液ｐＨ值以确定收获时间,可获得富含ＰＴ、ＦＨＡ、且活性均保持较高水平的培养液     

Surface Antigens of Smooth Brucellae

Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able to diffuse more readily.     

Effectiveness of immunizing donors with adsorbed tetanus anatoxin in relation to their genetic markers

Blood groups, including rhesus grouping, have been determined in 218 donors and HLA of loci A, B, C in 121 donors immunized with adsorbed tetanus toxoid (40 binding units). The presence of HLA Aw 32, B 8; HLA Bw 16, haplotypes A3 Bw 16 and A1, blank antigens of locus C is linked with the production of, respectively, high or low (less than or equal to 16 I.U./ml) antibody titers. The effectiveness of immunization is influenced by agglutinogen B, which is confirmed by its associations (in a complex with HLA Bw 16 or Bw 35) with low immune response.     

Serotype variation in Bordetella pertussis is governed by cis-acting elements

Bordetella pertussis contains two genes encoding the serospecific fimbrial subunit proteins 2 and 3 which are assembled into completed fimbriae, which elicit the formation of agglutinating antibodies. Expression of these agglutinogens can vary independently of each other. A gene library from a B. pertussis strain (fimbrial serotype 0.3) was probed with an oligonucleotide probe specific for fimbrial subunit genes. Three homologous genetic loci were identified; an active fim 3 gene, an inactive fim 2 gene and an unknown fim-homologous region. The fim 3 gene carried on a cosmid produced agglutinating fimbrial structures in B. parapertussis and in variants of B. pertussis which had lost the capacity to produce the agglutinogen. This indicated that cis-acting factors are associated with serotype variation in B. pertussis rather than the production of trans-acting repressor molecules.     

The cell mediated and humoral immune response to vaccination with acellular and whole cell pertussis vaccine in adult humans.

The cell mediated immune response (CMI) against pertussis antigens following vaccination with the traditional Danish whole cell pertussis vaccine (WC-P) and the Japanese acellular pertussis vaccine (A-PV) JNIH-3 was studied in four adult human volunteers. Vaccination with the A-PV induced an in vitro proliferative response of peripheral blood lymphocytes to pertussis toxin (PT) subunits S2-S4, S3-S4 and S5 and the filamentous hemagglutinin (FHA), and a better serological response to native PT, detoxified PT (dPT) and FHA than the WC-PV. The induced CMI and serological response were followed over a period of 17 weeks, and were not seen to decline during this period. Further, an in vitro proliferative response to Bordetella pertussis agglutinogen 2 and 3 were demonstrated using lymphocytes from recently and not-so-recently pertussis-vaccinated adults.     

Biological activities of pertussigen from Bordetella pertussis strains of various agglutinogen types

Pertussigen prepared from Bordetella pertussis strains of various agglutinogen types was found to have similar biological activities in mice, and to give precipitin lines of identity in agar diffusion tests. The doses required to induce histamine hypersensitivity ranged from 0.9 to 9.5 ng, and to induce leukocytosis from 124 to 190 ng. When pertussigen was detoxified by treatment with glutaraldehyde, it protected mice from intracerebral challenge with B. pertussis at doses of from 12.7 to 24.4 micrograms. The results showed that all smooth strains of B. pertussis produced pertussigen that was biologically and serologically similar, and that it was produced independently of fimbrial hemagglutinin.     

Blood groups of the mantled howler monkey (Alouatta palliata).

Fifty-two howler monkeys were tested for their human-type A-B-O blood groups. All were group B, as shown by the presence of B and H in their saliva, and anti-A in serum. The B-like agglutinogen of their red cells is common to all New World monkey species tested, and is of different origin and significance than their true A-B-O blood group. Differences among the B-like agglutinogens of the red cells of howler monkeys, marmosets, rabbits and humans group B were demonstrated, and limited tests have also been performed to study the biochemical basis of the anti-B reactions.     

Characteristics of antisera against periodate-resistant membrane antigens from Neisseria gonorrhoeae

Crude outer membrane (OM) was prepared by extraction of bacteria of the Neisseria gonorrhoeae strains 8551. V, and VII, with an EDTA-containing buffer. The preparations contained the lipopolysaccharide (LPS) and at least 10 proteins as shown by SDS-polyacrylamide gel electrophoresis. Immunization of rabbits with untreated OM resulted in production of antibodies against several antigens, including LPS. Antisera raised against periodate-treated OM did not contain antibodies against LPS. These latter antisera agglutinated heat-treated (100 degrees C, 60 min) gonoccal cells by means of antibodies to one or more common agglutinogens and against a strain-specific agglutinogen that was susceptible to digestion with proteolytic enzymes. Both side agglutination and a plate agglutination test could be used to detect antibodies against these agglutinogens.     

Serological characteristics of coagulase-positive staphylococci isolated from man and animals

A simplified method allowed Staphylococcus aureus, Staph. intermedius and coagulase-positive Staph. hyicus subsp. hyicus isolated from humans, dogs, monkey, sheep, poultry, rabbits, giant rats (Cricetomys gambianus) and other animals to be serotyped. The nine coagulase-positive staphylococcal strains of human origin possessed thermolabile and thermostable agglutinogens. Two strains of Staph. intermedius of human and canine origins examined had agglutinogen K1K2. The three Staph. aureus strains isolated from African giant rats (Cricetomys gambianus) had agglutinogens a5 and P common to them. The Staph. aureus strain isolated from a monkey belonged to serotype b1, c1, o and the caprine strain of Staph. hyicus subsp. hyicus was serotype a5, c1.     

Analysis of separate isolates of Bordetella pertussis repeated DNA sequences

Two independent isolates of a Bordetella pertussis repeated DNA unit were sequenced and shown to be an insertion sequence element with five nucleotide differences between the two copies. The sequences were 1053 bp in length with near-perfect terminal inverted repeats of 28 bp, had three open reading frames, and were each flanked by short direct repeats. The two insertion sequences showed considerable homology to two other B. pertussis repeated DNA sequences reported recently: IS481 and a 530 bp repeated DNA unit. The B. pertussis insertion sequence would appear to comprise a group of closely related sequences differing mainly in flanking direct repeats and the terminal inverted repeats. The two isolates reported here, which were from the adenylate cyclase and agglutinogen 2 regions of the genome, were numbered IS48lvl and IS48lv2 respectively.     

百日咳杆菌69KDa外膜蛋白的分离纯化及生物学特性研究

本文发展了一种从百日咳杆菌Ⅰ相菌株中纯化６９ＫＤａ外膜蛋白的简易方法,将细菌体经加热浸提、乙醇沉淀蛋白、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ５０柱层析精制而成。用ＳＤＳ－ＰＡＧＥ、免疫印迹、光密度仪扫描分析,证明纯化制剂为均一的、特异性的６９ＫＤａ外膜蛋白,其收率为５４．２％,纯度达９９．２％,每微克６９ＫＤａ蛋白制剂中的内毒素含量低于０．８５ＥＵ;ＰＴ残留量小于０．１０５ｎｇ。抗６９ＫＤａ蛋白抗血清能     

Immunochemical identification of the major cell surface agglutinogen of Acinetobacter calcoaceticus RAG-92

The immunochemical and immunocytochemical characteristics of three Acinetobacter calcoaceticus RAG strains were compared in order to clarify the relationship between antibody-induced agglutination and the production of polyanionic extracellular emulsifier (termed emulsan). In addition to the parent, RAG-92, two mutant strains were examined: (1) a non-agglutinating emulsan-producer (AB15), and (2) an agglutinating mutant (16TLU) defective in the production of emulsan. A combined genetic-immunochemical approach was employed. This included the comparison of crossed immunoelectrophoresis patterns of parent and mutant supernates and the effect of absorption of anti-whole cell antiserum with mutant cells. In addition, agglutinability and competition studies were performed as well as electron microscopic cytochemistry. The results demonstrated that three major antigenic components were associated with the cell surface and the supernate. Mutant cells were altered both in their cell surface properties and in their extracellular products. One antigenic component, termed component C3, was the major cell surface agglutinogen; this component was absent in non-agglutinating mutants. Component C3 may be identical with or attached to the 300 nm projections on the parent cell surface, but it is not directly related to the presence of emulsan. It appears that emulsan plays little or no role in the phenomenon of antibody-induced agglutination of this organism.     

Convection-free boundary sedimentation of dilute protein solutions

Use of the density gradient sedimentation velocity technique appears to be essential for the accurate determination of the mean sedimentation coefficients of dilute protein solutions. When performed on an analytical ultracentrifuge equipped with a photoelectric-scanning-absorption optical system, the density gradient sedimentation velocity technique has been shown to be particularly useful in studying the subunit association-dissociation equilibria of multisubunit enzyme systems. The time factor has been shown to be a major advantage of the density gradient sedimentation velocity technique, as opposed to the sedimentation equilibrium technique, in studying the subunit association-dissociation equilibria of multisubunit enzymes such as rabbit muscle apo-glyceraldehyde-3-phosphate dehydrogenase, which is very unstable in dilute solution.     

Band centrifugation of macromolecules in self-generating density gradients. II. Sedimentation and diffusion of macromolecules in bands

Three approaches to the simultaneous sedimentation and diffusion of hands or zones of noninteracting homogeneous macromolecules are examined: (1) The authors' method of moments: (2) the transport me of Sehumaker and Rosenbloom; and (3) the stochastic solution of the Lamm equation due to Gehatia and Katehalski. All three methods indicate that the motion of the maximum of the hand may be used to evaluate the sedimentation coefficient. The moment, method provides relations which appear to be useful for measuring diffusion coefficients. Relations are given for the analysis of resolved components. The problem of measuring sedimentation coefficients of macromolecules with concentration-dependent sedimentation coefficients is examined. Methods are described for evaluating the sedimentation coefficient in these systems and for obtaining the sedimentation coefficient at infinite dilution. Methods are described for determining the weight-average sedimentation coefficient in Multi-component systems, and the differential and integral distribution of sedimentation coefficients of macromolecules with low-diffusion coefficients.     

水体中病毒浓缩方法及其条件优化

本研究探讨了在自来水和污水两种不同的水体环境中, 三氯化铝沉淀法和正电荷滤膜法浓缩回收病毒的效率, 并比较应用不同的浓缩条件、洗脱物质时的病毒回收率, 从而建立有效的环境样本中病毒的浓缩方法。结果表明, 三氯化铝沉淀法在两种水体中的回收率均较高, 最高回收率分别达到96.0%和92.0%, 但正电荷滤膜法在两种水体中的回收率差别较大。在自来水中, 正电荷滤膜法的最高回收率为93.9%, 在污水中的最高回收率仅为69.9%, 这说明正电荷滤膜法适用于病毒含量较高且悬浮物等杂质较少的样本。在应用于自来水样时, 两种方法均可起到有效的病毒浓缩作用, 但在悬浮物等杂质较多的污水中, 三氯化铝法具有较好的回收效率。     

The sedimentation of amyloplasts in living statocytes of the dandelion flower stalk

Abstract The sedimentation of amyloplasts in inverted statocytes of the flower stalk of dandelion (Taraxacum officinale Weber) was followed using light microscopy. Much variety in sedimentation pattern was evident with complete sedimentation occurring in as little as 3–4 min to as much as a few h and individual amyloplasts showing barely perceptible sedimentation or sedimenting at speeds of up to 40 μm min^?1. It was also apparent that amyloplasts were subjected to considerable movement after they had sedimented. Large vacuoles and cytoplasmic streaming in statocytes were confirmed and are considered to greatly influence amy-loplast sedimentation pattern. The implications of these observations for the starch-statolith hypothesis of geotropism are discussed.