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Serologic parameters of cat scratch disease (CSD) were evaluated by Western blot analysis. Sera from patients with serologically confirmed CSD antigen were screened for immunoglobulin (Ig) isotype-specific as well as IgG subclass-specific reactivity against Bartonella henselae whole-cell antigen. Bartonella-negative control sera were used to determine baseline antibody activity. Heterogeneous B. henselae-specific IgG reactivity with numerous protein bands, ranging from >150 to <17 kDa, was observed. Though individual banding patterns were variable, one approximately 83-kDa B. henselae protein (Bh83) was immunoreactive with all CSD sera tested, suggesting it is a conserved antigen during infection. Bh83 was not recognized by reference human antisera against Rickettsia rickettsii, Chlamydia group positive, Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis, Ehrlichia chaffeensis, Mycoplasma pneumoniae, and Escherichia coli, although other cross-reactive proteins were evident. Significantly, CSD sera failed to recognize the 83-kDa protein when tested against Bartonella quintana antigen, though sera from B. quintana-infected patients did react to Bh83. This cross-reactivity suggests epitope conservation during infection with B. henselae or B. quintana. Western blot analysis further revealed similar banding patterns when B. henselae was reacted against the Ig isotypes IgG and IgG1 and both secretory and alpha chains of IgA. Neither IgM nor IgE reacted significantly to Bartonella antigen by our Western blot analysis. Dissection of the antibody response at the IgG subclass level indicated that prominent antigen recognition was limited to IgG1. These observations provide insight into induced immunity during CSD and provide evidence for conserved epitope expression during infection with B. henselae or B. quintana.

ABSTRACTAmong the species that compose the expanding genusBartonella, thus far only B. henselaeandB. quintanahave reportedly been isolated from humans in Europe. To evaluate the prevalence of Bartonellainfection in Sweden, we conducted a retrospective serological examination of 126 human serum samples. These samples were analyzed for antibodies toB. henselae, B. quintana, and B. elizabethae. Serum samples from 100 blood donors, who spanned the ages of 20 to 60 and had no apparent clinical signs of illness, were also studied as a control group. An immunoglobulin G indirect fluorescence antibody assay revealed 4 and 8.3% Bartonellapositivity rates for the blood donor and patient group, respectively, when a cutoff titer of =64 was chosen. Among the blood donors, four were seropositive to B. elizabethae; one of these also had concordant positive titer to B. henselae. In the patient group, 14 serum samples were positive against Bartonellaspp. These serum specimens represented nine patients. In three of these seropositive patients, paired serum samples displayed a fourfold increase in antibody titer to at least one of the three antigens. These three patients are discussed. In this report we also present a case study of a 60-year-old Swedish male with fatal myocarditis. Postmortem serological analysis revealed a high titer against B. elizabethae. PCR and nucleotide sequencing of the myocardial tissue from this patient, and of liver tissue from one of the other three patients, showed sequences similar to B. quintana. The age, geographical origin, animal contacts, and serological response pattern to the different Bartonellaantigens differed among the four patients. This study substantiates the presence ofBartonellaspp. in Sweden, documents the seroreactivity to three Bartonellaantigens in Swedish patients, and reports the first two cases of B. quintana-like infections in Sweden.

We have developed a system to study the interactions of diphtheria toxin with the cell surface using non‐toxic mutant proteins synthesized in vitro. Proteins obtained by N‐terminal deletions containing the whole B fragment bound strongly to cells. Deletions extending into the B fragment did not yield an autonomous binding domain. Loss of only the N‐terminal 3 kd of the B fragment significantly impaired the ability to recognize the receptor. This, together with previous reports that the C‐terminal end of the B fragment is required for binding, suggests that both ends of the B fragment are necessary for receptor recognition. Receptor bound diphtheria toxin undergoes a conformational change at pH less than 5.3 that results in translocation of the A fragment to the cytosol and the appearance of a B fragment‐derived 25 kd polypeptide (P25) resistant to externally applied protease. Only the B fragment was required for generation of P25. N‐terminal deletions of 130 amino acids or more resulted in proteins that gave rise to P25 at higher pH than full length toxin. Furthermore, a second protease‐inaccessible polypeptide of 18 kd (P18) was observed.

Diphtheria toxin B‐fragment binds to cell‐surface receptors and facilitates translocation of the enzymatically active A‐fragment to the cytosol. In this process the B‐fragment inserts into the plasma membrane and induces formation of cation‐selective channels. We examined the ability of a number of diphtheria toxin‐derived molecules translated in vitro to permeabilize cells. Two proteins consisting of the whole B‐fragment and small parts of the A‐fragment, and one protein comprising most of the B‐fragment alone, were more efficient than full‐length toxin in permeabilizing the plasma membrane to monovalent cations. Two shorter B‐fragment‐derived proteins, with 3 and 10 kd N‐terminal deletions, permeabilized the cells to sulfate and sucrose in addition to monovalent cations. The relationship between channel formation and toxin translocation is discussed.

Naturally occurring minerals and reagent-grade compounds were tested to determine their receptivity to microwave heating. The maximum temperature achieved for the samples and the time required to reach temperature are reported. A description of the microwave oven and a thermocouple technique that accurately measures and continuously records the temperature of samples during irradiation are reported. Extractive applications using the generated microwave data were investigated. Stress fracturing at mineral grain boundaries in a gangue matrix was shown. This should significantly affect grinding energy requirements and liberation properties. A method to produce a magnetic phase on pyrite surfaces, a method of using microwave heating to retort cinnabar concentrate, and a roast-leaching microwave treatment of chalcopyrite ore are described.

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

Hybridization-complementation studies indicated that mutations in multiple genes can render Chinese hamster cells resistant to the alkaloid translation inhibitor emetine. Two of the genes, emtA and emtB, recognized in Chinese hamster lung and ovary cell lines, respectively, are known to affect the ribosomes of the cells directly. Although mutations in a third gene, emtC, affect the translation apparatus of Chinese hamster peritoneal cells in vitro (Wasmuth et al., Mol. Cell. Biol. 1:58-65, 1981), the molecular product of the emtC locus remains to be determined. To study the molecular basis for genetic complementation among emetine-resistant Chinese hamster cell mutants, we analyzed ribosomal proteins elaborated by complementing, emetine-sensitive hybrid clones (EmtB X EmtA and EmtB X EmtC) and by emetine-resistant clones that segregated from the hybrids. The electrophoretic forms of ribosomal protein S14 (the emtB gene product) elaborated by these clones indicated that the EmtA and EmtC phenotypes are independent of the emtB locus and that the emtA and emtC loci are not chromosomally linked to emtB.