Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding casette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete less of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.     

Energy metabolism in the cyanobacterium Plectonema boryanum. Participation of the thylakoid photosynthetic electron transfer chain in the dark respiration of NADPH and NADH

The oxidation of NADPH and NADH was studied in the light and in the dark using sonically derived membrane vesicles and osmotically shocked spheroplasts. These two types of cell-free membrane preparations mostly differ in that the cell and thylakoid membranes are scrambled in the former type and that they are more or less separated in the latter type of preparations. In the light, using both kinds of preparations, each of NADPH and NADH donates electrons via the plastoquinone-cytochrome $\text{b}\text{c}$ redox complex (Qbc redox complex) to the thylakoid membrane-bound cytochrome c-553 preoxidized by a light flash and to methylviologen via Photosystem I. NADPH donates electrons to the thylakoid membrane via a weakly rotenone-sensitive dehydrogenase to a site that is situated beyond the 3(3′,4′-dichlorophenyl)-1,1-dimethylurea sensitive site and before plastoquinone. Ferredoxin and easily soluble cytoplasmic proteins are presumably not involved in light-mediated NADPH oxidation. Inhibitors of electron transfer at the Qbc redox complex as the dinitrophenylether of 2-iodo-4-nitrothymol, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2-n-heptyl-4-hydroxy-quinone-N-oxide are effective, but antimycin A and KCN are not. The oxidation of NADH showed comparable sensitivity to these inhibitors. However, the oxidation of NADH is antimycin-A-sensitive regardless of the kind of membrane preparation used, indicating that in this case electrons are donated to a different site on the thylakoid membrane. In the dark, NADPH and NADH donate electrons at sites that behave similar to those of light-mediated oxidation, indicating that the initial steps of electron transfer are situated at the thylakoid membranes. However, NADPH oxidation is in some cases not sensitive to inhibitors active at the Qbc redox complex. It is concluded that O₂ reduction takes place at two different sites, one partly developed in vitro, situated near the rotenone-sensitive NADPH dehydrogenase, and another, highly KCN-sensitive one, situated beyond the Qbc redox complex and used in vivo. The terminal oxygen-reducing step of NADPH and NADH oxidation in the dark showed a preparation-dependent sensitivity for KCN, more than 80% inhibition in sonically derived membrane vesicles and less than 30% inhibition in osmotically shocked spheroplasts. From this result we tentatively conclude that the highly KCN-sensitive oxidase is not necessarily located at the thylakoid membrane and could be located at the cytoplasmic membrane.     

Processing of McCoy cell cultures infected with Chlamydia trachomatis: sequential isolation of chlamydial elementary bodies and lipopolysaccharide

Abstract Triton X-100 (TX100) enhances the liberation of chlamydial elementary bodies (EB) from host cells and dissolves the host cell membrane. In the presence of TX100 only differential centrifugation is needed to isolate reasonably pure EBs. The remaining high-speed supernatant still contains a large part of the chlamydial lipopolysaccharide (LPS), which can be isolated with the standard phenol-chloroform-petroleum ether extraction.     

Population and formal genetics of the human C81(α-γ) polymorphism

Summary One hundred and ninety-six unrelated healthy individuals and 30 families with 75 offspring have been studied for the C81(-) polymorphism. The following allele frequencies were calculated: C81^*A=0.5536; C81^*B=0.4286; C81^*A1=0.0178. Observed and expected phenotype frequencies were in a good agreement according to the Hardy Weinberg law. No exceptions from the mode of inheritance were found. In family W the segregation of the rare allele C81^*A1 could be followed. Comparing the results of this study with previous data from Boston and Oslo, a combined technology including C8-dependent lysis and C8 structural variation is suggested for future investigations.     

Natural killer cells in mice and man and their possible biological significance

Conclusions Despite active and extensive research on human and murine NK cells in the last few years a number of basic questions remain to be answered. These include the nature of the antigen(s) on target cells to which NK cells react, the nature of the receptors on NK cells allowing the recognition, the mechanism of NK cell cytotoxicity, the exact relationship between NK cell activities in vivo and in vitro, genetic control of NK cells, involvement of NK cells in the field of bone marrow transplantation, and finally their involvement in immunosurveillance of malignancies. Although the etiology and biologic significance of NK cells remain obscure, there is suggestive evidence that these cells may be in volved in resistance to and immunosurveillance of malignancies, since there is experimental evidence for a correlation between in vivo and in vitro NK cell activities. The fact that athymic mice do not have a higher incidence of malignancies than conventional mice and express delayed appearance and higher resistance to virally and chemically induced tumors (Stutman, 1975) is consistent with a protective role of the NK cell effector mechanism. In addition, their spontaneous occurrence without any necessity for priming and their ability to recognize and kill malignant cells makes NK cells almost perfect candidates for the part of immunosurveillors. The similarity between bone marrow effector cells and NK cells also seems unlikely to be fortuitous. In summary, this interesting cell requires further attention.     

Mapping of nucleoside phosphorylase (Np-1) and esterase 10 (Es-10) on mouse chromosome 14

A method for detecting two alleles at Np-1 (nucleoside phosphorylase) and three alleles at Es-10 (esterase 10) from mouse blood by cellulose acetate electrophoresis is described. The allelic constitution at these loci for 44 inbred strains and stocks was determined. The location of Np-1 on chromosome 14 was established by backcross experiments in which alleles at Np-1 and Robertsonian translocations were segregating. Es-10 was shown to be linked to Np-1, and the following genetic map of Chr 14 was constructed: centromere-(8.9±4.0 cM)-[Np-1, Wc]-(10.2±1.9 cM)-Es-10-(15.5±3.7 cM)-s. The homologous human loci, NP and ES-D, are not linked.This work was supported by Contract E(11-1)-3267 with the Energy Research and Development Administration, by Contracts NO1-ES4-2156 and NO1-ES4-2159 with the National Institute of Environmental Health Sciences, and by Grants GM 19656 and GM 20919 from the National Institute of General Medical Sciences. D. A. K. was a participant in the 1975 Summer Program for College, Graduate, and Medical Students, which was supported, in part, by the Clark Foundation. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Comparison of storage proteins ofPhaseolus caracalla L. with the proteins of some other representatives of the genusPhaseolus

Storage proteins of the seeds (cotyledons) of the South-American speciesPhaseolus caracalla were compared by means of immunoelectrophoretic methods with other representatives of the genusPhaseolus. These proteins most resemble the proteins of the co-called tropical group (i.e. Ph. atropurpureus, Ph. geophilus, Ph. bracteatus, Ph. semierectus) and least the so-called American endemites (Ph. vulgaris, Ph. coccineus, Ph. acutifolius, Ph. lunatus), the main globulin of which is of a completely different specificity. The proteins ofPh. caracalla are less similar to the group of the so-called Asiatic species (Ph. aureus, Ph. calcaratus, Ph. angularis, Ph. aconitifolius, Ph. trilobus) including the analyzed representatives ofVigna sinensis; their main globulin is only partly similar to that ofPh. caracalla. Some considerations on the relationship ofPh. caracalla with the so-called tropical species is presented.     

Comparison of the protective ability of O and K immunity in mice after immunization withEscherichia coli

The protective abilities ofEscherichia coli O and K immunity against intraperitoneal infection was compared in CBA mice immunized with formalin-killed bacteria. The K13 immunity achieved seemed somewhat more effective than the O6 immunity. This was also reflected in a slightly more rapid clearance of a liveE. coli O6K13 bacteria in K13-immunized animals than in those immunized with O6. However, the protection achieved afterE. coli O6K2 and O2K1 immunization was related more to O immunity than to K immunity.     

Fetal cells in the maternal blood

Summary In an attempt to stimulate fetal cells in the maternal blood to mitotic division, peripheral blood lymphocytes were cultured from ten primiparous women and six multiparous women. In the case of the ten primiparous women, PWM was used to stimulate lymphocytes in 3- and 7-day cultures made at the 16th, 20th, 24th, and 28th week of gestation. Altogether, 10565 mitoses were analyzed after quinacrine staining of cells from five mothers who each subsequently gave birth to a male infant, and not a single XY mitosis was found.In the case of the multiparous women, lymphocyte cultures, with PHA or LPS as mitogen and MLC, were initiated between the 13th and 20th week of pregnancy. Four of the mothers were pregnant with a male child, and two with a female child. From cultures of each of the four mothers expecting a boy, a total of 9721 mitoses were analyzed after quinacrine staining, and not a single XY mitosis was found. However, one XY cell was found in the culture from one of the two women who delivered a girl. The XY mitosis probably originated from a pregnancy 8 months earlier which terminated in a male infant.In an attempt to culture and obtain good chromosome preparations from small numbers of cells, it was shown that a good mitotic response and good chromosome preparations could be obtained from as few as 6000 lymphocytes.