An RP4-prime containing a 285 kb fragment of rhizobium meliloti pSym megaplasmid: Structural characterization and utilization for genetic studies of symbiotic functions controlled by pSym

Summary We integrated the RP4 plasmid into a selected region of the pSym megaplasmid of Rhizobium meliloti 2011 by homologous recombination between pSym and a cloned fragment of pSym present in the RP4. This cointegrate was used to mobilize into Escherichia coli a Tn5 transposon located on pSym in the vicinity of the site of integration of the RP4. By this technique we obtained a series of RP4-primes that contained large fragments of the pSym megaplasmid and that were most probably generated by IS8 promoted deletions in the RP4-pSym cointegrate. One of them, pGMI42, which carries nitrogenase genes nifD and H as well as nodulation genes, was used for mutagenesis of the corresponding region of pSym after insertion of the Mu prophage into the tet gene. When various (pGMI-42:: Mu)::Tn7 were introduced into R. meliloti 2011 by conjugation, homologous recombination allowed insertion of Tn7 into pSym whereas the pGMI42::Mu was lost due to the suicide effect of Mu. In this way we obtained several symbiotic mutants deficient in either nodulation (Nod^-) or nitrogen fixation (Fix^-) in association with the host plant Medicago sativa.This paper is affectionately dedicated to the memory of Jean-Simon Julliot who initiated and inspired this work and who was killed by an avalanche on February 21, 1982     

The effect of ampicillin and tylosin on the faecal enterococci of healthy young chickens

Enterococcal isolates from young chickens were differentiated into one of three species, namely Enterococcus faecalis, Ent. faecium and Ent. gallinarum . The proportion of each species among the enterococcal population changed with time in birds not dosed with antibiotics. This pattern of change was modified in birds dosed with either tylosin or ampicillin even though ampicillin did not select for ampicillin resistance among the enterococcal population. A gradual increase in tylosin resistance was recorded with time among the enterococci of the 'undosed' control birds. This was associated with an increase in the proportion of Ent. faecium , a species commonly resistant to tylosin, among the enterococci of the birds as they grew older.     

Characterization of newly isolated monoclonal antibodies against MHC of a Japanese wild mouse

We have already developed nine B10.MOL congenic strains carrying H-2 haplotypes derived from Japanese wild mice, Mus musculus molossinus, with the C57BL/10 genetic background. To obtain monoclonal antibodies against the H-2 antigen of the Japanese wild mouse, we carried out cell fusion using spleen cells from the animal immunized with one of the B10.MOL strains, B10.MOL-SGR (H-2 ^wm7). As a result, 19 hybridomas producing monoclonal antibodies were produced. Analysis with the intro-H-2 recombinants derived from B10.MOL-SGR indicated that 8 of them reacted with the class I and II with the class II molecule. The class I antibodies were tested for their cross -reactivities on wild mice and on the panels of standard inbred and B10.MOL strains. Most of the antibodies reacted with both the Japanese wild mice and the other subspecies, including standard inbred, while two antibodies highly specific for the donor H-2K region reacted with only three wild-derived mice, two M. m. molossinus from Anj o and Shizuoka, Japan, and one M. m. domesticus from Pigeon, Canada. In addition, all of the other four antibodies reactive with the K antigen of B10.MOL-SGR also reacted with the same three wild mice. The wild mice belonging to different subspecies might share very similar H-2K antigenic determinants in spite of their genetic and geographical remoteness.     

Mechanism of proton-pumping in the cytochrome b/f complex

Several models have been proposed to interpret the mechanism of proton-pumping associated with the electron transfer reactions in the cytochrome b/f complex. Energetics considerations suggest that the proton pump is coupled to the oxidation of cytochrome b by plastoquinone. Experiments performed in living cells under anaerobic conditions suggest that proton-pumping can occur through two independent mechanisms. When the two b cytochromes are reduced prior to a flash illumination i.e. after a long dark anaerobic incubation (>10 minutes), proton-pumping is very likely associated with the reduction of a semiquinone by cyt b which occurs at a site close to the inner face of the membrane. The electrogenic phase is associated with the tranfer of protons via a transmembrane channel. This process is not inhibited by 2-n-nonyl-4-hydroxyquinoline N-oxide (NQNO). Under repetitive-flash or under aerobic conditions, proton-pumping occurs according to a modified Q-cycle mechanism, which is inhibited by NQNO.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

A reinvestigation of the Gn-RH (gonadotrophin-releasing hormone) systems in the goldfish brain using antibodies to salmon Gn-RH

Summary The organization of Gn-RH systems in the brain of teleosts has been investigated previously by immunohistochemistry using antibodies against the mammalian decapeptide which differs from the teleostean factor. Here, we report the distribution of immunoreactive Gn-RH in the brain of goldfish using antibodies against synthetic teleost peptide.Immunoreactive structures are found along a column extending from the rostral olfactory bulbs to the pituitary stalk. Cell bodies are observed within the olfactory nerves and bulbs, along the ventromedial telencephalon, the ventrolateral preoptic area and the latero-basal hypothalamus. Large perikarya are detected in the dorsal midbrain tegmentum, immediately caudal to the posterior commissure. A prominent pathway was traced from the cells located in the olfactory nerves through the medial olfactory tract and along all the perikarya described above to the pituitary stalk. In the pituitary, projections are restricted to the proximal pars distalis. A second immunoreactive pathway ascends more dorsally in the telencephalon and arches to the periventricular regions of the diencephalon. Part of this pathway forms a periventricular network in the dorsal and posterior hypothalamus, whereas other projections continue caudally to the medulla oblongata and the spinal cord. Lesions of the ventral preoptic area demonstrate that most of the fibers detected in the pituitary originate from the preoptic region.     

Phase II study of vaccinia melanoma cell lysates (VMCL) as adjuvant to surgical treatment of stage II melanoma

Summary Patients with stage II melanoma were vaccinated with vaccinia virus-induced melanoma cell lysates (VMCL). The vaccine contained viable vaccinia virus, membranous fragments and no intact nuclei. A number of antigens defined by monoclonal antibodies were detected in the vaccine including the ganglioside GD3 and DR antigens. Administration of the vaccine was associated with depression of natural killer cell activity against melanoma and K562 target cells in the first 3–6 months of treatment. Leucocyte dependent antibody (LDA) activity against melanoma cells was induced or increased in titre in approximately half of the patients studied. Continued vaccination was associated in a number of patients with a decrease in LDA titres. Studies on a small sample of patients revealed that this was associated with the development of serum factors which inhibited LDA activity. LDA activity appeared directed to non-MHC antigens on melanoma cells which were of at least two specificities. One specificity which was shared with antigens on a number of non-melanoma carcinoma cells was removed by absorption on fetal brain and may be similar to oncofetal antigens described by other workers. Reactivity against melanocytes was induced in some patients and may underlie the development of vitiligo in several patients. These results suggest that vaccines prepared from VMCL may be a favourable method for increasing immune responses against melanoma.     

Enumeration of chlorine-stressed organisms with acridine orange 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (AOINT)

Direct microscopic enumeration ofEnterobacter cloacae with the acridine orange 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride technique (AOINT) was compared with spread plate counts on nonselective media to establish the usefulness of the former technique in the enumeration of chlorine-stressed cells. Results indicate that the techniques are comparable when the organisms are not stressed. However, AOINT is more sensitive than are plate counts in the detection of chlorine-stressed cells.