Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Polyclonal plaque-forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti-trinitrophenyl (TNP) and anti-sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A-deficient mutant yielded many anti-TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures. These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column-filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage-depleted spleen cells responded to the organisms to an extent similar to that of the control. The 248βH organisms were less capable of stimulating spleen cells of 2-week-old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7-week-old) and aged (9-month-old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.     

Characterization of a glucuronyltransferase: neolactotetraosylceramide glucuronyltransferase from rat brain

The properties of a rat brain glucuronyltransferase, which is presumed to be associated with the biosynthesis of the HNK-1 epitope on sulfoglucuronyl glycolipids, are described. The enzyme required divalent cations for reaction, with maximal activity at 10mm Mn²⁺, and exhibited a dual optimum at pH 4–5 and pH 6 depending upon the buffer used, with the highest activity at pH 4.5 in MES buffer. This enzyme strictly recognized the Gal1-4GlcNAc terminal structure, and was highly specific for neolacto (type 2) glycolipids as acceptor. The enzyme was localized specifically in the brain, and was barely detected in other issues, including the thymus, spleen, liver, kidney, lung, and sciatic nerve fibres. Phosphatidylinositol and phosphatidylserine increased the enzymatic reaction 4.4- and 2.3-fold, respectively, whereas phosphatidylcholine slightly decreased the rate.Abbreviations GlcA glucuronic acid - Lc-PA₁₄ lactotetraose-phenyl-C₁₄H₂₉ - nLc-PA₁₄ neolactotetraose-phenyl-C₁₄H₂₉ - nLcOse₄-Cer neolactotetraosylceramide - NP-40 Nonidet P-40 - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SGGL sulfoglucuronyl glycolipid     

Adenosine 3′,5′-Cyclic Monophosphate-Deficient Mutants of Vibrio cholerae

Two adenosine 3',5'-cyclic monophosphate (AMP)-deficient mutants of Vibrio cholerae (biotype El Tor) were successfully isolated by nitrosoguanidine treatment followed by pencillin screening for pleiotropic sugar-negative clones. Exogenous cyclic AMP is required for the fermentation of sucrose, trehalose, fructose, maltose, and mannose but not of glucose, as well as for the formation of normal flagella and specific somatic antigens. A striking characteristic of the mutants is their growth behavior at higher temperatures. They cannot grow on TCBS selective plates at 37 C or higher unless they are provided with a supply of exogenous cyclic AMP, although they are capable of producing colonies on the same medium, even without cyclic AMP, at temperatures lower than 30 C. Since the mutants are converted to spheroplasts, spindle forms, and spiral filaments in cyclic AMP-free media at 37 C, and this phenomenon is stopped by the addition of cyclic AMP or a combination of 20% sucrose and 0.2% magnesium chloride, it is assumed that cyclic AMP is essential for the synthesis of the cell wall of V. cholerae at higher temperatures.     

Integration of Agrobacterium T-DNA into a tobacco chromosome: Possible involvement of DNA homology between T-DNA and plant DNA

Summary We established tobacco tumour cell lines from crown galls induced by Agrobacterium. Restriction fragments containing T-DNA/plant DNA junctions were cloned from one of the cell lines, which has a single copy of the T-DNA in a unique region of its genome. We also isolated a DNA fragment that contained the integration target site from nontransformed tobacco cells. Nucleotide sequence analyses showed that the right and left breakpoints of the T-DNA mapped ca. 7.3 kb internal to the right 25 by border and ca. 350 by internal to the left border respectively. When the nucleotide sequences around these breakpoints were compared with the sequence of the target, significant homology was seen between the region adjacent to the integration target site and both external regions of the T-DNA breakpoints. In addition, a short stretch of plant DNA in the vicinity of the integration site was deleted. This deletion seems to have been promoted by homologous recombination between short repeated sequences that were present on both sides of the deleted stretch. Minor rearrangements, which included base substitutions, insertions and deletions, also took place around the integration site in the plant DNA. These results, together with previously reported results showing that in some cases sequences homologous to those in T-DNA are present in plant DNA regions adjacent to left recombinational junctions, indicate that sequence homology between the incoming T-DNA and the plant chromosomal DNA has an important function in T-DNA integration. The homology may promote close association of both termini of a T-DNA molecule on a target sequence; then TDNA may in some cases be integrated by a mechanism at least in part analogous to homologous recombination.Shogo Matsumoto is on leave from Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd, Ogaki, Gifu-ken 503, Japan     

In vitro protein synthesis during germination and vernalization in winter wheat embryos

Protein synthesis during germination at 24?C and vernalizationat 4?C in winter wheat embryos were investigated with a cell-freesystem. During germination, the capacity for protein synthesisincreased in the early stage between 12 and 36 hr of imbibitionthen declined to a final low level between 48 and 72 hr. Thistransition was due to quantitative changes of the activitiesof ribosomal and supernatant fractions in the early stage andmainly to those of the supernatant fraction in the later stage.During vernalization, the capacity for protein synthesis continuedto decline over 15 to 60 days at 4?C. This transition was dueto the change in activity of the supernatant fraction; the activityof the ribosomal fraction was nearly constant. Electrophoretic analysis of in vitro products indicated thatthe high molecular weight proteins present in 12-hr embryoshad disappeared in 48-hr germinated wheat embryos and that theproducts in 24- and 36-hr embryos were types intermediate betweenthose of 12- and 48-hr embryos. The products in each vernalizedembryo resembled those in 24- and 36-hr germinated embryos.Therefore, it was concluded that the mRNA species for translationchanged during germination and vernalization in winter wheatembryos. (Received January 20, 1977; )     

Induction of micronucleated reticulocytes by potassium bromate and potassium chromate in CD-1 male mice.

Micronucleus tests of potassium bromate (KBrO3) and potassium chromate (K2CrO4) were conducted with peripheral blood reticulocytes (PB-RETs) of CD-1 male mice dose intraperitoneally. Peripheral blood cells collected from the tail were stained supravitally with acridine orange (AO) using AO-coated glass slides. Both KBrO3 and K2CrO4 induced micronuclei in PB-RETs in the same manner as in polychromatic erythrocytes of bone marrow.     

Streptococcal preparation OK-432 augments cytotoxic activity against an erythroleukemic cell line in humans

Summary The effects of OK-432, an inactivated and lyophilized preparation of a low-virulence strain of Streptococcus pyogenes, were evaluated on the cytotoxicity of lymphoid cells against a natural killer (NK)-sensitive erythroleukemic cell line K562 in patients with malignant diseases. When ten patients were treated postoperatively with daily injections of OK-432, a significant degree of augmentation in the cytotoxicity of peripheral blood lymphoid cells was evoked in all the patients, and the maximum level of cytotoxicity was on the third day after the beginning of the treatment. In spite of the successive daily administration, the level of cytotoxicity declined thereafter, but stayed higher than the pretreatment level. When OK-432 was injected IP in three patients with carcinomatous peritonitis, the cytotoxic activity of ascitic lymphoid cells was significantly enhanced. Cytotoxicity of in vitro-cultured lymphoid cells taken from peripheral blood of normal donors was also augmented by the addition of OK-432.     

Mechanism of silicate elution by hydrogen sulfide from bottom sediment in a brackish lake

Limnology - Highly concentrated dissolved silicate was detected in pore water from anoxic-reducing sediment in Lake Nakaumi, a brackish lake. Silicate concentration also simultaneously increased...