Experimental Salmonellosis VIII. Postinfective Immunity and Its Significance for Conferring Cellular Immunity

In the process of live-vaccine immunization of Salmonella enteritidis infection in mice, the relation between the number of bacteria in the organs of mice and their protecting effect was studied. Treatment with antibiotics was used to control the number of immunizing bacteria in the tissues. Mice, which were infected with 10(-5) mg (1,000 mouse MLD) of virulent S. enteritidis and treated with kanamycin simultaneously, acquired high antilethal resistance against infection with the same organisms. However, the administration of large amounts of kanamycin, which caused a rapid decrease in bacterial numbers in the organs of infected mice, was incapable of conferring immunity. This indicated the necessity of persistence of live bacteria in the host for the production of immunity. A large number of microorganisms were maintained for 53 weeks in a diffusion chamber inserted into the mouse abdominal cavity. The mice implanted with diffusion chambers containing large numbers of virulent S. enteritidis did not acquire antilethal resistance against infection with the same organisms, although agglutinins against S. enteritidis were observed in these mice. Agglutinin was also found in the fluid contained in diffusion chambers inserted into mice immunized with a killed vaccine of S. enteritidis. This indicated that antibody penetrated the membrane filter of diffusion chambers from outside to inside and vice versa. From these results, it is suggested that contact of live microorganisms with the host cell is necessary for conferring postinfective immunity in salmonellosis.     

Experimental Salmonellosis X. Cellular Immunity and Its Antibody in Mouse Mononuclear Phagocytes

A cell-associated antibody was detected in the peritoneal mononuclear phagocytes (referred to as monocytes) of mice hyperimmunized with live vaccine of Salmonella enteritidis, by use of immune transfer and immune adherence hemagglutination techniques. The cellular antibody inhibited the growth of a virulent strain of S. enteritidis with the aid of complement and lysozyme on nutrient agar plates. This type of bactericidal antibody could not be detected in the monocytes of mice immunized with killed vaccine of S. enteritidis. The antibody extracted from the peritoneal monocytes of mice hyperimmunized with live vaccine was identified as a macroglobulin by ultracentrifugal analysis.     

Drug Resistance of Enteric Bacteria XIII. Distribution of R Factors in Escherichia coli Strains Isolated from Livestock

Escherichia coli strains isolated from 151 swine and 108 fowl, which were kept at the Animal Health Center, Maebashi, Japan, were surveyed for drug resistance and distribution of R factors. All of the swine and 38% of the fowl excreted E. coli strains resistant to tetracycline, chloramphenicol, streptomycin, and sulfanilamide, or certain combinations thereof. Among 278 resistant cultures isolated from swine, 13% were found to be resistant to one antibiotic, whereas 87% were resistant to more than one antibiotic. Among these resistant strains, 40% carried R factors which were transferable by the usual conjugal process. The resistance patterns of these R factors included 36% which were singly resistant and 64% which were multiply resistant. Among 54 resistant cultures isolated from fowl, 24% were singly resistant and 76% were multiply resistant. Of the resistant strains from fowl, 22% carried R factors. The resistance patterns of R factors included 50% of the singly resistant type and 50% which were multiply resistant. In spite of feeding with dairy products containing only tetracycline, a high incidence of multiple resistance was observed in the E. coli strains and the R factors isolated from these animals.     

The reaction to c-banding of c-banded constituents during mitotic cycle ofCrepis capillaris

The reaction to C-banding was investigated throughout the mitotic cycle ofCrepis capillaris (2n=6): (1) 18–22 C-bodies or C-bands were found during mid telophase and interphase to prophase and metaphase, and also 12–14 at late anaphase to early telophase in the mitotic cycle. Fewer C-bands in late anaphase to early telophase were due to the absence of minute bands; (2) large and medium sized C-bands were strongly stained by Giemsa, while small and minute bands stained palely. It is suggested that inCrepis capillaris the difference of color in C-banded segments following Giemsa staining is referable to the amount of constitutive heterochromatin rather than to the difference in the condensation and decondensation; (3) the size of C-bodies changed during telophase to interphase and prophase. It is inferred that the extent of C-bodies is regulated by both the length of DNA sequences of constitutive heterochromatin and the amount of proteins combined with C-banded DNA. It was shown that the reaction to C-banding is neither due to the differential condensation of chromatin nor to a higher concentration of DNA in the C-banded regions, in the C-banding mechanism as has been suggested so far at least.     

Glial uptake system of GABA distinct from that of taurine in the bullfrog sympathetic ganglia

The kinetics and specificity of GABA and taurine uptake were studied in the bullfrog sympathetic ganglia. GABA uptake system consisted of simple saturable component and taurine uptake system consisted of two saturable components exclusive of non-saturable influx. Taurine unaffected GABA uptake while GABA inhibited taurine uptake competitively with theK _i/K_m ratio of 38. GABA (5.14 M) uptake was inhibited by -aminovaleric acid and slightly by 2,4-diaminobutyric acid (5 mM, each) among ten structural analogs. Taurine uptake under high-affinity conditions was most strongly suppressed by hypotaurine and -alanine competitively with theK _i/K_m ratio of 1.0 and 1.9, respectively. Autoradiography showed that glial cells were heavily labeled by both [³H]GABA and [³H]taurine. These results suggest that GABA is transported by a highly specific carrier system distinct from the taurine carrier and that taurine, hypotaurine, and -alanine may share the same high-affinity carrier system in the glial cells of the bullfrog sympathetic ganglia.     

Purification and spectral characterization of a 121-kilodalton phytochrome from etiolated pea (Pisum sativum L.) seedlings

Procedures for the purification of native phytochrome from etiolatedpea seedlings without the use of immuno-purification techniquesare described. Phytochrome (in the P_FR form) was purified bypolyethyleneglycol fractionation, adsorption to pentyl agaroseand batch elution, chromatography on DEAE-Sepharose, adsorptionto phenyl Toyopearl and batch elution, and chromatography onRed Toyopearl. The resulting phytochrome had specific absorbanceratios (SAR = A₆₆₆/A₂₈₀ of P_R) that ranged from 0.55 to 0.6.The subsequent chromatography on Sephacryl S-300 yielded verypure phytochrome with a SAR of 0.98. P_R and P_FR peaks in thedifference spectrum of the phytochrome were centered at 665and 730 nm, respectively. The spectral change ratio (Ar/Afr)of the difference spectrum was unchanged after the chromatographyon phenyl Toyopearl, and the value was 1.05–1.08, indicatingthat the spectral properties of this preparation were intact.The absorption spectra indicated that the peak absorbance ofP_FR was at 728–730 nm and that of P_R was at 666–667nm. These peak positions were essentially same as those obtainedwith the undegraded oat phytochrome. Incubation of the samplepurified on Sephacryl S-300 at 25?C for 5 h in either the P_Ror P_FR form did not result in degradation of the molecule. Therate of dark reversion of P_FR observed with the purified peaphytochrome was similar to that observed in vivo. The additionof dithionite had no effect on the reversion rate. ²Present address: Fuji-Gotenba, Research Lab. of Chugai PharmaceuticalCo. Ltd., Gotenba, Shizuoka, 412 Japan (Received February 22, 1990; Accepted May 28, 1990)     

Intergeneric hybridization betweenMonascus anka andAspergillus oryzae by protoplast fusion

Summary To breed industrially useful strains of a slow-growing, red-pigment-producing strain ofMonascus anka, protoplasts ofM. anka MAK1 (arg) andAspergillus oryzae AOK1 (met, thr) were fused. A mixture of protoplasts prepared from mycelia ofM. anka MAK1 treated with 2% Usukizyme and ofA. oryzae AOK1 treated with 2% Usukizyme and 0.2% NovoZym 234 was incubated with 30% (w/v) polyethylene glycol no. 6000. Heterokaryon fusants complementing the auxotrophies of both mutants were isolated on minimal medium, but segregated into red (MAK1) and white (AOK1) sectors after being cultured on a complete medium. After irradiation with UV light, the fusants gave stable heterozygous diploids that formed long white hyphae. These diploids, which had twice as much DNA in the nucleus as their parents, grew more rapidly than the parent strain YZT1, and produced ethanol earlier than the parents. Production of amylase, protease, and kojic acid by the fusants was intermediate in amount between that of the two parents.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.