Immunocytochemical localization of two peroxisomal enzymes of lipid beta-oxidation in specific granules of rat eosinophils

Peroxisomes contain a system for beta-oxidation of fatty acids which differs from the mitochondrial system and is associated with hydrogen peroxide formation. We show that two enzymes: enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase of the peroxisomal system are present in specific granules of rat eosinophils. Both enzyme proteins were purified from rat liver and monospecific antibodies were raised in rabbits. Eosinophils from peripheral blood and tissue eosinophils from the wall of intestine, fixed by glutaraldehyde and embedded in Epon were investigated. The postembedding immunocytochemical procedure with protein A-gold technique was used. The gold particles representing the antigenic sites for both enzymes were present only in specific granules of eosinophils with no immune deposits in mitochondria, nucleus and the cytoplasm. Although gold particles were found over the entire domain of the granule, the electron dense paracrystalline inclusions contained more gold than the granule matrix. Control preparations incubated with nonspecific IgG and protein A-gold complex alone were negative. These findings indicate that in specific granules of eosinophils both peroxisomal and lysosomal enzymes share the same intracellular compartment. The peroxisomal lipid beta-oxidation in eosinophils may be involved in generation of hydrogen peroxide, which has a crucial role in killing of metazoon parasites.     

Immunohistochemical identification of neurons containing corticotropin-releasing factor in the rat hypothalamus

A specific rabbit anti-CRF serum and the immunoperoxidase technique were used to show that CRF-containing neurons are mainly distributed in the paraventricular and supraoptic nuclei of the rat hypothalamus. In addition, immunoreactive neurons are scattered in other hypothalamic regions. These neurons are 20--30 micrometers in diameter. From the present and previous investigations it may be concluded that the hypothalamic magnocellular nuclei, i.e., paraventricular and supraoptic, and other hypothalamic accessory nuclei, are the producing sites not only for vasopressin and oxytocin, but also for corticotropin-releasing factor.     

Replication of Deoxyribonucleic Acid in Escherichia coli C Mutants Temperature Sensitive in the Initiation of Chromosome Replication

An Escherichia coli HF4704S mutant temperature sensitive in deoxyribonucleic acid (DNA) synthesis and different from any previously characterized mutant was isolated. The mutated gene in this strain was designated dnaH. The mutant could grow normally at 27 C but not at 43 C, and DNA synthesis continued for an hour at a decreasing rate and then ceased. After temperature shift-up, the increased amount of DNA was 40 to 50%. When the culture was incubated at 43 C for 70 min and then transferred to 27 C, DNA synthesis resumed after about 50 min, initiating synchronously at a fixed region on the bacterial chromosome. The initiation step in DNA replication sensitive to 30 mug of chloramphenicol per ml occurs synchronously before the resumption of DNA replication after the temperature shift-down, being completed about 30 min before the start of DNA replication. When the cells incubated at 27 C in the presence of 30 mug of chloramphenicol per ml after the temperature shift-down to 27 C were transferred to 43 C with simultaneous removal of the antibiotic, no resumption of DNA replication was observed. When the culture was returned to 43 C after being released from high-temperature inhibition at 30 min before the start of DNA replication, no recovery replication was observed; whereas at 20 min, the recovery of replication was observed. These results indicated that HF4704S was temperature sensitive in the initiation of DNA replication. Analysis of HF4704S, by an interrupted conjugation experiment, indicated that gene dnaH was located at about 64 min on the E. coli C linkage map. In E. coli S1814 (a K-12 derivative), which was a dnaH(ts) transductant from HF4704S (C strain) with phage P1, the mutated gene (dnaH) was demonstrated to be closely linked to the thyA marker by conjugation and P1 transduction experiments and to be distinct from genes dnaA through dnaG.     

R-Factor Mutant Capable of Specifying Hypersynthesis of Penicillinase

The physical characteristics of a mutant, R(M201-2), capable of conferring high and stable ampicillion resistance was analyzed. The R(M201-2) and its parent R-factor deoxyribonucleic acid (DNA) could be isolated as an extrachromosomal and covalently closed circular form. Their buoyant densities were both 1.712 g/cm(3), and their molecular weights were about 82 x 10(6) and 64 x 10(6), respectively, when measured by CsCl and sucrose density gradient analyses. The contour lengths by electron microscopy were 35.9 +/- 0.6 and 31.0 +/- 0.6 mum, respectively. By using the extracted R-factor DNA, the mutant and parent characters were transformable to another Escherichia coli strain. The mutant R factor showed an increased amount of DNA even after conjugal transfer to Proteus. An increase in the size of R-factor DNA was thus considered to be the cause of the high level of ampicillin resistance.     

Germination of Single Bacterial Spores

Changes in refractility and optical density occurring in individual spores of Bacillus cereus T and B. megaterium QM B1551 during germination were investigated by use of a Zeiss microscope photometer. The curves revealed that the germination process in single spores had two distinct phases; an initial rapid phase was followed by a second slower phase. Under the experimental condition employed, the first phase of germination of B. cereus spores lasted for approximately 75 +/- 15 sec, whereas the second phase lasted for 3 to 4.5 min. In B. megaterium spores, the first phase was observed to last for approximately 2 min and the second phase for more than 7 min. The duration of the second phase was dependent on conditions employed for germination. The kinetics of the first phase were strikingly similar under all conditions of physiological germination. Time-lapse phase-contrast microscopy of germinating spores also revealed the biphasic nature of germination. It was postulated that the first phase represents changes induced by an initial partial hydration of the spore and release into the medium of dipicolinic acid, whereas the second phase reflects degradation of the cortex and hydration of the core.     

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.