Immunocytochemical localization of lactoferrin in human neutrophils

Summary The subcellular localization of lactoferrin in human neutrophils was studied by an electron-microscopic immunoperoxidase method. This molecule was detected in small granules of blood polymorphonuclear leukocytes. A morphometrical analysis showed that there was no significant difference in the mean size between lactoferrin-positive and myeloperoxidase-negative granules. In contrast, the mean size of myeloperoxidase-positive granules was significantly larger than that of lactoferrin-positive granules. This indicates that lactoferrin is contained in the myeloperoxidase-negative, secondary, granules of human neutrophils. In immature bone marrow mononuclear neutrophils, lactoferrin was present in cytoplasmic granules of somewhat larger size than lactoferrin-positive granules of polymorphonuclear leucocytes. A morphometrical study showed that the mean size of lactoferrin-positive granules was significantly greater in immature bone marrow cells than in polymorphonuclear leucocytes. This indicates that lactoferrin-positive granules decrease in size as the cells mature. Besides cytoplasmic granules, lactoferrin was demonstrated in the Golgi complex and a part of the rough endoplasmic reticulum of immature bone marrow neutrophils, probably myelocytes and early metamyelocytes. These results show that lactoferrin is synthesized and packed into secondary granules in immature bone marrow neutrophils and therefore that the secondary granules are a type of secretory granule.     

Antibody-dependent cell-mediated cytotoxicity using a murine monoclonal antibody against human colorectal cancer in cancer patients

Summary Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by a murine monoclonal antibody against human colerectal carcinoma, antibody 19–9, with human effector cells was tested in 33 patients with various carcinomas, 16 patients with benign lesions, and 13 normal controls, using a 12-h ⁵¹Cr release assay using human colorectal cancer cells as targets. Peripheral blood mononuclear cells (PBM) from these groups of patients and normal controls achieved moderate levels of target cell lysis in the presence of the monoclonal antibody at the high effector to target cell ratio of 200:1. The ADCC activity of PBM in cancer patients was significantly higher than that in either normal persons or patients with benign lesions. Since the ADCC was shown to be mainly mediated by adherent monocytes in the PBM, ADCC activity of monocytes from cancer patients was compared to those from control groups at an effector to target cell ratio of 30:1. The results also showed that the lytic capacity of monocytes was significantly higher in cancer patients than that in the control populations.     

Mutagenicity of nitro- and amino-substituted phenazines in Salmonella typhimurium

The nitro- and amino-substituted phenazines were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98 and TA98NR. Of 7 tested nitrophenazines, 4 were mutagenic in the absence of a microsomal metabolic activation system (S9 mix) and were more mutagenic in TA98 than in TA98NR. The order of mutagenicity of nitrophenazines in TA98 is 1.7- less than 2- less than 2.8- less than 2.7-substituted phenazine. Of 7 tested amino derivatives, 4 exhibited mutagenic activity with S9 mix in TA98. 1-Nitro-, 1-amino, 1.6-dinitro-, 1.9-dinitro-, 1.6-diamino- and 1.9-diamino-phenazine were not mutagenic. As regards the relationship between mutagenic potency and chemical structure of the phenazines, the results suggested that structural requirements favoring mutagenic activity were the presence of substituents at the 2 and/or 7 position. Furthermore, 2.7-disubstituted phenazines were extremely mutagenic, 2.7-dinitrophenazine and 2.7-diaminophenazine induced 36,450 and 12,110 rev./nmole, respectively. In the preliminary study, 2.7-diaminophenazine was identified by gas chromatography/mass spectrometry from the reaction mixture of m-phenylenediamine and hydrogen peroxide.     

A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella typhimurium strains TA98 and TA100

'Classical nitroreductase' is an enzyme involved in the intracellular metabolic activation of mutagenic nitroarenes. The nitroreductase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1021 and YG1026) had more than 50 times higher nitrofurazone-reductase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic action of 2-nitrofluorene, 1-nitropyrene and 2-nitronaphthalene. These results indicate that the new strains permit the efficient detection of mutagenic nitroarenes.     

Effect of UV-B (290–320 nm) irradiation on growth and metabolism of cucumber cotyledons

Cucumber ( Cucumis sativus L. cv. Natsusairaku 3) seedlings were grown in a growth cabinet under UV-B (290–320 nm) irradiation (equivalent to the UV-B radiation normally incident at Tokyo, 36°N latitude, during clear sky conditions in mid-april on a weighted daily fluence basis) and a UV-B-free control condition. UV-B irradiation inhibited the growth of the cotyledons, i.e. the increase in area, and increase in fresh and dry weights of the cotyledons. The greatest inhibition rate was observed in the increase in area, causing a significant increase in specific leaf weight (the ratio of weight to area). UV-B irradiation had no significant effect on DNA and RNA contents in the cotyledons, but decreased protein content slightly. In contrast, the irradiation reduced the amounts of organic acids and soluble sugars, indicating that primary carbon metabolism was very sensitive to UV-B radiation. UV-B irradiation lowered the photosynthetic activity in the cotyledons without any effect on chlorophyll content and respiratory activity. These results indicate that UV-B radiation at the ambient level may act as a physiological stress in some UV-sensitive plants.     

Suppression of Inhibitory Effects of Copper on Enzymatic Activities by Copper-Binding Substances from Athyrium yokoscense Assayed in vitro

A metal-tolerant fern, Athyrium yokoscense, is capable of growingin highly copper-contaminated soil, but cupric chloride inhibitedthe activities of some enzymes extracted from the fern. Thefunction in the detoxification of copper of two copper-bindingsubstances was investigated by examination of their effectson various enzymes assayed in vitro, i.e. acid phosphatase (orthophosphoric-monoesterphosphohydrolase [acid optimum], EC 3.1.3.2 [EC] ), glucose-6-phosphatedehydrogenase (D-glucose-6-phosphate: NADP⁺ 1-oxidoreductase,EC 1.1.1.49 [EC] ) and isocitrate dehydrogenase (threo-Ds-isocitrate:NADP⁺ oxidoreductase [decarboxylating], EC 1.1.1.42 [EC] ). The twocopper-binding substances, whose apparent molecular weightsare 9.5 kDa and 2 kDa, were previously obtained from the solublecytoplasmic fraction of the fern root. The 9.5-kDa substance,which is a cysteine-rich peptide induced as a result of exposureof the fern to copper, was found to suppress almost entirelythe inhibitory effects of the metal on the enzymes. The suppressoractivity of the peptide was nearly as effective as that of ethylenediaminetetraaceticacid. The 2-kDa substance, which is also found in fern thathas not been exposed to copper, had a more modest suppressoractivity. These results indicate that the 9.5-kDa substancemay contribute to the copper-tolerance of the fern growing incopper-contaminated soil. (Received August 26, 1988; Accepted March 17, 1989)     

Longitudinal changes of dominance rank among the females of the Koshima group of Japanese monkeys

The changes of dominance rank among female Japanese monkeys of the Koshima group over a period of 29 years from 1957 were studied. The dominance rank order was relatively stable in the early population growing phase, while large scale-changes of dominance rank order occurred successively in the phase of population decrease brought about by the severe control of artificial feeding after 1972. Nevertheless, the rank order of several females of the highest status was stable. Furthermore, the reproductive success of these highest status females was high (Mori, 1979a;Watanabe et al., in prep.). Divergence of the dominance rank order fromKawamura's rules (Kawamura, 1958) was observed in the following respects: (1) Some females significantly elevated their rank depending on the leader males. (2) If mothers died when their daughters were still juveniles or nulliparous, the dominance rank of some of these offspring females was significantly lower than the mother's one. However 55% of daughters which lost their mothers at a young age inherited the mother's rank. (3) Dominance among sisters whose mother had died when at least one of the daughters was under 6 years old followed the rule of youngest ascendancy in 60% (Kawamura, 1958), and in 80% when both of the daughters were nulliparous at the mother's death. The mean rate of aggressive interactions for each female with subordinates to her was calculated by dividing the total aggressive interactions between the female in question and her subordinates by the number of subordinate females to the female in question. A female which showed a high rate of aggressive interactions with her subordinates was categorized as an “Attacker”, and a female showing a lower rate was categorized as a “Non-attacker”. Similarly, categories of “Attacked”, and “Non-attacked” were distinguished by using the rate of aggressive interactions with dominant females. Several females which were once categorized in one category in a year were repeatedly categorized in the same category over different years. The “Attacked” tended to be females of higher rank, and “Non-attackers” tended to be females of lower rank. “The second-higher-status females”, were “Attacked”, and their rank was unstable. In particular, females of lower rank within the lineage of the highest rank suffered this kind of severe status. Most of the daughters of these females showed a sharp drop of rank, and died when they were still at a young age, i.e. “the second-higher-status females” displayed low fitness. “Non-attackers” were significantly “Non-attacked”; i.e. they were females which showed a non-social attitude. Females which underwent a drop of rank tended to be “Non-attackers”. The most important factor which determined the females' rank was the memory of their dominance relations under the influence of their mother [dependent rank (Kawai, 1958)] in their early life during development. This finding corresponds well with the results in baboons obtained byWalter (1980); the target females of aggressive interactions by adolescent females were determined by the rank of the mothers when these adolescent females were born.     

Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of α-amylase

Summary In a continuous culture of Bacillus caldolyticus strain SP, which requires maltose as an inducer for production of -amylase in batch culture, a predominant mutant strain M1 which produced high amounts of -amylase in the absence of maltose in batch culture, developed. The change of cell population from strain SP to strain M1 in maltose-casitone medium was linear with time in the transient state after the change from batch to continuous culture at a dilution rate of 0.17 h^-1, and was completed in about 11 generations of bacterial growth. The dilution rate effect of continuous culture on -amylase activity was almost the same with both strains SP and M1. The maximum -amylase activity of 380 units/ml was observed at an intermediate dilution rate that was 11.5 times higher than -amylase activity at the end of a batch culture using the same medium. It was deduced that the enhancement of -amylase production in continuous culture was attributed partly to the predominant growth of a mutant strain with higher -amylase productivity.     

Purification and characterization of two serine isoacceptor tRNAs from bovine mitochondria by using a hybridization assay method.

For large scale preparation of mitochondrial tRNAs, a new hybridization assay method using synthetic oligodeoxyribonucleotide probes (16-17mer) complementary to individual tRNA sequences was developed and applied for the purification of two serine isoacceptor tRNAs (tRNASerAGY and tRNASerUCN) from bovine mitochondria. It is about 100 times more sensitive than the conventional aminoacylation assay method. 2-4 A260 units each of both tRNASer isoacceptors were purified from 17.5 kg of bovine liver, and they were characterized by means of nuclease digestion, melting profiles and aminoacylation activity. It is suggested that tRNASerUCN possesses the D loop/T loop interaction like usual L-shaped tRNAs, and that tRNASerAGY lacking almost an entire D arm is aminoacylated with an efficiency not very much lower than that of tRNASerUCN.