Complementation of an Enterococcus hirae (Streptococcus faecalis) mutant in the alpha subunit of the H+-ATPase by cloned genes from the same and different species

We isolated an Enterococcus hirae (formerly Streptococcus faecalis) mutant, designated MS117, in which ‘G’ at position 301 of the alpha-subunit gene of the F₁F₀ type of H⁺-ATPase was deleted. MS117 had low H⁺-ATPase activity, was deficient in the regulatory system of cytoplasmic pH, and was unable to grow at pH6.0. When the alpha-subunit gene of E. hirae H⁺-ATPase was ligated with the shuttle vector pHY300PLK at the downstream region of the tet gene of the vector, it was expressed without its own promoter in MS117, and the mutation of MS117 was complemented; the mutant harbouring the plasmid had the ability to maintain a neutral cytoplasm and grew at pH6.0. We next transformed MS117 with pHY300PLK containing the alpha-subunit gene of Bacillus megaterium F₁F₀-ATPase constructed in the same way. The transformant grew at pH 6.0, and the ATP hydrolysis activity was recovered. These results suggested that an active hybrid H⁺-ATPase containing the B. megaterium alpha subunit was produced, and that the hybrid enzyme regulated the enterococcal cytoplasmic pH, although the function of the B. megaterium enzyme did not include pH regulation. Thus, our present results support the previous proposal that the enterococcal cytoplasmic pH is regulated by the F₁F₀ type of H⁺-ATPase.     

Augmentation of antitumor immunity in regional lymph nodes by local immunotherapy

We have previously reported that the antitumor effect of OK-432, a streptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (OK-432/fbg) [1]. In order to elucidate the effects of this immunotherapy on regional lymph nodes (RLN), we carried out both morphological and functional analyses of the RLN from colonic cancer patients treated with OK-432/ fbg. Computer-aided morphometry revealed that the maximal cross-sectional areas and the broadest diameters of the RLN were significantly greater (p<0.01) in patients who had undergone local immunotherapy than in patients who had not. The component structures of RLN, such as sinus, follicle and paracortex, were all enlarged in the OK-432/fbg-treated patients, and necrosis of metastatic tumors was observed. RLN lymphocytes recovered from OK-432/fbg treated patients showed elevated reactivity to phytohemagglutinin (PHA) and the stimulation index was clearly higher than that of control patients. Flow cytometric analysis revealed a predominance of T-cells, especially CD4 subsets, and higher positivity for both CD25 and HLA-DR. Furthermore, RLN lymphocytes killed more effectively K562 and Daudi cells in the patients who had had immunotherapy. These results suggest that the effect of local immunotherapy with OK-432/fbg is not restricted to the site of injection but extends to the lymph nodes, and contributes to tumor regression through the augmentation of cellular immunity.Abbreviations RLN regional lymph node(s) - OK-432/fbg OK-432/fibrinogen solution - PHA phytohemagglutinin - NK natural killer - LAK lymphocyte activated killer     

The Effect of Unheated Bovine Serum on the Complement-Fixing Activity of Heat-Inactivated Bovine Antiserum with Homologous Antigen. IV. Chromatographic Studies

Unheated, non-dialyzed, normal bovine sera were fractionated by column chromatography on the cross-linked dextran, Sephadex G-25, and the fraction tested for "supplementing" properties, that is for complement-fixation augmenting activities when added to mixtures of heated bovine antiserum and homologous antigen. Supplementing activity was shown by precipitated fractions from earlier eluates with pH values below 7.2 and also by both supernatant and precipitated fractions of the later eluates with pH values from 7.6 to 8.1. The possibility is briefly discussed that certain alkaline protein substances of relatively lower molecular weight may be involved in the supplementing activities of the later fractions. Heating at 56 degrees C. for 30 min. destroyed the supplementing activity of each of these fractions. Some of the supplementing fractions proved to be anti-complementary, others were not or only slightly so. First component of complement, C(1)1, was detected in the precipitated fractions of certain of the earlier eluates with pH values below 6.5; second component of complement, C(1)2, was found exclusively in supernatant fractions of earlier eluates with pH values less than 6.2. Conglutinin was not separated from C(1)1 by this method.     

The effects of hemorrhagic shock on thyrotropin-releasing hormone and its receptors in discrete regions of rat brain

The effects of hemorrhagic shock on thyrotropin-releasing hormone (TRH) levels and its receptors were studied in different regions of the rat brain. Rats were bled for 30 min from the left femoral artery, and their mean arterial pressure was kept at 40 mmHg for the following hour. The rats were killed by decapitation. Rat brains were immediately removed and dissected into 7 regions. Hemorrhagic shock decreased TRH significantly in the frontal cortex, septum, hippocampus, and hindbrain but TRH was not changed in the striatum, hypothalamus, and midbrain. Hemorrhagic shock significantly decreased TRH receptor binding in the septum and hindbrain. Scatchard analysis of saturation isotherms of specific TRH binding showed that the decreased specific TRH binding in the hindbrain resulted not from an increase of the dissociation constant (Kd), but from a decrease in the maximum number of binding sites (Bmax). In the septum, the decrease in specific binding was due both to a decrease in Bmax and an increase in Kd. The findings indicate that TRH plays a role in the physiological response to hemorrhagic shock.     

Characterization of antibody against human liver guanase by immunoblotting and immunohistochemical staining of human liver guanase by a direct labeling antibody-enzyme method

Human liver guanase was purified and a specific antibody against it was raised in rabbits. The antiserum formed a single precipitin line with human liver extract, and also completely inhibited the activity of the liver enzyme. An immunoblotting study showed that the antibody bound specifically to one band of protein with guanase activity and not to other proteins. Therefore, we concluded that this antiserum against the liver enzyme was suitable for use in immunohistochemical demonstration of guanase. In tissue sections, the immunohistochemical reaction with this antibody was positive in the same locations as the histochemical guanase reaction with DAB (3,3'-diaminobenzidine tetrahydrochloride).     

Oxygen-Dependent Inhibition of Neutrophil Respiratory Burst by Nitric Oxide

Effect of nitric oxide (NO) on the respiratory burst of neutrophils was examined under different oxygen tensions. Phorbol myristate acetate (PMA) stimulated oxygen consumption and superoxide (O₂^-) generation in neutrophils by a mechanism which was inhibited reversibly by NO. The inhibitory effect of NO increased significantly with a decrease in oxygen tension in the medium. The inhibitory effect of NO was suppressed in medium containing oxyhemoglobin (HbO₂), a NO scavenging agent. In contrast, 3-morpholinosydnonimine (SIN-1), a compound that rapidly generates peroxynitrite (ONOO^-) from the released NO and O₂^-, slightly stimulated the PMA-induced respiratory burst. These results suggested that NO, but not ONOO, might reversibly inhibit superoxide generation by neutrophils especially at physiologically low oxygen tensions thereby decreasing oxygen toxicity particularly in and around hypoxic tissues.     

A Novel Class of Herbicides (Specific Inhibitors of Imidazoleglycerol Phosphate Dehydratase)

A new mode of herbicidal action was established by finding specific inhibitors of imidazoleglycerol phosphate dehydratase, an enzyme of histidine (His) biosynthesis. Three triazole phosphonates inhibited the reaction of the enzyme with Ki values of 40 [plus or minus] 6.5, 10 [plus or minus] 1.6, and 8.5 [plus or minus] 1.4 nM, respectively, and were highly cytotoxic to cultured plant cells. This effect was completely reversed by the addition of His, proving that the cytotoxicity was primarily caused by the inhibition of His biosynthesis. These inhibitors showed wide-spectrum, postemergent herbicidal activity at application rates ranging from 0.05 to 2 kg/ha.     

Rank and age related feeding strategy observed through field experiments in the Koshima group of Japanese macaques

The feeding process of Japanese monkeys on soy beans which were scattered over a sandy beach on Koshima islet was studied. Younger monkeys were able to pick up more beans when 8 kg of beans were divided and given two times (“two times feeding”) than when the whole amount (8 kg) of beans was given at one time (“one time feeding”). The effect of saturation of the food intake capacity in younger monkeys at the first feeding in “two times feeding” did not appear at the second feeding one hour later. The minutely intake of soy beans (feeding speed) for each age class was analyzed. The decline of feeding speed in adult females after the peak in “one time feeding” was not related to the decline in density of beans on the ground, and this decline was caused by saturation of their food intake capacity. Adult females were divided into four classes according to their dominance rank order: high, lower-high, higher-low, and low classes. The total amount of intake in “one time feeding” was far larger in the high class than in any of the other classes. The total amount of feeding in the first feeding of “two times feeding” increased in accordance with rise in the dominance rank class, and there was no relation to rank and total feeding amount in the second feeding of “two times feeding.” Differences existed in the process of feeding between the rank classes. The feeding speed of the low class was as high as that of the high class on the curve of minutely intake, while the low class stopped feeding much earlier than the high class. The lower-high class displayed a low feeding speed, and stopped feeding the latest. The order of the duration to stay and to feed in the feeding area was lower-high > high > higher-low > low, and this order did not change under the three different feeding conditions, “one time feeding,” and the first and second feedings of “two times feeding.” Adolescent females tended to stay the longest duration in the feeding area among all age classes. Both the lower-high class females and adolescent females had an unstable social status in the Koshima group, and their social status affected their feeding behaviors. The feeding behaviors were similar in attitude depending on social status, and are considered to be maintained for a fairly long time. The feeding strategy of the lower-high class, in staying a longer duration in the artificial feeding area, and departing later, may be effective under the artificial feeding conditions, but it may be a bad strategy in a natural habitat where the food is not so clumped as in artificial feeding, and where choice of other food patches is possible. The above results agree well with previous reports for the Koshima group, indicating that the rank of the lower-high class females was unstable (Mori et al., 1989), and that their reproductive success was low (Watanabe et al., 1992).