Intragranular co-storage of neuropeptide Y and arginine vasopressin in the paraventricular magnocellular neurons of the rat hypothalamus

Summary Certain populations of arginine vasopressin (AVP) neurons in the magnocellular paraventricular nucleus became immunoreactive for neuropeptide Y (NPY) when rats were treated with colchicine or monosodium glutamate (MSG). The co-storage of these peptides was examined by empooying a post-embedding electron-microscopic immunohistochemistry technique using goldlabeled antibodies to the two peptides. In colchicinetreated rats, the neuronal perikarya contained numerous secretory granules showing co-storage of the two peptides. The cells of the MSG-treated rats were characterized by having well-developed Golgi bodies with the granular structures also co-storing the two peptides, although the secretory granules in the perikarya were rather fewer than in the colchicine-treated rats. It is concluded that the destruction of the arcuate nucleus by MSG-treatment may potentiate the synthesis of NPY in AVP neurons, the synthesis of which is latent in intact animals.     

Significant antitumor effect of a synthetic lipid A analogue,DT-5461, on murine syngeneic tumor models

Summary The antitumor effect of a synthetic lipid A analogue, DT-5461, was investigated using syngeneic tumor models in mice. Intravenous injection of DT-5461 into mice transplanted with solid tumors of MethA fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis lung carcinoma (3LL), and colon adenocarcinomas 26 and 38 resulted in significant reductions in the weight of all tumors except Colon 26, with marked hemorrhagic necrosis of tumor tissues. Efficacy was almost equal to that of anEscherichia coli-type synthetic lipid A (compound 506), and also to those of some chemotherapeutics including Adriamycin, mitomycin C, fluorouracil and cisplatin. Furthermore, DT-5461 was more effective than other immunotherapeutics, including picibanil (OK-432) and lentinan. However, its antitumor effects were inferior to those of Adriamycin or OK-432 against the malignant ascites caused by intraperitoneal inoculation with MethA or with MH134 cells; life span was not prolonged by either intraperitoneal or intravenous administration. In addition, although DT-5461 showed direct inhibitory effects on the in vitro growth of MethA or MH134, these were much weaker than those of Adriamycin. These findings clearly indicated that DT-5461 with systemic administration is a highly effective antitumor agent on solid tumors, and suggest that the antitumor effect of DT-5461 with potent necrotizing activity might derive from indirect mechanisms related to the activation of host immune systems and not to the weak direct cytotoxicity.     

Different immunolocalizations of cathepsins B, H, and L in the liver

Different localizations of cathepsin B, H, and L in normal rat liver were revealed immunohistochemically with anticathepsin Fab'-horseradish peroxidase conjugates. Staining of cathepsin B was strong in the periportal sinusoids, possibly in Kupffer cells; and weaker in panlobular hepatocytes. Staining of cathepsin H was strong in panlobular hepatocytes, especially in the periphery of the cytoplasm, possibly representing the peribiliary dense bodies; and weaker in periportal sinusoidal cells, possibly Kupffer cells. Staining of cathepsin L was strongest in centrilobular hepatocytes and weaker in periportal sinusoidal cells, possibly Kupffer cells. These findings, revealed for the first time in the present study, show that the histologic and intracellular localizations of the three cathepsins are different, suggesting that they have different roles in degradation of exogenous and endogenous proteins.     

Direct evidence for nuclear and cytoplasmic colocalization of proteasomes (multiprotease complexes) in liver

Subcellular localization of the large multicatalytic protease complexes called proteasomes, which have been found in soluble fractions of various cells, was examined by biochemical, immunological, and immunohistological methods. Rat liver nuclei, purified by two different procedures, showed high activities for degrading [3H]methylcasein and various fluorogenic oligopeptides with neutral and weakly alkaline pH optima. On gel filtration, all of these peptidase activities were recovered in a single peak with the unusually large molecular weight of about 600,000. Properties of the proteolytic activity in crude extracts of the nucleus and the cytoplasm were very similar. Immunoelectrophoretic blot analysis showed the presence of appreciable concentrations of proteasomes with similar immunoreactivity in isolated nuclear and cytosolic fractions. Moreover, immunohistochemical staining of human liver showed that proteasomes were predominantly localized in the nuclear matrix but also were present diffusely in the cytoplasm of hepatocytes. These findings indicate the nuclear and cytoplasmic colocalization of proteasomes.     

Evaluation of antifungal volatile compounds on the basis of the elongation rate of a single hypha

A novel method is proposed for the evaluation of the activity of an antifungal agent administered as a gas. This system is composed of a batch-flow type reaction vessel, a gas flow system, and a microscopic observation system. The agar plate was prepared on the ceiling of the reaction vessel, and the mycelium of a fungus (Aspergillus niger or Rhizoctonia solani) was inoculated onto it. After preincubation at 25 degrees C for 24 h, the reaction vessel was connected to the gas flow system. An appropriate hypha was selected, and its elongation rate was measured. Then a sample holder containing an antifungal compound was inserted into the reaction vessel from the side hole to saturate the atmosphere inside with its vapor. The retardation or inhibition of the hypha elongation was observed on a television monitor and recorded on a video tape recorder. The antifungal compound was then removed, and the reaction vessel was flushed with air. If the hypha lived, it began to elongate again. By this method, antifungal activity of seven odor compounds could be evaluated quantitatively within several hours.     

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).     

Localization of arachidonate 12-lipoxygenase in parenchymal cells of porcine anterior pituitary

12-Lipoxygenases oxygenate arachidonic acid producing its 12S-hydroperoxy derivative and are well known as platelet and leukocyte enzymes. When a peroxidase-linked immunoassay of the enzyme according to the avidin-biotin method was applied to the cytosol fractions from various parts of porcine brain, a considerable amount of the enzyme was found in the anterior pituitary. The enzyme level (about 200 ng/mg cytosol protein) corresponded to about 6% of the enzyme content in porcine peripheral leukocytes. Posterior and intermediate lobes showed about one-tenth of the enzyme level of anterior pituitary. Other parts of porcine brain contained the 12-lipoxygenase in amounts below 7 ng/mg cytosol protein. The cytosol fraction (0.7 mg of protein) of anterior pituitary produced 12S-hydroxy-5,8,10,14-eicosatetraenoic acid from 25 microM arachidonic acid in about 34% conversion at 24 degrees C for 5 min, giving a specific enzyme activity about 3 nmol/min/mg protein. Furthermore, various octadecapolyenoic acids were oxygenated almost as fast as the arachidonate 12-oxygenation. When anterior pituitary was investigated immunohistochemically with anti-12-lipoxygenase antibody, most of the immunostained cells were certain parenchymal cells with granules, which were not blood cells. These biochemical and immunohistochemical results provide a good reason for considering that 12-lipoxygenase does play an important role in pituitary function.     

Acid release following activation of surf clam (Spisula solidissima) eggs.

Acid release was observed after activation of Spisula eggs with excess KCI. This acid release begins within 20 sec after the activation and continues for 9–15 min. The amount of acid released was 6.8 μmole per milliliter of packed eggs. In Ca-free or Na-free sea water, the acid release is completely inhibited; subsequent addition of the deficient ion leads to acid release and breakdown of germinal vesicles. These results suggest that Spisula eggs release protons after activation in a manner similar to that of sea urchin eggs, and that acid release with concomitant increase in cytoplasmic pH is probably a general event on activation of marine eggs.     

Respiration-dependent uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitochondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATPase activity was stimulated at higher concentrations of uncouplers.

2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response.

3. The addition of succinate, NADH or ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin.

4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10^-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin.

5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity.

6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.