Immunohistochemical Localization of Ca2+/Calmodulin-Dependent Protein Kinase II in Rat Brain and Various Tissues

Polyclonal antibodies against Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) of rat brain were prepared by immunizing rabbits and then purified by antigen-affinity column. The antibodies which recognized both subunits of the enzyme with Mrs 49K and 60K were used for the study on the distribution of CaM kinase II in formalin-fixed, paraffin-embedded tissues. In the brain, a light-microscopic study demonstrated strong immunoreactivity in neuronal somata and dendrites and weak immunoreactivity in nuclei. The densely stained regions included cerebral cortex, hippocampal formation, striatum, substantia nigra, and cerebellar cortex. In substantia nigra, neurites were stained, but not neuronal somata. Electron microscopy revealed that the immunoreactive product was highly concentrated at the postsynaptic densities. In addition to neurons, weak immunoreactivity was also demonstrated in glial cells, such as astrocytes and ependymal cells of ventricles and epithelial cells of choroid plexus. In other tissues, strong immunoreactivity was observed in the islet of pancreas and moderate immunoreactivity in skeletal muscle and kidney tubules. Immunoreactivity was demonstrated in all of the tissues tested. The results suggest that CaM kinase II is widely distributed in the tissues.     

Salt tolerance of the reed plant Phragmites communis

Reed plants ( Phragmites communis Trinius) were grown at NaCl concentrations up to 500 m M and their growth, mineral contents and leaf blade osmotic potential were determined. Addition of NaCl up to 300 m M did not affect growth significantly. Sucrose, Cl^-and Na⁺ concentrations in the shoots increased with the salinity of the medium and the shoot water content decreased. K⁺ always contributed most to the leaf osmotic potential. Even in the presence of 250 m M NaCl in the rooting medium, the leaf blade contained only 50 mM Na⁺, suggesting that the plants have an efficient mechanism for Na⁺ exclusion. ²²Na⁺ uptake experiments suggested that the retranslo-cation of absorbed Na⁺ from shoots to the rooting medium lowered the uptake of Na⁺.     

Immuno-Gold Localization of Nitrogenase in Root Nodules of Elaeagnus pungens Thunb

The molybdenum-iron component of nitrogenase (Mo-Fe component)was purified from soybean nodule bacteroids and antibody wasraised against it in rabbits. Antibody raised against the 53kDa polypeptide which was the major protein in the Mo-Fe componentfraction of soybean nitrogenase was confirmed to be specificto the nitrogenase by immunodiffusion and immunotitration. Thenitrogenase from root nodules of Elaeagnus pungens cross-reactedwith the antibody and appeared from the results of the immunodiffusionto be partially identical to soybean nitrogenase. Using the antibody, we examined intracellular localization ofnitrogenase in root nodules of Elaeagnus pungens, in which Frankiais present as a symbiont, by immuno-gold labelling. Thin sectionsof nodules of Elaeagnus pungens were first treated with anti-nitrogenasespecific antibody and then with colloidal gold-protein A asa marker. The gold particles were observed to be concentratedin the vesicles of the endophyte Frankia. This provides strongsupport for the existence E of nitrogenase in the vesicles.Furthermore, our results suggested that nitrogenase localizesin the hyphae of the endophyte Frankia in Elaeagnus pungensnodules. ¹Present address: Iwata Experiment Station, Japan Tobacco Inc.,Iwata-gun, Shizuoka 438, Japan. (Received March 9, 1988; Accepted July 28, 1988)     

Further technical considerations of the sleeve microanastomosis

In sleeve anastomoses, stenoses at the suture site have been the main concern. Mechanical dilatation is one way to prevent the stenosis, as suggested by Lauritzen. In the present study, 50 vessels (femoral and carotid) and 10 veins were used for sleeve anastomoses and the same numbers of vessels were used for conventional anastomoses (as control) to evaluate the effect of mechanical dilatation using resin corrosion cast (Mercox) because the Mercox cast facilitates three-dimensional stereoscopic views. Gradual dilatations around the suture sites were observed in seven carotid arteries, and three of seven resulted into aneurysm formation due to weakening of the inner vascular wall in the sleeve anastomosis. No dilatation or aneurysm was observed in the femoral arteries. Newly proliferating capillaries formed on the endothelial surfaces of the inner vascular walls around the suture sites after 4 weeks in the sleeve anastomoses. Operative time and endothelial trauma were markedly reduced with sleeve anastomoses. The gradual dilatation and aneurysm formation in the carotid arteries show that sleeve anastomoses should be used carefully for high-pressure arteries in clinical practice if mechanical dilatation is performed.     

Isolation and identification of prostaglandin I 2 stimulating factor from human plasma

To isolate and identify the plasma factor which stimulates prostaglandin I 2 production by rat aortic ring, a human plasma fraction which showed a major stimulating activity on prostaglandin I 2 production was purified by ultrafiltrate, Sephadex G-10 gel filtration and QAE-Sephadex column chromatography. The purified plasma factor was identified as acid by its ultraviolet and infrared absorption spectroscopy, and ¹H nmr and ¹³C nmr spectroscopy. The stimulating activity of the purified plasma factor and that of authentic uric acid coincided with each other. The stimulating potency of uric acid at its physiological concentration in human plasma (about 50 μg/ml) was half of the deproteinized human plasma, and was about 30 fold stronger than that of L-tryptophan, a cofactor of prostaglandin hyperoxidase.     

Modulation of cerebral acetylcholine metabolism by the dorsal diencephalic conduction system in the rat

We investigated the effects of interruption of the impulse flow in the habenulopeduncular pathways by local infusion of tetrodotoxin on the acetylcholine and choline content in selected dopamine rich regions in the forebrain and midbrain in rats. The tetrodotoxin infusion caused a marked increase in acetylcholine content in the medial frontal cortex, striatum and ventral tegmental area+interpeduncular nucleus, but not in the limbic area or the substantia nigra, whereas choline content was reduced only in both the striatum and ventral tegmental area+interpeduncular nucleus. There was an increase in 3,4-dihydroxyphenylacetic acid content in the striatum after the manipulation. These findings suggest that the dorsal diencephalic conduction system may be involved in the integration of the activity of cholinergic neurons in the forebrain and midbrain regions and striatal dopanine neurons may play a role in the modulation of cholinergic neurons.     

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.     

Sigma receptors in schizophrenic cerebral cortices

Antipsychotics represent high affinity for sigma receptors and sigma-like drugs often have the psychotomimetic properties. Besides, the receptors are unevenly distributed in human brain. These findings suggest that sigma receptors might be involved in the pathophysiology of schizophrenia. Sigma receptors in rat and human brain were measured with [³H]-1, 3, di-o-tolylguanidine (DTG) and non-specific binding of [³H]DTG was determined in the presence of 10^–5M haloperidol. Monovalent and divalent cations strongly inhibited [³H]DTG binding. Glutamate, aspartate and glycine also decreased the binding to human cerebral membranes. With post-mortem brain samples from 12 schizophrenics and 10 controls, sigma receptors were measured in 17 areas of cerebral cortex. Sigma receptors binding showed the regional differences in the cortex, but no significant differences between schizophrenics and controls were observed except the superior parietal cortex where the binding significantly increased in the schizophrenic group. These results suggest that sigma receptors in cerebral cortices might not be directly concerned with the pathophysiological role in schizophrenia.Dedicated to Dr. Morris Aprison. Received too late for publication in special issue.     

Antitumor effect of RBS (rice bran saccharide) on ENNG-induced carcinogenesis

We examined whether orally administered RBS (rice bran saccharide), prepared from rice bran by hot water extraction, increases immunocompetence, inhibits gastrointestinal carcinogenesis with N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) or shows an antitumor effect. After the administration of RBS, phytohemagglutinin (PHA)- and pokeweed mitogen (PWM)-stimulated blastogenesis of lymphocytes derived from the mesenteric lymph nodes and peripheral blood was enhanced, and the helper/ suppressor T-cell ratio was elevated, and migration activity of peritoneal macrophages was also increased in rats treated continuously with ENNG. ENNG-induced gastrointestinal carcinomas were observed in 43% of those administered RBS (ENNG-RBS) as compared with 88% in the control (ENNG) and 94% in the prednisolone (PRD) group (ENNG-PRD). The 12-month survival rate of rats bearing gastrointestinal cancer was 58% in the ENNG-RBS group as compared with 25% in the ENNG group and 15% in the ENNG-PRD group. RBS prevented the reduction in immunocompetence in the course of carcinogenesis, suppressed carcinogenesis, and prolonged the survival of rats with gastrointestinal cancer. Antitumor activities of RBS are thought to be a kind of host mediated action. The growth inhibition ratio of transplantable ENNG-induced cancer in Wistar rats was 42.1% in the RBS and 51.8% in the 5-FU group. Since little is known about the potent antitumor activity of -glucan, it would be interesting to consider the relationship between the structure and the biological activities of polysaccharides.