N-Acetyl-Aspartyl-Glutamate: Regional Levels in Rat Brain and the Effects of Brain Lesions as Determined by a New HPLC Method

Abstract: An isocratic HPLC method to measure endogenous N -acetyl-aspartyl-glutamate (NAAG) and N -acetyl-aspartate (NAA) is described. After removal of primary amines by passage of tissue extracts over AG-50 resin, the eluate was subject to HPLC anion-exchange analysis and eluted with phosphate buffer with absorbance monitored at 214 nm. The retention time for NAA was 5.6 min and for NAAG 11.4 min with a limit sensitivity of 0.1 nmol. The levels of NAA and NAAG were measured in 16 regions of rat brain and in heart and liver. NAAG was undetectable in heart and liver and exhibited 10-fold variation in concentration among brain regions; the highest levels were found in spinal cord. In contrast, low concentrations of NAA were detectable in heart and liver, and the regional distribution of NAA in brain varied only twofold. The regional distribution of NAA and NAAG correlated poorly. To assess the neuronal localization of these two compounds, the effects of selective brain lesions on their levels were examined. Decortication caused a 28% decrease in NAAG levels in the ipsi-lateral striatum while NAA decreased 38%. Kainate lesion of the striatum resulted in a 31% decrease in NAAG in the ipsilateral striatum, whereas NAA fell by 58%. Kainate lesion of the hippocampus resulted in significant decrements in NAAG and NAA in the hippocampus and septum. Transection of the spinal cord at midthorax resulted in a 51% decrease in NAAG levels immediately caudal and a 40% decrease immediately rostral to the lesion; however, NAA decreased only 30% in these areas. These results are consistent with a neuronal localization of NAAG in brain. Combined with the fact that NAAG interacts with a subpopulation of glutamate receptors, these results suggest that NAAG may serve as an excitatory neurotransmitter.     

Glutathione mixed disulfide inhibitors of the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase

Six glutathione-containing inhibitors of the human NADP-linked 15-hydroxyprostaglandin dehydrogenase have been isolated from placental homogenates. Glutathione disulfide is one of these inhibitors. Although the structures of the other five have not been fully elucidated, all are disulfides. Studies with these compounds and with other mixed disulfides have shown that the glutathione mixed disulfides of β-mercaptopyruvate, mercaptoacetate, and β-mercaptolactate are more effective inhibitors of the enzyme than are the glutathione-containing mixed disulfides isolated from placental homogenates. β-Mercaptolactate is particularly noteworthy because of its low K_j (0.13 μM). The results reported here suggest that the activity of the prostaglandin dehydrogenase may be influenced by various glutathione mixed disulfides.     

Ammonium ions prevent methylation of uridine to ribothymidine inAzotobacter vinelandii tRNA

It was shown that tRNA fromAzotobacter vinelandii grown in the presence of ammonium chloride lacks ribothymidine while that grown in the absence of the ammonium salt contains this modified nucleoside. [³²P]-Labelled tRNA from this organism grown in a medium containing the ammonium salt was digested with RNase T1 and the pseudouridinecontaining tetranucleotide, common to all tRNAs was isolated and analysed for the nucleoside replacing the ribothymidine. It was found to be uridine. Cells previously labelled with [³²P]-phosphate in the ammonium salt medium were washed and incubated in the ammonium saltfree medium to test whether ribothymidine would be formed upon removal of the ammonium ions. Methylation of the uridine did not take place.     

Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and beta-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate.

Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdown and intracellular Ca2+ ([Ca2+]i) mobilization by phorbol esters in platelets. In this study, we have examined the effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced granule secretion and correlated it with agonist-induced [Ca2+]i mobilization, arachidonate and thromboxane (Tx) release in human platelets. With increasing times of incubation with PMA (10 s-5 min), the rise in [Ca2+]i induced by thrombin and the TxA2 mimetic, U46619, was increasingly inhibited (90-100% with 5 min incubation) and, correlating with this, thrombin-induced [3H]arachidonate, TxB2 and beta-thromboglobulin (beta TG) release were also inhibited. In addition, the conversion of exogenously added arachidonate to TxB2 was inhibited (50-80%) by a 10 s-5 min pretreatment with PMA. However, secretion of 5-hydroxy[14C]tryptamine (5HT) induced by thrombin or U46619 was not inhibited by 10 s-2 min incubations with PMA and, on the contrary, with low agonist concentrations, was potentiated by PMA in the absence of a significant rise in [Ca2+]i or endogenous Tx formation, to levels significantly greater than or equal to the sum of that obtained when agonist and PMA were added separately. With longer times of incubation with PMA (5 min), these synergistic effects became less pronounced as inhibitory effects of PMA on agonist-induced [14C]5HT secretion became apparent. The results indicate that, while PMA may cause an inhibition of agonist-induced [Ca2+]i mobilization resulting in an inhibition of agonist-induced arachidonate, TxB2 and beta TG release, its effects on agonist-induced 5HT secretion may be complicated by [Ca2+]i-independent synergistic effects of agonist and PMA.     

Enzyme-linked immunosorbent assay for secalonic acid D

A competitive enzyme-linked immunosorbent assay was developed for the xanthone dimer secalonic acid D. The immunogen and enzyme marker were prepared by direct reaction of secalonic acid D with bovine serum albumin and horseradish peroxidase, respectively. The resultant conjugates were characterized by UV/VIS spectra and thin layer chromatography. The hapten:protein ratios in the conjugates were estimated by difference UV/VIS spectra and by fluorescent techniques. Immunization procedures were conducted utilizing New Zealand rabbits over a period of 12 weeks. The competitive enzyme-linked immunosorbent assay on microtiter plates showed that secalonic acid D was detectable within a range of 250–25 000 ng/assay.     

Polyamine depletion increases cellular ribonucleotide levels

Summary Depletion of the putrescine and spermidine content of Ehrlich ascites tumor cells by -difluoromethylornithine (DFMO) treatment results in at least a 1500-fold increase in the decarboxylated S-adenosylmethionine (deSAM) content. The accumulation of this adenine nucleoside occurs because of the absence of putrescine and spermidine to act as aminopropyl group acceptors in the spermidine and spermine synthase reactions and because of an increase in S-adenosylmethionine decarboxylase activity. The fact that the synthesis of deSAM continues in DFMO-treated cells makes the pathway an adenine trap. This prompted a study of the adenine nucleotide pools. High-performance liquid chromatographic analysis showed that the total adenine nucleotide pool increased, rather than decreased, as a result of DFMO treatment; the major contributors to the increase being ATP and ADP, which increased 2.6 and 1.9 times, respectively. The cellular content of other ribonucleotides increased as well, particularly that of UTP and CTP. When putrescine was added together with DFMO, the increases in cellular ribonucleotide contents were prevented, showing that they were indeed caused by polyamine depletion.     

Dye coupling in the organ of Corti

Summary Dye-coupling in an in vitro preparation of the supporting cells of the guinea-pig organ of Corti was evaluated by use of the fluorescent dyes, Lucifer Yellow, fluorescein and 6 carboxyfluorescein. Despite the presence of good electrical coupling in Hensen cells (coupling ratios >0.6) the spread of Lucifer yellow was inconsistent. Hensen cells are very susceptible to photoinactivation, i.e., cell injury upon illumination of intracellular dye; and this in conjunction with Lucifer Yellow's charge and K+-induced precipitability may account for its variability of spread. Fluorescein and 6 carboxyfluorescein, on the other hand, spread more readily and to a greater extent than Lucifer Yellow, often spreading to cell types other than those of Hensen. Dye spread is rapid, occurring within a few minutes. These results suggest that molecules of metabolic importance also may be shared by the supporting cells of the organ of Corti.     

A reinvestigation of the Gn-RH (gonadotrophin-releasing hormone) systems in the goldfish brain using antibodies to salmon Gn-RH

Summary The organization of Gn-RH systems in the brain of teleosts has been investigated previously by immunohistochemistry using antibodies against the mammalian decapeptide which differs from the teleostean factor. Here, we report the distribution of immunoreactive Gn-RH in the brain of goldfish using antibodies against synthetic teleost peptide.Immunoreactive structures are found along a column extending from the rostral olfactory bulbs to the pituitary stalk. Cell bodies are observed within the olfactory nerves and bulbs, along the ventromedial telencephalon, the ventrolateral preoptic area and the latero-basal hypothalamus. Large perikarya are detected in the dorsal midbrain tegmentum, immediately caudal to the posterior commissure. A prominent pathway was traced from the cells located in the olfactory nerves through the medial olfactory tract and along all the perikarya described above to the pituitary stalk. In the pituitary, projections are restricted to the proximal pars distalis. A second immunoreactive pathway ascends more dorsally in the telencephalon and arches to the periventricular regions of the diencephalon. Part of this pathway forms a periventricular network in the dorsal and posterior hypothalamus, whereas other projections continue caudally to the medulla oblongata and the spinal cord. Lesions of the ventral preoptic area demonstrate that most of the fibers detected in the pituitary originate from the preoptic region.     

Fine structure of the malaria parasite Plasmodium falciparum in human hepatocytes in vitro

Summary Recent advances in the ability to culture the hepatic forms of mammalian malaria parasites, particularly of the important human pathogen Plasmodium falciparum have provided novel opportunities to study the ultrastrucural organisation of the parasite in its natural host cell the human hepatocyte. In this electron-microscopic and immunofluorescence study we have found the morphology of both parasite and host cell to be well preserved. The exoerythrocytic forms, which may be found at densities of up to 100/cm², grow at rates comparable to that in vivo in the chimpanzee. In the multiplying 5- and 7-day schizogonic forms the ultrastructural organisation of the parasite bears striking resemblances to other mammalian parasites, e.g., the secretory activity and distribution of the peripheral vacuole system, but also homology with avian parasites, e.g., in nuclear and nucleolar structure and mitochondrial form. The latter homologies support earlier suggestions of the close phylogenetic relationship of P. falciparum with the avian parasites. Evidence is also presented showing the persistence of the cytoskeleton of the invasive sporozoite within the cytoplasm of the ensuing rapidly growing vegetative parasites.