Characterization of a Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Comparison with a 6S-Form

Abstract: 6R-l -erythro-Tetrahydrobiopterin (6R-BH₄) is a cofactor for aromatic l -amino acid hydroxylases and nitric oxide synthase. Recently, we have reported that independently of its cofactor activities, 6R-BH₄ acts from the outside of neurons in the brain to enhance the release of monoamine neurotransmitters such as dopamine. To characterize the pharmacological properties of the action, we examined the effects of 6S-BH₄, a diastereoisomer of 6R-BH₄, on dopamine release in the rat striatum by using brain microdialysis and compared its effects with those of 6R-BH₄. Perfusion of 6S-BH₄ or 6R-BH₄ through the dialysis probe increased extracellular dopamine levels (an index of in vivo dopamine release) concentration dependently; the maximal increase by 6S-BH₄, was one-sixth of that by 6R-BH₄. 6S-BH₄ increased extracellular DOPA levels in the presence of NSD 1015, an inhibitor of aromatic l -amino acid decarboxylase (an index of in vivo tyrosine hydroxylase activity), to an extent similar to the increase induced by 6R-BH₄. The increase in the DOPA levels induced by either of the pteridines was abolished after pretreatment of rats with α-methyl-p-tyrosine (an inhibitor of tyrosine hydroxylase). Under the same conditions, the 6S-BH₄-induced dopamine release was abolished, but most of the 6R-BH₄-induced increase persisted. Coadministration of 6S-BH₄ with 6R-BH₄ inhibited the increase in dopamine release induced by 6R-BH₄ alone. These results show that 6R-BH₄ stimulates dopamine release by acting at the specific recognition site on the neuronal membrane, and that 6S-BH₄ acts as an antagonist of 6R-BH₄ at this site, although it has cofactor activities.     

Localization of the antigen for the monoclonal antibody ②9F11-B-E4 interspecific cross-reaction in the freshwater triclads

The localization of the antigen for monoclonal antibody 9F11-B-E₄ was clarified by immuno-electron microscopy. The antigens were localized on the mitochondria and Golgi bodies in the male germ cells and on the secretory granules of various glands cells in the penis bulb and subepidermal parenchymal tissue of Phagocata vivida. The results of the interspecific cross-reaction tests with seven other freshwater triclads showed that these secretory granules are species-specific. A positive interspecific reaction was showed with Dugesia (family Dugesiidae), but not with Polycelis within the same family Planariidae which suggests the position of Phagocata within the Planariidae needs to be reassesed.     

Expression and control of an operon from an intracellular symbiont which is homologous to the groE operon.

Members of the genus Buchnera are intracellular symbionts harbored by the aphid bacteriocyte which selectively synthesize symbionin, a homolog of the Escherichia coli GroEL protein, in vivo. Symbionin and SymS, a GroES homolog, are encoded in the symSL operon. Northern blotting and primer extension analyses revealed that the symSL operon invariably gives rise to a bicistronic mRNA under the control of a heat shock promoter, though the amount of the symSL mRNA in the isolated symbiont did not increase in response to heat shock. The sigma32 protein that recognizes the heat shock promoter in E. coli was scarcely detected in Buchnera cells even after heat shock. Although the functionally essential regions of the Buchnera sigma32 protein were well conserved, the Buchnera rpoH gene did not complement an E. coli delta rpoH mutant. On the one hand, the A-T evolutionary pressure imposed on the Buchnera genome may have not only decreased the activity of its sigma32 but also ruined the nucleotide sequences necessary for the expression of rpoH; on the other hand, it may have facilitated expression of the symSL operon without activation by sigma32.     

DIVERSITY OF HALOGENATED SECONDARY METABOLITES IN THE RED ALGA LAURENCIA NIPPONICA (RHODOMELACEAE,CERAMIALES)1,2

Many morphologically similar, but chemically distinct, populations have been found in the marine red alga Laurencia nipponica Yamada (Rhodomelaceae, Ceramiales) growing in Japan. Each chemical type is characterized by a specific end-product of halogenated secondaly metabolite synthesis: chamigrane-type sesquiterpenoids such as prepacifenol and halochamigrene epoxide and C₁₅ bromoethers such as laurencin, laureatin, isoprelaurefucin, epilaurallene, and kumausallene. These seven types of secondary metabolite syntheses remained the same in the wild and under various culture conditions. Because bromoethers and terpenoids are probably synthesized by different metabolic pathways, it is virtually certain that different sets of enzymes participate in their synthesis. Prepacifenol- and laureatin-producing populations were selected as representatives of terpenoid and bromoether groups, respectively. F₁ tetrasporophytes derived from crosses between reciprocal, female and male gametophytes of prepacifenol- and laureatin-producing strains bore both types of metabolites, suggesting that the genes Producing these enzyme systems are encoded by nuclear genomes. The F₁ gametophytes resulting from the reciprocal crosses produced either prepacifenol or laureatin, and the four individuals derived from spore tetrads (a set of tetraspores derived from a single tetrasporangium) produced either prepacifenol or laureatin in a 1:1 ratio, indicating that genes participating in terpenoid synthsis and those participating in bromoether synthesis are on different loci of homologous chromosomes and are segregated at meiosis (tetrasporogenesis). One individual of this interpopulational F₁ gamtophyte produced both parental types of metabolite, perhaps indicating the occurrence of a recombination type. Natural hybrid individuals, including such recombination-type gametophytes, were found in a sympatric locality at which these two chemical types occur. F₁ tetrasporophytes derived from crosses between respective prepacifenol- and laureatin-producing strains and their F₁ gametohytes produced only parental-type metabolite-producing plants. These results indicate that the diverse chemical types can be referred to as races (chemical races).     

Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects

Isono, Shiroh, John E Remmers, Atsuko Tanaka, Yasuhide Sho,Jiro Sato, and Takashi Nishino. Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects.J. Appl. Physiol. 82(4):1319-1326, 1997.Anatomic abnormalities of the pharynx arethought to play a role in the pathogenesis of obstructive sleep apnea(OSA), but their contribution has never been conclusively proven. Thepresent study tested this anatomic hypothesis by comparing themechanics of the paralyzed pharynx in OSA patients and in normalsubjects. According to evaluation of sleep-disordered breathing (SDB)by nocturnal oximetry, subjects were divided into three groups: normalgroup (n = 17), SDB-1(n = 18), and SDB-2(n = 22). The static pressure-arearelationship of the passive pharynx was quantified under generalanesthesia with complete paralysis. Age and body mass index werematched among the three groups. The site of the primary closure was thevelopharynx in 49 subjects and the oropharynx in only 8 subjects.Distribution of the location of the primary closure did not differamong the groups. Closing pressure(PC) of the velopharynx forSDB-1 and SDB-2 groups (0.90 ± 1.34 and 2.78 ± 2.78 cmH₂O, respectively) wassignificantly higher than that for the normal group (3.77 ± 3.44 cmH₂O;P < 0.01). Maximal velopharyngealarea for the normal group (2.10 ± 0.85 cm²) was significantly greaterthan for SDB-1 and SDB-2 groups (1.15 ± 0.46 and 1.06 ± 0.75 cm², respectively). Theshape of the pressure-area curve for the velopharynx differed betweennormal subjects and patients with SDB, being steeper in slope nearPC in patients with SDB.Multivariate analysis of mechanical parameters and oxygen desaturationindex (ODI) revealed that velopharyngealPC was the only variable highly correlated with ODI. VelopharyngealPC was associated withoropharyngeal PC, suggestingmechanical interdependence of these segments. We conclude that thepassive pharynx is more narrow and collapsible in sleep-apneic patientsthan in matched controls and that velopharyngeal PC is the principal correlate ofthe frequency of nocturnal desaturations.

     

Oleic acid transformations by selected strains of Sphingobacterium thalpophilum and Bacillus cereus from composted manure

In a survey of 186 randomly selected microbial strains isolated from composted manure, 63 transformed oleic acid into three types of products: hydroxy fatty acid, fatty amide, and less polar oleyl lipid. Selection of oleic acid-transforming microorganisms was enhanced in nutrient agar supplemented with 0.1% (vol/vol) oleic acid at pH 7.2. Most of the 63 diverse isolates elicited inconsistent and poorly reproduced transformations. However, strains 142b (NRRL B-14797) transformed oleic acid to 10-hydroxystearic acid consistently, and strain 229b (NRRL B-14812) produced an octadecenamide. Taxonomic studies indicated that NRRL strain B-14797, possessing 1,3-dihydroxy-2-amino-15-methylhexadecane and sphinganine bases, was closely related to Sphingobacterium thalpophilum, and NRRL B-14812 was identified as Bacillus cereus.     

Hepatic dysfunction in primary hypothyroidism

Twenty-seven patients with primary hypothyroidism were studied to evaluate the relationship between hepatic function and thyroid hormone deficiency in this disorder. In hypothyroidism, hypergammaglobulinemia was found in 71%, elevated glutamic oxaloacetic transaminase (GOT) in 48%, high lactic dehydrogenase (LDH) in 58%, hypercholesteremia in 52% and low elimination rate constant of indocyanin green (KICG) in 44%. In each criterion of liver function, these patients were divided into two groups, normal level and abnormal level group, respectively. T3 and T4 in patients with abnormal levels of GOT, glutamic pyruvic transaminase (GPT), gamma-glutamyl transpeptidase (gamma-GTP), leucine aminopeptidase (LAP), alkaline phosphatase (ALP) and 45 minutes retention rate of bromsulphalein (BSP) were not different from those in the normal level group. However, T3 and T4 in patients with abnormal levels of LDH, cholesterol, cholinesterase (ChE) and KICG were lower than those in the normal level group. The abnormal KICG group had a statistically higher cardio-thoracic ratio (CTR) than the normal group (65.7 +/- 18.8% vs 50.4 +/- 8.3%, p less than 0.05). In patients with pericardial effusion, CTR was 65.9 +/- 14.6%, while that in patients without pericardial effusion was 49.9 +/- 7.5% (p less than 0.05). These abnormalities of liver function were normalized in all cases after hormone replacement therapy. Liver biopsy in three cases disclosed normal liver in two cases and mild infiltration of monocyte into Glisson's capsule in one case.(ABSTRACT TRUNCATED AT 250 WORDS)     

The chick oviduct in tissue culture. I. Initial characterization of growing primary oviduct tissue cultures

A simple three-enzyme treatment of collagenase, dispase and hyaluronidase on finely minced chick oviduct yields clumps of 50-150 cells. These cells attach to collagen-treated dishes and survive in culture for at least 2 weeks without subculturing. Oviduct cell cultures can also be induced to grow. Estradiol or epidermal growth factor (EGF) induce a 40% increase in cells in 4 days when cultures are grown in serum levels that do not support growth. Serum from estrogen-stimulated chicks promotes rapid cellular proliferation (doubling times of 1-2 days). Sera from estrogen withdrawn chicks, laying hen or horse do not support as rapid proliferation. The oviduct growth-promoting factors in serum from estrogen-stimulated chicks are not steroids or fibroblast growth factors (FGF). Removal of steroids from these sera by charcoal treatment or delipidization does not decrease the rate of growth. The addition of 1-100 nM estradiol does not increase a serum's ability to promote growth. Purified FGF or platelet-derived growth factor (PDGF) do not induce oviduct proliferation. These results were reproduced in oviduct cell cultures started from estrogen-stimulated and withdrawn chicks as well as laying hens. Thus the factors in serum from estrogen-stimulated chicks that promote rapid oviduct growth are induced by estrogen treatments in vivo, but do not seem to be only steroids.     

Hormonal effects on the development changes of mouse small intestinal glycolipids

The composition of intestinal glycosphingolipids during normal and hormone-perturbed development was investigated. The concentrations of glycosphingolipids of mouse small intestine were affected by the injection of thyroxine or cortisone during suckling and weaning periods. GDla was reduced by the hormonal treatment among major gangliosides, GM3, GM1 and GD1a, of mouse small intestine during the suckling period. In contrast, asialo GM1 was precociously produced by the treatment, which scarcely found in control suckling mouse small intestine. The results showed that these hormones were related to developmental alteration of small-intestinal glycolipids.     

Transmembrane control of laminin, lectin receptors and surface villi in teratocarcinoma-derived endodermal cells

Distribution of laminin on the surface of teratocarcinoma-derived parietal endoderm cells was studied by immuno-histochemical staining of the fixed specimen using affinity-purified anti-laminin antibody. Laminin was distributed on the basal surface of the cells, while treatment either with colchicine or with cytochalasin D (CD) resulted in a severely polarized distribution; laminin was seen only at one end of the cell. Treatment with both the reagents did not cause the severe polarization. Receptors for lectins and cell surface villi were polarized by treatment with CD but not by treatment with colchicine. These results suggest that laminin--or its cell surface receptor--is linked to both microfilament and microtubules and that the mode of transmembrane control for laminin is different from certain other cell surface components of the cells.