6R-L-erythro-5,6,7,8-tetrahydrobiopterin as a regulator of dopamine and serotonin biosynthesis in the rat brain

6R-L-Erythro-tetrahydrobiopterin (6R-BH4), the natural isomer of tetrahydrobiopterin, was synthesized from 7,8-dihydrobiopterin using dihydrofolate reductase. The effects of intracerebroventricular injection of 6R-BH4 on the biosyntheses of neurotransmitter monoamines in the rat brain were investigated by measuring accumulation of 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) after the inhibition of aromatic L-amino acid decarboxylase by NSD 1015 and the contents of metabolites of dopamine (DA) and 5-hydroxytryptamine (5-HT). The formation of DOPA and 5-HTP increased after the injection, reached a maximum level at about 1 h, then leveled off and reached a plateau over 2 h up to 8 h. The formation of DOPA and 5-HTP increased dose-dependently in the whole brain after the injection of 6R-BH4, and reached a plateau when the dose was more than 300 micrograms/rat. The enhancement was 100 and 70% for DOPA and 5-HTP, respectively. The formation of DOPA and 5-HTP increased in four brain regions, but the degree of stimulation was different among the brain regions. The contents of DA and 5-HT metabolites increased after the injection of 6R-BH4 in all brain regions tested, especially in the diencephalon and brain stem. The contents of DA and 5-HT increased slightly after the injection of 6R-BH4. These results suggest that 6R-BH4 concentration may be submaximal within DA and 5-HT neurons, and that an increase in 6R-BH4 in the brain enhances the biosyntheses of DA and 5-HT.     

Inactivation and sedimentation of mercury from organomercurials by Klebsiella pneumoniae

Abstract A susceptibility of 63 clinical isolates of Klebsiella pneumoniae to inorganic and organic mercuric compounds was determined. 18 of them were found to be resistant to fluorescein mercuric acetate (FMA) and merbromin (MB). Moreover, all the resistant strains inactivate the antibacterial effect of FMA. The changes in the amount of organic mercury at the time of inactivation of the drug and the structures of the end products were examined in detail with the plasmid-bearing strain JK9 and its transconjugants of Escherichia coli .
The results showed that FMA was inactivated by an intracellular enzyme produced inducively and was degraded to fluorescein (sodium salt, uranine), which led to the sedimentation of metallic mercury. The discovery of the genes conferring inducible organic mercury-inactivating enzymes determined by plasmids was the next step and their application in the recovery of metallic mercury from organomercurials is now imminent.     

Spider toxin (JSTX) on the glutamate synapse

A new neurotoxin (JSTX) was separated from spider (Nephila clavata, Joro spider) venom. JSTX irreversibly suppressed the excitatory postsynaptic potential (EPSP) and the glutamate potential in the lobster neuromuscular junction with high degree of specificity. The threshold concentration for suppressing EPSPs corresponds to a small fraction of the toxin in a venom gland, roughly estimated as low as 10(-10) M/l. 10(-10) M/l. In the giant synapse of squid stellate ganglion JSTX suppressed EPSPs without affecting the antidromic response. Glutamate-induced membrane depolarization was blocked by JSTX. In mammalian brain slice preparation, JSTX suppressed the orthodromic spike response but failed to affect on the antidromic spike in the hippocampal pyramidal neuron of CA1 and CA3 region. The above results strongly support the view that the squid giant synapse and synapses in the hippocampal pyramidal neuron are mediated by glutamate.     

R-Factor Mutant Capable of Specifying Hypersynthesis of Penicillinase

The physical characteristics of a mutant, R(M201-2), capable of conferring high and stable ampicillion resistance was analyzed. The R(M201-2) and its parent R-factor deoxyribonucleic acid (DNA) could be isolated as an extrachromosomal and covalently closed circular form. Their buoyant densities were both 1.712 g/cm(3), and their molecular weights were about 82 x 10(6) and 64 x 10(6), respectively, when measured by CsCl and sucrose density gradient analyses. The contour lengths by electron microscopy were 35.9 +/- 0.6 and 31.0 +/- 0.6 mum, respectively. By using the extracted R-factor DNA, the mutant and parent characters were transformable to another Escherichia coli strain. The mutant R factor showed an increased amount of DNA even after conjugal transfer to Proteus. An increase in the size of R-factor DNA was thus considered to be the cause of the high level of ampicillin resistance.     

Genetic analysis of streptomycin-resistance and-dependence inChlamydomonas

Summary Three non-chromosomal and two chromosomal genes which influence resistance to streptomycin are described. Each of the non-chromosomal factors,sr-500,sr-1500, andsd, exhibits uniparental inheritance, with all progeny receiving the factor when it is carried by the parent of mating-typeplus, and none when it is carried by the mating-typeminus parent. The streptomycin-dependence factor,sd, shows zygotic dominance when contributed by the mating-typeplus parent, but not when coming from the mating-typeminus parent, indicating that the uniparental transmission results from events occurring within the zygote early in maturation and well before meiosis. The chromosomal geneA interacts both with chromosomal and non-chromosomal genes at the biochemical level, but does not alter their patterns of inheritance.With 1 Figure in the TextThis paper is dedicated to ProfessorL. C. Dunn in gratitude to him as teacher and advisor, on the occasion of his retirement.This work was supported by grants from the U.S. Public Health Service and the National Science Foundation. The generosity and interest of ProfessorFrancis J. Ryan in providing laboratory space is gratefully acknowledged, as is the technical assistance of MissFran Yablonsky.     

High-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclodextrin-containing mobile phase

The high-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclodextrin-containing mobile phase is presented. Compared with conventional methods, inclusion chromatography gives much more satisfactory separation of derivatized bile acids in a short time. The application of this method to the separation of glycine-conjugated bile acids in human bile is also described.     

Reconstituted F1-ATPase complexes containing one impaired beta subunit are ATPase-active

Homogeneous populations of hybrid alpha 3 beta 3 gamma complexes of the thermostable F1-ATPase containing one, two, or three copies of the mutationally impaired beta subunits were produced using the solid phase reconstitution method. Two kinds of mutated beta subunits were used for the reconstitution, one of which lacked the ability to bind any adenine nucleotides. The complexes containing one impaired beta and two wild-type beta subunits retained a significant amount of ATPase activity with cooperative kinetics, whereas those containing two or three impaired beta subunits showed very little ATPase activity. These results imply that the catalysis of steady-state ATP hydrolysis can proceed even if one of the three beta subunits in F1-ATPase is not functional.     

Enzymatic Inactivation of Extracellular Carbonic Anhydrase and its Effect on K1/2 (CO2) for Photosynthesis in Chlorella ellipsoidea C-27

The ratio of the extracellular to the intracellular activityof carbonic anhydrase (CA) in cells of Chlorella ellipsoideaC-27, adapted to low levels of CO₂ for 24 h (low-CO₂ cells),was about one to one. Treatment of intact cells with PronaseP inactivated about one-half of the extracellular CA activitywithout affecting photosynthetic activity. The CA activity incell homogenates and in cell-wall ghosts liberated during celldivision was completely inactivated by the same treatment. Pretreatmentwith Glycosidase mix, Chitosanase and Macerozyme enhanced theinactivation of the CA activity in intact cells. These resultssuggest that extracellular CA is evenly distributed throughoutthe whole cell-wall region. The apparent K_1/2 for dissolved inorganic carbon (DIC) in low-CO₂cells doubled when extracellular CA was inactivated by treatmentwith Pronase P, but the K_1/2 obtained was still one-half ofthat in high-CO₂ cells. Photosynthetic ¹⁴CO₂-fixation in low-CO₂cells was enhanced by acetazolamide, whereas H¹⁴CO₃^–-fixationwas suppressed. The results suggest that CO₂ is a dominant substrateutilized by cells and that HCO₃^– is utilized after conversionto CO₂. The present results show that both intracellular andextracellular CA contribute to the increase in affinity forDIC during photosynthesis in low-CO₂ cells of Chlorella ellipsoideaC-27. (Received May 7, 1990; Accepted July 18, 1990)