Kinetic properties of a L-cysteine desulfhydrase-deficient mutant in the enzymatic formation of L-cysteine from D,L-ATC

Summary A mutant strain lacking in activity of L-cysteine desulfhydrase, a L-cysteine-decomposing enzyme, was screened after UV-treatment ofPseudomonas sp. CU6. The properties of the two strains, original and mutant, were compared on the basis of parameter values estimated from kinetic simulations of the enzymatic formation of L-cysteine from D,L-ATC. Both strains suffered from product inhibition, though inhibition was less for the mutant strain.     

Production of recombinant human glucagon in the form of a fusion protein in Escherichia coli; recovery of glucagon by sequence-specific digestion

Summary Recombinant human glucagon was succesfully produced with a high level of expression in Escherichia coli as a fusion protein with human interferon . The synthetic gene was designed to release glucagon, which does not contain glutamic acid residues, from fusion protein with the Staphylococcus aureus strain V8 protease that specifically cleaves the peptide bond on the carboxyl side of the glutamic acid residue. The resulting glucagon was purified to homogeneity by a combination of C₁₈ reverse-phase HPLC and ion-exchange HPLC. The yield of intact glucagon obtained from 11 of culture was approximately 12 mg. The structure of recombinant human glucagon was confirmed by HPLC and amino acid composition/sequence analyses. Offprint requests to: J. Ishizaki     

Partitioning of fluorescent phospholipid probes between different bilayer environments. Estimation of the free energy of interlipid hydrogen bonding.

Fluorescence spectroscopy has been used to monitor the partitioning of a series of exchangeable neutral phospholipid probes, labeled with carbazole, indolyl or diphenylhexatrienyl moieties, between large unilamellar vesicles containing 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1,2-dioleoyloxy-3-(trimethylammonio) propane (DOTAP) or N-hexadecyl-N-(9-octadecenyl)-N,N-dimethylammonium chloride (HODMA). Phosphatidylethanolamine (PE) probes desorb from POPC-containing vesicles at markedly slower rates than do phosphatidylcholine (PC) probes with the same acyl chains. The rate of probe desorption from such vesicles is progressively enhanced by successive N-methylations of the amino group but not by methylation of C-2 of the ethanolamine moiety, a modification that leaves unaltered the hydrogen-bonding capacity of the polar headgroup. By contrast, the rates of desorption of different probes (with the same acyl chains) from HODMA or from DOTAP vesicles are much more comparable and reflect no clear systematic influence of the headgroup hydrogen-bonding capacity. Equilibrium-partitioning measurements indicate that the relative affinities of different probes for PC-rich vesicles, in competition with HODMA or DOTAP vesicles, increase with increasing hydrogen-bonding capacity of the probe headgroup in the order PC less than N,N-dimethyl PE less than N-methyl PE less than PE approximately phosphatidyl-2-amino-1-propanol. From such partitioning data, we estimate that interlipid hydrogen-bonding interactions (in competition with lipid-water interactions) contribute roughly -300 cal mol-1 to the free energy of a PE molecule in a hydrated liquid-crystalline phospholipid bilayer; this free-energy contribution is somewhat smaller, but still significant, for N-mono- and dimethylated PE's.     

Identification of new urinary metabolites of trimeprazine in rats by gas chromatography—mass spectrometry

The metabolites of trimeprazine were identified in urine of rats by gas chromatography—mass spectrometry. After the oral administration of trimeprazine, the urinary metabolites were extracted with diethyl ether before or after hydrolysis with β-glucuronidase. The identified metabolites were N-demethyltrimeprazine, 3-hydroxytrimeprazine, N-demethyl-3-hydroxytrimeprazine and trimeprazine sulphoxide.     

Estimation of the degree of self-fertilization in Porphyra yezoensis (Bangiales,Rhodophyta)

Crosses between genotypically distinct thalli of the monoecious species Porphyra yezoensis were carried out using immature thallus fragments from green- and red-type color mutants and also wild-type thalli. As the genes governing the mutants are monogenic, recessive to the wild-type, and belong to the same linkage group, the degree of self-fertilization could be estimated based on the pigmentation of the resultant diploid conchocelis. The degree of self-fertilization in the cross between the green-type and the wild-type was 48.5–55.0%, and in the cross between the red-type and the wild-type was 45.1–56.5%. In the cross between the green- and red-type mutants, the degree of self-fertilization was 46.0–54.5% when the green-type was the female parent, and was 44.8–55.6% when the red-type was the female parent.     

Analysis of E. coli phoA-lacZ fusion gene expression inserted into a multicopy plasmid and host cell's chromosome

The production of beta-galactosidase from the E. coli phoA-lacZ fusion gene was studied to compare the gene expression behavior of two cloning methods: insertion to multicopy plasmids and integration into host cell's chromosome. The chromosome-integrating strain showed more tight control of fusion gene expression levels than the plasmid-containing strain. A 100-fold enhancement of specific beta-galactosidase activity in the former strain was achieved in response to changes of initial inorganic phosphate concentration from 1 to 0.1 mM, whereas a 26-fold increase was observed in the latter strain. The low degree of overexpression in the plasmid-bearing cells was due to a combination of factors including leaky expression in repressed conditions and limitation of biosynthetic machinery in derepressed conditions. In a mixture of inorganic and organic phosphates, inorganic phosphate levels in the medium exhibited oscillatory behavior. The oscillation of inorganic phosphate is attributed to selective usage of inorganic phosphate followed by hydrolysis of organic phosphate to inorganic by alkaline phosphatase. The fluctuation of inorganic phosphate levels also caused the oscillation of beta-galactosidase activity.     

Effects of heat treated cells of intestinal lactic acid bacteria in rats fed a deoxycholic acid diet

Effects of the administration of heat treated cells of intestinal lactic acid bacteria were examined in rats fed a deoxycholic acid (DCA) diet. Male Wistar rats were given a 0.25% DCA diet with or without the heat treated cells of Enterococcus faecalis AD 1001 (EFH-1) or Lactobacillus reuteri AD 0002 (LRH-2) for 4 weeks. Abnormal increases in serum GOT, GPT, UN and lipoproteins were observed in the rats fed the DCA diet. Furthermore, severe lesions in the kidney as well as in the liver were found in these rats. On the contrary, the increases in serum GOT, UN, VLDL and LDL were significantly suppressed, and markedly fewer lesions in the liver and the kidney were observed in the rats fed the DCA diet plus EFH-1 or LRH-2.     

Characteristics of a circadian pacemaker in the suprachiasmatic nucleus

Summary The nature of the circadian rhythms of the SCN in a hypothalamic island was examined in male rats by recording multiple unit activity from the SCN for longer durations. Successful continuous recording lasted up to 35 days. Neural activity of the SCN inside the island showed free-running rhythms whose periods were slightly longer than 24 h (Figs. 2, 3, Table 1). When the retino-hypothalamic pathway was spared, re-entrainment to a displaced light and dark cycle was attained following a transition period of a few days (Fig. 4). Phases of the rhythms shifted in a phase-dependent manner in response to single light pulses interrupting constant darkness (Fig. 5 and Fig. 6). These results suggest an endogenous nature of the circadian rhythm of the SCN within the hypothalamic island. Thus, neurons or neuronal networks in the SCN may have not only an inherent ability to generate a circadian rhythm, but also an intricate machinery to regulate its phase. Simultaneous recordings from the left and right SCN showed a slight but visible discrepancy in their phases between the two rhythms in 3 out of 12 cases (Fig. 7).Abbreviations LL constant light - LD light-dark - DD constant darkness - SCN Suprachiasmatic nucleus     

Direct infection of hepatocytes by sporozoites of Plasmodium berghei

To identify the unknown liver cell type initially invaded by sporozoites of mammalian malaria, young rats were inoculated intravenously with large numbers of Plasmodium berghei sporozoites obtained from infected Anopheles stephensi mosquitoes. Fine structural studies of liver specimens obtained from the rats within 2 min after inoculation demonstrated the presence of morphologically unaltered sporozoites in the cytoplasm of hepatocytes. Many sporozoites were also observed undergoing cytolysis within the lysophagosomes of Kupffer cells, as well as other phagocytic cells. These observations strongly suggest direct infection of the hepatocyte by the sporozoite.     

Interrelation of Two Forms of Ferredoxin-NADP+ Reductase with Different Molecular Weights

The heavier form of ferredoxin-NADP⁺ reductase was extractedfrom spinach leaves or chloroplasts and isolated as the majorfraction at high ionic strengths with ammonium sulfate or sodiumchloride. At low ionic strengths, the form with the higher molecularweight was relatively unstable and was converted gradually intothe lower form. We concluded that the enzyme exists in vivoas the form with the higher molecular weight. ¹Present address: Market Development Department, Shionogi &Co. LTD., 5-Sagisu, Fukushima-ku, Osaka 553, Japan. (Received December 16, 1980; Accepted January 19, 1981)