Formation of 12-oxo-trans-10-dodecenoic acid in chloroplasts from Thea sinensis leaves

Linolenic acid-[1-¹⁴C] was converted to 12-oxo-trans-10-dodecenoic acid, via 12-oxo-cis-9-dodecenoic acid by incubation with chloroplasts of Thea sinensis leaves. Thus, it was confirmed that linolenic acid is split into a C₁₂-oxo-acid, 12-oxo-trans-10-dodecenoic acid, and a C₆-aldehyde, trans-2-hexenal, leaf aldehyde, by an enzyme system in chloroplasts of tea leaves.     

A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.     

Excretion of peroxidase from horseradish hairy root in combination with ion supplementation

Summary The effect of ion-supplemented medium on peroxidase excretion from horseradish (Armoracia rusticana) hairy roots was studied. Supplementation of mannitol instead of ions revealed that the excretion was stimulated not by osmotic pressure in the medium but by ionic properties. Extracellular peroxidase activity per dry cell was proportionally correlated with the ionic strength of the cations. CaCl₂ or MgCl₂ was found to be the most effective agent for excretion among other combinations. CaCl₂ supplementation at the beginning of the culture caused higher peroxidase production in the medium without a significant loss of final cell mass compared with CaCl₂ addition during the culture. Repeated batch culture with 50 mM CaCl₂ supplementation allowed a continuous retention of cell viability over 149 days and produced a great amount of extracellular peroxidase, 12-fold higher than that achieved in a 40-day-old batch culture with 50 mM CaCl₂ supplementation. Correspondence to: T. Kobayashi     

Phosphoramidon inhibits the conversion of intracisternally administered big endothelin-1 to endothelin-1

It is suggested that endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). We examined the effects of intracisternal administration of big ET-1 on the cerebral arteries in the absence or presence of pretreatment with phosphoramidon, an inhibitor of ET converting enzyme, in anesthetized dogs. After intracisternal administration of big ET-1 (10 micrograms/dog), the caliber of the basilar artery on the angiogram was decreased to about 59% of the control. This was accompanied by a marked increase in immunoreactive ET in the cerebrospinal fluid. Systemic arterial pressure was markedly elevated following big ET-1 injection. All changes induced by big ET-1 were effectively prevented with phosphoramidon. These data suggest that intracisternally administered big ET-1 is converted to ET-1 and that the generated ET-1 produces cerebral vasospasm and hypertension. A phosphoramidon-sensitive metalloproteinase appears to contribute to this conversion.     

Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium

Summary A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na₂SeO₃ (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linkedN-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium.α1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9±1.5 pmol · h⁻¹ · mg⁻¹ protein) than cultured with FBS-containing media (18.2±1.2 pmol · h⁻¹ · mg⁻¹ protein). These results have indicated that the selective increase ofα1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.     

Superoxide dismutases enhance the rate of autoxidation of 3-hydroxyanthranilic acid

The autoxidation of 3-hydroxyanthranilate to cinnabarinate at 37 degrees C and at pH 7.4 is hastened by superoxide dismutase (SOD). The Cu,Zn-containing enzyme from bovine erythrocytes and the Mn-containing enzyme from Escherichia coli were equally effective in this regard; whereas the H2O2-inactivated Cu,Zn enzyme was ineffective. Catalase appears to augment the effect of superoxide dismutase, because it prevents the bleaching of cinnabarinate by H2O2. It follows that O2-, which is a product of the autoxidation, slows the net autoxidation by engaging in back reactions and that SOD increases the rate of autoxidation by removal of O2- and thus by prevention of these back reactions.     

Inhibition of rabbit liver fructose 1,6-biphosphatase by AMP: effect of temperature and physiological concentrations of cations and anions

Turkey gizzard smooth muscle myofibrils, the actin of which is composed of 75% smooth muscle γ-isoactin and 25% nonmuscle β-isoactin, were separated into an actomyosin and a cytoskeletal fraction. Isoelectric focusing analysis of the actomyosin actin showed it was 80% γ-isoactin and 20% β-isoactin. It thus appears that the major actin in the tissue is also the major form involved in force generation. When the cytoskeletal material was extracted with low-ionic-strength solution for 18 h at 4 °C, the actin released was 95% γ and 5% β compared with the 75:25 ratio found in the original cytoskeletal material. The extracted material revealed the presence of F-actin filaments and high-molecular-weight aggregates. Little of the material was in a low-molecular-weight form. On the other hand, extraction of the cytoskeletal material with 0.6 m KI resulted in the two isoactins being extracted in the same proportions in which they were found in the original cytoskeletal material. However, when this KI-extracted material was subsequently chromatographed on Bio-Gel A-5m equilibrated with 0.6 m KCl, the γ-isoactin migrated predominantly as a very high molecular weight form while the β-isomer moved in the lower-molecular-weight range of the elution profile. This aggregation behavior displayed by the γ-isoactin was not observed with the γ-isoactin in the actomyosin fraction. These results show that the two gizzard isoactins in the cytoskeletal residue behave very differently in response to various extraction media, and are consistent with possible differential isoactin utilization in gizzard smooth muscle.     

A case of supernumerary muscle bundle “M. tubero-femoralis fick” of the beceps femoris in a crab-eating monkey

A supernumerary muscle bundle “M. tubero-femoralis Fick” of the biceps femoris was found in a female crab-eating monkey in both hindlimbs.     

Contribution from the chirality of the growing chain to the enantiomer selectivity in activated-NCA polymerization of α-amino acid N-carboxyanhydride

As a model compound for the growing chain in the activated-NCA type of polymerization of α-amino acid N-carboxyanhydride (NCA), 3-[ω-acetylglycyl-poly(α-amino acid) acyl]-α-amino acid NCA (called the prepolymer) having various degrees of polymerization (DPs) was synthesized by the polymerization of Phe, Val, Glu(OEt), and Asp(OBzl) NCA in the presence of AcGly NCA by the tertiary amine. Activated (S)-Phe, Val, Glu(OEt), and Asp(OBzl) NCA were added to the terminal cyclic group of the corresponding (S)- or (R)- prepolymer, and the enantiomer selectivity in the reaction was investigated. With prepolymers having DPs ranging from 1 to 15, the addition reaction always took place preferentially between species having the same configuration, and the degree of the enantiomer selection increased with increasing DP of the prepolymer. With prepolymers having DP = 1 and 2, we found contributions from the chiral terminal unit and the chiral penultimate unit to the enantiomer selection, respectively. Prepolymer having DP = 5 was shown to take a β-type conformation, which led to higher enantiomer selection; and prepolymers having DP = 10 and 15 were shown to take an α-helix conformation, which led to much higher enantiomer selection than did the β-type conformation. In the present investigation the mechanisms of terminal-unit control, penultimate-unit control and conformational control of the enantiomer selection in the activated-NCA type of polymerization were clearly observed.     

Confirmation of the human thymidine kinase locus, 17q21 → 17qter,by means of a man-mouse somatic cell hybrid,D98/AH-2 X LMTK−Cl-1D

Summary A large metacentric marker chromosome, m20, in a line of human D98/AH-2 cells was identified by Q bands as being a translocation (1;17)(p36;q21). This was confirmed by means of somatic cell hybridization between D98/AH-2 and thymidine kinase (TK) deficient mouse cells. The hybrid clones by HAT selective system retained m20, indicating the presence of TK locus on this chromosome. The results also provide evidence that TK gene is located on the distal region of the breakpoint in 17q21 but not on 17q21 17pter.