Scale-up of hydrophobic interaction chromatography for purification of antitumor antibiotic SN-07

Scale-up of hydrophobic interaction chromatography for purification of SN-07 was studied. The height equivalent to a theoretical plate of the adsorbent gel (Sepabeads FP-PH 12) was kept constant for various superficial liquid velocities and column diameters. The efficiency of purification was also constant for the Sepabeads FP-PH 12 columns ranging from 16 mm to 113 mm in inner diameter. Effects of the ligand concentration on the adsorption capacity were also studied, and the adsorption capacity increased with an increase in the ligand concentration. These effects resulted in decreasing the required amount of salt to purify SN-07.     

Concentrations of catecholamines and indoleamines in the central nervous system of Wriggle mouse Sagami

1. The concentrations of norepinephrine (NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the central nervous system of Wriggle mouse Sagami (WMS), which is a new ataxic mutant mouse, were studied. 2. NE and MHPG levels were increased most remarkably in the cerebellum. 3. 5-HT and 5-HIAA levels were increased most remarkably in the brain stem and spinal cord. 4. The present results suggest enhancement of catecholamine and indoleamine metabolism in the cerebellum and bulbospinal cord, respectively, of the WMS, and these changes seem relevant to the specific motor dysfunction of the WMS.     

Morphological differences between secretory cells of wet and dry types of human ceruminous glands

Morphological differences between secretory cells of the wet and dry types of human ceruminous glands were examined. The heights of secretory cells varied from tall and medium to low in both wet- and dry-type glands. The two gland types differed in morphologic features of the tall cells and the cells of medium height. The Golgi apparatus was well developed in the tall cells and fairly well developed in the cells of medium height in the wet-type gland, whereas it was generally small in the corresponding cells of the dry type. Light granules were abundant in the tall cells and in the cells of medium height in the wet-type gland, whereas light granules were rare in these cells in the dry-type gland. Furthermore, the light granules in the wet-type gland cells were observed in close relation to a well-developed Golgi apparatus, and sometimes showed a morphologic appearance suggesting exocytosis. Apical protrusions, probably related to apocrine secretion, were generally large and round and bore "microvilli and light granules" or "very few microvilli and no light granules" in the tall cells of the wet-type gland. However, the protrusions of the tall cells of the dry-type gland were generally large and slender and possessed no microvilli and no granules. The protrusions were not observed in the cells of medium height or in low cells in either type of gland. The results show that eccrine secretion characterizes the wet-type gland, but it is not clearly evident in the dry-type gland. This differences may be related to differences in composition between the wet and dry cerumens.     

Determination of beta-phenylethylamine in catfish (Parasilurus asotus) tissues by gas chromatography/mass spectrometry

1. beta-Phenylethylamine (PEA) was detected and quantitated in tissues of the catfish, Parasilurus asotus, by very specific and sensitive gas chromatography/mass spectrometry. 2. The selected ion monitoring was made with a strong quasi-molecular ion of the pentafluoropropionic derivative of PEA in the positive chemical ionization mode. 3. PEA was found in all tissues tested ranging from 2.8 to 38.2 ng/g wet wt tissue. It was highest in the spinal cord, followed by the skin, brain and intestine.     

Antigen-specific augmentation factor involved in murine delayed-type footpad reaction. I. Nature of augmentation factor

A humoral factor capable of augmenting antigen-specific DTH has been found in the culture supernatant of immune spleen cells and erythrocyte antigen. In this study, a similar factor was identified in the sera of mice sensitized and elicited with heterologous erythrocytes, and the nature of this factor was investigated. Elicitation with antigen was essentially required for the production of the augmentation factor in sensitized mice. The factor showed antigen specificity and antigen-binding capacity. The activity was not assigned to immunoglobulins, as demonstrated by an absorption test with rabbit anti-mouse immunoglobulin-conjugated Sepharose. The activity was stable to heating at 56 degrees C for 30 min, to changes of pH from 3 to 10, and to treatment with trypsin or neuraminidase. The molecular weight of this factor was about 200,000 to 450,000.     

Antigen-specific augmentation of delayed-type hypersensitivity by a humoral factor in the culture supernatant of immune spleen cells

Supernatants were harvested from a 24-hr culture of immune mouse spleen cells and erythrocyte antigens. Delayed footpad reactions to such heterologous erythrocytes were augmented antigen specifically when the supernatants were transferred a few hours before immunization of recipients. The augmentation factor(s) contained in the supernatants may exert its effect on the induction phase of delayed-type hypersensitivity.     

Molecular basis of the protonophoric and uncoupling activities of the potent uncoupler SF-6847 ((3,5-di-tert-butyl-4-hydroxybenzylidene)malononitrile) and derivatives. Regulation of their electronic structures by restricted intramolecular rotation

We reported recently (Yoshikawa, K. and Terada, H. (1982) J. Am. Chem. Soc. 104, 7644–7646) that the potent uncoupler of oxidative phosphorylation SF-6847 ((3,5-di-tert-butyl-4-hydroxybenzylidene)malononitrile) shows unique intramolecular restricted rotation of the malononitrile moiety. In this study, values for the activation energy E_a of the restricted rotation of SF-6847 derivatives with the same alkyl chain R in both ortho positions of the phenolic hydroxyl group were determined from the temperature-dependent change in the¹H-NMR signals of their aromatic protons. The E_a values of the neutral forms of these derivatives were found to be the same irrespective of R, but those of the anionic forms increased with increase in the alkyl chain length of R. It was found that the restricted rotation of the malononitrile moiety regulates its electron-withdrawing ability in such a way as to keep the acid dissociability of these derivatives similar, overcoming the effect of steric hindrance by R. The protonophoric activity of these derivatives, in a phospholipid bilayer membrane and their uncoupling activity in rat-liver mitochondria were both found to depend on E_a of their anionic forms. The stability of the uncoupler anions regulated by the restricted rotation of the malononitrile group in a nonpolar membrane environment was found to be important for exhibition of these activities. The hydrophobicity of the anionic forms of these derivatives was suggested also to be affected by the intramolecular rotation.     

Alternating Dependency of Cytokinin Activity on the Number of Methylene Units in ω-Phenylalkyl Derivatives of Some Purine Cytokinins and 4-Substituted Pyrido[3,4-d]pyrimidine

ω-Phenylalkyl derivatives of N⁶-substituted adenine, N⁶-substituted adenosine and 6-alkoxy-purine with odd numbers of methylenes had relatively high activity, while the corresponding 2-methyl-4-substituted-aminopyrido[3,4-d]pyrimidines with even numbers of methylenes had high activity in an Amaranthus betacyanin test and lettuce seed germination test. These results suggest that the ω-phenyl groups of the cytokinins play a specific role for cytokinin-receptor binding and that the pyrido[3,4-d]pyrimidines interact with a receptor in a different binding mode.     

Bovine mitochondrial tRNAPhe, tRNASer (AGY) and tRNASer (UCN): preparation using a new detection method and their properties in aminoacylation

Bovine mitochondrial tRNAPhe, tRNASer (AGY), and tRNASer (UCN) possessing unusual structures were purified using a new hybridization assay system and their properties in aminoacylation were examined. Bovine mitochondrial phenyl-alanyl- and seryl-tRNA synthetases could aminoacylate the same amino acid-specific tRNAs obtained not only from the mitochondria but also from other sources such as E. coli, Thermus thermophilus, bovine and yeast cytosols and archaebacteria, Sulfolobus acidocaldarius. On the contrary, none of both bacterial and cytosolic synthetases could aminoacylate the same amino acid specific tRNAs from the heterologous sources with some exceptions. We consider that the bovine mitochondrial aminoacyl-tRNA synthetases have considerably simple recognition mechanism toward the substrate tRNAs compared with the non-mitochondrial ones. This mechanism may be correlated with the occurrence of structural varieties of the mitochondrial tRNA species with unusual structures.     

Separation of a Water-Soluble Adjuvant (MAF3) from Delipidated Cells of Mycobacterium tuberculosis Strain Aoyama B by Hydrogenolysis and Gel Filtration

A water-extract from hydrogenolyzed cells of Mycobacterium tuberculosis strain Aoyama B was separated into four portions (F-1 to F-4 fractions) by gel filtration with a Sephadex G-100 column. The third peak (called MAF3) eluted from the column was the most adjuvant-active fraction. The molecular weight determined by gel filtration was around 16 000 daltons. MAF3 consisted of heteropolymer(s) composed of approximately 76 to 79% neutral sugars (Ara, Gal, Man, and Glc) and 19% mucopeptide (MurN, GlcN, Glu, Ala, Dpm, Gly, Asp, Thr, Ser, Leu, Lys, Arg, His, Pro, Tyr, and Phe). The adjuvanticities of MAF3 and other fractions in water-in-oil emulsion were estimated by the enhancing effect on immune response to egg albumin (EA) in guinea pigs. MAF3 stimulated the production of humoral antibodies, particularly IgG2 antibody specific to the antigen, and induced delayed type hypersensitivity against EA in the skin and cornea of antigen-primed guinea pigs. These adjuvanticities of MAF3 were similar to the characteristics of mycobacterial cell wall in Freund's complete adjuvant.