Enzymatic Protein Carboxyl Methylation in Rat Posterior Pituitary: Neurophysins in Rapid-Turnover Pool Determine Methyl Accepting Capacity

In vitro stimulation of intact rat posterior pituitary by either veratridine or K+ depolarization results in the concomitant release of neurophysins and in a decrease (70-80%) in their carboxyl methylation as measured either with L-[methyl-3H]methionine in the intact lobes after stimulation or in their homogenates with [methyl-3H]S-adenosyl-L-methionine and purified protein carboxyl methyltransferase. A similar reduction in neurophysin methylation (60%) was observed when the arrival of newly synthesized neurophysins at the posterior pituitary was blocked by colchicine. Experimental data indicate that the reduction in neurophysin content of the lobes after 12 h of colchicine treatment (less than 7%) or after in vitro stimulation (about 10%) cannot account for the marked reduction in neurophysin methylation. The results suggest that the granule pool characterized by rapid turnover of neurophysins probably represents the major source of methyl acceptor proteins in the lobe. In spite of the marked reduction in neurophysin methyl accepting capacity observed after stimulation, there was no parallel increase in methyl accepting capacity of the released neurophysins. We propose that a neurophysin subfraction that might be associated with the membrane of releasable granules participates in the methylation reaction in situ.     

Liver glutathione S-transferase polymorphism in Japanese and its pharmacogenetic importance

Summary A total of 168 autopsy liver extracts from Japanese individuals were examined for the glutathione S-transferase (GST) isozymes by means of starch gel electrophoresis. The gene frequencies of GST1^*1, GST1^*2, and GST1^*0 in Japanese were 0.252, 0.057, and 0.691, respectively. GST1^*3 was detected as a rare variant allele. The incidence of GST1 0 in 41 liver biopsy samples from patients suffering from various liver diseases was investigated using polyacrylamide gel isoelectric focusing. The GST1 0 phenotype was found more frequently in livers with hepatitis and carcinoma than in control livers. The isozymes coded by different GST loci were partially purified and characterized to study their biochemical properties. The apparent Km values with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate for the isozymes at the GST1, GST2, GST3, and GST4 loci were 604, 1345, 776 and 591 M, respectively.     

Synthesis of acetone glycerol acyl esters by immobilized lipase ofMucor miehei

Summary The applications of immobilized lipase ofMucor miehei for the synthesis of acetone glycerol acyl ester from acetone glycerol and fatty acid, which is the first step for monoglyceride production was investigated. With a high oleic acid to acetone glycerol ratio (O/A, mol/mol), a high catalytic activity was observed under low water content in the reaction mixture. By the combination of high O/A ratio (>3) and removal of water which was produced during the reaction, the conversion degree was increased to almost 100%. With the O/A ratio of 3, the approximate half-life of the immobilized lipase and productivity of ester was estimated to be 20 days and 869 g product/g immobilized enzyme per 2 half-lives, respectively.     

Production of extracellular 5-aminolevulinic acid byClostridium thermoaceticum grown in minimal medium

Summary A growth associated formation of extracellular 5-aminolevulinic acid (ALA) was found in the homoacetogenesis of glucose byClostridium thermoaceticum grown in minimal defined medium. The growth and ALA production was enhanced by L-cysteine HCl both in complex medium (UM) and minimal defined medium (MDM). The amount of ALA produced extracellularly in MDM wasca. 15 mg/L after 90-h anaerobic cultivation (cell-mass: 1.5 g/l; glucose consumed: 20 g/l).     

Influence of Co2+, Ni2+ and Fe2+ on the production of tetrapyrroles by Methanosarcina barkeri

Summary The selective formation of three tetrapyrroles, Co-containing corrinoids, Ni-containing factor F₄₃₀ and Fe-containing cytochromes (haems) by Methanosarcina barkeri Fusaro (DSM 804) was achieved as a function of the concentrations of Co²⁺, Ni²⁺ and Fe²⁺ in a methanol minimmum medium. It was found that about 70% of the total tetrapyrroles synthesized was excreted into the culture supernatant. Hence, the continuous production of tetrapyrroles in a fixed-bed reactor (supporter: porous diatomaceous clay) was carried out at a dilution rate of 10 day^-1 (850 ml medium/85 ml column/day). The effluent discharged from the reactor contained the excreted tetrapyrroles, the concentrations of which were dependent upon the Co²⁺, Ni²⁺ and Fe²⁺ concentrations in the feed medium. The maximum productivities from the reactor (1 l basis) were 52 M corrinoids/day, 24 M F₄₃₀/day and 8 M haems/day, respectively.     

Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) shows sugar-binding specificity for L-fucose. A λgt11 expression library was constructed from A. aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL. An immunopositive clone carrying 1.3-kb EcoRI fragment was obtained. The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino-terminal amino acids. The 5′-terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA-1. Escherichia coli carrying pKA-1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.     

Local distribution of the lycaenid butterfly,Jalmenus evagoras,in response to host ants and plants

Summary The caterpillars of Jalmenus evagoras are tended by ants as they feed upon Acacia trees. In the area of Brisbane, Australia, J. evagoras require ants of the Iridomyrmex anceps species group; predation and parasitism are so intense that larvae and pupae deprived of attendant ants cannot survive (Pierce 1983). We investigated the efficiency with which J. evagoras locate and exploit the host ant resource by sampling 737 quadrats in 30 sampling grids and six study sites containing appropriate host plants; ants were collected at baits located in the center of each quadrat. J. evagoras was found in all habitats where I. anceps cooccurred with host Acacia. Nine of the ten sampling grids which had three or more I. anceps/Acacia host quadrats also had colonies of J. evagoras present (or immediately adjacent), including sites as far as 35 km apart. Of 19 sampling grids on which host quadrats were rare (i.e., less than three quadrats), none had J. evagoras (P<0.001). Within sample grids, I. anceps was distributed indepedently from Acacia trees, suggesting that they are not dependent for their survival on either Acacia or on J. evagoras. Within montane pasture habitats, I. anceps and at least one other ground-dwelling Iridomyrmex species were distributed in mutually exclusive ant mosaic territories which were stable during a one month period. I. anceps did not colonize or tend pupae of J. evagoras experimentally placed in adjacent territories of a different, nontending species of Iridomyrmex, demonstrating the integrity of territory boundaries. Sampling of ants in Acacia trees revealed that, in the absence of J. evagoras, Iridomyrmex workers are not common above ground level, and that their numbers decline in larger trees (P=0.02). In I. anceps territories, eight of nine J. evagoras pupae placed in trees over 3.0 m tall were not found after 24 h whereas all ten controls placed in low trees were found and tended (P=0.00012). This may explain why J. evagoras tends to oviposit in trees less than 2.0 m tall. An alternative hypothesis, that smaller trees have higher content of total nitrogen, and are threfore more nutritious, was not supported. We conclude that the local distribution and host tree selection by J. evagoras is dependent upon the distribution, patchiness, and foraging behavior of the host ant, I. anceps, and its spatial overlap with a number of species of host Acacia.     

Detection of tetrodotoxin by thin-layer chromatography/fast atom bombardment mass spectrometry

A new method for detection of tetrodotoxin (TTX) by thin-layer chromatography/fast atom bombardment (FAB) mass spectrometry was developed. TTX and/or related substances were separated by TLC on LHP-K high-performance precoated plates, with a solvent system of pyridine:ethyl acetate:acetic acid:water (15:5:3:4). The plates were subjected to positive FAB mass spectrometry, under scanning within a mass range from m/z 100 to 500. TTX was identified by selected ion-monitored chromatograms at m/z 320 (M + H)+ and 302 (M + H - H2O)+, along with full scan positive ion FAB mass spectrometry. The limit of detection for TTX was about 0.1 micrograms. TTX was also detected by cellulose acetate membrane electrophoresis/FAB mass spectrometry.