Population dynamics of a migrant skipper butterflyParnara guttata (Lepidoptera: Hesperiidae) II. Survival rates of immature stages in paddy fields

Population Ecology - Survival rates and mortality factors of a migrant skipperParnara guttata were censused in paddy fields in 4 localities of central and western Japan during 1975–1980, and...     

Cloning, bacterial expression, and unique structure of adenosylhomocysteine hydrolase-like protein 1, or inositol 1,4,5-triphosphate receptor-binding protein from mouse kidney

Adenosylhomocysteine hydrolase (SAHase)-like protein 1 (SAH-L), also called inositol 1,4,5-triphosphate receptor-binding protein (IRBIT) is a novel protein involved in fish embryo development and calcium release in mammalian cells through protein-protein interactions. To better understand its reaction mechanism, purified protein is indispensable. Here we describe a simple purification procedure and the unique properties of SAH-L. The cDNA was isolated from mouse kidney by RT-PCR and inserted into various pETtrade mark vectors. Escherichia coli harboring a plasmid coding for SAH-L with a C-terminal His-tag could solely produce a soluble protein. SAH-L purified through a Ni(2+) column gave M(r)s of 59,000 and 190,000 by SDS-PAGE and gel filtration, respectively, which is suggestive of a trimer, but chemical cross-linking experiments demonstrated a dimer. The incompatible M(r) values implicate an irregular structure of SAH-L. In fact, SAH-L was partially purified in a form lacking the 31 N-terminal residues, and was found to be extremely susceptible to proteases in the region around residue 70. The N-terminal polypeptide (residues 1-98) was also expressed as a soluble form and was trypsin-sensitive. Circular dichroism revealed a low alpha-helix content but not a randomly extended structure. Interestingly, SAH-L contained tightly bound NAD(+) despite showing no SAHase activity. The characterized properties of SAH-L and its N-terminal fragment present the notion that the structure of the protease-sensitive N-terminal region is relatively loose and flexible rather than compact, and which protrudes from the major SAHase-like domain. This structure is supposed to be favorable to interact with the IP(3) receptor.     

In situ RNA hybridization using Technovit resin in Arabidopsis thaliana

A protocol is described for RNA in situ hybridization using thin sections prepared by Technovit resin. Technovit is a widely used resin for histological examinations. Since it does not require time-consuming processes such as removal of the resin and can be performed without high temperature treatment, a high resolution of sections could be possible compared to other resins and paraffin. Thin sections (approximately 4 m) were made from inflorescences of Arabidopsis thaliana embedded in Technovit 8100 resin, and in situ hybridization was performed using the protocol described in this article. Hybridization signals were observed using LEAFY and other genes as probes, showing that this resin can be used for in situ analysis. In our experiments, the most important factor for a successful in situ hybridization pattern was to optimize the RNase A concentration after hybridization. We routinely used RNase A at a concentration of 2–5 ng/ml, a concentration much lower than that used for paraffin embedding method. Thus, the use of the Technovit resin for plant tissue embedding results in a faster protocol and greater quality than allowed by paraffin sections.     

Role of acetyl-L-carnitine in the brain: revealed by Bioradiography

To elucidate the role of acetyl-l-carnitine in the brain, we used a novel method, ‘Bioradiography,’ in which the dynamic process could be followed in living slices by use of positron-emitter labeled compounds and imaging plates. We studied the incorporation of 2-[¹⁸F]fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) into rat brain slices incubated in oxygenated Krebs-Ringer solution. Under the glucose-free condition, [¹⁸F]FDG uptake rate decreased with time and plateaued within 350 min in the cerebral cortex and cerebellum, and the addition of 1 or 5 mM acetyl-l-carnitine did not alter the [¹⁸F]FDG uptake rate. When a glutaminase inhibitor, 0.5 mM 6-diazo-5-oxo-l-norleucine (DON), was added under the normal glucose condition, [¹⁸F]FDG uptake rate decreased. Acetyl-l-carnitine (1 mM), which decreased [¹⁸F]FDG uptake rate, reversed this DON-induced decrease in [¹⁸F]FDG uptake rate in the cerebral cortex. These results suggest that acetyl-l-carnitine can be used for the production of releasable glutamate rather than as an energy source in the brain.     

Diene chirality and cotton effects of nonhomoannular cisoid conjugated dienes: circular dichroism and crystal structure of a steroidal 19-nor-1(10),9(11)-diene derived from Westphalen's diol diacetate

Nonhomoannular cisoid conjugated dienes exhibit negative lowest energy pi-->pi* Cotton effects when they have P diene chirality and positive CEs when they have M diene chirality. We investigated this relationship further with a variety of such dienes by MM2 conformational energy-minimization calculations and by an X-ray crystal structure of a steroidal 19 nor 1(10),9(11) diene. CEs are stronger when each double bond of the diene is endocyclic in a different ring and weaker when only one of the double bonds is endocyclic or when neither double bond is endocyclic. They are also stronger when axial allylic and homoallylic substituents with CH/pi interactions are present that exert consignate chirality contributions.     

Phylogenetic Analysis of Bacteria Preserved in a Permafrost Ice Wedge for 25,000 Years

Phylogenetic analysis of bacteria preserved within an ice wedge from the Fox permafrost tunnel was undertaken by cultivation and molecular techniques. The radiocarbon age of the ice wedge was determined. Our results suggest that the bacteria in the ice wedge adapted to the frozen conditions have survived for 25,000 years.     

PGH2 degradation pathway catalyzed by GSH-heme complex bound microsomal prostaglandin E2 synthase type 2: the first example of a dual-function enzyme

Prostaglandin E2 synthase (PGES) catalyzes the isomerization of PGH2 to PGE2. PGES type 2 (mPGES-2) is a membrane-associated enzyme, whose N-terminal section is apparently inserted into the lipid bilayer. Both intact and N-terminal truncated enzymes have been isolated and have similar catalytic activity. The recombinant N-terminal truncated enzyme purified from Escherichia coli HB101 grown in LB medium containing delta-aminolevulinate and Fe(NO3)3 has a red color, while the same enzyme purified from the same E. coli grown in minimal medium has no color. The red-colored enzyme has been characterized by mass, fluorescence, and EPR spectroscopies and X-ray crystallography. The enzyme is found to contain bound glutathione (GSH) and heme. GSH binds to the active site with six H-bonds, while a heme is complexed with bound GSH forming a S-Fe coordination bond with no polar interaction with mPGES-2. There is a large open space between the heme and the protein, where a PGH2 might be able to bind. The heme dissociation constant is 0.53 microM, indicating that mPGES-2 has relatively strong heme affinity. Indeed, expression of mPGES-2 in E. coli stimulates heme biosynthesis. Although mPGES-2 has been reported to be a GSH-independent PGES, the crystal structure and sequence analysis indicate that mPGES-2 is a GSH-binding protein. The GSH-heme complex-bound enzyme (mPGES-2h) catalyzes formation of 12(S)-hydroxy-5(Z),8(E),10(E)-heptadecatrienoic acid and malondialdehyde from PGH2, but not formation of PGE2. The following kinetic parameters at 37 degrees C were determined: KM = 56 microM, kcat = 63 s-1, and kcat/KM = 1.1 x 10(6) M-1 s-1. They suggest that mPGES-2h has significant catalytic activity for PGH2 degradation. It is possible that both GSH-heme complex-free and -bound enzymes are present in the same tissues. mPGES-2 in heme-rich liver is most likely to become the form of mPGES-2h and might be involved in degradation reactions similar to that of cytochrome P450. Since mPGES-2 is an isomerase and mPGES-2h is a lyase, mPGES-2 cannot simply be classified into one of six classes set by the International Union of Biochemistry and Molecular Biology.     

Production of Corynecins by Mutants Defective in Glycolipids Synthesis and Increased in Corynecin I

For the purpose of improving the productivity of corynecins (chloramphenicol analogs), the mutation study of the producer, Corynebacterium hydrocarboclastus, was concerned in this investigation with the isolation of glycolipids defective strains. The mutant KY 8834 possessed glycolipids less than one-third to one-tenth of the parent. Productivity of corynecins of the mutant was better than that of the parent, In addition, this character of the mutant was advantageous for the stirring fermentation, because aggregation of cells was remarkably diminished unlike the case of the parental strain.

Another mutant was isolated by mutagenesis of glycolipids defective mutant, KY 8834. The mutant, KY 8835, produced dominantly corynecin I (less toxic homolog to the producer strain). Thus, the productivity of corynecins was elevated to about two-fold of that of strain KY8834.     

On Further Study of Extracellular Accumulation of DNA by Hydrocarbon-utilizing Pseudomonas Species

Extracellular accumulation of high molecular weight DNA was further studied using Pseudomonas species. More efficient production was obtained by the use of glucose-grown seed culture and by controlling the broth-pH at around 6.0 for first 24 hr and then around 8.0 during the fermentation. The maximum yield was 5 to 6 g per liter of the broth culture, which corresponded to 10-fold of that reported in the previous work.

Purified DNA (4 × 10⁶ daltons) was obtained successfully by applying an aqueous biphase system of dextran-polyethyleneglycol and dextranase.

Significant release of DNA occurred only with cell lysis of H-paraffin-grown bacteria. The primary cause of rapid lysis was explained by the exhaustion of cellular glucose pool. Relation of DNA accumulation to the effect of rhamnolipids on cell membrane was also investigated.