Extraction and composition of polar lipids from the archaebacterium, Methanobacterium thermoautotrophicum: effective extraction of tetraether lipids by an acidified solvent

The usual Bligh and Dyer method could extract only a small part of the lipids of Methanobacterium thermoautotrophicum. When the water in the solvent was replaced by 5% trichloroacetic acid, the lipid recovery reached the maximum level, which was 6 times higher than that by the former method. The use of HCl (2 M) or disruption of cells was also effective but prolonged extraction with the HCl-containing solvent caused degradation of some phosphoglycolipids. Twenty-three spots of polar lipids were detected on a thin-layer chromatogram of the total lipid. These were 10 phospholipids (18%), 6 aminophospholipids (17%), 3 aminophosphoglycolipids (15%), 2 phosphoglycolipids (31%), and 2 glycolipids (19%). The predominant polar lipids were a highly polar phosphoglycolipid (PGL1, 30%) and a glycolipid (GL1a, 16%). The other major lipids included an aminophospholipid (PNL1a, 9%), and an aminophosphoglycolipid (PNGL1, 7%). The complete structure determination of PNL1a, GL1a, and PNGL1 is described in the accompanying paper. Acetolysis of the total lipids followed by acid methanolysis was required for the complete cleavage of polar head groups, releasing core residues of diphytanyl glycerol diether (C20 diether) and dibiphytanyl diglycerol tetraether (C40 tetraether). A densitometric assay of a thin-layer chromatogram showed that the ratio of C20 diether and C40 tetraether was 1:14. GLC analysis of alkyl chlorides prepared from the total lipid by BCl3 treatment showed that phytanyl (C20), biphytanyl (C40), and unidentified alkyl chains accounted for 10, 83, and 7 mol% of the total alkyl chains, respectively. Strong acid hydrolysis of the macromolecular residue obtained after lipid extraction gave a significant amount of C40 tetraether, which had probably been bound covalently to other substances in the cells.     

The occurrence of a premature form of egg-specific protein in vitellogenic follicles ofBombyx mori

Summary Analysis of yolk proteins of the silkworm,Bombyx mori, by SDS-polyacrylamide gel electrophoresis and immunoblotting showed that there was a developmental change in subunit composition of egg-specific protein; egg-specific protein consisting of 72 kDa subunits alone (premature form) was found in vitellogenic follicles, whereas the protein in mature eggs was composed of 72 kDa and 64 kDa subunits (mature form). The premature form of egg-specific protein was purified from young ovaries to homogeneity using a high performance liquid chromatography system. The purified protein had an apparent molecular mass of 225 kDa which could not be distinguished from that of the mature form. By circular dichroism analysis, both egg-specific proteins were estimated to have about 30% -helix and 20% -sheet, but the mature form showed a relatively rigid conformation in the aromatic region. The premature egg-specific protein purified from vitellogenic ovaries, consisted of three 72 kDa subunits, whereas mature egg-specific protein was composed of two 72 kDa subunits and one 64 kDa subunit. All of these subunits showed the same immunoreactivity towards antiserum raised against the mature form. An identical NH₂-terminal amino acid sequence was found in both 72 kDa polypeptides and 64 kDa polypeptide for the initial 10 amino acids.Abbreviations SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - ESP egg-specific protein - Vtn vitellin     

Cytogeographical study of four aneuploids ofCarex oxyandra Kudo in Japan

Chromosome numbers were determined for 342 clones ofCarex oxyandra collected from 35 localities in Hokkaido, Honshu, Shikoku and Kyushu, Japan. Four intraspecific aneuploids, 2n=18, 20, 24 and 26, were found. In meiotic division, only bivalent chromosomes were observed in all clones at metaphases I and II, suggesting that the aneuploids are established gamodemes. In the mitotic metaphase chromosomes, trimodal variation in chromosome length was observed. The 2n=26 clones found on Mt. Hiko had two particularly small chromosomes. The cytodemes with higher number of chromosomes are distributed in more southern areas of Japan.Carex oxyandra, therefore, accompanied with chromosome fragmentations, might spread the geographical distribution to the southern parts. The morphological characters of leaves, spikes, scales, perigynia and nuts were similar among the four cytodemes, except for the small leaves on plants from Yaku Island.     

Effect of gamma-aminobutyric acid on excitability of tubero-infundibular neurons in rat hypothalamic slices

The effect of gamma-aminobutyric acid (GABA) on antidromically identified tubero-infundibular (TI) neurons was examined in hypothalamic slices of ovariectomized female rats. Twenty antidromically evoked spikes were obtained in the medial basal hypothalamus, including the arcuate and ventromedial nuclei, by electrical stimulation of the median eminence. Sixteen of them had a notch in the rising phase and fractionation of the initial segment (IS)- and somatodendritic (SD)-spikes was elicited by repeated stimulation at frequencies higher than 10 Hz. The application of 0.5-1.5 mM GABA to the incubation medium inhibited SD spikes in 7 of these 16 neurons. The latency, amplitude and threshold of IS spikes were not affected by GABA except for one spike whose latency fluctuated. On the remaining 9 neurons having the notch, no effect of 5-10 mM GABA was discernible. Four of 20 antidromically evoked spikes, which had a smooth rising phase and a shorter duration, were not inhibited by 5-10 mM GABA, but a fluctuation of the latency was observed in one neuron. Fifteen neurons having spontaneous unit activity were also obtained in the arcuate nucleus and its adjacent area and tested with GABA. In 10 of the 15 neurons, spontaneous unit activity disappeared following 0.1-1.5 mM GABA perfusion, while the firing rate in the remaining 5 neurons was not affected by 5-10 mM GABA. These results provide evidence for a direct inhibitory effect of GABA on TI neurons and support the involvement of GABAergic neurons in regulating neuroendocrine functions.     

Biological Activity of 5-Hydroxymethyluracil and Its Deoxynucleoside in Noninfected and Phageinfected Bacillus subtilis

(14)C-hydroxymethyldeoxyuridine (dHMU) is specifically incorporated into the deoxyribonucleic acid (DNA) of bacteriophage SP8. Incorporation experiments demonstrate that the initiation of phage SP8 DNA synthesis occurs between 12.5 to 15 min after infection. Incorporation into host DNA does not occur. (14)C-dHMU can be used as an analytical tool for screening conditionally lethal phage mutants containing hydroxymethyluracil in their DNA to select those that are defective in DNA synthesis under restrictive conditions. The pyrimidine, (14)C-hydroxymethyluracil (HMU), is not incorporated into bacterial or phage DNA. Neither HMU nor dHMU can replace thymine as a growth requirement for Bacillus subtilis 168 Ind(-) Thy(-). HMU does not inhibit the utilization of thymine. Although dHMU inhibits deoxythymidine utilization, the inhibition is not competitive.     

Effect of Ca on composition of fat body of peanut seed

Peanut fruits were grown in nutrient media with or without Ca and in a soil with two Ca levels, from the 20th day after penetration of the gynophore. Seed weight was smaller in the nutrient medium without Ca than in the nutrient medium with Ca, and it was also smaller in the soil with 4 meq of exchangeable Ca (L treatment) than in the soil with 10 meq of exchangeable Ca (H treatment). The fat body of seeds from the Ca deficient medium and the L treatment had a decreased phospholipid content and an increased simple lipid content. In the seed from the H treatment, phosphatidylcholine increased from the 30th to 60th day, while caldiolipin decreased. The amount of triglyceride in the simple lipid content of fat body was decreased by Ca deficiency in the nutrient medium, whereas that of diglyceride was increased, but these effects were not observed in the fat body of the seed from the L treatment. No effect of Ca deficiency was observed in the fatty acid composition of triglyceride.     

A target protease activity of serpins in insect hemolymph

Two zymogens of the serine enzymes (prophenoloxidase activating enzyme and BAEEase, an enzyme hydrolyzing ethyl ester), which are thought to be components of prophenoloxidase cascade in silkworm (Bombyx mori) plasma, were activated through the action of microbial cell wall components. The two active enzymes of the zymogens were studied with regard to the regulation of their activities by two endogenous serpins (silkworm anti-trypsin and silkworm anti-chymotrypsin).

BAEEase activity was shown to be inactivated by silkworm antitrypsin, whereas the inactivation of prophenoloxidase activating enzyme by either of silkworm antitrypsin and silkworm antichymotrypsin could not be demonstrated under the experimental conditions.     

Cytogeography and meiotic chromosome configurations of six intraspecific aneuploids ofCarex conica Boott (Cyperaceae) in Japan

Chromosome numbers were determined for 340 plants ofCarex conica from 83 populations in Japan. Six aneuploids, 2n=32, 33, 34, 36, 37 and 38, were found. Plants with even diploid chromosome numbers 2n=32, 34, and 36 were the most common and had different geographical distributions. Individuals with 2n=32 were from islands in the Seto Inland Sea and nearby coastal areas of the Chugoku District of Honshu; those with 2n=34 were from the Kanto, Chubu and Kinki Districts of Honshu; those with 2n=36 were from the mountainous areas of Chugoku, Shikoku and Kyushu Districts. Canonical discriminant analysis of 17 morphological characters demonstrated that the plants with 2n=32 were clearly distinct from those with 2n=34 or 36. All four aneuploids with even chromosome numbers showed normal bivalent pairing at meiotic metaphase I and probably represent cytogenetically stable cytodemes. Plants with 2n=33 had one heteromorphic trivalent and 15 bivalents, indicating a structural mutation. At mitotic metaphase I, one chromosome was markedly larger than the others, suggesting that the 2n=33 plants arose from 2n=34 plants by fusion of two chromsomes. The plant with 2n=37 was intermediate in morphology betweenCarex conica (2n=36) andC. morrowii (2n=38) and probably originated as an interspecific hybrid between these species.     

Expression of the [beta]-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica,and Peony (Paeonia lactiflora) by Particle Bombardment

A [beta]-glucuronidase (GUS) gene that is under the control of the anther-specific LAT52 promoter of tomato (Lycopersicon esculentum) and the nopaline synthetase polyadenylation terminator was successfully expressed in pollen of Lilium longiflorum, Nicotiana tabacum, Nicotiana rustica, and Paeonia lactiflora using a pneumatic particle gun. The GUS gene in plasmid pBI221 was also expressed, to a lesser extent, in pollen of all of these species. The presence of methanol in the substrate solution for histochemical GUS assay and the incubation time in this solution influenced successful detection of GUS expression in bombarded pollen. Cytological analysis of GUS-expressing pollen of lily showed that introduced gold particles were seen in intracellular compartments of pollen, including the vegetative cytoplasm, vegetative nucleus, and generative cytoplasm.     

Ether polar lipids of methanogenic bacteria: structures, comparative aspects, and biosyntheses.

Complete structures of nearly 40 ether polar lipids from seven species of methanogens have been elucidated during the past 10 years. Three kinds of variations of core lipids, macrocyclic archaeol and two hydroxyarchaeols, were identified, in addition to the usual archaeol and caldarchaeol (for the nomenclature of archaeal [archaebacterial] ether lipids, see the text). Polar head groups of methanogen phospholipids include ethanolamine, serine, inositol, N-acetylglucosamine, dimethyl- and trimethylaminopentanetetrol, and glucosaminylinositol. Glucose is the sole hexose moiety of glycolipids in most methanogens, and galactose and mannose have been found in a few species. Methanogen lipids are characterized by their diversity in phosphate-containing polar head groups and core lipids, which in turn can be used for chemotaxonomy of methanogens. This was shown by preliminary simplified analyses of lipid component residues. Core lipid analysis by high-pressure liquid chromatography provides a method of determining the methanogenic biomass in natural samples. There has been significant progress in the biosynthetic studies of methanogen lipids in recent years. In vivo incorporation experiments have led to delineation of the outline of the synthetic route of the diphytanylglycerol ether core. The mechanisms of biosynthesis of tetraether lipids and various polar lipids, and cell-free systems of either lipid synthesis, however, remain to be elucidated. The significance and the origin of archaeal ether lipids is discussed in terms of the lipid composition of bacteria living in a wide variety of environments, the oxygen requirement for biosynthesis of hydrocarbon chains, and the physicochemical properties and functions of lipids as membrane constituents.