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In photosynthetic organisms, the photosynthetic membrane constitutes a scaffold for light-harvesting complexes and photosynthetic reaction centers. Three kinds of glycolipids, namely monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol, constitute approximately 80-90% of photosynthetic membrane lipids and are well conserved from tiny cyanobacteria to the leaves of huge trees. These glycolipids perform a wide variety of functions beyond biological membrane formation. In particular, the capability of adaptation to harsh environments through regulation of membrane glycolipid composition is essential for healthy growth and development of photosynthetic organisms. The genome analysis and functional genetics of the model seed plant Arabidopsis thaliana have yielded many new findings concerning the biosynthesis, regulation, and functions of glycolipids. Nevertheless, it remains to be clarified how the complex biosynthetic pathways and well-organized functions of glycolipids evolved in early and primitive photosynthetic organisms, such as cyanobacteria, to yield modern photosynthetic organisms like land plants. Recently, genome data for many photosynthetic organisms have been made available as the fruit of the rapid development of sequencing technology. We also have reported the draft genome sequence of the charophyte alga Klebsormidium flaccidum, which is an intermediate organism between green algae and land plants. Here, we performed a comprehensive phylogenic analysis of glycolipid biosynthesis genes in oxygenic photosynthetic organisms including K. flaccidum. Based on the results together with membrane lipid analysis of this alga, we discuss the evolution of glycolipid synthesis in photosynthetic organisms. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We characterized certain physiological functions of cyanobacterial monoglucosyldiacylglycerol using a Synechocystis sp. PCC 6803 mutant in which the gene for monoglucosyldiacylglycerol synthase had been disrupted and its function complemented by inclusion of an Arabidopsis monogalactosyldiacylglycerol synthase gene. By using this method, we prepared the first viable monoglucosyldiacylglycerol-deficient mutant of cyanobacterium and found that monoglucosyldiacylglycerol is not essential for its growth and photosynthesis under a set of "normal growth conditions" when monogalactosyldiacylglycerol is adequately supplied by the Arabidopsis monogalactosyldiacylglycerol synthase. The mutant had healthy thylakoid membranes and normal pigment content. The membrane lipid composition of the mutant was similar with that of WT except lack of monoglucosyldiacylglycerol and a slight increase in the level of phosphatidylglycerol at both normal and low temperatures. However, the ratio of unsaturated fatty acids in monogalactosyldiacylglycerol and digalactosyldiacylglycerol was reduced in the mutant compared with WT. Although the growth of the mutant was indistinguishable with that of WT at normal growth temperature, it was markedly retarded at low temperature compared with that of WT. Our data indicated the possibility that cyanobacterial monogalactosyldiacylglycerol-synthesis pathway might be required for the adequate unsaturation level of fatty acids in galactolipids and affect the low-temperature sensitivity., (Copyright © 2013 The Author. Published by Elsevier B.V. All rights reserved.)

Lemongrass is a widely used herb as a food flavoring, as a perfume, and for its analgesic and anti-inflammatory purposes; however, the molecular mechanisms of these effects have not been elucidated. Previously, we identified carvacrol from the essential oil of thyme as a suppressor of cyclooxygenase (COX)-2, a key enzyme for prostaglandin synthesis, and also an activator of peroxisome proliferator-activated receptor (PPAR), a molecular target for "lifestyle-related" diseases. In this study, we evaluated the essential oil of lemongrass using our established assays for COX-2 and PPARs. We found that COX-2 promoter activity was suppressed by lemongrass oil in cell-based transfection assays, and we identified citral as a major component in the suppression of COX-2 expression and as an activator of PPARα and γ. PPARγ-dependent suppression of COX-2 promoter activity was observed in response to citral treatment. In human macrophage-like U937 cells, citral suppressed both LPS-induced COX-2 mRNA and protein expression, dose-dependently. Moreover, citral induced the mRNA expression of the PPARα-responsive carnitine palmitoyltransferase 1 gene and the PPARγ-responsive fatty acid binding protein 4 gene, suggesting that citral activates PPARα and γ, and regulates COX-2 expression. These results are important for understanding the anti-inflammatory and anti-lifestyle-related disease properties of lemongrass., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Following previous cloning and expression studies of Xenopus aldolase C (brain-type) and A (muscle-type) cDNAs, we cloned here two Xenopus aldolase B (liver-type) cDNAs (XALDB1 and XALDB2, 2447 and 1490 bp, respectively) using two different liver libraries. These cDNAs had very similar ORF with only one conservative amino acid substitution, but 3'-UTR of XALDB1 contained ca. 1 kb of unrelated reiterated sequence probably ligated during library construction as shown by genomic Southern blot analysis. In adult, aldolase B mRNA (ca. 1.8 kb) was expressed strongly in kidney, liver, stomach, intestine, moderately strongly in skin, and very weakly in all the other tissues including muscles and brain, which strongly express aldolase A and C mRNAs, respectively. In oocytes and early embryos, aldolase A and C mRNAs occurred abundantly as maternal mRNAs, but aldolase B mRNA occurred only at a residual level, and its strong expression started only after the late neurula stage, mainly in liver rudiment, pronephros, epidermis and proctodeum. Thus, active expression of the gene for aldolase B, involved in dietary fructose metabolism, starts only later during development (but before the feeding stage), albeit genes for aldolases A and C, involved in glycolysis, are expressed abundantly from early stages of embryogenesis, during which embryos develop depending on yolk as the only energy source.

As the first examples among marine algal hemagglutinins, the primary structures of two hemagglutinins, named hypnin A-1 and A-2, from the red alga Hypnea japonica, were determined by Edman degradation. Both hemagglutinins were single-chain polypeptides composed of 90 amino acid residues including four half-cystines, all of which were involved in two intrachain disulfide bonds, Cys(5)-Cys(62) and Cys(12)-Cys(89). Hypnin A-1 and A-2 had calculated molecular masses of 9146.7 and 9109.7 Da which coincided with determined values, 9148 and 9109 Da, by electrospray ionization-mass spectrometry, respectively. Both hemagglutinins only differed from each other at three positions; Pro(19), Arg(31) and Phe(52) of hypnin A-1 as compared with Leu(19), Ser(31), and Tyr(52) of hypnin A-2. Approximately 43% of total residual numbers consisted of three kinds of amino acids: serine, glycine and proline. The hemagglutination activities were lost by reduction and alkylation of the disulfide bonds. The nature of the small-sized polypeptides, including disulfide bonds, may contribute to the extreme thermostability of the hemagglutinins. Sequence having overall similarity to hypnin A-1 or A-2 was not detected in databases. Unexpectedly, however, hypnins contained a motif similar to the alignment of the C-terminal conserved amino acids within carbohydrate-recognition domains of C-type animal lectins. Furthermore, interestingly, the hemagglutination activities were inhibited by a protein, phospholipase A-2 besides some glycoproteins, suggesting that hypnins may possess both a protein-recognition site(s) and a carbohydrate-recognition site(s).

A Xenopus aldolase C gene (XAClambda3-1), much longer (9.6 kb) than human and rat genes (3.7-3.6 kb), was isolated and characterized, and expression studies were performed using Xenopus embryos and A6 cells, a kidney cell line constitutively expressing aldolase C gene. The Xenopus gene contained nine exons, and in its proximal 5'-upstream region a GC box and a 16 bp long aldolase C-specific element (ACSE), and in addition, a CCAAT box and a TATA-like element, both missing in mammalian genes. The lacZ gene connected to the 5'-upstream region (1.6 kb) of the aldolase gene containing many potentially regulative sequence elements was expressed in embryos temporally and spatially like the endogenous aldolase C gene. Deletion experiments using embryos and A6 cells suggested that this 5'-upstream DNA contained in its distal part a region which negatively affected on its expression in embryos, but not in A6 cells. The proximal-most region contained a basal promoter (68 bp) essential for expression in both embryos and A6 cells. Deletion experiments using A6 cells failed to detect such regulative regions within the first intron (spanning ca. 4 kb). Analyses with mutated promoters in A6 cells revealed that the GC box was the crucial element in the basal promoter, although the TATA-like element appeared to have a slightly stimulative effect on the GC box functioning. Gel retardation and foot-printing assays revealed the occurrence in A6 cells of a nuclear factor(s) that binds specifically to the GC box. Since Xenopus aldolase C gene has several unique structural features, we expect that it will provide an interesting material for studying the evolution and developmental control of the aldolase C gene.

We obtained cDNA (XALDA; 1466 bp) for Xenopus laevis aldolase A gene (muscle-type), whose amino acid sequence had 88% similarity to those of mammalian aldolase A genes. XALDA mRNA occurred abundantly in skeletal muscle and at low levels also in other adult tissues, and such mRNA distribution was reflected in zymograms. In oocytes XALDA mRNA occurred at a relatively high level from stage I, and the mRNA level peaked at stage II, then decreased in later stages. XALDA mRNA in the full-grown oocyte was inherited as maternal mRNA throughout maturation and fertilization until midblastula stage, but its level became very low during gastrula and early neurula stages, and then increased greatly in later stages. While maternal XALDA mRNA was distributed uniformly in early embryos, mRNA zygotically expressed after late neurula stage occurred mainly in somites. In blastula animal caps XALDA mRNA occurred at a low level, but the expression was greatly enhanced by activin treatment. Thus, in Xenopus laevis aldolase A gene is actively transcribed in earlier phase of oogenesis, inherited as maternal mRNA in early embryos in a cell-type nonspecific way, then in later phases of embryogenesis, it is strongly expressed in somites with its concomitant ubiquitous expression at low levels in almost all the other cell types.

Previously, we reported that the 2.6 kDa peptide fragment extending from Arg-177 to Tyr-198 in rabbit skeletal muscle actin bound to actin itself and inhibited its polymerization, while the 9.1 kDa peptide extending from Ser-199 to Tyr-279 in actin did not. The 2.6 kDa segment of actin was reported to contain one of the important actin-actin contacts (Hori, K. and Morita, F. (1992) J. Biochem. 112, 401-408). In this paper, we show additional evidence that the rate of salt-induced increase in the fluorescence of pyrene-labeled actin was decreased in the presence of the 2.6 kDa peptide. Conventional actin filaments were only scarcely observed in the presence of the 2.6 kDa peptide under an electron microscope with a steady state of fluorescence increase. Furthermore, the 2.6 kDa peptide was found to sever F-actin into short filament fragments. The 9.1 kDa peptide, on the other hand, neither inhibited the fluorescence increment of pyrene-actin nor severed actin filaments. However, the 9.1 kDa peptide was found to increase the viscosity and fluorescence intensity of pyrene-G-actin and to form short actin filaments in the absence of salts. Contact sites in the 9.1 kDa segment in actin may have a different mode of interaction with adjacent actin promoters in actin filaments from that of the 2.6 kDa segment.

A full length cDNA clone (cXALD3) for Xenopus laevis aldolase mRNA, which exists abundantly in oocytes, was isolated from Xenopus laevis ovary cDNA library, and its nucleotide sequence was determined. The cDNA was 1.8 kb in length and encoded 363 amino acids. From the deduced amino acid sequence and the Northern blot analysis of the RNAs from several adult tissues, this clone was concluded to be a brain-type aldolase gene. The XALD3 mRNA level per egg or embryo was high during early oogenesis, but was markedly reduced during late oogenesis and was maintained at low level during early embryogenesis until it started to increase at the late neurula stage. The mRNA was also detected in testis. The characteristic change in the temporal pattern of expression and the distribution of XALD3 mRNA among different tissues suggest a possibility that brain type aldolase may play some important roles in gametogenesis and in neurulation.

As an approach for studying the function of chromosome proteins in DNA replication and gene expression, proteins remaining attached to Escherichia coli nucleoids were analyzed by two-dimensional polyacrylamide slab gel electrophoresis. Nucleoids were isolated by gentle lysis of the cells in the presence of a DNA counter-ion such as 1 M NaCl or 5 mM spermidine. In exponentially growing cells, about 100 proteins have been found to exist in the nucleoids. Kinetic studies indicated that the number of chromosome proteins remaining attached varied with time after synchronization. Based on the pattern of the variation, appearance, increase or disappearance of the 29 major proteins, nucleoid proteins were shown to be classified in six different groups (groups A--F). A strong correlation was observed between the variation of proteins belonging to group D and initiation of DNA synthesis or cell division.

E. coli expression plasmids for human aldolases A and B (EC 4.1.2.13) have been constructed from the pIN-III expression vector and their cDNAs, and expressed in E. coli strain JM83. Enzymatically active forms of human aldolase have been generated in the cells when transfected with either pHAA47, a human aldolase A expression plasmid, or pHAB 141, a human aldolase B expression plasmid. These enzymes are indistinguishable from authentic enzymes with respect to molecular size, amino acid sequences at the NH2- and COOH-terminal regions, the Km for substrate, fructose 1,6-bisphosphate and the activity ratio of fructose 1,6-bisphosphate/fructose 1-phosphate (FDP/F1P), although net electric charge and the Km for FDP of synthetic aldolase B differed from those for a previously reported human liver aldolase B. In addition, both the expressed aldolases A and B complement the temperature-sensitive phenotype of the aldolase mutant of E. coli h8. These data argue that the expressed aldolases are structurally and functionally similar to the authentic human aldolases, and would provide a system for analysis of the structure-function relationship of human aldolases A and B.

Escherichia coli nucleoids isolated in the presence of spermidine have been shown to contain DNA polymerase activity of which more than 95% was ascribed to DNA polymerase I and the rest to DNA polymerases II and III. It also has been found that DNA polymerases II and III or DNA polymerase III substituted entirely for DNA polymerase I or DNA polymerase I and II in the nucleoids isolated from E. coli mutants lacking the corresponding enzyme activities.

Western blot analyses of yolk proteins of the White Leghorn hens showed that an ovalbumin-like molecule was included in day 16 eggs but not in the ovarian follicles, and very little in newly deposited eggs. Northern hybridization, as well as in vitro translation, of poly(A)+ RNAs prepared from the yolk sac membranes of developing embryos gave no signal for ovalbumin messages. These results imply that the present ovalbumin-like protein of yolk has its origin in egg white, not being a de novo synthesis product in the yolk sac membranes.

Rat aldolase B gene was confirmed to have multiple transcriptional initiation sites; one site for the major transcript and at least two sites for the minor transcripts which are located at about 30 to 60 bases upstream of the major site. Although the transcription of the minor transcripts was entirely resistant to 1 microgram/ml of alpha-amanitin, which inhibits RNA polymerase II, it was completely blocked by 1000 micrograms/ml of the drug which inhibits RNA polymerase III. The box A- and B-like sequences, which are known to exist in the internal split promoter region of class III genes, were also found to be located around the initiation sites of two minor transcripts from the rat aldolase B gene. Analyses using deletion mutants of the promoter region of the gene suggested that the synthesis of the minor transcripts depends on the box A- and B-like sequences, but does not depend on a TATA promoter element. Northern blot analysis indicated that the minor transcripts are only a few percent of the total transcripts derived from the aldolase B gene. Some of the minor transcripts analyzed were shown to have a mature size in the poly(A)+ RNA fraction. Thus, the minor transcripts of rat aldolase B gene are apparently transcribed by RNA polymerase III, and subsequently normally processed and polyadenylated. The possible function of these minor transcripts will be discussed.