Organisms can essentially be classified according to two codon patterns.

We recently classified 23 bacteria into two types based on their complete genomes; "S-type" as represented by Staphylococcus aureus and "E-type" as represented by Escherichia coli. Classification was characterized by concentrations of Arg, Ala or Lys in the amino acid composition calculated from the complete genome. Based on these previous classifications, not only prokaryotic but also eukaryotic genome structures were investigated by amino acid compositions and nucleotide contents. Organisms consisting of 112 bacteria, 15 archaea and 18 eukaryotes were classified into two major groups by cluster analysis using GC contents at the three codon positions calculated from complete genomes. The 145 organisms were classified into "AT-type" and "GC-type" represented by high A or T (low G or C) and high G or C (low A or T) contents, respectively, at every third codon position. Reciprocal changes between G or C and A or T contents at the third codon position occurred almost synchronously in every codon among the organisms. Correlations between amino acid concentrations (Ala, Ile and Lys) and the nucleotide contents at the codon position were obtained in both "AT-type" and "GC-type" organisms, but with different regression coefficients. In certain correlations of amino acid concentrations with GC contents, eukaryotes, archaea and bacteria showed different behaviors; thus these kingdoms evolved differently. All organisms are basically classifiable into two groups having characteristic codon patterns; organisms with low GC and high AT contents at the third codon position and their derivatives, and organisms with an inverse relationship.

Codon evolution is governed by linear formulas.

When nucleotide (G, C, T and A) contents were plotted against each nucleotide, their relationships were clearly expressed by a linear formula, y = alphax + beta in the coding and non-coding regions. This linear relationship was obtained from the complete single-stranded DNA. Similarly, nucleotide contents at all three codon positions were expressed by linear regression lines based on the content of each nucleotide. In addition, 64 codon usages were also expressed by linear formulas against nucleotide content. Thus, the nucleotide content not only in coding sequence but also in non-coding sequence can be expressed by a linear formula, y = alphax + beta, in 145 organisms (112 bacteria, 15 archaea and 18 eukaryotes). Based on these results, the ratio of C/T, G/T, C/A or G/A one can essentially estimate all four nucleotide contents in the complete single-stranded DNA, and the determination of any ratio of two kinds of nucleotides can essentially estimate four nucleotide contents, nucleotide contents at the three different codon positions and codon distributions at 64 codons in the coding region. The maximum and minimum values of G content were approximately 0.35 and approximately 0.15, respectively, among various organisms examined. Codon evolution occurs according to linear formulas between these two values.

Modulation of interleukin-8 and nitric oxide synthase mRNA levels by interferon-gamma in macrophages stimulated with lignin derivatives and lipopolysaccharides.

It has been shown that interferon-gamma (IFN-gamma) plays a role in the regulation of interleukin-8 (IL-8), nitric oxide (NO), and tumor necrosis factor-alpha (TNF-alpha) secretion by macrophages stimulated with lignin derivatives, such as EP3, and lipopolysaccharides (LPS) [Cytokine 11 (1999) 571]. To examine the mechanism by which IFN-gamma affects secretion of these factors, EP3- or LPS-stimulated macrophages were treated with different concentrations of IFN-gamma, and mRNA levels of IL-8, nitric oxide synthase (NOS) and TNF-alpha were determined by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). As reported previously, stimulation of macrophages by EP3 or LPS dramatically induced the expression of IL-8, NOS, and TNF-alpha mRNAs. IFN-gamma clearly decreased the level of IL-8 mRNA in stimulated macrophages, although it did not affect the IL-8 mRNA level in unstimulated macrophages. In contrast, IFN-gamma appeared to increase the level of NOS mRNA both in unstimulated and stimulated macrophages. IFN-gamma, which increased the amount of TNF-alpha mRNA in unstimulated macrophages, showed no significant effect on the high level of TNF-alpha mRNA in stimulated macrophages. These results suggest that IFN-gamma causes changes in IL-8 and NO secretion by stimulated macrophages through its effects on the level of IL-8 and NO mRNA, respectively. Effects of IFN-gamma on TNF-alpha secretion by stimulated macrophages may be mediated by a different mechanism.

The classification of various organisms according to the free amino acid composition change as the result of biological evolution.

The free amino acid compositions in archaeobacteria, eubacteria, protozoa, blue-green alga, green alga, slime mold, plants and mammalian cells were analyzed, to investigate whether changes in their free amino acid compositions reflect biological evolution. Cell homogenates were treated with 80-90% ethanol to separate cellular proteins and free amino acids contained in the cells. Different patterns of the free amino acid compositions were observed in the various organisms. Characteristic differences were observed between plant and mammalian cells, and between archaeobacteria and eubacteria. The patterns of the free amino acid composition in blue-green alga, green alga, protozoa and slime mold differed from each other and from those of eubacteria and archaeobacteria. Rat hepatoma cells (R-Y121B) were cultured in Eagle's minimum essential medium (MEM) containing 5% serum or in a modified MEM lacking arginine, tyrosine and glutamine. No significant difference in the free amino acid composition was observed between the two cell groups cultured under two different conditions. It is suggested that the free amino acid composition reflects apparent biological changes as the result of evolution.

Inhibition by Agaricus blazei Murill fractions of cytopathic effect induced by western equine encephalitis (WEE) virus on VERO cells in vitro.

Anti-viral activities of Agaricus blazei Murill were investigated. The water extracts of the cultured mycelia and fruiting bodies were fractionated with different concentrations of ethanol. To several viruses which have cytopathic effects (CPE) on VERO cells, inhibition of these effects by the ethanol fractions was tested. Strong inhibition of CPE induced by western equine encephalitis (WEE) virus was observed in the mycelial fractions but not those of fruiting bodies.

Secretion of TNF-alpha, IL-8 and nitric oxide by macrophages activated with Agaricus blazei Murill fractions in vitro.

Water extracts of the mycelial culture and fruiting bodies of Agaricus blazei Murill were fractionated by ethanol precipitation using various ethanol concentrations. Original water extracts from mycelia (Fraction A-0) and fruiting bodies (Fraction B-0) induced TNF-alpha secretion by macrophages derived from rat bone marrow. Fractions B-4 and B-5 obtained from ethanol precipitation of fruiting bodies using 44% and 50% ethanol, respectively, and Fraction B-6 obtained from the supernatant at 50% ethanol markedly induced TNF-alpha secretion. Similar effects were observed in IL-8 secretion by macrophages. Regarding nitric oxide (NO), Fraction B-5 induced a significant increase in NO secretion and Fractions B-4 and B-6 induced slightly NO secretion. Northern blot analysis showed that the increases in cytokine- and NO secretion were due to an increase in cytokine mRNAs or NO synthase mRNA. Therefore, it is concluded that Agaricus blazei Murill components which activate macrophages result in the induction of cytokine- and NO secretion in vitro.

Conservation of the basic pattern of cellular amino acid composition of archaeobacteria during biological evolution and the putative amino acid composition of primitive life forms.

Previous studies showed that the cellular amino acid composition obtained by amino acid analysis of whole cells, differs such as eubacteria, protozoa, fungi and mammalian cells. These results suggest that the difference in the cellular amino acid composition reflects biological changes as the result of evolution. However, the basic pattern of cellular amino acid composition was relatively constant in all organisms examined. In the present study, we examined archaeobacteria, because they are considered important in understanding the relationship between biological evolution and cellular amino acid composition. The cellular amino acid compositions of Archaeoglobus fulgidus, Pyrococcus horikoshii, Methanobacterium thermoautotrophicum and Methanococcus jannaschii differed slightly from each other, but were similar to those determined from codon usage data, based on the complete genomes. Thus, the cellular amino acid composition reflects biological evolution. We suggest that primitive forms of life appearing on earth at the end of prebiotic evolution had a similar-cellular amino acid composition.

Isolation and characterization of the 5'-upstream and untranslated regions of the mouse type II iodothyronine deiodinase gene.

The type II iodothyronine deiodinase (D2) catalyzes the 5'-deiodination of thyroxine to yield the biologically active form, 3,3',5,-tri-iodothyronine, and is a member of the selenoproteins. We isolated a 17.5 kb mouse genomic clone containing the entire coding and 5'-untranslated regions of the D2 gene (mdio2). We also isolated the entire 5'-UTR of the mouse D2 cDNA, which was 753 bp in length and contained five ATG codons. An additional 258 bp ORF from the fourth ATG codon was found in the same reading frame as the coding region reported previously, and this additional ORF contained a TGA codon, which could encode selenocysteine. The proximal promoter of mdio2 contained a TATA box and several potential transcription factor-binding sequences, including CRE, C/EBP and GATA binding sites. The 1.3 kb 5'-upstream region exhibited a promoter activity by reporter assay using Mm5MT and JAR cells, which have a D2 transcript, but not HepG2 cells that have no detectable level of D2 transcript.

Conservation of the basic pattern of cellular amino acid composition during biological evolution in plants.

The cellular amino acid composition of plant cells was analyzed. The callus of carrot (Daucus carota), leaves of Torenia fournieri and protocomb-like body of Cymbidium, s.p. were examined as examples of plant cells. The cellular amino acid compositions differed in the plant cells, but their basic patterns were quite similar. It is concluded that the basic pattern of the cellular amino acid composition is conserved in all terrestrial organisms, including plants.

Biochemical and molecular biological evidence for the presence of type II iodothyronine deiodinase in mouse mammary gland.

In the present study we have obtained several lines of evidence indicating the presence of type II iodothyronine deiodinase (DII) in the mouse mammary gland. 5'-deiodinase activity in the mammary gland has an apparent K(m) value of 4.4 nM for T(4) and is inhibited by aurothioglucose but not by propylthiouracil. These characteristics are similar to those of DII in other tissues. We cloned a 1.4-kb cDNA, which contains the entire mouse DII coding region and has high homology with the rat DII cDNA, from the mammary gland and brain. Northern blot analysis showed the presence of 7.9 kb DII mRNA in the mammary gland and brain. The levels of DII activity and mRNA in lactating gland were significantly lower than those in virgin and pregnant glands, suggesting that DII is regulated at the pretranslational level. In addition, we found the low level of DII enzyme activity and transcript in various other mouse tissues.

Development of a Simple Culture Method for the Tissues Contaminated with Microorganisms and Application to Establishment of a Fish Cell line.

Cell cultures are good in vitro model systems in place of using living animals. In the present study, we developed a simple culture method in which tissues were pretreated with a low concentration of sodium hypochlorite solution (NaClO) to prevent not only bacteria but also fungi. Scales removed from a goldfish (Carassius auratus) body were treated with 70% ethanol and then with 0.3% of sodium hypochlorite solution, and cultured in vitro in an atmosphere containing 0.5% CO2. The doubling time of the established cells (GAKS) was 24 hr. The GAKS cells contained alkaline phosphatase activity (8.3+/-1.1 nmol/min/mg protein) and secreted 0.32+/-0.07 pg/ml endothelin during a 3 day culture of a full monolayer sheet.

Evolutionary changes reflected by the cellular amino acid composition.

Comparison of the amino acid composition of cell-proteins using 17 amino acids has been used to investigate the biological evolution of organisms such as bacteria, blue-green alga, green alga, fungi, slime mold, protozoa and vertebrates. The degree of difference in the amino acid ratios between any two groups reflects the degree of divergency in biological evolution. The amino acid composition of the Gram-negative bacteria (Escherichia coli, Klebsiella, Proteus, and Vibrio alginolyticus) was identical. However, the amino acid composition of Staphylococcus aureus and Bacillus subtilis, which are Gram-positive bacteria, differed from each other and from the Gram-negative bacteria. The amino acid composition of the blue-green alga (Cyanobacterium, Chroococidiopsis) was quite similar to that of E. coli. A marked difference in the amino acid composition was observed between E. coli and green alga (Chlorella), and significant differences were observed between E. coli and other organisms, such as fungi, protozoa (Tetrahymena), slime mold (Dictyostelium discoideum) and vertebrates. In conclusion, the change in cellular amino acid composition reflects the divergence which has occurred during biological evolution, whereas a basic pattern of amino acid composition is maintained in spite of a long period of evolutional divergence among the various organisms. Thus, it is proposed that the primitive life forms established at the end of prebiotic evolution had a similar amino acid composition.

Secretion of TNF-alpha, IL-8 and nitric oxide by macrophages activated with polyanions, and involvement of interferon-gamma in the regulation of cytokine secretion.

When macrophages derived from rat bone marrow were cultured in the presence of polyanions such as acetyl lignin (EP3), sulfonyl lignin (LS) or dextran sulfate (DS), the cells secreted TNF-alpha, IL-8 and nitric oxide (NO). EP3 had a dose-dependent effect on the secretion of TNF-alpha, IL-8 and NO. EP3 significantly affected secretion at concentrations greater than 5 microg/ml. The EP3 effect was at its maximum between concentrations of 50 and 100 microg/ml. LS and DS induced a slight increase in the secretion of cytokines and NO at a concentration of 100 microg/ml. The use of the reverse-transcription polymerase chain reaction (RT-PCR) showed that the increases in cytokine and NO secretion were due to an increase in cytokine mRNAs or NO synthase mRNA. Anti-TNF-alpha antibodies partially inhibited NO secretion by EP3-activated macrophages, although IL-8 secretion was independent of antibody treatment. The secretion of TNF-alpha and NO was also unaffected by the addition of anti-IL-8 antibodies. The addition of interferon-gamma (IFN-gamma) to the culture medium did not alter TNF-alpha and NO secretion by the EP3-activated macrophages, however, IL-8 secretion was increased when a low concentration of IFN-gamma (0.2 U/ml) was added, but was reduced in the presence of a high concentration of IFN-gamma (2000 U/ml). IFN-gamma produced similar effects on cytokine and NO secretion in macrophages activated with lipopolysaccharide (LPS). Therefore, it is concluded that macrophages treated with polyanions secrete cytokines and NO, and that INF-gamma is involved in the regulatory mechanism of cytokine and NO secretion.

Multinucleation and preservation of nucleolar integrity of macrophages.

Rat bone marrow-derived macrophages formed multinucleated giant cells spontaneously when cultured in slide glass chambers or when induced with the polyanion acetyl lignin. Nuclei in such cells tended to cluster in distinct rings. DNA fragmentation appeared to occur in multinucleated cells, as detected by 3' end-labeling. Southern blot analyses, using probes specific for nucleolar and non-nucleolar genes, indicated that chromatin DNA was fragmented whereas nucleolar DNA was relatively intact. Autoradiography revealed preservation, in multinucleated cells, of nucleoli into which radiolabeled uridine was incorporated. Multinucleated macrophages appeared to eventually fragment. Preserved integrity of nucleoli seems to be a feature of macrophage multinucleation, a process which apparently culminates in cell death.

Activation of macrophages by lactoferrin: secretion of TNF-alpha, IL-8 and NO.

When macrophages were cultured with lactoferrin, cytokines such as tumor necrosis factor (TNF-alpha), interleukin 8 (IL-8) and nitric oxide (NO) were secreted. Secretion of TNF-alpha peaked at 6 h of incubation in the presence of lactoferrin and then declined. About 80% of the maximum secretion of IL-8 was observed at 6 h of incubation. The concentration of IL-8 in the culture medium remained almost constant between 24-72 h. In contrast, no significant effect on NO secretion was observed at 6 h, but a significant effect was observed at 24 h and secretion gradually increased between 24-72 h. The effects of lactoferrin on the secretion of TNF-alpha, IL-8 and NO were dose-dependent and lactoferrin had a significant effect on secretion of at concentrations greater than 10 mg/ml. The use of reverse transcription-polymerase chain reaction (RT-PCR) showed that the results obtained were consistent with the cytokine secretion results. It is concluded that lactoferrin activates macrophages which result in the secretion of TNF-alpha, IL-8 and NO.

Function of conserved tryptophans in the Aspergillus niger glucoamylase 1 starch binding domain.

Nuclear magnetic resonance (NMR) and ultraviolet (UV) difference spectroscopy were used to assess the role of a number of tryptophan residues in the granular starch binding domain (SBD) of glucoamylase 1 from Aspergillus niger. Wild-type SBD and three variant (W563K, W590K, and W615K) proteins were produced using an A. niger expression system. Titration studies were conducted with beta-cyclodextrin (betaCD), a cyclic analogue of starch, as the ligand. The NMR studies show that the W563K and W590K variants only bind 1 equiv while the wild-type protein forms a 2:1 (ligand:protein) complex. It also clearly demonstrates the abolition of binding at site 1 and site 2 in W590K and W563K, respectively. UV difference spectroscopy was used to calculate dissociation constants with addition of betaCD: 14.4 microM (apparent) for the wild type, 28.0 microM for W563K, and 6.4 microM for W590K. The implication of this is that the two binding sites have unequal contributions to the overall binding of the SBD which may be related to functional differences between the two binding sites. The low stability of the third variant, W615K, suggests that this tryptophan is not involved in binding but has an essential structural role.

Solution structure of the granular starch binding domain of Aspergillus niger glucoamylase bound to beta-cyclodextrin.

BACKGROUND: Carbohydrate-binding domains are usually small and physically separate from the catalytic domains of hydrolytic enzymes. Glucoamylase 1 (G1) from Aspergillus niger, an enzyme used widely in the food and brewing industries, contains a granular starch binding domain (SBD) which is separated from the catalytic domain by a semi-rigid linker. The aim of this study was to determine how the SBD binds to starch, and thereby more generally to throw light on the role of carbohydrate-binding domains in the hydrolysis of insoluble polysaccharides. RESULTS: The solution structure of the SBD of A. niger G1 bound to beta-cyclodextrin (betaCD), a cyclic starch analogue, shows that the well-defined beta-sheet structure seen in the free SBD is maintained in the SBD-betaCD complex. The main differences between the free and bound states of the SBD are observed in loop regions, in or near the two starch-binding sites. The two binding sites, each of which binds one molecule of betaCD, are structurally different. Binding site 1 is small and accessible, and its structure changes very little upon ligand binding. Site 2 is longer and undergoes a significant structural change on binding. Part of this site comprises a flexible loop, which appears to allow the SBD to bind to starch strands in a range of orientations. CONCLUSIONS: The two starch-binding sites of the SBD probably differ functionally as well as structurally; site 1 probably acts as the initial starch recognition site, whereas site 2 is involved in specific recognition of appropriate regions of starch. The two starch strands are bound at approximately 90 degrees to each other. This may be functionally important, as it may force starch strands apart thus increasing the hydrolyzable surface, or alternatively it may localize the enzyme to noncrystalline (more hydrolyzable) areas of starch. The region of the SBD where the linker to the catalytic domain is attached is flexible, allowing the catalytic site to access a large surface area of the starch granules.

Delayed cytocidal effect of lignin derivatives on virally transformed rat fibroblasts.

When rat fibroblasts (Ad12-3Y1-Z19) transformed with adenovirus type 12 were cultured with lignin derivative (acetyl or sulfonyl), the cells grew for 2 to 3 days at the same rate as the control cells cultured without lignin derivative, then rapidly died. This cytocidal effect was independent of the cell population density. The lag time was longer than the doubling time (-24 h) of Ad12-3Y1-Z19 cells. Other polyanions such as dextran sulfate and glycosaminoglycans did not show significant inhibitory effect on Ad12-3Y1-Z19 cell growth. In order to determine whether or not this cytocidal effect is general for every cell line, we examined 14 cell lines derived from tumor tissues and normal tissues, and 8 cell lines transformed with viruses, chemical carcinogens, or oncogenes. Of these cell lines, many responded to lignin derivatives with inhibition of cell growth, while in some cell lines no inhibitory effect of lignin derivatives was observed. The cytocidal effect was observed in only Ad12-3Y1-Z19 cells. This may be a new type of cytocidal phenomenon.

Inhibitory effect of heparin on collagen fiber formation in hepatic cells in culture.

When M cells derived from rat liver and then transformed by treatment with 4-dimethylaminoazobenzene were cultured in vitro, the culture became covered with a collagen fiber network. When the M cells were cultured in the presence of more than 10 micrograms/ml heparin, no collagen fiber formation was observed. The inhibitory effect was evident at 5 micrograms/ml but was not significant at 1 microgram/ml. High performance liquid chromatography (HPLC) showed that about 50% decrease in hydroxyproline content occurred in the presence of 10 micrograms/ml heparin. The inhibition by heparin reached a plateau at 10 micrograms/ml. Other glycosaminoglycans such as heparan sulfate, keratan sulfate, chondroitin sulfate B and chondroitin sulfate C did not show a significant effect on hydroxyproline content. Modified heparins slightly decreased hydroxyproline content, but the collagen fibers were still observed. These results indicate that the native structure of heparin is important to attain the complete inhibition of collagen fiber formation; the basic structure, (-GlcUA or IdUA beta 1-4 GlcNSO3-)n, is important. HPLC, Northern blot analysis and Western blot analysis for rat type I collagen revealed that collagen synthesis is independent of heparin, but that collagen fiber formation is prevented by heparin.