Identification of receptor and heparin binding sites in fibroblast growth factor 4 by structure-based mutagenesis.

Fibroblast growth factors (FGFs) comprise a large family of multifunctional, heparin-binding polypeptides that show diverse patterns of interaction with a family of receptors (FGFR1 to -4) that are subject to alternative splicing. FGFR binding specificity is an essential mechanism in the regulation of FGF signaling and is achieved through primary sequence differences among FGFs and FGFRs and through usage of two alternative exons, IIIc and IIIb, for the second half of immunoglobulin-like domain 3 (D3) in FGFRs. While FGF4 binds and activates the IIIc splice forms of FGFR1 to -3 at comparable levels, it shows little activity towards the IIIb splice forms of FGFR1 to -3 as well as towards FGFR4. To begin to explore the structural determinants for this differential affinity, we determined the crystal structure of FGF4 at a 1.8-A resolution. FGF4 adopts a beta-trefoil fold similar to other FGFs. To identify potential receptor and heparin binding sites in FGF4, a ternary FGF4-FGFR1-heparin model was constructed by superimposing the FGF4 structure onto FGF2 in the FGF2-FGFR1-heparin structure. Mutation of several key residues in FGF4, observed to interact with FGFR1 or with heparin in the model, produced ligands with reduced receptor binding and concomitant low mitogenic potential. Based on the modeling and mutational data, we propose that FGF4, like FGF2, but unlike FGF1, engages the betaC'-betaE loop in D3 and thus can differentiate between the IIIc and IIIb splice isoforms of FGFRs for binding. Moreover, we show that FGF4 needs to interact with both the 2-O- and 6-O-sulfates in heparin to exert its optimal biological activity.

Activation of fgf4 gene expression in the myotomes is regulated by myogenic bHLH factors and by sonic hedgehog.

The Fgf4 gene encodes an important signaling molecule which is expressed in specific developmental stages, including the inner cell mass of the blastocyst, the myotomes, and the limb bud apical ectodermal ridge (AER). Using a transgenic approach, we previously identified overlapping but distinct enhancer elements in the Fgf4 3' untranslated region necessary and sufficient for myotome and AER expression. Here we have investigated the hypothesis that Fgf4 is a target of myogenic bHLH factors. We show by mutational analysis that a conserved E box located in the Fgf4 myotome enhancer is required for Fgf4-lacZ expression in the myotomes. A DNA probe containing the E box binds MYF5, MYOD, and bHLH-like activities from nuclear extracts of differentiating C2-7 myoblast cells, and both MYF5 and MYOD can activate gene expression of reporter plasmids containing the E-box element. Analyses of Myf5 and MyoD knockout mice harboring Fgf4-lacZ transgenes show that Myf5 is required for Fgf4 expression in the myotomes, while MyoD is not, but MyoD can sustain Fgf4 expression in the ventral myotomes in the absence of Myf5. Sonic hedgehog (Shh) signaling has been shown to have an essential inductive function in the expression of Myf5 and MyoD in the epaxial myotomes, but not in the hypaxial myotomes. We show here that expression of an Fgf4-lacZ transgene in Shh-/- embryos is suppressed not only in the epaxial but also in the hypaxial myotomes, while it is maintained in the AER. This suggests that Shh mediates Fgf4 activation in the myotomes through mechanisms independent of its role in the activation of myogenic factors. Thus, a cascade of events, involving Shh and bHLH factors, is responsible for activating Fgf4 expression in the myotomes in a spatial- and temporal-specific manner.

Induction of apoptosis in the human promyelocytic leukemia cell line HL60 by falconensone A and its derivatives, new polyenes.

Falconensones A and B are a new type of yellow pigment with structural similarity to retinoic acid isolated from the mycelial extract of ascomycetous fungi, Emericella falconensis or Emericella fruticulosa. In the present study we show that falconensone A alone induced apoptosis of HL60 human leukemia cells, while falconensone B, the 4'-nor-methyl derivative of falconensone A, had much lower activity. The synthetic derivatives of falconensone A, falconensone A p-bromophenylhydrazone and falconensone A dioxime, were more potent than natural falconensone A and B as far as the induction of apoptosis was concerned. The induction of apoptosis by the falconensones correlated with their inhibition of cell growth. In addition, falconensones A and B, and falconensone A dioxime, increased the generation of intracellular reactive oxygen species, while falconensone A p-bromophenylhydrazone was inactive. These results suggest that falconensone A, falconensone A p-bromophenylhydrazone and falconensone A dioxime are potential new apoptosis-inducing agents. The enhanced generation of reactive oxygen species in cells may be involved in apoptosis induced by falconensone A and falconensone A dioxime, but not by falconensone A p-bromophenylhydrazone. It is also suggested that the methyl residue at the 4' position of the falconensone A cyclopentenone ring may be essential for the induction of apoptosis. Based on these results, falconensone A and its derivatives may be clinically useful in the treatment of some leukemias.

cDNA-derived amino acid sequence of acetoacetyl-CoA synthetase from rat liver.

In order to examine the primary structure of acetoacetyl-CoA synthetase (acetoacetate-CoA ligase, EC 6.2.1.16; AA-CoA synthetase), the cDNA clone encoding this enzyme has been isolated from the cDNA library which was prepared from the liver of rat fed a diet supplemented with 4% cholestyramine and 0.4% pravastatin for 4 days. Nucleotide sequence analysis of cloned cDNA revealed that AA-CoA synthetase of rat liver contains an open reading frame of 2019 nucleotides, and the deduced amino acid sequence (672 amino acid residues) bears 25.0 and 38.9% homologies with acetyl-CoA synthetases of Saccharomyces cerevisiae and Archaeoglobus fulgidus, respectively.

Effects of development on acetoacetyl-CoA synthetase biosynthesis in rat liver.

In order to investigate the physiological role of acetoacetyl-CoA synthetase (acetoacetate-CoA ligase, EC 6.2.1.16), a cytosolic acetoacetate-activating enzyme, the effects of animal development on the activity and content of the enzyme were examined in rat liver. In male rats, the enzyme specific activity increased 21-fold at 4 weeks of age from that at 2 weeks of age, and then gradually decreased, while in female rats, it increased similarly to that of male rats, but further increased, reaching a maximum about 3-fold higher than that of male rats, at 6 weeks of age. The developmental patterns of the enzyme content correlated with that of the enzyme specific activity. These results indicate that changes in this enzyme activity and content during the developmental process might influence the rate of ketone body utilization for the formation of physiologically important lipidic substances in rat liver.

Biological activities of 1,1,6-trisubstituted indanes: beyond magainin 2.

MSI-78 is a peptide analog of naturally occurring magainin 2 isolated from the skin of Xenopus laevis. The peptide is known to have one of the strongest antibacterial activities in magainin 2 analogs against methicillin-resistant Staphylococcus aureus (MRSA). To find novel compounds superior to MSI-78, we have further designed, synthesizing 1,1-di(4-aminobutyl)-6-benzylindane (PM4) and 1,1-dibenzyl-6-(4-aminobutyl) indane (PM5), and tested their inhibitory ability of the growth of S. aureus. In an in vitro assay, PM4 showed the same antibacterial activity against the bacterium as MSI-78, and non-hemolytic activity against human red blood cells (RBCs) at the MIC (minimum inhibitory concentration) value, in contrast to the latter. On the other hand, PM5 showed stronger antibacterial activity than MSI-78, but being still accompanied with hemolysis at the MIC value. Otherwise, stronger decarboxylase activity for oxaloacetate was observed in PM5, rather than magainin 2 analogs or Oxaldie 1 as a control peptide, but not in PM4.

Induction of differentiation and apoptosis in human promyelocytic leukemia HL60 cell line by a new type of steroids.

Mer-NF8054X is a new type of steroid whose structure has been established as 11-oxo-18, 22-cycloergosta-6, 8(14)-diene-3beta, 5beta, 9beta, 23S-tetraol (an 18, 22-cycloergostane), which has been reported to have antifungal activity against Aspergillus fumigatus. However, other biological activities are unknown. Herein, we reported that Mer-NF8054X inhibited cell growth of HL60 human leukemia cells, when used either singly or in combination with retinoic acid (RA). In addition, Mer-NF8054X alone induced differentiation and apoptosis of HL60 cells. The induction of differentiation of HL60 cells by Mer-NF8054X was synergistic in combination with RA. On the other hand, Emesterone A, an analogue of Mer-NF8054X which is missing a hydroxy residue from the third position, showed much lower activity than Mer-NF8054X on the inhibition of cell growth and the induction of cell differentiation and apoptosis. However, Emesterone B, an analogue of Emesterone A which is missing a hydroxy residue from the fifth position, showed higher activity than Emesterone A but lower activity than Mer-NF8054X when examined for the inhibition of cell growth and the induction of cell differentiation and apoptosis. These results suggested that Mer-NF8054X and its analogs may be a new type of differentiation inducing agent. The hydroxy residue at the third position or fifth position in Mer-NF8054X may be necessary, but not essential, for inhibition of growth and induction of both differentiation and apoptosis of HL60 cells. In addition, Mer-NF8054X enhanced the differentiation of HL60 cells induced by RA. Based on these results, Mer-NF8054X may have utility in the clinic in combination with RA for leukemia patients.

Induction of differentiation in human promyelocytic leukemia cell line HL60 by a new type of polyenes, falconensone A and its derivatives.

Falconensones A and B are new type of yellow pigment isolated from the mycelial extract of ascomycetous fungi, Emericella falconensis or Emericella fruticulosa, whose structures are similar to retinoic acid (RA). To date, biological activities of falconensones have not been reported. Herein, we reported that falconensone A inhibited growth of HL60 human leukemia cells, when used either singly or in combination with RA. Falconensone A alone did not induce differentiation of HL60 cells. However, falconesone A enhanced the differentiation of HL60 cells induced by 10 nM RA, and its effect was synergistic. On the other hand, falconensone B, the 4'-nor-methyl derivative of falconensone A, showed much lower activity than falconensone A on the inhibition of cell growth. In addition, synthetic derivatives of falconensone A, falconensone A p-bromophenylhydrazone and falconensone A dioxime, were more potent on the inhibition of cell growth and the induction of differentiation than natural falconensones A and B. These compounds induced differentiation of HL60 cells into monocyte/macrophage-like cells, different from granulocyte-like cells induced by RA. These results suggest that falconensone A may be a new type of antiproliferative agent, and that the methyl residue at the 4' position of the cyclopentenone ring of falconensone A may be necessary for biological activity. In addition, falconensone A enhanced RA-induced differentiation of HL60 cells, while its derivatives alone showed growth inhibition and induction of differentiation of HL60 cells. Based on these results, falconensone A and its derivatives may have clinical utility in the treatment of leukemia.

Antibacterial activity of (+/-) 6-benzyl-1-(3-carboxypropyl) indane; a possible way to identify leading novel anti-H. pylori agents.

Magainin 2, isolated from the skin of the Xenopus laevis, is an antimicrobial peptide which reacts directly with the biological membrane to lyse various bacteria from negative and positive microorganisms. In a previous report, we showed that (+/-)1-(4-aminobutyl)-6-benzylindane (PM2), which mimicked the conformation of the side-chains of a complementary unit on the amino acid sequence of magainin 2 analogs, expressed the in vitro antibacterial activity not only against Helicobacter, pylori (ATCC43526, ATCC43579), but also against Escherichia coli (ATCC25922) and Staphylococcus aureus (ATCC25923). In addition, PM2 caused human blood red cells (RBCs) to lyse at the minimum inhibitory concentration (MIC) value. Based on the antibacterial activities of 9-phenylnonanoic acid (pC9c), we further synthesized (+/-)-6-benzyl-1-(3-carboxypropyl) indane (PM2c), which replaced a positive charge of PM2 with a negative one, and tested the biological activities. PM2c had the ability to inhibit the growth of H. pylori strains, but its activity to inhibit the growth of E. coli and S. aureus was not detected and weak, respectively. Moreover, PM2c showed non-hemolytic activity against RBCs at the MIC value. These results indicate the possibility that PM2c may be more useful than PM2 either alone or in combination with well-known therapeutic agents for the treatment of H. pylori infection.

On the antibacterial activity of normal and reversed magainin 2 analogs against Helicobacter pylori.

Magainin 2 is an antimicrobial peptide isolated from the skin of Xenopus laevis. We have tested the antibacterial activities of normal and reversed magainin 2 analogs against two strains of Helicobacter pylori (ATCC 43526, ATCC 43579), compared with those against Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). Among these analogs, MSI-78A showed the strongest activity against H. pylori. The MIC (minimum inhibitory concentration) values were almost the same as those against E. coli and S. aureus. No or lesser activity was observed in all the reversed peptides compared to the corresponding normal magainin 2 analogs. Based on the CD (circular dichroism) measurement, the more active peptide tends to show a higher alpha-content. The positively-charged five amino acids (KILKK) positioned at the C terminus on the amphipathical alpha-helical structure play important roles in exerting the strong activity against H. pylori. This indicates that the net charge of the cell surface in H. pylori may be more negative than that of E. coli, though both strains belong to the same genus.

Antibacterial activity of two alkylamines integrated an indane scaffold: mimicry of a complementary unit on magainin 2.

Based on the antibacterial activity of 9-phenylnonylamine (pC9a) against Escherichia coli (ATCC29522) and Staphylococcus aureus (ATCC25923), we have further tested the inhibitory ability of the growth of the bacteria by (+/-)1-(4-aminobutyl)-6-benzylindane (PM2) and (+/-)1-benzyl-6-(4-aminobutyl) indane (PM3), that is, two kinds of 1,6-disubstituted indanes. In an in vitro assay, they showed almost the same antibacterial activities against the bacteria as pC9a, as well as that of magainin 2 analogs (i.e., the peptides MSI-78 and 87-ISM), except in the case of 87-ISM against S. aureus. At the MIC (minimum inhibitory concentration) values, however, their killing rate of E. coli is actually quicker than pC9a. This indicates that an indane scaffold, used as a template to mimic a part of the alpha-helical structure of magainin 2, can accelerate the killing rate. At present, however, it is unknown whether either the hydrophobicity or the alpha-helical structure, or both, of the indane scaffold is involved in accelerating the rate. Moreover, these two indanes also showed stronger antibacterial activity against two strains of Helicobacter pylori (ATCC43526, ATCC43579) than either pC9a or magainin 2 related peptides.

Synthesis of reversed magainin 2 analogs enhanced antibacterial activity.

Magainin 2 is an antimicrobial peptide found in the skin of Xenopus laevis. To find a reversed peptide comparable to the antibacterial activity of magainin 2 analogs, we have synthesized three reversed analogs, the peptide 53D, 87-ISM and A87-ISM, corresponding to the normal peptide D35, MSI-78 and MSI-78A, respectively. We examined their ability to inhibit the growth of Escherichia coli and Staphylococcus aureus. Among the analogs, the A87-ISM, that is, the reverse of MSI-78A enhanced the amphiphilicity and the alpha-helical tendency of magainin 2, showed not only almost the same antibacterial activity against the bacteria as MSI-78A, but also stronger activity than other magainin 2 analogs. In addition, at the MIC (minimum inhibitory concentration) value, A87-ISM shows no hemolysis to human red blood cells, while both MSI-78 and MSI-78A cause strong hemolysis at the MIC value. This result indicates that a novel reversed peptide comparable or superior to normal magainin 2 analogs is available.

Tunicamycin in combination with retinoic acid synergistically inhibits cell growth while decreasing palmitoylation and enhancing retinoylation of proteins in the human breast cancer cell line MCF-7.

All-trans-Retinoic acid (RA) induces differentiation and inhibits growth of many tumor types. Whereas the RA nuclear receptors mediate genomic effects of RA, there also are many nongenomic effects that do not have defined mechanisms. Some nongenomic effects of RA may involve retinoylation (RA acylation), a posttranslational modification of proteins occurring in many eukaryotic cell lines including the human breast cancer cell line MCF-7. To gain further knowledge of the role(s) of retinoylation, we studied the effects of tunicamycin (TM), an inhibitor of both protein N-glycosylation and palmitoylation, on growth and retinoylation in MCF-7 cells. We found that RA or TM alone inhibited growth of MCF-7 cells. Combinations of RA and TM inhibited growth synergistically. TM increased retinoylation and decreased palmitoylation. These results suggest that increased retinoylation and decreased glycosylation and palmitoylation may play a role in the synergistic inhibition of cell growth by combinations of TM and RA in MCF-7 cells. Furthermore, our results suggest that combinations of TM and RA may have clinical utility.

Synthesis and biological activity of four kinds of reversed peptides.

We have synthesized four kinds of reversed peptides of various physiologically active peptides, which inhibit TNF (tumor necrosis factor) cytotoxicity, produce NGF (nerve growth factor), exert antimicrobial activity and inhibit cell attachment, respectively. They were examined for their biological activity in comparison with that of normal peptides, that is, naturally occurring peptides. The reversed peptides induce similar activities, but to a lesser extent than those of the normal peptides, respectively. These results indicate that there may be conformationally ambiguous binding in some of the naturally occurring ligand-protein interactions. This method may be useful as a tool to rapidly generate a novel lead peptide with the desired biological function from a naturally occurring active peptide.

[Detection of early cardiac dysfunction in patients with transfusion-dependent aplastic anemia and chronic iron overload in childhood. Stress-velocity relation as a sensitive index by echocardiography].

In order to detect early-stage left ventricular dysfunction, we examined a load-independent index, that is, the stress-velocity relation, by echocardiography in 13 patients with aplastic anemia including pure red cell anemia. Even when left ventricular contractility and pump function were within the normal range, its afterload had a tendency to increase in transfusion-dependent patients. The patients who had impairment of cardiac pump function died of congestive heart failure within one year after abnormal findings in stress-velocity relation were detected. Therefore, the stress-velocity relation is a sensitive, useful and noninvasive index for detecting asymptomatic myocardial dysfunction in patients with transfusion-dependency and chronic iron overload. It is necessary for these patients to undergo examinations of echocardiography at regular intervals. In case of abnormally in the stress-velocity relation, treatment for afterload mismatch and more effective chelation should be recommended to reduce the burden on the heart. Thereby, the heart is kept in better condition and the prognosis of these patients will be improved.

Human cDNA encoding DnaJ protein homologue.

We have cloned a cDNA encoding a DnaJ-like protein from the human fibrosarcoma HT-1080 cDNA library. This cDNA encodes a protein of 397 amino acid residues whose sequence shows 38.2% identity with the Escherichia coli DnaJ protein and 47.2% with the yeast DnaJ homologue along the entire length. Since the sequence contains the N-terminal domain region conserved in DnaJ family members and the four repeats of the Cys-X-X-Cys-X-Gly-X-Gly motif which are characteristic of DnaJ proteins, we conclude that this cDNA clone encodes the human homologue of DnaJ.