Hidetoshi Inagaki

Vertebrate homologs of tinman and bagpipe: Roles of the homeobox genes in cardiovascular development

In Drosophila, dorsal mesodermal specification is regulated by the homeobox genes tinman and bagpipe. Vertebrate homologs of tinman and bagpipe have been isolated in various species. Moreover, there are at least four different genes related to tinman in the vertebrate, which indicates that this gene has been duplicated during evolution. One of the murine homologs of tinman is the cardiac homeobox gene Csx or Nkx2.5. Gene targeting of Csx/Nkx2.5 showed that this gene is required for completion of the looping morphogenesis of the heart. However, it is not essential for the specification of the heart cell lineage. Early cardiac development might therefore be regulated by other genes, which may act either independently or in concert with Csx/Nkx2.5. Possible candidates might be other members of the NK2 class of homeobox proteins like Tix/Nkx2.6, Nkx2.3, nkx2.7, or cNkx2.8. Murine Tix/Nkx2.6 mRNA has been detected in the heart and pharyngeal endoderm (this study). Xenopus XNkx2.3 and chicken cNkx2.3 are expressed in the heart as well as in pharyngeal and gut endoderm. In contrast, murine Nkx2.3 is expressed in the gut and pharyngeal arches but not the heart. In zebrafish and chicken, two new NK-2 class homeoproteins, nkx2.7 and cNkx2.8, have been identified. Zebrafish nkx2.7 is expressed in both, the heart and pharyngeal endoderm. In the chicken, cNkx2.8 is expressed in the heart primordia and the primitive heart tube and becomes undetectable after looping. No murine homologs of nkx2.7 or cNkx2.8 have been found so far. The overlapping expression pattern of NK2 class homeobox genes in the heart and the pharynx may suggest a common origin of these two organs. In the Drosophila genome, the tinman gene is linked to another NK family gene named bagpipe. A murine homolog of bagpipe, Bax/Nkx3.1, is expressed in somites, blood vessels, and the male reproductive system during embryogenesis (this study), suggesting that this genes function may be relevant for the development of these organs. A bagpipe homolog in Xenopus, Xbap, is expressed in the gut masculature and a region of the facial cartilage during development. In this paper, we discuss molecular mechanisms of cardiovascular development with particular emphasis on roles of transcription factors.

Regulation and Role of the Mitochondrial Transcription Factor in the Diabetic Rat Heart

Abstract: To clarify the mechanism of abnormalities in mitochondrial expression and function in diabetic rat heart, we have studied the transcriptional activities of mitochondrial DNA using isolated intact mitochondria from the heart of either diabetic or control rats. The transcriptional activity of cardiac mitochondria isolated from diabetic rats decreased to 40% of the control level (P < 0.01. Consistently, in the heart of diabetic rats, the content of cytochrome b mRNA encoded by mitochondrial DNA was reduced to 50% of control (P < 0.01. This abnormal transcriptional activity of mitochondrial DNA could not be explained by mRNA or protein contents of mitochondrial transcription factor (mtTFA), but mtTFA binding to the promoter sequence of mitochondrial DNA, assessed by gel‐shift assay, was attenuated in diabetic rats. In contrast, the mRNA expression of nuclear‐encoded mitochondrial genes, such as ATP synthase‐β, was not affected by diabetes. Although O2 consumption of the mitochondria from diabetic rats was decreased, H2O2 production in these rats was increased compared with the control. Insulin treatment reversed all the abnormalities found in diabetic rats. These results clearly indicate that an impairment of binding activity of mtTFA to the promoter sequence has a key role in the abnormal mitochondrial gene expression, which might explain the mitochondrial dysfunction found in diabetic heart.

Isolation of rat mitochondrial transcription factor A (r-Tfam) cDNA

We have isolated rat mitochondrial transcription factor A (Tfam; formerly known as mtTFA) cDNA clones from a rat cerebellum cDNA library using human Tfam cDNA as a probe. The deduced amino acid sequence of r-Tfam shows 62% and 89% overall identity to human and mouse Tfam, respectively. We also show the presence of two r-Tfam isoforms in testis as for mouse. Our findings suggest that the mechanisms underlying transcription of mitochondrial genes are conserved among rat, mouse, and human.

A Large DNA-binding Nuclear Protein with RNA Recognition Motif and Serine/Arginine-rich Domain

cDNA species encoding a large DNA-binding protein (NP220) of 1978 amino acids was isolated from human cDNA libraries. Human NP220 binds to double-stranded DNA fragments by recognizing clusters of cytidines. Immunofluorescent microscopy with antiserum directed against NP220 revealed a punctate or “speckled” pattern and coiled body-like structures in the nucleoplasm of various human cell lines. These structures diffused in the cytoplasm during mitosis. Western blot analysis showed that NP220 is enriched in the lithium 3,5-diiodosalicylate-insoluble fraction of nuclei. The domain essential for DNA binding is localized in C-terminal half of NP220. Human NP220 shares three types of domains (MH1, MH2, and MH3) with the acidic nuclear protein, matrin 3 (Belgrader, P., Dey, R., and Berezney, R.(1991) J. Biol. Chem. 266, 9893-9899). MH1 is a 48-amino acid sequence near the N terminus of both human NP220 and rat matrin 3. MH2 is a 75-amino acid sequence homologous to the RNA recognition motifs of heterogeneous nuclear RNP I and L. It is repeated three times in NP220 and twice in matrin 3. MH3 is a 60-amino acid sequence at the C terminus of both NP220 and matrin 3. NP220 has an arginine/serine-rich domain commonly found in pre-mRNA splicing factors. Close to the domain essential for DNA binding, there are nine repeats of the sequence LVTVDEVIEEEDL. Thus, NP220 is a novel type of nucleoplasmic protein with multiple domains.

Pilosulin 5, a novel histamine-releasing peptide of the Australian ant, Myrmecia pilosula (Jack Jumper Ant).

Venom of the Australian ant species Myrmecia pilosula contains a number of allergenic peptides including pilosulins. To obtain novel cDNA clones that encode the pilosulin-related bioactive peptides, mRNA of M. pilosula species complex was subjected to RT-PCR in which the forward primer corresponds to a nucleotide sequence in the leader sequences of pilosulins. We isolated a cDNA clone encoding the novel bioactive peptide pilosulin 5. Tandem mass analysis was entirely consistent with the cDNA derived sequence, and indicated that pilosulin 5 is connected by a single disulfide bridge to create a dimmer peptide of 8546Da. Synthetic pilosulin 5 peptide caused a significant histamine release in a dose-dependent manner, and the mastoparan homologous region of pilosulin 5 was responsible for the activity.

INHIBITION OF MITOCHONDRIAL GENE EXPRESSION BY ANTISENSE RNA OF MITOCHONDRIAL TRANSCRIPTION FACTOR A (mtTFA)

Mitochondrial transcription factor A (mtTFA) plays an important role in regulating the expression of mitochondrial genes. To gain a better understanding of the relationships between mitochondrial gene expression and mtTFA, the mtTFA gene was inserted into a mammalian expression vector, both in the sense orientation and in the antisense orientation. After construction, these plasmids were transfected into COS‐7 cells. In antisense‐transformed cells, the expression of the cytochrome c oxidase subunit I and subunit III genes encoded by mitochondrial DNA was inhibited and there was an accompanying reduction of the level of mtTFA protein. These results provide direct evidence that the expression of mitochondrial genes is under the control of mtTFA.

Functional characterization of Kunitz-type protease inhibitor Pr-mulgins identified from New Guinean Pseudechis australis

Kunitz-type protease inhibitors, which consist of around 60 amino acid residues and three distinctive disulfide bridges, exhibit a broad range of physiological functions such as protease inhibitor and ion channel blocker. In this study, we identified cDNAs encoding Kunitz-type protease inhibitors, Pr-mulgins 1, 2 and 3, from the venom gland cDNA library of Papuan pigmy mulga snake (New Guinean Pseudechis australis). The deduced amino acid sequences of the Pr-mulgins are 92.4-99.3% identical with their orthologs in Australian P. australis. Pr-mulgin proteins were recombinantly prepared and subjected to inhibitory assays against proteases. Pr-mulgin 1 significantly affected matrix metalloprotease (MMP) 2; Pr-mulgins 2 and 3 showed potent inhibition to trypsin and plasma plasmin; and Pr-mulgin 2 inhibited α-chymotrypsin. Pr-mulgins 1, 2, and 3, however, had essentially no effect on Drosophila K(+) channels (Shaker) and rat K(+) channels (K(v) 1.1).

Induction of apoptosis in primary culture of rat hepatocytes by protease inhibitors

There is growing evidence that suggests the involvement of intracellular proteases in the process of apoptosis or programmed cell death. In this study, we have demonstrated that leupeptin, a cysteine protease inhibitor, can significantly increase the incidence of both apoptotic nuclear morphology change and internucleosomal DNA fragmentation in primary cultured hepatocytes in the absence of known apoptotic stimuli for hepatocytes. On the other hand, aspartic and serine protease inhibitors showed little or no effects on the apoptotic changes. In addition, we found that the apoptotic changes could be induced by chloroquine, an inhibitor of lysosomal proteolysis, but could not be induced by calpain inhibitors. These data suggest that inhibition of lysosomal cysteine proteases may induce apoptosis in primary cultured hepatocytes.

Enhanced activation of the transient receptor potential channel TRPA1 by ajoene, an allicin derivative

TRPA1 is a calcium-permeable, nonselective cation channel expressed in the dorsal root ganglion and trigeminal ganglia nociceptive neurons. It is activated by the pungent compounds in mustard oil (AITC, allyl isothiocyanate), cinnamon (cinnamaldehyde), garlic (allicin), and is believed to mediate the inflammatory actions of environmental irritants and proalgesic agents. Thiosulfinate (allicin) and isothiocyanate (AITC) compounds contain reactive electrophilic chemical groups that react with cysteine residues within the TRPA1 channel N terminus, leading to channel activation. Ajoene also contains reactive electrophilic chemical groups likely to target TRPA1 channel. Here, we have used voltage-clamp recordings to show that TRPA1-responses are enhanced by ajoene application in a Xenopus oocyte expression system. Though ajoene alone did not activate TRPA1, subsequent application of ajoene enhanced the AITC-, allicin- and depolarization-induced responses of TRPA1. Moreover, when increasing concentrations of ajoene were applied along with constant concentrations of allicin or AITC, stronger responses were elicited. These findings suggest that ajoene is a novel TRPA1 channel enhancer, operating in a channel-opening-dependent manner.

Influence of aldehyde groups on the thermostability of an immobilized enzyme on an inorganic support

Abstract The influence of glutaraldehyde cross-linking on the thermostability of an immobilized enzyme on an inorganic support is described. A thermostable β-galactosidase derived from a thermophile was covalently linked to a silanized porous inorganic support with glutaraldehyde. The effect of the amount of the immobilized enzyme on its thermostability was investigated at different temperatures and it was found that a combination of a low enzyme/support (g enzyme/g support) and high incubation temperature resulted in a considerable decrease in the residual activity. The thermostability of the immobilized enzyme under these conditions was improved by additional treatment with a solution of basic amino acids after immobilization procedure. These results suggest that the stability at high temperature of an immobilized enzyme by glutaraldehyde cross-linking is remarkably affected by free aldehyde groups remaining on the surface of an inorganic support. Therefore, it is important to eliminate free aldehyde groups for preventing the surface from influencing the enzymes at high incubation temperature.