Kinetic studies of site-directed mutational isomalto-dextranase-catalyzed hydrolytic reactions on a 27 MHz quartz-crystal microbalance

A quartz-crystal microbalance (QCM) technique was applied to analyze effects of site-directed mutagenesis of a glycosidase (isomalto-dextranase) on the hydrolysis mechanism of the substrate binding (k(on), k(off), and K(d)) and the catalytic process (k(cat)), separately, by using a dextran-immobilized QCM in buffer solution. D266N, D198N, and D313N mutants, which are predicted as critical residues of the isomalto-dextranase hydrolytic activity, dramatically decreased the apparent enzyme activity. The D266N mutant, however, did not change the substrate binding ability (K(d)), and the D198N and D313N mutants largely increased K(d) values due to the increase of k(off) and/or the decrease of k(on) values, as well as the negatively small k(cat) values. From these results, we estimate the reaction mechanism, in which Asp266 acts as only a general acid in the catalytic process, Asp198 acts as both nucleophile in the catalytic process and binding the substrate, and Asp313 acts as only the substrate binding.     

High production of methyl mercaptan by L-methionine-alpha-deamino-gamma-mercaptomethane lyase from Treponema denticola

Methyl mercaptan is derived from l-methionine by the action of l-methionine-alpha-deamino-gamma-mercaptomethane lyase (METase) and is a major component of oral malodor. This compound is highly toxic and is thought to play an important role in periodontal disease. We found that Treponema denticola, a member of the subgingival biofilm at periodontal disease sites, produced a large amount of methyl mercaptan even at low concentration of l-methionine. METase activity in a cell-free extract from T. denticola was detected by two-dimensional electrophoresis under non-denaturing conditions, and the protein spot that exhibited high METase activity was identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The identified gene produced a METase with a K(m) value for l-methionine (0.55mM) that is much lower than those of METases previously identified in the other organisms. This result suggests that T. denticola is an important producer of methyl mercaptan in the subgingival biofilm.     

Early role of CD4+ Th1 cells and antibodies in HER-2 adenovirus vaccine protection against autochthonous mammary carcinomas

HER-2 is an oncogenic tumor-associated Ag that is overexpressed in several human tumors including breast and ovarian cancer. The efficacy and mechanism of a HER-2-expressing recombinant adenoviral vaccine to protect against tumorigenesis was examined using HER-2 transgenic (BALB-neuT) mice, which develop spontaneous breast tumors in all 10 mammary glands, and also using a transplantable mouse tumor model. Vaccination beginning at 6-8 wk of age (through 19 wk of age) prevented development of spontaneous mammary tumors even after 50 wk, whereas the animals in the control groups had tumors in all mammary glands by 25 wk. Such long-term protection after the last boost has not been achieved previously in this transgenic mouse in which the oncogene is continuously spawning tumorigenesis. Using beta(2)-microglobulin-knockout, IFN-gamma-knockout, and B cell-deficient mice, CD4(+) and CD8(+) cell depletion, and Ab transfer studies, we show that induction of anti-HER-2/neu Abs are both necessary and sufficient for protection, and the IgG2a isotype is most effective. In contrast, CD8(+) T cells are not necessary at all, and CD4(+) T cells are necessary for only 36-48 h after immunization to provide help for B cells but not as effector cells. Equal protection in immunized mice deficient in FcgammaRI/III excluded an FcR-mediated mechanism. Anti-HER-2 serum not only inhibited growth of mammary tumor cell lines expressing HER-2 in vitro but also protected mice from tumors in vivo, suggesting a direct action of Ab on the tumor cells. Such a vaccine may provide Ab-mediated protection against HER-2-expressing breast cancers in humans.     

Identification of novel adrenomedullin in mammals: a potent cardiovascular and renal regulator

We have identified cDNA encoding a new member of the adrenomedullin (AM) family, AM2, for the first time in mammals (mouse, rat and human). The predicted precursor carried mature AM2 in the C-terminus, which had an intramolecular ring formed by an S-S bond and a possibly amidated C-terminus. Phylogenetic analyses clustered AM2 and AM into two distinct but closely related groups. Similarity of exon-intron structure and synteny of neighboring genes showed that mammalian AM2 is an ortholog of pufferfish AM2 and a paralog of mammalian AM. AM2 mRNA was expressed in submaxillary gland, kidney, stomach, ovary, lymphoid tissues and pancreas of mice, but not in adrenal and testis. Intravenous injection of synthetic mature AM2 decreased arterial pressure more potently than AM, and induced antidiuresis and antinatriuresis in mice. These results show that at least two peptides, AM and AM2, comprise an adrenomedullin family in mammals, and that AM2 may play pivotal roles in cardiovascular and body fluid regulation.     

Electronic spectral studies of dinuclear Cobalt(II) complexes with a phenol-based dinucleating ligand containing four methoxyethyl chelating arms

The electronic spectra of dinuclear cobalt(II) complexes [Co₂(bomp)(MeCO₂)₂]BPh₄ (1) and [Co₂(bomp)(PhCO₂)₂]BPh₄ (2) were studied [H(bomp): 2,6-bis[bis(2-methoxyethyl)aminomethyl]-4-methylphenol]; the spectral components obtained by Gaussian curve analysis were well simulated based on the angular overlap model using the aomx program. The first transition band ⁴T₁ → ⁴T₂(⁴F) of an octahedral high-spin cobalt(II) complex was found to be sensitive to the distortion around the cobalt(II) ion.     

Phosphorylation and stabilization of ATP binding cassette transporter A1 by synthetic amphiphilic helical peptides

To investigate structural requirement of helical apolipoprotein to phosphorylate and stabilize ATP-binding cassette transporter A1 (ABCA1), synthetic peptides (Remaley, A. T., Thomas, F., Stonik, J. A., Demosky, S. J., Bark, S. E., Neufeld, E. B., Bocharov, A. V., Vishnyakova, T. G., Patterson, A. P., Eggerman, T. L., Santamarina-Fojo, S., and Brewer, H. B. (2003) J. Lipid Res. 44, 828-836) were examined for these activities. L37pA, an L amino acid peptide that contains two class-A amphiphilic helices, and D37pA, the same peptide with all D amino acids, both removed cholesterol and phospholipid from differentiated THP-1 cells more than apolipoproteins (apos) A-I, A-II, and E. Both peptides also mediated lipid release from human fibroblasts WI-38 similar to apoA-I. L2D37pA, an L-peptide whose valine and tyrosine were replaced with D amino acids also promoted lipid release from WI-38 but less so with THP-1, whereas L3D37pA, in which alanine, lysine, and asparatic acid were replaced with D amino acids was ineffective in lipid release for both cell lines. ABCA1 protein in THP-1 and WT-38 was stabilized against proteolytic degradation by apoA-I, apoA-II, and apoE and by all the peptides tested except for L3D37pA, and ABCA1 phosphorylation closely correlated with its stabilization. The analysis of the relationship among these parameters indicated that removal of phospholipid triggers signals for phosphorylation and stabilization of ABCA1. We thus concluded that an amphiphilic helical motif is the minimum structural requirement for a protein to stabilize ABCA1 against proteolytic degradation.     

Protein phosphatase 2B dephosphorylates twitchin, initiating the catch state of invertebrate smooth muscle

"Catch" is the state where some invertebrate muscles sustain high tension for long periods at low ATP hydrolysis rates. Physiological studies using muscle fibers have not yet fully provided the details of the initiation process of the catch state. The process was extensively studied by using an in vitro reconstitution assay with several phosphatase inhibitors. Actin filaments bound to thick filaments pretreated with the soluble protein fraction of muscle homogenate and Ca2+ (catch treatment) in the presence of MgATP at a low free Ca2+ concentration (the catch state). Catch treatment with > 50 microm okadaic acid, > 1 microm microcystin LR, 1 microm cyclosporin A, 1 microm FK506, or 0.2 mm calcineurin autoinhibitory peptide fragment produced almost no binding of the actin filaments, indicating protein phosphatase 2B (PP2B) was involved. Use of bovine calcineurin (PP2B) and its activator calmodulin instead of the soluble protein fraction initiated the catch state, indicating that only PP2B and calmodulin in the soluble protein fraction are essential for the initiation process. The initiation was reproduced with purified actin, myosin, twitchin, PP2B, and calmodulin. 32P autoradiography showed that only twitchin was dephosphorylated during the catch treatment with either the soluble protein fraction or bovine calcineurin and calmodulin. These results indicate that PP2B directly dephosphorylates twitchin and initiates the catch state and that no other component is required for the initiation process of the catch state.     

Involvement of caspase-9 in execution of the maternal program of apoptosis in Xenopus late blastulae overexpressed with S-adenosylmethionine decarboxylase

We previously demonstrated that overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus early embryos induces execution of maternal program of apoptosis shortly after midblastula transition, which likely serves as a fail-safe mechanism of early development to eliminate physiologically damaged cells before they entering the gastrula stage. To determine how caspases are involved in this process, we microinjected peptide inhibitors and "dominant-negative forms" of caspase-9 and -1 into Xenopus fertilized eggs, and found that inhibitors of caspase-9, but not caspase-1, completely suppress SAMDC-induced apoptosis. The lysate of SAMDC-overexpressing late blastulae contained activity to cleave in vitro-synthesized [(35)S]procaspase-9, but not [(35)S]procaspase-1, and mRNA for caspase-9, but not caspase-1, occurred abundantly in the unfertilized egg as maternal mRNA. We also found that overexpression of caspase-9 and -1 equally executes the apoptosis, but the apoptosis executed by these mRNAs was only partially rescued by Bcl-2 and rescued embryos did not develop beyond neurula stage. These results indicate that activation of caspase-9 is a key step for execution of the maternally preset program of apoptosis in Xenopus early embryos.     

Circulation of type 1 vaccine-derived poliovirus in the Philippines in 2001

In 2001, highly evolved type 1 circulating vaccine-derived poliovirus (cVDPV) was isolated from three acute flaccid paralysis patients and one contact from three separate communities in the Philippines. Complete genomic sequencing of these four cVDPV isolates revealed that the capsid region was derived from the Sabin 1 vaccine strain but most of the noncapsid region was derived from an unidentified enterovirus unrelated to the oral poliovirus vaccine (OPV) strains. The sequences of the cVDPV isolates were closely related to each other, and the isolates had a common recombination site. Most of the genetic and biological properties of the cVDPV isolates were indistinguishable from those of wild polioviruses. However, the most recently identified cVDPV isolate from a healthy contact retained the temperature sensitivity and partial attenuation phenotypes. The sequence relationships among the isolates and Sabin 1 suggested that cVDPV originated from an OPV dose given in 1998 to 1999 and that cVDPV circulated along a narrow chain of transmission. Type 1 cVDPV was last detected in the Philippines in September 2001, and population immunity to polio was raised by extensive OPV campaigns in late 2001 and early 2002.