[Glycogenosis, glycogen storage disease (GSD)].
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Glycogen Storage Disease↗
Biomedical subjects
H Nakajima
Publications and source records attributed to H Nakajima.
Anomalous (preduodenal) portal vein: autosomal recessive mutation in AKR/J mice.
BACKGROUND AND PURPOSE: Anomalous (preduodenal) portal vein was found in AKR/J mice. It is a rare congenital malformation in humans, and to the authors' knowledge, has never been reported in laboratory animals. Morphology, clinical signs of disease, and heritability of this anomaly were examined. METHODS: Fifty-three strains of inbred mice (6,026 mice) in our mouse colony were examined for preduodenal portal vein and its association with clinical signs of disease (vomiting or abdominal pain) and other anomalies. Heritability also was tested by use of cross-backcross matings of AKR/J mice with clinically normal PT mice. RESULTS: The portal vein was found at the ventral side of the duodenum in most (98%) AKR/J mice, whereas it ran at the dorsal side of the duodenum in 52 other inbred mouse strains in our mouse colony. Clinical signs of disease and other congenital anomalies were not detected in this strain of mice, although position has a high association with other congenital anomalies in humans. Heritability testing of this anomaly in AKR/J mice indicated single autosomal recessive inheritance. CONCLUSIONS: Preduodenal portal vein found in AKR/J mice is a single autosomal recessive mutation, but is not associated with clinical signs of disease and other congenital malformations.
Animals↗
Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides.
The protein invasin expressed on the cell surface of the pathogenic bacteria Yersinia pseudotuberculosis mediates the entry of this bacterium into cultured mammalian cells. We have developed a system for expression of random peptides on the cell surface of Escherichia coli (E. coli) by creation of a fusion hybrid between a peptide and the invasin protein. The fusion protein constructs consist of part of the outer membrane domain of the invasin protein, six proline spacers, and a decamer of random peptides flanked by cysteine residues (CX(10)C). Peptides were constitutively expressed on the cell surface in the resulting random decamer peptide library, which we designated as ESPEL (E. coli Surface Peptide Expression Library). The ESPEL was systematically screened for its binding affinity toward human cultured cells. Several bacterial clones were identified whose binding to human cells was mediated by peptides expressed on the bacterial cell surface. Flow cytometric analysis showed that both the identified bacterial clones and these corresponding chemically synthesized peptides bound to human cells specifically. The techniques described provide a new method that uses E. coli random peptide library to select targeting peptides for mammalian cells without any knowledge of the human cellular receptors.
Adhesins, Bacterial↗
Antithrombogenicity of cultured endothelial cell-detached surface.
To examine the antithrombogenicity of cultured endothelial cell-detached surface, a simple hybrid vascular model tube consisting of a glass tube and endothelial cells was constructed. To detach the endothelial cells from the inner surface of the model tube, a steady shear stress of 2 or 8 N m(-2) was imposed onto the surface of endothelial cell monolayer by means of a coaxial double cylinder rotational-type apparatus. Coagulation of blood in contact with the endothelial cell-detached surface was examined using a damped oscillation rheometer. Coagulation of whole blood in the cell-detached tube occurred at about 40 min, which was almost the same as that in the endothelial cell-coated tube. A few platelets without shape change adhered to the endothelial cell-detached surface. These data suggest that the endothelial cell-detached surface may exhibit antithrombogenic and anticoagulant surfaces. Biochemical analyses showed that the glass surface, where endothelial cell was detached, was covered with components such as collagen type IV that is considered to be produced from the endothelial cells on the glass surface.
Journal Article↗
Ion implantation into collagen-coated surfaces for the development of small diameter artificial grafts.
Ion implantation into collagen (Type I) coated inner surfaces of test tubes with a length of 50 mm and an inner diameter of 2 and 3 mm were performed to develop hybrid type small diameter artificial vascular grafts. To obtain information about the cellular response and chemical and physical structure of those collagen surfaces, several experiments such as platelets adhesion test, endothelial cell culture, analysis of amino acids and animal study were performed. He(+) ion implanted collagen coated specimen exhibited cell attachment and inhibit platelet adhesion. From these results, it was assumed that He(+) ions broke the ligands that correspond to platelet, and the ligands that correspond to endothelial cell adhesion still existed after ion implantation. It was suggested that platelets and cell attachment could be control individually by ion implantation into collagen.
Journal Article↗
Dissociation and recombination between ligands and heme in a CO-sensing transcriptional activator CooA. A flash photolysis study.
CooA from Rhodospirillum rubrum is a transcriptional activator in which a heme prosthetic group acts as a CO sensor and regulates the activity of the protein. In this study, the electronic relaxation of the heme, and the concurrent recombination between ligands and the heme at approximately 280 K were examined in an effort to understand the environment around the heme and the dynamics of the ligands. Upon photoexcitation of the reduced CooA at 400 nm, electronic relaxation of the heme occurred with time constants of 0.8 and 1.7 ps. The ligand rebinding was substantially completed with a time constant of 6.5 ps, followed by a slow relaxation process with a time constant of 173 ps. In the case of CO-bound CooA, relaxation of the excited heme occurred with two time constants, 1.1 and 2.4 ps, which were largely similar to those with reduced CooA. The subsequent CO recombination process was remarkably fast compared with that of other CO-bound heme proteins. It was well described as a biphasic geminate recombination process with time constants of 78 ps (60%) and 386 ps (30%). About 10% of the excited heme remained unligated at 1.9 ns. The dynamics of rebinding of CO thus will help us to understand how the physiologically relevant diatomic molecule approaches the heme binding site in CooA with picosecond resolution.
Bacterial Proteins↗
Azido glycoside primer: a versatile building block for the biocombinatorial synthesis of glycosphingolipid analogues.
A lactoside primer, 12-azidododecyl beta-lactoside, was synthesized via the Koenigs-Knorr method by glycosylation of 1,12-dodecyldiol with perbenzoylated lactosyl bromide. The presence of the 2-O-acyl substituent in the donor gave the beta-lactoside, and an excess of acceptor ensured monoglycosylation of the diol. Mesylation of the omega-hydroxyl group in the aglycon, followed by displacement of the mesylate with azide and subsequent O-debenzoylation gave the desired omega-azidododecyl beta-lactoside. The azido glycoside primer was examined in mouse B16 melanoma cells for its feasibility as a building block for oligosaccharide biosynthesis. Uptake of the azido glycoside primer by B16 cells resulted in the sialylation of the galactose residue of the primer to give a glycosylated product having the same glycan as in ganglioside GM3. After 24 h incubation of B16 cells with the primers, the amount of sialylated omega-azidododecyl beta-lactoside primer was 75% of the amount of sialylated n-dodecyl beta-lactoside. However, after 48 h incubation, both primers gave equal amounts of the sialylated products. Interestingly, the remaining azido glycoside primer after 48 h incubation was 5.6-fold greater than that of the alkyl primer, indicating degradation of the alkyl primer to a larger extent than the omega-azido glycoside primer. The facile chemical synthesis and the efficient uptake in cells make the azido glycoside primer a versatile building block for the biocombinatorial synthesis of glycolipid oligosaccharides.
Animals↗
Redox properties and coordination structure of the heme in the co-sensing transcriptional activator CooA.
The CO-sensing transcriptional activator CooA contains a six-coordinate protoheme as a CO sensor. Cys(75) and His(77) are assigned to the fifth ligand of the ferric and ferrous hemes, respectively. In this study, we carried out alanine-scanning mutagenesis and EXAFS analyses to determine the coordination structure of the heme in CooA. Pro(2) is thought to be the sixth ligand of the ferric and ferrous hemes in CooA, which is consistent with the crystal structure of ferrous CooA (Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876-880). CooA exhibited anomalous redox chemistry, i.e. hysteresis was observed in electrochemical redox titrations in which the observed reduction and oxidation midpoint potentials were -320 mV and -260 mV, respectively. The redox-controlled ligand exchange of the heme between Cys(75) and His(77) is thought to cause the difference between the reduction and oxidation midpoint potentials.
Alanine↗
Identification of histidine 77 as the axial heme ligand of carbonmonoxy CooA by picosecond time-resolved resonance Raman spectroscopy.
The heme proximal ligand of carbonmonoxy CooA, a CO-sensing transcriptional activator, in the CO-bound form was identified to be His77 by using picosecond time-resolved resonance Raman spectroscopy. On the basis of the inverse correlation between Fe-CO and C-O stretching frequencies, we proposed previously that His77 is the axial ligand trans to CO [Uchida et al. (1998) J. Biol. Chem. 273, 19988-19992], whereas later a possibility of displacement of His77 by CO with retention of another unidentified axial ligand was reported [Vogel et al. (1999) Biochemistry 38, 2679-2687]. Although our previous resonance Raman study failed to detect the Fe-His stretching [nu(Fe-His)] mode of CO-photodissociated CooA of the carbonmonoxy adduct due to the rapid recombination, application of the picosecond time-resolved resonance Raman technique enabled us to observe a new intense line assignable to nu(Fe-His) at 211 cm(-)(1) immediately after photolysis, while it became nondiscernible after 100-ps delay. The low nu(Fe-His) frequency of photodissociated CooA indicates the presence of some strain in the Fe-His bond in CO-bound CooA. This and the rapid recombination of CO characterize the heme pocket of CooA. The 211 cm(-)(1) band was completely absent in the spectrum of the CO-photodissociated form of the His77-substituted mutant but the Fe-Im stretching band was observed in the presence of exogenous imidazole (Im). Thus, we conclude that His77 is the axial ligand of CO-bound CooA and CO displaces the axial ligand trans to His77 with retention of ligated His77 to activate CooA as the transcriptional activator.
Bacterial Proteins↗
A pathogenic role of Th2 responses on the severity of encephalomyelitis induced in mice by herpes simplex virus type 2 infection.
A pathogenic role of Th2 cells and their cytokine products (IL-4 and IL-10, Th2 cytokines) on the development of herpes simplex myelitis (HSM) was studied in mice exposed to footpad injection of herpes simplex virus type 2 (HSV-2). Morbidity and mortality of mice with HSM (HSM mice) increased when they were treated with a mixture of Th2 cytokines. Additionally, survival rates of HSM mice increased when they were treated with a mixture of mAbs for Th2 cytokines. As compared with HSM mice treated with saline, the growth of HSV-2 in spinal cords of HSM mice treated with the mixture of Th2 cytokines increased. Th2 cells (myelitis-associated Th2 cells, MTh2 cells) were demonstrated among cerebrospinal fluid cells from HSM mice. After the stimulation with HSV-2 antigen (Ag), MTh2 cells from HSM mice previously treated with the mixture of Th2 cytokines produced enhanced amounts of Th2 cytokines into their culture fluids, as compared with the amount of Th2 cytokines produced by MTh2 cells. Th2 cells were also demonstrated in mononuclear cells from spleens of HSM mice. When compared with HSM mice inoculated with splenic CD4(+) T cells from normal mice, morbidity and mortality of HSM mice inoculated with MTh2 cells markedly increased. These results indicated that the severity of HSM induced in mice by footpad injection of HSV-2 was influenced by MTh2 cells or Th2 cytokines released from these MTh2 cells. Th2 responses manifested in mice by HSV-2 infection may act as a pathogenic enhancer of HSM severities.
Adoptive Transfer↗
Caspase-dependent apoptosis by ectopic expression of E2F-4.
E2F is a family of transcription factors which regulates cell cycle and apoptosis of mammalian cells. E2F-1-3 localize in the nucleus, and preferentially bind pRb, while E2F-4 and 5 have no nuclear localization signal and preferentially bind p107/p130. E2F-6 suppresses the transcriptional activity of other E2F proteins. DP-1 and 2 are heterodimeric partners of each E2F protein. Using tetracycline-responsive promoters, here we compared the effects of ectopic expression of E2F-1, DP-1 and E2F-4 on cell cycle progression and apoptosis in Chinese hamster cell lines. We found that E2F-4, as well as DP-1 and E2F-1, induced growth arrest and caspase-dependent apoptosis. E2F-4 did not have a marked effect on cell cycle progression, while E2F-1 induced DNA synthesis of resting cells and DP-1 arrested cells in G1. Ectopic expression of E2F-4 did not activate E2F-dependent transcription. Our results suggest that expression of E2F-4 at elevated levels induces growth arrest and apoptosis of mammalian cells through a mechanism distinct from E2F-1 and DP-1.
Animals↗
Congenital deficiency of nitric oxide synthase 2 protects against endotoxin-induced myocardial dysfunction in mice.
BACKGROUND: Sepsis can be complicated by severe myocardial dysfunction and is associated with increased nitric oxide (NO) production by inducible NO synthase (NOS2). To investigate the role of NOS2 in endotoxin-induced myocardial dysfunction in vivo, we studied wild-type and NOS2-deficient mice. METHODS AND RESULTS: Serial echocardiographic parameters of myocardial function were measured before and at 4, 7, 16, and 24 hours after an endotoxin challenge. Seven hours after challenge with either endotoxin or saline, systemic and left ventricular pressures were measured, and the first derivative of left ventricular developed pressure (dP/dt), slope of the end-systolic pressure-dimension relationship (Slope(LVESPD)), and time constant of isovolumic relaxation (tau) were calculated. Endotoxin challenge in wild-type mice decreased left ventricular fractional shortening, velocity of circumferential shortening, dP/dt(max), Slope(LVESPD), and dP/dt(min) and increased time constant tau. Endotoxin-induced myocardial dysfunction was associated with increased ventricular NOS2 gene expression and cGMP concentrations. Seven hours after endotoxin challenge, NOS2-deficient mice had greater fractional shortening, dP/dt(max), and Slope(LVESPD) than did endotoxin-challenged wild-type mice. Measures of diastolic function, dP/dt(min) and time constant tau, were preserved in endotoxin-challenged NOS2-deficient mice. After endotoxin challenge in wild-type mice, early (3-hour) inhibition of NOS2 with L-N:(6)-(1-iminoethyl)lysine hydrochloride prevented, whereas later (7-hour) inhibition could not reverse, endotoxin-induced myocardial dysfunction. CONCLUSIONS: These results suggest that NOS2 is required for the development of systolic and diastolic dysfunction in murine sepsis.
Animals↗
Role of common cytokine receptor gamma chain (gamma(c))- and Jak3-dependent signaling in the proliferation and survival of murine mast cells.
The regulatory roles of the common cytokine receptor gamma chain (gamma(c))- and Jak3-dependent signaling in the proliferation and survival of mast cells were determined using gamma(c)-deficient (gamma(c)(-)) and Jak3-deficient (Jak3(-)) mice. Although the mast cells in gamma(c)(-) and Jak3(-) mice were morphologically indistinguishable from those in wild-type mice, the number of peritoneal mast cells was decreased in gamma(c)(-) and Jak3(-) mice as compared with that in wild-type mice. Among gamma(c)-related cytokines, interleukin (IL)-4 and IL-9, but not IL-2, IL-7, or IL-15, enhanced the proliferation and survival of bone marrow-derived mast cells (BMMCs) from wild-type mice. However, the effects of IL-4 and IL-9 were absent in BMMCs from gamma(c)(-) and Jak3(-) mice. In addition, IL-4Ralpha, gamma(c), and Jak3, but not IL-2Rbeta or IL-7Ralpha, were expressed in BMMCs. In contrast, IL-13 did not significantly induce the proliferation and survival of BMMCs even from wild-type mice, and IL-13Ralpha1 was not expressed in BMMCs. Furthermore, IL-4 phosphorylated the 65-kd isoform of Stat6 in BMMCs from wild-type mice but not from gamma(c)(-) and Jak3(-) mice. These results indicate that gamma(c)- and Jak3-dependent signaling is essential for IL-4- and IL-9-induced proliferation and survival of murine mast cells, that the effects of IL-4 are mediated by type I IL-4R and that type II IL-4R is absent on mast cells, and that IL-4 phosphorylates the 65-kd isoform of Stat6 in mast cells in a gamma(c)- and Jak3-dependent manner.
Animals↗
MAPK upstream kinase (MUK)-binding inhibitory protein, a negative regulator of MUK/dual leucine zipper-bearing kinase/leucine zipper protein kinase.
Mitogen-activated protein kinase upstream kinase/dual leucine zipper-bearing kinase/leucine-zipper protein kinase (MUK/DLK/ZPK) is a MAPKKK class protein kinase that induces JNK/SAPK activation. We report here a protein named MBIP that binds to MUK/DLK/ZPK. MUK-binding inhibitory protein (MBIP) contains two tandemly orientated leucine-zipper-like motifs with a cluster of basic amino acids located between the two motifs. MBIP interacts with one of the two leucine-zipper-like motifs of MUK/DLK/ZPK and inhibits the activity of MUK/DLK/ZPK to induce JNK/SAPK activation. Notably, no similar effect was observed with another JNK/SAPK-inducing MAPKKK, COT/Tpl-2, showing the specificity of MBIP action. Furthermore, the overexpression of MBIP partially inhibits the activation of JNK by 0.3 m sorbitol in 293T cells. Taken together, these observations indicate that MBIP can function as a regulator of MUK/DLK/ZPK, a finding that may provide a clue to understanding the molecular mechanism of JNK/SAPK activation by hyperosmotic stress.
3-Phosphoinositide-Dependent Protein Kinases↗
Lecithinized copper, zinc-superoxide dismutase ameliorates prolonged hypoxia-induced injury of cardiomyocytes.
Recent studies have suggested that prolonged hypoxia results in increased production of reactive oxygen species in cardiomyocytes, which leads to apoptosis of these cells. We previously showed that lecithinized recombinant human copper, zinc-superoxide dismutase (rhSOD) showed increased bioavailability through greater membrane affinity and a longer half-life than unmodified SOD. The purpose of this study was to investigate whether lecithinized SOD plays a protective role against hypoxic injury in cardiomyocytes. Cultured rat cardiomyocytes incubated with lecithinized SOD (100 U/ml), unmodified SOD (100 U/ml), or vehicle alone were subjected to hypoxia for up to 72 h. Lecithinized SOD, but not unmodified SOD, was successfully delivered intracellularly, which was verified by Western blot and confocal laser-scanning microscopy. Treatment of cells with lecithinized SOD significantly suppressed hypoxia-induced cell damage. Since lecithinized SOD also suppressed hypoxia-induced DNA fragmentation, the improved cell survival provided by lecithinized SOD is thought to be mediated by its antiapoptotic effect. In summary, lecithinization resulted in a facilitated rhSOD delivery into cultured cardiomyocytes, which reduced mortality of cardiomyocytes exposed to prolonged hypoxia.
Animals↗
Enhanced cardiomyocyte DNA synthesis during myocardial hypertrophy in mice expressing a modified TSC2 transgene.
Tuberous sclerosis complex (TSC) is a rare genetic disorder characterized by the appearance of benign tumors in multiple organs, including the heart. Disease progression is accompanied by homozygous mutation at 1 of 2 loci (designated TSC1 or TSC2), leading to the suggestion that these genes function as tumor suppressors. In this study, we generated a series of TSC2 cDNAs in which one or more structural motifs were deleted, with the hope that expression of the modified gene product would override the growth-inhibitory activity of the endogenous TSC2 gene product. Several of the modified cDNAs enhanced growth rate, increased endocytosis, and promoted aberrant protein trafficking when expressed in NIH-3T3 cells, thereby mimicking phenotypes typical of TSC2-deficient cells. Surprisingly, targeted expression of the most potent TSC2 cDNA to the heart did not perturb cardiac development. However, the level of cardiomyocyte DNA synthesis in adult transgenic mice was elevated >35-fold during isoproterenol-induced hypertrophy compared with their nontransgenic siblings. These results suggest that alteration of TSC2 gene activity in combination with beta-adrenergic stimulation can reactivate the cell cycle in a limited number of terminally differentiated adult cardiomyocytes.
3T3 Cells↗
Surfactant protein A exhibits inhibitory effect on eosinophils IL-8 production.
Eosinophils are believed to be one of the important sources of cytokines such as IL-8 at the site of allergic inflammation. It has been demonstrated that pulmonary surfactant protein A (SP-A) plays a potential role in modifying inflammation and the immune function. To verify the regulating effect of SP-A on eosinophil cytokine generation, we studied the effect of SP-A by determining of IL-8 production and expression stimulated with sIgA or PMA. SP-A purified from surfactant recovered from patients with alveolar proteinosis was added to eosinophils isolated by the negative selection method with immunomagnetic beads, and cultured for 24 h. The concentrations of IL-8 in the cell-free supernatants and cell lysates were then measured by ELISA. We also used a semiquantitative reverse transcriptase-polymerase chain reaction assay to detect the effect of SP-A on IL-8 mRNA expression. SP-A inhibited the secretion of IL-8 in a dose-dependent fashion. Suppression of IL-8 production by SP-A was significantly inhibited by SP-A antibody (PE10). SP-A also attenuated expression of IL-8 mRNA in eosinophils. These results indicate that SP-A might have the potential role to modify allergic inflammation by inhibiting IL-8 expression and production from eosinophils.
Antibodies↗
Blockade of JAK2 by tyrphostin AG-490 inhibits antigen-induced eosinophil recruitment into the mouse airways.
We studied the effect of tyrphostin AG-490, a specific Janus kinase 2 (JAK2) inhibitor, on antigen-induced eosinophil recruitment into the airways of sensitized mice and on IL-5-induced chemokinesis and adhesiveness of eosinophils. The in vivo administration of AG-490 prevented antigen-induced eosinophil infiltration in the airways of sensitized mice in a dose-dependent manner. However, the administration of AG-490 did not affect antigen-induced IL-5 production in the airways nor in vitro antigen-induced IL-5 production and T cell proliferation of spleen cells. Furthermore, AG-490 inhibited IL-5-induced chemokinesis and beta1-integrin adhesiveness of eosinophils in vitro. Because antigen-induced eosinophil recruitment into the airways is mediated by IL-5, these results indicate that JAK2 activation is critical for antigen-induced, IL-5-dependent mobilization of eosinophils into the tissue.
Animals↗