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Biomedical subjects

K Murata

Publications and source records attributed to K Murata.

At least 109 records · Page 6Linked to original sources
PubMed2000

Crystal structure of N-acyl-D-glucosamine 2-epimerase from porcine kidney at 2.0 A resolution.

The X-ray crystallographic structure of N-acyl-d-glucosamine 2-epimerase (AGE) from porcine kidney, which has been identified to be a renin-binding protein (RnBP), was determined by the multiple isomorphous replacement method and refined at 2.0 A resolution with a final R-factor of 16.9 % for 15 to 2.0 A resolution data. The refined structure of AGE comprised 804 amino acid residues (one dimer) and 145 water molecules. The dimer of AGE had an asymmetric unit with approximate dimensions 46 Ax48 Ax96 A. The AGE monomer is composed of an alpha(6)/alpha(6)-barrel, the structure of which is found in glucoamylase and cellulase. One side of the AGE alpha(6)/alpha(6)-barrel structure comprises long loops containing five short beta-sheets, and contributes to the formation of a deep cleft shaped like a funnel. The putative active-site pocket and a possible binding site for the substrate N-acetyl-d-glucosamine (GlcNAc) were found in the cleft. The other side of the alpha(6)/alpha(6)-barrel comprises short loops and contributes to the dimer formation. At the dimer interface, which is composed of the short loops and alpha-helices of the subunits, five strong ion-pair interactions were observed, which play a major role in the dimer assembly. This completely ruled out the previously accepted hypothesis that the formation of the RnBP homodimer and RnBP-renin heterodimer requires the leucine zipper motif present in RnBP.

Animals↗
PubMed2000

Structural determinants of water permeation through aquaporin-1.

Human red cell AQP1 is the first functionally defined member of the aquaporin family of membrane water channels. Here we describe an atomic model of AQP1 at 3.8A resolution from electron crystallographic data. Multiple highly conserved amino-acid residues stabilize the novel fold of AQP1. The aqueous pathway is lined with conserved hydrophobic residues that permit rapid water transport, whereas the water selectivity is due to a constriction of the pore diameter to about 3 A over a span of one residue. The atomic model provides a possible molecular explanation to a longstanding puzzle in physiology-how membranes can be freely permeable to water but impermeable to protons.

Amino Acid Sequence↗
PubMed2000

Inorganic Polyphosphate/ATP-NAD kinase of Micrococcus flavus and Mycobacterium tuberculosis H37Rv.

An enzyme with both inorganic polyphosphate [poly(P)]- and ATP-dependent NAD kinase activities was isolated from Micrococcus flavus. The enzyme was a dimer consisting of 34 kDa subunits, and was named poly(P)/ATP-NAD kinase. Internal amino acid sequences of the enzyme showed homologies with some function-unknown proteins released on the GenBank database. Among such proteins, hypothetical Rv1695 protein (Accession No. Z98268-16), which was encoded by a gene named "Rv1695" on genomic DNA of Mycobacterium tuberculosis H37Rv, was proposed to be poly(P)-dependent NAD kinase. By cloning and expression in Escherichia coli, Rv1695 was shown to encode poly(P)/ATP-NAD kinase and named ppnk. The ppnk product, recombinant-poly(P)/ATP-NAD kinase (Ppnk) was purified and characterized. The enzyme was a tetramaer consisting of 35 kDa subunits when expressed in E. coli. Poly(P)/ATP-NAD kinases of M. flavus and Ppnk of M. tuberculosis H37Rv specifically and completely phosphorylated NAD by utilizing commercially available poly(P)s and nucleoside triphosphates as phosphoryl donors.

Amino Acid Sequence↗
PubMed2000

1:1 complexes of dimethylthio- and ethylenedithio-tetrathiafulvalenothioquinone-1,3-dithiolemethides with CuBr2 as a new type of pi/d molecular system.

The reaction of dimethylthio- (1) and ethylenedithio-tetrathiafulvalenothioquinone-1,3-dithiolemethides (2) with CuBr2 gave 1:1 complexes between the donors and CuBr2, 1.CuBr2 and 2.CuBr2, in which the Cu atom of CuBr2 binds to the thiocarbonyl S atom in 1 and 2. The electrical conductivity (sigma) of 1.CuBr2 at room temperature was ca. 10(-5) S cm-1, while a comparatively high value of 4.0 S cm-1 was obtained for 2.CuBr2, whose temperature dependence of sigma exhibited, however, semiconducting behavior with a very small activation energy of 0.18 eV. The observed paramagnetic susceptibilities (chi p's) of the Cu complexes were composed of both a component due to the localized Cu spins obeying the Curie-Weiss law and a temperature-independent chi p due to the conducting pi electrons on the 1- or 2-stacked columns. From the Curie constants obtained, the degrees of intramolecular electron transfer from 1 and 2 to CuBr2 moieties were estimated at ca. 90% and 60%, respectively. The small, negative Weiss temperature suggest very weak antiferromagnetic interactions among the Cu spins on the CuBr2 moieties.

Journal Article↗
PubMed2000

v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation.

v-Crk induces cellular tyrosine phosphorylation and transformation of chicken embryo fibroblasts (CEF). We studied the molecular mechanism of the v-Crk-induced transformation. Experiments with Src homology (SH)2 and SH3 domain mutants revealed that the induction of tyrosine phosphorylation of cellular proteins requires only the SH2 domain, but both the SH2 and SH3 domains are required for complete transformation. Analysis of three well defined signaling pathways, the mitogen-activated protein kinase (MAPK) pathway, the Jun N-terminal kinase (JNK) pathway, and the phosphoinositide 3-kinase (PI3K)/AKT pathway, demonstrated that only the PI3K/AKT pathway is constitutively activated in v-Crk-transformed CEF. Both the SH2 and SH3 domains are required for this activation of the PI3K/AKT pathway in CEF. We also found that the colony formation of CEF is strongly induced by a constitutively active PI3K mutant, and that a PI3K inhibitor, LY294002, suppresses the v-Crk-induced transformation. These results strongly suggest that constitutive activation of the PI3K/AKT pathway plays an essential role in v-Crk-induced transformation of CEF.

Animals↗
PubMed2000

A role for Id-1 in the aggressive phenotype and steroid hormone response of human breast cancer cells.

The helix-loop-helix protein Id-1 inhibits the activity of basic helix-loop-helix transcription factors, and is an important regulator of cell growth and tissue-specific differentiation. We have shown (P. Y. Desprez et al., Mol. Cell. Biol., 18: 4577-4588, 1998) that ectopic expression of Id-1 inhibits differentiation and stimulates the proliferation and invasiveness of mouse mammary epithelial cells, and that there is a correlation between the levels of Id-1 protein and the aggressiveness of several human breast cancer cell lines. Here, we show that aggressive and metastatic breast cancer cells express high levels of Id-1 mRNA because of a loss of serum-dependent regulation that is mediated by a 2.2-kb region of the human Id-1 promoter. Three lines of evidence suggest that unregulated Id-1 expression may be an important regulator of the aggressive phenotype of a subset of human breast cancer cells: (a) a constitutively expressed Id-1 cDNA, when introduced into a nonaggressive breast cancer cell line (T47D), conferred a more aggressive phenotype, as measured by growth and invasiveness; (b) Id-1 was an important mediator of the effects of sex steroid hormones on T47D cell proliferation. Estrogen stimulated proliferation and induced Id-1 expression, whereas progesterone inhibited proliferation and repressed Id-1 expression. Progesterone repressed Id-1 expression, at least in part by repressing transcription. Most importantly, an antisense oligonucleotide that reduced Id-1 protein levels reduced the ability of estrogen to stimulate cell proliferation, whereas constitutive Id-1 expression rendered cells refractory to growth inhibition by progesterone; and (c) using a limited number of breast cancer biopsies, we showed that Id-1 was more frequently expressed in infiltrating carcinomas compared with ductal carcinomas in situ. Our results suggest that Id-1 can control the malignant progression of breast cancer cells, particularly that mediated by sex steroid hormones. Moreover, Id-1 has the potential to serve as a marker for aggressive breast tumors.

Animals↗
PubMed2000

Crystallization and preliminary X-ray crystallographic analysis of alginate lyase A1-II from Sphingomonas species A1.

Alginate lyase A1-II of Sphingomonas species A1 was purified and crystallized using the hanging drop vapor-diffusion method in 0.1 M Tris-HCl buffer containing 43% saturated ammonium sulfate, 8% polyethylene glycol 4000 and 0.2 M Li(2)SO(4) at pH 8.5 and 20 degrees C. The crystals are tetragonal and belong to the space group P4(3)2(1)2 or P4(1)2(1)2 with unit cell dimensions of a=b=144.07 and c=296.38 A. The diffraction data up to 2.8 A were collected by a synchrotron radiation source at SPring-8 in Japan.

Bacterial Proteins↗
PubMed2000

Complete nucleotide sequence of a plant tumor-inducing Ti plasmid.

Crown gall tumor disease in dicot plants is caused by Agrobacterium tumefaciens harboring a giant tumor-inducing (Ti) plasmid. Here, for the first time among agrobacterial plasmids, the nucleotide sequence of a typical nopaline-type Ti plasmid (pTi-SAKURA) was determined completely. In total, 195 open reading frames (ORFs) were estimated in the 206479 bp long sequence. 20 genes for conjugation, three for replication, 22 for pathogenesis and 37 for genetic colonization of host plants were found within two-thirds of the plasmid. These genes formed seven functional gene clusters with narrow inter-cluster spaces. In the remaining one-third of the plasmid, novel genes including homologs of mutT, Rhizobium nodQ and Sphingomonas ligE genes were found, which are likely to be responsible for the broad host range. Restriction fragment length variation indicates extreme plasticity of the part required for conjugational gene transfer and the above-mentioned one-third of the plasmid, even among closely related Ti plasmids.

Agrobacterium tumefaciens↗
PubMed2000

Impairment of antigen-presenting cell function in mice lacking expression of OX40 ligand.

OX40 expressed on activated T cells is known to be an important costimulatory molecule on T cell activation in vitro. However, the in vivo functional significance of the interaction between OX40 and its ligand, OX40L, is still unclear. To investigate the role of OX40L during in vivo immune responses, we generated OX40L-deficient mice and a blocking anti-OX40L monoclonal antibody, MGP34. OX40L expression was demonstrated on splenic B cells after CD40 and anti-immunoglobulin (Ig)M stimulation, while only CD40 ligation was capable of inducing OX40L on dendritic cells. OX40L-deficient and MGP34-treated mice engendered apparent suppression of the recall reaction of T cells primed with both protein antigens and alloantigens and a significant reduction in keyhole limpet hemocyanin-specific IgG production. The impaired T cell priming was also accompanied by a concomitant reduction of both T helper type 1 (Th1) and Th2 cytokines. Furthermore, antigen-presenting cells (APCs) derived from the mutant mice revealed an impaired intrinsic APC function, demonstrating the importance of OX40L in both the priming and effector phases of T cell activation. Collectively, these results provide convincing evidence that OX40L, expressed on APCs, plays a critical role in antigen-specific T cell responses in vivo.

Animals↗
PubMed2000

Convenient enzymatic resolution of alcohols using highly reactive, nonharmful acyl donors, 1-ethoxyvinyl esters

1-Ethoxyvinyl esters 3, a new type of acyl donors for enzymatic resolution of racemic alcohols, were disclosed to be superior to the contemporary major reagents, vinyl esters 1 and isopropenyl esters 2. Three features of 3 are noticeable: (1) 3 generates ethyl acetate as a single coproduct, which does not affect any enzymes, while acetaldehyde liberated from 1 deactivates some kinds of lipases. (2) The reactivity of 3 was not less than that of 1 and much higher than that of 2, and the optical purity of the products was as high as that of 1 and 2. Especially, it was generally observed that 3 showed higher reactivity than 1 for reactions using Candida rugosa lipases, one of the most commonly employed lipases, having liberal applicability to substrates but sensitive to acetaldehyde. Twelve examples of the kinetic resolution of racemic secondary alcohols (5 and 10) and one desymmetrization of meso-alcohol 7 were presented employing the acetate 3a or the octanoate 3b and four types of lipases. (3) A one-pot procedure for the preparation of 3 from the corresponding carboxylic acid and the subsequent enzymatic resolution of alcohols, which has not been reported using 1 or 2, was elucidated. The chemical and optical yields of the products by this procedure were similar to those obtained using isolated 3.

Journal Article↗
PubMed2000

Subclinical neurophysiological effects of lead: A review on peripheral, central, and autonomic nervous system effects in lead workers.

BACKGROUND: The peripheral nerve conduction velocity (NCV), distribution of nerve conduction velocities (DCV), somatosensory, visual, and brainstem auditory evoked potentials (SEP, VEP, and BAEP, respectively), event-related potential (P300), computerized static posturography with spectral analysis (postural balance), and electrocardiographic R-R interval variability (CV(RR)) with spectral analysis appear to be promising techniques for assessing subclinical effects of lead on the peripheral, central and autonomic nervous systems. This article presents an overview of research addressing subclinical neurophysiological effects of lead in workers exposed to lead. METHODS: We reviewed 102 articles to examine the effects and dose-effects relationships of lead on peripheral, central, and autonomic nervous system function together with reversibility of the effects, interaction between lead and other metals, and relative sensitivity and specificity of each technique. Background and methodology were also reviewed. RESULTS AND CONCLUSION: Available data suggest that, on a group basis, the reduction in the NCV, together with the effects on the P300 latency, postural balance, and CV(RR), occurs at a mean blood lead concentration (BPb) as low as 30-40 microg/dL; the effects on the latencies of the short-latency SEP, VEP, and BAEP, as well as the DCV, start at a BPb as low as 40-50 microg/dL. Further cross-sectional and preferably prospective studies by using each of those methods are needed to establish more precise dose-effects (and response) relationships of lead.

Autonomic Nervous System↗
PubMed2000

Immunohistochemical detection of parathyroid hormone-related protein in a squamous cell carcinoma arising from mature cystic teratoma causing humoral hypercalcemia of malignancy.

BACKGROUND: Humoral hypercalcemia of malignancy is a cancer-related hypercalcemia caused by the production of humoral factors by malignant cells in patients without bone metastases. Squamous cell carcinomas are the tumors most frequently associated with humoral hypercalcemia of malignancy, and parathyroid hormone-related protein (PTH-rP) is the main humoral factor implicated. Squamous cell carcinoma arising from mature cystic teratoma is a rare diagnosis itself, much less the description of associated hypercalcemia, despite the fact that the normal keratinocytes produce parathyroid hormone-related protein. CASE: We present a well-documented case of squamous cell carcinoma arising from mature cystic teratoma of the ovary, complicated by hypercalcemia in a patient with high levels of plasma parathyroid hormone-related protein and immunohistochemical evidence of parathyroid hormone-related protein expression by the tumor cells. CONCLUSION: In this case, the carcinoma cells had already produced PTH-rP in the primary tumor although the serum calcium levels had not been significantly high at surgery. It is therefore suggested that hypercalcemia may have occurred after PTH-rP production had overcome the homeostatic level during the terminal stage.

Aged↗
PubMed2000

Overexpression in Escherichia coli, purification, and characterization of Sphingomonas sp. A1 alginate lyases.

A bacterium Sphingomonas sp. A1 produces three kinds of alginate lyases [A1-I (66 kDa), A1-II (25 kDa), and A1-III (40 kDa)] from a single precursor, through posttranslational processing. Overexpression systems for these alginate lyases were constructed in Escherichia coli cells by controlling of the lyase genes under T7 promoter and terminator. Expression levels of A1-I, A1-II, and A1-III in E. coli cells were 3.50, 3.04, and 2.13 kU/liter of culture, respectively, and were over 10-fold higher than those in Sphingomonas sp. A1 cells. Purified A1-I, A1-II, and A1-III from E. coli cells were monomeric enzymes with molecular masses of 63, 25, and 40 kDa, respectively. The depolymerization pattern of alginate with A1-I and A1-II indicated that both enzymes cleaved the glycosidic bond of the polymer endolytically and by beta-elimination reaction. A1-II preferred polyguluronate rather than polymannuronate and released tri- and tetrasaccharides, which have unsaturated uronyl residues at the nonreducing terminal, from alginate as the major final products. A1-I acted equally on both homopolymers and produced di- and trisaccharides as the final products.

Alginates↗
PubMed2000

A study on the mechanism of the proton transport in bacteriorhodopsin: the importance of the water molecule.

The mechanism of proton transport around the Schiff base in bacteriorhodopsin was investigated by ab initio molecular orbital (MO) calculations. Computations were performed for the case where there is a water molecule between the Schiff base and the Asp residue and for the case where there is no water molecule. Changes in the atomic configuration and potential energy through the proton transport process were compared between two cases. In the absence of water, the protonated Schiff base was not stable, and a proton was spontaneously detached from the Schiff base. On the other hand, a stable structure of the protonated Schiff base was obtained in the presence of water. This suggests that the presence of a water molecule is required for stability in the formation of a protonated Schiff base.

Aspartic Acid↗
PubMed2000

Type IV collagenase and immunostaining of type IV collagen in human thyroid tumors.

OBJECTIVE: Required stages in the processes of malignant tumor invasion and metastasis are known to include the destruction of cell stroma and vascular basement membrane. It has been suggested that type IV collagenase can degrade type IV collagen, a main component of basement membrane. In our study, type IV collagenase activity was compared with the grade of destruction of type IV collagen by thyroid tumors. METHODS: Type IV collagenase activity was measured in human thyroid tissue obtained surgically from four patients with Graves' disease, four with follicular adenoma, six with papillary carcinoma, and four with follicular carcinoma. Six normal thyroid tissue specimens were also studied. The grade of destruction in the diseased regions of thyroid tumors and surrounding intact tissues were determined immunochemically by anti-type IV collagen antibody staining. RESULTS: Tumors with high type IV collagenase activity exhibited extracapsular invasion, lymph node metastasis and very weak discontinuity of immunostaining for type IV collagen from the basement membrane in diseased regions. In addition, surrounding intact tissues exhibited weak immunostaining for type IV collagen. Tumors with low type IV collagenase activity exhibited neither extracapsular invasion nor lymph node metastasis, but did exhibit weak immunostaining for type IV collagen from the basement membranes in diseased regions. However, the surrounding intact tissues had preserved type IV collagen in follicular basement membranes. CONCLUSION: These findings suggest that staining for type IV collagen can be considered a biochemical marker for prediction of the aggressiveness of invasion and metastasis.

Adenoma↗
PubMed2000

Bronchiolar disease: spectrum and radiological findings.

Two types of bronchiole, the terminal bronchiole and the respiratory bronchiole, have structural and functional differences. The former is characterized as a conducting airway and the latter is closely related to a gas-exchange function as it has numerous alveoli on the wall. Therefore, the diseases occurring at bronchiole demonstrate different pathological, radiological and clinical pictures depending on which bronchiole is mainly involved. The disease that mainly involves the conducting airway is appreciated as a small airway disease. Constrictive bronchiolitis is a well-recognized entity classified in this category. Whereas the disease mainly involves the respiratory bronchiole and distal alveolar space, it is recognized as an interstitial and parenchymal disease. BOOP or RB-ILD is classified in this category. These two types of bronchiolar diseases reveal the contrast clinical pictures, including incidence, causative disease, response to the treatment, prognosis, respiratory function test as well as the radiological findings. This pictorial essay will illustrate the radiological features of the varieties of bronchiolitis.

Alveolitis, Extrinsic Allergic↗
PubMed2000

Cyclic AMP specific phosphodiesterase activity and colon cancer cell motility.

To investigate mechanisms for regulation of intracellular cAMP involved in cancer cell invasion, phosphodiesterase (PDE) activity in a colon cancer cell line, DLD-1, was studied. Activities of PDE 2, 4, and 5 were detected in DLD-1 cells by pharmacological approach. Specific and cell permeable inhibitors for those PDEs were used to determine which PDE is responsible for cAMP turnover involved in cancer cell motility. Treatment of DLD-1 cells with rolipram and Ro-20-1724 inhibitors for PDE 4, elevated intracellular cAMP contents three to five times of control. EHNA, an inhibitor for PDE 2, and zaprinast. an inhibitor for PDE 5, did not affect cAMP levels. To assess cellular motility, we utilized chemotaxis assay. EHNA and zaprinast did not suppress serum-induced chemotaxis. In contrast, rolipram and Ro-20-1724, suppressed chemotaxis in a dose dependent fashion. These suggest that PDE 4 plays a critical role in regulating intracellular cAMP levels of colon cancer cells and is involved in cancer invasion. PDE 4 can be a novel target of anti-invasion drug.

3',5'-Cyclic-AMP Phosphodiesterases↗