Biomedical subjects

H Nakamura

Publications and source records attributed to H Nakamura.

At least 19 recordsLinked to original sources

PubMed1992

[Semidiscoid lateral meniscus].

We propose a new entity known as "semidiscoid lateral meniscus" of the knee. The diagnostic criteria for semidiscoid lateral meniscus is the appearance on a thin-sliced axial 3-D image of a crescent-shaped meniscus whose transverse width is within 11.6 mm to 14.3 mm on the coronal image. These numerical values were calculated by discriminant analysis. A retrospective review of MR examinations of the knees revealed 15 patients (15 knees) with this entity. These patients were our subjects. Of these 15 patients, complicated lateral meniscal tears were seen in only three cases. Nine knees were free from complications, and five were asymptomatic. Six patients were examined with MR on the contralateral side, and discoid lateral menisci were revealed in all cases. Thus semidiscoid lateral meniscus shows a cross-relationship with discoid menisci.

Adolescent↗

PubMed1992

[Abnormal chest shadow on CT in immunosuppressed patients].

An abnormal chest shadow was observed on CT scans in 25 cases of 23 immunosuppressed patients. Pulmonary disease was pathologically confirmed to be pneumocystis carinii pneumonia (PC pneumonia) in four patients, cytomegalovirus pneumonia (CMV pneumonia) in one, bacterial pneumonia in seven, fungal infection in three, miliary tuberculosis in one, leukemic infiltration in two, lymphangitis carcinomatosa in three, drug-induced pneumonitis in three, and ARDS in one. In almost all patients, especially those with infectious diseases such as PC pneumonia, CMV pneumonia, and bacterial pneumonia, the abnormal shadow was wide and visible in the bilateral lung fields. We presumed that such findings as lobular shadow, centrilobular shadow, and mosaic pattern reflected the extension of disease via the respiratory tract, and that those findings are typical of infectious diseases. Because such findings as abnormal linear shadow and swelling of a broncho-vascular bundle were very frequently recognized in patients with lymphangitis carcinomatosa and frequently recognized in those with drug-induced pneumonitis, these diseases may be distinguished from other diseases. An area of slightly increased density was frequently recognized in patients with PC pneumonia, bacterial pneumonia, and drug-induced pneumonitis. Such lesions were pathologically confirmed to be located in the interstitium and/or alveolus. CT was extremely useful in comprehending the character and extension of particular diseases among various diseases. As the number of patients studied was small, the utility of CT in immunosuppressed patients requires further investigation in a larger number of patients.

Adolescent↗

PubMed1992

[MR imaging evaluation of plica synoviallis mediopatellaris of the knee joint].

To evaluate the diagnostic ability of MR imaging for plica synoviallis mediopatellaris (PSM), we retrospectively reviewed the MR imaging findings of patellofemoral space in 20 knee joints of 11 patients. In all 20 knee joints, arthroscopy and MR imaging were available. MR imaging was performed with a 1.5 Tesla Magnetom (Siemens) using a round surface coil. Pulse sequences were SE (TR 600 ms/TE 26 ms), SE (TR 200 ms/TE 26, 70 ms) and FLASH (TR 450 ms/TE 15 ms/FA 90 degrees). In six of the 20 knees with PSM proved by arthroscopy, a low intensity band was shown above the medial condyle of the femur on both T1- and T2-weighted MR images, and on FLASH images this band was shown as intermediate intensity. In the other 14 knees with no PSM observed by arthroscopy, the low intensity band was not shown on MR imaging. In all 20 knees, a similar low intensity band was shown about 1 cm cranial to the medial condyle of the femur. This should not be diagnosed as PSM. The low intensity band seen on T1- and T2-weighted MR images and its anatomical relation to the medial condyle are important in diagnosing PSM.

Adolescent↗

PubMed1992

Structure-activity relationships of mu-conotoxin GIIIA: structure determination of active and inactive sodium channel blocker peptides by NMR and simulated annealing calculations.

A synthetic replacement study of the amino acid residues of mu-conotoxin GIIIA, a peptide blocker for muscle sodium channels, has recently shown that the conformation formed by three disulfide bridges and the molecular basicity, especially the one around the Arg13 residue, are important for blocking activity. In the present study, we determined the three-dimensional structure of an inactive analog, [Ala13]mu-conotoxin GIIIA, and refined that of the native toxin by NMR spectroscopy combined with simulated annealing calculations. The atomic root-mean-square difference of the mutant from the native conotoxin was 0.62 A for the backbone atoms (N, C alpha, C') of all residues except for the two terminal residues. The observation that the replacement of Arg13 by Ala13 does not significantly change the molecular conformation suggests that the loss of activity is not due to the conformational change but to the direct interaction of the essential Arg13 residue with the sodium channel molecules. In the determined structure, important residues for the activity, Arg13, Lys16, Hyp(hydroxyproline)17, and Arg19, are clustered on one side of the molecule, an observation which suggests that this face of the molecule associates with the receptor site of sodium channels. The hydroxyl group of Hyp17 is suggested to interact with the channel site with which the essential hydroxyl groups of tetrodotoxin and saxitoxin interact.

Alanine↗

PubMed1992

Inhibition of passive cutaneous anaphylaxis by synthetic human immunoglobulin E peptide fragments.

In an attempt to determine the regions responsible for type I immediate hypersensitivity, a total of 42 peptide fragments, which cover the CH3-CH4 domains in human immunoglobulin E (IgE), were chemically synthesized. Several peptide fragments located in the amino acid sequences Ala329-Thr357 and Arg419-Ala463, inhibited passive cutaneous anaphylaxis (PCA) in vivo. In order to pinpoint the sites responsible for the inhibition of the PCA reaction, various fragment peptides in these two regions were synthesized. As a result, residues Pro343-Leu348, Pro426-Thr433, and Ser456-Thr461 were suggested to be involved in type I immediate hypersensitivity.

Amino Acid Sequence↗

PubMed1992

Tumor necrosis factor modulation of expression of the cystic fibrosis transmembrane conductance regulator gene.

Based on the knowledge that expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be modulated at the transcriptional level, and that the CFTR gene promoter contains sequences homologous to elements in other promoters that respond to tumor necrosis factor-alpha (TNF), we evaluated the hypothesis that TNF might modulate CFTR gene expression in epithelial cells. Studies with HT-29 cells, a colon epithelium-derived tumor cell line known to express the CFTR gene, demonstrated that TNF downregulated CFTR mRNA transcript levels in a dose- and time-dependent fashion. Interestingly, nuclear run-on analyses demonstrated that TNF did not affect the rate of transcription of CFTR gene, but exposure of the cells to TNF did modify the stability of CFTR mRNA transcripts, resulting in a mRNA half-life that was reduced to 65% of the resting level. These observations suggest that CFTR gene expression can be modulated by TNF, at least in part, at the posttranscriptional level.

Blotting, Northern↗

PubMed1992

Thermostabilization of Escherichia coli ribonuclease HI by replacing left-handed helical Lys95 with Gly or Asn.

From the systematic replacements of amino acid residues of Escherichia coli ribonuclease HI with those of its thermophilic counterpart, the basic protrusion domain including region 6 (R6) from residues 91 to 95 was found to increase the structural stability of the mutant protein (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., and Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Further mutagenesis concentrating in the R6 region has revealed that replacements of Lys95 at the left-handed structure with Gly or Asn essentially enhances the protein stability. Gly and Asn substitutions stabilize the protein up to 1.9 kcal/mol and 0.9 kcal/mol in the free energy changes of unfolding, respectively. We propose that the amino acid substitution of left-handed non-Gly residue with Gly or Asn residue can be used as one of the general strategies to enhance protein stability, when such a non-Gly residue itself does not seriously contribute to protein stability.

Amino Acid Sequence↗

PubMed1992

Stabilization of Escherichia coli ribonuclease HI by strategic replacement of amino acid residues with those from the thermophilic counterpart.

Thermus thermophilus ribonuclease H is exceptionally stable against thermal and guanidine hydrochloride denaturations as compared to Escherichia coli ribonuclease HI (Kanaya, S., and Itaya, M. (1992) J. Biol. Chem. 267, 10184-10192). The identity in the amino acid sequences of these enzymes is 52%. As an initial step to elucidate the stabilization mechanism of the thermophilic RNase H, we examined whether certain regions in its amino acid sequence confer the thermostability. A variety of mutant proteins of E. coli RNase HI were constructed and analyzed for protein stability. In these mutant proteins, amino acid sequences in loops or terminal regions were systematically replaced with the corresponding sequences from T. thermophilus RNase H. Of the nine regions examined, replacement of the amino acid sequence in each of four regions (R4-R7) resulted in an increase in protein stability. Simultaneous replacements of these amino acid sequences revealed that the effect of each replacement on protein stability is independent of each other and cumulative. Replacement of all four regions (R4-R7) gave the most stable mutant protein. The temperature of the midpoint of the transition in the thermal unfolding curve and the free energy change of unfolding in the absence of denaturant of this mutant protein were increased by 16.7 degrees C and 3.66 kcal/mol, respectively, as compared to those of E. coli RNase HI. These results suggest that individual local interactions contribute to the stability of thermophilic proteins in an independent manner, rather than in a cooperative manner.

Amino Acid Sequence↗

PubMed1992

Methotrexate decreases thymidine kinase activity.

MTX cytotoxicity is not fully explained by its well-known inhibition of dihydrofolate reductase activity which leads to a decrease in the dTMP synthase reaction, since TdR kinase which converts TdR to dTMP could readily circumvent MTX action through this salvage activity. TdR kinase is of particular significance, since in various types of carcinoma cells its activity is orders of magnitude higher than that of dTMP synthase. To throw light on this problem, we tested the hypothesis that the impact of MTX treatment might in fact involve an inhibition or decrease in TdR kinase activity. Injection in rat of MTX (i.p.) decreased TdR kinase activity in a time- and dose-dependent fashion in liver (t1/2 = 46 h; IC50 = 95 mg/kg), bone marrow (t1/2 = 10 h; IC50 = 5 mg/kg) and rapidly growing transplantable hepatoma 3924A (t1/2 = 56 h; IC50 = 5 mg/kg). Injection in rat of cycloheximide (15 mg/kg, i.p.), an inhibitor of protein biosynthesis, rapidly decreased TdR kinase activity in the hepatoma (t1/2 = 3.6 h); activities of other purine and pyrimidine synthetic enzymes, dTMP synthase, IMP dehydrogenase, GMP reductase and GMP synthase, declined at a markedly slower rate (t1/2 = 11, 11.6, 12 and 22 h, respectively). MTX, by curtailing purine and pyrimidine biosynthesis, limits product of TdR kinase which is more sensitive to unopposed protein degradation than other enzymes of nucleic acid biosynthesis. TdR kinase is a newly discovered target of MTX treatment.

Animals↗

PubMed1992

The three-dimensional structure of guanine-specific ribonuclease F1 in solution determined by NMR spectroscopy and distance geometry.

Two-dimensional 1H-NMR studies have been performed on ribonuclease F1 (RNase F1), which contains 106 amino acid residues. Sequence-specific resonance assignments were accomplished for the backbone protons of 99 amino acid residues and for most of their side-chain protons. The three-dimensional structures were constructed on the basis of 820 interproton-distance restraints derived from NOE, 64 distance restraints for 32 hydrogen bonds and 33 phi torsion-angle restraints. A total of 40 structures were obtained by distance geometry and simulated-annealing calculations. The average root-mean-square deviation (residues 1-106) between the 40 converged structures and the mean structure obtained by averaging their coordinates was 0.116 +/- 0.018 nm for the backbone atoms and 0.182 +/- 0.015 nm for all atoms including the hydrogen atoms. RNase F1 was determined to be an alpha/beta-type protein. A well-defined structure constitutes the core region, which consists of a small N-terminal beta-sheet (beta 1, beta 2) and a central five-stranded beta-sheet (beta 3-beta 7) packed on a long helix. The structure of RNase F1 has been compared with that of RNase T1, which was determined by X-ray crystallography. Both belong to the same family of microbial ribonucleases. The polypeptide backbone fold of RNase F1 is basically identical to that of RNase T1. The conformation-dependent chemical shifts of the C alpha protons are well conserved between RNase F1 and RNase T1. The residues implicated in catalysis are all located on the central beta-sheet in a geometry similar to that of RNase T1.

Amino Acid Sequence↗

PubMed1992

Entropic stabilization of a mutant human lysozyme induced by calcium binding.

The stabilization mechanism of the mutant human lysozyme with a calcium binding site (D86/92) was investigated by using calorimetric approaches. By differential scanning calorimetry, the enthalpy change (delta H) in the unfolding of holo-D86/92 was found to be 6.8 kcal/mol smaller than that of the wild-type and apo-D86/92 lysozymes at 85 degrees C. However, the unfolding Gibbs energy change (delta G) of the holo mutant was 3.3 kcal/mol greater than the apo type at 85 degrees C, indicating a significant decrease of entropy (T delta S = 10.1 kcal/mol) in the presence of Ca2+. Subsequently, the Ca2+ binding process in the folded state of the mutant was analyzed by using titration isothermal calorimetry. The binding enthalpy change was estimated to be 4.5 kcal/mol, and delta G was -8.1 kcal/mol at 85 degrees C, which indicates that the binding was caused by a large increase in entropy (T delta S = 12.6 kcal/mol). From these analyses, the unfolded holo mutant was determined to bind Ca2+ with a binding delta G of -4.8 kcal/mol (delta H = -2.6 kcal/mol, T delta S = 2.2 kcal/mol) at 85 degrees C. Therefore, the major cause of stabilization of holo-D86/92 is the decrease in entropy of the peptide chain due to Ca2+ binding to the unfolded protein.

Amino Acid Sequence↗

PubMed1992

Chondrocytic differentiation of peripheral neuroectodermal tumor cell line in nude mouse xenograft.

We have established a cell line (KU-SN) from a peripheral neuroectodermal tumor originating in the left scapula of a 4-year-old girl. The original tumor was immunoreactive with antibodies for neurofilament proteins, neuron-specific enolase, vimentin, S100 protein, and beta 2-microglobulin. Dense core granules, 50-150 nm in diameter, were identified by electron microscopy. The cell line was established from tumor cells in metastatic lung fluid. KU-SN cells were immunoreactive with the antibodies for neurofilament proteins, vimentin, neuron-specific enolase, S100 protein, glial fibrillary acidic protein, cytokeratin, and carcinoembryonic antigen. Besides these neuronal features, KU-SN cells express type 2 collagen and insulin-like growth factor 1 receptor. The addition of insulin-like growth factor 1 (100 ng/ml) increased the growth rate of KU-SN cells 2.1-fold over control. Some cells were positive for Alcian blue and alkaline phosphatase staining. Cytogenetic analysis of KU-SN cells disclosed a reciprocal chromosomal translocation [t(11,22)]. Northern blot analysis of KU-SN cells demonstrated amplified expression of the c-myc gene but not the N-myc gene. When tumor cells were transplanted into nude mice, cartilage was formed. The cartilage was immunoreactive with the antibody for HLA-ABC, indicating that it was derived from the tumor cells, not from mouse tissue. Chondrocytic differentiation was not observed in xenografts of Ewing's sarcoma cell lines SK-ES or RD-ES or the peripheral neuroectodermal tumor cell line SK-N-MC. These results indicate that KU-SN cells represent primitive neural crest cells having the potential for chondrocytic differentiation.

Animals↗

PubMed1992

[Dynamic 3DFT FISP MR imaging of hepatocellular carcinoma].

Dynamic 3 dimensional Fourier transformation (3DFT) FISP MR imaging was performed in 5 patients with hepatocellular carcinoma before partial hepatectomy. Immediately after 0.1 mmol/kg of Gd-DTPA was administered intravenously, 3DFT FISP images (TR/TE/flip angle/slice thickness, 20 msec/8 msec/30 degrees/2-4 mm) were obtained every 30 seconds until 150 sec. We correlated dynamic MR images of the 5 patients with gross and microscopic findings. Some regions in the tumor corresponding to viable cells showed high intensity enhancement and other regions corresponding to necrotic regions showed no enhancement on the early phase images. We concluded that dynamic 3DFT FISP MR imaging which had good spatial resolution was useful in evaluating the vascularity of the tumor.

Aged↗

PubMed1992

Solution structure of a DNA-binding unit of Myb: a helix-turn-helix-related motif with conserved tryptophans forming a hydrophobic core.

The DNA-binding domain of the c-myb protooncogene product consists of three imperfect tandem repeats of 51 or 52 amino acids, each of which contains three conserved tryptophans, spaced 18 or 19 amino acids apart. The structure of the third repeat, which is essential for sequence-specific DNA binding, has been determined by NMR with distance geometry calculation. It includes three well-defined helices (residues 149-162, 166-172, and 178-187) maintained by a hydrophobic core that includes the three conserved tryptophans, together with two histidines. Helices 2 and 3 form a structure related to but distinct from a canonical helix-turn-helix motif. In particular, the turn between these helices is one amino acid longer than the corresponding turn in bacterial repressors and homeodomains and contains a proline residue. In addition, the architecture of the three helices is different from those of homeodomains and DNA-binding domains of bacterial repressors. Based on the present structure, the binding mode of Myb repeat 3 with a specific DNA is also discussed.

Amino Acid Sequence↗

PubMed1992

Expression of the human cystic fibrosis transmembrane conductance regulator gene in the mouse lung after in vivo intratracheal plasmid-mediated gene transfer.

As an approach to gene therapy for the respiratory manifestations of cystic fibrosis (CF), in vivo plasmid-mediated direct transfer of the normal CF transmembrane conductance regulator (CFTR) gene to the airway epithelium was investigated in mice. To evaluate the feasibility of this strategy, pRSVL, a plasmid composed of a firefly luciferase gene driven by the Rous sarcoma virus long terminal repeat (RSV-LTR), along with cationic liposomes was instilled into the trachea of C57BI/6NCR mice. With administration of 200-400 micrograms plasmid DNA, luciferase expression could be detected in the mouse lung homogenates for at least 4 wk. With this background, a CFTR expression plasmid vector (pRSVCFTR) constructed by replacing the luciferase cDNA from pRSVL with the normal human CFTR cDNA was evaluated in vivo in mice. Intratracheal instillation of pRSVCFTR with cationic liposomes followed by analysis of mouse lung RNA by polymerase chain reaction amplification (after conversion of mRNA to cDNA) using a RSV-LTR specific sense primer and a human CFTR-specific antisense primer demonstrated human CFTR mRNA transcripts from one day to 4 wk after instillation. Further, in vivo evaluation of beta-galactosidase activity after intratracheal administration of an E. coli lacZ gene expression plasmid vector directed by the cytomegalovirus promoter (pCMV beta) demonstrated that the airway epithelium was the major target of transfer and expression of the exogenous gene. These observations demonstrate successful plasmid-mediated gene transfer to the airway epithelium in vivo. This strategy may be feasible as a form of gene therapy to prevent the pulmonary manifestations of CF.

Animals↗

PubMed1992

[Differentiation between hepatoma and hemangioma by inversion recovery snapshot FLASH MR imaging with variable TIs].

To differentiate hepatoma from hemangioma, MR studies were performed for 18 patients with 21 untreated liver tumors (hepatoma 10, hemangioma 11). We obtained inversion recovery snapshot FLASH images of liver tumors with variable TIs (50, 100, 150, 200, 250, 300, 400, and 500 msec). 8 hemangiomas showed higher intensity than liver parenchyma on the images at 150 msec and less of TI, and lower intensity at 200 msec or more TI. In 7, signal intensity of hepatoma became low at 150 msec or less of TI or remained high with prolonged TI. Inversion recovery snapshot FLASH imaging with variable TIs will be useful for differentiating between hepatoma and hemangioma.

Adult↗

PubMed1992

Effect of cavity-modulating mutations on the stability of Escherichia coli ribonuclease HI.

The size of the cavity around Ser68 of Escherichia coli ribonuclease HI was modulated by amino acid substitutions to examine the effects on the stability of the enzyme. Five mutant proteins, Ser68----Gly, Ser68----Ala, Ser68----Thr, Ser68----Val and Ser68----Leu, were constructed. Each of the mutant proteins exhibited at least 40% of the enzyme activity of the wild-type protein. The stabilities of the mutant proteins were determined from urea-denaturation and thermal-denaturation curves. Among the five mutations, only the Ser----Val mutation resulted in an increase in the stability of the enzyme. The melting temperature, tm, at pH 3.0 of the mutant protein Ser68----Val was increased by 1.9 degrees C. Its free-energy change of unfolding in the absence of urea, delta G(H2O), and the midpoint of the denaturation curve, [D]1/2, were also increased by 5.4 kJ/mol and 0.18 M, respectively. The increase in the stability of the enzyme is probably due to the filling of the cavity space around Ser68 by valine. However, the mutation of Ser68 to glycine or leucine residues resulted in a considerable decrease in stability. In these cases, some conformational changes occur, as suggested by the CD and 1H-NMR spectra of these mutant proteins.

Base Sequence↗

PubMed1992

Increased interleukin 1 receptor, type I, at messenger RNA and protein level in skin fibroblasts from patients with systemic sclerosis.

Summary. To elucidate the mechanisms of the fibrosis in systemic sclerosis (SSc) through the action of a cytokine, interleukin 1 (IL-1), we studied the specific biologic and biochemical features of interleukin 1 receptor (IL-1R) as expressed on the surfaces of fibroblast cells in cultures from 3 SSc patients and 3 normal donors. 125I-IL-1 beta binding assays revealed a high density of IL-1R on the cell surfaces of SSc fibroblasts as compared to those of normal subjects. We also found an enhanced expression of IL-1R messenger RNA (mRNA) in SSc fibroblasts, using Northern blot or slot blot analysis. These findings indicate that the expression of IL-1R on SSc fibroblasts were spontaneously induced at the transcriptional level. It is suggested that SSc fibroblasts are more sensitive to IL-1, and that the signal transduction of IL-1 through IL-1R may be eventually involved in the fibrosis of SSc.

Cells, Cultured↗