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Biomedical subjects

S Wakatsuki

Publications and source records attributed to S Wakatsuki.

At least 55 records · Page 3Linked to original sources
PubMed1997

Ribosomal protein S7: a new RNA-binding motif with structural similarities to a DNA architectural factor.

BACKGROUND: The ribosome is a ribonucleoprotein complex which performs the crucial function of protein biosynthesis. Its role is to decode mRNAs within the cell and to synthesize the corresponding proteins. Ribosomal protein S7 is located at the head of the small (30S) subunit of the ribosome and faces into the decoding centre. S7 is one of the primary 16S rRNA-binding proteins responsible for initiating the assembly of the head of the 30S subunit. In addition, S7 has been shown to be the major protein component to cross-link with tRNA molecules bound at both the aminoacyl-tRNA (A) and peptidyl-tRNA (P) sites of the ribosome. The ribosomal protein S7 clearly plays an important role in ribosome function. It was hoped that an atomic-resolution structure of this protein would aid our understanding of ribosomal mechanisms. RESULTS: The structure of ribosomal protein S7 from Bacillus stearothermophilus has been solved at 2.5 A resolution using multiwavelength anomalous diffraction and selenomethionyl-substituted proteins. The molecule consists of a helical hydrophobic core domain and a beta-ribbon arm extending from the hydrophobic core. The helical core domain is composed of a pair of entangled helix-turn-helix motifs; the fold of the core is similar to that of a DNA architectural factor. Highly conserved basic and aromatic residues are clustered on one face of the S7 molecule and create a 16S rRNA contact surface. CONCLUSIONS: The molecular structure of S7, together with the results of previous cross-linking experiments, suggest how this ribosomal protein binds to the 3' major domain of 16S rRNA and mediates the folding of 16S rRNA to create the ribosome decoding centre.

Amino Acid Sequence↗
PubMed1997

Production, crystallization, and preliminary X-ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1-47) of troponin I.

Troponin is a ternary protein complex consisting of subunits TnC. TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli-expressed rabbit skeletal muscle TnC and fragment 1-47 of TnI, which is obtained by chemical cleavage of an E. coli-expressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex. Complex CI47 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P3(1)(2)21, with unit cell dimensions a = b = 48.2 A, c = 162 A. The asymmetric unit presumably contains one CI47 complex. Soaking with p-chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 A resolution, with completeness of 91% and R(merge) = 6.4%. The crystals of PCMBS-derivative should be suitable for X-ray studies using the multiple-wavelength anomalous diffraction technique. This is the first step for elucidating the structure of the full troponin complex.

Animals↗
PubMed1997

Alternative splicing of hMSH2 in normal human tissues.

hMSH2 is a homolog of bacterial mutS and yeast Msh2, a member of the group of mismatch repair genes whose products bind to mismatched regions of double-stranded DNA. We analyzed expression of hMSH2 in normal human organs by the polymerase chain reaction coupled with reverse transcription and found two novel types of alternatively spliced mRNAs that were expressed in normal human organs. One lacked exon 13, and the other lacked a portion from the second nucleotide of codon 633 to the second nucleotide of codon 719. In the latter transcript, intro 12 started with TA and ended with TT (TA-TT intron) which did not meet the GT-AG rule. Both types of transcript resulted in frameshifts which generated truncated hMSH2 proteins lacking the main part of the highly conserved region. The biological significance of the alternative splicing remains to be elucidated.

Adenosine Triphosphatases↗
PubMed1997

Thrombopoietin-responsive essential thrombocythaemia with myelofibrosis.

Platelet-derived growth factor (PDGF) and other cytokines released from megakaryocytes are thought to play a crucial role in the pathogenesis of myelofibrosis. We describe a patient with essential thrombocythaemia (ET) who developed myelofibrosis with an increased level of serum thrombopoietin (TPO). Recombinant human (rh) TPO stimulated the proliferation and spontaneous megakaryocyte colony formation of the neoplastic cells in the peripheral blood. Moreover, serum concentrations of PDGF, platelet factor 4, and beta-thromboglobulin were elevated and the production of these growth factors from the megakaryocyte progenitors was augmented with the addition of rhTPO in vitro. These results indicate that TPO may contribute to the development of myelofibrosis in ET.

Adult↗
PubMed1997

Isolation and characterization of a 230 kDa protein (p230) specifically expressed in fetal brains: its involvement in neurite outgrowth from rat cerebral cortex neurons grown on monolayer of astrocytes.

By screening with monoclonal antibodies (mAbs) raised against growth cone membrane fraction from fetal porcine brains, we have identified a 230 kDa antigen, termed p230. Western blot analysis of extracts from various tissues demonstrated that p230 is specifically expressed in brains, in which its expression is temporally restricted; it was especially prominent in the embryonic and the early postnatal stage, and decreased to subdetectable levels in the adult brain. Further characterization of p230 revealed that it is a peripherally-membrane associated, cell surface protein produced by astrocytes. Neurite outgrowth of E18 rat cerebral cortex neurons cultured on a monolayer of astrocytes was significantly reduced in the presence of anti-p230 polyclonal antibody. Partial amino acid sequences of p230 purified from fetal porcine brains were highly homologous to an extracellular matrix protein, tenascin-C. These lines of evidence suggest that p230, a tenascin-C-like molecule present in fetal porcine brains, plays important roles during early brain development, particularly in growth cone guidance.

Amino Acid Sequence↗
PubMed1996

Production and crystallization of lobster muscle tropomyosin expressed in Sf9 cells.

A new form of muscle tropomyosin crystal has been obtained, by employing new strategies in protein preparation and crystallization. Non-polymerizable tropomyosin was prepared by removing 11 amino acids at the C-terminus. The truncated tropomyosin was expressed in Sf9 insect cells by use of the baculovirus-based expression system, to obtain highly homogeneous protein preparations. By routinely monitoring homogeneity by mass spectrometry, we found that the homogeneity played a key role in obtaining good crystals. The crystal quality was also dependent on isoforms; the crystals raised from a slow muscle-specific isoform diffracted to a higher resolution, compared with a fast muscle-specific counterpart. For crystallization, a high concentration of organic solvent was used as the precipitant; in the presence of 35% DMSO, tetragonal crystals were formed, which belong to space group P4(3)(1)2(1)2 with cell constants of a=b=105.6 angstrom, c=506.9 angstrom. The crystals gave rise to reflections the intensities of which were characteristically determined by the transform of alpha-helical coiled-coil. Thus in the region of 10-5.5 angstrom resolut along the c*-axis, the reflections were weak. For accurate measurement of these reflection intensities, beam-line ID2 in ESRF Grenoble was advantageous owing to the high brilliance and a low background. There the crystals diffracted to beyond 3.0 A along the c*-axis, whereas along the a*-b*-plane reflections were limited to 6.6 angstrom. Data analysis is under way on a data set from a PtCl4 derivative.

Amino Acid Sequence↗
PubMed1996

Loss of human E-cadherin (ECD) correlated with invasiveness of transitional cell cancer in the renal pelvis, ureter and urinary bladder.

Loss or decreased expression of E-cadherin (ECD), which forms an epithelial junction complex that includes several other proteins and triggers signal transduction, may contribute to tumor progression. In the present study, we examined 90 transitional cell cancers (TCCs), 47 urinary bladder cancers and 43 ureteral or renal pelvic cancers, as well as TCC and papilloma cell lines to determine whether they express ECD. We classified ECD expression into normo-expression (like normal epithelial), decreased and loss of ECD staining on TCCs (urinary bladder, renal pelvic or ureteral). We found that low-stage TCCs expressed normal ECD in 68%, decreased of ECD in 20% and loss of ECD in 12%, whereas high-stage TCCs expressed 29%, 41% and 30% of ECD staining, respectively (P < 0.01). Furthermore, grade 1 TCCs were all estimated to show normo-expression, grade 2 TCCS expressed normal ECD in 49%, decreased of ECD in 41% and loss of ECD in 10% grade 3 TCCs classified as 20%, 30% and 50%, respectively (P < 0.01). Staining for cultured cell lines showed positive membranous staining for ECD in a benign papilloma cell line, RT4 and a TCC cell line, HT1376, but not in a TCC cell line, T24. Reverse-transcription polymerase chain reaction showed the presence of ECD and alpha-catenin mRNA in RT4 and HT1376, and only alpha-catenin in T24. Thus, it is more likely that decrease or loss of ECD might contribute to the malignant character of tumor cells and result in tumor progression.

Adult↗
PubMed1996

Primary meningeal lymphoma presenting solely with blindness: a report of an autopsy case.

A 64-year-old woman was admitted because of progressive paraplegia. She had a history of unexplained blindness which had developed gradually 1 year earlier. The contrast-enhanced CT showed a small mass at the sella turcica. The funduscopy showed merely atrophic discs. The lumbar puncture revealed marked pleocytosis (cell count: 10.4 x 10(9)/l) and a diagnosis of meningeal lymphoma was made by the cytology of the cerebrospinal fluid. No lymphomatous lesions were detected outside of the central nervous system (CNS) and the patient received whole brain irradiation together with intrathecal chemotherapy. However, there was no improvement in vision or other neurological activities. Eventually the patient died of interstitial pneumonia 2 months after admission. The autopsy revealed a residual B-cell lymphoma existing only in the leptomeninges of medulla oblongata. The optic nerves were atrophic macroscopically and massive gliosis was seen microscopically. In addition, cytomegalovirus infection was observed in the lungs and adrenal glands but not in the CNS. This patient must have had a primary meningeal lymphoma. The mechanism of the visual loss of this patient is discussed.

Atrophy↗
PubMed1995

The crystal structure of cyclin A.

BACKGROUND: Eukaryotic cell cycle progression is regulated by cyclin dependent protein kinases (CDKs) whose activity is regulated by association with cyclins and by reversible phosphorylation. Cyclins also determine the subcellular location and substrate specificity of CDKs. Cyclins exhibit diverse sequences but all share homology over a region of approximately 100 amino acids, termed the cyclin box. From the determination of the structure of cyclin A, together with results from biochemical and genetic analyses, we can identify which parts of the cyclin molecular may contribute to cyclin A structure and function. RESULTS: We have solved the crystal structure, at 2.0 A resolution, of an active recombinant fragment of bovine cyclin A, cyclin A-3, corresponding to residues 171-432 of human cyclin A. The cyclin box has an alpha-helical fold comprising five alpha helices. This fold is repeated in the C-terminal region, although this region shares negligible sequence similarity with the cyclin box. CONCLUSIONS: Analysis of residues that are conserved throughout the A, B, and E cyclins identifies two exposed clusters of residues, one of which has recently been shown to be involved in the association with human CDK2. The second cluster may identify another site of cyclin A-protein interaction. Comparison of the structure of the unbound cyclin with the structure of cyclin A complexed with CDK2 reveals that cyclin A does not undergo any significant conformational changes on complex formation. Threading analysis shows that the cyclin-box fold is consistent with the sequences of the transcription factor TFIIB and other functionally related proteins. The structural results indicate a role for the cyclin-box fold both as a template for the cyclin family and as a generalised adaptor molecule in the regulation of transcription.

Algorithms↗
PubMed1995

A Double Multilayer Monochromator at an ESRF Undulator for Microbeam Experiments.

A water-cooled double W/Si-multilayer monochromator has been operated at an ESRF low-beta undulator beam. For a fixed distance of the two multilayers the first-order Bragg reflection was at ~8 keV. The peak power density of the beam at the exit of the multilayers was ~1 W mm(-2) and the flux density of the first order after a 10 mum collimator was 4 x 10(5) photons s(-1) mum(-2) mA(-1.) The performance of the beam in microbeam diffraction has been tested on a 20 mum W wire. The observed pseudo-Laue pattern is discussed with respect to the multilayer spectrum.

Journal Article↗
PubMed1995

T cell-rich B cell lymphoma bearing Epstein-Barr virus in tumor cells: a case of IBL-T-like lesion following Lennert's lesion.

A case of T cell-rich B cell lymphoma (TCRBCL) with Epstein-Barr virus (EBV) infection in tumor cells is reported. A 50 year old male developed right cervical lymph node swelling in July 1988. Initial biopsy in April 1989 demonstrated many scattered Hodgkinoid atypical cells with Lennert's lesion. After partial remission following chemotherapy, the lymph nodes enlarged again, and a second biopsy in February 1991 showed an IBL-T-like lesion. Only a small number of Hodgkinoid atypical cells were still observed. After apparently, complete remission, the lesion soon recurred and the patient died in November 1992. Immunohistochemically the Hodgkinoid cells were positive for L26, but negative for LN2, LN3, UCHL-1, MT1, lysozyme, Ber-H2 and Leu-M1. Reactivity for immunoglobulins showed false-positive because of polyclonal staining. IgH monoclonality was detected by the polymerase chain reaction method in the first biopsied specimen, and by Southern blotting in the second biopsied snap-frozen specimen. Monoclonal TCR beta rearrangement was not detected. The Hodgkinoid atypical cells were positive for EBV-encoding RNA by in situ hybridization, and LMP-1 by immunostaining. Occasionally, EBV-bearing immunoblastic, medium sized, or small lymphocytic cells were also observed. This case indicates the possibility that EBV is related to the pathogenesis of TCRBCL.

Blotting, Southern↗
PubMed1995

Hemolytic-uremic syndrome due to deoxycoformycin: a report of the second case.

A hypercalcemic patient with adult T-cell leukemia was treated with deoxycoformycin (DCF) in a dose of 5 mg/m2 daily for three days. Several days later, severe thrombocytopenia appeared abruptly and then microangiopathic hemolytic anemia ensued. Subsequently, renal failure and hypertension developed, and the patient died seven weeks after DCF therapy. Needle necropsy of the kidney showed glomerular damage including swelling of endothelial cells, mesangiolysis and segmental collapse. This is the second reported case of hemolytic-uremic syndrome due to DCF.

Fatal Outcome↗
PubMed1994

Blue form of bacteriorhodopsin and its order-disorder transition during dehydration.

Freshly-prepared blue membranes from Halobacterium halobium, previously reported to be disordered, are shown to have a distinct crystal lattice structure, slightly different from the native form. The lattice of the blue form is disrupted irreversibly when dehydrated. The disorder process was observed using time-resolved small-angle X-ray diffraction and analyzed by radial autocorrelation functions. The diffraction peaks of the in-plane lattice first sharpen and increase due to improved membrane orientation, then the trimer lattice becomes disordered and the unit cell dimension decreases by 1.8 A. In contrast, dehydration of purple membranes does not disorder the lattice, and the unit cell dimension shrinks by only 1.0 A. Comparisons of radial autocorrelation functions for the blue membrane during drying show drastic loss of inter-trimer, long-range correlation while the intra-trimer, short-range correlations remain more or less unchanged. This suggests that the deionized protein trimers can maintain their overall structure during the dehydration, even though the lattice dimension decreases appreciably and the two-dimensional crystallinity is disrupted.

Bacteriorhodopsins↗
PubMed1994

Laue diffraction study on the structure of cytochrome c peroxidase compound I.

BACKGROUND: Cytochrome c peroxidase from yeast is a soluble haem-containing protein found in the mitochondrial electron transport chain where it probably protects against toxic peroxides. The aim of this study was to obtain a reliable structure for the doubly oxidized transient intermediate (termed compound I) in the reaction of cytochrome c peroxidase with hydrogen peroxide. This intermediate contains a semistable free radical on Trp191, and an oxyferryl haem group. RESULTS: Compound I was produced in crystals of yeast cytochrome c peroxidase by reacting the crystalline enzyme with hydrogen peroxide in a flow cell. The reaction was monitored by microspectrophotometry and Laue crystallography in separate experiments. A nearly complete conversion to compound I was achieved within two minutes of the addition of hydrogen peroxide, and the concentration of the intermediate remained at similar levels for an additional half an hour. The structure of the intermediate was determined by Laue diffraction. The refined Laue structure for compound I shows clear structural changes at the peroxide-binding site but no significant changes at the radical site. The photographs were processed with a new software package (LEAP), overcoming many of the former problems encountered in extracting structural information from Laue exposures. CONCLUSIONS: The geometry of the haem environment in this protein allows structural changes to be extremely small, similar in magnitude to those observed for the Fe2+/Fe3+ transition in cytochrome c. The results suggest that these molecules have evolved to transfer electrons with a minimal need for structural adjustment.

Amino Acid Sequence↗
PubMed1994

Laue and monochromatic diffraction studies on catalysis in phosphorylase b crystals.

The conversion of substrate, heptenitol, to product, beta-1-C-methyl, alpha-D-glucose-1-phosphate (heptulose-2-P), in crystals of glycogen phosphorylase b has been studied by Laue and monochromatic diffraction methods. The phosphorolysis reaction in the crystal was started following liberation of phosphate from a caged phosphate compound, 3,5-dinitrophenyl phosphate (DNPP). The photolysis of DNPP, stimulated by flashes from a xenon flash lamp, was monitored in the crystal with a diode array spectrophotometer. In the Laue diffraction experiments, data to 2.8 A resolution were collected and the first time shot was obtained at 3 min from the start of reaction, and data collection comprised three 800-ms exposures. Careful data processing of Laue photographs for the large enzyme resulted in electron density maps of almost comparable quality to those produced by monochromatic methods. The difference maps obtained from the Laue measurements showed that very little catalysis had occurred 3 min and 1 h after release of phosphate, and a distinct peak consistent with the position expected for phosphate, in the attacking position was observed. Data collection times with monochromatic crystallographic methods on a home source took 16 h for data to 2.3 A resolution. Sufficient phosphate was released from the caged phosphate in the crystal from 5 flashes with a xenon flashlamp within 1 min for the reaction to go to completion within the time scale of the monochromatic data collection procedures. The heptulose-2-P product complex has been refined and the model agrees with that obtained previously with the major difference that the interchange of an aspartic acid (Asp 283) by an arginine (Arg 569) was not observed at the catalytic site. This change is part of the activation process of glycogen phosphorylase and may not have taken place in the current experiments because the caged compound binds weakly at the inhibitor site, restricting conformational change, and because activators of the enzymic reaction were not present in the crystal. In experiments with monochromatic radiation in which low phosphate concentrations were generated either by fewer photons or by diffusion of known phosphate concentrations, mixtures of substrate and product were observed. It was not possible through crystallographic refinement at 2.3 A resolution to establish the fractional occupancies of the enzyme-substrate and enzyme-product complexes, but the results did indicate that the reaction was proceeding slowly, consistent with approximate calculations for the likely rate of the reaction in the crystal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗