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Biomedical subjects

K Yanagi

Publications and source records attributed to K Yanagi.

At least 19 recordsLinked to original sources
PubMed1992

Structure of a B-DNA decamer with a central T-A step: C-G-A-T-T-A-A-T-C-G.

The X-ray crystal structure analysis of the decamer C-G-A-T-T-A-A-T-C-G has been carried out to a resolution of 1.5 A. The crystals are space group P2(1)2(1)2(1), cell dimensions a = 38.60 A, b = 39.10 A, c = 33.07 A. The structure was solved by molecular replacement and refined with X-PLOR and NUCLSQ. The final R factor for a model with 404 DNA atoms, 108 water molecules and one magnesium hexahydrate cation is 15.7%. The double helix is essentially isostructural with C-G-A-T-C-G-A-T-C-G, with closely similar local helix parameters. The structure of the T-T-A-A center differs from that found in C-G-C-G-T-T-A-A-C-G-C-G in that the minor groove in our decamer is wide at the central T-A step rather than narrow, and the twist angle of the T-A step is small (31.1 degrees) rather than large. Whereas the tetrad model provides a convenient framework for discussing local DNA helix structure, it cannot be the entire story. The articulated helix model of DNA structure proposes that certain sequence regions of DNA show preferential twisting or bending properties, whereas other regions are less capable of deformation, in a manner that may be useful in sequence recognition by drugs and protein. Further crystal structure analyses should help to delineate the precise nature of sequence-dependent articulation in the DNA double helix.

Base Composition↗
PubMed1992

A packed-bed reactor utilizing porous resin enables high density culture of hepatocytes.

To enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes. Cubic PVF resins (2 x 2 x 2 mm, mean pore size: 100, 250 or 500 microns) were used as supporting substrates to immobilize hepatocytes. Two hundred and fifty cubes were packed in a cylindrical column, and 2.6-11.3 x 10(7) hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes. Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed. Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins. The highest density of immobilized hepatocytes attained with PVF resin was 1.2 x 10(7) cells/cm3 PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes.

Alanine Transaminase↗
PubMed1992

Induced CD25 expression in a human B-lymphoma cell line transfected with the Epstein-Barr virus nuclear antigen 2 gene.

Two EBV-negative human B-lymphoma cell lines, BJAB and DG75, were transfected with an Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) gene, which plays a critical role in the EBV-induced immortalization of primary B lymphocytes. Furthermore, DG75 cells were co-transfected with the EBNA-2 gene and a latent membrane protein (LMP) gene. Expression of eight surface antigens on the resultant EBNA-2-expressing cell clones was analyzed by flowcytometry. None of the EBNA-2-expressing cell clones derived from BJAB and DG75 showed a significant increase in the expression of cell surface marker CD23, of which enhancement by EBNA-2 in a different EBV-negative human B cell line, Louckes, was previously reported. Expression of CD25 (IL-2R/Tac) on cell surface, however, was induced in two of six DG75-derived cell clones. One of the two CD25-induced cell clones was expressing EBNA-2 only, and the other was co-expressing EBNA-2 and LMP. The results suggest that EBNA-2 has a potential to up-regulate CD25 independently of CD23 on human B cells.

Antigens, Surface↗
PubMed1992

Use of enzyme-linked immunosorbent assays with chimeric fusion proteins to titrate antibodies against Epstein-Barr virus nuclear antigen 1.

Two new enzyme-linked immunosorbent assays (ELISAs) with chimeric fusion polypeptides for the detection of human antibodies specific to Epstein-Barr virus nuclear antigen 1 (EBNA-1) are described. One is an indirect ELISA with affinity-purified beta-galactosidase-EBNA-1 fusion protein as the antigen. The other is a "sandwich" assay based on the use of anti-beta-galactosidase antibody to capture beta-galactosidase-EBNA-1 fusion proteins in bacterial extracts. A good correlation was shown between antibody titers determined by the ELISA with the EBNA-1 fusion proteins and those determined by a conventional anticomplement immunofluorescence test which is being widely performed with Raji cells for the purpose of research and clinical diagnosis. The advantage of the ELISAs for seroepidemiologic studies on Epstein-Barr virus was demonstrated by sensitive detection of marginal immunoglobulin G antibody to the EBNA-1 domain in serum samples from patients with infectious mononucleosis.

Antibodies, Viral↗
PubMed1992

Comparative sequence analyses of Epstein-Barr virus nuclear antigen-2 and -3B genes of a fresh Epstein-Barr virus-2 isolate.

The nucleotide sequence of Epstein-Barr virus (EBV) nuclear antigen (EBNA)-2 and EBNA-3B regions of an EBV-2 isolate in Japan was analyzed. The deduced amino acid sequences of EBNA-2 and EBNA-3B of the isolate and a prototype EBV-2 strain AG876 were identical, but the number of the repeated sequence in EBNA-3B was variable as that of IR-3 in EBNA-1. The remarkably well-conserved sequence seems to suggest that the structure of the EBNA-2 and EBNA-3B genes of EBV-2 is crucial for their function in virus replication.

Antigens, Viral↗
PubMed1992

[Coronary artery bypass surgery with bilateral internal mammary artery].

Increasing number of coronary artery bypass surgery has been performed utilizing more arterial grafts because of poor long term patency rate of saphenous vein grafts. The risk of bilateral internal mammary artery grafting was studied in two groups of patients who were matched for recognized risk factors such as year of operation, age, gender, extent of coronary artery disease, left ventricular function, completeness of myocardial revascularization, and history of congestive heart failure. The patient groups differed in the fact that they received on internal mammary artery graft or two internal mammary artery grafts. The operative mortality rate was zero in either group. Analysis of operative and postoperative morbidity demonstrated no significant differences except for a slight increase in transfusion requirement, rate of wound infection and use of catecholamine in the group receiving two internal mammary artery grafts (p = 0.1, 0.1 and 0.05, respectively). We conclude at this moment that bilateral internal mammary artery grafting does not increase surgical mortality but increases the use of catecholamine comparing with the ipsilateral internal mammary artery grafting. Since the cases analysed in this study are limited, definitive conclusion should be deferred.

Adult↗
PubMed1992

[Morbidity and mortality of coronary artery bypass surgery in patients 75 years of age or older].

A consecutive series of 30 patients 75 years of age and older who underwent isolated coronary artery bypass graftings during a 6 year period (from 1985 to 1990) was analyzed. This group was compared with a consecutive series of 512 patients under the age of 75 who underwent the same procedure during the same period. The elderly patients had a higher incidence of unstable angina pectoris, left main or triple vessel disease and depression of ejection fraction. There were no deaths in the hospital or within 30 days of operation (0%), but postoperative complication occurred in 26 cases (86.7%) in the elderly patients. Mean postoperative hospital stay was longer in the elderly patients than the younger ones (21.7 +/- 8.7 days, 18.9 +/- 5.9 days, respectively). The factors frequently noted in the elderly cases with major complications were emergency or urgent operation, history of congestive heart failure and diabetes. The factors associated with prolonged postoperative hospital stay in elderly cases were octogenarians, intraoperative blood transfusion, wound complications, perioperative myocardial infarction, pulmonary failure and low cardiac output state. It is concluded that CABG can be performed safely even in elderly patients by the proper postoperative management, in spite of having increased postoperative complications and resulting in a prolonged postoperative hospital stay.

Age Factors↗
PubMed1991

The structure of B-helical C-G-A-T-C-G-A-T-C-G and comparison with C-C-A-A-C-G-T-T-G-G. The effect of base pair reversals.

The crystal structure of the DNA decamer C-G-A-T-C-G-A-T-C-G has been solved to a resolution of 1.5 A, with a final R-factor of 16.1% for 5,107 two-sigma reflections. Crystals are orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 38.93 A, b = 39.63 A, c = 33.30 A, and 10 base pairs/asymmetric unit. The final structure contains 404 DNA atoms, 142 water molecules treated as oxygen atoms, and two Mg(H2O)6(2+) complexes. Decamers stack atop one another to simulate continuous helical columns through the crystal, as with three previously solved monoclinic decamers, but the lateral contacts between columns are quite different in the orthorhombic and monoclinic cells. Narrow and wide regions of the minor groove exhibit a single spine or two ribbons of hydration, respectively, and the minor groove is widest when BII phosphate conformations are opposed diagonally across the groove. Phosphate conformation, in turn, appears to have a base sequence dependence. Twist, rise, cup, and roll are linked as has been observed in the three monoclinic decamers and can be characterized by high or low twist profiles. In all five known decamer crystal structures and eight representative dodecamers, a high twist profile is observed with G-C and G-A steps whereas all other R-R steps are low twist profiles (R = purine). A-T and A-C steps are intermediate in character whereas C-A and C-G exhibit behavior that is strongly influenced by the profiles of the preceding and following steps. When sufficient data are in hand, sequence/structure relationships for all helix parameters probably should be considered in a 4-base pair context. At this stage of limited information the problem is compounded because there are 136 unique 4-base steps x-A-B-y in a double helix as compared with only 10 2-base steps A-B.

Crystallography↗
PubMed1991

Structure of the B-DNA decamer C-C-A-A-C-G-T-T-G-G and comparison with isomorphous decamers C-C-A-A-G-A-T-T-G-G and C-C-A-G-G-C-C-T-G-G.

The crystal structure of the DNA decamer C-C-A-A-C-G-T-T-G-G has been solved to a resolution of 1.4 A, and is compared with the 1.3 A structure of C-C-A-A-G-A-T-T-G-G and the 1.6 A structure of C-C-A-G-G-C-C-T-G-G. All three decamers crystallize isomorphously in space group C2 with five base-pairs per asymmetric unit, and with decamer double helices stacked atop one another along the c axis in a manner that closely approximates a continuous B helix. This efficient stacking probably accounts for the high resolution of the crystal data. Comparison of the three decamers reveals the following. (1) Minor groove width is more variable than heretofore realized. Regions of A.T base-pairs tend to be narrower than average, although two successive A.T base-pairs alone may not be sufficient to produce narrowing. The minor groove is wider in regions where BII phosphate conformations are opposed diagonally across the groove. (2) Narrow regions of minor groove exhibit a zig-zag spine of hydration, as was first seen in C-G-C-G-A-A-T-T-C-G-C-G, whereas wide regions show two ribbons of water molecules down the walls, connecting base edge N or O with sugar O-4' atoms. Regions of intermediate groove width may accommodate neither pattern of hydration well, and may exhibit a less regular pattern of hydration. (3) Base-pair stacking is virtually identical at equivalent positions in the three decamers. The unconnected step from the top of one decamer helix to the bottom of the next helix is a normal helix step in all respects, except for the absence of connecting phosphate groups. (4) BII phosphate conformation require the unstacking of the two bases linked by the phosphate, but do not necessarily follow as an inevitable consequence of unstacking. They have an influence on minor groove width as noted in point (1) above. (5) Sugar ring pseudorotation P and main-chain torsion angle delta show an excellent correlation as given by the equation: delta = 40 degrees cos (P + 144 degrees) + 120 degrees. Although centered around C-2'-endo, the conformations in these B-DNA helices are distributed broadly from C-3'-exo to O-4'-endo, unlike the tighter clustering around C-3'-endo observed in A-DNA oligomer structures.

Base Composition↗
PubMed1991

Analysis of local helix geometry in three B-DNA decamers and eight dodecamers.

Local variations in B-DNA helix structure are compared among three decamers and eight dodecamers, which contain examples of all ten base-pair step types. All pairwise combinations of helix parameters are compared by linear regression analysis, in a search for internal relationships as well as correlations with base sequence. The primary conclusions are: (1) Three-center hydrogen bonds between base-pairs occur frequently in the major groove at C-C, C-A, A-A and A-C steps, but are less convincing at C-C and C-T steps in the minor groove. The requirements for large base-pair propeller are (1) that the base-pair should be A.T rather than G.C, and (2) that it be involved in a major groove three-center hydrogen bond with the following base-pair. Either condition alone is insufficient. Hence, a large propeller is expected at the leading base-pair of A-A and A-C steps, but not at A-T, T-A, C-A or C-C steps. (2) A systematic and quantitative linkage exists between helix variables twist, rise, cup and roll, of such strength that the rise between base-pairs can hardly be described as an independent variable at all. Two typical patterns of behavior are observed at steps from one base-pair to the next: high twist profile (HTP), characterized by high twist, low rise, positive cup and negative roll, and low twist profile (LTP), marked by low twist, high rise; negative cup and positive roll. Examples of HTP are steps G-C, G-A and Y-C-A-R, where Y is pyrimidine and R is purine. Examples of LTP steps are C-G, G-G, A-G and C-A steps other than Y-C-A-R. (3) The minor groove is especially narrow across the two base-pairs of the following steps: A-T, T-A, A-A and G-A. (4) In general, base step geometry cannot be correlated solely with the bases that define the step in question; the two flanking steps also must be taken into account. Hence, local helix structure must be studied in the context, not of two base-pairs: A-B, but of four: x-A-B-y. Calladine's rules, although too simple in detail, were correct in defining the length of sequence over which a given perturbation is expressed. Whereas ten different two-base steps are possible, allowing for the identity of complementary sequences, there are 136 different four-base steps. Only 33 of these 136 four-base steps are represented in the decamer and dodecamer structures solved to date, and hence it is premature to try to set up detailed structural algorithms. (5) The sugar-phosphate backbone chains of B-DNA place strong limits on sequence-induced structural variation, damping down most variables within four or five base-pairs, and preventing purine-purine anti-anti mismatches from causing bulges in the double helix. Hence, although short-range sequence-induced deformations (or deformability) are observed, long-range deformations propagated down the helix are not to be expected.

Base Composition↗
PubMed1991

The domain of Epstein-Barr virus nuclear antigen 1 essential for binding to oriP region has a sequence fitted for the hypothetical basic-helix-loop-helix structure.

The domain of Epstein-Barr virus nuclear antigen 1 (EBNA-1) which is essential for binding to a region containing oriP, an episomal replication origin of EBV DNA, was analyzed by DNA binding assay with beta-galactosidase-EBNA-1 fusion proteins. It was revealed that a 159-amino acid (aa) domain, 460-618 aa, of EBNA-1 retained the oriP-binding activity and the domain's activity was abolished by a deletion of 29 aa from its amino-terminal end and by a 38 aa deletion from its carboxyl-end as well. One of five monoclonal antibodies against EBNA-1 specifically inhibited the binding of the beta-galactosidase-EBNA-1 fusion protein to the oriP region. The epitope recognized by the monoclonal antibody was mapped in the crucial 29 aa region. An analysis of the domain's putative secondary structure and a computer search of amino acid sequence homology indicated that the 159-aa domain contains the hypothetical basic-helix-loop-helix structure which is considered to be a common characteristic structure of a family of DNA binding proteins. Examinations of DNA binding activity of the other EBNA polypeptides with a series of fusion proteins and similar structural analyses of their amino acid sequences were also performed. This study suggests that EBNA-1 is a constituent of the family of DNA binding proteins which are involved in transcriptional regulation critical for cell differentiation or cell-type determination.

Amino Acid Sequence↗
PubMed1991

Analysis of antibody titers to Epstein-Barr virus nuclear antigens in sera of patients with Sjögren's syndrome and with rheumatoid arthritis.

To examine if Epstein-Barr virus (EBV) infection is associated with the two autoimmune diseases, Sjögren's syndrome and rheumatoid arthritis, IgG antibody titers of sera from patients with the disorders were evaluated for five constituents of EBV nuclear antigens (EBNA-1, -2, -3, -4, and -6). To circumvent interference by autoantibodies in the sera, fusion proteins synthesized in Escherichia coli were used as specific antigens. By ELISA the average IgG antibody titers to domains of the five EBNAs, especially the amino-terminal domain of EBNA-2, in sera of patients with Sjögren's syndrome were slightly higher than those in normal sera. The tendency of sera from Sjögren's syndrome patients to have higher reactivities to the EBNA domains was also observed by immunoblotting. By comparison, few heightened serologic responses to EBNAs were observed in sera from patients with rheumatoid arthritis. Further analyses are required to determine if EBV is associated with Sjögren's syndrome in any way.

Antibodies, Viral↗
PubMed1991

Goblet cell carcinoid of the vermiform appendix with ovarian metastasis mimicking mucinous cystadenocarcinoma.

We describe a case of goblet cell carcinoid of the vermiform appendix, which metastasized to the bilateral ovaries, uterus, vagina and peritoneum three years and four months after appendectomy. The appendiceal tumor showed transmural infiltration of carcinoembryonic antigen-positive goblet cell-type tumor cells, intermingled with a small number of argyrophilic cells immunoreactive for serotonin and chromogranin A. The presence of neurosecretory-type granules was confirmed ultrastructurally in some mucin-negative tumor cells. The pattern of proliferation was typical of carcinoid tumor originating from the lower-most part of the mucosa, and showed lymphatic permeation. The metastatic lesions in the pelvic organs showed either diffuse infiltration of goblet cell-type tumor cells or mucinous cystadenocarcinoma-like features, the latter being evident in the right ovary. No neuroendocrine component was identified in the metastatic deposits. The kinship of appendiceal goblet cell carcinoid to adenocarcinoma is discussed.

Adult↗
PubMed1991

Extracellular production of mouse interferon beta by the Bacillus subtilis alpha-amylase secretion vectors: antiviral activity and deduced NH2-terminal amino acid sequences of the secreted proteins.

Using two Bacillus subtilis alpha-amylase secretion vectors, mouse interferon-beta (IFN-beta) cDNA was expressed. Ten kinds of mature and hybrid forms of biologically active mouse IFN-beta proteins were synthesized in B. subtilis and were secreted into the culture medium. Similar amounts of proteins, which were determined by immunoblot analysis, were secreted from the B. subtilis transformants, while the antiviral activity in the medium was markedly different in each one of them. The highest antiviral activity was observed in a hybrid protein, in which eleven amino acids consisting of the prosequence of alpha-amylase (eight amino acids) and three amino acids encoding in the HindIII linker DNA were assumed to be extended in front of the Leu residue at position 6 of the mature form of mouse IFN-beta. Its activity was higher than that of the mature form of the mouse IFN-beta secreted from a B. subtilis transformant. These results indicate that the amino acid sequence around the NH2-terminal region of the mouse IFN-beta is closely related to the antiviral activity, but the NH2-terminal amino acid is not. The hybrid proteins of mouse IFN-beta show no interferon activity against a human cell line, HEL.

Amino Acid Sequence↗