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Biomedical subjects

K Sorimachi

Publications and source records attributed to K Sorimachi.

At least 37 records · Page 2Linked to original sources
PubMed1996

Polyanion induced preferential multinucleation in macrophages at a low level of TNF-alpha secretion.

When rat bone marrow macrophages were incubated with acetyl lignin (EP3) in the presence of a 10% solution of fetal bovine serum, the macrophages secreted tumor necrosis factor (TNF-alpha) in a dose-dependent manner. This was followed by macrophage multinucleation. EP3 was found to have a significant effect on TNF-alpha secretion at a minimum dose of 5 micrograms/ml and produced no significant further increase at levels above 50 micrograms/ml, while multinucleation was most active at 10 micrograms/ml. However, multinucleation did not occur at higher concentrations of EP3 (50 micrograms/ml and 100 micrograms/ml). Secretion of TNF-alpha was significantly reduced in the absence of fetal bovine serum, whereas multinucleation was very active, starting after 6 h of incubation. At concentrations of 100 micrograms/ml, sulfonyl lignin (LS) and dextran sulfate (DS) only induced low levels of TNF-alpha secretion from macrophages, but induced active multinucleation. The multinucleation induced by addition of LS or DS was inhibited by further addition of EP3. Thus, macrophage multinucleation was most active when a low level of TNF-alpha was secreted from the macrophages.

Animals↗
PubMed1995

Expression in Aspergillus niger of the starch-binding domain of glucoamylase. Comparison with the proteolytically produced starch-binding domain.

Glucoamylase 1 from Aspergillus niger is an economically important enzyme in many industrial processes. It hydrolyses granular starch and comprises two distinct domains, a catalytic and a starch-binding domain (SBD). We have transformed A. niger with an expression vector for the secretion of SBD for physico-chemical studies. This was achieved by introducing into the glucoamylase gene a short sequence encoding an endoproteolytic cleavage recognition site such that free SBD was secreted at yields up to 200 mg/l. Free SBD was also obtained by proteolytic digestion of full-length glucoamylase 1. Electrospray mass spectroscopy was used to determine the carbohydrate content of both SBDs. It revealed that the engineered one is more glycosylated: an average of three mannose residues compared to one for the proteolytically derived SBD. Sequencing results also suggest partial glycosylation for the three Thr residues involved (510, 511, 513). It is probable that the engineered SBD represents the true glycosylation level of the SBD in native glucoamylase. Binding of beta-cyclodextrin to the SBD was investigated. It was found that the stoichiometry and the spectral perturbation of Trp residues were identical for both SBDs, but the engineered SBD bound less strongly to the ligand. At high concentrations of beta-cyclodextrin relative to the estimated Kd values, the maximum absorbance changes were identical. The observed difference at low beta-cyclodextrin levels was probably due to the higher level of glycosylation of the expressed SBD. We conclude that the proteolytically derived and expressed starch binding domains both bind 2 mol beta-cyclodextrin/mol protein, but that the pattern of glycosylation and strength of binding are different.

Aspergillus niger↗
PubMed1995

1H and 15N assignments and secondary structure of the starch-binding domain of glucoamylase from Aspergillus niger.

1H and 15N NMR resonance assignments of the granular starch-binding domain (SBD) of glucoamylase from Aspergillus niger have been made by multi-dimensional homonuclear and heteronuclear NMR techniques. Secondary structure analysis based on chemical shifts, 1H-1H NOEs, coupling constants and backbone amide exchange data indicates the presence of a well-defined beta-sheet structure. This consists of one parallel and five antiparallel pairs of beta-strands forming two beta-sheets. Cis-trans isomerisation of proline residues and O-glycosylation of threonine residues are observed and compared between the proteolytically derived SBD fragment and the recombinant protein. Structural features of the SBD in solution were compared to the X-ray crystal structure of a homologous domain of cyclodextrin glycosyltransferase from Bacillus circulans. There are some differences in the locations of the start and end of beta-strands but overall the two structures are very similar. This study will form the basis for the structure determination of the granular SBD and of its complexes.

Amino Acid Sequence↗
PubMed1995

Secretion of TNF-alpha from macrophages following induction with a lignin derivative.

Macrophages derived from rat bone marrow were treated with macrophage colony stimulating factor (M-CSF) to obtain a sufficient number of cells for the tumor necrosis factor (TNF-alpha) assay. The present study has been designed to investigate whether the production of TNF-alpha, which induces multinucleated giant cell formation, is regulated by polyanions such as lignin derivatives. ELISA for TNF-alpha showed that the polyanion induced TNF-alpha production by macrophages. The secretion of TNF-alpha from the cells reached a maximum at 3-6 h, and then showed a slight decline. Northern blotting of TNF-alpha mRNA showed that the amount of TNF-alpha reached a maximum within 1 h of macrophage culture in the presence of a lignin derivative. On the other hand, TNF-alpha mRNA was undetectable in the control cells. It was concluded that stimuli such as that provided by lignin derivatives increases the amount of TNF-alpha mRNA, which is then followed by translation of TNF-alpha.

Animals↗
PubMed1995

Effect of tumor cell conditioned media on secretion of TNF-alpha by macrophages.

Conditioned medium obtained from rat liver (M cells) induced a greater than 3 fold increase in the proliferation of rat bone marrow macrophages. Non-specific esterase staining of the proliferated cells gave a positive result. The conditioned media obtained from 8 other established cell lines and 2 primary human cell cultures were also examined for their effects on cultured macrophages. All of the conditioned media tested increased macrophage proliferation. In addition, the conditioned media were examined to determine whether they induced TNF-alpha production in macrophages. TNF-alpha was not detected in the culture medium of the macrophages in the absence of conditioned medium derived from other cell cultures, but TNF-alpha production could be induced by the addition of these conditioned media. Conditioned media obtained from the cells of different species or tumor cell lines were more effective in inducing TNF-alpha production than those obtained from rat cell lines or normal tissue cell lines.

Animals↗
PubMed1994

Structure determination of extracellular fragments of amyloid proteins involved in Alzheimer's disease and Dutch-type hereditary cerebral haemorrhage with amyloidosis.

Amyloid deposition is a biochemical and histopathologic hallmark of various clinical forms of amyloidoses including Alzheimer's disease and the Dutch-type hereditary cerebral haemorrhage with amyloidosis. The self-aggregating peptides responsible for these irreversible deposits have been sequenced but the mechanisms involved in the aggregation processes are not well understood. In order to gain an understanding of the possible structures prior to self-association, the extracellular fragment of the Alzheimer amyloid protein (beta A4) responsible for the deposits (the 'native' fragment) and a mutant of this with a single residue substitution (which is responsible for deposits in the Dutch-type amyloidosis) were examined by 1H-NMR spectroscopy. Interproton distance constraints were derived from NMR experimental data and incorporated into tertiary structure calculations using a simulated annealing protocol. Solution conformations of the fragment peptides associated with the two forms of amyloidoses are presented and compared. Although in both peptides the existence of a mixture of conformations in equilibrium is likely, one such population of structures possesses a flexible N-terminus and a well defined C-terminal region. The latter region includes a helical segment and a terminal turn-like structure. These structural features may be important for the basis of amyloid formation. Comparison of the calculated structures of the two peptides revealed a conformationally different region arising from the conservative substitution of Gln22 for Glu22. This region may be responsible for altered binding in the mutant peptide, giving rise to the clinically different form of amyloidosis.

Alzheimer Disease↗
PubMed1993

Reversed-phase high-performance liquid chromatographic analysis of hydroxyproline and proline from collagen by derivatization with dabsyl chloride.

A high-performance liquid chromatographic method for the analysis of hydroxyproline and proline has been developed. The method is based on the derivatization of the secondary amino group with dabsyl-chloride after blocking of the primary amino group with o-phthalaldehyde. Dabsyl-hydroxyproline and dabsyl-proline were separated from other amino acids by high-performance liquid chromatography in the gradient elution mode, and eluted at 10.27 and 16.02 min, respectively. The correlations between the peak areas of dabsyl-hydroxyproline and dabsyl-proline were linear in the range from 20-200 pmol, with equations y = 1.10x - 0.80 (r = 0.999) and y = 1.12x - 0.52 (r = 0.999), respectively. The method was applied to the analysis of rat tail collagen, and the contents of hydroxyproline and proline were 1.55 +/- 0.04 and 2.03 +/- 0.04 nmol/micrograms, respectively.

Animals↗
PubMed1992

Differential responses of lignin derivatives between tumor and normal tissue derived cell lines; effects on cellular adhesion and cell growth.

When cell lines derived from tumor tissues were plated simultaneously with the lignin derivative or dextran sulfate in plastic culture dishes, the cells did not completely adhere to dishes and/or the shape of even the adhering cells changed significantly. On the other hand, using cell lines derived from normal tissues, no significant effect of the polyanions was observed. The present study revealed that the lignin derivatives demonstrated differential responses between cells derived from tumor tissues and from normal tissues, and that the lignin derivatives strongly inhibited the growth of mouse sarcoma cells (FRUKTO) and rat foetal cells (Ad12-3Y1-Z19) transformed with adeno virus type 12.

Animals↗
PubMed1992

Inhibition of fibroblast growth by polyanions; effects of dextran sulfate and lignin derivatives.

When macrophages prepared from rat bone marrow were cultured for more than one week, contaminating fibroblasts grew and formed large colonies which were observable with the naked eye after Giemsa staining. The addition of dextran sulfate or water-soluble lignin derivatives (acetyl or sulfonyl) to the culture medium almost completely inhibited the growth of fibroblasts at 10 micrograms/ml. These polyanions also inhibited the growth of chick embryo fibroblasts in a dose-dependent manner: at 5 micrograms/ml the inhibitory effect was 20-30% of the control and at 100 micrograms/ml it was 50-70%. In addition, these polyanions inhibited the mitosis of hepatocytes, although their inhibitory effects on the growth of hepatocytes were smaller than those observed on the growth of fibroblasts. On the other hand, these polyanions did not affect the growth of human urinary bladder carcinoma cells (HUB-4 and HUB-15). In conclusion, dextran sulfate and lignin derivatives may be useful in eliminating fibroblasts from certain cells.

Animals↗
PubMed1992

Activation of apoalkaline phosphatase by serum albumin with Zn2+ in rat hepatoma cells.

Reuber rat hepatoma cells (R-Y121B) cultured at 0.5% serum accumulated apoalkaline phosphatase in intact cells. When R-Y121B cells were cultured in the presence of bovine serum albumin, alkaline phosphatase activity increased in the cells, and the associated increase in enzyme activity differed amongst bovine serum albumin preparations. The treatment of bovine serum albumin with activated charcoal not only enhanced the effect of serum albumin on alkaline phosphatase activity, but also cancelled the differences due to different preparations of serum albumin. In contrast, no effect from serum albumin was observed in the increase of alkaline phosphatase activity in R-Y121B cell homogenates incubated at 37 degrees C. The activated-charcoal treatment of bovine serum albumin increased the amount of Zn2+ bound to the protein. When R-Y121B cells were cultured with bovine serum albumin, the concentration of Zn2+ in the cytosol fraction slightly increased. However, the effect of serum albumin on Zn2+ concentration in the cytosol fractions was independent of charcoal treatment. It was concluded that serum albumin with Zn2+ induces the activation of apoalkaline phosphatase due to Zn2+ binding.

Alkaline Phosphatase↗
PubMed1990

Multinucleation of macrophages with water-solubilized lignin derivatives in vitro.

When macrophages derived from bone marrow were incubated with lignin derivative at 10 micrograms/ml, they formed multinucleated giant cells containing more than sixty nuclei. Cine-microscopic observation revealed that the formation of the giant cells was due to cell fusion. This phenomenon was specific for primary-cultured macrophages, and was not observed with either a macrophage cell line or established cell lines derived from other tissues.

Animals↗
PubMed1990

Identification of a beta-turn in the tertiary structure of a peptide fragment of the Alzheimer amyloid protein.

In an attempt to understand the three-dimensional structure of the Alzheimer amyloid protein, a fragment decapeptide was studied by nuclear magnetic resonance spectroscopy. Using two-dimensional techniques, protons close in space were identified and their interproton distances determined. These distances were then used as input data for distance geometry calculations. The resulting three-dimensional structures suggest the presence of a beta-turn in the C-terminus of the decapeptide.

Amino Acid Sequence↗
PubMed1989

Inhibitory effect of dibutyryl cyclic adenosine monophosphate on the induction of alkaline phosphatase in human fetal liver cell line.

When human fetal liver cells (HuL-1-317), cultured continuously in a serum-free medium, were incubated with a combination of prednisolone, butyrate and a hypertonic concentration of NaCl at 37 degrees C, alkaline phosphatase activity increased. However, the addition of dibutyryl adenosine cyclic monophosphate (Bt2cAMP) to these agents inhibited the increase in alkaline phosphatase activity in a dose-dependent manner: the inhibitory effect of Bt2cAMP was significant at 0.05 mM, but disappeared at 0.01 mM. Both cycloheximide and actinomycin D inhibited the increase in alkaline phosphatase activity with the combination described above. Western blotting showed that this enzyme activity increase was a consequence of greater biosynthesis of enzyme molecules in HuL-1-317 cells, and that Bt2cAMP regulated the synthesis of enzyme molecules. We conclude that the changes in alkaline phosphatase activity under various conditions are based on the changes in the number of enzyme molecules in HuL-1-317 cells.

Alkaline Phosphatase↗