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Biomedical subjects

K Akasaka

Publications and source records attributed to K Akasaka.

At least 109 records · Page 6Linked to original sources
PubMed1994

Genomic organization of a gene encoding the spicule matrix protein SM30 in the sea urchin Strongylocentrotus purpuratus.

We report the characterization of a genomic clone containing portions of two tandemly arranged genes that encode a spicule matrix protein, SM30, of the sea urchin Strongylocentrotus purpuratus. The isolated 18.4-kilo-base genomic clone contains the complete genomic sequence of one SM30 gene, designated SM30-alpha, and a portion of another SM30 gene, designated SM30-beta. Southern blot analysis shows that SM30 protein is encoded by a small gene family of two to four members. RNase protection assays indicate that the SM30-alpha gene is expressed at the time of spicule formation in the sea urchin embryo. In addition, mapping of SM30-alpha shows that a large single intron interrupts the coding sequence. Comparison of the nucleic acid and amino acid sequences of the SM30-alpha genomic sequence and the previously isolated SM30 cDNA reveals them to be very similar, but not identical. We also demonstrate that 2.6 kilobases of upstream sequence of SM30-alpha are sufficient to direct primary mesenchyme cell-specific expression of a reporter gene construct.

Amino Acid Sequence↗
PubMed1994

Plasma peroxidized low-density lipoprotein with hydroperoxidized cholesteryl linoleates estimated in patients with familial hypercholesterolemia.

A study was conducted to ascertain the presence of peroxidized low-density lipoprotein (LDL) in plasma and to determine the chemical structure of the peroxidized LDL so that the mechanism by which vascular complications develop may be elucidated in patients with familial hypercholesterolemia (FH). Family trees showing hypercholesterolemia and moderate grade thickness of the Achilles' tendon were confirmed in all patients. Cholesteryl ester, triglycerides (TG), free fatty acid (FFA), free cholesterol (FC), and phospholipids in normal LDL were stained on a thin-layer chromatography (TLC) plate, but from peroxidized LDL, spot X1 between TG and FFA, and spot X2 between FFA and FC were identified clearly except for the lipids recognized in normal LDL on the TLC plates. From intermediate-density lipoprotein (IDL), LDL1, and LDL2 of FH patients, spot X1 was clearly recognized. Hydroperoxidized cholesteryl linoleate (HPO-CL) was estimated and identified from Cu(2+)-treated standard CL, Cu(2+)-treated LDL and LDL obtained from FH patients by means of high-pressure liquid chromatography, mass spectrometry, nuclear magnetic resonance, positive reaction of p-methoxydiphenylpyrenyl-phosphine and various chemical reactions. The percentage of HPO-CL to total lipids in LDL was in the order of IDL < LDL1 < LDL2 in FH patients, and it was markedly higher in FH patients when compared to healthy persons. Spot X1 obtained from Cu(2+)-treated CL, from Cu(2+)-treated normal LDL and from LDL of FH patients was chemically identical, consisting of at least 4 kinds of HPO-CL isomer. LDL2-cholesterol levels were higher in FH patients than those of LDL1-cholesterol, whereas in healthy persons LDL1 cholesterol levels were higher compared to those of LDL2 cholesterol. The capacity of LDL2 for peroxidization by Cu2+ was greater than that of LDL1 in healthy persons. Biological functions, such as acceleration of platelet aggregation, great internalization into macrophages and injuries of the arterial endothelia, were reported for LDL with HPO-CL in our previous experiments. Thus, the existence of peroxidized LDL with HPO-CL may be one of the important risks for vascular complications in FH patients.

Adult↗
PubMed1994

Application of pharmacokinetic studies to a novel antidepressant, E2011.

1. The original drug tested here, (5R)-3-[2-(3-cyanopropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxaz olidinone (ER-4539), exhibited strong MAO-A inhibitory activity in vitro, but its bioavailability in rat was very low. After ER-4539 was administered orally to dog, a metabolite was found in plasma. 2. The metabolite was isolated by hplc after incubation with dog liver microsomal preparations. Its structure, determined by ms and nmr analysis, was alpha-hydroxy-ER-4539. The configuration of the alpha-hydroxy metabolite was (S), determined in comparison with the authentic sample of (R) and (S) by hplc. The isolated metabolite had potent MAO-A inhibitory action in vitro, indicating that it would have antidepressant action. 3. (5R)-3-[2-((1S)-3-Cyano-1-hydroxypropyl)benzothiazol-6-yl]-5- methoxymethyl-2-oxazolidinone (E2011), the synthesized metabolite, has been improved in regard to biopharmaceutical characteristics in rat and dog.

Animals↗
PubMed1994

A long polypyrimidine:polypurine sequence in 5' flanking region of arylsulfatase gene of sea urchin embryo.

Sea urchin (Hemicentrotus pulcherrimus) arylsulfatase(Ars) gene contains a long (622 bp) polypyrimidine:polypurine (Pyr-Pur) sequence in its 5' flanking region. The Pyr-Pur sequence inserted into a plasmid was sensitive to S1 nuclease at a low acidic pH (pH 5) when the plasmid was negatively supercoiled. From the distribution pattern of S1 sites in the Pyr-Pur region it is concluded that a (CT)11:(GA)11 tract in this region could adopt an unusual DNA configuration distinct from the usual B-form. Another feature of the Pyr-Pur sequence is that this (CT)11:(GA)11 tract is sandwiched by two oligo(dC):oligo(dG) stretches (G-strings) that are located at almost an equal distance from both ends of the (CT)11:(GA)11 tract. Mobility shift assay and DNase-I footprinting revealed that the gastrula nuclei contain nuclear proteins that interact with two distinct oligo(dG):oligo(dG) tracts (G-strings) in the Pyr-Pur region. The possibility is suggested that G-strings may be related to formation and stabilization of an unusual DNA configuration of a (CT)11:(GA)11 tract.

Animals↗
PubMed1993

Simultaneous determination of hydroperoxides of phosphatidylcholine, cholesterol esters and triacylglycerols by column-switching high-performance liquid chromatography with a post-column detection system.

A method for the simultaneous determination of hydroperoxides of phosphatidylcholines (PC), triacylglycerols (TG) and cholesterol esters (CE) has been developed. A sample was separated into a combined TG and CE hydroperoxides fraction and a PC hydroperoxides fraction on a short silica column. The fractions were introduced into an ODS column and another silica column by a valve-switching device. The PC hydroperoxides were monitored by a post-column detection system with diphenyl-1-pyrenylphosphine, and the TG and CE hydroperoxides were monitored by another switching device. With this system, the hydroperoxides were determined at the picomole level within 32 min. Their detection limits were 2-4 pmol at a signal-to-noise ratio of 3, and the relative standard deviations of the peak areas were 1.6-3.1%. This method was successfully applied to determine lipid hydroperoxides in human plasma.

Cholesterol↗
PubMed1993

Determination of triacylglycerol and cholesterol ester hydroperoxides in human plasma by high-performance liquid chromatography with fluorometric postcolumn detection.

Cholesterol ester (ChE) and triacylglycerol (TG) hydroperoxides in human plasma were determined by high-performance liquid chromatography with postcolumn detection with diphenyl-1-pyrenylphosphine. Human plasma was extracted once with n-hexane. 2,6-Di-tert.-butyl-4-methylphenol and N-stearylcinnamide were added to human plasma before extraction as an antioxidation agent and an internal standard, respectively. The detection limits of both ChE and TG hydroperoxides were 1 pmol. The sample size was minimized to 250 microliters for each run. The recoveries of ChE and TG hydroperoxides from fresh plasma were ca. 90 and 80%, respectively. The relative standard deviations (n = 8) of their values in frozen human plasma were 5.4% (ChE hydroperoxides, 298 nM) and 5.7% (TG hydroperoxides, 267 nM). No TG hydroperoxides and 24.5 +/- 9.6 nM (n = 15) ChE hydroperoxides were detected in fresh human plasma. The relative standard deviation (n = 8) of ChE hydroperoxides values in fresh plasma was 5.8% (27.1 nM).

Cholesterol Esters↗
PubMed1993

Structure-activity relationship of a series of phenylureas linked to 4-phenylimidazole. Novel potent inhibitors of acyl-CoA:cholesterol O-acyltransferase with antiatherosclerotic activity. 2.

In our continuing search to find systemically bioavailable ACAT (acyl-CoA:cholesterol O-acyltransferase) inhibitors with more potent antiatherosclerotic effect than N-[2-(dimethylamino)-6-[3-(5-methyl-4-phenyl-1H-imidazol-1-yl)propoxy] phenyl]-N'-pentylurea (3), a series of phenylureas linked to 4-phenylimidazole were synthesized and evaluated for in vitro inhibitory activity toward both aortic and intestinal ACATs, and for in vivo hypocholesterolemic activity. The structure-activity relationships (SARs) were studied by strategic modification of five regions in the molecule of 3, i.e., by introducing functional groups or exchanging carbon atoms for heteroatoms. The SAR studies allowed us to select optimum substituents in the five regions, as follows. (1) Dimethylamino was convertible into nitro, methyl, ethyl, propyl, isopropyl, and chloro. On the basis of preliminary pharmacokinetic studies, the methyl group in the ortho-position of the phenylurea was selected. (2) Butyl, pentyl, isopentyl, and neopentyl were better substituents in the urea moiety. (3) Propoxy was the optimal moiety in the bridging portion. (4) Proton, methyl, ethyl, isopropyl, hydroxymethyl, and chloro were better substituents at the 5-position of the imidazole moiety. (5) An unsubstituted phenyl ring was selected as the phenyl group of phenylimidazole. The subsequent comparison studies of compounds containing various combinations of the optimum substituents in each region resulted in the selection of two compounds (67, 68) for further pharmacological and toxicological testing. These compounds were orally bioavailable, and possessed potent in vitro aortic ACAT inhibitory activity (IC50 = 0.16 and 0.012 microM, respectively) and in vivo cholesterol lowering effect (46% and 52% at 1 mg/kg po, respectively). In particular, 68 was 10-fold more potent in the in vitro aortic ACAT assay and 5-fold more potent with respect to hypocholesterolemic activity in vivo than 3.

Animals↗
PubMed1993

Identification of Ras farnesyltransferase inhibitors by microbial screening.

A microbial screen using a yeast strain with conditional deficiency in the GPA1 gene was carried out to search for inhibitors of protein farnesyltransferase (PFT). A strain of Streptomyces was found to produce active compounds named UCF1-A, UCF1-B, and UCF1-C. Structural determination of these compounds revealed that UCF1-C is identical to the known antibiotic, manumycin, whereas UCF1-A and UCF1-B are structurally related to manumycin. All three UCF1 compounds suppress the lethality of gpa1 disruption, with UCF1-C exhibiting the strongest activity. UCF1 inhibits yeast as well as rat brain PFT. Fifty percent inhibition of yeast PFT activity is observed with 5 microM UCF1-C. Kinetic analyses of the inhibition suggest that UCF1-C acts as a competitive inhibitor of PFT with respect to farnesyl pyrophosphate, exhibiting a Ki of 1.2 microM, whereas the same compound appears to act as a noncompetitive inhibitor of PFT with respect to the farnesyl acceptor, the Ras protein. UCF1-C shows significant activity to inhibit the growth of Ki-ras-transformed fibrosarcoma, raising the possibility of its use as an antitumor drug.

Alkyl and Aryl Transferases↗
PubMed1993

Interaction of subtilisin BPN' and recombinant Streptomyces subtilisin inhibitors with substituted P1 site residues.

Kinetic analysis was performed on the interaction between subtilisin BPN' and recombinant species of a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), of which the P1 site amino acid residue, Met73, was replaced by site-directed mutagenesis. The inhibitor constant, Ki, was determined from the residual enzyme activity by using a peptide substrate. The rate constant of binding, kon, and the rate constant of dissociation, koff, were determined from a progress curve of the substrate hydrolysis in the presence of the inhibitor by using newly derived equations. A recombinant SSI in which Met73 was replaced by Ile showed an affinity (1/Ki) toward subtilisin BPN' of only about 7% of that of the wild-type SSI, and the kinetic analysis revealed that the increase of koff was responsible for this difference. The affinity of other SSI mutants in which Met73 was replaced by Glu or Asp decreased significantly as pH became increasingly alkaline. The decrease in the affinity of these recombinants was due to the decrease of kon rather than the increase of koff. Stopped-flow studies revealed that the binding reaction was reconcilable with a two-step mechanism, and the kinetic parameters for each step were obtained for the binding of the enzyme and recombinant SSIs.

Bacterial Proteins↗
PubMed1993

Molecular cloning, sequencing, and mapping of the gene encoding protease I and characterization of proteinase and proteinase-defective Escherichia coli mutants.

Clones carrying the gene encoding a proteinase were isolated from Clarke and Carbon's collection, using a chromogenic substrate, N-benzyloxycarbonyl-L-phenylalanine beta-naphthyl ester. The three clones isolated, pLC6-33, pLC13-1, and pLC36-46, shared the same chromosomal DNA region. A 0.9-kb Sau3AI fragment within this region was found to be responsible for the overproduction of the proteinase, and the nucleotide sequence of the region was then determined. The proteinase was purified to homogeneity from the soluble fraction of an overproducing strain possessing the cloned gene. N-terminal amino acid sequencing of the purified protein revealed that the cloned gene is the structural gene for the protein, with the protein being synthesized in precursor form with a signal peptide. On the basis of its molecular mass (20 kDa), periplasmic localization, and substrate specificity, we conclude this protein to be protease I. By using the gene cloned on a plasmid, a deletion mutant was constructed in which the gene was replaced by the kanamycin resistance gene (Kmr) on the chromosome. The Kmr gene was mapped at 11.8 min, the gene order being dnaZ-adk-ush-Kmr-purE, which is consistent with the map position of apeA, the gene encoding protease I in Salmonella typhimurium. Therefore, the gene was named apeA. Deletion of the apeA gene, either with or without deletion of other proteinases (protease IV and aminopeptidase N), did not have any effect on cell growth in the various media tested.

Amino Acid Sequence↗
PubMed1993

[Assay of erythropoietin in serum with short term enzyme linked immunosorbent assay method--the clinical significance, Part 1: Relation to anemia in renal failure and hematological disorders].

With a newly developed enzyme linked immunosorbent assay kit TOYOBO Co. in which 2 anti-EPO monoclonal antibodies were used, we assayed EPO concentration in sera from normal adults, 168 patients with renal failure and 333 patients with hematological disorders. In the patients with renal failure, serum EPO level was normal (52.9%) or reduced (42.9%), and there was no correlation to their hematocrits. However, there was an increment in EPO concentration correlated to their severity of anemia in the most patients with hematological disorders, such as iron deficiency anemia (correlation coefficient r = -0.74), aplastic anemia (r = -0.89), leukemia (r = -0.81), and MDS (r = -0.65). On the other hand, EPO concentration in sera from all the untreated patients with polycythemia vera were significantly low level. But the concentrations of EPO from the patients successfully treated, with normal hematocrit were recovered to normal level. In the patients with secondary polycythemia, there were much varieties in EPO level. Assay of EPO in blood is important not only for diagnosis of polycythemia but also for the analysis of anemia and clinical use of EPO in vivo. The method described here is accurate and technically not complicated, and could be widely induced in most laboratories.

Adolescent↗
PubMed1993

[Assay of erythropoietin in serum with short term enzyme linked immunosorbent assay method--the clinical significance: Part 2--:Relation to serum iron, UIBC and ferritin in renal failure and hematological disorders].

With a newly developed short term enzyme linked immunosorbent assay kit (TOYOBO Co.), in which 2 kinds of anti-EPO monoclonal antibodies were used, we assayed EPO concentration in sera from patients with renal failure and hematological disorders. In this report, the EPO data were analysed in relation to serum iron concentrations, with ferritin and UIBC. In the patients with renal failure, there was no significant correlation between EPO concentration and serum iron, ferritin, nor UIBC concentration. On the other hand, in the patients with hematological disorders, there were two types. One was in patients with iron deficiency anemia, whose serum EPO was negatively correlated to serum iron (r = -0.64) and ferritin (r = -0.59), but positively related to UIBC (r = 0.27). The another was the pattern in patients with aplastic anemia, leukemia and MDS, whose serum EPO positively correlated to iron and ferritin but negatively correlated to UIBC. In the patients with aplastic anemia serum EPO had good correlation to serum iron (r = 0.62), ferritin (r = 0.60) and UIBC (r = -0.46). The relationship of EPO to iron in the patients with leukemia (r = 0.54), and EPO to ferritin in the patients with MDS (r = 0.42) show significantly positive correlation coefficient.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme-Linked Immunosorbent Assay↗
PubMed1993

[Determination of the standard level of serum erythropoietin in relation to hemoglobin concentration].

Serum erythropoietin (EP) concentration was measured by the recombinant EP-based radioimmunoassay and was examined to standardize the hemoglobin (Hb) related level of 144 normal control and 56 patients with iron deficiency anemia and hemolytic anemia excluding paroxysmal nocturnal hemoglobinuria. The standardization was achieved by logarithmic regression of the EP titier on Hb either by the two-phase linear form or by the third degree sigmoid form at a 95% confidence limit for each regression. The third degree regression was found to be preferable from the view point of both statistics and the negative feedback mechanism. The average and scattering of the deviation from the standard level thus determined of the disease groups indicated that the EP level is: (1) 12 fold higher than the standard level in 42 aplastic anemias (the most in excess and a few in standard). (2) three fold higher than that in 27 myelodysplastic syndromes (relatively higher dispersed state). (3) 29% of the standard level in 33 anemias associated with chronic renal failure (deficient state). (4) 105% of the extrapolated standard level in 22 polycythemia veras (standard state). The standardization of Hb-related Ep titer may provide new pathophysiological approaches in a variety of hematopoietic disorders.

Anemia, Hemolytic↗
PubMed1992

Terminal disorder: a common structural feature of the axial proteins of bacterial flagellum?

We report, based on proteolytic experiments and high resolution 1H nuclear magnetic resonance studies that the terminal regions of the monomeric hook protein are highly mobile and exposed to the solvent. The disordered parts of the hook protein span approximately the first 70 and the last 30 amino acid residues. Although the amino acid sequences of flagellin and hook protein do not resemble each other at all, both proteins have now been shown to contain large disordered terminal regions. Sequential similarities of flagellin and hook protein, especially near the NH2 and COOH termini, to other axial components of bacterial flagellum suggest that terminal disorder may be a common structural feature of the axial proteins of the bacterial flagellum.

Amino Acid Sequence↗
PubMed1992

High-performance liquid chromatography and post-column derivatization with diphenyl-1-pyrenylphosphine for fluorimetric determination of triacylglycerol hydroperoxides.

Triacylglycerol monohydroperoxides (TG-mHPO) were selectively detected at the picomole levels after post-column reaction with diphenyl-1-pyrenylphosphine (DPPP). TG-mHPO were separated on two types of reserved-phase columns, an ODS column and a phenylated silica gel column, which were useful for determining TG-mHPO at their molecular species levels and their class levels, respectively. After the separation, DPPP solution was mixed with the eluent followed by reaction in a stainless-steel coil 20 m x 0.5 mm I.D. at 80 degrees C, then the fluorescence intensity of DPPP oxide was measured (lambda ex. 352 nm, lambda em. 380 nm). Using these systems, TG-mHPO were determined in the range 2-1000 pmol. The relative standard deviations were 2.3-2.8%.

Chromatography, High Pressure Liquid↗
PubMed1992

Quantitative evaluation of water content in a solid protein by deuterium NMR.

A method for evaluating absolute water content in a solid protein based on deuterium NMR measurements in solution is described. By dissolving the hydrated solid protein, which has been specifically deuterium-labeled, into deuterium-depleted water and by comparing the deuterium NMR signal intensity of water (1H2HO) with that of the protein, the amount of water contained in the solid protein is evaluated quantitatively. The method requires a heat pretreatment of the protein sample in water of an enriched (e.g. 2%) deuterium composition for complete hydrogen exchange of the labile protons, and hence is applicable to a protein with a reasonably good reversibility of thermal unfolding. By utilizing this method, the absolute content of the bound water in a protein, Streptomyces subtilisin inhibitor (SSI), lyophilized for 8 h was determined to be 9.2%. The extent of hydration of solid SSI during its exposure to a deuterium-enriched water vapor could also be followed from the deuterium NMR signals in solution. In addition, solid state deuterium NMR measurements of SSI suggested that direct measurement of the natural abundance deuterium signal can give a reasonable estimate of the water content in a solid protein.

Bacterial Proteins↗
PubMed1992

Isolation of cDNAs for LCE and HCE, two constituent proteases of the hatching enzyme of Oryzias latipes, and concurrent expression of their mRNAs during development.

The hatching enzyme of medaka consists of two types of proteases (HCE, LCE). cDNA clones for LCE and HCE were isolated from a lambda gt11 cDNA library constructed with poly(A)+ RNA of Day 3 embryos. LCE cDNA is 936 bp long and contains an 813-bp open reading frame encoding a preproenzyme with a 20-amino-acid signal sequence, a 51-amino-acid propeptide, and a 200-amino-acid mature enzyme. For HCE, two distinct cDNAs (HCE21, HCE23) having nucleotide sequences with 92.8% similarity were obtained. These cDNAs contain open reading frames encoding preproenzymes of 279 and 270 amino acids, respectively. The mature enzyme forms of both consist of 200 amino acids, the similarity between them being 95.5%. On Northern blotting analysis, the transcripts of LCE and HCE genes were first detected coincidentally in Day 2 embryos shortly before the production of LCE and HCE, accumulated thereafter in parallel, and dramatically decreased after hatching. The amino acid sequence, the HExxH motif, which is known to constitute an active site in some Zn proteases, is also found in LCE and HCE. However, the sequence analyses strongly suggest that both the enzymes belong to the astacin (protease) family, being distinct from sea urchin hatching enzyme, which is reportedly similar to collagenase.

Amino Acid Sequence↗