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S Osawa

Publications and source records attributed to S Osawa.

At least 73 records · Page 4Linked to original sources
PubMed1995

Altered cAMP levels in retinas from transgenic mice expressing a rhodopsin mutant.

Transgenic mice expressing the rhodopsin mutant Pro347Ser (Serine 6) display retinal degeneration through apoptosis that is characteristic of the disease retinitis pigmentosa. By 5 weeks after birth, these mice have lost approximately 35% of their photoreceptor cells. Retinas from these mice showed higher levels of cAMP compared to the levels in retinas of normal mice. Our studies provide evidence that elevated cAMP is common to the apoptotic process that occurs in retinitis pigmentosa. In addition, in vitro studies demonstrate no differences in the ability of the mutant and the wild-type rhodopsin to activate transducin, the rod cell G protein, to be phosphorylated by rhodopsin kinase or to bind arrestin. Mutants of rhodopsin, including Pro347Ser, are mistargeted to the rod inner segment, raising the possibility that rhodopsin triggers apoptosis through activation of signaling pathways not normally under its control.

Animals↗
PubMed1995

Translation of synonymous codons in family boxes by Mycoplasma capricolum tRNAs with unmodified uridine or adenosine at the first anticodon position.

In Myocoplasma capricolum, codon family boxes except for arginine and threonine (CUN leucine, GUN valine, UCN serine, CCN proline, GCN alanine, and GGN glycine) have only a single tRNA species with the anticodon sequence UNN (N is U, C, A or G), the first nucleoside U being unmodified. Incorporation of the [3H]amino acid into the peptide fraction was examined with the M. capricolum cell-free translation system. Synthetic mRNA containing each of the respective amino acid codons in the coding frame was subjected to translation. The tRNAUNN translated all the family box codons with similar, if not equal, efficiencies. In M. capricolum, there are two species of threonine tRNA, tRNA(UGUThr) and tRNA(AGUThr), the first nucleoside U or A being unmodified. The tRNA(UGUThr) species translates codons ACA, ACG and ACU efficiently, and ACC only poorly. In contrast, the tRNA(AGUThr) species translates codons ACU, ACC and ACG efficiently and ACA poorly.

Adenosine↗
PubMed1995

Mitogen-activated protein kinase activation requires two signal inputs from the human anaphylatoxin C5a receptor.

The anaphylatoxin C5a receptor activates the Ras/Raf/mitogen-activated protein (MAP) kinase pathway in human neutrophils. The signal pathways involved in Ras/Raf/MAP kinase activation in response to C5a and other chemoattractant receptors is poorly understood. Stimulation of the C5a receptor expressed in HEK293 cells results in modest MAP kinase activation, which is inhibited by pertussis toxin-catalyzed ADP-ribosylation of G(i). Coexpression of the C5a receptor and the G16 alpha subunit (alpha 16) results in the G16-mediated activation of phospholipase C beta and a robust MAP kinase activation. Pertussis toxin treatment of C5a receptor/alpha 16-cotransfected cells inhibits C5a stimulation of MAP kinase activity approximately 60% relative to the control response. Similarly, the protein kinase C inhibitor, GF109203X inhibits activation of MAP kinase activation in C5a receptor/alpha 16-cotransfected cells by 60%; the protein kinase C inhibitor does not affect the modest C5a receptor response in the absence of alpha 16 expression. These results demonstrate that two independent signals are required for the maximal activation of MAP kinase by G protein-coupled receptors.

Antigens, CD↗
PubMed1995

Rhodopsin mutants discriminate sites important for the activation of rhodopsin kinase and Gt.

The cytoplasmic loops of rhodopsin, the rod cell photoreceptor, play important regulatory roles in the activation of both rhodopsin kinase and the rod cell G protein, Gt. A number of studies have identified domains in rhodopsin that are important for the activation of Gt. However, less is known concerning the cytoplasmic regions that regulate phosphorylation of the photoreceptor by rhodopsin kinase. To identify regions that participate in these processes, a series of alanine mutations were generated in the three cytoplasmic loops of rhodopsin and transiently expressed in HEK-293 cells. Membranes prepared from these cells were reconstituted with the opsin chromophore, 11-cis-retinal, and characterized for their ability to undergo light-dependent phosphorylation by rhodopsin kinase and to catalyze GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) binding to Gt. We have identified mutants that fall into three distinct categories: 1) those that show altered phosphorylation but normal Gt activation, such as T62A/V63A/Q64A and R147A/F148A/G149A in Loops I and II, respectively; 2) mutants that have reduced ability to activate Gt but are phosphorylated normally, including T242A/T243A and V250A/T251A/R252A in Loop III; and 3) mutants that affect both phosphorylation and Gt activation, including A233G/A234G/A235G and A233N/A234N/A235N in Loop III. The use of these two assays in parallel have allowed us to distinguish the presence of distinct functional domains within the cytoplasmic loops which are specific for interaction with rhodopsin kinase or Gt.

Alanine↗
PubMed1995

On codon reassignment.

Schultz and Yarus (J. Mol. Biol. 235:1377-1380, 1994) have proposed that reassignment of codons in the genetic code passes through a stage in which the codons are ambiguously translated. In contrast we state that such ambiguity would be deleterious, and that, to be reassigned, a codon, together with the tRNA that translates the codon, must first disappear from coding sequences, after which a tRNA appears with a mutated anticodon, and this enables the codon to reappear with a changed meaning. In the case of a stop codon, the relevant release factor must change so as to recognize it.

Codon↗
PubMed1995

[Automated analyzer of enzyme immunoassay].

Automated analyzers for enzyme immunoassay can be classified by several points of view: the kind of labeled antibodies or enzymes, detection methods, the number of tests per unit time, analytical time and speed per run. In practice, it is important for us consider the several points such as detection limits, the number of tests per unit time, analytical range, and precision. Most of the automated analyzers on the market can randomly access and measure samples. I will describe the recent advance of automated analyzers reviewing their labeling antibodies and enzymes, the detection methods, the number of test per unit time and analytical time and speed per test.

Alkaline Phosphatase↗
PubMed1995

Prostatic acid phosphatase assay with self-indicating substrate 2,6-dichloro-4-acetylphenyl phosphate.

We characterized six self-indicating substrates, synthesized as the derivative compounds of acetylphenyl phosphate, for serum prostatic acid phosphatase (PAP) activity. One of the substrates, 2,6-dichloro-4-acetylphenyl phosphate (DCAPP), is superior to others in terms of stability, affinity, and low Km for PAP. The hydrolyzed product, 2,6-dichloro-4-acetylphenol (DCAP), has a maximum absorption at 334.2 nm, a pKa of 4.15, and a molar absorptivity at 340 nm of 21,490 L.mol-1.cm-1 in citrate-HCl buffer, pH 5.4. PAP activity was assessed by subtracting tartaric acid-inhibited acid phosphatase activity from total acid phosphatase activity. Our assay system involving DCAPP is a unique kinetic method that shows good reproducibility, wide analytical dynamic range, and high specificity for PAP. Moreover, it is easily adaptable to automated analyzers because the product, DCAP, can be monitored at 340 nm.

Acid Phosphatase↗
PubMed1994

Effects of carboxyl-terminal truncation on the stability and G protein-coupling activity of bovine rhodopsin.

A number of studies have suggested that G protein-coupled receptors possess domains within the carboxyl terminus that are important for the catalytic activation of G proteins. To define these regions, truncation mutants were generated in the cDNA of bovine rhodopsin, the receptor responsible for visual signal transduction in the retinal rod cell. The mutants were expressed in HEK-293 cells and analyzed for their ability to bind the chromophore, 11-cis-retinal, and for activating Gt, the G protein of the rod cell regulated by rhodopsin. Removal of 38 carboxyl-terminal amino acids resulted in the production of a mutant (K311 stop) that does not bind 11-cis-retinal, has an abnormal pattern of glycosylation, and does not catalyze light-dependent binding of GTP gamma S to Gt, suggesting that it is unable to fold properly during biogenesis. However, a truncation mutant with only five additional amino acids (C316stop) coupled normally to Gt, using membranes from transfected cells, despite the fact that it lacked the "fourth cytoplasmic loop" formed by palmitoylation of cysteines-322 and -323. When C316stop is extracted from the membrane with detergent, only a fraction is able to bind 11-cis-retinal, but the fraction that binds retinal activates Gt normally. In contrast, detergent-solubilized wild-type rhodopsin and K325stop (a truncation mutant with the longest carboxyl terminus) both bind retinal and activate Gt normally. These data suggest that the proximal region of the carboxyl terminus is critical for the proper folding and stability of the rhodopsin molecule and that amino acids Cys316 to Ala348 are not necessary for the activation of Gt.

Amino Acid Sequence↗
PubMed1994

A new precipitation method with magnetic separation for high-density-lipoprotein cholesterol assay.

We describe a new precipitation method for high-density-lipoprotein cholesterol quantitation. The new method uses magnetic force instead of centrifugal force to separate high-density-lipoprotein from other lipoproteins that are fractioned with a precipitating reagent. The reagents used for the new method are the same as those for the conventional method except that magnetizable particles are included in the former. The magnetizable particles are used without any modifications. The correlation between the new and the centrifuge methods with dextran sulfate-magnesium chloride, sodium phosphotungstate-magnesium chloride and polyethylene glycol 6,000 were satisfactory (r = 0.990, 0.997 and 0.997, respectively). The new method, which can be combined with any precipitating reagents used in conventional methods, is very simple to perform and does not need any special equipment.

Chemical Precipitation↗
PubMed1994

The carboxyl terminus of bovine rhodopsin is not required for G protein activation.

Rhodopsin, the photoreceptor of mammalian rod cells, shares regions of structural homology with many G protein-coupled receptors. One of these domains is the "fourth cytoplasmic loop" formed by palmitoylation of two cysteines (Cys-322 and Cys-323) in the carboxyl terminus. Evidence from several laboratories suggests that this domain is important for the activation of the G protein for rhodopsin, Gt, and that it undergoes conformational changes upon exposure to light. Previously we reported that a truncation mutant with only six amino acids remaining at the proximal end of the carboxyl terminus was able to activate Gt, whereas a mutant lacking an additional five amino acids was misfolded and unable to bind retinal. In the present report, these six amino acids were mutated, to define their roles in the formation of a functional photoreceptor and in the activation of Gt. All of the point mutants displayed normal expression, post-translational processing, and Gt activation, suggesting that the fourth cytoplasmic loop in the carboxyl terminus does not play a major role in the activation of G proteins and that the specific amino acid sequence in this domain is not required for the production of a properly folded, functional photoreceptor.

Amino Acid Sequence↗
PubMed1993

Non-universal decoding of the leucine codon CUG in several Candida species.

It has been reported that CUG, a universal leucine codon, is read as serine in an asporogenic yeast, Candida cylindracea. The distribution of this non-universal genetic code in various yeast species was studied using an in vitro translation assay system with a synthetic messenger RNA containing CUG codons in-frame. It was found that CUG is used as a serine codon in six out of the fourteen species examined, while it is used for leucine in the remaining eight. The tRNA species responsible for the translation of codon CUG as serine was detected in all the six species in which CUG is translated as serine. The grouping according to the CUG codon assignments in these yeast species shows a good correlation with physiological classification by the chain lengths of the isoprenoid moiety of ubiquinone and the cell-wall sugar contained in the yeasts. The six Candida species examined in which CUG is used as serine belong to one distinct group in Hemiascomycetes.

Amino Acid Sequence↗
PubMed1993

Geranylgeranylacetone used as an antiulcer agent is a potent inducer of differentiation of various human myeloid leukemia cell lines.

Low concentrations of geranylgeranylacetone (GGA), known as an antiulcer agent (Teprenone), induces differentiation of various human myeloid leukemia cell lines. The cell lines examined in the present study were myeloblastic ML1, histiocytic U937, promyelocytic HL60, and multipotential K562. All of these cell lines were induced to differentiate by 20 microM GGA, as measured by NBT staining. Neither polyprenylacetones, with more or fewer isoprene units than the geranylgeranyl group, nor polyprenylalcohols had no differentiation-inducing activity. GGA used in combination with RA or TNF-alpha increased ML1 cell differentiation. The present results suggest that GGA may be a useful agent in differentiation therapy of leukemia.

Cell Differentiation↗
PubMed1993

Lack of peptide-release activity responding to codon UGA in Mycoplasma capricolum.

In Mycoplasma capricolum, a relative of Gram-positive eubacteria with a high genomic AT-content (75%), codon UGA is assigned to tryptophan instead of termination signal. Thus, in this bacterium the release factor 2 (RF-2), that recognizes UAA and UGA termination codons in eubacteria such as Escherichia coli and Bacillus subtilis, would be either specific to UAA or deleted. To test this, we have constructed a cell-free translation system using synthetic mRNA including codon UAA [mRNA(UAA)], UAG [mRNA(UAG)] and UGA [mRNA(UGA)] in-frame. In the absence of tryptophan, the translation of mRNA(UGA) ceased at UGA sites without appreciable release of the synthesized peptides from the ribosomes, whereas with mRNA(UAA) or mRNA(UAG) the bulk of the peptides was released. Upon addition of the E.coli S-100 fraction or B.subtilis S-100 fraction to the translation system, the synthesized peptides with mRNA(UGA) were almost completely released from the ribosomes, presumably because of the presence of RF-2 active to UGA in the added S-100 fraction. These data suggest that RF-2 is deleted or its activity to UGA is strongly weakened in M.capricolum.

Base Sequence↗
PubMed1993

Unassigned or nonsense codons in Micrococcus luteus.

We previously reported that in Micrococcus luteus, a Gram-positive eubacterium with high genomic G + C content, certain codons ending with A did not appear in coding frames, including termination sites, and tRNAs that translate these codons were not detected. These facts suggest that at least some of them are unassigned (nonsense) codons, i.e. not assigned to any amino acid or to any stop signal. We have investigated whether AGA and AUA, universal Arg and Ile codons, respectively, are really unassigned codons by using a cell-free extract prepared from M. luteus and synthetic messenger RNAs. Translation of synthetic mRNA containing in-frame AGA codons does not result in "read-through" to codons beyond the AGA codons, i.e. translation ceases at codon AGA. Essentially the same result was obtained with mRNA containing AUA in-frame. A sucrose-gradient centrifugation profile of the reaction mixture has shown that practically all of the peptides that have been synthesized are attached to 70 S ribosomes. When in-frame AGA or AUA codons are replaced by UGA codons in mRNA, no read-through occurs beyond UGA, just as in the case of AGA or AUA. However, the synthesized peptide is released from the 70 S ribosomes. These data suggest that AGA and AUA are unassigned codons and differ from UGA in that they are not used for termination.

Adenine Nucleotides↗
PubMed1993

Evolutionary changes in the genetic code.

1. The genetic code was thought to be identical ("universal") in all biological systems until 1981, when it was discovered that the coding system in mammalian mitochondria differed from the universal code in the use of codons AUA, UGA, AGA and AGG. 2. Many other differences have since been discovered, some in mitochondria of various phyla, others in bacteria, ciliated protozoa, algae and yeasts. 3. The original thesis that the code was universal and "frozen" depended on the precept that any mutational change in the code would be lethal, because it would produce widespread alterations in the amino acid sequences of proteins. Such changes would destroy protein function, and hence would be intolerable. 4. The objection was "by-passed" by nature. It is possible for a codon to disappear from mRNA molecules, often as a result of directional mutation pressure in DNA: thus all UGA stop codons can be replaced by UAA. 5. The missing UGA codon can then reappear when some UGG tryptophan codons mutate to UGA. The new UGA codons will be translated as tryptophan, as is the case in non-plant mitochondria and Mycoplasma. Therefore, no changes have taken place in the amino acid sequences of proteins. 6. Variations of this procedure have occurred, affecting various codons, and discoveries are still being made. The findings illustrate the evolutionary interplay between tRNA, release factors and codon-anticodon pairing.

Animals↗