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K Nakayama

Publications and source records attributed to K Nakayama.

At least 19 recordsLinked to original sources
PubMed1992

Proteolytic cleavages of proalbumin and complement Pro-C3 in vitro by a truncated soluble form of furin, a mammalian homologue of the yeast Kex2 protease.

We have recently purified and characterized a truncated soluble form of furin from which the predicted transmembrane domain and cytoplasmic tail were deleted (Hatsuzawa, K., Nagahama, M., Takahashi, S., Takada, K., Murakami, K., and Nakayama, K. (1992) J. Biol. Chem. 267, 16094-16099). Our results showed that furin resembles the yeast Kex2 protease with respect to both its enzymic properties and substrate specificity. Here we demonstrate that the soluble form of furin is capable of converting the precursors of albumin and the third component of complement (proalbumin and pro-C3, respectively) in vitro to mature proteins. Thus furin mimics the Ca(2+)-dependent proalbumin and pro-C3 convertases found in the Golgi membranes (Brennan, S. O., and Peach, R. J. (1988) FEBS Lett. 229, 167-170; Oda, K. (1992) J. Biol. Chem. 267, 17465-17471). Furthermore we show that the variant alpha 1-antitrypsin Pittsburgh, which is a specific inhibitor of the Golgi proalbumin convertase, inhibits not only the Golgi pro-C3 convertase, but also the soluble furin. These results suggest a role for furin in the cleavage of proproteins transported via the constitutive pathway.

Animals↗
PubMed1992

Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non-endocrine cell line, COS-7.

The amino acid sequence, Arg-4-X-3-Lys/Arg-2-Arg-1 decreases X+1, is thought to be a consensus processing site for a constitutive secretory pathway in non-endocrine cells. We created a mutant proinsulin DNA with a peptide structure of B chain-Arg-Arg-Lys-Arg-C peptide-Arg-Arg-Lys-Arg-A chain, which compares to the native proinsulin structure of B chain-Arg-Arg-C peptide-Lys-Arg-A chain. When the mutant insulin was expressed in a monkey kidney-derived cell line, COS-7, approximately 60% of the total immunoreactive insulin appeared as mature insulin in the culture medium. This conversion to the mature form was strikingly facilitated by co-expressing the mutant proinsulin with furin, a homologue of the yeast endoprotease, Kex2.

3-O-Methylglucose↗
PubMed1992

Cellular and peptide requirements for in vitro clonal deletion of immature thymocytes.

Thymocytes from DO10 T-cell-receptor transgenic mice undergo apoptosis, or programmed cell death, when chicken ovalbumin-(323-339) peptide is administered in vivo. Using DO10 mice thymocytes, we have now developed a simple in vitro model system that recapitulates the in vivo clonal-deletion process. When transgenic thymocytes were cocultured with fibroblasts, B cells, or thymic nurse cell lines (all bearing I-Ad) in the presence of chicken ovalbumin-(323-339), deletion of the transgenic TCR+CD4+CD8+ thymocytes was seen within 8-20 hr. Thymocytes designed to bear I-Ad on their surface could mediate the deletion themselves. Thus, thymocyte clonal deletion entirely depends on the stage at which the thymocytes are vulnerable to the onset of apoptosis, rather than on the nature of the peptide antigen-presenting cells. Furthermore, thymic nurse cell line TNC-R3.1 could cause deletion, strongly suggesting that some thymic epithelial/stromal components are potentially capable of participating in negative selection. In all cases examined, little deletion could be induced at a peptide concentration less than 10 nM, thus defining the minimum amount of peptide antigen required for negative selection. The peptide-dependent in vitro negative-selection system will allow further dissection of the molecular and cellular processes involved in clonal deletion due to apoptosis in the thymus.

Amino Acid Sequence↗
PubMed1992

Surfaces versus features in visual search.

Often implicit in the interpretation of visual search tasks is the assumption that the detection of targets is determined by the feature-coding properties of low-level visual processing. But higher level processes have also been implicated as visual search ability is enhanced in a depth plane or when two-dimensional shapes are interpreted as three-dimensional forms. Here we manipulate binocular disparity to degrade visual search, so that otherwise identical features become parts of surfaces through perceptual completion, rendering them less clearly distinguishable as targets and distractors. Our results indicate that visual search has little or no access to the processing level of feature extraction but must have as an input a higher level process of surface representation.

Humans↗
PubMed1992

Experiencing and perceiving visual surfaces.

A theoretical framework is proposed to understand binocular visual surface perception based on the idea of a mobile observer sampling images from random vantage points in space. Application of the generic sampling principle indicates that the visual system acts as if it were viewing surface layouts from generic not accidental vantage points. Through the observer's experience of optical sampling, which can be characterized geometrically, the visual system makes associative connections between images and surfaces, passively internalizing the conditional probabilities of image sampling from surfaces. This in turn enables the visual system to determine which surface a given image most strongly indicates. Thus, visual surface perception can be considered as inverse ecological optics based on learning through ecological optics. As such, it is formally equivalent to a degenerate form of Bayesian inference where prior probabilities are neglected.

Depth Perception↗
PubMed1992

Purification and characterization of furin, a Kex2-like processing endoprotease, produced in Chinese hamster ovary cells.

Furin, a mammalian homolog of the yeast Kex2 protease, is associated with Golgi membranes and is involved in cleavage of precursor proteins at sites marked by the Arg-X-Lys/Arg-Arg (RXK/RR) motif. We have recently shown that a furin mutant lacking the transmembrane domain can be secreted from cDNA-transfected cells with proteolytic activity for the fluorogenic peptide t-butoxycarbonyl-Arg-Val-Arg-Arg-4-methylcoumarin-7- amide. In this study, we purified and characterized the recombinant furin from the conditioned medium of these cells. Furin was purified as a mixture of 83- and 81-kDa forms and a 96-kDa form. The differences in molecular mass were not due to differences in molecular mass were not due to differences in glycosylation. Moreover, all forms had the same NH2-terminal sequence beginning at the residue after the Arg-Ala-Lys-Arg sequence. These data suggest that the three different forms may be produced by differential COOH-terminal processing of a furin molecule and that mature furin may be autocatalytically produced. Both enzyme preparations showed a pH optimum at 7.0, required Ca2+ for the activity, and showed essentially the same inhibitor profile. These properties resembled those of the Kex2 protease. Both preparations efficiently cleaved fluorogenic peptides with an RXK/RR sequence and moderately cleaved a peptide with an RXXR sequence, but did not cleave dibasic peptides. The sequence requirements determined in vitro were compatible with those determined by expression studies in cultured cells. These data unequivocally demonstrate that furin is an endogenous cellular protease responsible for cleavage of precursor proteins mainly at RXK/RR sites.

Amino Acid Sequence↗
PubMed1992

Consensus sequence for precursor processing at mono-arginyl sites. Evidence for the involvement of a Kex2-like endoprotease in precursor cleavages at both dibasic and mono-arginyl sites.

Many peptide hormones and neuropeptides are produced from larger, inactive precursors through endoproteolysis at sites usually marked by paired basic residues (primarily Lys-Arg and Arg-Arg), or occasionally by a monobasic residue (primarily Arg). Based upon data concerning processing of prorenin and its mutants around the native Lys-Arg cleavage site expressed in mouse pituitary AtT-20 cells, we present the following sequence rules that govern mono-arginyl cleavages: (a) a basic residue at the fourth (position -4) or the sixth (position -6) residue upstream of the cleavage site is required, (b) at position -4, Arg is more favorable than Lys, and (c) at position 1, a hydrophobic aliphatic residue is not suitable. These rules are compatible with those proposed by comparison of precursor sequences around mono-arginyl cleavage sites. We also provide evidence that precursor cleavages at mono-arginyl and dibasic sites can be catalyzed by the same Kex2-like processing endoprotease, PC1/PC3.

Amino Acid Sequence↗
PubMed1992

No requirement for p56lck in the antigen-stimulated clonal deletion of thymocytes.

Activation of protein-tyrosine kinases (PTKs) is required for signal transduction during T cell activation, although the pathway used during thymic selection is unknown. An in vitro system was established in which T cell receptor transgenic thymocytes underwent clonal deletion in response to peptide antigen. The effects of two PTK-specific inhibitors, herbimycin A and genistein, on the clonal deletion of immature thymocytes and the activation of mature thymocytes were examined. Clonal deletion occurred while T cell activation was inhibited and when no p56lck activity was evident. Thus, p56lck is not required for the antigen-stimulated step of clonal deletion of immature thymocytes, and negative selection proceeds via a distinct pathway.

Animals↗
PubMed1992

Sequence requirements for precursor cleavage within the constitutive secretory pathway.

We have recently demonstrated that the Arg-X-Lys/Arg-Arg sequence is a signal for precursor cleavage catalyzed by furin, a mammalian homologue of the yeast precursor-processing endoprotease Kex2, within the constitutive secretory pathway. In this study, we further examined sequence requirements for the constitutive precursor cleavage by expression of various prorenin mutants with amino acid substitutions around the native Lys-Arg cleavage site in Chinese hamster ovary cells. The results delineate the following sequence rules that govern the constitutive precursor cleavage. (a) A basic residue (Lys or Arg) at the 4th (position -4) or 6th (position -6) residue upstream of the cleavage site besides basic residues at positions -1 and -2 is necessary. (b) At position -2, a Lys residue is more preferable than Arg. (c) At position -4, an Arg residue is more preferable than Lys. (d) At position 1, a hydrophobic aliphatic amino acid is not suitable.

Amino Acid Sequence↗
PubMed1992

Identification of the fourth member of the mammalian endoprotease family homologous to the yeast Kex2 protease. Its testis-specific expression.

We used the polymerase chain reaction to identify a mouse testis cDNA that represented another member of a growing class of mammalian endoproteases involved in the processing of precursor proteins. This cDNA encoded a 655-residue protein, designated PC4, containing a bacterial subtilisin-like catalytic domain closely related to those of the recently characterized precursor-processing endoproteases, furin, PC1/PC3, PC2, and Kex2. Within this domain, the amino acid sequence of PC4 was 70, 58, 55, and 45% identical with those of mouse furin, mouse PC1/PC3, mouse PC2, and yeast Kex2, respectively. Northern blot analysis indicated that the PC4 mRNA was detectable only in the testes after the 20th day of postnatal development. Moreover, this message was mainly expressed in the round spermatids. These data suggest that PC4 represents a prime candidate for a precursor-processing endoprotease in the testicular germ cells and that its gene expression is regulated during spermatogenesis.

Amino Acid Sequence↗
PubMed1992

Recent duplication of the two human CD8 beta-chain genes.

Isolation of cDNA clones encoding the beta-chain of the human T cell surface glycoprotein CD8 revealed the presence of five distinct forms of cDNA resulting from alternative splicing. In the process of analysis of the gene organization, we found that there exist two recently duplicated genes for CD8 beta. These genes, designated CD8 beta 1 and beta 2, consist of nine and seven exons, respectively. The organization of CD8 beta 1 and beta 2 genes is almost identical except in their 3'-ends. There are nine nucleotide differences between the coding regions of the CD8 beta 1 and beta 2 genes in spite of the extremely high similarity of these genes which extends over the entire genes including introns. Pulse field gel analysis demonstrated that CD8 beta 1 and beta 2 genes are located more than 1.5 Mb apart. It was found that the CD8 beta 1 gene is approximately 25 kb upstream from the CD8 alpha gene in the same transcriptional orientation on chromosome 2. Although both CD8 beta 1 and beta 2 genes appear functional from the nucleotide sequence, the five distinct forms of CD8 beta cDNAs and corresponding mRNAs found in thymus, PBL, and leukemic cell line HPB-ALL are all derived by alternative splicing from CD8 beta 1 transcripts.

Amino Acid Sequence↗
PubMed1992

Deficiency of P62, a putative collagen receptor, in platelets from a patient with defective collagen-induced platelet aggregation.

Recently, we described a platelet antibody against a putative collagen receptor (P62), which was found in a patient with idiopathic thrombocytopenic purpura (ITP) (Blood 69:1712). We now report a deficiency of the P62 receptor in a young man whose platelets showed defective collagen-induced platelet aggregation. He had a mild bleeding tendency and slight thrombocytopenia. The results of coagulation and fibrinolysis studies were normal. The patient's platelets were partially unresponsive to collagen, although aggregation in response to ADP, thrombin, ristocetin, and calcium ionophore (A23187) was almost normal. Adhesion of his platelets to bovine collagen was markedly reduced. Addition of collagen caused no synthesis of thromboxane (TX)B2 in platelet rich plasma (PRP) from this patient. Furthermore, collagen produced no rise of cytosolic free calcium ([Ca2+]i) in fura2-loaded platelets. In contrast, thrombin caused TXB2 formation and an increase of [Ca2+]i in his platelets. These results suggest defective interaction between the platelets and collagen. The IgG from the ITP-patient induced irreversible aggregation in normal PRP, but caused no aggregation of the young man's platelets. Immunoblot studies showed that normal platelets had antigens with a molecular weight of 62 KDa under reducing conditions and of 57 KDa under nonreducing conditions. In contrast, the young man's platelets had no P62 band, although GPIa/IIa and thrombospondin were normally present. These results indicate that impaired collagen-induced aggregation in the patient's platelets was due to a deficiency of P62 and confirm that P62 may play a crucial role as a collagen receptor in platelet activation.

Adenosine Triphosphate↗
PubMed1992

OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharides.

The Saccharomyces cerevisiae och1 mutant shows a deficiency in the mannose outer chain elongation at the non-permissive temperature. We have cloned the OCH1 gene by complementation of temperature sensitive (ts) phenotype for growth. The integrant of OCH1 gene in the yeast chromosome can complement the ts phenotype and shows the same mapping position as that of the och1 mutation, indicating that the cloned gene is the true gene for mutation. The OCH1 gene disruptant is not lethal but ts for cell growth, and lacks mannose outer chains. The OCH1 gene sequence predicts a 55 kDa protein consisting of 480 amino acids. It contains four potential asparagine-linked (N-linked) glycosylation sites and a single transmembrane region near the N-terminus. In vitro translation/translocation analysis revealed that the large C-terminal region of the OCH1 protein is located at the lumenal side of microsomal membranes with some sugar modification, indicating a type II membrane topology. The OCH1 protein was detected in yeast membrane fractions as four forms of 58-66 kDa, which correspond to the size of a glycoprotein containing four N-linked sugar chains the length of which is almost the same or slightly larger than the inner core (Man8GlcNAc2) formed in the endoplasmic reticulum (ER). Finally, the OCH1 gene was found to encode a novel mannosyltransferase which specifically transfers [14C]mannose to the unique acceptor, the core-like oligosaccharide of cell wall mannan accumulated in the och1 disruptant.

Amino Acid Sequence↗
PubMed1992

Isolation of new temperature-sensitive mutants of Saccharomyces cerevisiae deficient in mannose outer chain elongation.

We have isolated two temperature-sensitive Saccharomyces cerevisiae mutants which exhibit a deficiency in mannose outer chain elongation of asparagine-linked oligosaccharide. The size of yeast glycoprotein, secretory form of invertase, of one mutant (och1) was slightly larger than that of the sec18 mutant at the non-permissive temperature, while that of the other mutant (och2) was almost the same as that of the sec18 mutant. Unlike sec mutants, the och mutants were not deficient in secretion of invertase. The och1 mutant showed a 2+:2- cosegregation with regard to the temperature sensitivity and mannose outer chain deficiency, suggesting that a single gene designated as OCH1 is responsible for these two phenotypes. The och1 mutant stopped its growth at the early stage of bud formation and rapidly lost its viability at the non-permissive temperature. The och1 mutation was mapped near the ole1 on the left arm of chromosome VII. The och1 mutant cells accumulated the external invertase containing a large amount of core-like oligosaccharides (Man9-10GlcNAc2) and a small amount of high mannose oligosaccharides (greater than Man50GlcNAc2) at the non-permissive temperature. Production of the active form of human tissue-type plasminogen activator was increased in the och1 mutant compared with the parental strain, suggesting the potential advantage of this mutant for the production of mammalian-type glycoproteins which lack mannose outer chains in yeast.

Asparagine↗
PubMed1992

Aromatic aldehydes and aromatic ketones open ATP-sensitive K+ channels in guinea-pig ventricular myocytes.

Patch-clamp techniques were used to study the effects of three carbonyl compounds, 3,4-dihydroxybenzaldehyde, 2,3-dihydroxybenzaldehyde, and 2,4-dihydroxyacetophenone, on the adenosine-5'-triphosphate(ATP)-sensitive K+ channel current (IK.ATP) in guinea-pig ventricular myocytes. 3,4-Dihydroxybenzaldehyde (0.5-1 mM) shortened the action potential duration, and this effect was inhibited by application of a specific blocker of IK.ATP, glibenclamide. The shortening of the action potential duration was shown to be caused by a time-independent outward current. In the cell-attached patch configuration, all three compounds activated a kind of single-channel current, which showed an inward rectification at positive potentials and which had a linear current/voltage relation at negative potentials, having a conductance of 90 pS. The current reversed at about 0 mV in symmetrical K+ concentrations on both sides of the membrane. In excised patches this current was blocked by internal application of ATP. Thus we identified this channel as IK.ATP. The activation effects of two aromatic aldehydes were stronger than that of the aromatic ketone. The effect of these compounds on IK.ATP was not reduced by addition of cysteine (10 mM). In inside-out patches, 3,4-dihydroxybenzaldehyde increased the activity of IK.ATP, which had been blocked by 0.5 mM MgATP in the presence of 0.5 mM ADP, but the activation effect was variable and much weaker than that in the cell-attached configuration, and was completely eliminated in the absence of ADP.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetophenones↗
PubMed1992

D1 dopamine receptor binding in mood disorders measured by positron emission tomography.

D1 dopamine receptor binding in mood disorders was studied by positron emission tomography (PET) using 11C-SCH23390. Ten patients with bipolar mood disorders and 21 normal controls were studied in the drug-free state. The patients were in euthymic (N = 6), depressed (N = 3) and manic (N = 1) states. Regional radioactivity in the brain was followed for 40 min by PET. A two-compartment model was used to obtain the binding potential (k3/k4) for the striatum and frontal cortex. The binding potentials for the frontal cortex for the patients were significantly lower than those for normal controls, whereas those for striatum were not significantly different. These findings suggest that D1 dopamine receptors in the frontal cortex may be in a different state in patients with bipolar mood disorders.

Adult↗
PubMed1992

Giant negative U waves in a patient with uncontrolled hypertension and severe hypokalemia.

A 66-year-old woman with a long history of hypertension had an electrocardiogram with giant negative U waves in left precordial leads despite hypokalemia. This seems to be the first report of giant negative U waves induced by uncontrolled hypertension with hypokalemia. The occurrence of negative U waves in the presence of profound hypokalemia is an important observation because it masks the electrocardiographic manifestation of hypokalemia.

Aged↗