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Biomedical subjects

K Tazaki

Publications and source records attributed to K Tazaki.

At least 19 recordsLinked to original sources

A receptor-like protein kinase with a lectin-like domain from lombardy poplar: gene expression in response to wounding and characterization of phosphorylation activity.

Plant receptor-like protein kinases (RLKs) are thought to be involved in various cellular processes mediated via signal transduction pathways. To clarify the initial step in such a signal transduction pathway in woody plants, we cloned a cDNA encoding PnLPK (a P opulus n igra var. italica lectin-like protein kinase) from lombardy poplar. The C-terminal region of the predicted PnLPK protein includes a protein kinase catalytic domain consisting of the 12 subdomains typical of the eukaryotic protein kinase superfamily. Following the signal peptide at the N-terminus, a domain that shows homology to legume lectins retains the putative Mn(2+)- and Ca(2+)-binding amino acids, which are highly conserved among lectin-related proteins. Because a putative hydrophobic transmembrane domain was localized between the lectin-like domain and the protein kinase domain, PnLPK was determined to be a member of the plant RLK subfamily with a lectin-like domain. Transcripts of the PnLPK gene accumulate in roots, mature leaves and calli of lombardy poplar, whereas only trace amounts of the transcripts are detectable in stems, young leaves and apical buds. Wounding of the young leaves increased the amount of PnLPK mRNA, but none of several phytohormones tested had any effect on the transcription of PnLPK. When incubated in the presence of divalent metal cations such as Mn(2+), the C-terminal catalytic domain of PnLPK showed significantly higher autophosphorylation activity than the full-length PnLPK protein. The phosphorylation activity of PnLPK was also detected using beta-casein as substrate. Phosphoamino acid analysis indicated that PnLPK is a serine/threonine kinase.

Amino Acid Sequence↗

Two patients with severe alcoholic hepatitis accompanied by hypercytokinemia and granulocytic hyperelastasemia, successfully treated by intravenous infusion of urinastarine (Miraclid).

Severe alcoholic hepatitis (SAH) is not simply a disease of the liver, but it also causes infection and multiple organ failure, and therefore carries an extremely poor prognosis. We report the successful treatment of two patients with SAH. Case 1: The patient was a 55-year-old man. He was a heavy drinker whose alcohol intake had increased for some 3 years to 1.8 L sake a day. Slight clouding of consciousness, fever, and jaundice were evident on his admission to our hospital. Laboratory data showed leukocytosis with a predominance of polymorphonuclear leukocytes, and a decline in coagulability. He tested negative for various hepatitis virus markers. With the diagnosis of SAH made, steroid pulse therapy and bilirubin adsorption therapy were administered. The jaundice improved and the interleukin-8 (IL-8) level decreased. Continuous intravenous infusion of urinastarine (Miraclid) normalized the granulocyte elastase level. Improvement was also seen in coagulability, ascites, icterus and consciousness. Case 2: The patient was a 49-year-old man. He was a heavy drinker whose alcohol intake had increased for 1 month. Fever, jaundice, ascites, and mild disturbance of consciousness were evident at the time of admission. Examination on admission diagnosed SAH. Bilirubin adsorption and continuous intravenous infusion of urinastarine were initiated. As a result, circulating IL-8 level was decreased and coagulability was improved. Therapy for granulocytic hyperelastasemia and hypercytokinemia supervening on SAH is a new effective approach on improvement in the disease.

Blood Coagulation↗

Multiple motor patterns in the stomatogastric ganglion of the shrimp Penaeus japonicus.

Motor patterns of the cardiac sac, the gastric and the pyloric network in the stomatogastric nervous system of the shrimp Penaeus japonicus, the most primitive decapod species, were studied. Single neurons can switch from the gastric or the pyloric pattern to the cardiac sac pattern. Some of the pyloric neurons fire with the gastric pattern. All of the gastric neurons fire with the pyloric pattern, unlike those in reptantians. Proctolin activates and modulates the cardiac sac and the pyloric rhythm, and promotes reconfiguration of the networks. Neurons of the three networks have so many interconnections that they construct a multifunctional neural network like those in Cancer. This network may function in different configurations under the appropriate conditions. Several modes of interactions between the networks found in different reptantian species can apply to the penaeidean shrimp. Such interactions are general features of the stomatogastric nervous system in decapods. Phylogenetic differences among the decapod infraorders are seen in the number and orientation of muscles and the innervation pattern of muscles. The multifunctional networks have existed in the most primitive decapod species, and types of configurations of the networks would have evolved to produce a wide range of motor patterns as the foregut structure has become complex.

Action Potentials↗

Studies by site-directed mutagenesis of the carbohydrate-binding properties of a bark lectin from Robinia pseudoacacia.

A bark lectin, RBL, from Robinia pseudoacacia (black locust), binds galactose-related sugars specifically. Recombinant RBL (rRBL) with a histidine tag was expressed in Escherichia coli, purified and characterized. rRBL agglutinated rabbit erythrocytes and the hemagglutination was inhibited by galactose and related sugars. To elucidate the mechanism of the binding of carbohydrate by RBL, 16 mutant rRBLs were produced by site-directed mutagenesis. The analysis of the mutants indicated that residues Phe130 and Asp87 play key roles in the binding of carbohydrate by RBL. When Thu215, Leu217 and Ser218 in the carboxy-terminal region were replaced by alanine, the respective replacements decreased the hemagglutinating activity. However, replacement by alanine of Glu219 did not decrease this activity. Three mutant rRBLs were generated by reference to the primary sequences of the proposed carbohydrate- and metal-binding regions of mannose-specific lectins. Although these rRBLs agglutinated rabbit erythrocytes, the hemagglutination was not inhibited by mannose. Substitution or insertion that yielded a partial sequence similar to those of L-fucose-specific lectins and hemagglutinin from Maackia amurensis resulted in a complete loss of the hemagglutinating activity of rRBL.

Animals↗

Radiolabeling of lectin from Robinia pseudoacacia by reductive methylation.

Lectin from the bark of Robinia pseudoacacia was radiolabeled by reductive methylation. Reductive methylation of the lectin with [14C]formaldehyde and a reducing agent, sodium borohydride, resulted in incorporation of 14C into the lectin but with considerable inactivation of carbohydrate binding activity. Dialysis of the labeled lectin against the buffer containing Ca2+ and Mn2+ partially restored the activity. Higher incorporation of 14C into the lectin was observed when sodium cyanoborohydride was used as a reducing agent. In this case, more than eighty percent of the labeled lectin retained carbohydrate binding activity. Autoradiography showed that the radiolabeled lectin had a molecular mass of 29 kDa, corresponding to that of the intact lectin subunit.

Carbon Radioisotopes↗

Sialylated oligosaccharide-specific plant lectin from Japanese elderberry (Sambucus sieboldiana) bark tissue has a homologous structure to type II ribosome-inactivating proteins, ricin and abrin. cDNA cloning and molecular modeling study.

Bark lectins from the elderberry species belonging to the genus Sambucus have a unique carbohydrate binding specificity for sialylated glycoconjugates containing NeuAc(alpha 2-6)Gal/GalNAc sequence. To elucidate the structure of the elderberry lectin, a cDNA library was constructed from the mRNA isolated from the bark tissue of Japanese elderberry (Sambucus sieboldiana) with lambda gt11 phage and screened with anti-S. sieboldiana agglutinin (SSA) antibody. The nucleotide sequence of a cDNA clone encoding full-length SSA (LecSSA1) showed the presence of an open reading frame with 1902 base pairs, which corresponded to 570 amino acid residues. This open reading frame encoded a signal peptide and a linker region (19 amino acid residues) between the two subunits of SSA, the hydrophobic (A-chain) and hydrophilic (B-chain) subunits. This indicates that SSA is synthesized as a preproprotein and post-translationally cleaved into two mature subunits. Homology searching as well as molecular modeling studies unexpectedly revealed that each subunit of SSA has a highly homologous structure to the galactose-specific lectin subunit and ribosome-inactivating subunit of plant toxic proteins such as ricin and abrin, indicating a close evolutionary relationship between these carbohydrate-binding proteins.

Abrin↗

237Np in hemp-palm leaves of Bontenchiku for fishing gear used by the Fifth Fukuryu-Maru: 40 years after "Bravo".

The alpha radioactive components in the Hemp-palm of Bontenchiku were determined with emphasis on the measurement low-level 237Np by alpha-ray spectrometry after chemical separation. Bontenchiku is a kind of fishing gear for long-line fishing used by the Fifth Fukuryu-Maru (Lucky Dragon). This gear was exposed to fallout from the second thermonuclear test explosion (Bravo) at Bikini Atoll in March 1954. The 237Np content in the Bontenchiku sample was determined to be 11.5 +/- 0.8 mBq g(-1), with an activity ratio of 237Np:239,240Pu and an atom ratio of 237Np:239Pu estimated to be (2.2 +/- 0.2) x 10(-3) and 0.42 +/- 0.04, respectively. The data showed the existence of a chain reaction of 238U and its ratio to be 237Np:239Pu, as well as the presence of 237U at the time of fallout from Bravo event in March 1954.

Neptunium↗

Expression of cDNA for a bark lectin of Robinia in transgenic tobacco plants.

A cDNA encoding a bark lectin of Robinia pseudoacacia was introduced into tobacco plants. The expression of the lectin cDNA under control of the 35S promoter was confirmed by Western blot analysis and a hemagglutination assay of extracts of transgenic plants. Western blot analysis revealed that the subunit of the lectin from tobacco had a molecular mass of 29 kDa. The sequence of nine amino acids from the N-terminus of the lectin from transgenic tobacco plants was identical to that of the bark lectin from Robinia, indicating that the lectin had been processed correctly at its N-terminus in tobacco. The molecular mass of the purified native lectin produced by tobacco plants was estimated to be 112 kDa by gel filtration on a column of Superdex 200. It is suggested that the lectin subunits assembled to form tetramers in transgenic tobacco plants.

Amino Acid Sequence↗

Binding of hemoglobin by Porphyromonas gingivalis.

In this study, we investigated whether Porphyromonas gingivalis can bind hemoglobin as an initial step in the acquisition of heme from hemoglobin. The binding of human hemoglobin by P. gingivalis cells was determined using [3H]hemoglobin. Hemoglobin binding occurred rapidly, reversibly and specifically. A Scatchard analysis of the binding data generated a linear plot, indicating a single population of binding proteins. The apparent Kd was 1.0 +/- 0.19 x 10(-6) M and there were 3.2 +/- 0.76 x 10(4) binding sites per cell. Hemoglobin binding was inhibited by unlabeled human hemoglobin but not by hemin and protoporphyrin IX. The binding was only partially inhibited by human serum albumin, transferrin, lactoferrin, catalase and cytochrome c. These results suggest that the ligand recognized by the binding protein may not be the heme moiety. The binding of hemoglobin considerably increased when the organisms were grown under hemin-limited conditions. Hemoglobin bound to outer membrane proteins extracted from P. gingivalis cells on a dot blot binding assay and binding ability was lost after heating bacterial proteins. These results suggest that P. gingivalis cells interact with human hemoglobin through specific binding sites on their surfaces as a preliminary step in iron acquisition.

Bacterial Outer Membrane Proteins↗

Effect of concentration of compounds containing iron on the growth of Porphyromonas gingivalis.

We examined the effect of the concentration of various types of iron molecules on the regulation of growth of Porphyromonas gingivalis. Bacterial growth was monitored spectrophotometrically. The hemin-depleted cells of P. gingivalis 381 were incubated in the basal medium plus test substrates such as hemoglobin, hemin, transferrin and various inorganic iron compounds. The relationship between the specific growth rate of organisms and the concentration of iron-containing compounds was determined. The value of Ks, a parameter analogous to the Michaelis-Menten constant, was estimated. P. gingivalis 381 showed a Ks value of 3.85, 4.91 and 0.0017 microM for hemin, transferrin and hemoglobin, respectively. However, the inorganic iron compounds tested did not support growth of P. gingivalis. These findings suggest that P. gingivalis utilizes hemoglobin as an iron source much more effectively than other iron-containing compounds under an iron-limited environment.

Culture Media↗

Interaction of Porphyromonas gingivalis with transferrin.

In this study, we characterized the binding of transferrin to Porphyromonas gingivalis using a classical receptor-binding assay, and examined the relationship between the binding and availability of transferrin for the growth of P. gingivalis. The binding of 125I-labeled human transferrin to P. gingivalis occurred rapidly, reversibly and specifically. Scatchard analysis yielded a Kd of 1.37 +/- 0.16 microM and an apparent number of 1.13 +/- 0.26 x 10(5) receptors per cell. The binding of transferrin was much increased when organisms were grown in iron-limited conditions. Among the species of black-pigmented anaerobic.rods, those strains of P. gingivalis which had high transferrin-binding activity exhibited unrestricted growth following the addition of transferrin to the hemin-free culture medium. On the other hand, the presence of transferrin in the culture medium did not support unrestricted growth of organisms that had low transferrin-binding activity. These results suggest that the binding of transferrin to P. gingivalis cells may be a preliminary step in iron acquisition, which allows them to survive in the healthy periodontal environment.

Animals↗

Estimation of motility of subgingival microflora using high speed shutter camera and image analysis system.

This paper reports on the development of a motility measuring system for human subgingival microflora using a high speed shutter camera and an image analysis system. The paper first presents the method for estimating the motility of subgingival microflora by the total number of pixels and then describes the evaluation of the total number of pixels as an indicator of bacterial motility. Our results with this system demonstrate that the motility of subgingival microflora can be expressed as the total number of pixels.

Bacterial Physiological Phenomena↗

Cloning of a lectin cDNA and seasonal changes in levels of the lectin and its mRNA in the inner bark of Robinia pseudoacacia.

A cDNA clone encoding a lectin was isolated by immunological screening of an expression library prepared from poly(A)+ RNA from the inner bark of Robinia pseudoacacia. The cDNA clone (RBL104) had an open reading frame of 858 bp that encoded a polypeptide with a predicted molecular weight of 31210. This molecular weight corresponded closely to that of a polypeptide immunoprecipitated from products of translation in vitro of the poly(A)+ RNA. Thus, RBL104 appeared to be a full-length cDNA. The N-terminal amino acid sequence of the purified lectin protein matched a portion of the predicted amino acid sequence. It appeared that the lectin was synthesized as a precursor that consisted of a putative signal peptide of 31 amino acids and a mature polypeptide of 255 amino acids. Southern blot analysis of the genomic DNA revealed that the lectin was encoded by a small multigene family. The lectin was mostly localized in the axial and ray parenchymal cells of the inner bark. A small amount of lectin was also found in the axial and ray parenchymal cells of the xylem. The lectin accumulated in the inner bark in September, remained at high levels during the winter and disappeared in May. The mRNA for the lectin was detected from August to the following March. The appearance and disappearance of the mRNA were observed prior to those of the lectin protein.

Amino Acid Sequence↗

Characterization of coaggregation and fibrinogen-binding by Porphyromonas gingivalis.

We have examined whether the adhesin of Porphyromonas gingivalis which aggregates Streptococcus oralis contributes to its fibrinogen-binding. Various properties of coaggregation between P. gingivalis and S. oralis were compared with those of fibrinogen-binding to P. gingivalis cells. The coaggregation activity was measured by a turbidimetric method and the fibrinogen-binding activity was determined by using 125I-fibrinogen. Both the activities showed maximum values in the pH range from 5.0 to 6.0 and they were inhibited by arginine and lysine. Heating treatment of P. gingivalis 381 cells at 75 degrees C abrogated the activities. However, treatment of P. gingivalis 381 cells with proteinase K considerably reduced the fibrinogen-binding activity but not the coaggregation activity. Metal ions such as Zn2+ and Cu2+ inhibited the coaggregation activity but enhanced the fibrinogen-binding activity. The results also indicated that P. gingivalis strains 381, ATCC 33277 and SU-3 showed relatively higher activities both in coaggregation and fibrinogen-binding, whereas P. gingivalis strains W83, 51 and 165 showing lower fibrinogen-binding activity than P. gingivalis 381 did not coaggregate with S. oralis ATCC 9811. These findings suggest that coadhesin of P. gingivalis with S. oralis is not be associated with its fibrinogen-binding.

Adhesins, Bacterial↗

Glutamatergic motoneurons in the stomatogastric ganglion of the mantis shrimp Squilla oratoria.

1. Transmitters of motoneurons in the stomatogastric ganglion (STG) of Squilla were identified by analyzing the excitatory neuromuscular properties of muscles in the posterior cardiac plate (pcp) and pyloric regions. 2. Bath and iontophoretic applications of glutamate produce depolarizations in these muscles. The pharmacological experiments and desensitization of the junctional receptors elucidate the glutamatergic nature of the excitatory junctional potentials (EJPs) evoked in the constrictor and dilator muscles. The reversal potentials for the excitatory junctional current (EJC) and for the glutamate-induced current are almost the same. 3. Some types of dilator muscle show sensitivity to both glutamate and acetylcholine (ACh) exogenously applied. The pharmacological evidence and desensitization of the junctional receptors indicate the glutamatergic nature of neuromuscular junctions in these dually sensitive muscles. The reversal potentials for the EJC and for the ACh-induced current are not identical. 4. Glutamate is a candidate as an excitatory neuro-transmitter at the neuromuscular junctions which the STG motoneurons named PCP, PY, PD, LA and VC make with the identified muscles. Kainic and quisqualic acids which act on glutamate receptors are potent excitants of these muscles. Extrajunctional receptors to ACh are present in two types of the muscle innervated by LA and VC. 5. Neurotransmitters used by the STG motoneurons of stomatopods are compared to those of decapods.

Acetylcholine↗

Mechanisms underlying burst generation of the pyloric muscle in the mantis shrimp, Squilla oratoria.

The pyloric constrictor muscles of the stomach in Squilla can generate spikes by synaptic activation via the motor nerve from the stomatogastric ganglion. Spikes are followed by slow depolarizing afterpotentials (DAPs) which lead to sustained depolarization during a burst of spikes. 1. The frequency of rhythmic bursts induced by continuous depolarization is membrane voltage-dependent. A brief depolarizing or hyperpolarizing pulse can trigger or terminate bursts, respectively, in a threshold-dependent manner. 2. The conductance increases during the DAP response. The amplitude of DAP decreases by imposed depolarization, whereas it increases by hyperpolarization. DAPs from successive spikes sum to produce a sustained depolarizing potential capable of firing a burst. 3. The spike and DAP are reduced in amplitude by decreasing [Ca]o, enhanced by Sr2+ or Ba2+ substituted for Ca2+, and blocked by Co2+ or Mn2+. DAPs are selectively blocked by Ni2+, and the spike is followed by a hyperpolarizing afterpotential. 4. The spike and DAP are prolonged by intracellular injection of the Ca2+ chelator EGTA. A hyperpolarizing afterpotential is abolished by EGTA and enhanced by increasing [Ca]o. The DAP is diminished in Na(+)-free saline and reduced by tetrodotoxin. 5. It is concluded that the muscle fiber is endowed with endogenous oscillatory properties and that the oscillatory membrane events result from changes of a voltage- and time-dependent conductance to Ca2+ and Na+ and a Ca2+ activated conductance to K+.

Animals↗

Characterization of Ca current underlying burst formation in lobster cardiac ganglion motorneurons.

1. The anterior motorneurons of the cardiac ganglion of Homarus americanus were ligated less than 300 microns from the soma. This removes impulse-generating membrane and sites of synaptic input while preserving the ability of the soma to generate the burst-forming potentials termed "driver potentials" regenerative, slow (250-ms duration) depolarizations (to -20 mV) in response to brief, depolarizing stimuli. At stimulus intervals corresponding to rates of bursting observed in spontaneously active, intact ganglia (0.3-1.2/s), driver potential amplitude increases with increasing stimulus interval. 2. A two-electrode voltage clamp was used to characterize inward current observable from the ligated neurons in tetrodotoxin (TTX)-tetraethylammonium (TEA)-containing salines. The amplitude of inward current shows a hyperbolic relation to [Ca]o that is well fitted by a form of the Michaelis-Menten equation. Inward current is maintained but not augmented when Ca2+ is replaced by Ba2+ or Sr2+. It is concluded that the inward current, to be referred to as ICa, is mediated by voltage-dependent Ca channels. 3. Contamination of ICa by early outward current (IA) was evaluated by addition of 4-aminopyridine (4-AP, 4 mM). In the presence of 4-AP, the net inward current is increased and the potential at which maximum ICa occurs is shifted 10 mV more positive. 4. Subtraction of outward currents recorded in Mn2(+)-containing saline from overall currents in the absence of Mn2+ provided another means to separate inward from outward current. I-V curves from such "Mn-subtracted" records show ICa approaches a saturating value for steps to -5 mV and more depolarized. The time to peak ICa is voltage dependent. The largest inward currents (up to 240 nA) and minimal time to peak (4 ms) are observed for steps from holding potentials of -50 to -60 mV. 5. Decline of ICa during depolarized steps observed in Mn-subtracted records represents inactivation rather than development of competing outward current. Inactivation is slow and incomplete; the rate and fractional amount of inactivation are not directly voltage dependent. Nonsubtracted responses to 500-ms depolarizations to potentials evoking little outward current show that an initial rapid decline of ICa (tau approximately 40 ms) is followed at approximately 80 ms by a slower phase of decline (tau approximately 180 ms). With repetitive clamps, the early phase proved labile.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗