Biomedical subjects
Publications and source records attributed to K Tsuzuki.
Clostridium botulinum 125I-labeled Cl neurotoxin bound to NG108 hybridoma cell line. Unlabeled type Cl neurotoxin inhibited the binding of the labeled Cl toxin but neither types D nor E toxin. 125I-labeled type D neurotoxin bound to rat brain synaptosomes but did not bind to NG108 cells. It is suggested that receptors for types C and D or E toxins on neuronal cell membranes are different.
Large defects in the lumbosacral and perineal regions were reconstructed with bilateral gluteal thigh flaps. These two patients demonstrate the versatility of the flap, which also was used as a sensory flap in one patient.
Chromosomal DNA was extracted from toxigenic Clostridium butyricum strain BL6340 isolated from a case of infant botulism. After digestion by EcoRI, a DNA fragment of about 1 kbp was cloned into Escherichia coli using lambda gt11, and was subcloned into pUC118. The E. coli cells transformed with this cloned fragment produced a 33 kDa protein which reacted with monoclonal antibodies recognizing the light chain (Lc) component of botulinum type E toxin. The nucleotide sequence of the cloned fragment was determined. The sequence was similar to that from botulinum type E toxin gene fragments previously determined by our laboratory (strains Mashike, Otaru and Iwanai). Several highly homologous sequences among the botulinum type A, C, E, butyricum and tetanus toxin genes were found in both translated and untranslated regions. These results suggest that the toxin gene of C. butyricum may have evolved by transfer from C. botulinum.
The toxigenicity of Clostridium botulinum type C1 is mediated by specific bacteriophages. DNA was extracted from one of these phages. Two DNA fragments, 3 and 7.8 kb, which produced the protein reacting with antitoxin serum were cloned by using bacteriophage lambda gt11 and Escherichia coli. Both DNA fragments were then subcloned into pUC118 plasmids and transferred into E. coli cells. The nucleotide sequences of the cloned DNA fragments were analyzed by the dideoxy chain termination method, and their gene products were analyzed by Western immunoblot. The 7.8-kb fragment coded for the entire light chain component and the N terminus of the heavy chain component of the toxin, whereas the 3-kb fragment coded for the remaining heavy chain component. The entire nucleotide sequence for the light chain component was determined, and the derived amino acid sequence was compared with that of tetanus toxin. It was found that the light chain component of C1 toxin possessed several amino acid regions, in addition to the N terminus, that were homologous to tetanus toxin.
1. Two different types of kainate response were recorded in cultured rat hippocampal neurons with the use of the whole-cell and outside-out configurations of the patch-clamp technique. 2. There was an outward rectification in the current-voltage (I-V) plot of the kainate-induced current (type I response) in relatively large neurons bearing a morphological resemblance to young pyramidal cells. In smaller neurons with elliptical somata and fine neurites, the kainate response was characterized by a remarkable inward rectification in the I-V plot of the kainate-induced current and a significant permeability to Ca2+ (type II response). 3. Both type I and type II responses were negligible below 2 microM and almost saturated at 500 microM kainate. The concentrations producing half-maximal responses and the Hill coefficients were 68 microM and 1.76 and 56 microM and 1.21 for type I and type II responses, respectively. Both responses were suppressed similarly by the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). 4. The mean single-channel conductance (gamma) of the type II kainate response was estimated, from the relation between the whole-cell mean currents and current variances, to be 8.7 pS. The power spectrum for the current noise was fitted with the sum of two Lorentzians with cutoff frequencies (fc) of 61.1 +/- 1.4 and 327.8 +/- 10.5 Hz (n = 12).(ABSTRACT TRUNCATED AT 250 WORDS)
Although both posteroventral cochlear nucleus (PVCN) and dorsal cochlear nucleus (DCN) are innervated by the descending branch of auditory nerve fibers, their intrinsic morphological organizations are so different that their physiological roles are expected to be different in signal processing. Temporal information coding of acoustic signals in the cochlear nucleus was examined by using stimuli of "tone-burst-trains (TBT)". Responses of cochlear nucleus neurons of anesthetized cats were recorded either intracellularly or extracellularly. Responses of the neurons to TBT stimuli were classified into "adaptive type" and "non-adaptive type". The "adaptive type" neurons were mainly recorded from PVCN. Responses of these neurons to TBT stimuli decayed exponentially, because of short-term adaptation, in the subsequent tone-bursts. These neurons faithfully preserve the adaptative behavior of auditory nerve fibers. On the contrary, the "non-adaptive type" neurons were mainly found in DCN. They showed variety of responses to TBT stimuli including facilitation, disinhibition and inhibition depending on duration and/or interval of tone-bursts. Our results suggest that some "non-adaptive type" neurons, showing facilitative and/or inhibitory responses to TBT stimuli, act as temporal filters that extract temporal information from acoustic signals.
Simple, efficient syntheses of jietacin A, a nematocidal antibiotic, and its analogs have been developed in order to study structure-activity relationships. A series of alpha,beta-unsaturated azoxy compounds was prepared from phenylselenomethyl azoxy compounds as key intermediates and its nematocidal activity was determined.
Two DNA fragments, 3 kbp and 7.8kbp, which encode the type C1 botulinum neurotoxin gene, were obtained from toxigenic bacteriophage DNA by treatment with a restriction enzyme. They were cloned into the plasmid vectors for nucleotide sequence determination. The nucleotide sequence contained a single open reading frame coding for 1,291 amino acids corresponding to a polypeptide with a molecular weight of 149,000. The amino acid sequence of the C1 toxin has a few regions highly homologous with tetanus toxin.
The author treated 456 cases of port-wine stains using a tunable dye laser (577-nm wavelength, 1-microsecond pulse, 2-3-mm diameter spot), and followed up 22 cases for a year or more after the initial treatment. The treatment was regarded as effective in 80.5% of the abnormal lesions. The results were influenced by location: good results were achieved in the neck region and face, but results in the extremities were poor. The dye laser can achieve selective injury to abnormal vessels of port-wine stains while inflicting less damage to the overlying dermis than the argon laser. Therefore, dye laser treatment for port-wine stains is more effective than argon laser treatment.
A monitoring procedure using a Doppler flowmeter for assessment of patency after microvascular surgery is reported. Thirty-two free flaps were monitored using this procedure. There were three problematic cases in which flap circulation was not evaluated by the Doppler flowmeter. One involved the existence of another artery beneath the pedicle. The second was one of distal necrosis of the flap caused by impairment of peripheral blood circulation, in spite of patency of the pedicle. The third case involved venous occlusion of the pedicle, that was clinically detected by evaluating flap color, and the flap was salvaged. Checking beneath the pedicle prior to anastomosis was instituted after the first case cited, and there has not been a similar complication. The procedure has been proved to be useful in flap monitoring.
Chromosomal DNAs were extracted from toxigenic three Clostridium botulinum type E strains isolated from food-borne botulism. After digestion by EcoRI, the fragments were cloned into Escherichia coli by using bacteriophage lambda gt11 and screened with monoclonal antibody recognizing the light chain component of botulinum type E toxin. The fragments (about 1 kbp size) cloned from each strain were recloned into a plasmid vector pUC118. The E. coli cells transformed with the recombinant plasmids produced 33 kDa protein with or without IPTG (isopropyl-beta-D-thiogalactopyranoside) which reacted with the monoclonal antibody. The nucleotide sequences of the cloned EcoRI fragments from the three type E strains were identical and contain the 5'-terminal region of the type E toxin gene. It was also found that there exist several highly homologous nucleotide sequences among the botulinum types A, C and E, and tetanus toxin genes in both translated and untranslated regions.
1. N-methyl-D-aspartate (NMDA)-, quisqualate- and kainate-induced currents were recorded in cultured rat hippocampal neurones using the whole-cell voltage-clamp technique. To isolate the inward currents carried by Ca2+ and other divalent cations (Sr2+, Ba2+, Mn2+ and Mg2+), both Na+ and K+ in the control external solution were replaced with the impermeant cation N-methylglucamine (NMG). 2. Replacement of Na+, K+ and Ca2+ with NMG abolished NMDA-, quisqualate- and kinate-induced inward currents. In Na(+)-, K(+)-free (abbreviated simply as Na(+)-free) solution containing 10 mM-Ca2+ NMDA caused prominent inward currents at -60 mV. In this solution with the internal solution containing 165 mM-Cs+, the reversal potential of the NMDA-induced current was -5.0 +/- 0.7 mV (n = 36), indicating a value of PCa/PCs = 6.2 for the ratio of the permeability coefficients of Ca2+ and Cs+ according to the constant-field equation. 3. NMDA elicited inward current responses at -60 mV in Na(+)-, Ca2(+)-free solution containing 10 mM-Sr2+, Ba2+, or Mn2+, but not in Na(+)-free, 10 mM-Mg2+ solution. On the basis of reversal potential measurements, the permeability sequence of NMDA receptor channels among the divalent cations was determined to be Ba2+ (1.2) greater than Ca2+ (1.0) greater than Sr2+ (0.8) greater than Mn2+ (0.3) much greater than Mg2+ (less than 0.02). 4. The reversal potential of the quisqualate-induced current was more negative than -80 mV in Na(+)-free, 10 mM-Ca2+ solution, indicating a value of PCa/PCs less than 0.18. 5. Kainate-induced current responses were classified into two types. In the type I response the reversal potential of the kainate-induced current was more negative than -80 mV in Na(+)-free, 10 mM-Ca2+ solution, indicating that the Ca2+ permeability of this type of kainate channel is as low as that of the quisqualate channel. In the neurones which showed a type I response, there was a tendency of outward rectification in the current-voltage plots of the kainate response in control solution. 6. In the type II response kainate caused prominent inward currents at -60 mV in Na(+)-free, 10 mM-Ca2+ solution. The reversal potential was -23.3 +/- 5.6 mV (n = 17), indicating a permeability ratio PCa/PCs = 2.3. In the neurones which showed a type II response, a remarkable inward rectification was observed in the current-voltage plots of the kainate response in control solution. 7. Type II kainate channels showed relatively poor selectivity among divalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)
In Clostridium botulinum types C and D, phage conversion to toxin and hemagglutinin (HA) production has been reported. DNA was extracted from a converting type C Stockholm phage, c-st, and a fragment (7.8 kilobase pairs) coding for the parts of both toxin and HA was cloned. The gene for HA was recloned, and the complete nucleotide sequence was determined. The molecular mass of this gene product was 33 kilodaltons, and it showed HA activity. The HA preparation partially purified from a type C Stockholm culture demonstrated two major bands (33 and 53 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reducing agent. The amino acid sequence of the N terminus of the 33-kilodalton component of the native HA preparation, which was determined by a direct protein microsequencing procedure, was identical to that deduced from the nucleotide sequence of cloned HA gene. These data indicate that the cloned gene product (33 kilodaltons) is an important component of HA.
1. The effects of increasing K+ concentration in Mg2(+)-free extracellular solution on N-methyl-D-aspartate (NMDA)-induced current were studied in cultured rat hippocampal neurons with the use of the whole-cell and outside-out configurations of the patch-clamp technique. 2. When the K+ concentration in the external solution was increased by replacement of Na+ with isomolar K+, the amplitude of the NMDA-induced current decreased in a concentration-dependent manner. The effect of K+ was almost saturated at 100 mM, when the NMDA response was reduced to 12% of that in K(+)-free, 150 mM Na+ solution. Increasing the external K+ concentration did not affect either the kainate- or quisqualate-induced current in these experimental conditions. 3. Increase in the external K+ concentration reduced the NMDA-induced current almost equally over the whole range of membrane potential tested (-60-30 mV). The reversal potential of the NMDA-induced current was not significantly shifted by the replacement of Na+ with K+. 4. A rise in the external K+ concentration to 100 mM did not reduce the single-channel conductance of the NMDA channel, whereas it reduced the mean open time to about two-thirds of that in the control external solution. 5. The suppressed activation of the NMDA receptor channel in high-K+ environments may have a functional significance to alleviate entry of toxic Ca2+ into neurons of the CNS in pathological conditions such as hypoxia and ischemia.
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The calcium permeability of receptor channels activated by quinolinic acid (QUIN) in cultured rat hippocampal neurons was investigated using the whole-cell voltage-clamp method. In Na+-free, 10 mM Ca2+ solution with the internal solution containing 165 mM Cs+, QUIN elicited prominent inward currents at -60 mV, and the reversal potential of the QUIN-induced current was -5.8 +/- 1.2 mV, indicating that QUIN-activated channels are highly permeable to Ca2+ (permeability ratio PCa2+/PCs+ = 5.9). This result was substantiated by microfluorometry using fura-2, which revealed that QUIN caused a marked increase in the intracellular Ca2+ concentration even after the voltage-dependent Ca2+ channels had been suppressed by La3+.
Voltage-dependent calcium (Ca2+) currents in cultured rat hippocampal neurons were studied with the whole-cell recording mode of the patch-clamp technique. On the basis of the voltage-dependence of activation, kinetics of inactivation and pharmacology, 3 types of Ca2+ currents were distinguished. The low-threshold Ca2+ current (Il) was activated at -60 mV, and completely inactivated during a 100-ms depolarization to -40 mV (time constant: tau = 16 +/- 1 ms). The high-threshold currents (Ih), which were activated at -20 mV, could be separated into two types. The high-threshold, fast inactivating current (Ih,f) decayed quickly during a maintained depolarization (tau = 33 +/- 3 ms at 0 mV), whereas the high-threshold, slowly inactivating current (Ih,s) decayed with a much slower time constant (tau = 505 +/- 42 ms at 0 mV). The inactivations of Ih,f and Ih,s exhibited different time- and voltage-dependencies. Nickel ions (Ni2+, 25 microM) markedly suppressed Il, but little affected Ih. Cadmium ions (Cd2+, 10 microM) almost completely suppressed Ih, but left a small amount of Il. Lanthanum ions (La3+, 10 microM) almost completely suppressed both Il and Ih. Ih,s was sensitive to block by the dihydropyridine antagonist nicardipine (10 microM).