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Biomedical subjects

K Kusano

Publications and source records attributed to K Kusano.

At least 91 records · Page 5Linked to original sources

Galactosylceramide and galactosylsphingosine loading studies in cultured skin fibroblasts in human and murine globoid cell leukodystrophy.

Cell level studies of 3H-galactosylceramide(GalCer) and 3H-galactosyl sphingosine (GalSph) have been carried out in cultured skin fibroblasts from human and murine globoid cell leukodystrophy (GLD). GalCer loading studies disclosed that the hydrolysis rates of GalCer in human control and GLD were 72% and 45%, respectively, and those from the murine control and GLD cells were 77% and 21%, respectively, on the 5th day of culture. On the other hand, GalSph loading studies showed that the hydrolysis rate of GalSph in the human control and GLD were 40% and 10%, respectively, and those from murine control and GLD cells were 38% and 10% on the 12th day of culture. These data suggest that both GalCer and GalSph degradations were impaired in cell level in human and murine GLD. Furthermore, when radioactive 3H-GalSph was loaded into cultured fibroblasts from murine and human GLD, 3H-GalCer band was formed via GalSph. These data strongly suggest that GalCer could be synthesized through the GalSph route as a minor pathway at least in cultured skin fibroblasts, although the major pathway to synthesize GalCer should be via ceramide.

Animals↗

Simultaneous measurements of endogenous and deuterium-labelled tracer variants of androstenedione and testosterone by capillary gas chromatography-mass spectrometry.

A capillary gas chromatographic-mass spectrometric method for the simultaneous determination of androstenedione and testosterone in human plasma using [19,19,19-2H3]androstenedione and [19,19,19-2H3]testosterone as internal standards is described. For calculation of plasma androstenedione and testosterone, peak heights were measured by selected-ion monitoring of the molecular ions of the heptafluorobutyryl derivatives of androstenedione and [2H3]androstenedione (m/z 482 and 485) and of testosterone and [2H3]testosterone (m/z 680 and 683). The isotope dilution method needed no complex corrections for contributions and provides a sensitive and reliable technique with good accuracy, precision and reproducibility.

Androstenedione↗

Mechanical changes associated with synaptic transmission in the mammalian superior cervical ganglion.

Mechanical changes in the superior cervical ganglion (SCG) of the guinea pig, evoked by stimulating the pre- or postganglionic nerve, were determined by using a mechano-electric transducer. The mechanical change evoked by either one of these nerves was monophasic, an initial phase of rapid swelling (about 1 dyn/cm2) being followed by a slow relaxation. The mechanism of these mechanical changes was examined by treating the preparations with agents which are known to suppress or facilitate synaptic transmission in the SCG. Mechanical changes elicited by preganglionic nerve stimulation were suppressed by agents that reversibly block ganglionic transmission (Ca2(+)-free high Mg2+, d-tubocurarine, hexamethonium, TEA, TTX). On the other hand, agents which are known to facilitate synaptic transmission (high Ca2+) increased both the amplitude and the duration of swelling. In a Ba2(+)-containing medium, a large, long-lasting shrinkage of the ganglion was observed. It is suggested that the mechanical changes recorded in this study are associated with postsynaptic electro-genesis.

Animals↗

Heat generation associated with synaptic transmission in the mammalian superior cervical ganglion.

By use of a thermal detector constructed with a thin polyvinylidene fluoride film (PVDF), heat production in the superior cervical ganglion (SCG) of the guinea pig was examined. A single electric shock applied to the preganglionic nerve evokes a temperature rise of approximately 1.5 x 10(-6) deg. The thermal responses summate when the preganglionic nerve is stimulated repetitively. The amplitude of the thermal response is increased when the preparation is treated with a high Ca2+ medium. Treatment with agents that block ganglionic transmission (high Mg2+, d-tubocurarine, hexamethonium, TTX) reversibly suppresses thermal response. It is thus concluded that the thermal responses described in this paper are generated by the physico-chemical events underlying postsynaptic electrogenesis in the SCG cells.

Animals↗

Sequence and expression of a frog brain complementary DNA encoding a kainate-binding protein.

Excitatory amino acids (EAAs) are important neurotransmitters in the vertebrate central nervous system. Electrophysiological and ligand-binding studies indicate that at least three different receptor subtypes for EAAs exist--N-methyl-D-aspartate, kainate and quisqualate receptor subtypes--on the basis of the preferred agonist of the receptors. We recently purified a kainate-binding protein (KBP) from frog (Rana pipiens berlandieri) brain by domoic acid (a high-affinity kainate analogue) affinity chromatography, and showed that the kainate-binding activity was associated with a protein of relative molecular mass 48,000 (Mr 48 K). The pharmacological properties and the anatomical distribution of KBP were consistent with those of a kainate receptor-ionophore complex. We have now isolated a complementary DNA encoding KBP of Mr 48 K. The deduced amino-acid sequence of the KBP has similar hydrophobic profiles to those found in other ligand-gated ion channel subunits, and shows some amino-acid sequence similarities to the corresponding regions of brain nicotinic acetylcholine receptor subunits. Localization of the KBP messenger RNAs by in situ hybridization histochemistry is compatible with the results of immunohistochemistry and receptor autoradiography studies. COS-7 cells transfected with the cDNA encoding the KBP show high-affinity kainate-binding activity with pharmacological properties similar to those of the biochemically purified KBP. These results provide the first molecular characterization of an EAA-binding site and raise the possibility that the KBP cDNA encodes a ligand-binding subunit of a kainate receptor-ionophore complex.

Amino Acid Sequence↗

Pharmacological characterization and region-specific expression in brain of the beta 2- and beta 3-subunits of the rat GABAA receptor.

The cDNA for a third beta-subunit of the rat GABAA receptor has been cloned using another beta-subunit, which we had previously cloned [(1989) FEBS Lett. 246, 145-148], as a probe. The approximately 8-kb cDNA for this beta-subunit (termed beta 2) encodes a protein of 474 amino acid residues that shares approximately 80% sequence identity with the rat and bovine beta 1- and beta 3-subunits. Coexpression of the cloned beta-subunit cDNA with the alpha 1-subunit cDNA of the rat GABAA receptor in Xenopus oocytes produced a functional receptor and Cl- channel with pharmacological characteristics of a GABAA receptor. In contrast to interchanging alpha-subunits [(1988) Nature 335, 76-79], exchange of beta 2- or beta 3-subunits in an alpha 1/beta receptor complex did not markedly alter the pharmacological properties of expressed receptors. In situ hybridization histochemistry with synthetic subunit-specific oligo-deoxynucleotide probes revealed a region-specific expression of alpha 1-, beta 2- and beta 3-subunit mRNAs in the rat central nervous system. These observations provide an additional molecular basis for the functional heterogeneity in the GABAA receptor complex.

Animals↗

Plasmid-mediated lethality and plasmid multimer formation in an Escherichia coli recBC sbcBC mutant. Involvement of RecF recombination pathway genes.

Apparent plasmid instability, i.e. progressive plasmid loss in a bacterial culture growing in the absence of selection for the plasmid, in an Escherichia coli recBC sbcBC mutant was investigated with two different ColE1 derivatives (pMB9 and pBR322) and a mini-F plasmid. The instability was most striking for pMB9 and much less, but still significant, for pBR322 and the mini-F. It was also dependent upon a subset of the genes involved in the RecF recombination pathway: in addition to the previously reported recA, recF and recJ mutations, a recO and a recQ mutation showed a total and a partial suppression, respectively, of the instability. Other recF-family mutations, recN and ruv, were without such an effect. Population analyses of the recBC sbcBC strain carrying pMB9 or the mini-F, as carried out by plating and Coulter counting, revealed marked loss of viability in plasmid-carrying cells, strongly implicating plasmid-mediated cell death in the apparent defect in plasmid maintenance. Analysis of intracellular plasmid DNA by pulsed-field gel electrophoresis combined with the in-agarose cell lysis technique showed that the instability was associated with the formation of plasmid multimers, with a good correlation between the degree of the instability and the amount of the multimers. The multimer formation was also dependent on the same subset of the RecF pathway genes as in the instability phenomenon. These results strongly suggest that the lethality is somehow caused by the multimer formation. Various DNase treatments of cell lysates showed that such multimers of pMB9 DNA comprised molecules of exonuclease-sensitive and exonuclease-resistant types. It was inferred that the former class, which showed electrophoretic mobilities corresponding to plain linear duplexes of approximately 200 x 10(3) to 2200 x 10(3) base-pairs, represented linear multimers possibly carrying circular structures at one end. The latter class, which remained in the origin, was thought to consist of circular multimers and/or linear multimers protected by circular structures at both ends against exonucleolytic attack.

DNA Replication↗

Intestinal-like alkaline phosphatase expressed in normal human adult kidney.

Human adult kidney was found to contain not only the 'tissue-unspecific alkaline phosphatase' but also another alkaline phosphatase isozyme. By use of monoclonal antibodies specific for human intestinal alkaline phosphatase, this kidney isozyme was purified to homogeneity by immunoaffinity chromatography. The structural and kinetic properties of the enzyme were compared with those of the other alkaline phosphatase isozymes expressed in normal human tissues, i.e. the placental, intestinal, meconial (fetal), liver and kidney isozymes. The new kidney isozyme was clearly different from both the tissue-unspecific and the adult intestinal alkaline phosphatase as regards isoelectric point, molecular mass and peptide maps after cyanogen bromide cleavage, but it was found to be identical to the meconial alkaline phosphatase. The results demonstrate simultaneous expression of two alkaline phosphatase isozymes in human kidney, one of which is normally related only to the fetal intestine.

Alkaline Phosphatase↗

Cloning and expression of a novel rat GABAA receptor.

Two full-length cDNA clones encoding alpha- and beta-subunits of a GABAA receptor have been isolated from a rat cerebral cortex cDNA library. The mature alpha-subunit protein consists of 428 amino acids with a calculated Mr of 48,680. This protein is highly homologous (approximately 99% amino acid identity) with the bovine brain alpha 1-subunit receptor [(1988) Nature 335, 76-79]. The mature rat beta-subunit receptor is a 448 amino acid polypeptide and shares approximately 80% amino acid identity with the previously characterized bovine GABAA receptor beta-subunit [(1987) Nature 328, 221-227]. Co-expression of the cloned DNA in Xenopus oocytes produces a functional receptor and ion channel with pharmacological characteristics of a GABAA receptor. GABAA alpha- and beta-subunit mRNA is detectable in the cortex, cerebellum and hippocampus.

Amino Acid Sequence↗

Rapid mechanical and thermal changes in the garfish olfactory nerve associated with a propagated impulse.

Mechanical and thermal changes associated with a propagated nerve impulse were determined using the garfish olfactory nerve. Production of an action potential was found to be accompanied by swelling of the nerve fibers. The swelling starts nearly at the onset of the action potential and reaches its peak at the peak of the action potential. There is a decrease in the length of the fibers while an impulse travels along the fibers. The time-course of the initial heat was determined at room temperature using heat-sensors with a response-time of 2-3 ms. Positive heat production was found to start and reach its peak nearly simultaneously with the action potential. The rise in temperature of the nerve was shown to be 23 (+/- 4) mu degrees C. In the range between 10 degrees and 20 degrees C, the temperature coefficient of heat production is negative, primarily due to prolongation of the period of positive heat production at low temperatures. The amount of heat absorbed during the negative phase varies widely between 45 and 85% of the heat evolved during the positive phase. It is suggested that both mechanical and thermal changes in the nerve fibers are associated with the release and re-binding of Ca-ions in the nerve associated with action potential production.

Action Potentials↗

Three types of chemical modification-effects induced by various chemical reagents on the glutamate receptors in molluscan neurons.

The effects of specific protein modifying reagents on the dose (Glu)-response (delta G) relationship of the glutamate-hyperpolarizing (Glu-H) receptors in molluscan neurons (Onchidium verruculatum) were analyzed. The effects could be classified into three types. Type I, parallel shift of the dose-response curve towards higher concentration by modification of COO- groups by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The double reciprocal plots of the dose-response curves before and after the treatment indicated competitive inhibition type modification. Type II, a decrease in the slope of the dose-response curve through modification of -SH by N-ethylmaleimide and -NH3 groups by trinitrobenzenesulfonic acid. The double reciprocal plots indicated non-competitive inhibition type modification. The combined effects of Type I and Type II, after modification of arginyl residues with diacetyl trimer. The modification was most effective when applied during an activated state of receptors and channels by Glu. EEDQ had an irreversible after-effect on Glu-H type Onchidium neurons to activate an additional Na+ permeability increase in Glu induced hyperpolarizing response. Pretreatment of N-acetylimidazole (NAI), Glu-H (including weak D) receptors in Helix aspersa produced no significant difference in Glu response. However, simultaneous application of NAI and Glu induced an additional Na+ permeability increase, probably by modification of tyrosyl residues. This indicates the greatest effectiveness of the NAI modification during the activated state of the receptors and the channels by Glu. It is suggested that the Glu-H receptor protein possesses both negatively and positively charged residues, containing COO- and arginyl+ in the receptive site, and that several amino acid resides (SH, -NH, arginyl, and tyrosyl) act in the Glu-activated receptors and channels as their subsites. The role of the subsites in the receptors and channels is discussed.

Animals↗

Lateral hypothalamic modulation of oral sensory afferent activity in nucleus tractus solitarius neurons of rats.

This study was designed to examine whether the sensory afferents from the anterior part of the tongue are modulated by activity of the lateral hypothalamic area (LHA) at the level of the nucleus tractus solitarius (NTS) in rats. The electrical activity of the NTS neurons was recorded extracellularly, and they were classified as gustatory, thermal, or mechanical neurons in accordance with their responsiveness to tongue stimulation by taste solutions, by warm and cool water, and by stroking the tongue surface. Sixty-two percent of the neurons were polysynaptically activated by electrical stimulation of the LHA. When a single conditioning stimulus of the LHA was applied prior to the test electrical stimulation of the tongue at various conditioning-test intervals, the activity of the gustatory neurons was facilitated by 30 to 80% of their control level for a period of approximately 20 to 150 msec. The activity of the mechanical and thermal neurons was suppressed for a 50- to 300-msec period. These results demonstrate the existence of modulatory effects from the LHA on the NTS neurons, which receive sensory information from the tongue.

Afferent Pathways↗

Lateral hypothalamic modulation of the gustatory-salivary reflex in rats.

It is well recognized that the basic mechanism for the gustatory-salivary reflex is located in the lower brainstem and that suprabulbar structures possibly influence this mechanism. This study is designed to evaluate the neurophysiological mechanism underlying the effect of the lateral hypothalamic area (LHA) on the bulbar gustatory-salivary reflex. Submandibular salivary secretion and the electrical activity of the preganglionic parasympathetic fibers innervating the submandibular and sublingual glands were recorded in anesthetized rats. Stimulation of the tongue with high concentrations of chemical solutions (1 M and 2 M NaCl and 0.01 M and 0.05 M HCl) and/or pinching the tongue with a small clamp induced a profuse salivary secretion (3 to 28.5 microliter/5 min) recorded from a unilateral submandibular gland. The preganglionic fibers consisted of three types: taste-related fibers, which increased their firing rate by taste stimuli; pinch-related fibers, which increased their firing rate by pinching; and unidentified fibers, which did not respond to taste or pinching stimulations of the tongue. Electrical stimulation of the ipsilateral LHA caused the secretion of a small amount of saliva (1.5 microliter/5 min), and it appeared that taste-related fibers more often received polysynaptic connections from the LHA than other types of fibers. Conditioning stimulation of the LHA increased the magnitude of impulse discharges by the test stimulation of the tongue in the taste-related fibers. Therefore, the results of this study demonstrate that the LHA enhances the activity of the gustatory-salivary reflex in the lower brainstem.

Animals↗