Biomedical subjects

T Kusakabe

Publications and source records attributed to T Kusakabe.

At least 19 recordsLinked to original sources

PubMed1995

Analysis of the in vitro translation product of a novel-type Drosophila melanogaster aldolase mRNA in which two carboxyl-terminal exons remain unspliced.

Drosophila melanogaster generates three different types of aldolase mRNAs from a single gene by selective usage of the triplicate exons 4 (4 alpha, 4 beta, and 4 gamma), which encode three different isozymes having respective carboxyl termini. We have found the presence of a novel-type mRNA (named alpha beta) in which two final exons, 4 alpha and 4 beta, were retained unspliced. Herein, a cDNA clone containing the alpha beta sequence was inserted into pINIII and expressed in an Escherichia coli system. The product, which exhibited aldolase activity, was found to be isozyme alpha from the primary structure and the enzymological properties, with the 4 alpha sequence alone being present as the carboxyl terminus. In tissues of D. melanogaster, the production of mRNA encoding exon 4 alpha is known to be restrained to a low level. This may be understood by the fact that the aldolase gene of this species does not have a typical poly(A) signal at the 3' end in exon 4 alpha. Instead, the transcript-encoding exons, 4 alpha and 4 beta, might be produced when AATATA, which resides downstream of the coding frame in exon 4 beta, is recognized as a poly(A) signal during RNA processing.

Alternative Splicing↗

PubMed1995

Sequence preference for strand cleavage of gapped duplexes by dynemicin A: possible mechanism of sequence-dependent double-stranded breaks.

A double-stranded DNA cleavage mechanism by a novel enediyne type antitumor antibiotic, dynemicin A, has been investigated through sequence-dependent strand breakage of a series of duplexes containing a single nucleotide gap. We found that (1) dynemicin A breaks specifically at the 3'-shifted position by one base opposite the gap, (2) the strong cleavage is detected at 5'-Pu_Pu/3'-PyPuPy sequences, and (3) dynemicin H (aromatized form of dynemicin A) gives only a small inhibition effect (20%) on the cleavage of gapped duplex by dynemicin A. The long half-life of aromatization of dynemicin A (118 min, in the presence of DNA) obtained from HPLC analysis provides enough time for the second cleavage. The present results strongly indicate a two-step mechanism for the double-stranded DNA scission of dynemicin A. Namely, this double-stranded break is caused by two drug molecules, each of which cuts one DNA strand.

Alkenes↗

PubMed1995

Effects of acetylcholine and adrenaline on axoplasmic transport at different regions of mouse superior cervical ganglion cells in culture.

Adrenaline and acetylcholine (ACh) were applied locally at three different positions in cultured superior cervical ganglion cells, i.e., cell body, neurite, and growth cone and the effects on the axoplasmic transport were measured with a video-enhanced microscope. Local ACh application to the cell body, neurite, and growth cone caused the same decreasing effect, but the effects of local adrenaline application were different from each other. Local adrenaline application to the cell body and growth cone caused an increase of axoplasmic transport, but local application at the neurite caused no effect. These data may indicate that there was a lack of beta 2 adrenergic receptors in the neurite. Desensitization of axoplasmic transport was also examined in the SCG neurons. Repetitive adrenaline application to the cell body caused desensitization to the stimulus of adrenaline application.

Acetylcholine↗

PubMed1995

Calcitonin gene-related peptide and substance P in the pharynx and lung of the bullfrog, Rana catesbeiana.

Indirect double immunofluorescence labelling in the pharynx and lung of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of two neuropeptides. In the pharynx, immunoreactive calcitonin gene-related peptide (CGRP) and substance P (SP) were localized in nerve fibers distributed within and just beneath the ciliated epithelium. In the lung, CGRP and SP were localized in nerve fibers in five principal locations: 1) within the smooth muscle layer in the interfaveolar septa; 2) in the luminal thickened edges of the septa; 3) around the pulmonary vasculature; 4) within, and 5) under the ciliated epithelium. Within the smooth muscle layer in the septa, luminal thickened septa, and around blood vessels, almost all fibers showed coexistence of CGRP and SP. Within and just beneath the ciliated epithelium in the thickened septa, all fibers showed coexistence of CGRP and SP. No immunoreactivity for vasoactive intestinal polypeptide, neuropeptide Y, galanin, somatostatin, FMRFamide, and leucine- and methionine-enkephalins was detected in the nerve fibers within the larynx and the lung. Together with our previous data, the present findings suggest that peptidergic mechanisms are involved in the regulation of amphibian respiratory systems throughout their life.

Animals↗

PubMed1995

Distribution of galanin-immunoreactive nerve fibers in the carotid labyrinth of the bullfrog, Rana catesbeiana: Comparison with substance P-immunoreactive fibers.

Immunoreactivity of galanin (GAL) was detected in the nerve fibers distributed within the intervascular stroma of the bullfrog carotid labyrinth. GAL-immunoreactive fibers are numerous, and some are close to the sinusoidal plexus. Most GAL fibers appear as thin processes with some varicosities. A combination of indirect double immunofluorescence labelling and image processing clearly demonstrated that the distribution pattern of GAL fibers is different from that of SP fibers. This indicates that GAL and SP do not coexist in the same nerve fibers. The role of GAL fibers may be different from that of previously reported neuropeptides (substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y, and others) as a neuromodulator in controlling vascular tone of the labyrinth.

Animals↗

PubMed1995

Structures of cDNAs encoding the muscle-type and non-muscle-type isozymes of lamprey fructose bisphosphate aldolases and the evolution of aldolase genes.

Nearly full-length cDNA clones for muscle-type and non-muscle-type aldolase mRNAs were cloned from lambda gt10 cDNA libraries constructed from skeletal muscle and liver mRNAs of lamprey (Entosphenus japonicus). The cDNA-M8 has 2,240 bp carrying an open reading frame of 1,089 bp which encodes 362 amino acids without the amino terminal methionine, while the cDNA-L3 is 1,761 bp in length and has an open reading frame of 1,092 bp, which encodes 363 amino acids without the methionine. We designated the cDNA clones M8 and L3 as the muscle-type and non-muscle-type aldolase cDNAs, respectively. The entire amino acid sequences deduced from cDNA-M8 and -L3 show a high degree of identity to one another (76%) and also to vertebrate aldolases A (74-76%), B (68-70%), and C (71-76%) and Drosophila melanogaster aldolases alpha, beta, and gamma (66-67%). Northern blot analyses using the 3'-noncoding sequences of cDNA-M8 and -L3 as hybridization probes indicated that the muscle-type mRNA is expressed mainly in the skeletal muscle, heart muscle, brain, and some other tissues, but probably not in liver, while the non-muscle-type mRNA is expressed mainly in the liver and also in brain and other tissues, except for the heart muscle. Phylogenetic analyses showed that both muscle-type and non-muscle-type aldolases of lamprey resemble one another and might share a common ancestor with vertebrate aldolases A and C, but they are not direct ancestors of vertebrate aldolases.

Amino Acid Sequence↗

PubMed1995

Peptidergic innervation in the amphibian carotid labyrinth.

The amphibian carotid labyrinth, which corresponds to the mammalian carotid body and carotid sinus, is innervated by nerve fibers containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), FMRFamide (FMRF), and somatostatin (SOM). SP, CGRP, VIP, and NPY immunoreactive varicose fibers are more densely distributed in the peripheral portion of the carotid labyrinth than FMRF and SOM fibers. The time of appearance of SP, CGRP, and VIP is different for each. First CGRP fibers, then SP fibers appear at an early stage of larval development, and finally VIP fibres are detected at a later stage of larval development. Most SP fibres show coexistence with CGRP, and some SP fibres which show coexistence with NPY immunoreactivity are assumed to be continuous with those demonstrating VIP immunoreactivity. This indicates the possibility of coexistence of four different peptides in the same nerve fibers within the labyrinth. In various vasculatures of mammals, it has been shown that SP, CGRP, VIP, and NPY have a vasoactive nature in relation to the vascular smooth muscle cells. On this basis, it seems that the target of the peptidergic innervation in the amphibian carotid labyrinth is the smooth muscle cells which are abundantly distributed in the intervascular stroma. Accordingly, the peptidergic innervation may be involved in the vascular regulatory function of the labyrinth, although the possibility that these peptides participate in the chemoreception cannot be ruled out. In addition, the vascular regulatory function of the labyrinth may be modulated by the interaction of multiple neuropeptides.

Amphibians↗

PubMed1994

Coexistence of substance P, neuropeptide Y, VIP, and CGRP in the nerve fibers of the carotid labyrinth of the bullfrog, Rana catesbeiana: a double-labelling immunofluorescence study in combination with alternate consecutive sections.

Double immunohistochemical staining with rhodamine- and fluorescein isothiocyanate (FITC)-conjugated antisera revealed the coexistence of substance P (SP) and neuropeptide Y (NPY), and SP and calcitonin gene-related peptide (CGRP) in most nerve fibers in the intervascular stroma of the carotid labyrinth of the bullfrog, Rana catesbeiana, although there were a few fibers which showed only SP- or NPY-immunoreactivity. Approximately one third of SP-immunoreactive fibers also showed coexistence with vasoactive intestinal polypeptide (VIP)-immunoreactivity, and a few fibers contained VIP without SP. The combination of the double immunofluorescence technique and alternate consecutive sections further demonstrated the possible coexistence of SP, VIP, NPY, and CGRP. This coexistence of four different peptides in the same nerve fibers was proved by the following two evident facts: 1) some SP fibers which demonstrated coexistence with NPY-immunoreactivity were assumed to be continuous with those showing VIP-immunoreactivity, and 2) almost all of the SP fibers showed coexistence with CGRP-immunoreactivity. By this reasoning, nearly one third of SP fibers may demonstrate coexistence with NPY-, VIP-, and CGRP-immunoreactivities. These multiple peptides might be involved in vascular regulatory function, which is a possible function of the amphibian carotid labyrinth.

Animals↗

PubMed1994

Human aldolase B: liver-specific properties of the isozyme depend on type B isozyme group-specific sequences.

A series of chimeric enzymes between two human aldolases A, B or C were constructed to identify the molecular regions responsible for isozyme-specific functions. Chimeras constructed between aldolases A and B were AB34 (an AB chimera connected at position 34), ABA34-306 and ABA212-306 (the ABA chimeras). Those between aldolases B and C are BC243, BC263 and BC306 (the BC chimeras connected at positions as indicated), as well as CB55, CB243, CB263 and CB306 (the CB chimeras connected at positions as indicated), CBC55-263 (a CBC chimera), and BCB55-193, BCB55-306, BCB79-193 and BCB79-306 (the BCB chimeric enzymes). Through the analysis of the properties of these chimeras, it was found that for aldolase B, isozyme B group-specific sequences (IGSs)-1 and -4 were required for exerting type B-specific functions, while the IGSs-2 and -3 enhanced, in collaboration with the IGS-1, the catalytic activity of aldolase B. In addition, the alpha/beta-barrel and the restricted stretches, which were not specified but occupied two distinct regions spanning the amino acid positions 108-137 (designated connector 1) and 243-306 (designated connector 2), were found to be indispensable for showing full catalytic activity of aldolase B.

Amino Acid Sequence↗

PubMed1994

Distribution of substance P-containing and catecholaminergic nerve fibers in the rabbit carotid body: an immunohistochemical study in combination with catecholamine fluorescent histochemistry.

The distribution of substance P (SP)-immunoreactive nerve fibers in the rabbit carotid body was studied in combination with catecholamine autofluorescence images of sections where SP immunoreactivity was confirmed. Immunoreactivity for SP was found in nerve fibers distributed in the parenchyma of the carotid body. No glomus cells with SP immunoreactivity were observed in the carotid body. On comparing the distribution of SP-immunoreactive fibers with the catecholamine autofluorescence image in a single section, most SP fibers appeared associated with the fluorescent glomus cells, and were located around clusters of them. These results support the suggestion that SP fibers in the cat and rat carotid bodies are involved in chemosensory mechanisms. Furthermore, a survey of the present results and previous ones reported by other workers indicates that SP may be an essential neuropeptide in chemoreceptor organs in most vertebrates from amphibians on upwards evolutionally. In addition, the courses of some catecholaminergic fibers precisely agreed with those of some SP fibers. This suggests that certain sympathetic nerve fibers also contain SP.

Animals↗

PubMed1993

Immunohistochemical coexistence of calcitonin gene-related peptide and substance P in the nerve fibers of the internal gills of bullfrog (Rana catesbeiana) larvae.

A double-labelling immunofluorescence study of the internal gills of larval bullfrogs revealed coexistence of calcitonin gene-related peptide (CGRP) and substance P (SP) in the same nerve fibers in the gill tufts. More than about 95% of CGRP fibers showed coexistence of SP, although some fibers contained CGRP without SP. In the branchial muscle, all CGRP fibers demonstrated coexistence of SP. These findings suggest that both CGRP- and SP-containing fibers may use both peptides as neuromodulators of active ion transport and gill movements.

Animals↗

PubMed1993

Coexistence of substance P and calcitonin gene-related peptide in the nerve fibers of the carotid labyrinth of the bullfrog, Rana catesbeiana.

Double immunohistochemical staining with fluorescein isothiocyanate (FITC)- and rhodamine-conjugated antisera revealed the coexistence of substance P (SP) and calcitonin gene-related peptide (CGRP) in most nerve fibers in the intervascular stroma of the carotid labyrinth of the bullfrog, Rana catesbeiana. A few fibers contained SP without CGRP. The results suggest that the vascular regulatory function, which is one of the possible functions of the carotid labyrinth, may be controlled in part by the interaction of SP and CGRP.

Animals↗

PubMed1993

Mechanism of inhibitory action of capsaicin on particulate axoplasmic transport in sensory neurons in culture.

The inhibitory effect of capsaicin on axoplasmic transport in cultured dorsal root ganglion cells was analyzed by video-enhanced contrast microscopy. Capsaicin inhibited particle transports in a dose-dependent manner, irrespective of the diameter of axons. The effect of capsaicin was reversible at low concentrations. Capsaicin affected both the anterograde and retrograde transport. Large organelles were more sensitive to capsaicin than small ones in the retrograde transport. An experiment using calcium-sensitive dye, Fura 2, indicated that capsaicin raised the intraneuronal free calcium concentration preceding the inhibition of the transport. Electron microscopy revealed that microtubules and neurofilaments are disorganized and disoriented by capsaicin. We reached a conclusion that capsaicin inhibits fast axoplasmic transport of both anterograde and retrograde directions in all types of somatosensory neurons in culture by disorganizing intraaxonal cytoskeletal structures, through the elevated intracellular Ca2+ concentration.

Animals↗

PubMed1993

Distribution of FMRFamide-immunoreactive nerve fibers in the carotid labyrinth of the bullfrog, Rana catesbeiana in corresponding differential interference-contrast (Nomarski) images.

Immunoreactivities for FMRFamide and substance P (SP) in the carotid labyrinth of the bullfrog were detected using the peroxidase-antiperoxidase method, and the results compared with corresponding differential interference-contrast (Nomarski) images. Colocalization of both peptides was determined by the indirect double immunofluorescence method. Immunoreactivities for FMRFamide and SP were found in nerve fibers distributed in the intervascular stroma of the labyrinth. The FMRFamide-immunoreactive fibers were less numerous than the SP-immunoreactive fibers. In the Nomarski image, FMRFamide-fibers were recognized in relief, with most of them located near the walls of blood vessels. All FMRFamide-fibers coexisted with SP. The results suggest that FMRFamide-immunoreactive fibers are also involved in local vascular regulation of the carotid labyrinth.

Animals↗

PubMed1992

Tunicate muscle actin genes. Structure and organization as a gene cluster.

We have isolated and determined the complete nucleotide sequences of two genes, HrMA4a and HrMA2, which encode the same muscle actin protein of the tunicate Halocynthia roretzi. HrMA4a and HrMA2 contain three exons, and the genes have intron-exon splice junctions at the same positions. The 5' flanking region of HrMA4a gene contains several potential regulatory elements. A TATA box is located at -30 and a CArG box found in regulatory region of vertebrate muscle-specific genes is located at -116. Seven E-box consensus sequences (CANNTG) known as binding sites for vertebrate myogenic determination factors are found within a 500 base-pair portion of the 5' flanking region of HrMA4a gene. HrMA4a and HrMA2 are separated by 1600 bases in genomic DNA and transcribed in the same direction. In addition to these genes, we have identified three other actin genes encoding muscle-type actins. All five actin genes are located in a 30 x 10(3) base-pair region of the genome and aligned in the same direction. This is the first report of a cluster of "vertebrate-type" muscle actin genes. The consensus sequences of 5' flanking region are conserved among these five genes, suggesting that the expression of the genes is controlled coordinately. This may be advantageous for the accumulation of considerable amounts of actin proteins in rapidly developing embryos of this animal.

Actins↗

PubMed1992

Ultrastructural characteristics of glomus cells in the external carotid artery during larval development and metamorphosis in bullfrogs, Rana catesbeiana.

Electron microscopic observations of the external carotid artery in the larvae of the bullfrog, Rana catesbeiana, showed that glomus cells are present in the subendothelial stroma of the septum between the expanded region of the external carotid artery and the carotid arch. There were some differences in the ultrastructure of the glomus cells at each stage of larval development. At the early stages (stages I, III, V, X), most glomus cells were isolated and free from the covering of a supporting cell. The cytoplasm of the glomus cells contained fewer dense-cored vesicles. No synaptic junctions were observed. At the middle stages (stages XV, XX, XXI), some glomus cells showed a tendency to form small clusters. Between adjacent cells in a cluster, gap junctions were often observed. The number of dense-cored vesicles increased remarkably. Intimate apposition of the glomus and smooth muscle cells (g-s connection) was also observed. Nerve terminals containing clear vesicles were observed in synaptic contact with glomus cells at this phase. At the metamorphic climax (stages XXII-XXV), in addition to g-s connections, the glomus cells made intimate apposition to the cells around the glomus cells. The afferent synapses described in other amphibians were not encountered in this study. These findings suggest that the glomus cells at the early stages of development are nonfunctional, the vascular regulation via the g-s connection starts at the middle stages, and the chemoreception starts after metamorphosis.

Animals↗

PubMed1992

Intimate apposition of the glomus and smooth muscle cells (g-s connection) in the carotid labyrinth of juvenile bullfrogs.

Intimate apposition of the glomus and smooth muscle cells (g-s connection) was found in almost all glomus cells of the carotid labyrinths in juvenile bullfrogs, Rana catesbeiana. There were three types of g-s connection: between thin processes (0.1-0.2 micron in width) without dense-cored vesicles of glomus cells and smooth muscle cells; between thick processes (1.0-1.5 microns in width) with dense-cored vesicles of glomus cells and smooth muscle cells; and between the tongue-like projections of smooth muscle cells and the flat surface of the glomus cell. In some cases, a single glomus cell made g-s connections with several smooth muscle cells. Exocytosis often occurs at the g-s connection. Afferent and efferent synapses were found on the glomus cells with g-s connections. Reciprocal synapses were also observed. On the basis of these findings, the second and third types of g-s connection are presumed to participate in vascular regulation.

Animals↗

PubMed1992

Ontogeny of substance P-, CGRP-, and VIP-containing nerve fibers in the amphibian carotid labyrinth of the bullfrog, Rana catesbeiana. An immunohistochemical study.

The ontogeny of substance P, CGRP (calcitonin gene-related peptide), and VIP (vasoactive intestinal polypeptide) containing nerve fibers in the carotid labyrinth of the bullfrog, Rana catesbeiana, was examined by the peroxidase-antiperoxidase method. The time of appearance of these three peptides was different for each. First, CGRP fibers appeared in the wall of the carotid arch and external carotid arteries, and in a thin septum between these two arteries at an early stage of larval development (stage III). At stage V, substance P immunoreactive fibers appeared, and VIP fibers were detected at the early metamorphic stage (stage XXII). Up to the completion of metamorphosis, the number of these fibers remained low. From 1 to 5 weeks after metamorphosis, substance P, CGRP, and VIP fibers increased in number to varying degrees. By 8 weeks after metamorphosis, the distribution and abundance of these fibers closely resembled those of the adults. Some CGRP and VIP immunoreactive glomus cells were found at the stages immediately before and after the completion of metamorphosis. These findings suggest that substance P, CGRP, and VIP fibers during larval development and metamorphosis may be nonfunctional, and start to participate in vascular regulation only after metamorphosis. The transient CGRP and VIP in some glomus cells may be important for the development of the labyrinth, or may take part in vascular regulation through the close apposition of the glomus and smooth muscle cells (g-s connection).

Animals↗