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Biomedical subjects

K Watabe

Publications and source records attributed to K Watabe.

At least 145 records · Page 8Linked to original sources

Tactile-like corpuscles in neurofibromas: immunohistochemical demonstration of S-100 protein.

Tactile-like corpuscles in neurofibroma tissues were examined by the indirect immunoperoxidase method using rabbit anti-S-100 sera. In all five cases examined, positive staining was observed in their constituent cells, both in the flattened cytoplasm and nuclei. On examination of the normal peripheral nervous system, positive staining was exclusively confined to Schwann cells in the nerve bundles and Schwann-related cells such as lamellar cells in the Meissner corpuscle, whereas staining was negative in perineurial cells. Electron-microscopic examination showed ultrastructural similarity between constituent cells of the tactile-like corpuscles in neurofibroma and lamellar cells in the Meissner corpuscle. These findings may indicate Schwannian nature of the tactile-like corpuscles.

Adolescent↗

Protein-primed initiation of phage phi 29 DNA replication.

We recently reported the development of an in vitro replication system for bacteriophage phi 29 DNA. We have used this system for the isolation of replication activity associated with gene 3 protein (terminal protein) from phi 29-infected Bacillus subtilis cells. We utilized two assay systems: (i) DNA replication dependent on phi 29 DNA with the 5' end covalently linked to terminal protein (DNA-protein) and (ii) the formation of complex between the terminal protein and dAMP. The DNA-replication and the complex-forming activities were purified together through all steps. The complex of terminal protein and dAMP formed in the purified fraction was shown to serve as an effective primer for successive chain elongation in the presence of dNTPs by a pulse-chase experiment. The protein fraction purified from cells infected with a temperature-sensitive phi 29 mutant in gene 3 was thermolabile compared to the wild-type activity in the assay system for complex formation. This shows that the purified fraction having replication activity includes the gene 3 product of phi 29. Both the DNA replication and the complex formation activities are highly specific for phi 29 DNA-protein as template. The product analysis of elongated DNA revealed that the replication starts at both termini of the phi 29 genome. These results are consistent with the basic elements of the protein-priming model for the initiation of linear DNA synthesis.

Bacillus subtilis↗

Visual afferents to norepinephrine-containing neurons in cat locus coeruleus.

A total of 208 single neurons were extracellularly recorded in the locus coeruleus (LC) of 11 cats. In later histofluorescence studies, greenish fluorescent LC neurons, from which we believed to have recorded well-isolated action potentials, were always found in or close to the center of red fluorescent halo due to an injected dye which marked the recording site. One hundred twelve of these 208 neurons were further subjected to electrical stimulation of the dorsal bundle of ascending axons originating from the norepinephrine (NE)-containing LC neurons and thus activated antidromically with a mean latency of 8.9 ms (the remaining neurons were lost before this examination). The mean conduction velocity was 1.2 m/s. Furthermore, it is suggested that 22% of thus antidromically identified NE neurons in the cat LC had an ascending axon of the conduction velocity faster than 2.4 m/s. This finding may be related with an electron-microscopic observation which indicated the presence of myelinated catecholamine (CA) axons, though not many in number, in the cat visual cortex. Responses by the NE-containing LC neurons to various natural visual stimuli, such as flashlight, moving and stationary light-slit, multiple spots, and gratings were examined. It turned out, however, that flash alone was effective to activate LC neurons. The latency of flash evoked activity was between 48 and 96 ms (N = 12; mean: 60 ms). Furthermore, the following areas in the central visual pathway were electrically stimulated to activate LC neurons orthodromically: the optic chiasm (OX), the dorsal lateral geniculate nucleus (LGN), the superior colliculus (SC), and the visual cortex (VC). The range and the mean of the latency for orthodromic responses were as follows: OX (N = 36, 8.4-42 ms; mean: 21 ms); LGN (N = 17, 6.0-17 ms; mean: 8.1 ms); SC (N = 12, 3.6-12 ms; mean: 5.6 ms); VC (N = 10, 7.8-40 ms; mean: 16.4 ms). The long latency of these orthodromic responses and its wide distribution suggest that afferents to the LC from the above-mentioned visual structures are most likely polysynaptic in nature. The extensive input convergence, including acoustic and nociceptive afferents, and the polysynaptic connection in each afferent pathway indicated a strong similarity between the afferent connectivity of NE-containing LC neurons revealed in the present study and that known for reticular formation neurons. Then, we would like to suggest that visual signals from the eyes impinge upon the NE-containing LC neurons via the reticular formation and that the afferents from the LGN, the SC, and the VC also join this common path through the reticular formation to reach the LC.

Animals↗

In vitro replication of bacteriophage phi 29 DNA.

We have been studying the mechanisms of linear DNA replication by using Bacillus bacteriophage phi 29 as a model system. To isolate and characterize the proteins required for phi 29 DNA replication, we have developed a cell-free replication system. A cell-free extract prepared from phi 29-infected Bacillus subtilis catalyzes the semiconservative replication of phi 29 DNA, but only if exogenous phi 29 DNA-protein complex is used as the template. This template consists of linear duplex DNA with a 30,000-dalton terminal protein attached covalently to both 5' ends. Replication starts nonsimultaneously at or near both ends of the template. The extract also catalyzes the specific binding between dATP and the phi 29 terminal protein. Thus, the in vitro system closely mimics the in vivo replication of phi 29 DNA. This system should allow characterization of the phi 29 DNA replication machinery.

Bacillus subtilis↗

In vitro conversion of S13 viral DNA in phage particles to the double-stranded DNA.

The conversion of single-stranded DNA in S13 intact phage particles to the double-stranded replicative form DNA was observed in cell extracts prepared from Escherichia coli H560 (S13s, polA, endA) cells lysed with lysozyme and the non-ionic detergent, Brij 58. The DNA product, which associated with a rapidly sedimenting component, was identified as RFII-DNA with a gap by sedimentation analysis. The conversion was inhibited by N-ethylmaleimide, but not by rifampicin, nicotinamide mononucleotide or polymyxin B. The dnaB gene product was involved in the replicative system. Similar extracts prepared from a S13-resistant E. coli strain K12W6 also catalyzed this synthesis.

Bacterial Proteins↗

Involvement of host cell gene products in conversion of bacteriophage S13 single-stranded DNa to duplex replicative form DNA in vitro.

The single-stranded circular DNA of bacteriophage S13 was converted to the duplex replicative form DNA by soluble extracts from Escherichia coli strain H560 (polA, endA) in vitro. The maximal conversion required four deoxyribonucleoside triphosphates, Mg2+, exogenous S13 DNA and ATP, but not CTP, UTP, or GTP. The conversion was blocked by N-ethylmaleinimide but not by rifampicin. The product was identified as a gapped duplex replicative form DNA. Using extracts from some thermosensitive mutants of E. coli defective in DNA replication, we found that dnaB and dnaC gene products are involved in the conversion stage of single-stranded DNA to duplex DNA in vitro.

Coliphages↗

Protein synthesis in the isolated forespores from sporulating cells of Bacillus subtilis.

Developing forespores were isolated from Bacillus subtilis at different stages of sporulation and protein synthesis in the forespore compartment was examined. Pulse-labeling experiments indicated that [14C]phenylalanine was continuously incorporated into the sporangium throughout sporulation, and at t5 (early stage V of sporulation) 58% of the radioactivity was located in the forespore compartment. Significantly high incorporation of [14C]phenylalanine was observed when the isolated forespores at t5 were incubated with the corresponding mother-cell cytoplasmic fraction or an amino acid mixture. About 73% of the radioactivity incorporated into the isolated forespore at t5 was found in the cytoplasmic fraction and 26% in the membranous fraction. Analysis by sodium dodecyl sulfate-gel electrophoresis showed that the 14C-labeled cytoplasmic protein had a molecular weight of about 20,000, and that a protein having the same molecular weight was present in the t5 forespore as a slight protein band and also in the mature spore as a clear protein band. Gel electrophoresis also revealed that the 14C-labeled membranous-soluble protein (prepared by solubilization with detergents) had broad peaks with molecular weights of about 74,000, 33,000, 20,000, and 12,000.

Bacillus subtilis↗

Presynaptic inhibition of tooth pulp afferents in the trigeminal nucleus during REM sleep.

In order to find whether presynaptic inhibition of tooth pulp afferents is enhanced in any phase of sleep or wakefulness, the orthodromic mass response of the trigeminal nucleus to electrical stimulation of the ipsilateral tooth pulp and the antidromic mass response of the tooth pulp to stimulation of the trigeminal nucleus were recorded in the cat. Both responses were stable in amplitude during different phases of sleep and wakefulness. However, attenuation of the orthodromic response and augmentation of the antidromic one were sometimes observed during the burst of rapid eye movements during paradoxical sleep, suggesting that presynaptic inhibition is massively enhanced only on those occasions.

Afferent Pathways↗