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We describe a low-cost, small, remotely triggerable LED device for wireless control of transcranial optical stimulation of cortical neurons, for use in freely moving mice. The device is easily mountable on the head of a mouse with a high-polymer block. Using the Thy1-ChR2-YFP transgenic mice, we demonstrate that the device is capable of remotely triggering muscle twitches upon activation of the primary motor cortex in freely moving conditions., (Copyright © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Objective: To clarify whether the ratio of tone burst vestibular evoked myogenic potential (VEMP) amplitude to galvanic (electric) VEMP amplitude can be a useful indicator of peripheral vestibular disorders, especially labyrinthine disorders., Methods: Twelve healthy volunteers and 12 patients with endolymphatic hydrops (EH) were enrolled in this study. VEMP was recorded using 500 Hz short tone bursts (135dBSPL, rise/fall time 1 ms, plateau time 2 ms) and galvanic stimulation (3 mA, 1 ms). Amplitudes of p13-n23 (tone burst) and p13g-n23g (galvanic) were corrected using background muscle activity. We defined the ratio of the corrected amplitude of p13-n23 to the corrected amplitude of p13g-n23g as TGratio., Results: The mean+/-SD of logarithmic value of TGratio (LTGratio) of healthy volunteers was 0.34+/-0.12. LTGratios in patients were 0.002+/-0.24 on the affected side and 0.34+/-0.14 on the unaffected side. LTGratio on the affected side of patients was significantly smaller than the ratio on the unaffected side and the ratio of healthy volunteers. Among the 12 patients with EH, 8 patients (66%) had significantly decreased LTGratio (smaller than mean-2SD of healthy volunteers)., Conclusions: Patients with EH had decreased ratio of tone burst VEMP amplitude to galvanic VEMP amplitude., Significance: TGratio (or LTGratio) could be a new promising parameter of VEMP as it can be calculated from results of one side.

Endothelin-1 (ET-1), a 21-amino acid peptide secreted by the epithelium and core mesenchyme in the branchial arches as well as vascular endothelium, is involved in craniofacial and cardiovascular development through endothelin receptor type-A (EdnrA) expressed in the neural crest-derived ectomesenchyme. Here we show that ET-1(-/-) mutant mice exhibit a homeotic-like transformation of the lower jaw to an upper jaw. Most of the maxillary arch-derived components are duplicated and replaced mandibular arch-derived structures, resulting in a mirror image of the upper and lower jaws in the ET-1(-/-) mutant. As for hyoid arch-derivatives, the ventral structures are severely affected in comparison to the dorsal ones in the ET-1(-/-) mutant. Correspondingly, the expression of Dlx5 and Dlx6, Distalless-related homeobox genes determining the ventral identity of the anterior branchial arches, and of the mandibular marker gene Pitx1 is significantly downregulated in the ET-1(-/-) mutant, whereas the expression of Dlx2 and the maxillary marker gene Prx2 is unaffected or rather upregulated. These findings indicate that the ET-1/EdnrA signaling may contribute to the dorsoventral axis patterning of the branchial arch system as a mediator of the regional intercellular interactions.

Effects of sinusoidal grating stimulus presented outside the classical receptive field (CRF) on neuronal responses were studied in the primary visual cortex of anaesthetized cats. Among 101 cells electrophysiologically recorded, the predominant effect of the stimulus in the receptive field surround (SRF) was the suppression of responses to the CRF stimulation, and the SRF grating suppressed them up to 56% of the responses (44% suppression) to the CRF stimulus alone. The strong suppression was observed more often in layer II/III cells than in other layers and in complex cells more often than in simple cells. The modulatory effects by SRF stimulus might be enhanced by the cortical recurrent excitation particularly in the superficial layers. We also examined whether the modulation by the surround grating exhibits a differential effect according to the presence or absence of figure-ground segregation in the stimulus configuration. For this purpose, effects of stimulus configuration with orientation-, direction-contrast or relative spatial phase difference between CRF and SRF stimuli (figure-ground segregated configuration) were compared with those of uniform configuration of stimulus (non-segregated configuration). There was a population of cells, which exhibited significantly stronger suppression with non-segregated configuration than with figure-ground segregated configuration. Such differential modulation of response by the SRF stimulus in the primary visual cortex is a possible basis of perceptual figure-ground segregation.

Objectives: To show that galvanic-evoked responses on the sternocleidomastoid muscle (SCM) are useful for differentiating labyrinthine lesions from retro-labyrinthine lesions in patients with an absence of click-evoked vestibulo-collic reflexes., Methods: We studied the average responses in the unrectified electromyographic (EMG) activities of the SCM to galvanic stimulation (3mA, 1ms). The cathodal electrode was on the mastoid, and the anodal electrode was on the forehead. Twenty-two healthy subjects and 28 patients with vestibular disorders were studied. All of the 28 patients showed the unilateral absence of vestibulo-collic reflexes evoked by 95dBnHL clicks on the affected side., Results: In healthy subjects mastoid-forehead galvanic stimulation produced a positive-negative biphasic EMG response at short latency on the SCM ipsilateral to the cathodal electrode. All patients with labyrinthine lesions showed biphasic EMG responses even in the affected side. In contrast, almost all patients with retro-labyrinthine lesions (16/18) showed no response or a decreased response on the affected side., Conclusions: These results suggest that galvanic-evoked myogenic responses on the SCM may be useful in the differential diagnosis of labyrinthine lesions from retro-labyrinthine lesions in patients with an absence of vestibulo-collic reflexes evoked by clicks.

Mammalian melanins exist in two chemically distinct forms; the brown to black eumelanins and the yellow to reddish pheomelanins. Eumelanins are derived from copolymerization of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Eumelanins can be quantified by HPLC analysis of the oxidation product pyrrole-2,3,5-tricarboxylic acid (PTCA) and by our spectrophotometric method (Sp.EM). We also developed a spectrophotometric method for assaying the total amount of eu- and pheomelanins by dissolving them in Soluene-350 (TM). In addition, we previously showed that Sp.EM/TM and PTCA/TM ratios are significant parameters in characterizing eumelanins produced in follicular melanocytes. The objectives of this study were (1) to clarify the significance of Sp.EM/TM and PTCA/TM ratios in synthetic eumelanins and (2) to apply these methods to characterizing natural eumelanins with various DHI/ DHICA ratios and molecular sizes. The results obtained show that (1) the Sp.EM/TM ratio of synthetic eumelanins increases as polymerization proceeds, (2) the Sp.EM/TM and PTCA/TM ratios in copolymers of DHI and DHICA correlate to the percentage content of DHICA-derived units, and (3) combination of the Sp.EM/TM and PTCA/TM ratios serves to estimate the DHICA content and the degree of polymerization in natural eumelanins.

Mammalian melanins exist in two chemically distinct forms: the brown to black eumelanins and the yellow to reddish pheomelanins. Melanogenesis is influenced by a number of genes, the levels of whose products determine the quantity and quality of the melanins produced. To examine the effects of various coat-color genes on the chemical properties of melanins synthesized in the follicular melanocytes of mice, we have introduced new methods to solubilize differentially pheomelanins and brown-type eumelanins. We applied these and previously developed high-performance liquid chromatography and spectrophotometric methods for assaying eu- and pheomelanins to characterize melanins in various mutant mice: black, lethal yellow, viable yellow, agouti, brown, light, albino, dilute, recessive yellow, pink-eyed dilution, slaty, and silver. It was demonstrated that 1) complete solubilization of melanins in Soluene-350 is a convenient method to estimate the total amount of eu- and pheomelanins, 2) lethal yellow, viable yellow, and recessive yellow hairs contain almost pure pheomelanins, and 3) melanins from brown, light, silver, and pink-eyed black hairs share chemical properties in common that are characterized by partial solubility in strong alkali. We suggest that 1) the brown-type eumelanins have lower degrees of polymerization than the black-type eumelanins, and 2) slaty hair melanin contains a greatly reduced ratio of 5,6-dihydroxyindole-2-carboxylic acid-derived units as compared with black and other eumelanic hair melanins. These results indicate that our methodology, high-performance liquid chromatography and spectrophotometric methods combined, may be useful in chemically characterizing melanin pigments produced in follicular melanocytes.

Most of the natural melanin pigments consist of not only indolic eumelanin but also sulfur-containing pheomelanin. Previous methods for spectrophotometric assay of melanins use solubilization in alkaline media; a major disadvantage of these procedures is that they do not distinguish between eumelanin and pheomelanin. A spectrophotometric method for assaying eumelanin in tissue samples is described. Sepia melanin serves as a standard. Hair and melanoma samples were hydrolyzed in hot hydriodic acid, and insoluble eumelanic pigments were solubilized in hot sodium hydroxide in the presence of hydrogen peroxide and analyzed for absorbance at 350 nm (A350). The detection limit of eumelanin was ca. 2 micrograms. Eumelanins prepared from dopa, 5,6-dihydroxyindole and its carboxy derivative gave similar A350 values. Mixed-type melanins prepared from dopa and various ratios of cysteine gave A350 values inversely proportional to their sulfur contents. Excellent correlations were observed between A350 values and contents of pyrrole-2,3,5-tricarboxylic acid, an oxidation product specific for eumelanin, in hair samples from sheep and humans of various colors and in melanomas and eyes from mice. The present method provides a specific and direct measurement of eumelanin contents in tissue samples.

By using a set of 476 ordered DNA clones (in lambda phage vector) that covers the entire chromosome of Escherichia coli K12, we have made an exhaustive survey of tRNA genes in the E. coli genome. Ultraviolet-irradiated bacteria were separately infected with each of the 476 clones and the RNA molecules produced upon infection were labeled with 32P. The labeled tRNAs were separated by gel electrophoresis and then characterized by fingerprinting analysis. Fifty-nine of the 476 clones produced tRNAs, including adjacent overlapping ones that share the same tRNA genes. The products of all the previously mapped tRNA genes (about 60, to date) were detected according to their expected positions, and 19 more tRNA genes were newly elucidated. These new tRNA genes were identified by sequencing the DNA from relevant regions of the clones; the DNA sequences were scanned for the stretches that could be folded into the familiar cloverleaf structure and the transcription units were deduced by predicting the promoters and terminators. The total complement of the tRNA genes in E. coli K12 was 78 for 45 tRNA (or 41 anticodon) species, distributed in 40 different transcription units throughout the chromosome. In addition, a gene for selenocysteine tRNA was detected by hybridization and mapped to a specific DNA segment. A comprehensive tRNA gene map of E. coli was constructed, including the selenocysteine tRNA gene. All the tRNA genes encode the 3' CCA, and in several cases the terminal 19 nucleotides (including the 3' CCA) of a tRNA gene is repeated several times. Finally, in the present study the sites for a long inversion (approx. 800 x 10(3) base-pairs, around the oriC region) in Kohara's library was determined to be within the 23 S-5 S regions in rrnD and rrnE, revealing the exchange of combinations of spacer and distal tRNA genes between these two ribosomal RNA operons.

The nucleotide sequence (25,320 base-pairs) of a part of the large single-copy region of chloroplast DNA from the liverwort Marchantia polymorpha was determined. This region encodes putative genes for four tRNAs, isoleucine tRNA(CAU), arginine tRNA(CCG), proline tRNA(UGG) and tryptophan tRNA(CCA); eight photosynthetic polypeptides, the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL), 51,000 Mr photosystem II chlorophyll alpha apoprotein (psbB), apocytochrome b-559 polypeptides (psbE and psbF), 10,000 Mr phosphoprotein (psbH), cytochrome f preprotein (petA), cytochrome b6 polypeptide (petB), and cytochrome b6/f complex subunit 4 polypeptide (petD); 13 ribosomal proteins (L2, L14, L16, L20, L22, L23, L33, S3, S8, S11, S12, S18 and S19); initiation factor 1 (infA); ribosome-associating polypeptide (secX); and alpha subunit of RNA polymerase (rpoA). Functionally related genes were located in several clusters in this region of the genome. There were two ribosomal protein gene clusters: rpl23-rpl2-rps19-rpl22-rps3-rpl16-+ ++rpl14-rps8-infA-secX-rps11-rpoA, with a gene arrangement similar to that of the Escherichia coli S10-spc-alpha operons, and the rps12'-rpl20-rps18-rpl33 cluster. There were gene clusters encoding photosynthesis components such as the psbB-psbH-petB-petD and the psbE-psbF clusters. Thirteen open reading frames, ranging in length from 31 to 434 amino acid residues, remain to be identified.

An Escherichia coli DNA fragment containing an Su+6 amber suppressor gene (supP) was cloned into a lambda gt lambda Ch vector by the shotgun method, selecting a Su+6 transducing phage lambda pSu+6. Through prophage integration followed by induction occurring at the transducing region of the lambda pSu+6 in Su- E. coli, a counterpart transducing phage carrying the wild-type allele (Su degrees 6) was isolated (lambda pSu degrees 6). The fingerprint of a tRNA encoded by lambda pSu degrees 6 was identical to that of an unidentified tRNAE previously reported (Ikemura & Ozeki, 1977). The cloverleaf structure of this tRNA was determined by combining the results of tRNA analysis and DNA sequencing of the gene. Judging from the anticodon of 5'-CAA-3', Su degrees 6 tRNA was identified as a new type of leucine isoacceptor in E. coli. Unlike other suppressors analyzed, Su+6 tRNA differed by two nucleotides from Su degrees 6 tRNA; one at the anticodon (CAA to CUA) and the other at the junction of D- and anticodon-stem (A27 to G27). DNA sequence analysis revealed that a single stretch of tRNA is flanked by the putative sequences of promoter and terminator. Thus a single copy of the Su degrees 6 tRNA gene constitutes a solitary tRNA transcription unit. Southern blotting showed only one copy of Su degrees 6 tRNA gene per haploid genome of E. coli. Since this single gene can mutate to the Su+6 suppressor, the Su degrees 6 leucine tRNA may be accounted as a dispensable species among the leucine isoacceptor tRNAs. Two possible open reading frames are found immediately following the Su degrees 6 tRNA gene.

The nucleotide sequence (56,410 base-pairs) of the large single-copy region of chloroplast DNA from the liverwort Marchantia polymorpha has been determined. The sequence starts from one end (JLA) of the large single-copy region and encompasses genes for 21 tRNAs, six ATPase subunits (atpA, atpB, atpE, atpF, atpH and atpI), two photosystem I polypeptides (psaA and psaB), four photosystem II polypeptides (psbA, psbC, psbD and psbG), five ribosomal proteins (rps2, rps4, rps7, rps'12 and rps14), and three RNA polymerase subunits (rpoB, rpoC1 and rpoC2). In addition, we detected 18 open reading frames ranging from 29 to 2136 amino acid residues long, four of which share significant amino acid sequence homology to those of an Escherichia coli malK protein (designated mbpX), human mitochondrial ND2 (ndh2) and ND3 (ndh3) of a respiratory chain NADH dehydrogenase, or a bacterial antenna protein of a light-harvesting complex (lhcA). Sequence analysis suggests that four tRNA genes and six protein genes might be split by introns; they are trnG(UCC), trnK(UUU), trnL(UAA), trnV(UAC), atpF, ndh2, rpoC1, rps'12, ORF135 and ORF167. In the large single-copy region described here, the gene organization deduced is highly conserved with respect to that of higher plants, but an inversion of some 30,000 base-pairs flanked by trnL(CAA) and trnD(GUC) was seen between the liverwort and tobacco chloroplast genomes.

We characterized the genes in the regions of large inverted repeats (IRA and IRB, 10,058 base-pairs each) and a small single copy (SSC 19,813 bp) of chloroplast DNA from Marchantia polymorpha. The inverted repeat (IR) regions contain genes for four ribosomal RNAs (16 S, 23 S, 4.5 S and 5 S rRNAs) and five transfer RNAs (valine tRNA(GAC), isoleucine tRNA(GAU), alanine tRNA(UGC), arginine tRNA(ACG) and asparagine tRNA(GUU)). The gene organization of the IR regions in the liverwort chloroplast genome is conserved, although the IR regions are smaller (10,058 base-pairs) than any reported in higher plant chloroplasts. The small single-copy region (19,813 base-pairs) encoded genes for 17 open reading frames, a leucine tRNA(UAG) and a proline tRNA(GGG)-like sequence. We identified 12 open reading frames by homology of their coding sequences to a 4Fe-4S-type ferredoxin protein, a bacterial nitrogenase reductase component (Fe-protein), five human mitochondrial components of NADH dehydrogenase (ND1, ND4, ND4L, ND5 and ND6), two Escherichia coli ribosomal proteins (S15 and L21), two putative proteins encoded in the kinetoplast maxicircle DNA of Leishmania tarentolae (LtORF 3 and LtORF 4), and a bacterial permease inner membrane component (encoded by malF in E. coli or hisQ in Salmonella typhimurium).

The first attempt to study crystal structures of tRNA by electron microscopy is described. Sufficiently thin crystals were prepared from yeast tRNAphe. The thickness of the thinnest was estimated at 130 A corresponding to a bilayer of the molecules. The L-shaped structure seemed to be maintained even after the negative staining with uranyl acetate. Optically filtered images from electron micrographs were compared with those simulated from the drawing of the molecular model by optical transform. The results suggest that the observed images reflect the real molecular arrangements within the crystal lattice although the shape of tRNA molecules seems to be somewhat modified by the uneven staining.

Transducing phages of lambda carrying suppressors, lysT (Su+ beta), supG and and supL, were isolated in vivo. Upon infection with each of these phages, the production of tRNALys and tRNAVal1 was markedly enhanced. Fingerprint analysis of these tRNAs revealed that they consisted of normal tRNALys, mutant tRNALys and tRNAVal1 in equimolar ratios. The mutant tRNALys carried a single-base alteration at the anticodon, from 5'-UUU-3' to 5'-UUA-3', which makes it an ochre suppressor. DNA sequence analysis of the entire transducing fragment (730 base-pairs) of lambda pSu+ beta revealed that three tRNA genes are tightly clustered within a transcription unit in the following order; i.e. promoter-(48 base-pairs)-wild-type tRNALys-(132 base-pairs)-tRNAVal1-(2 base-pairs)-Su+ beta tRNALys-. In wild-type bacteria there are two identical tRNALys genes in one operon. Although we have shown that in Su+ beta it is the distal tRNALys that has been mutated to the ochre suppressor by a single base change at the anticodon (U36 to A36), we have not determined which of the two genes bears the supG or the supL mutation. The sequences following both tRNALys genes are highly homologous: both are about 100 base-pairs long and both terminate with an 18 base-pair sequence homologous to the last 18 bases of each tRNA. The sequences of tRNALys and tRNAVal1 are also very similar. Thus, including the 3'-portions of these tRNA genes, the 18 base-pair sequence is more or less periodically repeated five times in the DNA sequence.