Thermomechanical performance of continuous carbon fibre composite materials produced by a modified 3D printer.

First of all, this article aimed to evidence the role of a modified printer developed for continuous carbon fibre reinforced PolyAmide (cCF/PA6-I) together with the use of a fully open slicing step on the printing quality and the longitudinal/transverse tensile and in-plane shear properties. A comprehensive assessment of the microstructure and properties with a similar material (cCF/PA6-I), but produced with a commercial printer (i.e., Markforged® MarkTwo) has been achieved. Our customised printer and the open slicer used have made possible to better control the print conditions (i.e., layer height and distance between filaments), to reduce the porosity from more than 10% to about 2% and improve the mechanical properties. Moreover, the understanding of the behaviour of these 3D printed composites with wide-ranging external temperatures is mandatory for future use in a severe environment and/or development of new thermally active 4D printed composites. The 3D printed cCF/PA6-I composites have been then thermomechanically characterised along different printing directions (0, 90 and ± 45°) from -55 to +100 °C. Unlike the longitudinal properties that hardly change with temperature, the transverse and in-plane shear stiffness and strength of these 3D printed composites were particularly sensitive to temperature variations, with decreases of 25-30% and 30-55%, respectively. This was due to the high sensitivity of the polymer matrix, the fibre/matrix and interfilament interfaces when the composites were loaded along those directions, because damages induced by internal thermal stresses. Fractography has also been carried out to reveal damage mechanisms.

Cerebello-thalamo-cortical projections to the posterior parietal cortex in the macaque monkey.

The cerebello-thalamo-posterior parietal cortical projections were investigated electrophysiologically and morphologically in macaque monkeys. In anesthetized monkeys, electrical stimulation of every cerebellar nucleus evoked marked surface-positive, depth-negative (s-P, d-N) cortical field potentials in the superior parietal lobule and the cortical bank of the intraparietal sulcus, but no responses in the inferior parietal lobule. Tract-tracing experiments combining the anterograde method with the retrograde one indicated that the interposed and lateral cerebellar nuclei projected to the posterior parietal cortex mainly through the nucleus ventral lateralis caudalis of the thalamus. The significance of the projections is discussed in connection with cognitive functions.

Cortical field potentials associated with audio-initiated vocalization in monkeys.

Five monkeys vocalizing at self-pace (self-paced vocalization) were well trained to vocalize in response to a monkey call (audio-initiated vocalization). Field potentials associated with audio-initiated vocalizations were recorded by using electrodes which were implanted chronically on the surface and at a 2.0-3.0 mm depth in various cortical areas. A surface-negative (s-N), depth-positive (d-P) potential (at about 70 ms latency after stimulus onset) was recorded in the rostral bank of the inferior limb of the arcuate sulcus in the left hemisphere, in which an insignificant potential was associated with self-paced vocalizations. An s-N, d-P slow potential which occurred in the motor and somatosensory cortices with a latency of about 300 ms after stimulus, started about 700 ms before vocalizations. The duration and amplitude of this potential was substantially the same with those of the potential which occurred with self-paced vocalizations. Reaction times from stimulus onset to vocalization start were variable, but were about 0.9s on the average. The findings were discussed in connection with reaction-time hand movements.

Exploring a channel to the active site of copper/topaquinone-containing phenylethylamine oxidase by chemical modification and site-specific mutagenesis.

Copper amine oxidase contains an organic redox cofactor, 2,4, 5-trihydroxyphenylalaninequinone (topaquinone, TPQ), derived by the post-translational modification of a specific tyrosyl residue. To identify amino acid residues participating in the biogenesis of TPQ in the recombinant phenylethylamine oxidase from Arthrobacter globiformis, we have modified the copper/TPQ-less apoenzyme and the copper/TPQ-containing holoenzyme with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-F). In the apoenzyme modification, the Cu2+-dependent, self-processing formation of the TPQ cofactor was retarded in accordance with the amount of NBD incorporated. The holoenzyme was also rapidly inactivated by incubation with NBD-F. The inactivation was prevented almost completely in the presence of an oxidation product from phenylethylamine, phenylacetaldehyde. Furthermore, the reaction of an inhibitor, phenylhydrazine, with TPQ was much slower in the NBD-labeled holoenzyme than in the native holoenzyme. Sequence analysis of the NBD-labeled holoenzyme has identified Lys184 and Lys354 as the labeled sites. The two Lys residues are located close to the entrance to a channel, which has been found by recent X-ray crystallographic studies to be suitable for the movement of substrates and products to and from the Cu2+/TPQ-active site buried in the protein interior (Wilce, M. C. J., et al. (1997) Biochemistry 36, 16116-16133). However, site-specific mutant enzymes for Lys184, Lys354, and the neighboring invariant His355 had normal capacities for the TPQ formation in apoenzyme. These residues were also found to be dispensable for catalytic activity of holoenzyme. Thus, modification of Lys184 and Lys354 with NBD-F presumably causes structural perturbations of the substrate channel or steric hindrance for the access of small molecules to the active site through the channel.

Pontine neurons which relay projections from the superior colliculus to the posterior vermis of the cerebellum in the cat: distribution and visual properties.

Extracellular unit recording revealed that the dorsolateral pontine nucleus (DLPN) and nucleus reticularis tegmenti pontis (NRTP) of the cat constituted pontine relays for transmission of visual information from the superior colliculus (SC) to the posterior vermis (lobules VI and VII) of the cerebellum. The relay neurons in DLPN/NRTP responded to moving visual stimuli with large receptive fields (23-75 degrees ) which occupied mainly the contralateral hemifield including the fovea. These neurons were usually more responsive to large-sized stimuli than to discrete-spot stimuli, had direction selectivity, and showed preference to visual motion at a relatively high speed. No clear differences in visual properties were observed between DLPN and NRTP. After a local injection of lidocaine into SC, the visual responses in DLPN/NRTP transiently disappeared, indicating that DLPN/NRTP received the visual inputs through SC.

Cerebello-cerebral projections onto the ventral part of the frontal cortex of the macaque monkey.

The cerebellar projections to the ventral part of the frontal cortex were investigated in the Japanese monkey (Macaca fuscata). In anesthetized monkeys, stimulation of either the lateral or interposed cerebellar nucleus evoked marked surface-negative and depth-positive potentials in the face motor area (Mf) and the upper part of the ventral premotor area (PMv) on the contralateral side; responses were hardly recorded in the lower part of PMv including the areas related to the laryngeal movements. Stimulation of the medial cerebellar nucleus induced no significant responses in these areas. The results of the combined anterograde and retrograde tracing study indicated that the cerebellar nuclei projected to Mf and the upper part of PMv mainly through the nucleus ventralis posterior lateralis pars oralis and area X of the thalamus, respectively.

Motivation-dependent activity in the dorsolateral part of the prefrontal cortex in the monkey.

Field potentials were recorded on self-paced hand and mouth movements with implanted electrodes on the surface and at 2.0-3.0 mm depth in respective cortical areas in seven monkeys. Surface-negative (s-N), depth-positive (d-P) premovement slow potentials were recorded in the prefrontal cortex only when the movements were performed with an intense motivation for a reward. Such a motivation-dependent activity was mainly obtained in the rostral bank of the arcuate sulcus on both sides (except for the inferior limb of the arcuate sulcus in the left hemisphere). Three of the seven monkeys were also tasked with visuo-initiated hand movements. As motivation for reward decreased, s-N, d-P potentials at a latency of about 80 ms after stimulus onset became gradually smaller in the rostral bank of the arcuate sulcus in the right hemisphere. These facts suggest that motivation-dependent activity is represented in the dorsolateral part of the prefrontal cortex in monkeys, and that the cortical part is involved in motivational functions as well as in cognitive functions.

Studies on integrative functions of the human frontal association cortex with MEG.

Our MEG studies on the human frontal association cortex are briefly reviewed. (1) The no-go potential was first found at go/no-go reaction-time hand movement task with discrimination between different colour light stimuli in the prefrontal cortex of monkeys. The potential was recorded in human subjects with EEG over the scalp, but its current dipoles could be localized only by use of MEG, in the dorsolateral part of the frontal association cortex in both cerebral hemispheres. The function for no-go decision and subsequent suppressor action was thus substantiated in the human frontal cortex. (2) Utterance of a short noun in Japanese was found to be initially preceded by an activity in the lower lateral part of the frontal lobe and then by that around the central sulcus. The area of the former, often in both hemispheres, appears to correspond to Broca's motor speech centre and that of the latter, always in both hemispheres, to correspond to the motor-somatosensory cortices. (3) Intensive and continuous concentration on mental calculation and some 'abstract' thinking for a few minutes were often associated with magnetic theta (5-7 Hz) wave bursts in the frontal part of the scalp. Dipole fitting suggested that the electrical current dipoles occur successively and scattered in wide areas of the frontal lobe on both sides. They are to be called "frontal mental theta wave", revealing dynamic and active participation of the frontal lobe in mental functions.

Role of conserved Asn-Tyr-Asp-Tyr sequence in bacterial copper/2,4, 5-trihydroxyphenylalanyl quinone-containing histamine oxidase.

Copper amine oxidase contains a covalently bound quinonoid cofactor, 2,4,5-trihydroxyphenylalanyl quinone (TPQ), which is synthesized by post-translational modification of a specific tyrosyl residue occurring in the highly conserved sequence, Asn-Tyr-(Asp/Glu)-Tyr. To elucidate the role(s) of the conserved sequence in the biogenesis of TPQ, each of the corresponding residues at positions 401-404 in the recombinant histamine oxidase from Arthrobacter globiformis has been replaced with other amino acids by site-directed mutagenesis. When Asn-401 was changed to Asp or Gln, the rate of TPQ formation by copper-dependent self-processing was 10(3)- to 10(4)-fold slower than in the wild-type enzyme. When Tyr-402 was replaced by Phe, TPQ was not formed at all, showing that Tyr-402 is essential as the precursor to TPQ. In contrast, Asp-403 could be replaced by Glu without changes in the rate of TPQ formation, whereas its replacement by Asn led to a marked decrease. Furthermore, when Tyr-404 was changed to Phe, TPQ was formed swiftly on incubation with copper ions, but the TPQ enzyme exhibited very low activity with altered substrate specificity. These results collectively indicate that a very rigorous structural motif is required for efficient formation of TPQ and for the catalytic activity in the active site of copper amine oxidases.

cDNA cloning of IX/X-BP, a heterogeneous two-chain anticoagulant protein from snake venom.

IX/X-bp is an anticoagulant protein isolated from the venom of the habu snake (Trimeresurus flavoviridis). It is a heterogeneous two-chain protein linked by an interchain S-S bond. We prepared a cDNA library from the venom gland of the habu snake in the vector pSPORT1. cDNA clones containing the coding sequences for IX/X-bp were isolated and sequenced to determine the structure of the proprotein of IX/X-bp. All cDNA clones containing coding sequences of either chain of IX/X-bp consisted of the 5'-end noncoding bases, the first ATG codon, a typical signal peptide sequence that was immediately followed by mature protein sequence that corresponded to one of the chains, a stop codon, the 3'-end noncoding bases, a polyadenylation signal, and a poly(A)+ region. These data indicate that the gene for each chain of the two-chain protein is transcribed and translated separately.

Biosynthesis of topa quinone cofactor in bacterial amine oxidases. Solvent origin of C-2 oxygen determined by Raman spectroscopy.

Resonance Raman spectroscopy is an excellent technique for providing structural information on the 2,4, 5-trihydroxyphenylalanine quinone (TPQ) cofactor in copper-containing amine oxidases. This technique has been used to investigate the copper- and O2-dependent biosynthesis of the TPQ cofactor in phenylethylamine oxidase (PEAO) and histamine oxidase from Arthrobacter globiformis. Incubation of the holoenzyme in H218O causes frequency shifts at 1684(-26) cm-1 in PEAO and at 1679(-28) cm-1 in histamine oxidase, allowing this feature to be assigned to the C=O stretch of a single carbonyl group at the C-5 position. When apoprotein is reacted with Cu(II) and O2 in the presence of H218O, the resultant holoproteins show increased shifts of -3 to -6 cm-1 in a number of other vibrational modes, particularly at 411 and 1397 cm-1. Because these small shifts persist when the H218O-regenerated protein is back-exchanged into H216O, they can be assigned to oxygen isotope substitution at the C-2 postion. No isotope shifts are observed when apoprotein is regenerated with Cu(II) in the presence of 18O2. Thus, it is concluded that the C-2 oxygen atom of TPQ originates from H2O rather than O2. The isotope dependence of the 1397-cm-1 mode allows it to be assigned to the C O moiety at the C-2 position, with its low frequency being indicative of only partial double bond character. Similar frequency shifts due to 18O at C-2 are observed in the resonance Raman spectra of H218O-regenerated PEAO after derivatization of the C-5 carbonyl with either p-nitrophenylhydrazine (-5 cm-1 at 480 cm-1) or methylamine (-5 cm-1 at 1301 cm-1). Taken together, these results indicate that the TPQ cofactor in the native enzyme has substantial electron delocalization between the C-2 and C-4 oxygens and that only the C-5 oxygen has predominantly C=O character.

Introduction of macromolecules into living Dictyostelium cells by electroporation.

An attempt was made to optimize conditions for introduction of macromolecules into Dictyostelium cells by electroporation. The amount of fluorescein-labeled bovine serum albumin (FITC-BSA) introduced into cells was measured by fluorometry after extraction of FITC-BSA from cells with detergent. The amount increased as the applied voltage and capacitance of the discharger were increased. However, the survival of cells decreased at higher voltages and elevated capacitance. FITC-BSA was introduced into 80-90% of treated cells. FITC-BSA at 0.25 mg/ml was introduced into cells under optimum conditions when the concentration of the extracellular protein was 2.5 mg/ml. Several discharges in sequence improved the extent of introduction of FITC-BSA although viability decreased. There was a linear correlation between final intracellular concentration and the initial extracellular concentration of FITC-BSA, suggesting the possible quantitative introduction of the protein into cells. The membrane pores that opened during electroporation closed within 2.5 sec after the discharge. FITC-labeled dextran with molecular weights of less than 5 x 10(5) were able to pass through these pores. Our results show that electroporation provides a quantitative and reproducible method for introduction of macromolecules into living Dictyostelium cells.

Motor speech centres in the frontal cortex.

Activities of the frontal cortices in both cerebral hemispheres preceding utterance of a short word were recorded and analyzed with multichannel SQUID gradiometers. Light stimuli of two different colours of 500 ms duration were delivered in front of a subject at random time intervals and in irregular order of the different colours. The subject should respond to either of the stimuli by uttering a short word, e.g., 'en' [en] ('round' in Japanese) or another by a short simple voice without meaning as a word, e.g., 'e' [e]. The initial sounds of both voices are to be the same, i.e., 'e' [e] in these examples. The 37 gradiometers covering either the left or the right frontal-parietal part of the hemisphere recorded different magnetic fields between the word and the simple voice. Magnetic fields averaged 100 times at the onset of the stimuli revealed that the utterance of a word is preceded by significant magnetic field changes at a peak latency of 120-165 ms from the onset of light stimuli, whereas the utterance of a simple voice is not preceded by such changes. At a peak latency of 160-190 ms, about 20-40 ms before the start of perioral EMGs, both the utterances are commonly preceded by magnetic field changes. Dipole fittings based on these magnetic fields suggest that the earlier magnetic fields reflect electrical activities in the ventral lateral part of the frontal association area, usually in both left and right hemispheres, and that the later fields represent those in the sensorimotor area in both the hemispheres. That part of the frontal association area appears to be the centre for organizing words to speak and to correspond possibly to the Broca's speech area.

Spectroscopic studies on the mechanism of the topa quinone generation in bacterial monoamine oxidase.

Electron paramagnetic resonance (EPR), circular dichroism (CD), and optical absorption spectroscopies have been used to investigate the copper-dependent autoxidation process generating the 6-hydroxydopa (topa) quinone cofactor in the recombinant phenethylamine oxidase from Arthrobacter globiformis. The cupric ion bound to the copper/topa quinone-less, inactive enzyme is first reduced to Cu(I), as inferred from the spectroscopic features observed under strictly anaerobic conditions. Cu(I) is also detectable chemically with a Cu(I)-specific chelating agent, bathocuproinedisulfonate. Introduction of a limited amount of oxygen then leads to the formation of a paramagnetic species (g = 2.004) that is stable for over several to 10 min but vanishes swiftly upon addition of sufficient oxygen. Strikingly, the hyperfine EPR structure of the organic radical is almost identical with that of the topa semiquinolamine observed in the copper/topa quinone-containing, active enzyme anaerobically reduced with substrate. Concomitant with the generation of topa quinone exhibiting characteristic optical absorption and CD bands under fully aerobic conditions, the bound copper finally shows EPR signals typical of nonblue type II Cu(II) and optical absorption around 700 nm with negative CD above 700 nm. None of these spectral changes are evoked in the binding of Cu(II) to the Tyr382-->Phe mutant enzyme, indicating that the precursor Tyr382 to topa quinone participates in the initial reduction of bound copper and serves as the origin of the transiently formed semiquinone radical. The prosthetic cupric ion plays an essential role, by changing its redox state, in the oxidative modification of the tyrosyl phenol ring, leading to topa quinone.

Copper/topa quinone-containing histamine oxidase from Arthrobacter globiformis. Molecular cloning and sequencing, overproduction of precursor enzyme, and generation of topa quinone cofactor.

The gene coding for histamine oxidase has been cloned and sequenced from a Coryneform bacterium Arthrobacter globiformis. The deduced amino acid sequence consists of 684 residues with a calculated molecular mass of 75,109 daltons and shows a high overall identity (58%) with that of phenethylamine oxidase derived from the same bacterial strain. Although the sequence similarities are rather low when compared with copper amine oxidases from other organisms, the consensus Asn-Tyr-Asp/Glu sequence, in which the middle Tyr is the precursor to the quinone cofactor (the quinone of 2,4,5-trihydroxyphenylalanine, topa) covalently bound to this class of enzymes, is also conserved in the histamine oxidase sequence. To identify the quinone cofactor, an overexpression plasmid has been constructed for the recombinant histamine oxidase. The inactive enzyme purified from the transformed Escherichia coli cells grown in a copper-depleted medium gained maximal activity upon stoichiometric binding of cupric ions. Concomitantly with the enzyme activation by copper, a brownish pink compound was generated in the enzyme, which was identified as the quinone of topa by absorption and resonance Raman spectroscopies of the p-nitrophenylhydrazine-derivatized enzyme and found at the position corresponding to the precursor Tyr (Tyr-402). Therefore, the copper-dependent autoxidation of a specific tyrosyl residue operates on the formation of the topa quinone cofactor in this enzyme, as recently demonstrated with the precursor form of phenethylamine oxidase (Matsuzaki, R., Fukui, T., Sato, H., Ozaki, Y., and Tanizawa, K. (1994) FEBS Lett. 351, 360-364).

Generation of the topa quinone cofactor in bacterial monoamine oxidase by cupric ion-dependent autooxidation of a specific tyrosyl residue.

The quinone of 2,4,5-trihydroxyphenylalanine (topa), recently identified as the covalently bound redox cofactor in copper amine oxidases, is encoded by a specific tyrosine codon. To elucidate the mechanism of its formation, the recombinant phenylethylamine oxidase of Arthrobacter globiformis has been overproduced in Escherichia coli and purified in a Cu(2+)-deficient form. The inactive precursor enzyme thus obtained was dramatically activated upon incubation with Cu2+, concomitantly with the formation of the topa quinone at the position corresponding to Tyr382, occurring in the tetrapeptide sequence highly conserved in this class of enzymes. The topa quinone was produced only under aerobic conditions, but its formation required no external enzymatic systems. These findings demonstrate the Cu(2+)-dependent autooxidation of a specific tyrosyl residue to generate the topa quinone cofactor.

Dynamic activities of the frontal association cortex in calculating and thinking.

We found 5-7 Hz magnetic theta waves in the frontal association cortex of adult human subjects during calculation and musical imagination by using 37-channel SQUID gradiometers. Simultaneous recording from the left and right cerebral hemispheres with two sets of 37-channel gradiometers revealed that the theta activity appeared in a waxing and waning manner in the frontal cortices of both hemispheres during the mental exercises. Electrical current dipoles for the theta waves were estimated to occur repeatedly and scatteringly in various parts of the frontal lobes of both hemispheres during continuous and intense mental exercises for 2 min. The results suggest a dynamic mode of activities in the frontal association cortex during mental effort such as calculating and thinking.

Cloning and sequencing of phenylethylamine oxidase from Arthrobacter globiformis and implication of Tyr-382 as the precursor to its covalently bound quinone cofactor.

The gene of Arthrobacter globiformis encoding a quinoprotein, phenylethylamine oxidase, has been cloned and sequenced. In the deduced amino acid sequence comprising 638 residues is a tetrapeptide sequence, Asn-Tyr-Asp-Tyr, which has been found to be highly conserved in other copper amine oxidase. Mutation of the former Tyr (Tyr-382) of the recombinant enzyme into Phe resulted in the complete loss of catalytic activity and disappearance of the quinone compound that is specifically detected in the wild-type enzyme, suggesting that Tyr-382 is the precursor to the covalently-bound cofactor, most probably topa quinone. Furthermore, the expression of the active, quinone-containing enzyme in Escherichia coli cells was markedly dependent on the presence of Cu2+ ions in the culture medium, and the inactive, Cu2(+)-deficient enzyme produced without Cu2+ ions could be converted to the active quinone form by reconstitution with Cu2+ ions.

No-go activity in the frontal association cortex of human subjects.

'No-go potential', specific to the no-go reaction in go/no-go reaction-time hand movement with discrimination between different colour light stimuli, was recorded with electrodes chronically implanted in the prefrontal cortex of monkeys. Similar potentials were widely distributed over frontal-parietal parts of the human scalp on the same no-go reaction. In this report, MEG measurement was made over the scalp of five healthy subjects in order to localize current sources responsible for the scalp potential. We found in- and outflow of magnetic fields over the dorsolateral frontal parts of the head, contra- and ipsilateral to the operant hand. These magnetic fields were most reasonably interpreted to be due to current dipoles localized in the dorsolateral parts of frontal lobes in both contra- and ipsilateral hemispheres, presumably in the prefrontal-premotor areas. No-go decision and subsequent suppression of voluntary movements are suggested to be one of the functional features of the human frontal association cortex, as in the case of monkeys.