Mechanical behavior of in-situ alloyed Ti6Al4V(ELI)-3 at.% Cu lattice structures manufactured by laser powder bed fusion and designed for implant applications.

In the present study, cellular lattice structures for implant applications are reported for the first-time incorporating copper directly by in-situ alloying in the laser powder bed fusion process. The aim to incorporate 3 at.% Cu into Ti6Al4V(ELI) is selected for improved antibacterial properties while maintaining appropriate mechanical properties. Previously, topologically optimized Ti6Al4V(ELI) lattice structures were successfully designed, manufactured and studied for implant applications. The development of a new alloy produced by in-situ alloying of elemental powder mixture of Ti6Al4V(ELI) and pure Cu powders was used here for the production of identical lattice structures with improved antibacterial properties. One of the same as-designed CAD models was used for the manufacturing of these lattices compared to previous work on pure Ti6Al4V(ELI) lattices, making direct comparison of mechanical properties possible. Similar manufacturability highlights the applicability of this alloying technique to other lattice designs. Microstructural characterization was performed by optical and electron microscopies, as well as microCT. Mechanical characterization was performed by means of compression tests and hardness measurements. Results showed that in-situ alloying with copper leads to the formation of localized Cu-rich regions, refinement of martensitic phase and the formation of CuTi2 intermetallic precipitates, which increased the hardness and strength of the material. Deviations in wall thickness between the as-designed and as-manufactured lattices led to anisotropy of the mechanical properties of the lattices. Higher compressive strength values were obtained when thicker walls were oriented along the loading direction. Nevertheless, alloying with Cu had a higher impact on the compressive strength of lattice structure than the wall thickness deviations. The direct in-situ alloying of copper in Ti6Al4V(ELI) is a promising route for direct manufacturing of antibacterial implants.

Expression of serine protease inhibitors in epidermal keratinocytes is increased by calcium but not 1,25-dihydroxyvitamin D3 or retinoic acid.

BACKGROUND: In human skin, the serine proteases kallikrein-related peptidase (KLK)5 and KLK7 degrade corneodesmosome proteins, leading to desquamation. Serine protease activity of the skin is tightly regulated by the interplay between such proteases and serine protease inhibitors, including lymphoepithelial Kazal-type related inhibitor (LEKTI), encoded by SPINK5; secretory leucocyte peptidase inhibitor (SLPI); and elafin. Expression of KLK5 and KLK7 is controlled and upregulated by stimulants such as calcium, 1,25-dihydroxyvitamin D3 [1,25(OH)2 VD3 ] and retinoic acid (RA). OBJECTIVES: To understand the effect of calcium, 1,25(OH)2 VD3 and RA on the expression of serine protease inhibitors in epidermal keratinocytes. METHODS: We stimulated normal human epidermal keratinocytes (NHEKs) with high calcium, 1,25(OH)2 VD3 or RA, and then analysed the expression of serine protease inhibitors using quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay and immunocytofluorescence. We also analysed trypsin- and chymotrypsin-like serine protease activities in stimulated NHEKs. RESULTS: High calcium, but not 1,25(OH)2 VD3 or RA, significantly induced the expression of LEKTI, SLPI and elafin at both transcript and protein levels in NHEKs. These inductions were time- and dose-dependent. The activities of trypsin- and chymotrypsin-like serine proteases were significantly up- and downregulated by high calcium, respectively, in NHEKs. CONCLUSIONS: High calcium, but not 1,25(OH)2 VD3 or RA, increases the expression of serine protease inhibitors in epidermal keratinocytes. Our findings contribute to the understanding of the mechanisms by which serine protease activities are regulated by serine proteases and related inhibitors in epidermal keratinocytes.

Identification of ST2A1 as a rat brain neurosteroid sulfotransferase mRNA.

A hydroxysteroid sulfotransferase (ST2A1) was identified as a form mediating neurosteroid sulfation in rat brain. The sole expression among known rat ST2A forms was indicated by brain RT-PCR. All nucleotide sequences of seven ST2A cDNA clones isolated from brain matched completely with that of hepatic ST2A1. The recombinant ST2A1 protein mediated neurosteroid sulfation. These data strongly suggest a functional role of ST2A1 as a neurosteroid sulfotransferase in rat brain.

Identification of the opener and closer of the pharyngoesophagus in laryngectomees.

BACKGROUND: In esophageal and tracheoesophageal speakers, the neoglottis acts not only as the orifice of the digestive tract but also as the airway and the voice source. The opening and closing mechanism is thought to be essential for these functions. It is not known, however, whether there is any active muscular control of neoglottal opening and closing. Examinations have been carried out to find the physiological background of the opening and closing of the neoglottis. In this paper, we present our findings of the opening and closing mechanism of the neoglottis and discuss them with reviewing the previous studies. SUBJECTS AND METHODS: Subjects were volunteer esophageal speakers. Neoglottal width, EMG of the inferior pharyngeal constrictor (IPC) and that of the geniohyoid muscle (GH) were recorded simultaneously during swallowing and air intake for esophageal speech. RESULTS: At rest, the neoglottis was closed by the mucosal protrusion in the posterior wall of the pharyngoesophagus. During swallowing, the neoglottis was widely opened. Traction of the anterior pharyngeal wall anteriorly by the GH and reciprocal suppression of the IPC activity was thought to be the mechanism of the neoglottal opening in this case. However, such simple reciprocity was not observed during air intake, although transient opening of the neoglottis was commonly observed. The GH showed increased activity at neoglottal opening as it did during swallowing. The IPC was. on the other hand, continuously activated, and no significant suppression was observed. During air intake, the muscle is assumed to play a role in maintaining the shape and tension of the posterior mucosal protrusion which acts as a voice source in subsequent phonation. CONCLUSIONS: The GH and the IPC were found to open and close the neoglottis, respectively. Their activities were not always clearly reciprocal in the various functions of the neoglottis.

Role of parabrachial nucleus in submandibular salivary secretion induced by bitter taste stimulation in rats.

When rats lick a bitter taste solution such as quinine-hydrochloride, they secrete profuse amounts of saliva. The salivation has a higher flow rate than that induced by other qualities of taste stimulation: sweet, salty, and sour. The present study is aimed to clarify the neural mechanism of the quinine-evoked salivation by means of behavioral, neuroanatomical, and electrophysiological experiments. Behaviorally, submandibular salivary secretion and rejection behavior (gaping) were observed in normal rats, as well as in rats chronically decerebrated at the precollicular level. In chronically decerebrate rats, these quinine-evoked reactions were strongly suppressed by destruction of the medial part of the parabrachial nucleus, including the so-called taste area, and ventral part of the parabrachial nucleus, including the pontine reticular formation. Neuroanatomical study using a retrograde tracer, Fluoro-gold, revealed that the neurons sending their axons to the superior salivatory nucleus, parasympathetic secretory center, were located mainly in the pontine reticular formation ventral to the parabrachial nucleus, not in the parabrachial taste area. Extracellular neural activity was recorded from the parabrachial region in decerebrate rats, and responsiveness to taste stimulation, jaw movements, and electrical stimulation of the superior salivatory nucleus was examined. Neurons responsive to both taste stimulation and antidromic stimulation of the superior salivatory nucleus were found in the pontine reticular formation ventral to the parabrachial nucleus, which responded well to quinine and HCl taste stimuli. Neurons in the parabrachial taste area could respond to four qualities of taste stimulation, but not to antidromic stimulation of the salivary center. These results suggest that aversive taste information from the parabrachial taste area reaches the salivary secretory center via the reticular formation ventral to the parabrachial nucleus.

Abdominal vagi mediate c-Fos expression induced by X-ray irradiation in the nucleus tractus solitarii of the rat.

The mechanism of induction of emesis by X-ray irradiation remains largely unknown. The purpose of the present research was to clarify the neuronal basis of the induction of nausea induced by X-ray irradiation analyzing c-Fos expression in the nucleus tractus solitarii (NTS) as a marker of cellular excitation. We confirmed that the dose of X-ray irradiation (4 Gy) used for the present research could actually induce nausea by preliminary measurement of kaolin intake. Induction of c-Fos immunoreactivity in the NTS was observed in the animals that received X-ray irradiation of the whole body. The mean number of c-Fos positive cells in the animals that received irradiation was significantly larger than that in the non-irradiated animals. Partial exposure of the abdomen to X-rays showed significantly greater c-Fos expression than that of the head. These results indicated the presence of a certain route for transmitting information from the periphery toward the central nervous system by X-ray irradiation. The number of c-Fos positive cells induced by X-ray irradiation in animals vagotomized at the subdiaphragmatic level was lower than that in sham-operated animals. Animals receiving a serotonin subtype three (5-HT3, 5-hydroxytryptamine) receptor antagonist (tropisetron, ICS 205-930, 3-tropanyl-indole-3-carboxylate) showed a significant reduction in c-Fos protein expression compared to animals receiving a vehicle. These results strongly suggested that X-ray irradiation activates 5-HT3 receptors on the terminals of the abdominal vagal nerves to excite the afferent pathway, thereby inducing emesis.

Water stimulation of the posterior oral cavity induces inhibition of gastric motility.

The response of gastric motility to the administration of water and saline in the larynx and epiglottis was investigated in urethan-chloralose anesthetized rats. Administration of water inhibited motility of the distal stomach, but 0.15 M NaCl did not induce the inhibitory response. Bilateral sectioning of the superior laryngeal nerve (SLN) abolished the inhibitory response induced by water. Bilateral cervical vagotomies abolished the inhibitory responses, although spinal transection did not affect the inhibitory response. These inhibitory responses have been observed in immobilized animals. The degree of inhibition by water and hypotonic saline was negatively correlated with the sodium concentration. In contrast, the degree of inhibition to hypertonic saline was positively correlated with the sodium concentration. The proximal stomach also showed a reduction in intragastric pressure in response to the administration of water. These findings suggest that water-responsive afferent neurons in the SLN suppress gastric motility via the vagal efferent nerve.

Facilitation of gastric motility induced by portal infusion of hyper- and hypotonic solution in rats.

The effects of the portal infusion of hyper- and hypotonic solution on gastric motility in rats were investigated. The infusion of hypertonic saline into the portal vein (portal infusion) elicited a significant enhancement of gastric contractile activity. The portal infusion of water also produced this enhancement. However, the portal infusion of isotonic saline showed no significant enhancement; nor did the infusion of water and hypertonic saline into the jugular vein. Sectioning of the hepatic branch of the vagus nerve (hepatic vagus) eliminated the enhanced responses of the gastric motility. It is therefore concluded that hepatoportal osmoreceptive afferent signals affect the gastric motility by way of the hepatic vagus. These effects on osmolarity revealed that hypotonic stimulation is more effective than hypertonic stimulation for the enhancement of motility. Sectioning of the dorsal subdiaphragmatic vagus, which includes the dorsal gastric and celiac branch, did not eliminate these responses. Sectioning of the ventral gastric vagus, in contrast, did eliminate the responses. These results suggest that vagal preganglionic neurons in the left dorsal motor nucleus of the vagus play a role in enhancement of gastric motility observed in the present research.

Effects of GABA on neurons of the gustatory and visceral zones of the parabrachial nucleus in rats.

Response characteristics of neurons in the gustatory and visceral zone of the parabrachial nucleus (PBN) to gamma-aminobutyric acid (GABA) were examined using whole cell recordings in brain slices of the rat. Based on the recording site, neurons were divided into three groups: neurons in the dorsolateral quadrant of the PBN (DL-neurons), neurons in the dorsomedial quadrant of the PBN (DM-neurons) and neurons in the ventromedial quadrant of the PBN (VM-neurons). Recordings were made from 44 DL-, 43 DM-, 39 VM-neurons. Superfusion of GABA resulted in a concentration-dependent reduction in input resistance in 67.5% of the neurons in the PBN (73.1% of the DL-, 62.5% of the DM-, 66.7% of the VM-neurons). No obvious difference of the concentration-response curve was found among three groups. The mean reversal potential of the GABA effect was about -74 mV and no significant differences were observed among three groups of neurons. The GABA response was partly or completely blocked by the GABAA antagonist bicuculline in all neurons tested. Superfusion of the GABAA agonist muscimol resulted in a decrease of the input resistance in all neurons tested. It was concluded that GABA functions as an inhibitory neurotransmitter in both gustatory and visceral part of the PBN, mediated in part, by GABAA receptors.

Differences in the intrinsic membrane characteristics of parabrachial nucleus neurons processing gustatory and visceral information.

Whole-cell current-clamp recordings were made from neurons in the rat parabrachial nucleus (PBN) in three rostro-caudal brain slices. During recording the neurons were located in one of four quadrants of the PBN. Successful recordings were obtained from neurons in three of these quadrants termed the dorsolateral (DL), dorsomedial (DM) and ventromedial (VM) quadrants. Recordings were made of the intrinsic membrane properties and repetitive discharge characteristics of 58 neurons in the DL, 60 neurons in the DM, and 54 neurons in the VM-quadrants. The input resistance of the neurons in the DL quadrant was significantly lower and the membrane time constant significantly shorter than that of the neurons in the DM- and VM-quadrants. The mean action potential duration of the VM-quadrant neurons was significantly longer than that of both DL- and DM-quadrant neurons. The discharge frequency in response to a 1500 ms 100 pA current pulse of the DL quadrant neurons was significantly lower than that of the neurons in the other two quadrants. The latency of action potential initiation following a 100 pA depolarizing current pulse was significantly longer for DL quadrant neurons compared to neurons in the other two quadrants. Neurons were divided into groups based on their response to a long depolarizing current pulse immediately preceded by a hyperpolarizing current pulse. In all three rostro-caudal slices of the PBN, the largest populations of neurons were in Group II and Group III. The results demonstrate that neurons in different locations in the PBN have different membrane and repetitive discharge properties. These different PBN locations receive inputs from the visceral and gustatory regions of the NST. It is possible therefore that the differences in properties of the PBN neurons may relate to the type of sensory information that they process.

[A case of Paget's disease with progressive sensorineural hearing loss involved in the bilateral bony labyrinths].

Paget's disease of the bone is a chronic, progressive disease of unknown etiology characterized by abnormal bony resorption and deposition. It is a common skeletal disease in Europe and North America, while in Japan it is very rare. Paget's disease of the temporal bone has been reported to cause hearing loss frequently. We report a 50-year-old woman with Paget's disease who had progressive bilateral sensorineural hearing loss and right vestibular dysfunction. CT and 3 Dimensional CT (3D-CT) images demonstrated resorption of the entire bony labyrinth and its surroundings on both sides and that the temporal bone elsewhere remained intact. Bone scans revealed disease symmetrically in the bilateral otic capsules. Previous studies indicated that the bone changes in Paget's disease in the petrous pyramid begin in areas best supplied with marrow tissue and that the otic capsule is relatively spared until advanced changes are present in the remainder of the petrous pyramid. But, this patient mainly had foci in the bilateral otic capsules and the pattern was similar to cochlear otosclerosis. 3D-CT was useful for differentiation of Paget's disease and cochlear otosclerosis. The pattern of the affected areas indicated that this is a very rare situation even in the reports of Europe and North America, where the disease is rather common.

Effects of portal infusion of hypotonic- and hypertonic solutions on neuronal activity in the rat dorsal motor nucleus of the vagus.

The present experiment was designed to elucidate the characteristics of the response of neurons in the dorsal motor nucleus of the vagus (DMV) to stimulation of the hepatoportal area by hypotonic as well as hypertonic solutions. Responses of 81 neurons that exhibited an antidromic response to electrical stimulation of the ventral gastric vagus were recorded in the left DMV in urethane-chloralose anesthetized rats. The effects on these 81 neurons of portal infusion of hypertonic saline (3.6% NaCl) and of pure water were examined. The discharge rates of 16 neurons increased in response both to portal infusion of hypertonic saline and to that of water. Portal infusion of 0.9% NaCl produced no changes in firing rates. Their discharge rates of seven neurons increased in response to portal infusion of hypertonic saline but not to that of water. The other 58 neurons did not respond to these stimuli. Jugular infusion of water produced no response. Therefore, the responses to portal infusion of water appear to be derived from activation of the hepatoportal receptors. These results indicate that a certain fraction of DMV neurons respond similarly to portal infusions of hypertonic and hypotonic saline. It is possible that there exist some reflex arcs that mediate a similar response to both an increase and a decrease in portal blood osmolarity (or Na+ concentration), namely, a suppression of absorption.

Glucose-responsive neurons in the brainstem.

AREA POSTREMA: The influence on feeding behavior caused by ablation of the area postrema (AP) in rodents indicates the participation of this structure in the control of ingestion. Two types of glucose responsive neurons were identified in the AP: one is characterized by increasing the discharge rate in response to glucose (glucoreceptor type) and the other by decreasing the discharge rates in response to glucose (glucose sensitive type). These glucose responsive neurons may participate in glycemic homeostasis. NUCLEUS OF SOLITARY TRACT: The glucose responsive neurons exist within the caudal portion of nucleus of the solitary tract (NTS), a relay station in visceral afferents. Two types similar to the AP were also recognized. It is confirmed that hepatic glucose sensitive afferents terminate on some of the glucose sensitive neurons. This convergence may serve as a fail-safe mechanism. In addition, the NTS involving complex neural networks of excitatory and inhibitory interneurons may be concerned with integration of glycemic information. DORSAL MOTOR NUCLEUS OF VAGUS: Some neurons within the dorsal motor nucleus of the vagus (DMV) were identified as the glucose responsive ones. Both types were also recognized. It is confirmed by antidromic activation that these glucose responsive DMV neurons send their axons toward the gastric or coeliac branch that innervates either the stomach, intestine or pancreas. Some of the DMV neurons may subserve an enteroceptor function by themselves. They may also play a role in the brainstem neural control of glycemic homeostasis as the fail-safe mechanism.

A new cis-acting element for RNA replication within the 5' noncoding region of poliovirus type 1 RNA.

Mouse cells expressing the human poliovirus receptor (PVR-mouse cells) as well as human HeLa cells are susceptible to poliovirus type 1 Mahoney strains and produce a large amount of progeny virus at 37 degrees C. However, the virus yield is markedly reduced at 40 degrees C in PVR-mouse cells but not in HeLa cells. The reduction in virus yield at 40 degrees C appears to be due to a defective initiation process in positive-strand RNA synthesis (K. Shiroki, H. Kato, S. Koike, T. Odaka, and A. Nomoto, J. Virol. 67:3989-3996, 1993). To gain insight into the molecular mechanisms involved in this detective process, naturally occurring heat-resistant (Hr)-mutants which show normal growth ability in PVR-mouse cells even at 40 degrees C were isolated from a virus stock of the Mahoney strain and their mutation sites that affect the phenotype were identified. The key mutation was a change from adenine (A) to guanine (G) at nucleotide position (nt) 133 within the 5' noncoding region of the RNA. This mutation also gave an Hr phenotype to the viral plus-strand RNA synthesis in PVR-mouse cells. Mutant Mahoney strains with a single point mutation at nt 133 (A to G, C, or T or deletion) were investigated for their ability to grow in PVR-mouse cells at 40 degrees C. Only the mutant carrying G at nt 133 showed an Hr growth phenotype in PVR-mouse cells. These results suggest that a host cellular factor(s) interacts with an RNA segment around nt 133 of the plus-strand RNA or the corresponding region of the minus-strand RNA, contributing to efficiency of plus-strand RNA synthesis.

Chemosensitivity of neurons in the dorsal motor nucleus of the vagus that responded to portal infusion of hypertonic saline in rats.

Neural responses in the dorsal motor nucleus of the vagus (DMV) to topical administrations of sodium and portal infusions of hypertonic saline were investigated electrophysiologically by using multibarrel electrodes in anesthetized rats. Of 102 neurons that showed antidromic response to electrical stimulation of the ventral gastric vagus or the accessory celiac vagus, 51 neurons increased and 13 neurons decreased their discharge rates in response to the electrophoretic administration of sodium. The other 38 neurons did not respond to this stimulation. The portal infusion of hypertonic saline elicited neural responses of some DMV neurons whose axons are involved into either the ventral gastric or the accessory celiac vagus. Further, effects of the topical administration and the portal infusion of hypertonic saline were examined on 33 neurons. Typical response was characterized by an increase in discharge rate responding to both of the portal infusion and the topical administration. In conclusion, the DMV neurons receiving the afferent inputs from hepatoportal osmoreceptors may have an enteroceptor function detecting the change in osmotic pressure of their environment.

Effects of gastric distension and electrical stimulation of dorsomedial medulla on neurons in parabrachial nucleus of rats.

The effects of gastric distension and electrical stimulation of the dorsomedial medulla on neurons within the parabrachial nucleus (PB) were investigated electrophysiologically in urethane-chloralose anesthetized rats. Among 74 neurons tested, electrical stimulation of the nucleus of the solitary tract (NTS) excited 30 neurons (excitatory neurons) and inhibited 14 neurons (inhibitory neurons). Fourteen neurons increased and 12 neurons decreased their discharge rates in response to gastric distension. Twenty-two neurons responded to both electrical stimulation of the NTS and gastric distension. Both excitatory and inhibitory neurons showed either an increase or a decrease in discharge rate responding to gastric distension. Furthermore, three neurons that decreased their discharge rates and two neurons that increased their discharge rates during gastric distension also responded to intravenous administration of metaraminol indicating some effect of baroreceptor activation on the neural activity. The responses of another 49 neurons in the PB to electrical stimulation of area postrema and gastric distension were analyzed. Electrical stimulation of the AP excited 14 neurons and inhibited only one neuron. Five neurons increased and seven neurons decreased their discharge rates in response to gastric distension. Only one inhibitory neuron responded to gastric distension. These observations suggested that the PB neurons received gastric mechanoreceptive inputs from the NTS.

Formation of chloroamines from styrene under conditions mimicking those of water "chlorination" treatment.

The reaction of styrene with sodium hypochlorite in the presence of ammonium ion in 0.3 M phosphate buffer (pH 6.0) at room temperature afforded N-chloro-1-phenyl-2-chloroethylamine and N,N-dichloro-1-phenyl-2-chloroethylamine. 1-Phenyl-2-chloroethylamine hydrochloride, obtained as the reduction product of the two N-chloroamines, was shown to be mutagenic in the Salmonella typhimurium test.

Effect of topical administration of glucose on neurons innervating abdominal viscera in dorsal motor nucleus of vagus in rats.

Neural responses in the dorsal motor nucleus of the vagus (DMV) to topical administration of glucose were investigated electrophysiologically using multibarrel electrodes in anesthetized rats. Of 52 neurons that showed the antidromic response to the ventral gastric vagus, 4 neurons increased and 14 neurons decreased their discharge rates in response to topical administration of glucose from the multibarrel pipette. Thirty-four neurons did not respond to this administration. Of 4 neurons that showed the antidromic response to the accessory celiac vagus, 1 neuron increased and 1 neuron decreased their discharge rates in response to topical administration of glucose. Two neurons did not respond to this administration. These results suggest that some DMV neurons innervating the abdominal viscera may have an enteroceptor function detecting the change in glucose concentration of their environment.

Effect of portal infusion of hypertonic saline on neurons in the dorsal motor nucleus of the vagus in the rat.

Effects of hepatoportal osmo-receptive (or sodium-receptive) afferents on neurons within the dorsal motor nucleus of the vagus (DMV) were investigated electrophysiologically in urethane-chloralose anesthetized rats. Responses of 56 spontaneously active neurons to antidromic stimulation of the ventral trunk of the subdiaphragmatic vagus were recorded in the left DMV. Among them, 35 neurons were inhibited by electrical stimulation of the hepatic branch of the vagus nerve (inhibitory neurons), except two neurons that were slightly excited. Effects of portal infusion of 3.6% NaCl were examined on 26 inhibitory neurons. Sixteen neurons increased their discharge rates and one neuron decreased its discharge rate in response to portal infusion of hypertonic saline. Thirty-five right DMV neurons responded to electrical stimulation of the dorsal trunk of the subdiaphragmatic vagus were inhibited by electrical stimulation of the hepatic branch of the vagus. Four neurons were excited by this stimulation. Relatively smaller number of neurons (5 out of 22 inhibitory neurons) increased their discharge rates in response to portal infusion of hypertonic saline. In conclusion, the response of DMV neuron observed in this experiment was characterized by increasing the frequency of spike discharges in response to portal infusion of hypertonic saline. However, these neurons were inhibited by electrical stimulation of the hepatic branch of the vagus nerve. These results suggest that the hepatoportal osmo-receptive afferents may be conveyed to the DMV via inhibitory synapses.