Characterization of anti-P monoclonal antibodies directed against the ribosomal protein-RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice.

Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus.

Occlusion and brain function: mastication as a prevention of cognitive dysfunction.

Research in animals and humans has shown that mastication maintains cognitive function in the hippocampus, a brain area important for learning and memory. Reduced mastication, an epidemiological risk factor for the development of dementia in humans, attenuates spatial memory and causes hippocampal neurons to deteriorate morphologically and functionally, especially in aged animals. Active mastication rescues the stress-attenuated hippocampal memory process in animals and attenuates the perception of stress in humans by suppressing endocrinological and autonomic stress responses. Active mastication further improves the performance of sustained cognitive tasks by increasing the activation of the hippocampus and the prefrontal cortex, the brain regions that are essential for cognitive processing. Abnormal mastication caused by experimental occlusal disharmony in animals produces chronic stress, which in turn suppresses spatial learning ability. The negative correlation between mastication and corticosteroids has raised the hypothesis that the suppression of the hypothalamic-pituitary-adrenal (HPA) axis by masticatory stimulation contributes, in part, to preserving cognitive functions associated with mastication. In the present review, we examine research pertaining to the mastication-induced amelioration of deficits in cognitive function, its possible relationship with the HPA axis, and the neuronal mechanisms that may be involved in this process in the hippocampus.

Ion shower milling: its application to cell membrane removal.

A thickness of about 100 angstroms of the cell membrane of an isolated single freeze-dried neuron of the snail can be etched off with an ion shower milling machine. The calcium content of the cell membrane area was more than one-fifth of the whole cell. The calcium content of the cell membrane area increased during pentylenetetrazole-induced bursting activity.

Intracellular calcium: its movement during pentylenetetrazole-induced bursting activity.

The intracellular calcium concentration in the cytoplasm decreased and the calcium concentration near the cell membrane increased during bursting activity induced by pentylenetetrazole in snail neuron. Incubation in medium containing cobalt chloride or lanthanum chloride did not change this tendency, which suggests that this calcium distribution change is due to the stored calcium in the subcellular structure moving toward the cell membrane.

Cervical sympathectomy causes alveolar bone loss in an experimental rat model.

BACKGROUND AND OBJECTIVE: Periodontal disease, a pathological destructive inflammatory condition, is characterized by alveolar bone loss. Recent studies have suggested a correlation between the sympathetic nervous system and bone remodeling. To confirm the importance of the sympathetic nervous system in bone resorption, we investigated the effects of superior cervical ganglionectomy and oral challenge with Porphyromonas gingivalis on alveolar bone loss in rats. MATERIAL AND METHODS: Rats were divided into three groups: group A underwent a sham operation as the control group; group B underwent superior cervical ganglionectomy; and group C underwent a sham operation and oral challenge with P. gingivalis. Horizontal alveolar bone loss was evaluated by measuring the distance between the cemento-enamel junction and the alveolar bone crest. Cytokine gene expression in the gingival tissues was assessed using reverse transcription-polymerase chain reaction analyses. The furcation areas of the mandibular molars were examined histologically. RESULTS: Both superior cervical ganglionectomy and oral challenge with P. gingivalis resulted in accelerated alveolar bone loss. Gingival tissues in the superior cervical ganglionectomy group showed increased expression of the cytokines interleukin-1 alpha, tumor necrosis factor-alpha and interleukin-6. The density of neuropeptide Y-immunoreactive fibers was decreased following superior cervical ganglionectomy. Osteoclasts were observed in the superior cervical ganglionectomy and P. gingivalis-challenged groups. CONCLUSION: Both superior cervical ganglionectomy and oral challenge with P. gingivalis induced alveolar bone loss. These results provide new information on the occurrence of alveolar bone loss, in that both oral challenge with P. gingivalis and superior cervical ganglionectomy are important accelerating factors for alveolar bone loss. Thus, we suggest that the sympathetic nervous system is linked with the prevention of alveolar bone loss.

Dynorphin-A immunoreactive terminals on the neuronal somata of rat mesencephalic trigeminal nucleus.

Dynorphin-A-like immunoreactivity was investigated in the rat mesencephalic trigeminal nucleus (Mes 5) at the light and electron microscopic levels. Dynorphin-A immunoreactive fibers and puncta, likely representing nerve terminals, were observed throughout rostrocaudal extension of the Mes 5 at the light microscopic level. Within the rostrocaudal extension, more abundant fibers and puncta were localized in the midbrain-pontine junction and pontine areas than in the midbrain area. At the electron microscopic level, dynorphin-A immunoreactive synapses were observed on the somata of Mes 5. Dynorphin-A-like immunoreactivity tended to be restricted to dense-cored vesicles in the synapses. These results suggest that dynorphin-A-containing fiber systems affect mastication through the Mes 5.

Soft-diet feeding decreases dopamine release and impairs aversion learning in Alzheimer model rats.

To examine the effects of soft-diet feeding on the dopaminergic system in a model rat for Alzheimer's disease (AD), we measured dopamine release in the hippocampus using a microdialysis approach and assessed learning ability and memory using step-through passive avoidance tests. Furthermore, we immunohistochemically examined the ventral tegmental area (VTA), which is the origin of hippocampal dopaminergic fibers using tyrosine hydroxylase (TH), a marker enzyme for the dopaminergic nervous system. Feeding a soft diet decreased dopamine release in the hippocampus and impaired learning ability and memory in AD model rats in comparison with rats fed a hard diet; however, TH-immunopositive profiles in the VTA seemed not to be notably different between rats fed a soft diet and those fed a hard diet. These observations suggest that soft-diet feeding enhances the impairment of learning ability and memory through the decline of dopamine release in the hippocampus in AD rats.

Isolation and characterization of a human thiamine pyrophosphokinase cDNA.

A human thiamine pyrophosphokinase cDNA clone (hTPK1) was isolated and sequenced. When the intact hTPK1 open reading frame was expressed as a histidine-tag fusion protein in Escherichia coli, marked enzyme activity was detected in the bacterial cells. The hTPK1 mRNA was widely expressed in various human tissues at a very low level, and the mRNA content in cultured fibroblasts was unaffected by the thiamine concentration of the medium. The chromosome localization of the hTPK1 gene was assigned to 7q34.

Morphological aspects of the postnatal development of submandibular glands in male rats: involvement of apoptosis.

We studied the involvement of the apoptotic mechanism(s) in cell differentiation in the developing male rat submandibular gland using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-labeling) assay in combination with light and electron microscopy. Whereas the proacinar cells were completely transformed into acinar cells within 2 weeks after birth, starting on postnatal Day 21, the terminal tubule cells formed vacuoles that disappeared by postnatal Day 35. During this period, positive TUNEL reactivity was seen in the terminal tubule cells, and electron microscopic analysis showed that certain morphological features of apoptosis, including fragmentation of nuclei and the presence of apoptotic bodies in the cytoplasm, were present in and restricted to the terminal tubule cells. These results indicate that, in addition to an autophagocytosis-mediated mechanism, apoptosis may also be involved in reducing the number of terminal tubule cells during postnatal development in the submandibular gland.

Evidence for involvement of dysfunctional teeth in the senile process in the hippocampus of SAMP8 mice.

In order to evaluate the involvement of dysfunctional teeth in age-related deficits in hippocampal function, we examined the effect of removal of molar teeth (molarless condition) on neuronal degeneration and glial fibrous acidic protein (GFAP) expression in the hippocampus and on learning ability in a water maze test in young, middle-aged, and aged accelerated senescence-prone mice (SAMP8). The molarless condition enhanced an age-dependent decrease in both learning ability and the number of neurons in the hippocampal CA1 subfield and the age-dependent increase in the number and hypertrophy of GFAP-labeled astrocytes in the same subfield. These observations suggest that the molarless condition may be involved in the senile process in the hippocampus in SAMP8 mice.

Involvement of pentylenetetrazole in synapsin I phosphorylation associated with calcium influx in synaptosomes from rat cerebral cortex.

To determine precisely how pentylenetetrazole (PTZ) is involved in the biochemical processes at the presynaptic nerve terminal, the effect of PTZ, under various conditions, on the phosphorylation of synapsin I (previously called protein I) was investigated, using 32Pi in synaptosomes from rat cerebral cortex. PTZ markedly stimulated the incorporation of 32P into this protein as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography, but it failed to stimulate protein phosphorylation in Ca2+-free medium containing ethylene glycol bis-(beta-aminoethylether)-N',N'-tetraacetic acid (EGTA). Moreover, the PTZ-stimulated synapsin I phosphorylation was reversed by addition of EGTA sufficient to chelate all external free Ca2+. PTZ also stimulated synaptosomal accumulation of Ca2+. The PTZ-stimulatory effects of both synapsin I phosphorylation and synaptosomal accumulation of Ca2+ were inhibited markedly by tetrodotoxin as well as by cobalt chloride and lanthanum chloride. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7, strongly) and N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5, weakly) reduced the PTZ-stimulatory effect on synapsin I phosphorylation by about 75 and 15%, respectively, whereas these antagonists had essentially no effect on PTZ-stimulated synaptosomal accumulation of Ca2+. These results suggest that PTZ causes the influx of Ca2+ into the presynaptic nerve terminal secondary to the elevated Na+ and is consequently involved in the synapsin I phosphorylation step, facilitating the Ca2+/calmodulin-mediated presynaptic event leading to seizure discharge.

Pentylenetetrazole suppresses the potassium current in Euhadra neurons which is coupled with Ca2+/calmodulin-dependent protein phosphorylation.

The Ca2+/calmodulin-dependent protein phosphorylation-related mechanism underlying pentylenetetrazole (PTZ)-induced reduction of delayed potassium current (IKD) was examined in identified Euhadra neurons. PTZ gradually reduced peak IKD in a dose-dependent manner, as well as an inhibition of Ca2+/calmodulin-dependent protein phosphorylation. Similar effects were observed by a general protein kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpoperazine, whose saturating dose occluded the action of PTZ on the IKD. Intracellular injection of Ca2+/calmodulin-dependent protein kinase II transiently restored the PTZ-suppressed IKD nearly to the pre-PTZ level, whereas either CaCl2 or calmodulin, injected in the same way, had little effect. However, this restoration was not detectable in the presence of N-(6-aminohexyl)-5-chloronaphthalenesulfonamide, a calmodulin inhibitor, in the perfusate. These results suggest that PTZ suppresses the potassium current coupled with Ca2+/calmodulin-dependent protein phosphorylation.

Membrane properties and intracellular biochemical processes during vasopressin-induced bursting activity in snail neurons.

To elucidate the mechanism generating bursting activity, the effect of arginine vasopressin (AVP) was studied electrophysiologically and biochemically in ganglionic preparations from the snail, Euhadra peliomphala. AVP caused bursting activity which is accompanied by the development of a negative slope resistance (NSR) region in the current-voltage (I-V) curve of the identified neurons. Similar effects were observed by application of veratridine, dibutyryl cyclic AMP and isobutylmethylxanthine. Both the bursting activity and the I-V relation induced by AVP were markedly inhibited by reduction of extracellular Na+ but not by Co2+-substituted Ca2+-free saline. This hormone also caused the following intracellular biochemical alterations: elevation in the cyclic AMP levels; stimulation of adenylate cyclase and Ca2+-dependent protein kinase activities; and promotion of Ca2+ release from the intracellular reservoir, lysosome-like granules. These results suggest that AVP-induced bursting activity is mediated through intracellular biochemical processes.

Modification of noradrenergic innervation in the cerebellum of mutant rats with Purkinje cell degeneration (jaundiced Gunn rats).

In heterozygous (Jj) and homozygous Gunn rats (jj), cerebellar noradrenergic innervation was examined using immunohistochemical, neurochemical and electrophysiological techniques. Immunohistochemical analysis using an antiserum against tyrosine hydroxylase (TH) revealed a marked enhancement in immunoreactivity largely in the granular layer and the whole nuclei in the jj cerebellum, resulting from an increase in TH-immunoreactive varicose fibers forming synapse-like structures on the somata and dendrites of granule cells or nuclear neurons. The concentration of norepinephrine in both the cortical and nuclear regions of the jj cerebellum was significantly higher than that in the control, whereas no significant difference of this total amount was observed between the jj and Jj cerebella. Injection of norepinephrine into the Jj cerebellar nuclei reduced the firing rate of spontaneous unitary discharges of neurons in the interpositus nucleus. These findings suggest that the the jj cerebellum causes an enhancement of the noradrenergic innervation which may possibly be one of its characteristic alterations.

Age-associated changes in the dopamine synthesis as determined by GTP cyclohydrolase I inhibitor in the brain of senescence-accelerated mouse-prone inbred strains (SAMP8).

Our objective in this study was to elucidate the mechanism underlying the decrease in dopamine (DA) levels in the brain with ageing We administered 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I to senescence-accelerated mouse-prones (SAMP8), to inhibit DA and serotonin syntheses, and following immunohistochemical staining, analyzed the immunoreactive intensities (IR-Is) for DA in the nigrostriatal dopaminergic neurons by microphotometry. The DA-IR-Is in the substantia nigra pars compacta and neostriatum of young mice (2 months old) reached a minimal value 3 h after DAHP administration and returned to the control value 12 h after the administration. However, in aged mice (10 months old), the minimal value was reached 6 h after the administration and the value remained at approximately 70 and 80% of the control value at 24 and 72 h, respectively, after DAHP administration. The results suggest that DA turnover is lower in aged mice than in young mice.

Impairment of spatial memory and changes in astroglial responsiveness following loss of molar teeth in aged SAMP8 mice.

In order to evaluate the mechanism(s) responsible for senile impairment of cognitive function as a result of reduced mastication, the effects of the loss of the molar teeth (molarless condition) on the hippocampal expression of glial fibrous acidic protein (GFAP) and on spatial memory in young adult and aged SAMP8 mice were studied using immunohistochemical and behavioral techniques. Aged molarless mice showed a significantly reduced learning ability in a water maze test compared with age-matched control mice, while there was no difference between control and molarless young adult mice. Immunohistochemical analysis showed that the molarless condition enhanced the age-dependent increase in the density and hypertrophy of GFAP-labeled astrocytes in the CA1 region of the hippocampus. These effects increased the longer the molarless condition persisted. When the extracellular K+ concentration ([K+]o) was increased from 4 to 40 mM for hippocampal slices in vitro, the mean increase in the membrane potential was about 57 mV for fine, delicate astrocytes, the most frequently observed type of GFAP-positive cell in the young adult mice, and about 44 mV for the hypertrophic astrocytes of aged mice. However, there was no significant difference in resting membrane potential between these cell types. The data suggest that an impairment of spatial memory and changes in astroglial responsiveness occur following the loss of molar teeth in aged SAMP8 mice.

Induction of epileptic seizure activity by a specific protein from cobalt-induced epileptogenic cortex of rats.

To evaluate the protein-related mechanism for epileptogenesis, we examined protein behavior during cobalt-induced epileptic seizure activity and the effect of injection of cobalt-induced proteins into cerebral cortex on electrocorticographic discharge in rats. Cobalt-induced epileptic seizure activity caused an increase in two proteins that migrated at about 84 kDa (P84) and 70-71 kDa (P70) and a decrease in a protein of about 57 kDa (P57) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Among these proteins, P70 evoked epileptic discharges on the electroencorticograph and behavioral seizure, when it was intracortically injected into the motor region of the normal rat cerebrum. The data suggest that P70 may be a factor for epileptogenesis.

Glutamate elicits an outward K+ current which is normally suppressed by a Ca2+/calmodulin-dependent protein kinase II.

The inhibitory action of glutamate (Glu) was examined in identified Euhadra neurons, using the voltage-clamp method in combination with the pressure injection technique. Glu elicited a slow outward K+ current (Glu current) whose amplitude was dose-dependent. This current was inhibited by exogenous Ca2+/calmodulin-dependent protein kinase II (CaMKII) and is enhanced by a specific CaMKII inhibitor. However, no significant changes in the Glu current were observed when the catalytic subunit of protein kinase A (PKA) or the protein kinase C (PKC) fragment (530-558) was intracellularly applied; or using a PKA inhibitor or a PKC inhibitor. Neither the antagonists of the Glu receptor, D-2-amino-5-monophosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3 dione and kynurenic acid, nor the G protein blockers, pertussis toxin and chorela toxin, had any significant effect on the Glu current. These results indicate that Glu opens the CaMKII-suppressing K+ channels, suggesting a novel Glu-induced inhibitory mechanism.

Intracellularly applied anti-P70 antibody blocks the induction of abnormal membrane properties by pentylenetetrazole in identified Euhadra neurons.

Using the voltage-clamp technique combined with pressure injection, we have studied the action of pentylenetetrazole (PTZ) on identified Euhadra neurons by examining how the PTZ-induced changes in membrane properties are affected by an antibody against P70, a protein found in the experimentally-induced epileptogenic cortex of rats. Intracellular injection of anti-P70 antibody blocked the induction by PTZ; bursting activity with both of development of negative slope resistance region in the steady state 1-V curve and a reduction in the delayed outward potassium current. These results suggest a novel mechanism of action for PTZ, involving intracellular protein(s) which react with anti-P70 antibody.

The 70-kDa epileptic cortical protein elicits bursting activity accompanied by a reduction of outward current in Euhadra neurons which is inhibited by an antibody against this protein.

The effect on membrane properties of 70-kDa protein (namely P70), a specific protein found in cobalt-induced epileptic cortex of rats, was examined electrophysiologically in identified neurons of the snail, Euhadra peliomphala. Injection of P70 into the neurons elicited a bursting activity resembling the paroxysmal depolarization shift seen in mammalian epileptic neurons and a reduction of the outward current, which were suppressed by Ca2(+)-free saline or tetraethylammonium. The injection of P70-antibody into neurons, preceding the P70 injection, markedly inhibited the bursting activity and the reduced outward current elicited by P70. These findings suggest that P70 causes a reduction of Ca2(+)-dependent potassium conductance which may be one of the mechanisms generating epileptic bursting activity.