Decellularized Human Dental Pulp as a Scaffold for Regenerative Endodontics.

Teeth undergo postnatal organogenesis relatively late in life and only complete full maturation a few years after the crown first erupts in the oral cavity. At this stage, development can be arrested if the tooth organ is damaged by either trauma or caries. Regenerative endodontic procedures (REPs) are a treatment alternative to conventional root canal treatment for immature teeth. These procedures rely on the transfer of apically positioned stem cells, including stem cells of the apical papilla (SCAP), into the root canal system. Although clinical success has been reported for these procedures, the predictability of expected outcomes and the organization of the newly formed tissues are affected by the lack of an available suitable scaffold that mimics the complexity of the dental pulp extracellular matrix (ECM). In this study, we evaluated 3 methods of decellularization of human dental pulp to be used as a potential autograft scaffold. Tooth slices of human healthy extracted third molars were decellularized by 3 different methods. One of the methods generated the maximum observed decellularization with minimal impact on the ECM composition and organization. Furthermore, recellularization of the scaffold supported the proliferation of SCAP throughout the scaffold with differentiation into odontoblast-like cells near the dentinal walls. Thus, this study reports that human dental pulp from healthy extracted teeth can be successfully decellularized, and the resulting scaffold supports the proliferation and differentiation of SCAP. The future application of this form of an autograft in REPs can fulfill a yet unmet need for a suitable scaffold, potentially improving clinical outcomes and ultimately promoting the survival and function of teeth with otherwise poor prognosis.

Apoptotic surface delivery of K+ channels.

Apoptosis in cortical neurons requires efflux of cytoplasmic potassium mediated by a surge in Kv2.1 channel activity. Pharmacological blockade or molecular disruption of these channels in neurons prevents apoptotic cell death, while ectopic expression of Kv2.1 channels promotes apoptosis in non-neuronal cells. Here, we use a cysteine-containing mutant of Kv2.1 and a thiol-reactive covalent inhibitor to demonstrate that the increase in K+ current during apoptosis is due to de novo insertion of functional channels into the plasma membrane. Biotinylation experiments confirmed the delivery of additional Kv2.1 protein to the cell surface following an apoptotic stimulus. Finally, expression of botulinum neurotoxins that cleave syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) blocked upregulation of surface Kv2.1 channels in cortical neurons, suggesting that target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins support proapoptotic delivery of K+ channels. These data indicate that trafficking of Kv2.1 channels to the plasma membrane causes the apoptotic surge in K+ current.

Altered expression of potassium channel subunit mRNA and alpha-dendrotoxin sensitivity of potassium currents in rat dorsal root ganglion neurons after axotomy.

Previous studies have raised the possibility that a decrease in voltage-gated K+ currents may contribute to hyperexcitability of injured dorsal root ganglion (DRG) neurons and the emergence of neuropathic pain. We examined the effects of axotomy on mRNA levels for various Kv1 family subunits and voltage-gated K+ currents in L4-L5 DRG neurons from sham-operated and sciatic nerve-transected rats. RNase protection assay revealed that Kv1.1 and Kv 1.2 mRNAs are highly abundant while Kv1.3, Kv1.4, Kv1.5 and Kv1.6 mRNAs were detected at lower levels in L4-L5 DRGs from sham and intact rats. Axotomy significantly decreased Kv1.1, Kv1.2, Kv1.3 and Kv1.4 mRNA levels by approximately 35%, approximately 60%, approximately 40% and approximately 80%, respectively, but did not significantly change Kv1.5 or Kv1.6 mRNA levels. Patch clamp recordings revealed two types of K+ currents in small-sized L4-L5 DRG neurons: sustained delayed rectifier currents elicited from a -40 mV holding potential and slowly inactivating A-type currents that was additionally activated from a -120 mV holding potential. Axotomy decreased both types of K+ currents by 50-60% in injured DRG neurons. In addition, axotomy increased the alpha-dendrotoxin sensitivity of the delayed rectifier, but not slow A-type K+ currents in injured DRG neurons. These results suggest that Kv1.1 and Kv1.2 subunits are major components of voltage-gated K+ channels in L4-L5 DRG neurons and that the decreased expression of Kv1-family subunits significantly contributes to the reduction and altered kinetics of Kv current in axotomized neurons.

Effect of non-aqueous phase liquid on biodegradation of PAHs in spilled oil on tidal flat.

Biodegradation rates of polycyclic aromatic hydrocarbons (PAHs) in spilled oil stranded on tidal flats were studied using model reactors to clarify the effects of NAPL on the biodegradation of PAHs in stranded oil on tidal flat with special emphasis on the relationship between dissolution rates of PAHs into water and viscosity of NAPL. Biodegradation of PAHs in NAPL was limited by the dissolution rates of PAHs into water. Biodegradation rate of chrysene was smaller than that for acenaphthene and phenanthrene due to the smaller dissolution rates. Dissolution rates of PAHs in fuel oil C were smaller those in crude oil due to high viscosity of fuel oil C. Therefore, biodegradation rates of PAHs in fuel oil C were smaller than those in crude oil. Biodegradation rates of PAHs in NAPL with slow decrease rate like fuel oil C were slower than those in NAPL with rapid decrease like crude oil. The smaller decrease rate of fuel oil C than crude oil was due to higher viscosity of fuel oil C. Therefore, not only the dissolution rate of PAHs but also the decrease rates of NAPL were important factors for the biodegradation of PAHs.

Cryoglobulinemia in Japanese patients with chronic hepatitis C virus infection: host genetic and virological study.

Essential cryoglobulinemia is associated closely with hepatitis C virus (HCV) infection. The mechanism responsible for occurrence of the disease is unclear. The aim of this study was to investigate pathogenetic roles of HCV in cryoglobulinemia. One hundred sixty-seven consecutive patients with HCV were studied clinically by HCV grouping, HCV RNA levels, GBV-C/HGV, HCV quasispecies (target region was hypervariable region-1) and HLA polymorphism. The quasispecies in cryoprecipitate were compared with those in supernatant. The results of HLA polymorphism of patients with cryoglobulinemia were compared with those without cryoglobulinemia and healthy controls. The frequency of HCV-related cryoglobulinemia was 71 of 167 (42.5%). Patients with cirrhosis (36 of 63, 57.1%) had cryoglobulinemia more frequently than those with chronic hepatitis (35 of 104, 33.7%, P < 0.01). No significant differences were not found between the two groups (patients with and without cryoglobulinemia) in age, gender, HCV grouping, HCV RNA level and frequency of GBV-C/HGV. HCV was found quantitatively and clonally more frequently in the cryoprecipitate than in the supernatant. HLA polymorphism presented no significant differences among three groups. The stage of liver disease is one of pathogenetic factors. The greater the presence of HCV quasispecies in cryoprecipitate than in the supernatant indicates that various antigen presentations play an important role in the formation of cryoglobulin, whereas HLA typing dose not seem to contribute to the development of cryoglobulinemia.

[Effect of an oral appliance for obstructive sleep apnea syndrome].

The purpose of this study was to evaluate the effect of an oral appliance (Sleep Splint) for obstructive sleep apnea syndrome (OSAS) patients, and to examine problems with this therapy. Fixed in the oral cavity, the Sleep Splint intends to reduce the occurrence of apnea and prevent blockage of the upper airway during sleep. In this study, the therapy using Sleep Splint was done for 50 patients (41 men and 9 women) who had been introduced by the physician diagnosing with OSAS. Sleep monitoring around an oral appliance mounting was carried out, and the effect was evaluated. In addition, the ESS was used in respect of the evaluation of the daytime sleepiness. However, four patients stopped using the oral appliance due to serious discomfort in the maxillofacial region, and they were excluded from the study. Forty-six patients were classified into three groups by the seriousness of OSAS, or into two groups according to body fatness, and the difference of the effect between each group was compared. The results obtained were as follows. 1. As to the Apnea hypopnea index (AHI), Apnea hypopnea density, Oxygen desaturation, and ESS, there was statistically significant improvement regardless of the seriousness of OSAS and body fatness. 2. About all patients except for the patient group of the mild cases (AHI < 10), the occurrence of snoring showed the statistically significant improvement. These results indicated that oral appliance therapy was an effective treatment for OSAS.

Independent regulation of cardiac Kv4.3 potassium channel expression by angiotensin II and phenylephrine.

Hypertrophied cardiac myocytes exhibit prolonged action potentials and decreased transient outward potassium current (I(to)). Because Kv4.3 is a major contributor to I:(to), we studied regulation of its expression in neonatal rat cardiac myocytes in response to the known stimulators of cardiac myocyte hypertrophy, angiotensin II (Ang II) and phenylephrine (PE). RNase protection assays and immunoblots revealed that Ang II and PE each downregulate Kv4.3 mRNA and protein. However, although PE induces a faster and more extensive hypertrophic response than Ang II, the PE effect on Kv4.3 mRNA develops slowly and is sustained, whereas Ang II rapidly and transiently decreases Kv4.3 mRNA expression. Turnover measurements revealed that Kv4.3 mRNA is very stable, with a half-life >20 hours. This suggests that Ang II must destabilize the channel mRNA. In contrast, PE does not affect the rate of Kv4.3 mRNA degradation. To test for transcriptional regulation, the 5' flanking region of the rat Kv4.3 gene was cloned, and Kv4.3 promoter-reporter constructs were expressed in cardiac myocytes. Whereas Ang II was found to have no effect on transcription, PE inhibits Kv4.3 promoter activity. Pharmacological experiments also indicate that PE and Ang II act independently to downregulate Kv4.3 gene expression. Thus, regulation of Kv4.3 gene expression is not a simple secondary response to hypertrophy. Rather, Ang II and PE use different mechanisms to decrease Kv4.3 channel expression in neonatal rat cardiac myocytes.

Kvbeta subunits increase expression of Kv4.3 channels by interacting with their C termini.

Auxiliary Kvbeta subunits form complexes with Kv1 family voltage-gated K(+) channels by binding to a part of the N terminus of channel polypeptide. This association influences expression and gating of these channels. Here we show that Kv4.3 proteins are associated with Kvbeta2 subunits in the brain. Expression of Kvbeta1 or Kvbeta2 subunits does not affect Kv4.3 channel gating but increases current density and protein expression. The increase in Kv4.3 protein is larger at longer times after transfection, suggesting that Kvbeta-associated channel proteins are more stable than those without the auxiliary subunits. This association between Kv4.3 and Kvbeta subunits requires the C terminus but not the N terminus of the channel polypeptide. Thus, Kvbeta subunits utilize diverse molecular interactions to stimulate the expression of Kv channels from different families.

Increased base change mutations at G:C pairs in Escherichia coli deficient in endonuclease III and VIII.

Various types of mutation induced by oxidative DNA damage, induced by hydrogen peroxide and riboflavin photosensitization, were determined in Escherichia coli (E. coli) mutants deficient in endonuclease III (endo III) and endonuclease VIII (endo VIII). The majority of hydrogen peroxide-induced and spontaneous mutations consisted of G:C to A:T and to T:A base changes, shown on the mutation assay system by a reversion at a specific site of the lacZ gene. Base changes were also localized at G:C pairs in the mutation of the supF gene, induced by riboflavin photosensitization, which specifically yields 7,8-dihydro-8-oxoguanine (8-oxoG). G:C to T:A and to C:G transversions dominated in both mutants. These results suggest that endo III and endo VIII are involved in the repair of oxidative lesions of guanine.

Differentiation of mouse hepatitis viruses in animal facilities in Japan by use of nucleotide analysis of the nucleocapsid gene.

The nucleotide sequences of the coding region of the nucleocapsid (N) gene of 12 mouse hepatitis virus (MHV) strains recently found in animal facilities in Japan were analyzed. Nucleotide sequencing was performed directly on polymerase chain reaction (PCR) products amplified by reverse transcription (RT) and polymerase chain reaction (RT-PCR) analysis from fecal samples or isolated viruses. Phylogenetic analysis of these MHV strains along with those reported previously indicated that sequence analysis of the N gene was a useful tool for differentiation of MHV strains,although most MHV strains in Japanese facilities were phylogenetically close. Results suggested that interchange of mice infected with MHV among facilities provided opportunities of introduction of MHV into otherwise MHV-free facilities and that the source of MHV infection could be traced by use of nucleotide analysis of the N gene.

Extremely low frequency magnetic fields suppress the reduction of germination rate of Arabidopsis thaliana seeds kept in saturated humidity.

The effects of an extremely low frequency magnetic field (ELFMF) on the germination of plant seeds were examined. The decrease in the germination activity of the seeds of Arabidopsis thaliana WS kept in saturated humidity and high temperature (37 degrees C) was suppressed by the exposure to a 400 mT ELFMF.

TRH regulates Kv1.5 gene expression through a Galphaq-mediated PLC-independent pathway.

Thyrotropin-releasing hormone (TRH) decreases transcription of the Kv1.5 K(+) channel gene in GH(3) pituitary cells. Here, we examine whether TRH utilizes Gq activated phospholipase C, Gs or Gi to produce this response. We report that expression of constitutively active Galphaq mimicked and occluded the TRH effect. In contrast, expression of activated Galpha(S) or Galpha(i2) had no effect on Kv1. 5 mRNA expression. Furthermore, pertussis and cholera toxins failed to block the TRH-induced decrease in channel mRNA. Surprisingly, despite the role of Gq, the phospholipase C inhibitor U73122 did not alter down-regulation of channel mRNA by TRH, although it abolished the TRH-induced increase in intracellular [Ca(2+)] and up-regulation of c-fos mRNA. Furthermore, depletion of an intracellular Ca(2+) pool or inhibition of protein kinase C did not block the TRH-induced decrease in Kv1.5 mRNA. These results indicate that TRH-induced down-regulation of Kv1.5 gene expression is mediated by Galphaq proteins, but does not require PLC activation.

Surface expression of Kv1 channels is governed by a C-terminal motif.

Voltage-gated K(+) channel subunits must reach the plasma membrane to repolarize action potentials. Yet the efficiency of cell surface targeting varies among Kv subunits with some requiring auxiliary subunits for optimal expression. Here we identify a conserved motif located in the variable C-terminal region of Kv1 channels that controls the efficiency of functional channel expression. Variations among wild type channels in the optimal sequence VXXSL produce differences in distribution and the requirement for auxiliary subunits. Furthermore, deletion of this motif decreases subunit glycosylation and surface localization but does not prohibit subunit multimerization. Finally, the action of the essential sequence is shown to be independent of the chaperone effect of Kvbeta subunits. Thus, the newly identified C-terminal motif governs processing and cell surface expression of Kv1 voltage-gated K(+) channels.

Surface expression of Kv1 voltage-gated K+ channels is governed by a C-terminal motif.

The normal rhythmic beating of the heart relies on tight control of expression of voltage-gated ion channels in the plasma membrane of cardiac myocytes. Recently, a conserved motif was identified near the C-terminus of Kv1 voltage-gated K+ channels that is required for efficient processing and surface expression. Furthermore, variations in the motif account for differences among normal channels in localization and the requirement for auxiliary subunits for robust expression. Thus, this motif is a key regulator of cell surface expression of Kv1 family K+ channels.