Analysis of interaction between intracellular spermine and transient receptor potential canonical 4 channel: multiple candidate sites of negatively charged amino acids for the inward rectification of transient receptor potential canonical 4

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.

Osteogenesis imperfecta and combined orthodontics and orthognathic surgery: a case report on two siblings

Osteogenesis imperfecta is a heterogeneous group of connective tissue diseases that is predominantly characterized by bone fragility and skeletal deformity. Two siblings with undiagnosed type I osteogenesis imperfecta underwent orthognathic surgery for the treatment of facial asymmetry and mandibular prognathism. The authors report two cases of combined orthodontics and orthognathic surgery in patients with type I osteogenesis imperfecta, mandibular prognathism, and facial asymmetry.

Osteogenesis imperfecta and combined orthodontics and orthognathic surgery: a case report on two siblings

Osteogenesis imperfecta is a heterogeneous group of connective tissue diseases that is predominantly characterized by bone fragility and skeletal deformity. Two siblings with undiagnosed type I osteogenesis imperfecta underwent orthognathic surgery for the treatment of facial asymmetry and mandibular prognathism. The authors report two cases of combined orthodontics and orthognathic surgery in patients with type I osteogenesis imperfecta, mandibular prognathism, and facial asymmetry.

Osteogenesis imperfecta and combined orthodontics and orthognathic surgery: a case report on two siblings

Osteogenesis imperfecta is a heterogeneous group of connective tissue diseases that is predominantly characterized by bone fragility and skeletal deformity. Two siblings with undiagnosed type I osteogenesis imperfecta underwent orthognathic surgery for the treatment of facial asymmetry and mandibular prognathism. The authors report two cases of combined orthodontics and orthognathic surgery in patients with type I osteogenesis imperfecta, mandibular prognathism, and facial asymmetry.

Digital technology in orthognathic surgery: virtual surgical planning and digital transfer

No abstract available.

Identification of phospholipase C β downstream effect on transient receptor potential canonical 1/4, transient receptor potential canonical 1/5 channels

Gα(q)-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P₂) depletion. When PI(4,5)P₂ depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gα(q)-phospholipase C β (Gα(q)-PLCβ) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCβ activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca²⁺ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca²⁺ due to Ca²⁺ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P₂ depletion.

Englerin A-sensing charged residues for transient receptor potential canonical 5 channel activation

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.

Timing of orthognathic surgery: paradigm shift by surgery-first approach?

No abstract available.

Dental implant treatment after healing of bisphosphonate-related osteonecrosis of the jaw (BRONJ) in the same region: a case report

Although pathophysiology, incidence, and factors associated with the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ) and management strategies for patients treated with bisphosphonates or patients with BRONJ are well-established, few guidelines or recommendations are available for patients with a history of successfully healed BRONJ. We present a case of successful dental implant treatment after healing of BRONJ in the same region of the jaw, and speculate that implant placement is possible after healing of BRONJ surgery in select cases.

A Study of Core Humanistic Competency for Developing Humanism Education for Medical Students

The authors conducted a survey on essential humanistic competency that medical students should have, and on teaching methods that will effectively develop such attributes. The participants consisted of 154 medical school professors, 589 medical students at Seoul National University College of Medicine, 228 parents, and 161 medical school and university hospital staff. They answered nine questions that the authors created. According to the results, all groups chose "morality and a sense of ethics," a "sense of accountability," "communication skills," and "empathic ability" were selected as essential qualities. According to the evaluation on the extent to which students possess each quality, participants believed students had a high "sense of accountability" and "morality," whereas they thought students had low "empathic ability," "communicate," or "collaborate with others". In terms of effective teaching methods, all sub-groups preferred extracurricular activities including small group activities, debates, and volunteer services. With regard to the speculated effect of humanism education and the awareness of the need for colleges to offer it, all sub-groups had a positive response. However the professors and students expressed a relatively passive stance on introducing humanism education as a credited course. Most participants responded that they preferred a grading method based on their rate of participation, not a relative evaluation. In order to reap more comprehensive and lasting effects of humanism education courses in medical school, it is necessary to conduct faculty training, and continuously strive to develop new teaching methods.

Unilateral intraoral vertical ramus osteotomy and sagittal split ramus osteotomy for the treatment of asymmetric mandibles

In surgery for facial asymmetry, mandibles can be classified into two types, rotational and translational, according to the required mandibular movements for surgery. During surgery for rotational mandibular asymmetry, a bilateral sagittal split ramus osteotomy (BSSRO) may cause a large bone gap between the proximal and distal segments as well as condylar displacement, resulting in a relapse of the temporomandibular joint disorder, especially in severe cases. The intraoral vertical ramus osteotomy has an advantage, in this respect, because it causes less rotational displacement of the proximal segment on the deviated side and even displaced or rotated condylar segments may return to their original physiologic position. Unilateral intraoral vertical ramus osteotomy (UIVRO) on the short side combined with contralateral SSRO was devised as an alternative technique to resolve the spatial problems caused by conventional SSRO in cases of severe rotational asymmetry. A series of three cases were treated with the previously suggested protocol and the follow-up period was analyzed. In serial cases, UIVRO combined with contralateral SSRO may avoid mediolateral flaring of the bone segments and condylar dislocation, and result in improved condition of the temporomandibular joint. UIVRO combined with contralateral SSRO is expected to be a useful technique for the treatment of rotational mandibular asymmetry.

Surgical treatment planning for the two subtypes of mandibular asymmetry

No abstract available.

Differential alternative splicing of human transglutaminase 4 in benign prostate hyperplasia and prostate cancer

Transglutaminase 4 is a member of enzyme family that catalyzes calcium-dependent posttranslational modification of proteins. Although transglutaminase 4 has been shown to have prostate-restricted expression pattern, little is known about the biological function of transglutaminase 4 in human. To gain insight into its role in prostate, we analyzed the expression status of human transglutaminase 4 in benign prostate hyperplasia (BPH) and prostate cancer (PCa). Unexpectedly, RT-PCR and nucleotide sequence analysis showed four alternative splicing variants of transglutaminase 4: transglutaminase 4-L, -M (-M1 and -M2) and -S. The difference between transglutaminase 4-M1 and -M2 is attributed to splicing sites, but not nucleotide size. The deduced amino acid sequences showed that transglutaminase 4-L, -M1 and -M2 have correct open reading frames, whereas transglutaminase 4-S has a truncated reading frame. RT-PCR analysis of clinical samples revealed that transglutaminase 4-M and -S were detected in all tested prostate tissue (80 BPH and 48 PCa). Interestingly, transglutaminase 4-L was found in 56% of BPH (45 out of 80) and only in 15% of PCa (7 out of 48). However, transglutaminase 4-L expression did not correlate with serum prostate-specific antigen (PSA) level, prostate volumes or PSA densities. These results will provide a clue to future investigation aiming at delineating physiological and pathological roles of human transglutaminase 4.

Transglutaminase 2 inhibits apoptosis induced by calciumoverload through down-regulation of Bax

An abrupt increase of intracellular Ca2+ is observed in cells under hypoxic or oxidatively stressed conditions. The dysregulated increase of cytosolic Ca2+ triggers apoptotic cell death through mitochondrial swelling and activation of Ca2+-dependent enzymes. Transglutaminase 2 (TG2) is a Ca2+-dependent enzyme that catalyzes transamidation reaction producing cross-linked and polyaminated proteins. TG2 activity is known to be involved in the apoptotic process. However, the pro-apoptotic role of TG2 is still controversial. In this study, we investigate the role of TG2 in apoptosis induced by Ca2+-overload. Overexpression of TG2 inhibited the A23187-induced apoptosis through suppression of caspase-3 and -9 activities, cytochrome c release into cytosol, and mitochondria membrane depolarization. Conversely, down-regulation of TG2 caused the increases of cell death, caspase-3 activity and cytochrome c in cytosol in response to Ca2+-overload. Western blot analysis of Bcl-2 family proteins showed that TG2 reduced the expression level of Bax protein. Moreover, overexpression of Bax abrogated the anti-apoptotic effect of TG2, indicating that TG2-mediated suppression of Bax is responsible for inhibiting cell death under Ca2+-overloaded conditions. Our findings revealed a novel anti-apoptotic pathway involving TG2, and suggested the induction of TG2 as a novel strategy for promoting cell survival in diseases such as ischemia and neurodegeneration.

Optimized Immunohistochemical Analysis of Cerebellar Purkinje Cells Using a Specific Biomarker, Calbindin D28k

Cerebellar Purkinje cells (PCs) play a crucial role in motor functions and their progressive degeneration is closely associated with spinocerebellar ataxias. Although immunohistochemical (IHC) analysis can provide a valuable tool for understanding the pathophysiology of PC disorders, the method validation of IHC analysis with cerebellar tissue specimens is unclear. Here we present an optimized and validated IHC method using antibodies to calbindin D28k, a specific PC marker in the cerebellum. To achieve the desired sensitivity, specificity, and reproducibility, we modified IHC analysis procedures for cerebellar tissues. We found that the sensitivity of staining varies depending on the commercial source of primary antibody. In addition, we showed that a biotin-free signal amplification method using a horseradish peroxidase polymer-conjugated secondary antibody increases both the sensitivity and specificity of ICH analysis. Furthermore, we demonstrated that dye filtration using a 0.22 micrometer filter eliminates or minimizes nonspecific staining while preserving the analytical sensitivity. These results suggest that our protocol can be adapted for future investigations aiming to understand the pathophysiology of cerebellar PC disorders and to evaluate the efficacy of therapeutic strategies for treating these diseases.

Sweet syndrome initially manifesting odontogenic buccal cellulitis; Report of a case

Immunocytochemical detection of HPV16 E7 in cervical smear

Cervical cancer is characterized by a long period of preclinical dysplasia or carcinoma in situ progressing into invasive cancer. Although Papanicolaou (Pap) smear test has contributed significantly to the early detection of precursor lesions, the cytological screening has inherent problems that produce considerable false negative/positive results. Since the infection of high-risk type of human papillomavirus (HPV) is strongly associated with cervical cancer, we investigated the feasibility of an immunostaining test to detect cells infected by HPV in cervical smear. We produced monoclonal antibodies against HPV16 E7 in mice by repeated injections with the recombinant HPV16 E7. Western blot analysis and immunocytochemical assay demonstrated that the selected monoclonal antibody, mAb (130-9-7), reacts specifically with cultured cervical cancer cell lines infected by HPV16. Specific staining was observable with the HPV16-positive smear specimens obtained from the cervical cancer patients, whereas no staining was detected with the HPV-negative smear specimens. To achieve the desired sensitivity, specificity and reproducibility, we modified and optimized the conventional immunocytochemical procedure for cervical smear specimens. Our results suggest that this immunostaining method for detecting high-risk HPV in cervical smear may be used as a strategy to distinguish a high-risk group, especially those patients with low grade cytological abnormality.

Methylene Blue-stained Interstitial Cells are Electrically Active in the Myenteric Board Freshly Prepared from the Murine Small Intestine

Many gastrointestinal muscles show electrical oscillation, so-called "slow wave", originated from interstitial cells of Cajal (ICCs). Thus, a technique to freshly isolate the cells is indispensable to explore the electrophysiological properties of the ICCs. To apply an enzyme solution on the serosal surface for cell isolation, the intestine was inverted and 0.02% trypsin solution and 0.04% collagenase solution were applied to serosal cavity. After the enzyme treatment, mucosal layer was removed and longitudinal muscle layer was gently separated from the rest of tissue. The thin layer was stretched in the recording chamber and mounted on an inverted microscope. Using beta-escine, perforated whole cell patch clamp technique was used. Under a microscope, the tissue showed smooth muscle cells and interstitial cells around the myenteric plexus. Under voltage clamp condition, three types of membrane potential were recorded. One group of interstitial cells, which were positive to methylene blue and CD34, showed spontaneous outward current. These cells had bipolar shape and were considered as fibroblast-like cells because of their peculiar shape and arrangement. Another group, positive to c-kit and methylene blue, showed spontaneous inward current. These cells had more rounded shape and processes and were considered as ICCs. The third, positive to c-kit and had granules containing methylene blue, showed quiet membrane potentials under the voltage-clamp mode. These cells appeared to be resident macrophages. Therefore, in the freshly isolated thin tissue preparation, methylene blue could easily identify three types of cells rather than morphological properties. Using this method, we were able to study electrical properties of fibroblast and residential macrophage as well as myenteric ICCs.

Clinical significance of anti-filaggrin antibody recognizing uncitrullinated filaggrin in rheumatoid arthritis

Filaggrin is expressed in the cornified layer of epidermis and known to be one of the antigenic targets in rheumatoid arthritis. Although the citrulline residue in filaggrin is thought to be an antigenic determinant recognized by autoantibodies, the diagnostic sensitivity of synthetic citrullinated peptide is variable. To investigate the implication of anti-filaggrin antibodies recognizing uncitrullinated filaggrin in rheumatoid arthritis, we assayed antibody titers using unmodified recombinant filaggrin in the sera from 73 patients with rheumatoid arthritis, 150 patients with other connective tissue diseases and 70 normal controls. We also performed the correlation analysis between antibody titers and the clinical variables in patients with rheumatoid arthritis. Titers of IgG anti-filaggrin antibodies were significantly higher in rheumatoid arthritis patients compared to normal controls (P=0.02), but not in patients with osteoarthritis, ankylosing spondylitis or systemic lupus erythematosus. IgG anti-filaggrin antibodies were more frequently found in patients with rheumatoid arthritis compared to normal controls (12.3% vs 1.4% respectively, P=0.04). An anti-filaggrin antibody titer was correlated with visual analogue scale of pain, tender joint count, Ritchie articular index or C-reactive protein, but not with anti-nuclear antibody or rheumatoid factor. These results suggest that anti-filaggrin antibody recognizes the uncitrullinated filaggrin as an antigen and its titer correlates with clinical parameters, explaining the variable sensitivity of anti-filaggrin antibody test.

Different inhibition characteristics of intracellular transglutaminase activity by cystamine and cysteamine

The treatment of cystamine, a transglutaminase (TGase) inhibitor, has beneficial effects in several diseases including CAG-expansion disorders and cataract. We compared the inhibition characteristics of cystamine with those of cysteamine, a reduced form of cystamine expected to be present inside cells. Cystamine is a more potent inhibitor for TGase than cysteamine with different kinetics pattern in a non- reducing condition. By contrast, under reducing conditions, the inhibitory effect of cystamine was comparable with that of cysteamine. However, cystamine inhibited intracellular TGase activity more strongly than cysteamine despite of cytoplasmic reducing environment, suggesting that cystamine itself inhibits in situ TGase activity by forming mixed disulfides.