TRPC expression in human periodontal ligament cells and the periodontal tissue of periodontitis mice: a preliminary study.

BACKGROUND: Transient receptor potential canonical (TRPC) channels are non-selective cationic channels with permeability to Ca2+ and Na+. Despite their importance, there are currently few studies on TRPC in the periodontal ligament (PDL) and bone cells in the dental field. To provide biological information regarding TRPC in PDL cells and periodontal tissue, we evaluated TRPC channels expression in the osteoblast differentiation of PDL cells and periodontitis-induced tissue. Human PDL cells were cultured in osteogenic differentiation media for 28 days, and the expression of Runx2, osteocalcin (OCN), and TRPC1, 3, 4, and 6 was evaluated by real-time PCR. In ligature-induced periodontitis mice, the alveolar bone and osteoid areas, the osteoclast number, and the expression of Runx2, OCN, TRPC3, and TRPC6 was evaluated by H&E staining, TRAP staining, and immunohistochemistry, respectively. RESULTS: In the PDL cell differentiation group, TRPC6 expression peaked on day 7 and TRPC3 expression generally increased during differentiation. During the 28 days of periodontitis progression, alveolar bone loss and osteoclast numbers increased compared to the control group during the experimental period and the osteoid area increased from day 14. TRPC6 expression in the periodontitis group increased in the PDL area and in the osteoblasts compared to the control group, whereas TRPC3 expression increased only in the PDL area on days 7 and 28. CONCLUSIONS: These results indicate changes of TRPC3 and TRPC6 expression in PDL cells that were differentiating into osteoblasts and in periodontitis-induced tissue, suggesting the need for research on the role of TRPC in osteoblast differentiation or periodontitis progression.

Periodontitis deteriorates renal fibrosis and macrophage infiltration in rats with chronic kidney disease.

OBJECTIVE: The objective of this study was to examine the effect of periodontitis on renal function and morphology in rats with or without nephrectomy (Nx)-induced chronic kidney disease (CKD). METHODS: Rats were divided into sham surgery (Sham), Sham with tooth ligation (ShamL), Nx, and NxL groups. Periodontitis was induced by tooth ligation at 16-week olds. Creatinine, alveolar bone area, and renal histopathology were analyzed at 20-week olds. RESULTS: Creatinine did not differ between the Sham and ShamL groups or between the Nx and NxL groups. The ShamL and NxL groups (both p = 0.002) had less alveolar bone area than the Sham group. The NxL group had fewer glomeruli than the Nx group (p < 0.000). The periodontitis groups demonstrated more tubulointerstitial fibrosis (Sham vs. ShamL p = 0.002, Nx vs. NxL p < 0.000) and macrophage infiltration (Sham vs. ShamL p = 0.002, Nx vs. NxL p = 0.006) than the groups without periodontitis. Only the NxL group had greater renal TNFα expression than the Sham group (p < 0.003). CONCLUSIONS: These suggest that periodontitis increases renal fibrosis and inflammation in the presence or absence of CKD but does not affect renal function. Periodontitis also increases TNFα expression in the presence of CKD.

Alveolar bone and tibia responses to hormonal and mineral abnormalities in rats with chronic kidney disease: A pilot study.

OBJECTIVES: As the impact of chronic kidney disease (CKD) severity on different bone types remains unclear, we induced increasing levels of CKD severity in a rat model and investigated hormone and mineral levels as well as alveolar and tibia bone histomorphology. METHODS: Rats were divided into sham operation (sham), 4/6 nephrectomy (4/6Nx), 5/6Nx, and 4/6Nx with hyperphosphorous (HP) diet (4/6NxHP). At week 20, BUN, FGF23, PTH, and P were estimated in plasma. Bone parameters were evaluated by microCT, and osteoclasts and osteoid areas were evaluated by TRAP and H&E stains, respectively. RESULTS: The 4/6NxHP and 5/6Nx groups had elevated PTH, and the 4/6NxHP group alone had elevated P. Compared to the 4/6Nx group, the 4/6NxHP group demonstrated increased FGF23 and P. In the alveolar bone, the 4/6NxHP group had reduced bone volume and BMD compared to the sham and 4/6Nx groups. In the tibia cortical bone, bone surface density was higher in the 4/6NxHP group compared to the sham group. Tibia cortical bone volume was negatively correlated with FGF23 and P. Moreover, alveolar bone volume was negatively correlated with FGF23, PTH, and P. CONCLUSIONS: Our results demonstrate that hormone and mineral levels vary with CKD severity, and alveolar bone loss strongly correlates with these hormone and mineral alterations.

Distribution of neutrophil and monocyte/macrophage populations induced by the CXCR4 inhibitor AMD3100 in blood and periodontal tissue early after periodontitis induction.

CXCR4, a CXCL12 receptor, is expressed on epithelial cells, fibroblasts, and inflammatory cells. The CXCR4-CXCL12 interaction is related to the migration of neutrophils and monocytes/macrophages. Periodontitis, an inflammatory disease mainly caused by gram-negative bacteria, is characterized by infiltration of circulating inflammatory cells and alveolar bone (AB) loss. To investigate whether CXCR4 is involved in the distribution of neutrophils and monocytes/macrophages early after periodontitis induction, we examined the effects of AMD3100 (AMD), a CXCR4 antagonist, in ligature-induced periodontitis mice and LPS-injected air pouch mice. The periodontitis study was accomplished in control (C), periodontitis (P), and P + AMD groups. Periodontitis was induced by ligation of the mandibular first molar. AMD was intraperitoneally administered daily beginning the day before ligation until sacrifice on the third day after ligation. The air pouch study was accomplished in C, lipopolysaccharide (LPS), and LPS + AMD groups. Air pouches on mice backs were formed by subcutaneous injection of sterilized air. AMD was administered and then LPS was injected into the air pouch. For the detection of neutrophils and monocytes/macrophages in blood and air pouch exudates, flow cytometry was performed with anti-Ly6G/anti-CD11b antibodies (Abs) and anti-CD115 Ab, respectively. In periodontal tissue, Ly6G+ cells and CD115+ cells were counted by immunohistological analysis. AB loss was estimated by the periodontal ligament area in the furcation. In the periodontitis study, the P group showed higher numbers of Ly6G+ cells and CD115+ cells in blood and periodontal tissue than the C group. The P + AMD group showed a greater number of Ly6G+ cells and CD115+ cells in blood, but not in periodontal tissue compared to the P group. There was no difference in AB loss between the P and P + AMD groups. In the air pouch study, the LPS group had higher levels of Ly6G+ CD11b+ cells and CD115+ cells in both blood and exudates than the C group. The number of these cells in the LPS + AMD group was higher in blood than in the LPS group, but not in the exudates. The CXCR4 antagonist further increased neutrophil and monocyte/macrophage populations in the blood, but did not alter the levels in the periodontal tissue and exudates in mice with periodontitis and LPS-injected air pouches. These results suggest that during inflammatory conditions such as periodontitis, CXCR4 is involved in the distribution of neutrophils and monocytes/macrophages in the blood, but not in inflamed peripheral tissues.

Lipopolysaccharides of Fusobacterium nucleatum and Porphyromonas gingivalis increase RANKL-expressing neutrophils in air pouches of mice.

Increases of neutrophils and osteoclasts are pathological changes of periodontitis. RANKL is an osteoclast differentiation factor. The effect of periodontopathogen LPS on RANKL-expressing neutrophils has not been clarified yet. We evaluated numerical changes of RANKL-expressing neutrophils in air pouches of mice injected with LPSs of Fusobacterium nucleatum and Porphyromonas gingivalis. Mice with air pouches were assigned into saline (C)-, E. coli LPS- (Ec LPS)-, F. nucleatum LPS (Fn LPS)-, P. gingivalis LPS (Pg LPS)-, and Fn LPS and Pg LPS (Fn + Pg LPS)-injected groups. CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils in blood and air pouch exudates were determined by flow cytometry. In blood, compared to the C group, the Fn LPS group showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils whereas the Pg LPS group showed no significant differences. These increases in the Fn LPS group were not different to those in the Ec LPS group. In exudates, Fn LPS and Pg LPS groups showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils compared to the C group. Increased levels in the Fn LPS group were not different to those in the Ec LPS group, but Pg LPS group was lower than those in the Ec LPS group. In blood and exudates, the Fn + Pg LPS group showed no difference in levels of these neutrophils compared to the Ec LPS group. LPSs of F. nucleatum and P. gingivalis increased RANKL-expressing neutrophils although the degrees of increases were different. These suggest that periodontopathogen LPS can act as a stimulant to increase RANKL-expressing neutrophils.

The presence of neutrophils causes RANKL expression in periodontal tissue, giving rise to osteoclast formation.

BACKGROUNDS AND OBJECTIVE: Increased neutrophil infiltration and osteoclast formation are key characteristics of periodontitis. The effect of these neutrophils on osteoclast formation in periodontitis remains unclear. Therefore, we investigated the effects of neutrophils on osteoclast formation in a neutrophil-deficient mouse model of periodontitis. METHODS: Anti-Ly6G antibody (Ab) was used for neutrophil depletion in two mouse models: periodontitis and air pouch. In the periodontitis experiments, mice were divided into PBS-administered control (C), control Ab-administered periodontitis (P), and anti-Ly6G Ab-administered periodontitis (P + Ly6G) groups. Periodontitis was induced by ligature of mandibular first molars. In the air pouch experiments, mice were divided into PBS-administered (C), LPS and control Ab-administered (LPS), and LPS and anti-Ly6G Ab-administered (LPS + Ly6G) groups. Neutrophil migration into air pouches was induced by LPS injection. Flow cytometry was used to examine CD11b+ Ly6G+ neutrophils in the blood of periodontitis mice and CD11b+ Ly6G+ RANKL+ neutrophils in exudates of air pouch mice. In periodontal tissue, Ly6G+ neutrophil and RANKL+ cell numbers in periodontal ligament and alveolar bone areas were estimated using immunohistochemistry, osteoclast numbers were measured using TRAP assay, and alveolar bone loss was determined by H&E staining. RESULTS: In blood, CD11b+ Ly6G+ neutrophils were found in greater percentage in the P group than in the C group on days 3 and 7. However, the percentage of neutrophils was lower in the P + Ly6G group than in the C and P groups. In periodontal tissue, the numbers of Ly6G+ neutrophils and RANKL+ cells were lower in the P + Ly6G group than in the P group on day 3. Ly6G+ neutrophil numbers decreased more in the P + Ly6G group than in the P group on day 7, but RANKL+ cell numbers did not decrease in the P + Ly6G group. In exudates, the number of CD11b+ Ly6G+ RANKL+ neutrophils was greater in the LPS group than in the C and LPS + Ly6G groups. On days 3 and 7, the numbers of osteoclasts and alveolar bone loss were greater in periodontal tissue in the P and P + Ly6G groups than in the C group. Interestingly, there were fewer osteoclasts in the P + Ly6G group than in the P group on day 3. CONCLUSION: Neutrophil deficiency caused a reduction in numbers of both RANKL+ cells and osteoclasts in periodontitis-induced tissues only on day 3. Furthermore, in the LPS-injected air pouch model, neutrophil deficiency reduced the influx of RANKL+ neutrophils. These findings suggest that the presence of neutrophils induces RANKL expression and could induce osteoclast formation in the early stages of periodontitis.

The clinicopathologic significance of Notch3 expression in prostate cancer.

The Notch3 signaling pathway plays an important role in oncogenesis, tumor maintenance, and resistance to chemotherapy in human cancers. However, its role in prostate cancer (PC) is less clear. In this study, we investigated a total of 142 PC patients who underwent radical prostatectomy and examined the expression of Notch3 in PC cells using immunohistochemistry on tissue microarrays and evaluated their clinicopathological significance. The overexpression of Notch3 was observed in 22 (15.5%) out of 142 PC cases. The overexpression of Notch3 was significantly associated with lymph node metastasis (P = 0.013), higher pT stages (P = 0.033), higher pathological tumor stages (P = 0.034), and higher grades groups (P = 0.025). However, the overexpression of Notch3 was not correlated with lympho-vascular invasion, neural invasion, extra-prostatic extension, or the serum prostate-specific antigen level. This study demonstrates that Notch3 plays an oncogenic function in PC and the overexpression of Notch3 is correlated with invasiveness, metastasis, and higher Gleason grades, reflecting the features of aggressive tumors in PC, and could be an important biomarker and a possible therapeutic target. Further studies evaluating the association between Notch3 expression and survival are required.

Simvastatin attenuates tibial bone loss in rats with type 1 diabetes and periodontitis.

BACKGROUND: Diabetes induces long bone loss and aggravation of periodontitis-induced alveolar bone loss. Simvastatin (SIM), which is a lipid-lowering agent is known to have an anabolic effect on bone. Therefore, we investigated effect of SIM on tibial and alveolar bone loss in type 1 diabetic rats with periodontitis. METHODS: Rats were divided into control (C), diabetes with periodontitis (DP), and diabetes with periodontitis treated with SIM (DPS) groups. DP and DPS groups were intravenously injected with streptozotocin (50 mg/kg), and C group was injected with citrate buffer. Seven days later (day 0), periodontitis was induced by ligatures of mandibular first molars. DP and DPS groups were orally administered vehicle or SIM (30 mg/kg) from day 0 to days 3, 10, or 20. Alveolar and tibial bone loss was measured using histological and m-CT analysis alone or in combination. Osteoclast number and sclerostin-positive osteocytes in tibiae were evaluated by tartrate-resistant acid phosphatase and immunohistochemical staining, respectively. Glucose, triglyceride (TG), cholesterol (CHO), and low-density lipoprotein (LDL) were evaluated. RESULTS: Consistent with diabetes induction, the DP group showed higher glucose and TG levels at all timepoints and higher CHO levels on day 20 than C group. Compared to the DP group, the DPS group exhibited reduced levels of glucose (day 3), TG (days 10 and 20), CHO, and LDL levels (day 20). Bone loss analysis revealed that the DP group had lower bone volume fraction, bone mineral density, bone surface density, and trabecular number in tibiae than C group at all timepoints. Interestingly, the DPS group exhibited elevation of these indices at early stages compared to the DP group. The DPS group showed reduction of osteoclasts (day 3) and sclerostin-positive osteocytes (days 3 and 20) compared with the DP group. There was no difference in alveolar bone loss between DP and DPS groups. CONCLUSIONS: These results suggest that SIM attenuates tibial, but not alveolar bone loss in type 1 diabetic rats with periodontitis. Moreover, attenuation of tibial bone loss by SIM may be related to inhibition of osteoclast formation and reduction of sclerostin expression.

Intermittent PTH administration improves alveolar bone formation in type 1 diabetic rats with periodontitis.

BACKGROUND: Periodontitis is an infectious disease that manifests as alveolar bone loss surrounding the roots of teeth. Diabetes aggravates periodontitis-induced alveolar bone loss via suppression of bone formation. Intermittent parathyroid hormone (PTH) administration displays an anabolic effect on bone. In this study, we investigated the effect of intermittent PTH administration on alveolar bone loss in type 1 diabetic rats with periodontitis. METHODS: Rats were divided into control (C), periodontitis (P), periodontitis treated with PTH (P + PTH), diabetes with periodontitis (DP), and diabetes with periodontitis treated with PTH (DP + PTH) groups. To induce type 1 diabetes, rats were injected with streptozotocin and periodontitis was induced bilaterally by applying ligatures to the mandibular first molars for 30 days. During the experimental period, the P + PTH and DP + PTH groups were subcutaneously injected with PTH (40 µg/kg) three times per week, whereas the C, P, and DP groups were injected with citrate buffer. To observe the mineralization of the alveolar bone, the DP and DP + PTH groups were injected with calcein on days 10 and 27, and with alizarin red on day 20. Thirty days after ligation, histological findings and fluorescence labeling were analyzed in the furcations of the mandibular first molars. Sclerostin-positive osteocytes were assessed by immunohistochemical analyses. RESULTS: The DP groups had smaller areas of alveolar bone than the other groups, and the DP + PTH group had a larger alveolar bone area than the DP group. The DP group had less osteoid formation than the C group, whereas the DP + PTH had greater osteoid formation than the DP group. Fluorescence labeling results revealed that the DP + PTH group had more mineral deposition on the alveolar bone than the DP group. The DP + PTH group exhibited lower percentage of sclerostin-positive osteocytes in alveolar bone than the DP group. CONCLUSIONS: Intermittent PTH administration diminishes alveolar bone loss and sclerostin expression in osteocytes, but increases osteoid formation and mineralization, suggesting that intermittent PTH administration attenuates diabetes-aggravated alveolar bone loss by the induction of bone formation. PTH-induced bone formation may be related to the regulation of osteocytic sclerostin expression in type 1 diabetic rats with periodontitis.

Tumor necrosis factor-α antagonist diminishes osteocytic RANKL and sclerostin expression in diabetes rats with periodontitis.

Type 1 diabetes with periodontitis shows elevated TNF-α expression. Tumor necrosis factor (TNF)-α stimulates the expression of receptor activator of nuclear factor-κB ligand (RANKL) and sclerostin. The objective of this study was to determine the effect of TNF-α expression of osteocytic RANKL and sclerostin in type 1 diabetes rats with periodontitis using infliximab (IFX), a TNF-α antagonist. Rats were divided into two timepoint groups: day 3 and day 20. Each timepoint group was then divided into four subgroups: 1) control (C, n = 6 for each time point); 2) periodontitis (P, n = 6 for each time point); 3) diabetes with periodontitis (DP, n = 8 for each time point); and 4) diabetes with periodontitis treated with IFX (DP+IFX, n = 8 for each time point). To induce type 1 diabetes, rats were injected with streptozotocin (50 mg/kg dissolved in 0.1 M citrate buffer). Periodontitis was then induced by ligature of the mandibular first molars at day 7 after STZ injection (day 0). IFX was administered once for the 3 day group (on day 0) and twice for the 20 day group (on days 7 and 14). The DP group showed greater alveolar bone loss than the P group on day 20 (P = 0.020). On day 3, higher osteoclast formation and RANKL-positive osteocytes in P group (P = 0.000 and P = 0.011, respectively) and DP group (P = 0.006 and P = 0.017, respectively) than those in C group were observed. However, there was no significant difference in osteoclast formation or RANKL-positive osteocytes between P and DP groups. The DP+IFX group exhibited lower alveolar bone loss (P = 0.041), osteoclast formation (P = 0.019), and RANKL-positive osteocytes (P = 0.009) than that of the DP group. On day 20, DP group showed a lower osteoid area (P = 0.001) and more sclerostin-positive osteocytes (P = 0.000) than P group. On days 3 and 20, the DP+IFX group showed more osteoid area (P = 0.048 and 0.040, respectively) but lower sclerostin-positive osteocytes (both P = 0.000) than DP group. Taken together, these results suggest that TNF-α antagonist can diminish osteocytic RANKL/sclerostin expression and osteoclast formation, eventually recovering osteoid formation. Therefore, TNF-α might mediate alveolar bone loss via inducing expression of osteocytic RANKL and sclerostin in type 1 diabetes rats with periodontitis.

[Needs assessment of a core curriculum for residency training].

PURPOSE: The core curriculum in graduate medical education (GME) is an educational program that covers the minimum body of knowledge and skills that is required of all residents, regardless of their specialty. This study examined the opinions of stakeholders in GME regarding the core curriculum. METHODS: A questionnaire was administered at three tertiary hospitals that were affiliated with one university; 192 residents and 61 faculty members and attending physicians participated in the survey. The questionnaire comprised six items on physician competency and the needs for a core curriculum. Questions on subjects or topics and adequate training years for each topics were asked only to residents. RESULTS: Most residents (78.6%) and faculty members (86.9%) chose "medical expertise" as the "doctor's role in the 21st century." In contrast, communicator, manager, and collaborator were recognized by less than 30% of all participants. Most residents (74.1%) responded that a core curriculum is "necessary but not feasible," whereas 68.3% of faculty members answered that it is "absolutely needed." Regarding subjects that should be included in the core curriculum, residents and faculty members had disparate preferences- residents preferred more "management of a private clinic" and "financial management," whereas faculty members desired "medical ethics" and "communication skills." CONCLUSION: Residents and faculty members agree that residents should develop a wide range of competencies in their training. However, the perception of the feasibility and opinions on the contents of the core curriculum differed between groups. Further studies with larger samples should be conducted to define the roles and professional competencies of physicians and the needs for a core curriculum in GME.

Fine needle aspiration cytology of hepatic hydatid cyst: a case study.

Hydatid cysts (echinococcosis) are caused by an infestation with larval tapeworms of the genus Echinococcus. The disease is extensively distributed worldwide, and it has been rarely reported in Korea. We describe the cytologic features of a case of hepatic hydatid cyst in a 28-year-old male. Computed tomography revealed a cystic mass in the right lobe of the liver. A right hemihepatectomy was performed. The aspirated fluid from the hepatic cystic mass was clear. The smears showed protoscolices, hooklets, and a laminated membrane.

Imprint cytology of soft tissue myoepithelioma: a case study.

Soft tissue myoepithelioma is a rare neoplasm composed of myoepithelial cells. Here, we describe the cytologic features of soft tissue myoepithelioma arising on the right forearm in an 18-year-old man. The excised tumor (3.0×1.8×1.5 cm) was well-demarcated, yellow-gray, soft, and myxoid. The cytologic smears showed round to spindle, epithelioid, and plasmacytoid cells in the myxoid background. The nuclei were uniform, round to ovoid, with finely distributed chromatin and eosinophilic or pale cytoplasm. The tumor cells demonstrated immunoreactivity for cytokeratin (AE1/AE3), epithelial membrane antigen, S100 protein, and glial fibrillary acidic protein. Electron microscopy showed intermediate filaments, desmosomes, and basal lamina.

Imaging of renal and prostate carcinoma with refractive index radiology.

AIM: Having better edge enhancement and penetrating power, refractive index radiology is suitable for the imaging of weakly absorbing objects such as tissue specimens. In this study the potential of refractive index radiology was evaluated for the imaging of renal cell carcinoma (RCC) and prostate cancer (PCA). METHODS: Specimens were cut in 3 mm and 4 microm thickness for X-ray radiology and hematoxylin and eosin (HE) staining, respectively. Radiographic images of RCC and PCA were obtained using the synchrotron hard X-rays from the 7B2 beam-line of the Pohang Light Source (PLS). The imaging technique applied was phase-contrast radiology based on the refraction enhancement mechanism. The resulting radiographic images were analyzed in correlation with those of optical microscopy. RESULTS: Using unmonochromatized hard X-rays, it was possible to obtain images with clear edge enhancement and relatively large field of view (6 cm x 6 cm). Even with overlapping signals from thick samples (more than 700-fold thicker than microscopic images), radiographic images clearly showed histological information of organelles in normal kidney such as glomeruli, tubules, and collecting ducts. Histological information of RCC including tumor subtypes and minute changes such as cystic degeneration could be identified without difficulty. The radiographic images of the prostate were comparable with those of low magnification optical microscopy, providing good visualization of normal microstructures such as adenoma, smooth muscle, and normal glands, or differentiation of tiny tumors from surrounding normal tissues. CONCLUSIONS: These results suggest the potential of refractive index radiology to provide a new way of imaging biological tissues with low absorption contrast such as RCC and PCA.

Substrate binding and catalytic mechanism of human choline acetyltransferase.

Choline acetyltransferase (ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine from choline and acetyl-CoA, and its presence is a defining feature of cholinergic neurons. We report the structure of human ChAT to a resolution of 2.2 A along with structures for binary complexes of ChAT with choline, CoA, and a nonhydrolyzable acetyl-CoA analogue, S-(2-oxopropyl)-CoA. The ChAT-choline complex shows which features of choline are important for binding and explains how modifications of the choline trimethylammonium group can be tolerated by the enzyme. A detailed model of the ternary Michaelis complex fully supports the direct transfer of the acetyl group from acetyl-CoA to choline through a mechanism similar to that seen in the serine hydrolases for the formation of an acyl-enzyme intermediate. Domain movements accompany CoA binding, and a surface loop, which is disordered in the unliganded enzyme, becomes localized and binds directly to the phosphates of CoA, stabilizing the complex. Interactions between this surface loop and CoA may function to lower the KM for CoA and could be important for phosphorylation-dependent regulation of ChAT activity.

Surface-entropy reduction used in the crystallization of human choline acetyltransferase.

Human choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine (ACh) from choline and acetyl-CoA. A crystal structure of human ChAT has been a long-standing goal in the neuronal signalling field. Milligram quantities of pure ChAT can be purified [Kim et al. (2005), Protein Expr. Purif. 40, 107-117], but exhaustive crystallization efforts failed to produce any crystals suitable for high-resolution structural studies. To obtain high-quality crystals of human ChAT, a truncation was made in a large poorly conserved loop region and high-entropy side chains were removed from the surface of the protein. The resulting 'entropy-reduced' ChAT (MR = 68.1 kDa) crystallizes readily and reproducibly and the crystals diffract X-rays to approximately 2.2 A. The availability of these crystals will allow us to study the structure of human ChAT on its own as well as in complex with its substrates and inhibitor molecules, leading to a greater understanding of its catalytic mechanism and regulation.

Two methods for large-scale purification of recombinant human choline acetyltransferase.

Choline acetyltransferase (ChAT) catalyzes the transfer of an acetyl group from acetyl-CoA to choline to produce the neurotransmitter acetylcholine (ACh). We have produced large quantities of pure human ChAT using two different bacterial expression systems. In the first, ChAT is fused to a chitin-binding domain via a self-cleavable linker allowing the release of ChAT without the use of proteases. In the second, ChAT is fused to a hexahistidine (His6) tag at the N-terminus with a linker incorporating a TEV protease cleavage site. In both cases, pure ChAT was produced that has a final specific activity of approximately 50 micromol ACh/min/mg and is suitable for structural characterization. Analysis of purified ChAT by Western blots and mass spectrometry revealed that the C-terminal 15 amino acids were slowly removed by endogenous proteolytic activity, to produce a stable 615 residue protein. Furthermore, we show that purified recombinant human ChAT is highly prone to oxidation, leading to the formation of covalent dimers and/or a loss of catalytic activity. Kinetic parameters of our purified proteins were obtained and, when compared to previously published constants for human placental ChAT, we found that recombinant human ChAT displays lower values for Michaelis and inhibition constants for ACh, which may be due to the complete absence of post-translational modifications.

Protein kinase C isoforms differentially phosphorylate human choline acetyltransferase regulating its catalytic activity.

Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons; regulation of its activity or response to physiological stimuli is poorly understood. We show that ChAT is differentially phosphorylated by protein kinase C (PKC) isoforms on four serines (Ser-440, Ser-346, Ser-347, and Ser-476) and one threonine (Thr-255). This phosphorylation is hierarchical, with phosphorylation at Ser-476 required for phosphorylation at other serines. Phosphorylation at some, but not all, sites regulates basal catalysis and activation. Ser-476 with Ser-440 and Ser-346/347 maintains basal ChAT activity. Ser-440 is targeted by Arg-442 for phosphorylation by PKC. Arg-442 is mutated spontaneously (R442H) in congenital myasthenic syndrome, rendering ChAT inactive and causing neuromuscular failure. This mutation eliminates phosphorylation of Ser-440, and Arg-442, not phosphorylation of Ser-440, appears primarily responsible for ChAT activity, with Ser-440 phosphorylation modulating catalysis. Finally, basal ChAT phosphorylation in neurons is mediated predominantly by PKC at Ser-476, with PKC activation increasing phosphorylation at Ser-440 and enhancing ChAT activity.

[Task analysis of Korean transplantation nurse practitioner].

PURPOSE: This study was designed to create the job description of Korean transplantation nurse practitioner and examine performance frequencies, criticality, and difficulties of task elements. METHOD: The sample consisted of 63 nurses and coordinators who performed duties related to transplantation at medical center in Korea. A survey method was used, and the questionnaire included frequencies, criticality, and difficulties of task elements in job description by the DACUM method. Using SPSS WIN 10.0, descriptive statistics such as frequency distribution, means, and standard deviations were conducted to examine the subject's general characteristics, the frequencies, criticality, and difficulties of task performance. RESULT: The job description of transplantation nurse practitioners revealed 5 duties, 22 tasks, and 85 task elements. On the all five duties, the averages of the performance frequency, criticality, and difficulty were 2.41, 3.38, and 2.78, meaning that the respondents rarely perform the 5 duties, but consider them critical and easy to perform. CONCLUSION: The job description of the transplantation nurse practitioner included duty, task, and task element and definition of job completed. Thus we recommended a data based trial to confirm and validate the information gathered.